Research ArticleParainfluenza Virus Type 1 Induces Epithelial IL-8Production via p38-MAPK Signalling

Miguel Aacutengel Galvaacuten Morales Carlos Cabello Gutieacuterrez Fidencio Mejiacutea NepomucenoLeticia Valle Peralta Elba Valencia Maqueda and Mariacutea Eugenia Manjarrez Zavala

Department of Virology and Mycology Research National Institute of Respiratory Diseases ldquoIsmael Cosio VillegasrdquoCalzada de Tlalpan 4502 Colonia Seccion XVI Delegacion Tlalpan 14080 Mexico DF Mexico

Correspondence should be addressed to Miguel Angel Galvan Morales ygalvan2000yahoocommx

Received 12 December 2013 Revised 11 May 2014 Accepted 23 May 2014 Published 11 June 2014

Academic Editor Michael H Kershaw

Copyright copy 2014 Miguel Angel Galvan Morales et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Human parainfluenza virus type 1 (HPIV-1) is the most common cause of croup in infants The aim of this study was to describemolecularmechanisms associatedwith IL-8 production duringHPIV-1 infection and the role of viral replication inMAPK synthesisand activation An in vitro model of HPIV-1 infection in the HEp-2 and A549 cell lines was used a kinetic-based ELISA for IL-8 detection was also used phosphorylation of the mitogen-activated protein kinases (MAPKs) was identified by Western blotanalysis and specific inhibitors for each kinase were used to identify which MAPK was involved Inactivated viruses were used toassess whether viral replication is required for IL-8 production Results revealed a gradual increase in IL-8 production at differentselected times when phosphorylation of MAPK was detected The secretion of IL-8 in the two cell lines infected with the HPIV-1is related to the phosphorylation of the MAPK as well as viral replication Inhibition of p38 suppressed the secretion of IL-8 in theHEp-2 cells No kinase activation was observed when viruses were inactivated

1 Introduction

Human parainfluenza virus type 1 (HPIV-1) which is a mem-ber of the family Paramyxoviridae is an enveloped virus witha single-stranded RNA genome [1 2] HPIV-1 infects the up-per and lower respiratory tract and causes acute respiratoryinfections (ARIs) ranging from mild infections such as thecommon cold and laryngitis to severe infections such ascroup pneumonia and bronchiolitis HPIV-1 is responsiblefor almost half of all hospitalizations due to ARIs both inpatients younger than 5 years old and in the elderly addition-ally HPIV-1 is the most common cause of infectious laryngo-tracheitis (croup) in children [3ndash6]The therapy used to treatsymptoms of inflammation is based on glucocorticoid andephedrine also humidifying the airway however this is notalways effective [7ndash9] The pathogenic mechanisms activatedby HPIV-1 during infection are largely unknown Local re-sponse mechanisms have been described in which innate and

adaptive defence systems participateThere is no evidence in-dicating that mitogenic signal activation is required in theearly stages of infection [10 11] IL-8 is a mediator responsiblefor the recruitment of neutrophils that participate in the localinflammatory infiltrate contributing to airway closure [1213] It has been reported that epithelial cells alveolar macro-phages and neutrophils secrete IL-8 [14ndash17] Other authorshave reported that infection with respiratory syncytial virus(RSV) varicella-zoster virus and smallpox virus activatesIL-8 secretion without viral replication [18 19] These obser-vations indicate that the interaction between the virus and itsreceptor is sufficient to promote the signalling pathways thatactivate the IL-8 gene [20] however replication is necessaryin other viruses such as vaccinia virus and rhinovirus [21ndash23] It has been shown that viruses have different effects on theregulation of IL-8 expression and secretionThemost promi-nent examples include the filovirusesMarburg and Ebola andarenaviruses such as Lassa and Junin Other viruses such as

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2014 Article ID 515984 12 pageshttpdxdoiorg1011552014515984

2 Journal of Immunology Research

RSV adenovirus vaccine virus and herpes virus secrete IL-8immediately [24 25]

The primary signalling pathways that elicit a response bychemokines are theMAPKs and transcription factor NF kap-pa B pathways MAPKs are a family of proteins that activatekinases through a cascade of intracellular phosphorylationevents and signal transduction from the cell surface to thenucleus They are composed of three well-characterized sub-families extracellular signal-regulated kinases (ERK12) c-Jun N-terminal kinases (JNKs) and p38 mitogen-activatedproteinkinases (p38) Each subfamily includes a kinase thatsequentially acts on three proteinsMEKKMEK andMAPKEach protein is activated through phosphorylation TheMAPK family substrates in the cytoplasm and nucleus in-clude additional kinases transcription factors phospholi-pases and cytoskeletal proteins ERK12 is associated withanabolic processes such as cell division growth and differ-entiation while JNK and p38 are associated with cellularresponses to stress conditions death and inflammation [26ndash28]

The molecular mechanisms in which larynx epithelialcells play a role and their active involvement in the inflam-matory infiltration response to infection by HPIV-1 throughproduction of small chemoattractants that recruit neutrophilshave not been investigated sufficiently to generate a strategythat counteracts pathogenesis and to determine whether viralreplication is necessary In this study an in vitro modelof HPIV-1 infection of HEp-2 and A549 cells was used tosimulate the upper and lower airways The aim of this studywas to determine whether viral replication induces the IL-8secretion and MAPK kinase signalization

2 Materials and Methods

21 Cell Lines TheHEp-2 human laryngeal epithelioma type2 cells (ATCCCCL-23USAwhich reported a contaminationwithHeLa cells) andA549 human alveolar type II-like epithe-lial cells (ATCC CCL-185 USA) were acquired from Ameri-can Type Culture Collection (Manassas VA USA) Cell lineswere propagated in 25 cm3 flasks (Nunc Thermo ScientificInc Turnberry USA) and Petri dishes with Eaglersquos MinimalEssential Medium (MEM) supplemented with 10 foetalbovine serum and antibiotics (Penicillin 100UmL Strepto-mycin 100 120583gmL Amphotericin-B 1 120583gmL) from InvitrogenLife Technologies (Gaithersburg MD) Cells were main-tained at 37∘C in 5 CO

2and were stored and grown in

accordance with the manufacturerrsquos instructions

22 HPIV-1 Propagation Virus strain was obtained fromAmerican Type Culture Collection Manassas VA USA(ATCC number VR-94 strain C35 HPIV-1) Confluentmonolayers of HEp-2 and A549 cells in 25 cm3 flasks wereinoculated with HPIV-1 and incubated at 37∘C in 5 CO

2

until a cytopathic effect was observed 48 hours after inocula-tion (formation of syncytia) The virus was titrated by plaqueassay using methylcellulose One aliquot of the virus wastransferred to 18mL cryotubes after titration and stored atminus70∘C and another aliquot was used to infect cell monolayers

at a 001 multiplicity of infection (MOI) Uninfected HEp-2and A549 cell monolayers were used as controls

23 HPIV-1 Inactivation A portion of HPIV-1 (5mL of virusat a titre of 6 times 106 PFU) in serum-free medium (RPMI) wasplaced in a sealed box and irradiated at a distance of 10 cmfor 10 minutes with optimal radiation levels (1200 units times100 120583Jcm2) using an FB-UVXL 1000UV cross-linker FisherBiotech (Thermo Fisher Scientific Inc KY USA) as previ-ously described [29] Inactivationwas verified by plaque assayon monolayers of HEp-2 and A549 cells Haemagglutinationassays were performed in 96-well plates to verify the haemag-glutinin activity ofHPIV-1 using serial dilutions ofO-positivehuman blood (1 2 1 4 etc) [30] Results are expressed inhemagglutinating units (HAU) [31 32]

24 Quantitation of IL-8 HEp-2 andA549 cells were infectedwith live and inactive HPIV-1 at an MOI of 001 supernatantwas collected at different times (30 45 60 90 and 120 min-utes and 12 and 24 hours) and uninfected HEp-2 and A549cells were used as controls IL-8 concentrations were quan-titated by ELISA according to the procedure described byKit-tigul et al (1998) [33] using anti-human CXCL8IL-8 mono-clonal antibodies fromRampDSystems Inc (MinneapolisMNUSA) Optical density at 450 nm was measured on an ELISAreader spectrophotometer from Thermo Labsystems (SantaRosa CA USA and Helsinki Finland)

25 Reverse Transcription-Polymerase Chain Reaction Theexpression of mRNA for IL-8 was determined by reversetranscription-polymerase chain reaction (RT-PCR) TotalRNA was isolated by using SV Total RNA Isolation System(Promega Madison USA) following the manufacturerrsquos pro-tocol A 05 120583g portion of total RNA was reverse-transcribedfor subsequent PCR amplification for each pair of primersin a volume of 20120583L including 200U M-MLV RT reversetranscriptase (Invitrogen CA USA) 50U of RNase inhibitor(Sigma) 02120583g of random primer 10mM of dNTP Mix(Invitrogen CA USA) and 5x First-Strand Buffer (250mMTris-HCl pH 83 375mMKCl 15mMMgCl

2) provided by

Life Technologies The reaction was incubated at 42∘C for50min A 20120583L portion of the RT products was then broughtto a volume of 20120583L containing 10mM of each dNTP1 U of Taq polymerase (Promega Madison USA) 10 pmolof both the upstream and downstream PCR primers and1 x PCR buffer (proprietary formulation supplied at pH 85containing blue dye and yellow dye) provided by PromegaThe primers for IL-8 are as follows forward 51015840-ATG ACTTCC AAG CTG GCC GTG GCT-31015840 and reverse 51015840-TCTCAG CCC TCT TCA AAA ACT TCT-31015840 with a product of289 bps [34] For glyceraldehyde-3-phosphate dehydrogenase(GAPDH) the forward primer was 51015840-CCAGCCGAGCCACATCGCTC-3 and the reverse primer was 51015840-ATGAGCCC-CAGCCTTCTCCAT-31015840 giving a 390 bps PCR product TheGAPDH gene was amplified in the same reaction to serveas the reference gene Amplification was carried out in aBiometra personal thermal cycler after an initial denaturationat 94∘C for 3minThis was followed by 35 cycles of PCR using

Journal of Immunology Research 3

the following temperature and time profile denaturation at94∘C for 40 s primer annealing at 58∘C for 40 s primerextension at 72∘C for 1min and a final extension of 72∘C for6min The PCR products were visualised by electrophoresison a 2 agarose gel and visualized by UV transillumination

26 Detection of MAPK Phosphorylation (ERK12 JNKandor p38) HEp-2 and A549 cells were infected with liveor inactive HPIV-1 at an MOI of 001 with the same kineticsas that described above in the previous assays Cells were har-vested and total protein was extracted and quantified by theBradford method Cells were boiled at 100∘C for 10 minutesin a buffer solution supplemented with 120573-mercaptoethanolThe obtained proteins were separated on 15 SDS-PAGEgels and transferred to a 045 120583m nitrocellulose membranefrom Bio-Rad Laboratories (Hercules CA) for 35 hoursand Western blot assays were performed Transfer efficiencywas determined by staining with 01 Ponceau red in 5glacial acetic acid from Sigma-Aldrich (St Louis MO)and measurements were performed using Laemmlirsquos SDS-PAGE system [35] Membranes were washed blocked andprobed overnight with MAPK phospho-ERK12 (sc-81492)MAPK ERK12 (sc-292838) phospho-p38 MAPK (sc-7973)p38MAPK (sc-535) phospho-JNK (sc-135642) and JNK (sc-571) antibodies (1 3 000 dilution) purchased from Santa CruzBiotechnology (Santa Cruz CA) The bound antibody wascoated with a peroxidase-conjugated goat anti-mouse IgGsecondary antibody (Sigma-Aldrich) (1 3 000) for 2 hoursMembranes were washed and incubated with a luminalchemiluminescent substrate from Santa Cruz Biotechnologybefore exposure to X-ray film The band intensity wasquantified using the Quantity One (Bio-Rad) Image Analysissoftware

27 IL-8 Quantitation in the Presence of Kinase InhibitorsFor the best reproduction of larynx conditions assays wereconducted using HEp-2 cells that were pretreated with thekinase inhibitors PD980559 for ERK12 SB203580lowast for p38MAPK and SP600125lowast for JNK fromCalbiochemMilliporelowast(San Diego CA) The concentrations used were from 5 to20120583M (13 to 52 120583L) in DMSO the same DMSO concen-tration was added to control cells and was incubated at37∘C One mL of HPIV-1 at an MOI of 001 was addedand infected cells were incubated under the same conditionsSupernatants were collected at the selected times and IL-8concentrations were determined Supernatants from HEp-2cells without inhibitors collected at the same infection timewere used as controls When the MAPK inhibitor wasused cells were pretreated with the inhibitor 1 hour beforeinfection Because MAPK inhibitors were diluted in DMSOequal concentrations of DMSO were added to the untreatedinfected cells as control After treatment with the inhibitorIL-8 concentrations were measured and supernatant wascollected at the different times tested

28 Quantitation of the Bands Theanalysis done in theWest-ern blot bands was performed in the Quantity One softwarefrom Bio-Rad Individual quantification of the bands was

A549 cells withHPIV-1 UV

Control

HEp-2 cells withHPIV-1 UV

A549 cells withHPIV-1 live

HEp-2 cells withHPIV-1 live

(c)

(a)(b)

UFP = nfd (log)4(10)(1 times 105) = 4 times 106

UFP = 4000000 (mL)

6 times 106 = 25 cm boxfor an moi of 01

10minus1 10minus2 10minus3 10minus4 10minus5

Figure 1 Lytic plaque assay The number of viral particles wasdetermined by dilution In a 24-well plate with HEp-2 and A549cell cultures each lytic plaque that develops is initially related toa single infectious virion particle This method was performed todetermine the viability of both viruses live and inactive (a) refersto the cell type and the characteristic of the virus (inactive and live)(b) corresponds to dilutions ranging from 10minus1 to 10minus5 and (c) refersto plaque-forming units This assay was performed in triplicate

made from the intensity of light generated in them due to thefluorescence emitted in the radiographic plate

29 Statistical Analyses Data were tested for normality(Shapiro-Wilk) and since data did not show normal distribu-tion Kruskal-Wallis test was used to compare the differenttreatments The assays were performed in triplicate and theresults were statistically analysed using Statistica Software 50(Stat Soft Tulsa Oklahoma) A 119875 value lt005 was consideredto indicate a statistically significant difference

3 Results

31 Evaluation of HPIV-1 Inactivation The inactivatedviruses were created to demonstrate whether viral replicationwas involved in the activation of the secretion of IL-8 or viralstructure alone was sufficient to generate the response to thischemokineThe virus was inactive with UV radiation plaqueassays were performed to verify that the virus was foundinactive and will not be replicated

Exponential virus dilutions are presented in Figure 1 todetermine the number of infectious particles (a)The first tworows correspond to the viral activity with the inactive viruswhere there was no replication due to the absence of PFU Inthe remaining rows of live virus activity in both cell types wasevaluated Panel (b) corresponds to viral dilutions and panel(c) refers to the number of infective particles in a dilution typecellular

Because the inactive virus did not replicate haemaggluti-nation tests were made showing that inactive virus particleshad the same ability to agglutinate erythrocytes and the active

4 Journal of Immunology Research

Table 1 IL-8 secretion (pgmL) in HEp-2 and A549 cells infected with live and inactive virus

Time HEp-2 live virus A549 live virus HEp-2 inactive virus A549 inactive virus SD SEM Chi square 119875 CVUninfected cells 0004a 0001a 0002a 0027a 0018 0005 371 029 20749301015840 3901b 4704a 002c 006c 2265 654 1038 001 10516451015840 5503b 6211a 003c 003c 3069 886 935 002 10474601015840 10415a 9272b 001c 004c 5157 1489 1038 001 10474901015840 13238a 13507a 133b 100b 6924 1999 946 002 102651201015840 21008a 20172b 002c 001c 10756 3105 935 002 1044712 hours 26572a 23984b 001c 001c 13236 3821 943 002 1047124 hours 14506a 12930b 205c 200c 7083 2045 946 002 10174abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

L LUV

(a)

(b)

1 1

1 2

1 4

1 8

1 16

1 32

25120583L125 120583L

625 120583L

3125 120583L

175120583L

0875120583L

PBS 50120583L

1 HAU = 2 times 105 ivp1 32 = 0875mL of virus

for moi of 01(0875)(6 times 105)2 times 105

HPIV-1

Control (mdash)Erythrocytes 50120583L

Figure 2Haemagglutinating capacity of both live and inactive viruswas determined in a 96-well plate (a) Serial dilutions of the virus1 2 1 32 (b) hemagglutinating units (HAU) L corresponds to thelive virus lane and UV corresponds to the inactive virus This assaywas performed in triplicate

virus (L) In both lytic and haemagglutination assays expo-nential dilutions were prepared to determine the virus viabil-ity (Figure 2)

These tests are often used to demonstrate the viral repli-cation for the presence of hemagglutinin glycoprotein or via-ble structures

32 IL-8 Secretion in Cell Lines Infected with Live and InactiveVirus IL-8 secretion was quantified duringHPIV-1 infectionsuggesting that infection of cell lines with live HPIV-1induced a gradual increase in IL-8 secretion The highest IL-8 secretion in HEp-2 and A549 was observed at 12 hoursafter infection whereas the cells with inactive virus showeda marginal response at 24 hours (205 and 20) The higher

secretion of IL-8 was found using infected HEp-2 cellsfollowed by infected A549 cells (Table 1) compared withthe cells with inactive virus where a minimal response wasachieved In the case of HEp-2 cells and A549 cells treatedwith inactive virus no IL-8 secretion was observed neither inthe uninfected cells used as controls

33 Reverse Transcription-Polymerase Chain Reaction ThemRNA expression of IL-8 in A549 andHEp-2 cells was deter-mined by RT-PCR analysis In the analysis uninfected cellscells infected with HPIV-infected cells and HPIV-1 inactivewere used The increased expression of IL-8 originated frominfected cells followed by uninfected cells and the inactivatedvirus and the expression in the latter two was almost equalInfection was performed at different times and behavior foreach was similar in both cell lines (Figure 3) GAPDH wasconstitutively expressed in both cell lines and expression didnot vary by infection

34 HPIV-1 Infection Stimulates MAPK Phosphorylation Toestablish whether the inactive virus stimulated the phospho-rylation of MAPKs (ERK JNK and p38) Western blot testswere made (Figure 4) The two different cell lines HEp-2 cellline for the upper portion of the tract and the A549 for thebottom line of the same tract were used to simulate the respir-atory tract

