induction of cxc chemokines in a 549 airways epithelial cells by trypsin in crs

Clinical and Experimental Immunology doi:10.1111/j.1365-2249.2006.03089.x

534

© 2006 British Society for Immunology,

Clinical and Experimental Immunology

,

144:

534–542

et al.

Accepted for publication March 2006

Correspondence: Florian Sachse MD, ENT

Department, Universitätsklinikum Münster,

Kardinal-von-Galen Ring 10, 48149 Münster,

Germany.

E-mail: [email protected]

OR IG INAL ART I C L E

Induction of CXC chemokines in A

549

airway epithelial cells by trypsin and staphylococcal proteases

-

a possible route for neutrophilic inflammation in chronic rhinosinusitis

F. Sachse,* C. von Eiff,

†

W. Stoll,* K. Becker

†

and C. Rudack*

*Department of Otorhinolaryngology, Head and

Neck Surgery, University Hospital Münster,

Germany, and

†

Institute of Medical Microbiology,

University Hospital Münster, Germany

Summary

While various microorganisms have been recovered from patients withchronic rhinosinusitis, the inflammatory impact of virulence factors, in par-ticular proteases from

Staphylococcus aureus

and coagulase negative staphy-lococci on the nasal epithelium, has not yet been investigated. Expression ofCXC chemokines was determined in the epithelium of patients with chronicrhinosinusitis by immunohistochemistry. In a cell culture system of A549 res-piratory epithelial cells, chemokine levels were quantified by enzyme-linkedimmunosorbent assay (ELISA) after stimulation with supernatants originat-ing from three different staphylococcal strains or with trypsin, representing aserine protease. Inhibition experiments were performed with prednisolone,with the serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonylfluo-ride (AEBSF) and with the nuclear transcription factor (NF)-

κκκκΒΒΒΒ

inhibitor(2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulphonyl]-2-propenenitrite (BAY)11–7085

.

Electromobility shift assays (EMSA) were used to demonstrate NF-

κκκκ

B-dependent protein synthesis. CXC chemokines interleukin (IL)-8, growth-related oncogene alpha (GRO-

αααα

) and granulocyte chemotactic protein-2(GCP-2) were expressed in the patients’ epithelium whereas epithelial cell-derived neutrophil attractant 78 (ENA-78) was rarely detected. In A549 cells,chemokines IL-8, ENA-78 and GRO-

αααα

but not GCP-2 were induced by trypsinand almost equal levels were induced by staphylococcal supernatants. IL-8,GRO-

αααα

and ENA-78 synthesis was suppressed almost completely by AEBSFand BAY 11–7085, whereas prednisolone reduced chemokine levels differen-tially dependent on the supernatant added. CXC chemokines were detectablein the epithelium of patients with chronic rhinosinusitis. Staphylococcalserine proteases induced CXC chemokines in A549 cells, probably by the acti-vation of proteases activated receptors, and thus might potentially be involvedin neutrophilic inflammation in chronic sinusitis.

Keywords:

A549, chemokines, PAR, sinusitis,

Staphylococcus aureus

Introduction

Sinusitis is an inflammatory process involving one or moresinuses, and is divided commonly into acute, recurrent andchronic forms [1]. Nasal polyposis (NP) represents a severesubform of chronic rhinosinusitis and is characterized clin-ically by nasal polyps originating from the middle nasalmeatus and/or from the anterior ethmoid. In most cases ofbilateral diffuse NP histology revealed a marked tissue infil-tration with eosinophils and to a lesser degree with neutro-phils and other inflammatory cells. In contrast, in tissuespecimens of patients suffering from chronic rhinosinusitis

without nasal polyps (CRS), more neutrophils than eosino-phils were detected [2], assuming that CRS and NP are dif-ferent entities of chronic rhinosinusitis. Another strikingphenomenon reveals that permanent cure of patients suffer-ing from NP, in particular, cannot be achieved, neither withsinus surgery nor with application of steroids, and the rea-sons are therefore unknown. As in allergic rhinitis andasthma, little is known about the role of neutrophils. Here,it has been assumed that neutrophil chemokines areregulated differently by steroids and that failure of thisregulation might be one reason for persistence of lungneutrophilia.

Induction of CXC chemokines in A549 airway epithelial cells



,

144:

534–542

535

Because more than 50% of neutrophils were activateddue to neutrophil-elastase positivity [2] in NP and CRS, wefocused on possible routes of regulation mechanisms ofneutrophil-attracting CXC chemokines. For this purpose wetook the presence of bacteria in the upper airway intoaccount, as the impact of these microorganisms on theonset of chronic rhinosinusitis still remains unclear. Recentreports revealed that a wide spectrum of bacterial specieshas been recovered from patients with chronic rhinosinusi-tis, among them

Staphylococcus aureus,

coagulase-negativestaphylococci (CoNS),

Pseudomonas aeruginosa

and anaero-bic bacteria [3–5]. While it has been demonstrated that

S. aureus

enterotoxins may act as superantigens, therebyinducing a topical multi-clonal IgE-formation as well as asevere, possibly steroid-insensitive eosinophilic inflamma-tion in NP [6], further research is warranted to study theeffects of other major staphylococcal virulence factors suchas proteases on the airway epithelium in chronic rhinosi-nusitis. In particular, soluble serine proteases are known toinitiate and maintain inflammatory mechanisms by activat-ing specific G protein-coupled receptors, defined as pro-tease activated receptors (PARs). Activation of PARs leads toG-protein regulated gene transcription responses that inturn induce the production of cytokines and chemokines[7].

As a first step, we investigated expression profiles of CXCchemokines growth-related oncogene alpha (GRO-

α

/CXCL1), epithelial cell-derived neutrophil attractant 78(ENA-78/CXCL5), granulocyte chemotactic protein-2(GCP-2/CXCL6) and interleukin (IL)-8/CXCL8 in the nasalepithelium of patients suffering from chronic rhinosinusitis.These factors were selected because the epithelial layer of thenasal/paranasal mucosa represents the first line of defenceagainst microorganisms, and induction of CXC chemokineshas been observed in bacterial-induced airway inflammation[8].

Subsequently, in a cell culture model of A549 airway epi-thelial cells known to express PAR-1 to PAR-4 [9], IL-8,ENA-78, GRO-

α

and GCP-2 expression was induced by thePAR-2/PAR-4 agonist trypsin and quantified after 6 and24 h. In addition, three different staphylococcal superna-tants were used to investigate their impact on the expres-sion of CXC chemokines in A549 airway epithelial cells.Finally, chemokine responses were modulated by the serineprotease inhibitor 4-(2-aminoethyl)-benzenesulphonylfluo-ride (AEBSF) or by prednisolone and the involvement ofnuclear transcription factor

κΒ

(NF-

κΒ

) on trypsin- andstaphylococcal-mediated expression of neutrophil chemok-ines was investigated by electromobility shift assays(EMSA).

Materials and methods

Trypsin, and unless otherwise stated, all other reagents used,were purchased from Sigma (Deisenhofen, Germany).

Patients

Fifteen CRS specimens [recovered from seven males, meanage

±

standard deviation (s.d.), 52·60

±

24·93 years, eightfemales, 55·71

±

18·71, all of them non-allergic, non-smok-ers], 15 NP specimens (eight males, 65·60

±

16·92, sevenfemales 57·80

±

10·43, all of them non-allergic, non-smokers, no aspirin-sensitivity) and 15 turbinate mucosa(TM) specimens (seven males, 40·15

±

18·72, eight females36·86

±

12·86, all of them non-allergic, non-smokers) wereembedded in paraffin.

Chronic rhinosinusitis was diagnosed according to themedical history with corresponding clinical symptoms, apreoperative computerized tomography (CT) scan andan endoscopic examination of the nasal cavity. Thosecases in which nasal polyps were present in the middlenasal meatus were classified as NP. TM was obtainedfrom patients undergoing septoplasty with no history ofrhinosinusitis. No patient had undergone sinus surgerypreviously. Allergy was excluded by negative skin-pricktests to common inhalant allergens and radio allergy sor-bent test (RAST). No patient had been treated with sys-temic/topical steroids or antibiotics 4 weeks prior tosurgery. Informed consent was obtained from all patientsand the study was approved by the ethics committee ofthe University of Muenster, Germany.

Immunohistochemistry

Paraffin was resolved from sections using decreasing con-centrations of ethanol. Subsequent steps were performedaccording to the instructions of the Dako-LSAB kit (Dako-LSAB

+

system HRP kit (Universal), no. K0679; Dako Ham-burg, Germany) as described previously for chemokines.Briefly, after inhibition of the endogenous peroxidase,polyclonal antibodies were added (IL-8: polyclonal rabbit–human IL-8 antibody, 1 : 50, AHC0881, Biosource, Solin-gen, Germany; ENA-78: polyclonal goat–human ENA-78antibody, 1 : 200, AF254, R&D, Wiesbaden, Germany; GRO-

α

: polyclonal goat–human antibody GRO-

α

, 1 : 50, SC-1374, Santa Cruz, Heidelberg, Germany; GCP-2: polyclonalgoat–human GCP-2 antibody, 1 : 100, SC-5813, SantaCruz). These primary antibodies were replaced by a non-immune serum of the first antibodies for the negativecontrol.

A universal biotinylated link-antibody served as secondaryantibody. Subsequently, streptavidin peroxidase (streptavi-din conjugated to horseradish peroxidase, HRPOD, Dako)was added. Sections were then incubated with diaminoben-zidine (DAB) chromogen (Dako) substrate for 5 min. Withregard to GRO-

α

and GCP-2, AEC chromogen substrate(Dako) was added for 30 min. Finally, sections were washedwith Mayer’s haematoxylin (Merck, Darmstadt, Germany)for 20 s.

F. Sachse

et al.

536



,

144:

534–542

Microscopic quantification of stained cells

Positive-stained cells were investigated analysing the surfaceepithelium of TM (

n

=

15), CRS (

n

=

15) and NP (

n

=

15) bytwo independent investigators. Stained cells were counted infour randomly observed microscopic fields at 1000

×

magni-fication and divided by the total number of epithelial cells inthat field. The mean of the four ratios was the count for eachsample. Data were analysed using the non-parametricMann–Whitney

U

-test and shown as means

±

s.d. The levelof significance was set at

P

<

0·05 (*).

Cultivation of A549 cells

A549 cells (ATTC CCL-185) were obtained from the Amer-ican Type Culture Collection (Manassas, VA, USA). Cellswere grown in Hams F12K medium (

gibco

Life Technolo-gies, Eggenstein, Germany) and supplemented with 10%fetal calf serum (FCS), 100 U/ml penicillin and 100

µ

g/mlstreptomycin (Sigma). A549 cells were cultured at 37

°

C,gassed with 5% CO

2

and grown to 80% confluence. Finally,they were split using trypsin (0·1%).