Western blot assays were performed to determine thephosphorylation status of theMAPKs ERK12 JNK and p38In HEp-2 cells infection with live HPIV-1 promoted earlyERK12 phosphorylation beginning at 60minutes of interac-tion (Figures 5(a) and 5(b) band intensities were quantifiedthrough densitometric analysis) Phosphorylation increasedat 90 and 120 minutes However ERK phosphorylation washigher at 12 hours than at any other selected time and de-creased at 24 hours In the case of inactivated virus the 12-hour band was only in lane 3 because no evidence of phos-phorylation was observed in the kinetics selected times Thesame criteria were applied to the Western blot figures only aband corresponding to a 12-hour interaction was observedwith no increase in phosphorylation before and after this timepoint Controls consisted of uninfected cells which showedno significant changes

Journal of Immunology Research 5

Uninfected Uninfected

Inoculated withHPIV-1

UV inactivated

Inoculated withHPIV-1

UV inactivated

Infected withHPIV-1

Infected withHPIV-1

GPDHGPDH

MW White

Cell A549

289pb

289pb

289pb

289pb

289pb

289pb

390pb390pb

HEp-2 cell

60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24 MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

(min) (hrs) (min) (hrs)

Figure 3 IL8 expression by RT-PCR analysis in HEp2 and A549 cells infected uninfected and the HPVI-1 inactiveThe enhanced expressionof IL8 was given in infected cells HPVI-1 GAPDH expression was not different by infection

HEp-2 cell

p-ERKvirus UV

p-ERKvirus UV

virus UVp-JNK

virus UVp-JNK

virus UVp-p38

virus UVp-p38

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

A549 cell

Figure 4 Kinetics of expression of MAPKs in HEp-2 and A549 cells after inoculation with HPIV-1 virus inactive ERK12 JNK and p38phosphorylation showed no time points analyzed during 30min to 24 hrs Ctl 2nd lane (minus) refers to the proteins of uninfected cells The Ctl(+) corresponds to the proteins from cells infected with live virus (90min) Antibodies used are described inMaterials andMethodsWesternblot assays were performed in triplicate

In A549 cells infected with live HPIV-1 the detection ofERK12 phosphorylation began at 60 minutes of interactiona small phosphorylation signal was observed at 90minutes ofinteraction (Figures 5(c) and 5(d)) increasing slightly at 120minutes and remaining constant until the 24-hour time pointPhosphorylation was slightly detected for the inactivatedvirus and control samples In controls (uninfected cells)inactive virus samples and samples thatwere incubated for 30and 45 minutes phosphorylation was only slightly detectedWith inactive viruses the result was similar to the previousWestern blot (Figures 5(c) lane 4 and 5(d) lane 3)

JNK phosphorylation in HEp-2 cells also began at 60minutes and gradually increased until the 90-minute timepoint but decreased at 120 minutes By 12 and 24 hours phos-phorylation ceased (Figures 6(a) and 6(b)) As inHEp-2 cellsJNK phosphorylation in A549 cells began at 60 minutes in-creased at 120minutes and decreased at 12 and 24 hours withno further increase (Figures 6(c) and 6(d) lanes 9 and 10)

Likewise p38 phosphorylation in the HEp-2 cell linebegan at 30 minutes (Figures 7(a) and 7(b)) increased againat 120 minutes and subsequently remained stable until the24-hour time point In A549 cells p38 phosphorylation levelswere similar beginning at 30 minutes (Figures 7(c) and 7(d))

but with a gradual increase from the 90-minute time pointand achieving a maximum at 12 and 24 hours

35 IL-8 Secretion Decreased with HPIV-1 Infection throughp38 Inhibition Thekinase inhibition assaywas performed onthe cell line HEp-2 derived from human laryngeal epithe-lioma given that is a suitable target for the in vitro virus assayOur results suggest that p38 ERK12 and JNK are activatedfollowing HPIV-1 infectionTherefore to determine whetherthese kinases are required for IL-8 gene expression ELISAswere performed using the supernatant from HEp-2 cells pre-treated with ERK12 JNK and p38 kinase inhibitors andinfected with HPIV-1 at the following time points 45 60 and90 minutes and 12 and 24 hours (Figure 9) Results demon-strate that the p38 inhibitor repressed IL-8 production uponviral infection whereas ERK12 and JNK MAPK inhibitorsdid not inhibit IL-8 production (Figures 9(a) and 9(b)) sug-gesting that p38 MAPK signalling is involved in IL-8 pro-duction Figure 9(c) depicts the average of three experimentswith HEp-2 cells infected with HPIV-1 and inhibitors Con-trols without inhibitors for each kinase were included for alltime points

6 Journal of Immunology Research

UVCtlMp

pERK-12

HEp-2

ERK-12

30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pERK HEp-2

30998400 45998400 60998400 90998400 120998400 12h 24h

Den

sity

(IN

Tm

m2)

(b)

A549

pERK-12

ERK-12

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 pERK A549

30998400 45998400 60998400 90998400 120998400 12h 24hD

ensit

y (I

NT

mm2)

(d)

Figure 5 ERK phosphorylationDetection of phosphorylation of ERK12 byWestern blot in protein extracts of HEp-2 andA549 cells infectedwith HPIV-1 live at different time points (a) phospho-ERK was detected with p-ERK12 (sc-81492) antibody as a loading control and totalERK12 (sc-292838) in HEp-2 cells (b) densitometric analysis of ERK12 protein concentrations (c) phospho-ERK and total ERK12 proteinconcentrations in A549 cells (d) densitometric analysis of protein concentrations Mp molecular weight CTL control and UV (inactivevirus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the three separate experiments

Table 2 IL-8 production (pgmL) in infected HEp-2 cells treated with ERK12 JNK and p38 kinase inhibitors

Time ERK12 JNK P38 Uninfected cells SD SEM Chi square 119875 CV451015840 4188a 4371a 006b 5004a 2094 604 874 003 6171601015840 10216a 9527a 1110c 9248b 7526 1125 946 002 5178901015840 13925a 14391a 3973b 13585a 4551 1314 843 003 39681201015840 17971a 16971a 5948b 18199a 5430 1568 761 005 367512 hours 24308a 24005a 15278b 24607a 4133 1193 658 008 187424 hours 13639ab 14450a 12951b 13707ab 650 188 746 005 474abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

Treating the cells with JNK and ERK12 inhibitors did notimpair IL-8 production but treating cells with an inhibitor ofp38 MAPK significantly impaired IL-8 secretion in most ofthe incubation times (before 24 h) ERK12 and JNK inhib-itors were not different from the control group (Table 2)

Total inhibitory dose MAPKs (ERK12 JNK and p38)were evaluated showing that the optimal concentration tosuppress phosphorylation was 20120583M (Figure 8)

4 Discussion

In the present study an in vitromodel with two cell lines thatwere infected with HPIV-1 at different time points was usedto determine whether an increase in IL-8 secretion occurredOur assay showed that IL-8 secretion gradually increasedduring the time points assessed

These results are consistent with reports that other respi-ratory viruses including influenza A virus (H3N2) and RSVinduce IL-8 secretion from bronchial epithelial cells [36 37]

It is known that IL-8 is responsible for the recruitmentof neutrophils and macrophages and that it performs otherfunctions such as adhesion and degranulation Likewise thepresence of IL-8 in inflammatory infiltrates in lung tissues hasbeen reported Studies performed with different viruses haverevealed that epithelial cells infected with specific virusesrelease IL-8 without requiring viral replication as whatoccurs in RSV infections [18 19] Contact with the cellularreceptor is sufficient to trigger IL-8 secretion whereas otherviruses only promote IL-8 synthesis during replication [2123] In this study assays using both live and inactivatedHPIV-1 were performed to determine whether replication-independent IL-8 release occurs with HPIV-1 a virus from

Journal of Immunology Research 7

p-JNK

HEp-2

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pJNK HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

A549

p-JNK

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)Time

180

0

60

UVControl

120

240

300 pJNK A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 6 JNK phosphorylation Detection of JNK phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infectedwith live and inactive HPIV-1 at different time points and JNK phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) phospho-JNK was detectedwith monoclonal antibody sc-135642 and the total JNK sc-571 in HEp-2 cells (b) densitometric analysis of JNK protein concentrations (c)phospho-JNK and total protein concentrations in A549 cells (d) densitometric analysis of the protein concentrations Mp molecular weightCTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the threeseparate experiments

the same family as RSV The results indicate that contact be-tween the virus and target cell is insufficient even when hae-magglutination activity is intact in the inactivated virus andviral replication is required to stimulate IL-8 secretion

Previous reports have shown that IL-8 secretion is in-volved in MAPK pathway activation [12 16] These kinasesrespond to different ligands and stimuli and are involved insignalling pathways and cellular processes such as prolifer-ation differentiation inflammation immune response andapoptosis

HPIV-1 does not use the JNK pathway for IL-8 activationwhereas p38 and ERK are phosphorylated when IL-8 ispresent in the supernatant after infection

The results of this study showed that HPVI-1 does not usethe JNK pathway for activation of IL-8 while theMAPKs p38and ERK are phosphorylated by the production of IL-8 afterthe infection similar results have been described after RSVinfection (virus belonging to the Paramyxoviridae family) inhuman lung endothelial cells where ERK phosphorylationenabled IL-8se creation [37]

Therefore to define which kinases are required for IL-8secretion the same assays were performed using specificinhibitors for each kinaseThe results showed that even in thepresence of specific inhibitors of ERK and JNK IL-8 was stillsecreted but there was no secretion in the presence of p38

inhibitor In this case a complete suppression of IL-8 secre-tionwas observedwhich suggests that p38MAPK is the path-way through which HPIV-1 induces IL-8 production

It is known that the inhibitor SB203580 is a potent cell-permeable selective and reversible inhibitor of p38120572 and 120573isoforms Inhibition is competitive with ATP and requiressubstrates to accommodate the fluorophenyl ring structure ofthe pyridinyl imidazole in their ATP-binding pocket Struc-tures of other p38 isoforms JNKs and ERK12 do not allowinhibitor binding at equivalent positions [38 39]

Many members of the Paramyxovirus family have beenshown to be potent inducers of chemokines of theCXC familysuch as interleukin-8 (IL-8) including respiratory syncytialvirus (RSV) Sendai virus human parainfluenza virus type 2(HPIV-2) HPIV-3 and Newcastle disease virus [40]

Paramyxoviruses have two glycoproteins that are impor-tant during infection The receptor-binding glycoprotein isHN in parainfluenza virus and mumps virus H in measlesvirus andG in pneumoviruses (RSV andmetapneumovirus)The second glycoprotein is the fusion protein (F) which catal-yses membrane fusion (viral and cellular) It has been sug-gested that HN glycoproteins form a complex that is initiatedby the binding HN activating the fusion protein that actsimmediately in such a way that the virus can enter the cellHowever it must be noted that glycoprotein HN acts as both

8 Journal of Immunology Research

p-p38

HEp-2

p38

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 p-p38 HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

p-p38

p38

A549UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 p-p38 A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 7 p38 phosphorylation Detection of p38 phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infected withlive and inactivated HPIV-1 at different time points and p38 phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) Total protein concentrationsin HEp-2 cells Phospho-p38 was detected with monoclonal antibody sc-7973 and the total p38 sc-535 in HEp-2 cells (b) densitometricanalysis of p38 protein concentrations (c) phospho-p38 and total protein concentrations in A549 cells (d) densitometric analysis of theprotein concentrations Mp molecular weight CTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are themeans for triplicate measurements from the three separate experiments

PD980559 (120583M)

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

minus + + + +

minus + + + +

minus + + + +

SP600125 (120583M)

SB203580 (120583M)

p-ERK 12

p-JNK

p-p38

Figure 8 Inhibitors of ERK12 PD980559 JNK SP600125 and p38 SB203580 inhibited the expression of each of theMAPKs in the HEp-2 cellline treated cell 1 hour before infection and maintained for 120 minutes after the test The experiments were performed by adding inhibitorat concentrations from 5120583M to 20 120583M (13 to 52 120583L resp) Protein levels were detected by Western blot Immunoblotting was done withmonoclonal antibody specific for protein phosphorylated technique that was described in ldquoMaterials and Methodsrdquo The extracted proteins(minus) represent HEp-2 cells without infecting and the proteins (+) refer to cells infected with the HPIV-1The tests were performed in triplicate

Journal of Immunology Research 9

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

ERKControl

(a)IL

-8 (p

gm

L)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

JNKControl

(b)

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

lowast

lowast

lowast

lowast

p38Control

lowast P gt 005

(c)

Figure 9 Kinetic inhibition ofMAPKs ELISA quantitation of IL-8 concentrations in cells pretreated with ERK12 PD980559 JNK SP600125and SB203580 inhibitors Control is HEp-2 cells infected with live HPIV-1 (a) ERK12 inhibitor assay is displayed in red cells treated with theinhibitor (b) JNK inhibitor assay green occurs in cells treated with the inhibitor (c) p38 inhibitor assay purple occurs in cells treated withinhibitor Data are the means plusmn SD for triplicate measurements from the three separate experiments

a haemagglutinin (HA) and a neuraminidase (NA) bindingto sialic acidmolecules through its HA activity mediating theenzymatic cleavage of sialic acid and promoting fusionthrough its neuraminidase activityThis dual activity suggeststhat coexpressed HN and F form a complex Glycoprotein Hof morbillivirus only has haemagglutinin activity and gly-coprotein G has neither of these two activities [41ndash44]

Therefore these glycoproteins exhibit significant differencesSuch differences are likely related to signalling pathways thatare activated when viruses interact with the cell AlthoughHPIV-1 and RSV belong to the same family the two viruseshave different glycoproteins which may explain why RSVuses ERK kinase andHPIV-1 uses p38 kinase Following virusbinding to its cellular receptor the extracellular stimulus is

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Journal of Immunology Research

RSV adenovirus vaccine virus and herpes virus secrete IL-8immediately [24 25]

The primary signalling pathways that elicit a response bychemokines are theMAPKs and transcription factor NF kap-pa B pathways MAPKs are a family of proteins that activatekinases through a cascade of intracellular phosphorylationevents and signal transduction from the cell surface to thenucleus They are composed of three well-characterized sub-families extracellular signal-regulated kinases (ERK12) c-Jun N-terminal kinases (JNKs) and p38 mitogen-activatedproteinkinases (p38) Each subfamily includes a kinase thatsequentially acts on three proteinsMEKKMEK andMAPKEach protein is activated through phosphorylation TheMAPK family substrates in the cytoplasm and nucleus in-clude additional kinases transcription factors phospholi-pases and cytoskeletal proteins ERK12 is associated withanabolic processes such as cell division growth and differ-entiation while JNK and p38 are associated with cellularresponses to stress conditions death and inflammation [26ndash28]

The molecular mechanisms in which larynx epithelialcells play a role and their active involvement in the inflam-matory infiltration response to infection by HPIV-1 throughproduction of small chemoattractants that recruit neutrophilshave not been investigated sufficiently to generate a strategythat counteracts pathogenesis and to determine whether viralreplication is necessary In this study an in vitro modelof HPIV-1 infection of HEp-2 and A549 cells was used tosimulate the upper and lower airways The aim of this studywas to determine whether viral replication induces the IL-8secretion and MAPK kinase signalization

2 Materials and Methods

21 Cell Lines TheHEp-2 human laryngeal epithelioma type2 cells (ATCCCCL-23USAwhich reported a contaminationwithHeLa cells) andA549 human alveolar type II-like epithe-lial cells (ATCC CCL-185 USA) were acquired from Ameri-can Type Culture Collection (Manassas VA USA) Cell lineswere propagated in 25 cm3 flasks (Nunc Thermo ScientificInc Turnberry USA) and Petri dishes with Eaglersquos MinimalEssential Medium (MEM) supplemented with 10 foetalbovine serum and antibiotics (Penicillin 100UmL Strepto-mycin 100 120583gmL Amphotericin-B 1 120583gmL) from InvitrogenLife Technologies (Gaithersburg MD) Cells were main-tained at 37∘C in 5 CO

2and were stored and grown in

accordance with the manufacturerrsquos instructions

22 HPIV-1 Propagation Virus strain was obtained fromAmerican Type Culture Collection Manassas VA USA(ATCC number VR-94 strain C35 HPIV-1) Confluentmonolayers of HEp-2 and A549 cells in 25 cm3 flasks wereinoculated with HPIV-1 and incubated at 37∘C in 5 CO

2

until a cytopathic effect was observed 48 hours after inocula-tion (formation of syncytia) The virus was titrated by plaqueassay using methylcellulose One aliquot of the virus wastransferred to 18mL cryotubes after titration and stored atminus70∘C and another aliquot was used to infect cell monolayers

at a 001 multiplicity of infection (MOI) Uninfected HEp-2and A549 cell monolayers were used as controls

23 HPIV-1 Inactivation A portion of HPIV-1 (5mL of virusat a titre of 6 times 106 PFU) in serum-free medium (RPMI) wasplaced in a sealed box and irradiated at a distance of 10 cmfor 10 minutes with optimal radiation levels (1200 units times100 120583Jcm2) using an FB-UVXL 1000UV cross-linker FisherBiotech (Thermo Fisher Scientific Inc KY USA) as previ-ously described [29] Inactivationwas verified by plaque assayon monolayers of HEp-2 and A549 cells Haemagglutinationassays were performed in 96-well plates to verify the haemag-glutinin activity ofHPIV-1 using serial dilutions ofO-positivehuman blood (1 2 1 4 etc) [30] Results are expressed inhemagglutinating units (HAU) [31 32]

24 Quantitation of IL-8 HEp-2 andA549 cells were infectedwith live and inactive HPIV-1 at an MOI of 001 supernatantwas collected at different times (30 45 60 90 and 120 min-utes and 12 and 24 hours) and uninfected HEp-2 and A549cells were used as controls IL-8 concentrations were quan-titated by ELISA according to the procedure described byKit-tigul et al (1998) [33] using anti-human CXCL8IL-8 mono-clonal antibodies fromRampDSystems Inc (MinneapolisMNUSA) Optical density at 450 nm was measured on an ELISAreader spectrophotometer from Thermo Labsystems (SantaRosa CA USA and Helsinki Finland)

25 Reverse Transcription-Polymerase Chain Reaction Theexpression of mRNA for IL-8 was determined by reversetranscription-polymerase chain reaction (RT-PCR) TotalRNA was isolated by using SV Total RNA Isolation System(Promega Madison USA) following the manufacturerrsquos pro-tocol A 05 120583g portion of total RNA was reverse-transcribedfor subsequent PCR amplification for each pair of primersin a volume of 20120583L including 200U M-MLV RT reversetranscriptase (Invitrogen CA USA) 50U of RNase inhibitor(Sigma) 02120583g of random primer 10mM of dNTP Mix(Invitrogen CA USA) and 5x First-Strand Buffer (250mMTris-HCl pH 83 375mMKCl 15mMMgCl