Bacterial strains

For stimulation experiments, the well-characterized

S.aureus

laboratory strains Newman and COL and the refer-ence

S. epidermidis

strain DSM 20044 were used.

S. aureus

COL, a methicillin-resistant strain (

mecA

gene-positive),was tested polymerase chain reaction (PCR)-positive forenterotoxin B gene (

seb

), whereas the Newman strain waspositive for enterotoxin A gene (

sea

) [10]. In a previousstudy using an

in vitro

assay for protease activity in cul-ture supernatants,

S. aureus

Newman produced 240 U/mgprotein or 180 U/1

×

10

8

colony-forming units (CFU) [11].The

S. epidermidis

strain used is superantigen negative[12].

Stimulation of A549 cells

Prior to stimulation, cells were incubated with Hams F12Kmedium devoid of FCS for 24 h. Characterization and purityof A549 cells was examined by inverted-phase contrastmicroscopy. Cell viability assessed by trypan blue exclusionwas greater than 95% in all experiments. Staphylococcalstrains were grown in 150 ml tryptic soy bouillon (TSB) for10–12 h, adjusted to OD

=

1·0 with sterile TSB, an aliquotremoved for CFU determination (5

×

10

8

−

1

×

10

9

), and thebacteria removed by centrifugation (3000

g

for 20 min, 4

°

C).Supernatants of

S. aureus

COL and

S. epidermidis

DSM20044 were added in a dilution of 1 : 5. Supernatants of

S aureus

Newman D2C (ATCC 25904) were employed in adilution of 1 : 10.

Inhibition was performed with prednisolone (Merck) at aconcentration of 10

µ

M

or with the serine protease inhibitor

AEBSF [4-(2-aminoethyl)-benzenesulphonyl fluoride] at aconcentration of 1 m

M

.BAY 11–7085 [(2E)-3-4-1,1-dimethylethylphenylsulpho-

nyl-2-propenenitrile], which represents an inhibitor of

Ν

F-

κΒ

, was employed at a concentration of 10 m

m

to block NF-

κ

B dependent synthesis of CXC chemokines.

Protein measurements

After 6 and 24 h, supernatants were harvested and enzyme-linked immubosorbent assay (ELISA) tests for IL-8 (detec-tion range

>

3·5 pg/ml), ENA-78 (detection range

>

15 pg/ml), GRO-

α

(detection range

>

10 pg/ml) and GCP-2 (detec-tion range

>

1·6 pg/ml) were performed in duplicate accord-ing to the manufacturer’s instructions (R&D Systems,Wiesbaden, Germany). The optical density of the sampleswas measured at a wavelength of 450 nm. Protein levels weretested using the non-parametric Mann–Whitney

U

-test.Results were shown as mean

±

s.d.

P

-values

<

0·05 (*) wereconsidered statistically significant.

EMSA

The assay was performed as described recently with minormodifications [13]. Briefly, after treatment of A549 with

S.aureus

Newman and trypsin for 30 and 60 min, cells werewashed with phosphate-buffered saline (PBS) and lysed in60

µ

l of total cell extract buffer (20 m

m

HEPES pH 7·9,350 m

M

NaCl, 1 m

M

MgCl

2

, 0·5 m

M

ethylenediamine tet-raacetic acid (EDTA), 0·1 m

m

ethylene-bis(

β

-aminoethylether)-N,N,N

′

,N

′

-tetraacetic acid, 20% (v/v) glycerol, 1%(v/v) Nonidet P-40, 0·5 m

M

dithiothreitol (DTT), 2 m

M

phenylmethylsulphonyl fluoride (PMSF), 50

µ

g/ml aproti-nin, 50

µ

g/ml leupeptin). The lysate-containing proteinswas cleared by centrifugation and the supernatant wasstored at

−

80

°

C after the protein concentration had beendetermined. The binding reaction was carried out in 20 µl ofthe mixture containing 4 µl of cell extract (10 µg protein),4 µl of 5× binding buffer (20 mM HEPES, pH 7·7, 50 mMKCl, 1 mM DTT, 2·5 mM MgCl2 and 20% Ficoll), 2 µgpoly(dI-dC) (Roche Diagnostics, Mannheim, Germany) as anon-specific competitor DNA, 2 µg of bovine serum albu-min (BSA), and 80 000 counts per minute (cpm) of thelabelled oligonucleotide. After 20 min of incubation at roomtemperature, the samples were electrophoresed through a4% non-denaturing polyacrylamide gel. The gel was thendried and exposed to Kodak BioMax XAR film. To verifyspecific NF-κB activity, competitive EMSA with a 30-foldexcess of unlabelled oligonucleotide was used in the bindingreaction and allowed to react for 20 min before adding [32P]-end-labelled oligonucleotide.

The NF-κB-binding oligonucleotide (Santa Cruz Biotech-nology) was end-labelled using [γ-32P] ATP (HartmannAnalytic, Braunschweig, Germany) by T4 polynucleotidekinase (Fermentas Life Sciences, St Leon-Rot, Germany)according to the manufacturer’s instructions. The labelled


© 2006 British Society for Immunology, Clinical and Experimental Immunology, 144: 534–542 537

oligonucleotide was purified using the nucleotide removalkit (Qiagen, Hilden, Germany).

Results

Immunohistochemistry of nasal epithelium

IL-8-, GRO-α- and GCP-2-stained cells were increased sig-nificantly in the epithelium of CRS patients compared toTM. A similar pattern of IL-8, GRO-α and GCP-2 expressionwas found for NP. However, IL-8 and GCP-2 expression inNP was not significantly different from that in TM. ENA-78expression was rarely observed and did not differ signifi-cantly between CRS, NP and TM. GCP-2 expression wasfound to be increased in the epithelium of CRS compared toNP (Figs 1 and 2).

A549 cells release IL-8, GRO-αααα and ENA-78, but not GCP-2

Trypsin is a serine protease and activates PAR-2 and PAR-4 bycleaving the receptor. A significant increase of IL-8, ENA-78and GRO-α production after 6 and 24 h was noted. Highestresponses were determined for ENA-78 followed by IL-8 after

Fig. 1. Epithelial expression of interleukin (IL)-

8 in (a), expression of growth-related oncogene

alpha (GRO-α) in (b), expression of epithelial

cell-derived neutrophil attractant 78 (ENA-78)

in (c) and expression of granulocyte chemotactic

protein-2 (GCP-2) in (d) in surgical specimens

from chronic rhinosinusitis (CRS) patients. (e)

Lower degree of IL-8 expression in turbinate

mucosa (control).

(b)(a)

(c) (d)

(e)

Fig. 2. Expression of neutrophil chemokines in the nasal epithelium.

Data represent mean ratios (stained cells/total epithelial cells) of turbi-

nate mucosa (TM, n = 15), chronic rhinosinusitis (CRS, n = 15) and

nasal polyposis (NP, n = 15). Cells were counted in four randomly

selected high-power fields at 1000× magnification. The level of signifi-

cance was set at P < 0·05.

TM

Sta

ined

cel

ls/to

tal e

pith

elia

l cel

ls

0·0

0·2

0·4

0·6

0·8

1·0

IL-8 ENA-78 GRO-αGCP-2

**

*

*

CRS NP

F. Sachse et al.

538 © 2006 British Society for Immunology, Clinical and Experimental Immunology, 144: 534–542

24 h. GRO-α production increased slightly between 6 and24 h, but remained low compared to ENA-78 (Fig. 3).

In contrast to IL-8, GRO-α and ENA-78, only very low butyet detectable amounts of GCP-2 were released upon stimu-lation with trypsin. In order to stimulate the synthesis ofGCP-2, IL-1β and lipopolysaccharide (LPS) were added tothe cell culture. It was found that GCP-2 synthesis wasweakly driven up by the classical proinflammatory cytokineIL-1β, but not by LPS stimulation (results not shown).

Staphylococcal supernatants induce IL-8, ENA-78 and GRO-αααα expression in A549 cells

Addition of S. aureus COL supernatants resulted in a signif-icant increase in the production of IL-8, ENA-78 and GRO-

α after 6 and 24 h. Again, the strongest chemokine responseswere determined for ENA-78 after 24 h. Interestingly,addition of S. epidermidis DSM20044 and S. aureus COLsupernatants caused a stronger IL-8, ENA-78 and GRO-αresponse after 6 and 24 h than addition of S. aureus Newmansupernatants (Fig. 4).

Chemokine production is inhibited by prednisolone, AEBSF and BAY 11–7085

Production of IL-8, ENA-78 and GRO-α was reduced signif-icantly by prednisolone, AEBSF and BAY 11–7085 in allexperiments (Fig. 5). Prednisolone displayed different effi-cacy in the inhibition of chemokines induced by staphylo-coccal supernatants.

Fig. 3. Release of CXC chemokines from A549

cells in response to trypsin. Trypsin-induced

chemokine expression in A549 cells after 6 and

24 h (enzyme-linked immunosorbent assay).

Bars represent means ± standard deviation of

four independent experiments performed in

duplicate. The level of significance was set at

P < 0·05.Hours

6

Che

mok

ine

conc

entr

atio

n (p

g/m

l)

0

500

1000

1500

2000

*

Hours60

500100015002000250030003500

*

*

Hours6

0

200

400

600

800

1000*

*

*

Che

mok

ine

conc

entr

atio

n (p

g/m

l)

Che

mok

ine

conc

entr

atio

n (p

g/m

l)

IL-8 ENA-78 GRO-αControlTrypsin

ControlTrypsin

ControlTrypsin

24 24 24

Fig. 4. Induction of interleukin (IL)-8, epithelial

cell-derived neutrophil attractant 78 (ENA-78)

and growth-related oncogene alpha (GRO-α) by

stapylococcal supernatants. Induction of neutro-

phil chemokines by staphylococcal supernatants

in A549 cells after 6 and 24 h (enzyme-linked

immunosorbent assay). Bars represent

means ± standard deviation of four independent

experiments performed in duplicate. The level of

significance was set at P < 0·05.

6-h Stimulation

Che

mok

ine

conc

entr

atio

n (p

g/m

l)

0

500

1000

1500

2000

250024-h Stimulation

Che

mok

ine

conc

entr

atio

n (p

g/m

l)

0

2 000

4 000

6 000

8 000

10 000

12 000

14 000

IL-8 IL-8

** * *

*

*

**

**

** *

*

*

**

*

S. epidermidis DSM S. aureus Newman

S. aureus COL

Controls

ENA-78 GRO-α ENA-78 GRO-α

Fig. 5. Inhibition of interleukin (IL)-8, epithelial cell-derived neutrophil attractant 78 (ENA-78) and growth-related oncogene alpha (GRO-α) synthesis

in A549 cells by prednisolone, 4-(2-aminoethyl)-benzenesulphonylfluoride (AEBSF) and BAY 11–7085. Data represent mean percentages ± standard

deviation of four independent experiments after 12 h of stimulation with bacterial supernatants and addition of prednisolone, AEBSF or BAY 11–7085.