2) provided by

Life Technologies The reaction was incubated at 42∘C for50min A 20120583L portion of the RT products was then broughtto a volume of 20120583L containing 10mM of each dNTP1 U of Taq polymerase (Promega Madison USA) 10 pmolof both the upstream and downstream PCR primers and1 x PCR buffer (proprietary formulation supplied at pH 85containing blue dye and yellow dye) provided by PromegaThe primers for IL-8 are as follows forward 51015840-ATG ACTTCC AAG CTG GCC GTG GCT-31015840 and reverse 51015840-TCTCAG CCC TCT TCA AAA ACT TCT-31015840 with a product of289 bps [34] For glyceraldehyde-3-phosphate dehydrogenase(GAPDH) the forward primer was 51015840-CCAGCCGAGCCACATCGCTC-3 and the reverse primer was 51015840-ATGAGCCC-CAGCCTTCTCCAT-31015840 giving a 390 bps PCR product TheGAPDH gene was amplified in the same reaction to serveas the reference gene Amplification was carried out in aBiometra personal thermal cycler after an initial denaturationat 94∘C for 3minThis was followed by 35 cycles of PCR using

Journal of Immunology Research 3

the following temperature and time profile denaturation at94∘C for 40 s primer annealing at 58∘C for 40 s primerextension at 72∘C for 1min and a final extension of 72∘C for6min The PCR products were visualised by electrophoresison a 2 agarose gel and visualized by UV transillumination

26 Detection of MAPK Phosphorylation (ERK12 JNKandor p38) HEp-2 and A549 cells were infected with liveor inactive HPIV-1 at an MOI of 001 with the same kineticsas that described above in the previous assays Cells were har-vested and total protein was extracted and quantified by theBradford method Cells were boiled at 100∘C for 10 minutesin a buffer solution supplemented with 120573-mercaptoethanolThe obtained proteins were separated on 15 SDS-PAGEgels and transferred to a 045 120583m nitrocellulose membranefrom Bio-Rad Laboratories (Hercules CA) for 35 hoursand Western blot assays were performed Transfer efficiencywas determined by staining with 01 Ponceau red in 5glacial acetic acid from Sigma-Aldrich (St Louis MO)and measurements were performed using Laemmlirsquos SDS-PAGE system [35] Membranes were washed blocked andprobed overnight with MAPK phospho-ERK12 (sc-81492)MAPK ERK12 (sc-292838) phospho-p38 MAPK (sc-7973)p38MAPK (sc-535) phospho-JNK (sc-135642) and JNK (sc-571) antibodies (1 3 000 dilution) purchased from Santa CruzBiotechnology (Santa Cruz CA) The bound antibody wascoated with a peroxidase-conjugated goat anti-mouse IgGsecondary antibody (Sigma-Aldrich) (1 3 000) for 2 hoursMembranes were washed and incubated with a luminalchemiluminescent substrate from Santa Cruz Biotechnologybefore exposure to X-ray film The band intensity wasquantified using the Quantity One (Bio-Rad) Image Analysissoftware

27 IL-8 Quantitation in the Presence of Kinase InhibitorsFor the best reproduction of larynx conditions assays wereconducted using HEp-2 cells that were pretreated with thekinase inhibitors PD980559 for ERK12 SB203580lowast for p38MAPK and SP600125lowast for JNK fromCalbiochemMilliporelowast(San Diego CA) The concentrations used were from 5 to20120583M (13 to 52 120583L) in DMSO the same DMSO concen-tration was added to control cells and was incubated at37∘C One mL of HPIV-1 at an MOI of 001 was addedand infected cells were incubated under the same conditionsSupernatants were collected at the selected times and IL-8concentrations were determined Supernatants from HEp-2cells without inhibitors collected at the same infection timewere used as controls When the MAPK inhibitor wasused cells were pretreated with the inhibitor 1 hour beforeinfection Because MAPK inhibitors were diluted in DMSOequal concentrations of DMSO were added to the untreatedinfected cells as control After treatment with the inhibitorIL-8 concentrations were measured and supernatant wascollected at the different times tested

28 Quantitation of the Bands Theanalysis done in theWest-ern blot bands was performed in the Quantity One softwarefrom Bio-Rad Individual quantification of the bands was

A549 cells withHPIV-1 UV

Control

HEp-2 cells withHPIV-1 UV

A549 cells withHPIV-1 live

HEp-2 cells withHPIV-1 live

(c)

(a)(b)

UFP = nfd (log)4(10)(1 times 105) = 4 times 106

UFP = 4000000 (mL)

6 times 106 = 25 cm boxfor an moi of 01

10minus1 10minus2 10minus3 10minus4 10minus5

Figure 1 Lytic plaque assay The number of viral particles wasdetermined by dilution In a 24-well plate with HEp-2 and A549cell cultures each lytic plaque that develops is initially related toa single infectious virion particle This method was performed todetermine the viability of both viruses live and inactive (a) refersto the cell type and the characteristic of the virus (inactive and live)(b) corresponds to dilutions ranging from 10minus1 to 10minus5 and (c) refersto plaque-forming units This assay was performed in triplicate

made from the intensity of light generated in them due to thefluorescence emitted in the radiographic plate

29 Statistical Analyses Data were tested for normality(Shapiro-Wilk) and since data did not show normal distribu-tion Kruskal-Wallis test was used to compare the differenttreatments The assays were performed in triplicate and theresults were statistically analysed using Statistica Software 50(Stat Soft Tulsa Oklahoma) A 119875 value lt005 was consideredto indicate a statistically significant difference

3 Results

31 Evaluation of HPIV-1 Inactivation The inactivatedviruses were created to demonstrate whether viral replicationwas involved in the activation of the secretion of IL-8 or viralstructure alone was sufficient to generate the response to thischemokineThe virus was inactive with UV radiation plaqueassays were performed to verify that the virus was foundinactive and will not be replicated

Exponential virus dilutions are presented in Figure 1 todetermine the number of infectious particles (a)The first tworows correspond to the viral activity with the inactive viruswhere there was no replication due to the absence of PFU Inthe remaining rows of live virus activity in both cell types wasevaluated Panel (b) corresponds to viral dilutions and panel(c) refers to the number of infective particles in a dilution typecellular

Because the inactive virus did not replicate haemaggluti-nation tests were made showing that inactive virus particleshad the same ability to agglutinate erythrocytes and the active

4 Journal of Immunology Research

Table 1 IL-8 secretion (pgmL) in HEp-2 and A549 cells infected with live and inactive virus

Time HEp-2 live virus A549 live virus HEp-2 inactive virus A549 inactive virus SD SEM Chi square 119875 CVUninfected cells 0004a 0001a 0002a 0027a 0018 0005 371 029 20749301015840 3901b 4704a 002c 006c 2265 654 1038 001 10516451015840 5503b 6211a 003c 003c 3069 886 935 002 10474601015840 10415a 9272b 001c 004c 5157 1489 1038 001 10474901015840 13238a 13507a 133b 100b 6924 1999 946 002 102651201015840 21008a 20172b 002c 001c 10756 3105 935 002 1044712 hours 26572a 23984b 001c 001c 13236 3821 943 002 1047124 hours 14506a 12930b 205c 200c 7083 2045 946 002 10174abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

L LUV

(a)

(b)

1 1

1 2

1 4

1 8

1 16

1 32

25120583L125 120583L

625 120583L

3125 120583L

175120583L

0875120583L

PBS 50120583L

1 HAU = 2 times 105 ivp1 32 = 0875mL of virus

for moi of 01(0875)(6 times 105)2 times 105

HPIV-1

Control (mdash)Erythrocytes 50120583L

Figure 2Haemagglutinating capacity of both live and inactive viruswas determined in a 96-well plate (a) Serial dilutions of the virus1 2 1 32 (b) hemagglutinating units (HAU) L corresponds to thelive virus lane and UV corresponds to the inactive virus This assaywas performed in triplicate

virus (L) In both lytic and haemagglutination assays expo-nential dilutions were prepared to determine the virus viabil-ity (Figure 2)

These tests are often used to demonstrate the viral repli-cation for the presence of hemagglutinin glycoprotein or via-ble structures

32 IL-8 Secretion in Cell Lines Infected with Live and InactiveVirus IL-8 secretion was quantified duringHPIV-1 infectionsuggesting that infection of cell lines with live HPIV-1induced a gradual increase in IL-8 secretion The highest IL-8 secretion in HEp-2 and A549 was observed at 12 hoursafter infection whereas the cells with inactive virus showeda marginal response at 24 hours (205 and 20) The higher

secretion of IL-8 was found using infected HEp-2 cellsfollowed by infected A549 cells (Table 1) compared withthe cells with inactive virus where a minimal response wasachieved In the case of HEp-2 cells and A549 cells treatedwith inactive virus no IL-8 secretion was observed neither inthe uninfected cells used as controls

33 Reverse Transcription-Polymerase Chain Reaction ThemRNA expression of IL-8 in A549 andHEp-2 cells was deter-mined by RT-PCR analysis In the analysis uninfected cellscells infected with HPIV-infected cells and HPIV-1 inactivewere used The increased expression of IL-8 originated frominfected cells followed by uninfected cells and the inactivatedvirus and the expression in the latter two was almost equalInfection was performed at different times and behavior foreach was similar in both cell lines (Figure 3) GAPDH wasconstitutively expressed in both cell lines and expression didnot vary by infection

34 HPIV-1 Infection Stimulates MAPK Phosphorylation Toestablish whether the inactive virus stimulated the phospho-rylation of MAPKs (ERK JNK and p38) Western blot testswere made (Figure 4) The two different cell lines HEp-2 cellline for the upper portion of the tract and the A549 for thebottom line of the same tract were used to simulate the respir-atory tract

Western blot assays were performed to determine thephosphorylation status of theMAPKs ERK12 JNK and p38In HEp-2 cells infection with live HPIV-1 promoted earlyERK12 phosphorylation beginning at 60minutes of interac-tion (Figures 5(a) and 5(b) band intensities were quantifiedthrough densitometric analysis) Phosphorylation increasedat 90 and 120 minutes However ERK phosphorylation washigher at 12 hours than at any other selected time and de-creased at 24 hours In the case of inactivated virus the 12-hour band was only in lane 3 because no evidence of phos-phorylation was observed in the kinetics selected times Thesame criteria were applied to the Western blot figures only aband corresponding to a 12-hour interaction was observedwith no increase in phosphorylation before and after this timepoint Controls consisted of uninfected cells which showedno significant changes

Journal of Immunology Research 5

Uninfected Uninfected

Inoculated withHPIV-1

UV inactivated

Inoculated withHPIV-1

UV inactivated

Infected withHPIV-1

Infected withHPIV-1

GPDHGPDH

MW White

Cell A549

289pb

289pb

289pb

289pb

289pb

289pb

390pb390pb

HEp-2 cell

60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24 MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

(min) (hrs) (min) (hrs)

Figure 3 IL8 expression by RT-PCR analysis in HEp2 and A549 cells infected uninfected and the HPVI-1 inactiveThe enhanced expressionof IL8 was given in infected cells HPVI-1 GAPDH expression was not different by infection

HEp-2 cell

p-ERKvirus UV

p-ERKvirus UV

virus UVp-JNK

virus UVp-JNK

virus UVp-p38

virus UVp-p38

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

A549 cell

Figure 4 Kinetics of expression of MAPKs in HEp-2 and A549 cells after inoculation with HPIV-1 virus inactive ERK12 JNK and p38phosphorylation showed no time points analyzed during 30min to 24 hrs Ctl 2nd lane (minus) refers to the proteins of uninfected cells The Ctl(+) corresponds to the proteins from cells infected with live virus (90min) Antibodies used are described inMaterials andMethodsWesternblot assays were performed in triplicate

In A549 cells infected with live HPIV-1 the detection ofERK12 phosphorylation began at 60 minutes of interactiona small phosphorylation signal was observed at 90minutes ofinteraction (Figures 5(c) and 5(d)) increasing slightly at 120minutes and remaining constant until the 24-hour time pointPhosphorylation was slightly detected for the inactivatedvirus and control samples In controls (uninfected cells)inactive virus samples and samples thatwere incubated for 30and 45 minutes phosphorylation was only slightly detectedWith inactive viruses the result was similar to the previousWestern blot (Figures 5(c) lane 4 and 5(d) lane 3)

JNK phosphorylation in HEp-2 cells also began at 60minutes and gradually increased until the 90-minute timepoint but decreased at 120 minutes By 12 and 24 hours phos-phorylation ceased (Figures 6(a) and 6(b)) As inHEp-2 cellsJNK phosphorylation in A549 cells began at 60 minutes in-creased at 120minutes and decreased at 12 and 24 hours withno further increase (Figures 6(c) and 6(d) lanes 9 and 10)

Likewise p38 phosphorylation in the HEp-2 cell linebegan at 30 minutes (Figures 7(a) and 7(b)) increased againat 120 minutes and subsequently remained stable until the24-hour time point In A549 cells p38 phosphorylation levelswere similar beginning at 30 minutes (Figures 7(c) and 7(d))

but with a gradual increase from the 90-minute time pointand achieving a maximum at 12 and 24 hours

35 IL-8 Secretion Decreased with HPIV-1 Infection throughp38 Inhibition Thekinase inhibition assaywas performed onthe cell line HEp-2 derived from human laryngeal epithe-lioma given that is a suitable target for the in vitro virus assayOur results suggest that p38 ERK12 and JNK are activatedfollowing HPIV-1 infectionTherefore to determine whetherthese kinases are required for IL-8 gene expression ELISAswere performed using the supernatant from HEp-2 cells pre-treated with ERK12 JNK and p38 kinase inhibitors andinfected with HPIV-1 at the following time points 45 60 and90 minutes and 12 and 24 hours (Figure 9) Results demon-strate that the p38 inhibitor repressed IL-8 production uponviral infection whereas ERK12 and JNK MAPK inhibitorsdid not inhibit IL-8 production (Figures 9(a) and 9(b)) sug-gesting that p38 MAPK signalling is involved in IL-8 pro-duction Figure 9(c) depicts the average of three experimentswith HEp-2 cells infected with HPIV-1 and inhibitors Con-trols without inhibitors for each kinase were included for alltime points

6 Journal of Immunology Research

UVCtlMp

pERK-12

HEp-2

ERK-12

30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pERK HEp-2

30998400 45998400 60998400 90998400 120998400 12h 24h

Den

sity

(IN

Tm

m2)

(b)

A549

pERK-12

ERK-12

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 pERK A549

30998400 45998400 60998400 90998400 120998400 12h 24hD

ensit

y (I

NT

mm2)

(d)

Figure 5 ERK phosphorylationDetection of phosphorylation of ERK12 byWestern blot in protein extracts of HEp-2 andA549 cells infectedwith HPIV-1 live at different time points (a) phospho-ERK was detected with p-ERK12 (sc-81492) antibody as a loading control and totalERK12 (sc-292838) in HEp-2 cells (b) densitometric analysis of ERK12 protein concentrations (c) phospho-ERK and total ERK12 proteinconcentrations in A549 cells (d) densitometric analysis of protein concentrations Mp molecular weight CTL control and UV (inactivevirus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the three separate experiments

Table 2 IL-8 production (pgmL) in infected HEp-2 cells treated with ERK12 JNK and p38 kinase inhibitors

Time ERK12 JNK P38 Uninfected cells SD SEM Chi square 119875 CV451015840 4188a 4371a 006b 5004a 2094 604 874 003 6171601015840 10216a 9527a 1110c 9248b 7526 1125 946 002 5178901015840 13925a 14391a 3973b 13585a 4551 1314 843 003 39681201015840 17971a 16971a 5948b 18199a 5430 1568 761 005 367512 hours 24308a 24005a 15278b 24607a 4133 1193 658 008 187424 hours 13639ab 14450a 12951b 13707ab 650 188 746 005 474abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

Treating the cells with JNK and ERK12 inhibitors did notimpair IL-8 production but treating cells with an inhibitor ofp38 MAPK significantly impaired IL-8 secretion in most ofthe incubation times (before 24 h) ERK12 and JNK inhib-itors were not different from the control group (Table 2)

Total inhibitory dose MAPKs (ERK12 JNK and p38)were evaluated showing that the optimal concentration tosuppress phosphorylation was 20120583M (Figure 8)

4 Discussion

In the present study an in vitromodel with two cell lines thatwere infected with HPIV-1 at different time points was usedto determine whether an increase in IL-8 secretion occurredOur assay showed that IL-8 secretion gradually increasedduring the time points assessed

These results are consistent with reports that other respi-ratory viruses including influenza A virus (H3N2) and RSVinduce IL-8 secretion from bronchial epithelial cells [36 37]

It is known that IL-8 is responsible for the recruitmentof neutrophils and macrophages and that it performs otherfunctions such as adhesion and degranulation Likewise thepresence of IL-8 in inflammatory infiltrates in lung tissues hasbeen reported Studies performed with different viruses haverevealed that epithelial cells infected with specific virusesrelease IL-8 without requiring viral replication as whatoccurs in RSV infections [18 19] Contact with the cellularreceptor is sufficient to trigger IL-8 secretion whereas otherviruses only promote IL-8 synthesis during replication [2123] In this study assays using both live and inactivatedHPIV-1 were performed to determine whether replication-independent IL-8 release occurs with HPIV-1 a virus from

Journal of Immunology Research 7

p-JNK

HEp-2

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pJNK HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

A549

p-JNK

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)Time

180

0

60

UVControl

120

240

300 pJNK A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 6 JNK phosphorylation Detection of JNK phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infectedwith live and inactive HPIV-1 at different time points and JNK phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) phospho-JNK was detectedwith monoclonal antibody sc-135642 and the total JNK sc-571 in HEp-2 cells (b) densitometric analysis of JNK protein concentrations (c)phospho-JNK and total protein concentrations in A549 cells (d) densitometric analysis of the protein concentrations Mp molecular weightCTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the threeseparate experiments

the same family as RSV The results indicate that contact be-tween the virus and target cell is insufficient even when hae-magglutination activity is intact in the inactivated virus andviral replication is required to stimulate IL-8 secretion

Previous reports have shown that IL-8 secretion is in-volved in MAPK pathway activation [12 16] These kinasesrespond to different ligands and stimuli and are involved insignalling pathways and cellular processes such as prolifer-ation differentiation inflammation immune response andapoptosis

HPIV-1 does not use the JNK pathway for IL-8 activationwhereas p38 and ERK are phosphorylated when IL-8 ispresent in the supernatant after infection

The results of this study showed that HPVI-1 does not usethe JNK pathway for activation of IL-8 while theMAPKs p38and ERK are phosphorylated by the production of IL-8 afterthe infection similar results have been described after RSVinfection (virus belonging to the Paramyxoviridae family) inhuman lung endothelial cells where ERK phosphorylationenabled IL-8se creation [37]

Therefore to define which kinases are required for IL-8secretion the same assays were performed using specificinhibitors for each kinaseThe results showed that even in thepresence of specific inhibitors of ERK and JNK IL-8 was stillsecreted but there was no secretion in the presence of p38

inhibitor In this case a complete suppression of IL-8 secre-tionwas observedwhich suggests that p38MAPK is the path-way through which HPIV-1 induces IL-8 production

It is known that the inhibitor SB203580 is a potent cell-permeable selective and reversible inhibitor of p38120572 and 120573isoforms Inhibition is competitive with ATP and requiressubstrates to accommodate the fluorophenyl ring structure ofthe pyridinyl imidazole in their ATP-binding pocket Struc-tures of other p38 isoforms JNKs and ERK12 do not allowinhibitor binding at equivalent positions [38 39]

Many members of the Paramyxovirus family have beenshown to be potent inducers of chemokines of theCXC familysuch as interleukin-8 (IL-8) including respiratory syncytialvirus (RSV) Sendai virus human parainfluenza virus type 2(HPIV-2) HPIV-3 and Newcastle disease virus [40]

Paramyxoviruses have two glycoproteins that are impor-tant during infection The receptor-binding glycoprotein isHN in parainfluenza virus and mumps virus H in measlesvirus andG in pneumoviruses (RSV andmetapneumovirus)The second glycoprotein is the fusion protein (F) which catal-yses membrane fusion (viral and cellular) It has been sug-gested that HN glycoproteins form a complex that is initiatedby the binding HN activating the fusion protein that actsimmediately in such a way that the virus can enter the cellHowever it must be noted that glycoprotein HN acts as both

8 Journal of Immunology Research

p-p38

HEp-2

p38

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 p-p38 HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

p-p38

p38

A549UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 p-p38 A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 7 p38 phosphorylation Detection of p38 phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infected withlive and inactivated HPIV-1 at different time points and p38 phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) Total protein concentrationsin HEp-2 cells Phospho-p38 was detected with monoclonal antibody sc-7973 and the total p38 sc-535 in HEp-2 cells (b) densitometricanalysis of p38 protein concentrations (c) phospho-p38 and total protein concentrations in A549 cells (d) densitometric analysis of theprotein concentrations Mp molecular weight CTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are themeans for triplicate measurements from the three separate experiments

PD980559 (120583M)

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

minus + + + +

minus + + + +

minus + + + +

SP600125 (120583M)

SB203580 (120583M)

p-ERK 12

p-JNK

p-p38

Figure 8 Inhibitors of ERK12 PD980559 JNK SP600125 and p38 SB203580 inhibited the expression of each of theMAPKs in the HEp-2 cellline treated cell 1 hour before infection and maintained for 120 minutes after the test The experiments were performed by adding inhibitorat concentrations from 5120583M to 20 120583M (13 to 52 120583L resp) Protein levels were detected by Western blot Immunoblotting was done withmonoclonal antibody specific for protein phosphorylated technique that was described in ldquoMaterials and Methodsrdquo The extracted proteins(minus) represent HEp-2 cells without infecting and the proteins (+) refer to cells infected with the HPIV-1The tests were performed in triplicate

Journal of Immunology Research 9

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

ERKControl

(a)IL

-8 (p

gm

L)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

JNKControl

(b)

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

lowast

lowast

lowast

lowast

p38Control

lowast P gt 005

(c)

Figure 9 Kinetic inhibition ofMAPKs ELISA quantitation of IL-8 concentrations in cells pretreated with ERK12 PD980559 JNK SP600125and SB203580 inhibitors Control is HEp-2 cells infected with live HPIV-1 (a) ERK12 inhibitor assay is displayed in red cells treated with theinhibitor (b) JNK inhibitor assay green occurs in cells treated with the inhibitor (c) p38 inhibitor assay purple occurs in cells treated withinhibitor Data are the means plusmn SD for triplicate measurements from the three separate experiments

a haemagglutinin (HA) and a neuraminidase (NA) bindingto sialic acidmolecules through its HA activity mediating theenzymatic cleavage of sialic acid and promoting fusionthrough its neuraminidase activityThis dual activity suggeststhat coexpressed HN and F form a complex Glycoprotein Hof morbillivirus only has haemagglutinin activity and gly-coprotein G has neither of these two activities [41ndash44]

Therefore these glycoproteins exhibit significant differencesSuch differences are likely related to signalling pathways thatare activated when viruses interact with the cell AlthoughHPIV-1 and RSV belong to the same family the two viruseshave different glycoproteins which may explain why RSVuses ERK kinase andHPIV-1 uses p38 kinase Following virusbinding to its cellular receptor the extracellular stimulus is

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

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Oxidative Medicine and Cellular Longevity

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Journal of Immunology Research 3

the following temperature and time profile denaturation at94∘C for 40 s primer annealing at 58∘C for 40 s primerextension at 72∘C for 1min and a final extension of 72∘C for6min The PCR products were visualised by electrophoresison a 2 agarose gel and visualized by UV transillumination

26 Detection of MAPK Phosphorylation (ERK12 JNKandor p38) HEp-2 and A549 cells were infected with liveor inactive HPIV-1 at an MOI of 001 with the same kineticsas that described above in the previous assays Cells were har-vested and total protein was extracted and quantified by theBradford method Cells were boiled at 100∘C for 10 minutesin a buffer solution supplemented with 120573-mercaptoethanolThe obtained proteins were separated on 15 SDS-PAGEgels and transferred to a 045 120583m nitrocellulose membranefrom Bio-Rad Laboratories (Hercules CA) for 35 hoursand Western blot assays were performed Transfer efficiencywas determined by staining with 01 Ponceau red in 5glacial acetic acid from Sigma-Aldrich (St Louis MO)and measurements were performed using Laemmlirsquos SDS-PAGE system [35] Membranes were washed blocked andprobed overnight with MAPK phospho-ERK12 (sc-81492)MAPK ERK12 (sc-292838) phospho-p38 MAPK (sc-7973)p38MAPK (sc-535) phospho-JNK (sc-135642) and JNK (sc-571) antibodies (1 3 000 dilution) purchased from Santa CruzBiotechnology (Santa Cruz CA) The bound antibody wascoated with a peroxidase-conjugated goat anti-mouse IgGsecondary antibody (Sigma-Aldrich) (1 3 000) for 2 hoursMembranes were washed and incubated with a luminalchemiluminescent substrate from Santa Cruz Biotechnologybefore exposure to X-ray film The band intensity wasquantified using the Quantity One (Bio-Rad) Image Analysissoftware

27 IL-8 Quantitation in the Presence of Kinase InhibitorsFor the best reproduction of larynx conditions assays wereconducted using HEp-2 cells that were pretreated with thekinase inhibitors PD980559 for ERK12 SB203580lowast for p38MAPK and SP600125lowast for JNK fromCalbiochemMilliporelowast(San Diego CA) The concentrations used were from 5 to20120583M (13 to 52 120583L) in DMSO the same DMSO concen-tration was added to control cells and was incubated at37∘C One mL of HPIV-1 at an MOI of 001 was addedand infected cells were incubated under the same conditionsSupernatants were collected at the selected times and IL-8concentrations were determined Supernatants from HEp-2cells without inhibitors collected at the same infection timewere used as controls When the MAPK inhibitor wasused cells were pretreated with the inhibitor 1 hour beforeinfection Because MAPK inhibitors were diluted in DMSOequal concentrations of DMSO were added to the untreatedinfected cells as control After treatment with the inhibitorIL-8 concentrations were measured and supernatant wascollected at the different times tested

28 Quantitation of the Bands Theanalysis done in theWest-ern blot bands was performed in the Quantity One softwarefrom Bio-Rad Individual quantification of the bands was

A549 cells withHPIV-1 UV

Control

HEp-2 cells withHPIV-1 UV

A549 cells withHPIV-1 live

HEp-2 cells withHPIV-1 live

(c)

(a)(b)

UFP = nfd (log)4(10)(1 times 105) = 4 times 106

UFP = 4000000 (mL)

6 times 106 = 25 cm boxfor an moi of 01

10minus1 10minus2 10minus3 10minus4 10minus5

Figure 1 Lytic plaque assay The number of viral particles wasdetermined by dilution In a 24-well plate with HEp-2 and A549cell cultures each lytic plaque that develops is initially related toa single infectious virion particle This method was performed todetermine the viability of both viruses live and inactive (a) refersto the cell type and the characteristic of the virus (inactive and live)(b) corresponds to dilutions ranging from 10minus1 to 10minus5 and (c) refersto plaque-forming units This assay was performed in triplicate

made from the intensity of light generated in them due to thefluorescence emitted in the radiographic plate

29 Statistical Analyses Data were tested for normality(Shapiro-Wilk) and since data did not show normal distribu-tion Kruskal-Wallis test was used to compare the differenttreatments The assays were performed in triplicate and theresults were statistically analysed using Statistica Software 50(Stat Soft Tulsa Oklahoma) A 119875 value lt005 was consideredto indicate a statistically significant difference

3 Results

31 Evaluation of HPIV-1 Inactivation The inactivatedviruses were created to demonstrate whether viral replicationwas involved in the activation of the secretion of IL-8 or viralstructure alone was sufficient to generate the response to thischemokineThe virus was inactive with UV radiation plaqueassays were performed to verify that the virus was foundinactive and will not be replicated

Exponential virus dilutions are presented in Figure 1 todetermine the number of infectious particles (a)The first tworows correspond to the viral activity with the inactive viruswhere there was no replication due to the absence of PFU Inthe remaining rows of live virus activity in both cell types wasevaluated Panel (b) corresponds to viral dilutions and panel(c) refers to the number of infective particles in a dilution typecellular

Because the inactive virus did not replicate haemaggluti-nation tests were made showing that inactive virus particleshad the same ability to agglutinate erythrocytes and the active

4 Journal of Immunology Research

Table 1 IL-8 secretion (pgmL) in HEp-2 and A549 cells infected with live and inactive virus

Time HEp-2 live virus A549 live virus HEp-2 inactive virus A549 inactive virus SD SEM Chi square 119875 CVUninfected cells 0004a 0001a 0002a 0027a 0018 0005 371 029 20749301015840 3901b 4704a 002c 006c 2265 654 1038 001 10516451015840 5503b 6211a 003c 003c 3069 886 935 002 10474601015840 10415a 9272b 001c 004c 5157 1489 1038 001 10474901015840 13238a 13507a 133b 100b 6924 1999 946 002 102651201015840 21008a 20172b 002c 001c 10756 3105 935 002 1044712 hours 26572a 23984b 001c 001c 13236 3821 943 002 1047124 hours 14506a 12930b 205c 200c 7083 2045 946 002 10174abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

L LUV

(a)

(b)

1 1

1 2

1 4

1 8

1 16

1 32

25120583L125 120583L

625 120583L

3125 120583L

175120583L

0875120583L

PBS 50120583L

1 HAU = 2 times 105 ivp1 32 = 0875mL of virus

for moi of 01(0875)(6 times 105)2 times 105

HPIV-1

Control (mdash)Erythrocytes 50120583L

Figure 2Haemagglutinating capacity of both live and inactive viruswas determined in a 96-well plate (a) Serial dilutions of the virus1 2 1 32 (b) hemagglutinating units (HAU) L corresponds to thelive virus lane and UV corresponds to the inactive virus This assaywas performed in triplicate

virus (L) In both lytic and haemagglutination assays expo-nential dilutions were prepared to determine the virus viabil-ity (Figure 2)

These tests are often used to demonstrate the viral repli-cation for the presence of hemagglutinin glycoprotein or via-ble structures

32 IL-8 Secretion in Cell Lines Infected with Live and InactiveVirus IL-8 secretion was quantified duringHPIV-1 infectionsuggesting that infection of cell lines with live HPIV-1induced a gradual increase in IL-8 secretion The highest IL-8 secretion in HEp-2 and A549 was observed at 12 hoursafter infection whereas the cells with inactive virus showeda marginal response at 24 hours (205 and 20) The higher

secretion of IL-8 was found using infected HEp-2 cellsfollowed by infected A549 cells (Table 1) compared withthe cells with inactive virus where a minimal response wasachieved In the case of HEp-2 cells and A549 cells treatedwith inactive virus no IL-8 secretion was observed neither inthe uninfected cells used as controls

33 Reverse Transcription-Polymerase Chain Reaction ThemRNA expression of IL-8 in A549 andHEp-2 cells was deter-mined by RT-PCR analysis In the analysis uninfected cellscells infected with HPIV-infected cells and HPIV-1 inactivewere used The increased expression of IL-8 originated frominfected cells followed by uninfected cells and the inactivatedvirus and the expression in the latter two was almost equalInfection was performed at different times and behavior foreach was similar in both cell lines (Figure 3) GAPDH wasconstitutively expressed in both cell lines and expression didnot vary by infection

34 HPIV-1 Infection Stimulates MAPK Phosphorylation Toestablish whether the inactive virus stimulated the phospho-rylation of MAPKs (ERK JNK and p38) Western blot testswere made (Figure 4) The two different cell lines HEp-2 cellline for the upper portion of the tract and the A549 for thebottom line of the same tract were used to simulate the respir-atory tract

Western blot assays were performed to determine thephosphorylation status of theMAPKs ERK12 JNK and p38In HEp-2 cells infection with live HPIV-1 promoted earlyERK12 phosphorylation beginning at 60minutes of interac-tion (Figures 5(a) and 5(b) band intensities were quantifiedthrough densitometric analysis) Phosphorylation increasedat 90 and 120 minutes However ERK phosphorylation washigher at 12 hours than at any other selected time and de-creased at 24 hours In the case of inactivated virus the 12-hour band was only in lane 3 because no evidence of phos-phorylation was observed in the kinetics selected times Thesame criteria were applied to the Western blot figures only aband corresponding to a 12-hour interaction was observedwith no increase in phosphorylation before and after this timepoint Controls consisted of uninfected cells which showedno significant changes

Journal of Immunology Research 5

Uninfected Uninfected

Inoculated withHPIV-1

UV inactivated

Inoculated withHPIV-1

UV inactivated

Infected withHPIV-1

Infected withHPIV-1

GPDHGPDH

MW White

Cell A549

289pb

289pb

289pb

289pb

289pb

289pb

390pb390pb

HEp-2 cell

60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24 MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

(min) (hrs) (min) (hrs)

Figure 3 IL8 expression by RT-PCR analysis in HEp2 and A549 cells infected uninfected and the HPVI-1 inactiveThe enhanced expressionof IL8 was given in infected cells HPVI-1 GAPDH expression was not different by infection

HEp-2 cell

p-ERKvirus UV

p-ERKvirus UV

virus UVp-JNK

virus UVp-JNK

virus UVp-p38

virus UVp-p38

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

A549 cell

Figure 4 Kinetics of expression of MAPKs in HEp-2 and A549 cells after inoculation with HPIV-1 virus inactive ERK12 JNK and p38phosphorylation showed no time points analyzed during 30min to 24 hrs Ctl 2nd lane (minus) refers to the proteins of uninfected cells The Ctl(+) corresponds to the proteins from cells infected with live virus (90min) Antibodies used are described inMaterials andMethodsWesternblot assays were performed in triplicate

In A549 cells infected with live HPIV-1 the detection ofERK12 phosphorylation began at 60 minutes of interactiona small phosphorylation signal was observed at 90minutes ofinteraction (Figures 5(c) and 5(d)) increasing slightly at 120minutes and remaining constant until the 24-hour time pointPhosphorylation was slightly detected for the inactivatedvirus and control samples In controls (uninfected cells)inactive virus samples and samples thatwere incubated for 30and 45 minutes phosphorylation was only slightly detectedWith inactive viruses the result was similar to the previousWestern blot (Figures 5(c) lane 4 and 5(d) lane 3)

JNK phosphorylation in HEp-2 cells also began at 60minutes and gradually increased until the 90-minute timepoint but decreased at 120 minutes By 12 and 24 hours phos-phorylation ceased (Figures 6(a) and 6(b)) As inHEp-2 cellsJNK phosphorylation in A549 cells began at 60 minutes in-creased at 120minutes and decreased at 12 and 24 hours withno further increase (Figures 6(c) and 6(d) lanes 9 and 10)

Likewise p38 phosphorylation in the HEp-2 cell linebegan at 30 minutes (Figures 7(a) and 7(b)) increased againat 120 minutes and subsequently remained stable until the24-hour time point In A549 cells p38 phosphorylation levelswere similar beginning at 30 minutes (Figures 7(c) and 7(d))

but with a gradual increase from the 90-minute time pointand achieving a maximum at 12 and 24 hours

35 IL-8 Secretion Decreased with HPIV-1 Infection throughp38 Inhibition Thekinase inhibition assaywas performed onthe cell line HEp-2 derived from human laryngeal epithe-lioma given that is a suitable target for the in vitro virus assayOur results suggest that p38 ERK12 and JNK are activatedfollowing HPIV-1 infectionTherefore to determine whetherthese kinases are required for IL-8 gene expression ELISAswere performed using the supernatant from HEp-2 cells pre-treated with ERK12 JNK and p38 kinase inhibitors andinfected with HPIV-1 at the following time points 45 60 and90 minutes and 12 and 24 hours (Figure 9) Results demon-strate that the p38 inhibitor repressed IL-8 production uponviral infection whereas ERK12 and JNK MAPK inhibitorsdid not inhibit IL-8 production (Figures 9(a) and 9(b)) sug-gesting that p38 MAPK signalling is involved in IL-8 pro-duction Figure 9(c) depicts the average of three experimentswith HEp-2 cells infected with HPIV-1 and inhibitors Con-trols without inhibitors for each kinase were included for alltime points

6 Journal of Immunology Research

UVCtlMp

pERK-12

HEp-2

ERK-12

30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pERK HEp-2

30998400 45998400 60998400 90998400 120998400 12h 24h

Den

sity

(IN

Tm

m2)

(b)

A549

pERK-12

ERK-12

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 pERK A549

30998400 45998400 60998400 90998400 120998400 12h 24hD

ensit

y (I

NT

mm2)

(d)

Figure 5 ERK phosphorylationDetection of phosphorylation of ERK12 byWestern blot in protein extracts of HEp-2 andA549 cells infectedwith HPIV-1 live at different time points (a) phospho-ERK was detected with p-ERK12 (sc-81492) antibody as a loading control and totalERK12 (sc-292838) in HEp-2 cells (b) densitometric analysis of ERK12 protein concentrations (c) phospho-ERK and total ERK12 proteinconcentrations in A549 cells (d) densitometric analysis of protein concentrations Mp molecular weight CTL control and UV (inactivevirus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the three separate experiments

Table 2 IL-8 production (pgmL) in infected HEp-2 cells treated with ERK12 JNK and p38 kinase inhibitors