Values of inhibited chemokine production were referred to corresponding means of experiments without addition of inhibitors. Inhibition was significant

in all cases (P < 0·0.05). Trypsin (Tryp), Staphylococcus aureus COL (COL), S. epidermidis DSM20044 (DSM), S. aureus Newman (New).

Tryp

Inhi

bitio

n of

che

mok

ine

synt

hesi

s (%

)

0

20

40

60

80

100

Prednisolone (10 µM) AEBSF (1 mM) BAY-11 (10 mM)

Inhi

bitio

n of

che

mok

ine

synt

hesi

s (%

)

0

20

40

60

80

100

Inhi

bitio

n of

che

mok

ine

synt

hesi

s (%

)

0

20

40

60

80

100IL-8 ENA-78 GRO-α

COL DSM New Tryp COL DSM New Tryp COL DSM New



Addition of AEBSF caused almost total inhibition of IL-8,ENA-78 and GRO-α responses induced by S. epidermidisDSM20044 and S. aureus Newman, whereas S. aureus COLstimulation was inhibited by 50–60% only. Remarkably,induction of IL-8, ENA-78 and GRO-α by staphylococcalsupernatants was inhibited more effectively by AEBSF thanby prednisolone, thus implying protease-mediated chemok-ine synthesis (Fig. 5). Addition of BAY 11–7085, an inhibitorof NF-κB, caused almost complete blockage of chemokinesynthesis independent of the applied staphylococcalsupernatant.

Toxic effects of the applied inductors and inhibitors wereconsidered observing the morphology of A549 cells after24 h of stimulation. Shrinkage of cells was always visible.Desquamation of cells was pronounced if cells were stimu-lated with S. aureus COL or S. aureus Newman. Cell deathwas not observed in our experiments, with the exception ofa low rate of 3% in the case of AEBSF.

Trypsin and S. aureus Newman signalling in human nasal mucosa cells via transcription factor NF-κκκκB

EMSAs were performed with a [32P]-end-labelled oligonu-cleotide containing the NF-κB binding site. The PAR-2 andPAR-4 agonist trypsin (lane 4) showed a protein DNA com-plex band at the same level as the positive control [tumournecrosis factor (TNF)-α, lane 1]. Supernatants of S. aureusNewman showed a similar protein complex to TNF-α andtrypsin. AEBSF caused an intensity loss of the band (lanes 5and 7), indicating a reduction of the protein DNA complex.Pretreatment of the cells with prednisolone caused a reducedintensity of the band (lanes 3 and 8) (Fig. 6).

Our data reveal that specific PAR-2 and PAR-4 stimulus,trypsin and supernatants of S. aureus Newman induced acti-vation of NF-κB in A549 cells.

Discussion

The aetiology of chronic rhinosinusitis is still poorly under-stood. Most recently, findings point at bacterial or microbialproducts that may be involved in the initiation or mainte-nance of chronic rhinosinusitis by the modulation ofproinflammatory mediators and the initiation of tissueremodelling [14–16]. Paranasal sinuses are believed to besterile, although the upper respiratory tract, anterior naresand nasopharynx are colonized with numerous microorgan-isms forming the transient and resident microflora, amongthem staphylococci, streptococci and Corynebacterium spe-cies [17]. While CoNS are the most commonly reportedmicroorganisms in patients with chronic rhinosinusitis,their significance is still a matter of debate [3,4,18–23].

Generally, the virulence of S. aureus and in part of CoNS isdetermined by cell wall proteins as well as secreted toxins andenzymes. Concerning S. epidermidis, an extracellular metal-loprotease with elastase activity has been detected [24]. Fur-thermore, an extracellular serine protease is involved in theprocessing of epidermin, structurally an antibiotic peptideknown to be important for colonization mechanisms onmucosal surfaces [23].

With regard to S. aureus, extra-bacterial proteins that con-tribute to its virulence comprise adhesion proteins such asthe microbial surface components recognizing adhesivematrix molecules (MSCRAMMs), a broad range of exotoxins(e.g. superantigens, exfoliative toxins and pore-forming tox-ins) and enzymes (e.g. proteases, lipases and nucleases)[25,26]. Gene expression of virulence factors is very complexand regulated mainly at the transcriptional level. Factors thatinfluence gene expression are regulated largely by two-component regulatory systems such as the agr (accessorygene regulator) system. Examples of agr-regulated genesinclude S. aureus haemolysins (hla, hlb, hld, hlg), TSST-1 (tst)and enterotoxins B-C [27,28]. Moreover, gene expression ofseveral proteases (slp A, B, C, D, E, F genes) and of the serineprotease SspA has been shown to be agr-regulated [29,30].Dunman et al. have identified more than 100 genes that areup-regulated and 34 that are down-regulated by the agr-system, reflecting the enormous complexity of virulencedeterminants in S. aureus [31]. Although it has been dem-onstrated that S. aureus enterotoxins may contribute to thepathogenesis of nasal polyposis [6,32,33], little is knownabout the role of other staphylococcal virulence factors suchas serine proteases.