Time ERK12 JNK P38 Uninfected cells SD SEM Chi square 119875 CV451015840 4188a 4371a 006b 5004a 2094 604 874 003 6171601015840 10216a 9527a 1110c 9248b 7526 1125 946 002 5178901015840 13925a 14391a 3973b 13585a 4551 1314 843 003 39681201015840 17971a 16971a 5948b 18199a 5430 1568 761 005 367512 hours 24308a 24005a 15278b 24607a 4133 1193 658 008 187424 hours 13639ab 14450a 12951b 13707ab 650 188 746 005 474abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

Treating the cells with JNK and ERK12 inhibitors did notimpair IL-8 production but treating cells with an inhibitor ofp38 MAPK significantly impaired IL-8 secretion in most ofthe incubation times (before 24 h) ERK12 and JNK inhib-itors were not different from the control group (Table 2)

Total inhibitory dose MAPKs (ERK12 JNK and p38)were evaluated showing that the optimal concentration tosuppress phosphorylation was 20120583M (Figure 8)

4 Discussion

In the present study an in vitromodel with two cell lines thatwere infected with HPIV-1 at different time points was usedto determine whether an increase in IL-8 secretion occurredOur assay showed that IL-8 secretion gradually increasedduring the time points assessed

These results are consistent with reports that other respi-ratory viruses including influenza A virus (H3N2) and RSVinduce IL-8 secretion from bronchial epithelial cells [36 37]

It is known that IL-8 is responsible for the recruitmentof neutrophils and macrophages and that it performs otherfunctions such as adhesion and degranulation Likewise thepresence of IL-8 in inflammatory infiltrates in lung tissues hasbeen reported Studies performed with different viruses haverevealed that epithelial cells infected with specific virusesrelease IL-8 without requiring viral replication as whatoccurs in RSV infections [18 19] Contact with the cellularreceptor is sufficient to trigger IL-8 secretion whereas otherviruses only promote IL-8 synthesis during replication [2123] In this study assays using both live and inactivatedHPIV-1 were performed to determine whether replication-independent IL-8 release occurs with HPIV-1 a virus from

Journal of Immunology Research 7

p-JNK

HEp-2

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pJNK HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

A549

p-JNK

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)Time

180

0

60

UVControl

120

240

300 pJNK A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 6 JNK phosphorylation Detection of JNK phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infectedwith live and inactive HPIV-1 at different time points and JNK phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) phospho-JNK was detectedwith monoclonal antibody sc-135642 and the total JNK sc-571 in HEp-2 cells (b) densitometric analysis of JNK protein concentrations (c)phospho-JNK and total protein concentrations in A549 cells (d) densitometric analysis of the protein concentrations Mp molecular weightCTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the threeseparate experiments

the same family as RSV The results indicate that contact be-tween the virus and target cell is insufficient even when hae-magglutination activity is intact in the inactivated virus andviral replication is required to stimulate IL-8 secretion

Previous reports have shown that IL-8 secretion is in-volved in MAPK pathway activation [12 16] These kinasesrespond to different ligands and stimuli and are involved insignalling pathways and cellular processes such as prolifer-ation differentiation inflammation immune response andapoptosis

HPIV-1 does not use the JNK pathway for IL-8 activationwhereas p38 and ERK are phosphorylated when IL-8 ispresent in the supernatant after infection

The results of this study showed that HPVI-1 does not usethe JNK pathway for activation of IL-8 while theMAPKs p38and ERK are phosphorylated by the production of IL-8 afterthe infection similar results have been described after RSVinfection (virus belonging to the Paramyxoviridae family) inhuman lung endothelial cells where ERK phosphorylationenabled IL-8se creation [37]

Therefore to define which kinases are required for IL-8secretion the same assays were performed using specificinhibitors for each kinaseThe results showed that even in thepresence of specific inhibitors of ERK and JNK IL-8 was stillsecreted but there was no secretion in the presence of p38

inhibitor In this case a complete suppression of IL-8 secre-tionwas observedwhich suggests that p38MAPK is the path-way through which HPIV-1 induces IL-8 production

It is known that the inhibitor SB203580 is a potent cell-permeable selective and reversible inhibitor of p38120572 and 120573isoforms Inhibition is competitive with ATP and requiressubstrates to accommodate the fluorophenyl ring structure ofthe pyridinyl imidazole in their ATP-binding pocket Struc-tures of other p38 isoforms JNKs and ERK12 do not allowinhibitor binding at equivalent positions [38 39]

Many members of the Paramyxovirus family have beenshown to be potent inducers of chemokines of theCXC familysuch as interleukin-8 (IL-8) including respiratory syncytialvirus (RSV) Sendai virus human parainfluenza virus type 2(HPIV-2) HPIV-3 and Newcastle disease virus [40]

Paramyxoviruses have two glycoproteins that are impor-tant during infection The receptor-binding glycoprotein isHN in parainfluenza virus and mumps virus H in measlesvirus andG in pneumoviruses (RSV andmetapneumovirus)The second glycoprotein is the fusion protein (F) which catal-yses membrane fusion (viral and cellular) It has been sug-gested that HN glycoproteins form a complex that is initiatedby the binding HN activating the fusion protein that actsimmediately in such a way that the virus can enter the cellHowever it must be noted that glycoprotein HN acts as both

8 Journal of Immunology Research

p-p38

HEp-2

p38

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 p-p38 HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

p-p38

p38

A549UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 p-p38 A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 7 p38 phosphorylation Detection of p38 phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infected withlive and inactivated HPIV-1 at different time points and p38 phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) Total protein concentrationsin HEp-2 cells Phospho-p38 was detected with monoclonal antibody sc-7973 and the total p38 sc-535 in HEp-2 cells (b) densitometricanalysis of p38 protein concentrations (c) phospho-p38 and total protein concentrations in A549 cells (d) densitometric analysis of theprotein concentrations Mp molecular weight CTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are themeans for triplicate measurements from the three separate experiments

PD980559 (120583M)

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

minus + + + +

minus + + + +

minus + + + +

SP600125 (120583M)

SB203580 (120583M)

p-ERK 12

p-JNK

p-p38

Figure 8 Inhibitors of ERK12 PD980559 JNK SP600125 and p38 SB203580 inhibited the expression of each of theMAPKs in the HEp-2 cellline treated cell 1 hour before infection and maintained for 120 minutes after the test The experiments were performed by adding inhibitorat concentrations from 5120583M to 20 120583M (13 to 52 120583L resp) Protein levels were detected by Western blot Immunoblotting was done withmonoclonal antibody specific for protein phosphorylated technique that was described in ldquoMaterials and Methodsrdquo The extracted proteins(minus) represent HEp-2 cells without infecting and the proteins (+) refer to cells infected with the HPIV-1The tests were performed in triplicate

Journal of Immunology Research 9

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

ERKControl

(a)IL

-8 (p

gm

L)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

JNKControl

(b)

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

lowast

lowast

lowast

lowast

p38Control

lowast P gt 005

(c)

Figure 9 Kinetic inhibition ofMAPKs ELISA quantitation of IL-8 concentrations in cells pretreated with ERK12 PD980559 JNK SP600125and SB203580 inhibitors Control is HEp-2 cells infected with live HPIV-1 (a) ERK12 inhibitor assay is displayed in red cells treated with theinhibitor (b) JNK inhibitor assay green occurs in cells treated with the inhibitor (c) p38 inhibitor assay purple occurs in cells treated withinhibitor Data are the means plusmn SD for triplicate measurements from the three separate experiments

a haemagglutinin (HA) and a neuraminidase (NA) bindingto sialic acidmolecules through its HA activity mediating theenzymatic cleavage of sialic acid and promoting fusionthrough its neuraminidase activityThis dual activity suggeststhat coexpressed HN and F form a complex Glycoprotein Hof morbillivirus only has haemagglutinin activity and gly-coprotein G has neither of these two activities [41ndash44]

Therefore these glycoproteins exhibit significant differencesSuch differences are likely related to signalling pathways thatare activated when viruses interact with the cell AlthoughHPIV-1 and RSV belong to the same family the two viruseshave different glycoproteins which may explain why RSVuses ERK kinase andHPIV-1 uses p38 kinase Following virusbinding to its cellular receptor the extracellular stimulus is

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Journal of Immunology Research

Table 1 IL-8 secretion (pgmL) in HEp-2 and A549 cells infected with live and inactive virus

Time HEp-2 live virus A549 live virus HEp-2 inactive virus A549 inactive virus SD SEM Chi square 119875 CVUninfected cells 0004a 0001a 0002a 0027a 0018 0005 371 029 20749301015840 3901b 4704a 002c 006c 2265 654 1038 001 10516451015840 5503b 6211a 003c 003c 3069 886 935 002 10474601015840 10415a 9272b 001c 004c 5157 1489 1038 001 10474901015840 13238a 13507a 133b 100b 6924 1999 946 002 102651201015840 21008a 20172b 002c 001c 10756 3105 935 002 1044712 hours 26572a 23984b 001c 001c 13236 3821 943 002 1047124 hours 14506a 12930b 205c 200c 7083 2045 946 002 10174abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

L LUV

(a)

(b)

1 1

1 2

1 4

1 8

1 16

1 32

25120583L125 120583L

625 120583L

3125 120583L

175120583L

0875120583L

PBS 50120583L

1 HAU = 2 times 105 ivp1 32 = 0875mL of virus

for moi of 01(0875)(6 times 105)2 times 105

HPIV-1

Control (mdash)Erythrocytes 50120583L

Figure 2Haemagglutinating capacity of both live and inactive viruswas determined in a 96-well plate (a) Serial dilutions of the virus1 2 1 32 (b) hemagglutinating units (HAU) L corresponds to thelive virus lane and UV corresponds to the inactive virus This assaywas performed in triplicate

virus (L) In both lytic and haemagglutination assays expo-nential dilutions were prepared to determine the virus viabil-ity (Figure 2)

These tests are often used to demonstrate the viral repli-cation for the presence of hemagglutinin glycoprotein or via-ble structures

32 IL-8 Secretion in Cell Lines Infected with Live and InactiveVirus IL-8 secretion was quantified duringHPIV-1 infectionsuggesting that infection of cell lines with live HPIV-1induced a gradual increase in IL-8 secretion The highest IL-8 secretion in HEp-2 and A549 was observed at 12 hoursafter infection whereas the cells with inactive virus showeda marginal response at 24 hours (205 and 20) The higher

secretion of IL-8 was found using infected HEp-2 cellsfollowed by infected A549 cells (Table 1) compared withthe cells with inactive virus where a minimal response wasachieved In the case of HEp-2 cells and A549 cells treatedwith inactive virus no IL-8 secretion was observed neither inthe uninfected cells used as controls

33 Reverse Transcription-Polymerase Chain Reaction ThemRNA expression of IL-8 in A549 andHEp-2 cells was deter-mined by RT-PCR analysis In the analysis uninfected cellscells infected with HPIV-infected cells and HPIV-1 inactivewere used The increased expression of IL-8 originated frominfected cells followed by uninfected cells and the inactivatedvirus and the expression in the latter two was almost equalInfection was performed at different times and behavior foreach was similar in both cell lines (Figure 3) GAPDH wasconstitutively expressed in both cell lines and expression didnot vary by infection

34 HPIV-1 Infection Stimulates MAPK Phosphorylation Toestablish whether the inactive virus stimulated the phospho-rylation of MAPKs (ERK JNK and p38) Western blot testswere made (Figure 4) The two different cell lines HEp-2 cellline for the upper portion of the tract and the A549 for thebottom line of the same tract were used to simulate the respir-atory tract

Western blot assays were performed to determine thephosphorylation status of theMAPKs ERK12 JNK and p38In HEp-2 cells infection with live HPIV-1 promoted earlyERK12 phosphorylation beginning at 60minutes of interac-tion (Figures 5(a) and 5(b) band intensities were quantifiedthrough densitometric analysis) Phosphorylation increasedat 90 and 120 minutes However ERK phosphorylation washigher at 12 hours than at any other selected time and de-creased at 24 hours In the case of inactivated virus the 12-hour band was only in lane 3 because no evidence of phos-phorylation was observed in the kinetics selected times Thesame criteria were applied to the Western blot figures only aband corresponding to a 12-hour interaction was observedwith no increase in phosphorylation before and after this timepoint Controls consisted of uninfected cells which showedno significant changes

Journal of Immunology Research 5

Uninfected Uninfected

Inoculated withHPIV-1

UV inactivated

Inoculated withHPIV-1

UV inactivated

Infected withHPIV-1

Infected withHPIV-1

GPDHGPDH

MW White

Cell A549

289pb

289pb

289pb

289pb

289pb

289pb

390pb390pb

HEp-2 cell

60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24 MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

(min) (hrs) (min) (hrs)

Figure 3 IL8 expression by RT-PCR analysis in HEp2 and A549 cells infected uninfected and the HPVI-1 inactiveThe enhanced expressionof IL8 was given in infected cells HPVI-1 GAPDH expression was not different by infection

HEp-2 cell

p-ERKvirus UV

p-ERKvirus UV

virus UVp-JNK

virus UVp-JNK

virus UVp-p38

virus UVp-p38

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

A549 cell

Figure 4 Kinetics of expression of MAPKs in HEp-2 and A549 cells after inoculation with HPIV-1 virus inactive ERK12 JNK and p38phosphorylation showed no time points analyzed during 30min to 24 hrs Ctl 2nd lane (minus) refers to the proteins of uninfected cells The Ctl(+) corresponds to the proteins from cells infected with live virus (90min) Antibodies used are described inMaterials andMethodsWesternblot assays were performed in triplicate

In A549 cells infected with live HPIV-1 the detection ofERK12 phosphorylation began at 60 minutes of interactiona small phosphorylation signal was observed at 90minutes ofinteraction (Figures 5(c) and 5(d)) increasing slightly at 120minutes and remaining constant until the 24-hour time pointPhosphorylation was slightly detected for the inactivatedvirus and control samples In controls (uninfected cells)inactive virus samples and samples thatwere incubated for 30and 45 minutes phosphorylation was only slightly detectedWith inactive viruses the result was similar to the previousWestern blot (Figures 5(c) lane 4 and 5(d) lane 3)

JNK phosphorylation in HEp-2 cells also began at 60minutes and gradually increased until the 90-minute timepoint but decreased at 120 minutes By 12 and 24 hours phos-phorylation ceased (Figures 6(a) and 6(b)) As inHEp-2 cellsJNK phosphorylation in A549 cells began at 60 minutes in-creased at 120minutes and decreased at 12 and 24 hours withno further increase (Figures 6(c) and 6(d) lanes 9 and 10)

Likewise p38 phosphorylation in the HEp-2 cell linebegan at 30 minutes (Figures 7(a) and 7(b)) increased againat 120 minutes and subsequently remained stable until the24-hour time point In A549 cells p38 phosphorylation levelswere similar beginning at 30 minutes (Figures 7(c) and 7(d))

but with a gradual increase from the 90-minute time pointand achieving a maximum at 12 and 24 hours

35 IL-8 Secretion Decreased with HPIV-1 Infection throughp38 Inhibition Thekinase inhibition assaywas performed onthe cell line HEp-2 derived from human laryngeal epithe-lioma given that is a suitable target for the in vitro virus assayOur results suggest that p38 ERK12 and JNK are activatedfollowing HPIV-1 infectionTherefore to determine whetherthese kinases are required for IL-8 gene expression ELISAswere performed using the supernatant from HEp-2 cells pre-treated with ERK12 JNK and p38 kinase inhibitors andinfected with HPIV-1 at the following time points 45 60 and90 minutes and 12 and 24 hours (Figure 9) Results demon-strate that the p38 inhibitor repressed IL-8 production uponviral infection whereas ERK12 and JNK MAPK inhibitorsdid not inhibit IL-8 production (Figures 9(a) and 9(b)) sug-gesting that p38 MAPK signalling is involved in IL-8 pro-duction Figure 9(c) depicts the average of three experimentswith HEp-2 cells infected with HPIV-1 and inhibitors Con-trols without inhibitors for each kinase were included for alltime points

6 Journal of Immunology Research

UVCtlMp

pERK-12

HEp-2

ERK-12

30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pERK HEp-2

30998400 45998400 60998400 90998400 120998400 12h 24h

Den

sity

(IN

Tm

m2)

(b)

A549

pERK-12

ERK-12

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 pERK A549

30998400 45998400 60998400 90998400 120998400 12h 24hD

ensit

y (I

NT

mm2)

(d)

Figure 5 ERK phosphorylationDetection of phosphorylation of ERK12 byWestern blot in protein extracts of HEp-2 andA549 cells infectedwith HPIV-1 live at different time points (a) phospho-ERK was detected with p-ERK12 (sc-81492) antibody as a loading control and totalERK12 (sc-292838) in HEp-2 cells (b) densitometric analysis of ERK12 protein concentrations (c) phospho-ERK and total ERK12 proteinconcentrations in A549 cells (d) densitometric analysis of protein concentrations Mp molecular weight CTL control and UV (inactivevirus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the three separate experiments

Table 2 IL-8 production (pgmL) in infected HEp-2 cells treated with ERK12 JNK and p38 kinase inhibitors

Time ERK12 JNK P38 Uninfected cells SD SEM Chi square 119875 CV451015840 4188a 4371a 006b 5004a 2094 604 874 003 6171601015840 10216a 9527a 1110c 9248b 7526 1125 946 002 5178901015840 13925a 14391a 3973b 13585a 4551 1314 843 003 39681201015840 17971a 16971a 5948b 18199a 5430 1568 761 005 367512 hours 24308a 24005a 15278b 24607a 4133 1193 658 008 187424 hours 13639ab 14450a 12951b 13707ab 650 188 746 005 474abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

Treating the cells with JNK and ERK12 inhibitors did notimpair IL-8 production but treating cells with an inhibitor ofp38 MAPK significantly impaired IL-8 secretion in most ofthe incubation times (before 24 h) ERK12 and JNK inhib-itors were not different from the control group (Table 2)

Total inhibitory dose MAPKs (ERK12 JNK and p38)were evaluated showing that the optimal concentration tosuppress phosphorylation was 20120583M (Figure 8)

4 Discussion

In the present study an in vitromodel with two cell lines thatwere infected with HPIV-1 at different time points was usedto determine whether an increase in IL-8 secretion occurredOur assay showed that IL-8 secretion gradually increasedduring the time points assessed

These results are consistent with reports that other respi-ratory viruses including influenza A virus (H3N2) and RSVinduce IL-8 secretion from bronchial epithelial cells [36 37]

It is known that IL-8 is responsible for the recruitmentof neutrophils and macrophages and that it performs otherfunctions such as adhesion and degranulation Likewise thepresence of IL-8 in inflammatory infiltrates in lung tissues hasbeen reported Studies performed with different viruses haverevealed that epithelial cells infected with specific virusesrelease IL-8 without requiring viral replication as whatoccurs in RSV infections [18 19] Contact with the cellularreceptor is sufficient to trigger IL-8 secretion whereas otherviruses only promote IL-8 synthesis during replication [2123] In this study assays using both live and inactivatedHPIV-1 were performed to determine whether replication-independent IL-8 release occurs with HPIV-1 a virus from

Journal of Immunology Research 7

p-JNK

HEp-2

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pJNK HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

A549

p-JNK

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)Time

180

0

60

UVControl

120

240

300 pJNK A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 6 JNK phosphorylation Detection of JNK phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infectedwith live and inactive HPIV-1 at different time points and JNK phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) phospho-JNK was detectedwith monoclonal antibody sc-135642 and the total JNK sc-571 in HEp-2 cells (b) densitometric analysis of JNK protein concentrations (c)phospho-JNK and total protein concentrations in A549 cells (d) densitometric analysis of the protein concentrations Mp molecular weightCTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the threeseparate experiments

the same family as RSV The results indicate that contact be-tween the virus and target cell is insufficient even when hae-magglutination activity is intact in the inactivated virus andviral replication is required to stimulate IL-8 secretion

Previous reports have shown that IL-8 secretion is in-volved in MAPK pathway activation [12 16] These kinasesrespond to different ligands and stimuli and are involved insignalling pathways and cellular processes such as prolifer-ation differentiation inflammation immune response andapoptosis

HPIV-1 does not use the JNK pathway for IL-8 activationwhereas p38 and ERK are phosphorylated when IL-8 ispresent in the supernatant after infection

The results of this study showed that HPVI-1 does not usethe JNK pathway for activation of IL-8 while theMAPKs p38and ERK are phosphorylated by the production of IL-8 afterthe infection similar results have been described after RSVinfection (virus belonging to the Paramyxoviridae family) inhuman lung endothelial cells where ERK phosphorylationenabled IL-8se creation [37]

Therefore to define which kinases are required for IL-8secretion the same assays were performed using specificinhibitors for each kinaseThe results showed that even in thepresence of specific inhibitors of ERK and JNK IL-8 was stillsecreted but there was no secretion in the presence of p38

inhibitor In this case a complete suppression of IL-8 secre-tionwas observedwhich suggests that p38MAPK is the path-way through which HPIV-1 induces IL-8 production

It is known that the inhibitor SB203580 is a potent cell-permeable selective and reversible inhibitor of p38120572 and 120573isoforms Inhibition is competitive with ATP and requiressubstrates to accommodate the fluorophenyl ring structure ofthe pyridinyl imidazole in their ATP-binding pocket Struc-tures of other p38 isoforms JNKs and ERK12 do not allowinhibitor binding at equivalent positions [38 39]

Many members of the Paramyxovirus family have beenshown to be potent inducers of chemokines of theCXC familysuch as interleukin-8 (IL-8) including respiratory syncytialvirus (RSV) Sendai virus human parainfluenza virus type 2(HPIV-2) HPIV-3 and Newcastle disease virus [40]

Paramyxoviruses have two glycoproteins that are impor-tant during infection The receptor-binding glycoprotein isHN in parainfluenza virus and mumps virus H in measlesvirus andG in pneumoviruses (RSV andmetapneumovirus)The second glycoprotein is the fusion protein (F) which catal-yses membrane fusion (viral and cellular) It has been sug-gested that HN glycoproteins form a complex that is initiatedby the binding HN activating the fusion protein that actsimmediately in such a way that the virus can enter the cellHowever it must be noted that glycoprotein HN acts as both

8 Journal of Immunology Research

p-p38

HEp-2

p38

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 p-p38 HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

p-p38

p38

A549UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 p-p38 A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 7 p38 phosphorylation Detection of p38 phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infected withlive and inactivated HPIV-1 at different time points and p38 phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) Total protein concentrationsin HEp-2 cells Phospho-p38 was detected with monoclonal antibody sc-7973 and the total p38 sc-535 in HEp-2 cells (b) densitometricanalysis of p38 protein concentrations (c) phospho-p38 and total protein concentrations in A549 cells (d) densitometric analysis of theprotein concentrations Mp molecular weight CTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are themeans for triplicate measurements from the three separate experiments

PD980559 (120583M)

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

minus + + + +

minus + + + +

minus + + + +

SP600125 (120583M)

SB203580 (120583M)

p-ERK 12

p-JNK

p-p38

Figure 8 Inhibitors of ERK12 PD980559 JNK SP600125 and p38 SB203580 inhibited the expression of each of theMAPKs in the HEp-2 cellline treated cell 1 hour before infection and maintained for 120 minutes after the test The experiments were performed by adding inhibitorat concentrations from 5120583M to 20 120583M (13 to 52 120583L resp) Protein levels were detected by Western blot Immunoblotting was done withmonoclonal antibody specific for protein phosphorylated technique that was described in ldquoMaterials and Methodsrdquo The extracted proteins(minus) represent HEp-2 cells without infecting and the proteins (+) refer to cells infected with the HPIV-1The tests were performed in triplicate

Journal of Immunology Research 9

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

ERKControl

(a)IL

-8 (p

gm

L)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

JNKControl

(b)

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

lowast

lowast

lowast

lowast

p38Control

lowast P gt 005

(c)

Figure 9 Kinetic inhibition ofMAPKs ELISA quantitation of IL-8 concentrations in cells pretreated with ERK12 PD980559 JNK SP600125and SB203580 inhibitors Control is HEp-2 cells infected with live HPIV-1 (a) ERK12 inhibitor assay is displayed in red cells treated with theinhibitor (b) JNK inhibitor assay green occurs in cells treated with the inhibitor (c) p38 inhibitor assay purple occurs in cells treated withinhibitor Data are the means plusmn SD for triplicate measurements from the three separate experiments

a haemagglutinin (HA) and a neuraminidase (NA) bindingto sialic acidmolecules through its HA activity mediating theenzymatic cleavage of sialic acid and promoting fusionthrough its neuraminidase activityThis dual activity suggeststhat coexpressed HN and F form a complex Glycoprotein Hof morbillivirus only has haemagglutinin activity and gly-coprotein G has neither of these two activities [41ndash44]

Therefore these glycoproteins exhibit significant differencesSuch differences are likely related to signalling pathways thatare activated when viruses interact with the cell AlthoughHPIV-1 and RSV belong to the same family the two viruseshave different glycoproteins which may explain why RSVuses ERK kinase andHPIV-1 uses p38 kinase Following virusbinding to its cellular receptor the extracellular stimulus is

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Journal of Immunology Research 5

Uninfected Uninfected

Inoculated withHPIV-1

UV inactivated

Inoculated withHPIV-1

UV inactivated

Infected withHPIV-1

Infected withHPIV-1

GPDHGPDH

MW White

Cell A549

289pb

289pb

289pb

289pb

289pb

289pb

390pb390pb

HEp-2 cell

60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24 MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

MW White 60 90 120 12 24

(min) (hrs) (min) (hrs)

Figure 3 IL8 expression by RT-PCR analysis in HEp2 and A549 cells infected uninfected and the HPVI-1 inactiveThe enhanced expressionof IL8 was given in infected cells HPVI-1 GAPDH expression was not different by infection

HEp-2 cell

p-ERKvirus UV

p-ERKvirus UV

virus UVp-JNK

virus UVp-JNK

virus UVp-p38

virus UVp-p38

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

MW Ctlminus Ctl+ 30998400 60998400 90998400 120998400 12 24hrs

A549 cell

Figure 4 Kinetics of expression of MAPKs in HEp-2 and A549 cells after inoculation with HPIV-1 virus inactive ERK12 JNK and p38phosphorylation showed no time points analyzed during 30min to 24 hrs Ctl 2nd lane (minus) refers to the proteins of uninfected cells The Ctl(+) corresponds to the proteins from cells infected with live virus (90min) Antibodies used are described inMaterials andMethodsWesternblot assays were performed in triplicate

In A549 cells infected with live HPIV-1 the detection ofERK12 phosphorylation began at 60 minutes of interactiona small phosphorylation signal was observed at 90minutes ofinteraction (Figures 5(c) and 5(d)) increasing slightly at 120minutes and remaining constant until the 24-hour time pointPhosphorylation was slightly detected for the inactivatedvirus and control samples In controls (uninfected cells)inactive virus samples and samples thatwere incubated for 30and 45 minutes phosphorylation was only slightly detectedWith inactive viruses the result was similar to the previousWestern blot (Figures 5(c) lane 4 and 5(d) lane 3)

JNK phosphorylation in HEp-2 cells also began at 60minutes and gradually increased until the 90-minute timepoint but decreased at 120 minutes By 12 and 24 hours phos-phorylation ceased (Figures 6(a) and 6(b)) As inHEp-2 cellsJNK phosphorylation in A549 cells began at 60 minutes in-creased at 120minutes and decreased at 12 and 24 hours withno further increase (Figures 6(c) and 6(d) lanes 9 and 10)

Likewise p38 phosphorylation in the HEp-2 cell linebegan at 30 minutes (Figures 7(a) and 7(b)) increased againat 120 minutes and subsequently remained stable until the24-hour time point In A549 cells p38 phosphorylation levelswere similar beginning at 30 minutes (Figures 7(c) and 7(d))

but with a gradual increase from the 90-minute time pointand achieving a maximum at 12 and 24 hours

35 IL-8 Secretion Decreased with HPIV-1 Infection throughp38 Inhibition Thekinase inhibition assaywas performed onthe cell line HEp-2 derived from human laryngeal epithe-lioma given that is a suitable target for the in vitro virus assayOur results suggest that p38 ERK12 and JNK are activatedfollowing HPIV-1 infectionTherefore to determine whetherthese kinases are required for IL-8 gene expression ELISAswere performed using the supernatant from HEp-2 cells pre-treated with ERK12 JNK and p38 kinase inhibitors andinfected with HPIV-1 at the following time points 45 60 and90 minutes and 12 and 24 hours (Figure 9) Results demon-strate that the p38 inhibitor repressed IL-8 production uponviral infection whereas ERK12 and JNK MAPK inhibitorsdid not inhibit IL-8 production (Figures 9(a) and 9(b)) sug-gesting that p38 MAPK signalling is involved in IL-8 pro-duction Figure 9(c) depicts the average of three experimentswith HEp-2 cells infected with HPIV-1 and inhibitors Con-trols without inhibitors for each kinase were included for alltime points

6 Journal of Immunology Research

UVCtlMp

pERK-12

HEp-2

ERK-12

30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pERK HEp-2

30998400 45998400 60998400 90998400 120998400 12h 24h

Den

sity

(IN

Tm

m2)

(b)

A549

pERK-12

ERK-12

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 pERK A549

30998400 45998400 60998400 90998400 120998400 12h 24hD

ensit

y (I

NT

mm2)

(d)

Figure 5 ERK phosphorylationDetection of phosphorylation of ERK12 byWestern blot in protein extracts of HEp-2 andA549 cells infectedwith HPIV-1 live at different time points (a) phospho-ERK was detected with p-ERK12 (sc-81492) antibody as a loading control and totalERK12 (sc-292838) in HEp-2 cells (b) densitometric analysis of ERK12 protein concentrations (c) phospho-ERK and total ERK12 proteinconcentrations in A549 cells (d) densitometric analysis of protein concentrations Mp molecular weight CTL control and UV (inactivevirus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the three separate experiments

Table 2 IL-8 production (pgmL) in infected HEp-2 cells treated with ERK12 JNK and p38 kinase inhibitors

Time ERK12 JNK P38 Uninfected cells SD SEM Chi square 119875 CV451015840 4188a 4371a 006b 5004a 2094 604 874 003 6171601015840 10216a 9527a 1110c 9248b 7526 1125 946 002 5178901015840 13925a 14391a 3973b 13585a 4551 1314 843 003 39681201015840 17971a 16971a 5948b 18199a 5430 1568 761 005 367512 hours 24308a 24005a 15278b 24607a 4133 1193 658 008 187424 hours 13639ab 14450a 12951b 13707ab 650 188 746 005 474abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

Treating the cells with JNK and ERK12 inhibitors did notimpair IL-8 production but treating cells with an inhibitor ofp38 MAPK significantly impaired IL-8 secretion in most ofthe incubation times (before 24 h) ERK12 and JNK inhib-itors were not different from the control group (Table 2)

Total inhibitory dose MAPKs (ERK12 JNK and p38)were evaluated showing that the optimal concentration tosuppress phosphorylation was 20120583M (Figure 8)

4 Discussion

In the present study an in vitromodel with two cell lines thatwere infected with HPIV-1 at different time points was usedto determine whether an increase in IL-8 secretion occurredOur assay showed that IL-8 secretion gradually increasedduring the time points assessed

These results are consistent with reports that other respi-ratory viruses including influenza A virus (H3N2) and RSVinduce IL-8 secretion from bronchial epithelial cells [36 37]

It is known that IL-8 is responsible for the recruitmentof neutrophils and macrophages and that it performs otherfunctions such as adhesion and degranulation Likewise thepresence of IL-8 in inflammatory infiltrates in lung tissues hasbeen reported Studies performed with different viruses haverevealed that epithelial cells infected with specific virusesrelease IL-8 without requiring viral replication as whatoccurs in RSV infections [18 19] Contact with the cellularreceptor is sufficient to trigger IL-8 secretion whereas otherviruses only promote IL-8 synthesis during replication [2123] In this study assays using both live and inactivatedHPIV-1 were performed to determine whether replication-independent IL-8 release occurs with HPIV-1 a virus from

Journal of Immunology Research 7

p-JNK

HEp-2

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pJNK HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

A549

p-JNK

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)Time

180

0

60

UVControl

120

240

300 pJNK A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 6 JNK phosphorylation Detection of JNK phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infectedwith live and inactive HPIV-1 at different time points and JNK phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) phospho-JNK was detectedwith monoclonal antibody sc-135642 and the total JNK sc-571 in HEp-2 cells (b) densitometric analysis of JNK protein concentrations (c)phospho-JNK and total protein concentrations in A549 cells (d) densitometric analysis of the protein concentrations Mp molecular weightCTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the threeseparate experiments

the same family as RSV The results indicate that contact be-tween the virus and target cell is insufficient even when hae-magglutination activity is intact in the inactivated virus andviral replication is required to stimulate IL-8 secretion

Previous reports have shown that IL-8 secretion is in-volved in MAPK pathway activation [12 16] These kinasesrespond to different ligands and stimuli and are involved insignalling pathways and cellular processes such as prolifer-ation differentiation inflammation immune response andapoptosis

HPIV-1 does not use the JNK pathway for IL-8 activationwhereas p38 and ERK are phosphorylated when IL-8 ispresent in the supernatant after infection

The results of this study showed that HPVI-1 does not usethe JNK pathway for activation of IL-8 while theMAPKs p38and ERK are phosphorylated by the production of IL-8 afterthe infection similar results have been described after RSVinfection (virus belonging to the Paramyxoviridae family) inhuman lung endothelial cells where ERK phosphorylationenabled IL-8se creation [37]

Therefore to define which kinases are required for IL-8secretion the same assays were performed using specificinhibitors for each kinaseThe results showed that even in thepresence of specific inhibitors of ERK and JNK IL-8 was stillsecreted but there was no secretion in the presence of p38

inhibitor In this case a complete suppression of IL-8 secre-tionwas observedwhich suggests that p38MAPK is the path-way through which HPIV-1 induces IL-8 production

It is known that the inhibitor SB203580 is a potent cell-permeable selective and reversible inhibitor of p38120572 and 120573isoforms Inhibition is competitive with ATP and requiressubstrates to accommodate the fluorophenyl ring structure ofthe pyridinyl imidazole in their ATP-binding pocket Struc-tures of other p38 isoforms JNKs and ERK12 do not allowinhibitor binding at equivalent positions [38 39]

Many members of the Paramyxovirus family have beenshown to be potent inducers of chemokines of theCXC familysuch as interleukin-8 (IL-8) including respiratory syncytialvirus (RSV) Sendai virus human parainfluenza virus type 2(HPIV-2) HPIV-3 and Newcastle disease virus [40]

Paramyxoviruses have two glycoproteins that are impor-tant during infection The receptor-binding glycoprotein isHN in parainfluenza virus and mumps virus H in measlesvirus andG in pneumoviruses (RSV andmetapneumovirus)The second glycoprotein is the fusion protein (F) which catal-yses membrane fusion (viral and cellular) It has been sug-gested that HN glycoproteins form a complex that is initiatedby the binding HN activating the fusion protein that actsimmediately in such a way that the virus can enter the cellHowever it must be noted that glycoprotein HN acts as both

8 Journal of Immunology Research

p-p38

HEp-2

p38

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 p-p38 HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

p-p38

p38

A549UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 p-p38 A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 7 p38 phosphorylation Detection of p38 phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infected withlive and inactivated HPIV-1 at different time points and p38 phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) Total protein concentrationsin HEp-2 cells Phospho-p38 was detected with monoclonal antibody sc-7973 and the total p38 sc-535 in HEp-2 cells (b) densitometricanalysis of p38 protein concentrations (c) phospho-p38 and total protein concentrations in A549 cells (d) densitometric analysis of theprotein concentrations Mp molecular weight CTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are themeans for triplicate measurements from the three separate experiments

PD980559 (120583M)

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

minus + + + +

minus + + + +

minus + + + +

SP600125 (120583M)

SB203580 (120583M)

p-ERK 12

p-JNK

p-p38

Figure 8 Inhibitors of ERK12 PD980559 JNK SP600125 and p38 SB203580 inhibited the expression of each of theMAPKs in the HEp-2 cellline treated cell 1 hour before infection and maintained for 120 minutes after the test The experiments were performed by adding inhibitorat concentrations from 5120583M to 20 120583M (13 to 52 120583L resp) Protein levels were detected by Western blot Immunoblotting was done withmonoclonal antibody specific for protein phosphorylated technique that was described in ldquoMaterials and Methodsrdquo The extracted proteins(minus) represent HEp-2 cells without infecting and the proteins (+) refer to cells infected with the HPIV-1The tests were performed in triplicate

Journal of Immunology Research 9

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

ERKControl

(a)IL

-8 (p

gm

L)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

JNKControl

(b)

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

lowast

lowast

lowast

lowast

p38Control

lowast P gt 005

(c)

Figure 9 Kinetic inhibition ofMAPKs ELISA quantitation of IL-8 concentrations in cells pretreated with ERK12 PD980559 JNK SP600125and SB203580 inhibitors Control is HEp-2 cells infected with live HPIV-1 (a) ERK12 inhibitor assay is displayed in red cells treated with theinhibitor (b) JNK inhibitor assay green occurs in cells treated with the inhibitor (c) p38 inhibitor assay purple occurs in cells treated withinhibitor Data are the means plusmn SD for triplicate measurements from the three separate experiments

a haemagglutinin (HA) and a neuraminidase (NA) bindingto sialic acidmolecules through its HA activity mediating theenzymatic cleavage of sialic acid and promoting fusionthrough its neuraminidase activityThis dual activity suggeststhat coexpressed HN and F form a complex Glycoprotein Hof morbillivirus only has haemagglutinin activity and gly-coprotein G has neither of these two activities [41ndash44]