We therefore investigated the impact of staphylococcalproteases on the induction of CXC chemokines in A549 air-way epithelial cells as epithelial cells are not consideredmerely as a barrier against microorganisms and environ-mental influences, but are also capable to modulate inflam-matory responses actively by the generation of cytokines andchemokines which, in turn, attract leucocytes at sites ofinflammation [34]. Previous studies have revealed that theepithelium of the respiratory reacts against luminal antigens

Fig. 6. Activation of nuclear transcription factor (NF)-κB in A549 cells.

Lane 1: tumour necrosis factor (TNF)-α (positive control), lane 2: neg-

ative control. Lanes 3–5: stimulation with trypsin and inhibition with

prednisolone (+ prds) or 4-(2-aminoethyl)-benzenesulphonylfluoride

(AEBSF). Lanes 6–8: stimulation with Staphylococcus aureus Newman

and inhibition with prednisolone or AEBSF. Prds: prednisolone.

NF-κΒ

1 2 3 4 5 6 7 8

TNF-α Trypsin

+AEBSF+Prds. +AEBSF

Newman

+Prds.

F. Sachse et al.


and environmental factors by the production of proinflam-matory signals as, for instance, IL-8 synthesis [35,36].

In the present study we could demonstrate that the PAR-2agonist trypsin significantly increased IL-8, ENA-78 andGRO-α responses in A549 airway epithelial cells after 6 and24 h of stimulation, as demonstrated previously by others[9,37]. Stimulation of A549 cells with trypsin was associatedwith the activation of NF-κB as shown by EMSA, andchemokine expression was completely inhibited by the NF-κB inhibitor BAY 11–7085. These results indicated thattrypsin-induced chemokine expression was dependent onNF-κB, as demonstrated by Page and coworkers [38].

To investigate the impact of staphylococcal proteases onchemokine release, we performed inhibition experimentsusing the serine protease inhibitor AEBSF. Chemokineexpression could almost be suppressed by AEBSF, moreeffective in experiments applying S. aureus Newman and inS. epidermidis DSM20044 than in those using S. aureus COL,thus indicating the importance of bacterial serine proteasesas stimulators of chemokine synthesis in our cell culturemodel. Protease activation has been considered importantfor airway inflammation and airway remodelling. Amongthe four different PARs identified so far, PAR-2 has beenexceedingly characterized in recent years and was found tobe increased in the epithelial cells of patients with asthma[39]. Expression of CXC chemokines as a result of activationof PARs by staphylococcal proteases might therefore repre-sent a possible mechanism to explain neutrophilic airwayinflammation in chronic rhinosinusitis.

Prednisolone was tested for its ability to abolish chemok-ine synthesis, as topical corticosteroids are recommended forthe treatment of acute and chronic rhinosinusitis [1,15]. Wehave shown previously that treatment of nasal polyps withprednisolone resulted in a down-regulation of IL-5 [40].Even production of inflammatory mediators and recruit-ment of leucocytes in response to a Gram-negative bacterialtoxin and staphylococcal enterotoxin have been shown to besensitive to glucocorticoid treatment [41]. In this study,addition of prednisolone caused a reduction but not a com-plete suppression of chemokine production, in particularusing S. aureus COL and S. epidermidis as bacterial strains.Very recently, it has been demonstrated that IL-8 expressionwas even increased by methylprednisolone in asthmatic air-way mucosa, whereas eosinophil and CC chemokine expres-sion was decreased [42]. These results reflect the currentdiscussion about the dual nature of corticosteroids exertingdifferential effects on the regulation of chemokines. In addi-tion, partial inhibition with prednisolone might also beexplained by other chemokine-inducing factors such aslipoteichoic acids (LTA) which might also be present in sta-phylococcal supernatants.

In contrast to IL-8, GRO-α and ENA-78, no substantialquantities of GCP-2 protein were produced by A549 cells. Toexamine other proinflammatory stimuli that are capable ofinducing GCP-2, we used IL-1β and LPS in a concentration-

dependent manner [43] (results not shown). Even high con-centrations of LPS did not lead to a significant release ofGCP-2. IL-1β at a concentration of 10 ng/ml induced ENA-78, GRO-α, followed by IL-8 and GCP-2, which is in accor-dance with the results of other stimulation experiments inendothelial cells, human umbilical vein endothelial cells(HUVECs) and A549 cells [44]. Despite the fact that theproduction of GCP-2 by most cell types is weak in vitro [45],the detection of GCP-2 by immunohistochemistry is pro-nounced in the nasal epithelium, as it is in other forms oftissue [44]. By contrast, IL-8 is weakly detected by immuno-histochemistry while it is produced at high levels in fibro-blasts, endothelial cells and monocytes [45]. ENA-78 wasrarely detectable in our immunohistochemistry, whereasabundant ENA-78 expression in A549 cells was expected, asit was originally cloned from this cell line [46]. The specificGCP-2 staining in the nasal epithelium suggests that thefunction of GCP-2 is different from that of IL-8 and ENA-78,despite their structural relationship. Our findings supportthe notion that the chemokine network shows complemen-tarity rather than redundancy, as assumed by others [44].

To summarize, we demonstrated epithelial expression ofCXC chemokines in chronic rhinosinusitis using immuno-histochemistry. In a cell culture system of A549 cells, whichare known to express PAR-1 to PAR-4, we showed PAR-2-and PAR-4-dependent stimulation of the neutrophilchemokines IL-8, GRO-α and ENA-78 by trypsin. Additionof the serine protease AEBSF to supernatants of differentstaphylococci revealed that soluble serine proteases areinvolved in chemokine regulation, and thus might activatePARs. In conclusion, bacterial serine proteases might beinvolved in neutrophilic airway inflammation in chronicrhinosinusitis.

References

1 Meltzer EO, Hamilos DL, Hadley JA et al. Rhinosinusitis: establish-

ing definitions for clinical research and patient care. J Allergy Clin

Immunol 2004; 114:155–212.

2 Rudack C, Sachse F, Alberty J. Chronic rhinosinusitis − need for

further classification? Inflamm Res 2004; 53:111–7.

3 Hsu J, Lanza DC, Kennedy DW. Antimicrobial resistance in bac-

terial chronic sinusitis. Am J Rhinol 1998; 12:243–8.

4 Biel MA, Brown CA, Levinson RM et al. Evaluation of the micro-

biology of chronic maxillary sinusitis. Ann Otol Rhinol Laryngol

1998; 107:942–5.

5 Brook I. Sinusitis − overcoming bacterial resistance. Int J Pediatr

Otorhinolaryngol 2001; 58:27–36.

6 Zhang N, Gevaert P, van Zele T et al. An update on the impact of

Staphylococcus aureus enterotoxins in chronic sinusitis with nasal

polyposis. Rhinology 2005; 43:162–8.

7 Reed CE, Kita H. The role of protease activation of inflammation

in allergic respiratory diseases. J Allergy Clin Immunol 2004;

114:997–1008.

8 Ratner AJ, Lysenko ES, Paul MN, Weiser JN. Synergistic proinflam-

matory responses induced by polymicrobial colonization of epi-

thelial surfaces. Proc Natl Acad Sci USA 2005; 102:3429–34.



9 Asokananthan N, Graham PT, Fink J et al. Activation of protease-

activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-

8, and prostaglandin E2 release from human respiratory epithelial

cells. J Immunol 2002; 168:3577–85.

10 Becker K, Roth R, Peters G. Rapid and specific detection of toxi-

genic Staphylococcus aureus: use of two multiplex PCR enzyme

immunoassays for amplification and hybridization of staphylococ-

cal enterotoxin genes, exfoliative toxin genes, and toxic shock syn-

drome toxin 1 gene. J Clin Microbiol 1998; 36:2548–53.

11 Jonsson IM, von Eiff C, Proctor RA, Peters G, Ryden C, Tarkowski

A. Virulence of a hemB mutant displaying the phenotype of a Sta-

phylococcus aureus small colony variant in a murine model of septic

arthritis. Microb Pathog 2003; 34:73–9.

12 Becker K, Haverkamper G, von Eiff C, Roth R, Peters G. Survey of

staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic

shock syndrome toxin 1 gene in non-Staphylococcus aureus species.

Eur J Clin Microbiol Infect Dis 2001; 20:407–9.

13 Buddenkotte J, Stroh C, Engels IH et al. Agonists of proteinase-

activated receptor-2 stimulate upregulation of intercellular cell

adhesion molecule-1 in primary human keratinocytes via activa-

tion of NF-kappa B. J Invest Dermatol 2005; 124:38–45.

14 Rudack C, Stoll W, Bachert C. Cytokines in nasal polyposis, acute

and chronic sinusitis. Am J Rhinol 1998; 12:383–8.

15 Bachert C, Hormann K, Mosges R et al. An update on the diagnosis

and treatment of sinusitis and nasal polyposis. Allergy 2003;

58:176–91.

16 Watelet JB, Bachert C, Claeys C, van Cauwenberge P. Matrix met-

alloproteinases MMP-7, MMP-9 and their tissue inhibitor TIMP-1:

expression in chronic sinusitis vs nasal polyposis. Allergy 2004;

59:54–60.

17 Gwaltney JM Jr, Savolainen S, Rivas P et al. Comparative effective-

ness and safety of cefdinir and amoxicillin-clavulanate in treat-

ment of acute community-acquired bacterial sinusitis. Cefdinir

Sinusitis Study Group. Antimicrob Agents Chemother 1997;

41:1517–20.

18 Doyle PW, Woodham JD. Microbiology and histopathology of

chronic ethmoiditis. J Otolaryngol 1991; 20:445–7.

19 Hoyt WH III. Bacterial patterns found in surgery patients with

chronic sinusitis. J Am Osteopath Assoc 1992; 92:1992.

20 Brook I, Frazier EH. Correlation between microbiology and previ-

ous sinus surgery in patients with chronic maxillary sinusitis. Ann

Otol Rhinol Laryngol 2001; 110:148–51.

21 Finegold SM, Flynn MJ, Rose FV et al. Bacteriologic findings

associated with chronic bacterial maxillary sinusitis in adults. Clin

Infect Dis 2002; 35:428–33.

22 Jiang RS, Lin PK, Lin JF. Correlation between bacteriology and

computed tomography staging for chronic sinusitis. J Laryngol

Otol 2005; 119:193–7.

23 von Eiff C, Peters G, Heilmann C. Pathogenesis of infections due to

coagulase-negative staphylococci. Lancet Infect Dis 2002; 2:677–

85.

24 Teufel P, Gotz F. Characterization of an extracellular metallopro-

tease with elastase activity from Staphylococcus epidermidis. J Bac-

teriol 1993; 175:4218–24.

25 Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;

339:520–32.

26 Vaudaux P, Francois P, Bisognano C et al. Increased expression of

clumping factor and fibronectin-binding proteins by hemB

mutants of Staphylococcus aureus expressing small colony variant

phenotypes. Infect Immun 2002; 70:5428–37.

27 Cheung AL, Bayer AS, Zhang G, Gresham H, Xiong YQ. Regulation

of virulence determinants in vitro and in vivo in Staphylococcus

aureus. FEMS Immunol Med 2004; 40:1–9.

28 Bronner S, Monteil H, Prevost G. Regulation of virulence determi-

nants in Staphylococcus aureus: complexity and applications. FEMS

Microbiol Rev 2004; 28:183–200.

29 Chan PF, Foster SJ. Role of SarA in virulence determinant produc-

tion and environmental signal transduction in Staphylococcus

aureus. J Bacteriol 1998; 180:6232–41.

30 Reed SB, Wesson CA, Liou LE et al. Molecular characterization of

a novel Staphylococcus aureus serine protease operon. Infect

Immun 2001; 69:1521–7.

31 Dunman PM, Murphy E, Haney S et al. Transcription profiling-

based identification of Staphylococcus aureus genes regulated by the

agr and/or sarA loci. J Bacteriol 2001; 183:7341–53.

32 Seiberling KA, Conley DB, Tripathi A et al. Superantigens and

chronic rhinosinusitis: detection of staphylococcal exotoxins in

nasal polyps. Laryngoscope 2005; 115:1580–5.

33 Bernstein JM, Kansal R. Superantigen hypothesis for the early

development of chronic hyperplastic sinusitis with massive nasal

polyposis. Curr Opin Otolaryngol Head Neck Surg 2005; 13:39–44.

34 Kunkel SL, Standiford T, Kasahara K, Strieter RM. Interleukin-8

(IL-8): the major neutrophil chemotactic factor in the lung. Exp

Lung Res 1991; 17:17–23.

35 DiMango E, Zar HJ, Bryan R, Prince A. Diverse Pseudomonas

aeruginosa gene products stimulate respiratory epithelial cells to

produce interleukin-8. J Clin Invest 1995; 96:2204–10.

36 Skerrett SJ, Liggitt HD, Hajjar AM, Ernst RK, Miller SI, Wilson CB.

Respiratory epithelial cells regulate lung inflammation in response

to inhaled endotoxin. Am J Physiol Lung Cell Mol Physiol 2004;

287:L143–L152.

37 Dulon S, Cande C, Bunnett NW, Hollenberg MD, Chignard M,

Pidard D. Proteinase-activated receptor-2 and human lung epithe-

lial cells: disarming by neutrophil serine proteinases. Am J Respir

Cell Mol Biol 2003; 28:339–46.

38 Page K, Hughes VS, Odoms KK, Dunsmore KE, Hershenson MB.

German cockroach proteases regulate interleukin-8 expression via

nuclear factor for interleukin-6 in human bronchial epithelial cells.

Am J Respir Cell Mol Biol 2005; 32:225–31.

39 Knight DA, Lim S, Scaffidi AK et al. Protease-activated receptors in

human airways: upregulation of PAR-2 in respiratory epithelium

from patients with asthma. J Allergy Clin Immunol 2001; 108:797–

803.

40 Rudack C, Bachert C, Stoll W. Effect of prednisolone on cytokine

synthesis in nasal polyps. J Interferon Cytokine Res 1999; 19:1031–

5.

41 Schramm R, Thorlacius H. Staphylococcal enterotoxin B-induced

acute inflammation is inhibited by dexamethasone: important role

of CXC chemokines KC and macrophage inflammatory protein 2.

Infect Immun 2003; 71:2542–7.

42 Fukakusa M, Bergeron C, Tulic MK et al. Oral corticosteroids

decrease eosinophil and CC chemokine expression but increase

neutrophil, IL-8, and IFN-gamma-inducible protein 10 expression

in asthmatic airway mucosa. J Allergy Clin Immunol 2005;

115:280–6.

43 Froyen G, Proost P, Ronsse I et al. Cloning, bacterial expression

and biological characterization of recombinant human granulo-

cyte chemotactic protein-2 and differential expression of granulo-

cyte chemotactic protein-2 and epithelial cell-derived neutrophil

activating peptide-78 mRNAs. Eur J Biochem 1997; 243:762–9.

F. Sachse et al.


44 Gijsbers K, Van Assche G, Joossens S et al. CXCR1-binding

chemokines in inflammatory bowel diseases: down-regulated IL-8/

CXCL8 production by leukocytes in Crohn’s disease and selective

GCP-2/CXCL6 expression in inflamed intestinal tissue. Eur J

Immunol 2004; 34:1992–2000.

45 Wuyts A, Struyf S, Gijsbers K et al. The CXC chemokine GCP-

2/CXCL6 is predominantly induced in mesenchymal cells by

interleukin-1beta and is down-regulated by interferon-gamma:

comparison with interleukin-8/CXCL8. Lab Invest 2003; 83:23–

34.

46 Walz A, Burgener R, Car B, Baggiolini M, Kunkel SL, Strieter RM.

Structure and neutrophil-activating properties of a novel inflam-

matory peptide (ENA-78) with homology to interleukin 8. J Exp

Med 1991; 174:1355–62.

induction of cxc chemokines in a 549 airways epithelial cells by trypsin in crs

Documents