Therefore these glycoproteins exhibit significant differencesSuch differences are likely related to signalling pathways thatare activated when viruses interact with the cell AlthoughHPIV-1 and RSV belong to the same family the two viruseshave different glycoproteins which may explain why RSVuses ERK kinase andHPIV-1 uses p38 kinase Following virusbinding to its cellular receptor the extracellular stimulus is

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Journal of Immunology Research

UVCtlMp

pERK-12

HEp-2

ERK-12

30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pERK HEp-2

30998400 45998400 60998400 90998400 120998400 12h 24h

Den

sity

(IN

Tm

m2)

(b)

A549

pERK-12

ERK-12

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 pERK A549

30998400 45998400 60998400 90998400 120998400 12h 24hD

ensit

y (I

NT

mm2)

(d)

Figure 5 ERK phosphorylationDetection of phosphorylation of ERK12 byWestern blot in protein extracts of HEp-2 andA549 cells infectedwith HPIV-1 live at different time points (a) phospho-ERK was detected with p-ERK12 (sc-81492) antibody as a loading control and totalERK12 (sc-292838) in HEp-2 cells (b) densitometric analysis of ERK12 protein concentrations (c) phospho-ERK and total ERK12 proteinconcentrations in A549 cells (d) densitometric analysis of protein concentrations Mp molecular weight CTL control and UV (inactivevirus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the three separate experiments

Table 2 IL-8 production (pgmL) in infected HEp-2 cells treated with ERK12 JNK and p38 kinase inhibitors

Time ERK12 JNK P38 Uninfected cells SD SEM Chi square 119875 CV451015840 4188a 4371a 006b 5004a 2094 604 874 003 6171601015840 10216a 9527a 1110c 9248b 7526 1125 946 002 5178901015840 13925a 14391a 3973b 13585a 4551 1314 843 003 39681201015840 17971a 16971a 5948b 18199a 5430 1568 761 005 367512 hours 24308a 24005a 15278b 24607a 4133 1193 658 008 187424 hours 13639ab 14450a 12951b 13707ab 650 188 746 005 474abcMeans with no common superscript in a row differ (119875 lt 005) SD standard deviation SEM standard error of themean119875 probability value CV coefficientof variation

Treating the cells with JNK and ERK12 inhibitors did notimpair IL-8 production but treating cells with an inhibitor ofp38 MAPK significantly impaired IL-8 secretion in most ofthe incubation times (before 24 h) ERK12 and JNK inhib-itors were not different from the control group (Table 2)

Total inhibitory dose MAPKs (ERK12 JNK and p38)were evaluated showing that the optimal concentration tosuppress phosphorylation was 20120583M (Figure 8)

4 Discussion

In the present study an in vitromodel with two cell lines thatwere infected with HPIV-1 at different time points was usedto determine whether an increase in IL-8 secretion occurredOur assay showed that IL-8 secretion gradually increasedduring the time points assessed

These results are consistent with reports that other respi-ratory viruses including influenza A virus (H3N2) and RSVinduce IL-8 secretion from bronchial epithelial cells [36 37]

It is known that IL-8 is responsible for the recruitmentof neutrophils and macrophages and that it performs otherfunctions such as adhesion and degranulation Likewise thepresence of IL-8 in inflammatory infiltrates in lung tissues hasbeen reported Studies performed with different viruses haverevealed that epithelial cells infected with specific virusesrelease IL-8 without requiring viral replication as whatoccurs in RSV infections [18 19] Contact with the cellularreceptor is sufficient to trigger IL-8 secretion whereas otherviruses only promote IL-8 synthesis during replication [2123] In this study assays using both live and inactivatedHPIV-1 were performed to determine whether replication-independent IL-8 release occurs with HPIV-1 a virus from

Journal of Immunology Research 7

p-JNK

HEp-2

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pJNK HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

A549

p-JNK

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)Time

180

0

60

UVControl

120

240

300 pJNK A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 6 JNK phosphorylation Detection of JNK phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infectedwith live and inactive HPIV-1 at different time points and JNK phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) phospho-JNK was detectedwith monoclonal antibody sc-135642 and the total JNK sc-571 in HEp-2 cells (b) densitometric analysis of JNK protein concentrations (c)phospho-JNK and total protein concentrations in A549 cells (d) densitometric analysis of the protein concentrations Mp molecular weightCTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the threeseparate experiments

the same family as RSV The results indicate that contact be-tween the virus and target cell is insufficient even when hae-magglutination activity is intact in the inactivated virus andviral replication is required to stimulate IL-8 secretion

Previous reports have shown that IL-8 secretion is in-volved in MAPK pathway activation [12 16] These kinasesrespond to different ligands and stimuli and are involved insignalling pathways and cellular processes such as prolifer-ation differentiation inflammation immune response andapoptosis

HPIV-1 does not use the JNK pathway for IL-8 activationwhereas p38 and ERK are phosphorylated when IL-8 ispresent in the supernatant after infection

The results of this study showed that HPVI-1 does not usethe JNK pathway for activation of IL-8 while theMAPKs p38and ERK are phosphorylated by the production of IL-8 afterthe infection similar results have been described after RSVinfection (virus belonging to the Paramyxoviridae family) inhuman lung endothelial cells where ERK phosphorylationenabled IL-8se creation [37]

Therefore to define which kinases are required for IL-8secretion the same assays were performed using specificinhibitors for each kinaseThe results showed that even in thepresence of specific inhibitors of ERK and JNK IL-8 was stillsecreted but there was no secretion in the presence of p38

inhibitor In this case a complete suppression of IL-8 secre-tionwas observedwhich suggests that p38MAPK is the path-way through which HPIV-1 induces IL-8 production

It is known that the inhibitor SB203580 is a potent cell-permeable selective and reversible inhibitor of p38120572 and 120573isoforms Inhibition is competitive with ATP and requiressubstrates to accommodate the fluorophenyl ring structure ofthe pyridinyl imidazole in their ATP-binding pocket Struc-tures of other p38 isoforms JNKs and ERK12 do not allowinhibitor binding at equivalent positions [38 39]

Many members of the Paramyxovirus family have beenshown to be potent inducers of chemokines of theCXC familysuch as interleukin-8 (IL-8) including respiratory syncytialvirus (RSV) Sendai virus human parainfluenza virus type 2(HPIV-2) HPIV-3 and Newcastle disease virus [40]

Paramyxoviruses have two glycoproteins that are impor-tant during infection The receptor-binding glycoprotein isHN in parainfluenza virus and mumps virus H in measlesvirus andG in pneumoviruses (RSV andmetapneumovirus)The second glycoprotein is the fusion protein (F) which catal-yses membrane fusion (viral and cellular) It has been sug-gested that HN glycoproteins form a complex that is initiatedby the binding HN activating the fusion protein that actsimmediately in such a way that the virus can enter the cellHowever it must be noted that glycoprotein HN acts as both

8 Journal of Immunology Research

p-p38

HEp-2

p38

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 p-p38 HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

p-p38

p38

A549UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 p-p38 A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 7 p38 phosphorylation Detection of p38 phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infected withlive and inactivated HPIV-1 at different time points and p38 phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) Total protein concentrationsin HEp-2 cells Phospho-p38 was detected with monoclonal antibody sc-7973 and the total p38 sc-535 in HEp-2 cells (b) densitometricanalysis of p38 protein concentrations (c) phospho-p38 and total protein concentrations in A549 cells (d) densitometric analysis of theprotein concentrations Mp molecular weight CTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are themeans for triplicate measurements from the three separate experiments

PD980559 (120583M)

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

minus + + + +

minus + + + +

minus + + + +

SP600125 (120583M)

SB203580 (120583M)

p-ERK 12

p-JNK

p-p38

Figure 8 Inhibitors of ERK12 PD980559 JNK SP600125 and p38 SB203580 inhibited the expression of each of theMAPKs in the HEp-2 cellline treated cell 1 hour before infection and maintained for 120 minutes after the test The experiments were performed by adding inhibitorat concentrations from 5120583M to 20 120583M (13 to 52 120583L resp) Protein levels were detected by Western blot Immunoblotting was done withmonoclonal antibody specific for protein phosphorylated technique that was described in ldquoMaterials and Methodsrdquo The extracted proteins(minus) represent HEp-2 cells without infecting and the proteins (+) refer to cells infected with the HPIV-1The tests were performed in triplicate

Journal of Immunology Research 9

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

ERKControl

(a)IL

-8 (p

gm

L)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

JNKControl

(b)

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

lowast

lowast

lowast

lowast

p38Control

lowast P gt 005

(c)

Figure 9 Kinetic inhibition ofMAPKs ELISA quantitation of IL-8 concentrations in cells pretreated with ERK12 PD980559 JNK SP600125and SB203580 inhibitors Control is HEp-2 cells infected with live HPIV-1 (a) ERK12 inhibitor assay is displayed in red cells treated with theinhibitor (b) JNK inhibitor assay green occurs in cells treated with the inhibitor (c) p38 inhibitor assay purple occurs in cells treated withinhibitor Data are the means plusmn SD for triplicate measurements from the three separate experiments

a haemagglutinin (HA) and a neuraminidase (NA) bindingto sialic acidmolecules through its HA activity mediating theenzymatic cleavage of sialic acid and promoting fusionthrough its neuraminidase activityThis dual activity suggeststhat coexpressed HN and F form a complex Glycoprotein Hof morbillivirus only has haemagglutinin activity and gly-coprotein G has neither of these two activities [41ndash44]

Therefore these glycoproteins exhibit significant differencesSuch differences are likely related to signalling pathways thatare activated when viruses interact with the cell AlthoughHPIV-1 and RSV belong to the same family the two viruseshave different glycoproteins which may explain why RSVuses ERK kinase andHPIV-1 uses p38 kinase Following virusbinding to its cellular receptor the extracellular stimulus is

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Journal of Immunology Research 7

p-JNK

HEp-2

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 pJNK HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

A549

p-JNK

JNK

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)Time

180

0

60

UVControl

120

240

300 pJNK A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 6 JNK phosphorylation Detection of JNK phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infectedwith live and inactive HPIV-1 at different time points and JNK phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) phospho-JNK was detectedwith monoclonal antibody sc-135642 and the total JNK sc-571 in HEp-2 cells (b) densitometric analysis of JNK protein concentrations (c)phospho-JNK and total protein concentrations in A549 cells (d) densitometric analysis of the protein concentrations Mp molecular weightCTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are the means for triplicate measurements from the threeseparate experiments

the same family as RSV The results indicate that contact be-tween the virus and target cell is insufficient even when hae-magglutination activity is intact in the inactivated virus andviral replication is required to stimulate IL-8 secretion

Previous reports have shown that IL-8 secretion is in-volved in MAPK pathway activation [12 16] These kinasesrespond to different ligands and stimuli and are involved insignalling pathways and cellular processes such as prolifer-ation differentiation inflammation immune response andapoptosis

HPIV-1 does not use the JNK pathway for IL-8 activationwhereas p38 and ERK are phosphorylated when IL-8 ispresent in the supernatant after infection

The results of this study showed that HPVI-1 does not usethe JNK pathway for activation of IL-8 while theMAPKs p38and ERK are phosphorylated by the production of IL-8 afterthe infection similar results have been described after RSVinfection (virus belonging to the Paramyxoviridae family) inhuman lung endothelial cells where ERK phosphorylationenabled IL-8se creation [37]

Therefore to define which kinases are required for IL-8secretion the same assays were performed using specificinhibitors for each kinaseThe results showed that even in thepresence of specific inhibitors of ERK and JNK IL-8 was stillsecreted but there was no secretion in the presence of p38

inhibitor In this case a complete suppression of IL-8 secre-tionwas observedwhich suggests that p38MAPK is the path-way through which HPIV-1 induces IL-8 production

It is known that the inhibitor SB203580 is a potent cell-permeable selective and reversible inhibitor of p38120572 and 120573isoforms Inhibition is competitive with ATP and requiressubstrates to accommodate the fluorophenyl ring structure ofthe pyridinyl imidazole in their ATP-binding pocket Struc-tures of other p38 isoforms JNKs and ERK12 do not allowinhibitor binding at equivalent positions [38 39]

Many members of the Paramyxovirus family have beenshown to be potent inducers of chemokines of theCXC familysuch as interleukin-8 (IL-8) including respiratory syncytialvirus (RSV) Sendai virus human parainfluenza virus type 2(HPIV-2) HPIV-3 and Newcastle disease virus [40]

Paramyxoviruses have two glycoproteins that are impor-tant during infection The receptor-binding glycoprotein isHN in parainfluenza virus and mumps virus H in measlesvirus andG in pneumoviruses (RSV andmetapneumovirus)The second glycoprotein is the fusion protein (F) which catal-yses membrane fusion (viral and cellular) It has been sug-gested that HN glycoproteins form a complex that is initiatedby the binding HN activating the fusion protein that actsimmediately in such a way that the virus can enter the cellHowever it must be noted that glycoprotein HN acts as both

8 Journal of Immunology Research

p-p38

HEp-2

p38

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 p-p38 HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

p-p38

p38

A549UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 p-p38 A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 7 p38 phosphorylation Detection of p38 phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infected withlive and inactivated HPIV-1 at different time points and p38 phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) Total protein concentrationsin HEp-2 cells Phospho-p38 was detected with monoclonal antibody sc-7973 and the total p38 sc-535 in HEp-2 cells (b) densitometricanalysis of p38 protein concentrations (c) phospho-p38 and total protein concentrations in A549 cells (d) densitometric analysis of theprotein concentrations Mp molecular weight CTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are themeans for triplicate measurements from the three separate experiments

PD980559 (120583M)

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

minus + + + +

minus + + + +

minus + + + +

SP600125 (120583M)

SB203580 (120583M)

p-ERK 12

p-JNK

p-p38

Figure 8 Inhibitors of ERK12 PD980559 JNK SP600125 and p38 SB203580 inhibited the expression of each of theMAPKs in the HEp-2 cellline treated cell 1 hour before infection and maintained for 120 minutes after the test The experiments were performed by adding inhibitorat concentrations from 5120583M to 20 120583M (13 to 52 120583L resp) Protein levels were detected by Western blot Immunoblotting was done withmonoclonal antibody specific for protein phosphorylated technique that was described in ldquoMaterials and Methodsrdquo The extracted proteins(minus) represent HEp-2 cells without infecting and the proteins (+) refer to cells infected with the HPIV-1The tests were performed in triplicate

Journal of Immunology Research 9

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

ERKControl

(a)IL

-8 (p

gm

L)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

JNKControl

(b)

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

lowast

lowast

lowast

lowast

p38Control

lowast P gt 005

(c)

Figure 9 Kinetic inhibition ofMAPKs ELISA quantitation of IL-8 concentrations in cells pretreated with ERK12 PD980559 JNK SP600125and SB203580 inhibitors Control is HEp-2 cells infected with live HPIV-1 (a) ERK12 inhibitor assay is displayed in red cells treated with theinhibitor (b) JNK inhibitor assay green occurs in cells treated with the inhibitor (c) p38 inhibitor assay purple occurs in cells treated withinhibitor Data are the means plusmn SD for triplicate measurements from the three separate experiments

a haemagglutinin (HA) and a neuraminidase (NA) bindingto sialic acidmolecules through its HA activity mediating theenzymatic cleavage of sialic acid and promoting fusionthrough its neuraminidase activityThis dual activity suggeststhat coexpressed HN and F form a complex Glycoprotein Hof morbillivirus only has haemagglutinin activity and gly-coprotein G has neither of these two activities [41ndash44]

Therefore these glycoproteins exhibit significant differencesSuch differences are likely related to signalling pathways thatare activated when viruses interact with the cell AlthoughHPIV-1 and RSV belong to the same family the two viruseshave different glycoproteins which may explain why RSVuses ERK kinase andHPIV-1 uses p38 kinase Following virusbinding to its cellular receptor the extracellular stimulus is

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Journal of Immunology Research

p-p38

HEp-2

p38

UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(a)

180

0

60

UVControlTime

120

240

300 p-p38 HEp-2

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(b)

p-p38

p38

A549UVCtlMp 30998400 45998400 60998400 90998400 120998400 12h 24h

(c)

180

0

60

UVControlTime

120

240

300 p-p38 A549

Den

sity

(IN

Tm

m2)

30998400 45998400 60998400 90998400 120998400 12h 24h

(d)

Figure 7 p38 phosphorylation Detection of p38 phosphorylation in HEp-2 and A549 cells infected with HPIV-1 Cells were infected withlive and inactivated HPIV-1 at different time points and p38 phosphorylation was detected Whole cell lysates were prepared for Westernblotting (as described in Materials and Methods) to detect the phosphorylated (active) forms of the kinases (a) Total protein concentrationsin HEp-2 cells Phospho-p38 was detected with monoclonal antibody sc-7973 and the total p38 sc-535 in HEp-2 cells (b) densitometricanalysis of p38 protein concentrations (c) phospho-p38 and total protein concentrations in A549 cells (d) densitometric analysis of theprotein concentrations Mp molecular weight CTL control and UV (inactive virus) that corresponds to 12 hours in path 3 Data are themeans for triplicate measurements from the three separate experiments

PD980559 (120583M)

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

5120583M 10120583M 15120583M 20120583M

minus + + + +

minus + + + +

minus + + + +

SP600125 (120583M)

SB203580 (120583M)

p-ERK 12

p-JNK

p-p38

Figure 8 Inhibitors of ERK12 PD980559 JNK SP600125 and p38 SB203580 inhibited the expression of each of theMAPKs in the HEp-2 cellline treated cell 1 hour before infection and maintained for 120 minutes after the test The experiments were performed by adding inhibitorat concentrations from 5120583M to 20 120583M (13 to 52 120583L resp) Protein levels were detected by Western blot Immunoblotting was done withmonoclonal antibody specific for protein phosphorylated technique that was described in ldquoMaterials and Methodsrdquo The extracted proteins(minus) represent HEp-2 cells without infecting and the proteins (+) refer to cells infected with the HPIV-1The tests were performed in triplicate

Journal of Immunology Research 9

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

ERKControl

(a)IL

-8 (p

gm

L)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

JNKControl

(b)

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

lowast

lowast

lowast

lowast

p38Control

lowast P gt 005

(c)

Figure 9 Kinetic inhibition ofMAPKs ELISA quantitation of IL-8 concentrations in cells pretreated with ERK12 PD980559 JNK SP600125and SB203580 inhibitors Control is HEp-2 cells infected with live HPIV-1 (a) ERK12 inhibitor assay is displayed in red cells treated with theinhibitor (b) JNK inhibitor assay green occurs in cells treated with the inhibitor (c) p38 inhibitor assay purple occurs in cells treated withinhibitor Data are the means plusmn SD for triplicate measurements from the three separate experiments

a haemagglutinin (HA) and a neuraminidase (NA) bindingto sialic acidmolecules through its HA activity mediating theenzymatic cleavage of sialic acid and promoting fusionthrough its neuraminidase activityThis dual activity suggeststhat coexpressed HN and F form a complex Glycoprotein Hof morbillivirus only has haemagglutinin activity and gly-coprotein G has neither of these two activities [41ndash44]

Therefore these glycoproteins exhibit significant differencesSuch differences are likely related to signalling pathways thatare activated when viruses interact with the cell AlthoughHPIV-1 and RSV belong to the same family the two viruseshave different glycoproteins which may explain why RSVuses ERK kinase andHPIV-1 uses p38 kinase Following virusbinding to its cellular receptor the extracellular stimulus is

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Journal of Immunology Research 9

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

ERKControl

(a)IL

-8 (p

gm

L)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

JNKControl

(b)

IL-8

(pg

mL)

150

0

50

Time

100

200

250

300

45998400 60998400 90998400 120998400 12h 24h

lowast

lowast

lowast

lowast

p38Control

lowast P gt 005

(c)

Figure 9 Kinetic inhibition ofMAPKs ELISA quantitation of IL-8 concentrations in cells pretreated with ERK12 PD980559 JNK SP600125and SB203580 inhibitors Control is HEp-2 cells infected with live HPIV-1 (a) ERK12 inhibitor assay is displayed in red cells treated with theinhibitor (b) JNK inhibitor assay green occurs in cells treated with the inhibitor (c) p38 inhibitor assay purple occurs in cells treated withinhibitor Data are the means plusmn SD for triplicate measurements from the three separate experiments

a haemagglutinin (HA) and a neuraminidase (NA) bindingto sialic acidmolecules through its HA activity mediating theenzymatic cleavage of sialic acid and promoting fusionthrough its neuraminidase activityThis dual activity suggeststhat coexpressed HN and F form a complex Glycoprotein Hof morbillivirus only has haemagglutinin activity and gly-coprotein G has neither of these two activities [41ndash44]

Therefore these glycoproteins exhibit significant differencesSuch differences are likely related to signalling pathways thatare activated when viruses interact with the cell AlthoughHPIV-1 and RSV belong to the same family the two viruseshave different glycoproteins which may explain why RSVuses ERK kinase andHPIV-1 uses p38 kinase Following virusbinding to its cellular receptor the extracellular stimulus is

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Journal of Immunology Research

transmitted to the nucleus to activate the expression of var-ious genes involved in a given biological activity The virusactivates these pathways according to the coevolution of theparasite-host complex and it is likely that the virus also cantake advantage of the activation of the pathway to provide abetter environment and thus freely replicate Viruses such asherpesviruses [12] hepatitis B virus [11] HIV [45] influenzavirus [36] Coxsackie virus and vaccinia virus activate theMAPK pathway particularly ERK in the early stages of in-fection In this study we found thatMAPKs are activated dur-ing the early stages of infection (60minutes) It is possible thatthe activation of the p38 kinase pathway following HPIV-1infection plays a similarly essential role during other stages ofinfection Importantly the stimulus by HPIV-1 increased andwas maintained up to 24 hours therefore it was detectable atthe first stages of infection even when cytopathic effect wasclearly visible in at least 90 of the culture Several authorshave asserted that kinase activation could be a requirementfor signalling regulation associated with different biologicalfunctions during infection with other viruses [22] For exam-ple the first events during HIV-1 infection depend on theMAPKERK pathway which improves viral infectivity [45]

The mechanism by which HPIV-1 stimulates p38 activa-tion needs to be further investigated However a product ofany of the virus genes may be involved in the process con-sidering that no kinase activation was observed when viruseswere UV-irradiated Additionally it is possible that MAPKsalso influence virus replication promoting the synthesis ofsome proteins necessary for the virus

HPIV-1 infection causes inflammation of the larynx andtrachea or croup in children Children with croup are regu-larly admitted to the emergency department with their livesat risk due to airway closure Currently the treatment forpatients withmoderate to severe croup is oral dexamethasoneat 06mgkg (maximum 10ndash12mg) doses which acts on theseand additional cell types involved in the immune response[46] However data show that p38 MAPK inhibitors may re-verse glucocorticoid insensitivity and have beneficial effectson glucocorticoid concentrations in patients with asthma aswell as airway inflammation in addition to its role as a treat-ment substitute [47]

A better understanding ofHPIV-1 pathogenesis will guidethe design of strategies and therapies allowing the inhibitionof excessive cell recruitment and IL-8 synthesis to reduce theuncontrolled inflammatory process For now the exactmech-anism in inflammatory and immune processes must be de-fined and further research is required to understand whetherthese pathways are significant for HPIV-1 pathogenesis

5 Conclusions

An in vitromodel with two cell lines infected with HPIV-1 atdifferent time points was used to determine whether an in-crease in IL-8 production occurred The results showed thatIL-8 production gradually increased during the time pointsassessed and that viral replication is required for IL-8 secre-tion It was found that MAPKs are activated during the earlystages of infection No kinase activation was observed when

viruses were inactivated suggesting that a product of any ofthe virus genes may be involved in the process When a p38inhibitor was used IL-8 concentrations decreased suggestingthat HPIV-1 induces IL-8 secretion through p38 Addition-ally it is possible that MAPKs also affect virus replicationpromoting the synthesis of some proteins necessary for thevirus However further studies are needed to understandwhether these pathways are significant for HPIV-1 pathogen-esis in order to develop strategies and therapies allowing theinhibition of excessive cell recruitment and IL-8 synthesis toreduce the uncontrolled inflammatory process

Conflict of Interests

Theauthors declare they have no conflict of interests and haveno direct financial relationship with any commercial identitymentioned in the paper that might lead to a conflict of in-terests

Acknowledgments

The authors acknowledge the financial assistance of CONA-CYT This paper is part of the doctoral thesis of the studentin the Biomedical Research Institute of the National Auto-nomous University of MexicoThe authors thank Dr PatriciaTato Z and Dr Gladis del Carmen Fragoso G for their in-sightful comments on the paper

References

[1] H Schomacker A Schaap-Nutt P L Collins and A CSchmidt ldquoPathogenesis of acute respiratory illness caused byhuman parainfluenza virusesrdquo Current Opinion in Virology vol2 no 3 pp 294ndash299 2012

[2] C B Hall ldquoRespiratory syncytial virus and parainfluenza virusrdquoThe New England Journal of Medicine vol 344 no 25 pp 1917ndash1928 2001

[3] M E Manjarrez E Montufar D P Rosete R Chapela ICalderon and J Villalba ldquoInfeccion en pacientes con infla-macion y enfermedad pulmonar obstructiva cronica o asmardquoRevista del Instituto Nacional de Enfermedades Respiratoriasvol 12 no 2 pp 101ndash106 1999

[4] M E Manjarrez D P Rosete M Rincon J Villalba A Crav-ioto and R Cabrera ldquoComparative viral frequency in Mexicanchildren under 5 years of agewith andwithout upper respiratorysymptomsrdquo Journal of Medical Microbiology vol 52 no 7 pp579ndash583 2003

[5] A Marx T J Torok R C Holman M J Clarke and L J-Anderson ldquoPediatric hospitalizations for croup (laryngotra-cheobronchitis) biennial increases associated with humanparainfluenza virus 1 epidemicsrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1423ndash1427 1997

[6] M E Counihan D K Shay R C Holman S A Lowther andL J Anderson ldquoHuman parainfluenza virus-associated hospi-talizations among children less than five years of age in theUnited Statesrdquo Pediatric Infectious Disease Journal vol 20 no7 pp 646ndash653 2001

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Journal of Immunology Research 11

[7] M Dobrovoljac and G C Geelhoed ldquoHow fast does oraldexamethasone work in mild to moderately severe croup Arandomized double-blinded clinical trialrdquo Emergency MedicineAustralasia vol 24 no 1 pp 79ndash85 2012

[8] M G Ottolini D D Porter J C G Blanco and G A PrinceldquoA cotton ratmodel of human parainfluenza 3 laryngotracheitisvirus growth pathology and therapyrdquo Journal of InfectiousDiseases vol 186 no 12 pp 1713ndash1717 2002

[9] J D Cherry ldquoState of the evidence for standard-of-care treat-ments for croup are we where we need to berdquo The PediatricInfectious Disease Journal vol 24 no 11 pp S198ndashS201 2005

[10] M Yoshizumi H Kimura Y Okayama et al ldquoRelationshipsbetween cytokine profiles and signaling pathways (I120581B kinaseand p38 MAPK) in parainfluenza virus-infected lung fibrob-lastsrdquo Frontiers in Microbiology vol 1 article 124 2010

[11] K Parhar A Ray U Steinbrecher C Nelson and B Salh ldquoThep38 mitogen-activated protein kinase regulates interleukin-1120573-induced IL-8 expression via an effect on the IL-8 promoter inintestinal epithelial cellsrdquo Immunology vol 108 no 4 pp 502ndash512 2003

[12] X Li D Liang X Lin E S Robertson and K Lan ldquoKaposissarcoma-associated herpesvirus-encoded latency-associatednuclear antigen reduces interleukin-8 expression in endothelialcells and impairs neutrophil chemotaxis by degrading nuclearp65rdquo Journal of Virology vol 85 no 17 pp 8606ndash8615 2011

[13] A K J Chan J M Langley and J C LeBlanc ldquoInterobservervariability of croup scoring in clinical practicerdquo Paediatrics andChild Health vol 6 no 6 pp 347ndash351 2001

[14] J F Bermejo-Martin D Bernardo M Dominguez-Gil etal ldquoInterleukin (IL)-1120573 IL-6 and IL-8 in nasal secretions acommon role for innate immunity in viral bronchial infectionin infantsrdquoTheBritish Journal of Biomedical Science vol 63 no4 pp 173ndash175 2006

[15] R Garofalo M Sabry M Jamaluddin et al ldquoTranscriptionalactivation of the interleukin-8 gene by respiratory syncytialvirus infection in alveolar epithelial cells nuclear translocationof the RelA transcription factor as a mechanism producingairway mucosal inflammationrdquo Journal of Virology vol 70 no12 pp 8773ndash8781 1996

[16] M Z Norzila K Fakes R L Henry J Simpson and P GGibson ldquoInterleukin-8 secretion and neutrophil recruitmentaccompanies induced sputum eosinophil activation in childrenwith acute asthmardquo The American Journal of Respiratory andCritical Care Medicine vol 161 no 3 pp 769ndash774 2000

[17] D S Bischoff J-H Zhu N S Makhijani and D T YamaguchildquoAcidic pH stimulates the production of the angiogenic CXCchemokine CXCL8 (interleukin-8) in human adult mesenchy-mal stem cells via the extracellular signal-regulated kinasep38 mitogen-activated protein kinase and NF-120581B pathwaysrdquoJournal of Cellular Biochemistry vol 104 no 4 pp 1378ndash13922008

[18] R Arnold B Humbert H Werchau H Gallati and W KonigldquoInterleukin-8 interleukin-6 and soluble tumour necrosis fac-tor receptor type release from a human pulmonary epithe-lial cell line (A549) exposed to respiratory syncytial virusrdquoImmunology vol 82 no 1 pp 126ndash133 1994

[19] N Desloges C Schubert M H Wolff and M Rahaus ldquoVari-cella-zoster virus infection induces the secretion of interleukin-8rdquo Medical Microbiology and Immunology vol 197 no 3 pp277ndash284 2008

[20] B Konig T Krusat H J Streckert and W Konig ldquoIL-8 releasefrom human neutrophils by the respiratory syncytial virus isindependent of viral replicationrdquo Journal of Leukocyte Biologyvol 60 no 2 pp 253ndash260 1996

[21] D J Hall M E Bates L Guar M Cronan N Korpi andP J Bertics ldquoThe role of p38 MAPK in rhinovirus-inducedmonocyte chemoattractant protein-1 production bymonocytic-lineage cellsrdquo Journal of Immunology vol 174 no 12 pp 8056ndash8063 2005

[22] A A Andrade P N G Silva A C T C Pereira et al ldquoThevaccinia virus-stimulated mitogen-activated protein kinase(MAPK) pathway is required for virusmultiplicationrdquoBiochem-ical Journal vol 381 no 2 pp 437ndash446 2004

[23] S L JohnstonA Papi P J Bates J GMastronardeMMMon-ick and G W Hunninghake ldquoLow grade rhinovirus infectioninduces a prolonged release of IL-8 in pulmonary epitheliumrdquoJournal of Immunology vol 160 no 12 pp 6172ndash6181 1998

[24] I S Lukashevich R Maryankova A S Vladyko et alldquoLassa and Mopeia virus replication in human monocytesmacrophages and in endothelial cells different effects on IL-8and TNF-alpha gene expressionrdquo Journal of Medical Virologyvol 59 no 4 pp 552ndash560 1999

[25] J L Booth K M Coggeshall B E Gordon and J P MetcalfldquoAdenovirus type 7 induces interleukin-8 in a lung slice modeland requires activation of Erkrdquo Journal of Virology vol 78 no8 pp 4156ndash4164 2004

[26] R J Davis ldquoThe mitogen-activated protein kinase signal trans-duction pathwayrdquo Journal of Biological Chemistry vol 268 no20 pp 14553ndash14556 1993

[27] L Chang and M Karin ldquoMammalian MAP kinase signallingcascadesrdquo Nature vol 410 no 6824 pp 37ndash40 2001

[28] C Dong R J Davis and R A Flavell ldquoMAP kinases in theimmune responserdquo Annual Review of Immunology vol 20 pp55ndash72 2002

[29] R Wahid M J Cannon and M Chow ldquoVirus-specific CD4+and CD8+ cytotoxic T-cell responses and long-term T-cellmemory in individuals vaccinated against poliordquo Journal ofVirology vol 79 no 10 pp 5988ndash5995 2005

[30] D H Clarke and J Casals ldquoTechniques for hemagglutina-tion and hemagglutination-inhibition with arthropod-bornevirusesrdquo The American Journal of Tropical Medicine andHygiene vol 7 no 5 pp 561ndash573 1958

[31] E Trybala B Svennerholm T Bergstrom S Olofsson S Jeans-son and J L Goodman ldquoHerpes simplex virus type 1-inducedhemagglutination glycoprotein C mediates virus binding toerythrocyte surface heparan sulfaterdquo Journal of Virology vol 67no 3 pp 1278ndash1285 1993

[32] W Wiriyarat H Lerdsamran P Pooruk et al ldquoErythrocytebinding preference of 16 subtypes of low pathogenic avianinfluenza and 2009 pandemic influenza A (H1N1) virusesrdquoVeterinary Microbiology vol 146 no 3-4 pp 346ndash349 2010

[33] L Kittigul S Suthachana C Kittigul and V Pengruan-grojanachai ldquoImmunoglobulin M-capture biotin-streptavidinenzyme-linked immunosorbent assay for detection of anti-bodies to dengue virusesrdquo The American Journal of TropicalMedicine and Hygiene vol 59 no 3 pp 352ndash356 1998

[34] Y M Zhu S J Webster D Flower and P J Woll ldquoInterleukin-8CXCL8 is a growth factor for human lung cancer cellsrdquo TheBritish Journal of Cancer vol 91 no 11 pp 1970ndash1976 2004

[35] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Journal of Immunology Research

[36] S Matsukura F Kokubu H Noda H Tokunaga and MAdachi ldquoExpression of IL-6 IL-8 and RANTES on humanbronchial epithelial cells NCI-H292 induced by influenza virusArdquo Journal of Allergy and Clinical Immunology vol 98 no 6 pp1080ndash1087 1996

[37] W Chen M M Monick A B Carter and GW HunninghakeldquoActivation of ERK2 by respiratory syncytial virus in A549 cellsis linked to the production of interleukin 8rdquo Experimental LungResearch vol 26 no 1 pp 13ndash26 2000

[38] S J Ajizian B K English and E A Meals ldquoSpecific in-hibitors of p38 and extracellular signal-regulated kinase mito-gen-activated protein kinase pathways block inducible nitricoxide synthase and tumor necrosis factor accumulation inmurine macrophages stimulated with lipopolysaccharide andinterferon-120574rdquo Journal of Infectious Diseases vol 179 no 4 pp939ndash944 1999

[39] A Kalmes J Deou A W Clowes and G Daum ldquoRaf-1 isactivated by the p38mitogen-activated protein kinase inhibitorSB203580rdquo FEBS Letters vol 444 no 1 pp 71ndash74 1999

[40] V A Young P J Dillon and G D Parks ldquoVariants of theParamyxovirus Simian virus 5 with accelerated or delayed viralgene expression activate proinflammatory cytokine synthesisrdquoVirology vol 350 no 1 pp 90ndash102 2006

[41] K J Henrickson ldquoParainfluenza virusesrdquo Clinical MicrobiologyReviews vol 16 no 2 pp 242ndash264 2003

[42] Y Matsuoka J Curran T Pelet D Kolakofsky R Ray and RW Compans ldquoThe P gene of human parainfluenza virus type1 encodes P and C proteins but not a cysteine-rich V proteinrdquoJournal of Virology vol 65 no 6 pp 3406ndash3410 1991

[43] C Ah-Tye S Schwartz K Huberman E Carlin and AMoscona ldquoVirus-receptor interactions of human parainfluenzaviruses types 1 2 and 3rdquo Microbial Pathogenesis vol 27 no 5pp 329ndash336 1999

[44] K J Henrickson and L L Savatski ldquoAntigenic structure func-tion and evolution of the hemagglutinin-neuraminidase pro-tein of human parainfluenza virus type 1rdquo Journal of InfectiousDiseases vol 176 no 4 pp 867ndash875 1997

[45] X Yang Y Chen and D Gabuzda ldquoERK MAP kinase linkscytokine signals to activation of latent HIV-1 infection bystimulating a cooperative interaction of AP-1 and NF-120581BrdquoJournal of Biological Chemistry vol 274 no 39 pp 27981ndash27988 1999

[46] K K Rittichier ldquoThe role of corticosteroids in the treatment ofcrouprdquoTreatments in RespiratoryMedicine vol 3 no 3 pp 139ndash145 2004

[47] E Irusen J GMatthews A Takahashi P J Barnes K F Chungand I M Adcock ldquop38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces itsactivity role in steroid-insensitive asthmardquo Journal of Allergyand Clinical Immunology vol 109 no 4 pp 649ndash657 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom