respiratory syncytial virus deficient in soluble g protein induced an increased proinflammatory...

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Virology 330 (20

Respiratory syncytial virus deficient in soluble G protein induced an

increased proinflammatory response in human lung epithelial cells

Ralf Arnold*, Brigitte Kfnig, Hermann Werchau, Wolfgang Kfnig

Institute of Medical Microbiology, Otto-von-Guericke-University, Magdeburg, Germany

Received 27 May 2004; returned to author for revision 4 August 2004; accepted 5 October 2004

Abstract

Respiratory syncytial virus (RSV) is worldwide the single most important respiratory pathogen in infancy and early childhood. The G

glycoprotein of RSV, named attachment protein, is produced by RSV-infected lung epithelial cells in both a membrane-anchored (mG

protein) and a soluble form (sG protein) that is secreted by the epithelial cell. Currently, the biological role of the sG protein in primary

RSV infection is still elusive. Therefore, we analyzed the inflammatory response of human lung epithelial cells (A549) infected either with

wild-type RSV (RSV-WT) or a spontaneous mutant thereof deficient in the production of secreted G protein (RSV-DsG). Our data reveal

that RSV-DsG, in comparison to RSV-WT, induced an increased cell surface expression of ICAM-1 on A549 cells and an enhanced

release of the chemokines IL-8 and RANTES after 20 h postinfection. The increased protein expression pattern correlated with an

enhanced mRNA level encoding for ICAM-1, IL-8, and RANTES, respectively. Furthermore, epithelial cells infected with RSV-DsG

showed a more increased binding activity of the transcription factor NF-nB when compared to RSV-WT. In contrast, the mutant RSV-DsG

replicated less efficiently in A549 cells than RSV-WT. Our data suggest that RSV, in the course of an ongoing infection, reduces by the

production of sG protein the detrimental inflammatory response evolved by the infected resident lung epithelial cell and thereby supports

its own replication.

D 2004 Elsevier Inc. All rights reserved.

Keywords: ICAM-1; RANTES; IL-8; Inflammation; NF-nB; RSV; A549 cell; Soluble G protein

Introduction

Respiratory syncytial virus (RSV) is worldwide the

major cause of serious lower respiratory disease in

infancy and early childhood (McIntosh, 1997). By the

age of 2 years, nearly every child becomes infected with

RSV, and due to incomplete immunity adults get

reinfected throughout their life (Hall and McCarthy,

2000; Falsey and Walsh, 2000). The epithelial cell of

the respiratory mucosa is the main target for RSV.

Evidence accumulated that epithelial cells infected with

0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2004.10.004

* Corresponding author. Current address: Institute of Medical Micro-

biology, Otto-von-Guericke-University, Leipzigerstr. 44, 39120 Magde-

burg, Germany. Fax: +49 391 6713384.

E-mail address: [email protected] (R. Arnold).

RSV play an active role in orchestrating the early

inflammatory and immune responses of the host (Doma-

chowske et al., 2001). The release of chemokines, i.e.,

interleukin-8 (IL-8), regulated upon activation, normal T-

cell-expressed and -secreted (RANTES), macrophage

inflammatory protein-1a (MIP-1a), monocyte chemoattrac-

tant protein-1 (MCP-1) (Arnold et al., 1994; Olszewska-

Pazdrak et al., 1998), and the expression of the intercellular

adhesion molecule-1 (ICAM-1) (Arnold and Konig, 1996)

are important and early epithelial responses following RSV

infection. Inflammatory cells are then chemotactically

recruited along the chemotaxin gradient from the local

blood vessels via the mucosal tissue into the lumen of the

alveolar space (Everard et al., 1994). Especially polymor-

phonuclear neutrophil granulocytes (PMN) are subsequently

responsible for the acute inflammation of the lung (Wang

and Forsyth, 2000).

04) 384–397

R. Arnold et al. / Virology 330 (2004) 384–397 385

The envelope of RSV contains three transmembrane

surface proteins, i.e., the fusion glycoprotein (F protein),

responsible for fusion of the viral envelope with the plasma

membrane of the host target cell (Collins and Mottet,

1991), the G glycoprotein (G protein), mediating attach-

ment of the virus particle to the target cell (Levine et al.,

1987), and the small SH protein with not well-understood

functions (Bukreyev et al., 1997). The G and F glyco-

proteins are the major neutralization and protective antigens

(Routledge et al., 1988). The G protein rich in serine,

threonine, and proline residues is a highly glycosylated

type II transmembrane protein with an apparent Mr of 90

kDa (Collins et al., 2001). The specific immune response

elicited by the G protein appears to be linked to the

pathogenesis of RSV: Immunization of mice with a

vaccinia virus recombinant expressing the G protein

induces a CD4+ T-cell response with an increased TH2

component and development of an eosinophilic pulmonary

infiltrate (Openshaw, 1995). Evidence accumulated that an

epitope in the region of amino acid 184 to 198 is essential

for the induction of detrimental lung eosinophilia and

increased lung pathology (Sparer et al., 1998; Tebbey et al.,

1998; Varga et al., 2000). Recently, Tripp et al. (2001)

found that this central conserved region contains a CX3C

motif at amino acid positions 182 to 186 that is able to

compete with fractalkine for binding to CX3CR1, the

fractalkine receptor, thereby interfering with the fractalkine-

mediated leukocyte chemotaxis. Moreover, in a mouse

vaccination model, this CX3C motif was shown to be

responsible for some aspects of enhanced pulmonary

disease after live RSV infection (Haynes et al., 2003).

Konig et al. (1996) have shown that purified membrane-

anchored G protein (mG protein) modulates the cytokine

release of IL-10, IL-12, and TNF-a from PBMC.

Beside the mG protein, a smaller soluble form of the G

protein (sG protein) with an apparent Mr of 82 kDa is also

produced by infected cells (Hendricks et al., 1987). It is

generated by translation initiation at a second AUG in the

mRNA and subsequent proteolytic trimming (Hendricks et

al., 1988; Roberts et al., 1994). The sG protein is already

shed during the first 12 h postinfection, when mG protein is

still not detectable (Hendricks et al., 1988), suggesting that

sG protein may play some role during the early innate

immune response. With regard to its biological role in

primary RSV infection, only few data are available

demonstrating that the sG protein can bind via its conserved

CX3C motif, like the mG protein, to the fractalkine receptor

(CX3CR1) thereby inducing migration of leukocytes (Tripp

et al., 2001). Recently, it has been shown by barometric

whole-body plethysmography in a primary mouse infection

model that a spontaneous variant of human RSV only

deficient in the production of the sG protein (RSV-DsG)

induced a more increased airway inflammation and impaired

lung function when compared with the wild-type strain

(RSV-WT) expressing both mG and sG protein, respectively

(Schwarze and Schauer, 2004). Therefore, we hypothesized

whether the sG protein might have some influence on the

inflammatory epithelial cell response during the first 20 h

postinfection. Since A549 lung epithelial cells serve as a

well-established in vitro infection model for analyzing

proinflammatory epithelial cell responses in the course of

an ongoing RSV infection, we used A549 cells in our

infection studies. We analyzed the cell surface expression of

ICAM-1 and the release of the chemokines IL-8 and

RANTES from A549 cells infected either with RSV-WT

or RSV-DsG, respectively.

Our data show that the expression of proinflammatory

molecules (IL-8, RANTES, ICAM-1) and the binding

activity of the transcription factor NF-nB was more

increased 20 h postinfection when A549 cells were infected

with RSV-DsG instead of RSV-WT. In contrast, the mutant

RSV-DsG replicated less efficiently in A549 cells, suggest-

ing that sG protein might act as a viral evasion factor during

an ongoing innate immune response.

Results

Characterization of the RSV-DsG mutant

To demonstrate that RSV-DsG was only deficient in the

production of sG protein but still able to synthesize mG

protein, we performed immunoblot analysis for detection

of G protein. Since RSV was propagated in HEp-2 cells,

we used this cell line in our immunoblot studies. HEp-2

cells were infected with RSV-WT and RSV-DsG, respec-

tively, and cultured for 36 h. As shown in Fig. 1A, the

RSV-infected HEp-2 cells expressed the mG protein with

an apparent Mr of 90 kDa irrespective of whether they

were infected with RSV-WT (lane 2) or the mutant RSV-

DsG (lane 3). The immunoblots were probed with anti-G

protein mAb (12E2, 2 Ag/ml) (Roder et al., 2000). Next,

we analyzed the presence of sG in the cell supernatant 20

h postinfection. After this culture time, the sG protein

accounts for nearly 80% of the G protein released into the

medium (Hendricks et al., 1988). The sG protein was

enriched by means of ConA-sepharose extraction. As

presented in Fig. 1B, cell supernatants enriched for the

sG protein and analyzed by anti-G protein immunoblot

analysis revealed that only cell supernatant obtained from

RSV- WT-infected cells (lane 3) contained the sG protein

with an apparent Mr of 82 kDa. The supernatant from

RSV-DsG-infected cells, enriched for sG protein (lane 2),

did not contain any sG protein. Since supernatants were

precleared by high-speed centrifugation from infectious

virus particles and detached cells, the prepared cell

supernatants were not confounded by mG protein. In this

regard, we obtained no mG protein immunoblot signal in

the range of 90 kDa. For control, we coelectrophoresed

mG protein (lane 1), purified from RSV-WT-infected HEp-

2 cells 36 h postinfection. The different migration rates

of mG protein and sG protein due to their different

Fig. 1. Western blot analysis of membrane-anchored G protein (mG), soluble G protein (sG) and virus protein expression in HEp-2 cells infected with RSV-WT

and RSV-DsG. (A) Prepared cell lysates (10 Ag) from HEp-2 cells were separated by SDS-PAGE (8%) and analyzed by anti-G protein immunoblot. Mock-

infected cells (lane 1) and cells infected (m.o.i. = 3) with RSV-WT (lane 2) and RSV-DsG (lane 3), respectively, were cultured for 36 h. The molecular masses

(in kDa) of the coelectrophoresed molecular weight markers are depicted on the left (lane M). (B) mG protein (1 Ag) purified from HEp-2 cells infected with

RSV-WT for 36 h (lane 1) and sG protein (40 Al eluate) concentrated by ConA-sepharose 4B affinity chromatography from HEp-2 cell supernatants 20 h

following infection with RSV-DsG (lane 2) and RSV-WT (lane 3), respectively, were assessed by SDS-PAGE (6%). (C) HEp-2 cell lysates prepared from cells

infected with RSV-WT (lane 1) and RSV-DsG (lane 2) (m.o.i. = 3) 36 h postinfection were separated by SDS-PAGE (12%) and analyzed by anti-RSV

polyclonal antibody. Shown are representative blots out of three.

R. Arnold et al. / Virology 330 (2004) 384–397386

molecular weights are obvious on the performed 6% SDS-

PAGE. Thus, only mG protein was produced by RSV-

DsG-infected cells in contrast to cells infected with the

wild-type strain of RSV, which synthesized both the

membrane-anchored and the secreted form of G protein.

In order to demonstrate that both virus strains did not

differ with regard to the expression profile of other virus

proteins, we stained whole cell lysates prepared from RSV-

infected HEp-2 cells with a polyclonal anti-RSV Ab. As

shown in Fig. 1C, both RSV-WT and RSV-DsG showed an

identical virus protein expression pattern.

ICAM-1 expression on A549 cells infected with RSV-DsG

and RSV-WT

We and others have shown that RSV infection leads to

an increase of ICAM-1 cell surface expression on lung

epithelial cells (Arnold and Konig, 1996; Domachowske et

al., 2001). Thus, we thought to define the role of sG protein

in regulating RSV-induced ICAM-1 expression on infected

human lung epithelial cells. Confluent monolayers of A549

cells were infected with RSV at a m.o.i. of 1 and 3. We

analyzed ICAM-1 cell surface expression 20 h postinfection

since, as mentioned above, at that time the sG protein

accounts for nearly 80% of the G protein released into the

cell supernatant (Hendricks et al., 1988). As shown in Figs.

2A and B, the infection with RSV-DsG induced in

comparison to RSV-WT a more pronounced ICAM-1

expression pattern on A549 cells. The ICAM-1 expression

increased in a RSV-dose dependent manner (Fig. 2C). But

regardless of the infection dose used, the mutant RSV-DsG

induced always significantly more ICAM-1 expression on

A549 cells than RSV-WT. When infection time proceeded

up to 48 h, the RSV-induced expression of ICAM-1 was

similar when A549 cells were either infected (m.o.i. = 3)

with RSV-WT or RSV-DsG (RSV-WT: 1677 F 140 MFI;

RSV-DsG: 1317 F 184 MFI). Cells treated with UV-

inactivated RSV, i.e., RSV-WT and RSV-DsG, respectively,

did not show any ICAM-1 up-regulation, suggesting that

replication of both strains was a prerequisite for RSV-

induced ICAM-1 expression (data not shown). To study

whether the more increased ICAM-1 cell surface expression

on RSV-DsG-infected A549 cells might be a direct

consequence of a more increased cellular ICAM-1 mRNA

level, we determined the ICAM-1 mRNA level in a

quantitative manner. As presented in Fig. 2D, during the

first 24 h postinfection, the cells infected with RSV-DsG

possessed a higher amount of ICAM-1 mRNA when

directly compared with cells infected with RSV-WT.

Induction of RANTES release from A549 cells by RSV-DsG

and RSV-WT

It has been shown that human lung epithelial cells

infected with RSV in vitro or in vivo are able to secrete the

C–C chemokine RANTES into the cell supernatant

(Olszewska-Pazdrak et al., 1998). To compare the ability

of RSV-WT and RSV-DsG to induce the release of

RANTES from A549 cells, we analyzed the amount of

RANTES in the cell supernatants 20 h postinfection. The

cell supernatants harvested from RSV-DsG-infected cells

contained significantly more RANTES than supernatants

from A549 cells infected with RSV-WT (Fig. 3A). In

contrast, when the amount of RANTES was determined 48

h postinfection, both RSV-WT and RSV-DsG did not differ

with regard to their capability to induce the release of

RANTES from A549 cells (RSV-WT: 3.2 F 0.3 ng/ml;

RSV-DsG: 3.0 F 0.2 ng/ml). Similar to the experiments

concerning ICAM-1 gene expression, the expression of the

RANTES gene was quantitatively determined. Our data

show that the enhanced release of RANTES from A549

cells infected with RSV-DsG correlated with a more

pronounced RANTES mRNA level 20 h postinfection

(Fig. 3B).

Fig. 2. Effect of RSV-WT and RSV-DsG on ICAM-1 expression in A549 cells. The cell surface expression of ICAM-1 was measured by flow cytometry

analysis (A–C) and the cellular ICAM-1 mRNA amount was determined by quantitative real-time RT-PCR (D). (A, B) FACS histograms are shown as

representative results demonstrating different cell surface expression of ICAM-1 induced by RSV-WT (A) and RSV-DsG (B) 20 h postinfection (m.o.i. = 3).

(C) Cells infected at a different m.o.i. (1 and 3) showed a virus dose-dependent up-regulation of ICAM-1 20 h postinfection. Data are shown from mock-

infected cells (black bars), RSV-WT-infected cells (open bars), and RSV-DsG-infected cells (shaded bars), respectively. Results are means F SEM (n = 9).

Significant differences from mock-infected cells (black bars) are indicated by *P b 0.01. Significant differences from RSV-WT-infected cells (open bars) are

indicated by #P b 0.01. (D) ICAM-1 mRNA levels were analyzed 6–24 h postinfection (m.o.i. = 3) by TaqMan real-time RT-PCR. The housekeeping gene

GAPDH was used for normalization. The data are means of two independent experiments.


Induction of IL-8 release from A549 cells by RSV-DsG and

RSV-WT

Evidence accumulated that A549 cells infected with RSV

secrete a substantial amount of IL-8 in a time- and RSV-

dose dependent manner (Arnold et al., 1994). Therefore, we

also analyzed the RSV-induced IL-8 synthesis in A549 cells.

Similar to the RANTES secretion pattern, RSV-DsG

induced a markedly increased release of IL-8 when

compared with RSV-WT-infected cells (Fig. 4A). These

results were obtained irrespective of the virus dose used.

As was already shown for the cell surface expression of

ICAM-1 and secretion of RANTES, when RSV-infected

cells (m.o.i. = 3) were incubated up to 48 h postinfection,

both virus strains induced a similar release of IL-8 (RSV-

WT: 45 F 4.9 ng/ml; RSV-DsG: 39 F 3.7 ng/ml). By using

real-time RT-PCR, we observed a significantly more

increased IL-8 mRNA level when A549 cells were infected

with RSV-DsG instead of RSV-WT (Fig. 4B).

Activation of NF-jB by RSV-DsG and RSV-WT

Following infection of human lung epithelial cells with

RSV, especially the activation of the transcription factor

NF-nB leading to an increased gene expression of ICAM-1,

IL-8, and RANTES has been recently reported (Chini et al.,

1998; Garofalo et al., 1996; Thomas et al., 1998). To

determine whether the observed increased mRNA levels

encoding for ICAM-1, IL-8 and RANTES subsequent to

infection with RSV-DsG might be associated with a further

increased activation of NF-nB, we analyzed the binding

activity of Rel A (p65) and NF-nBI (p50) in A549 cells by

means of a quantitative NF-nB binding assay. The

activation of NF-nB was determined after 6 and 20 h

postinfection because RSV activates NF-nB late during

infection. The binding activity of p65 and p50 in RSV-

infected A549 cells is presented in Fig. 5. When compared

to uninfected cells (medium control) both transcription

factors showed a markedly increased binding activity in

A549 cells 24 h after infection with RSV-WT. Also, when

determined 6 h postinfection, a small but detectable

increased binding activity of p65 and p50 was observed.

However, with regard to activation of NF-nB (p65/p50)

binding, RSV-DsG was approximately twice as effective as

RSV-WT (Figs. 5A,B). A nuclear extract prepared from

Jurkat cells (supplied by the manufacturer) served as an

internal positive control (Figs. 5A,B: see control). Se-

quence specificity of the test was monitored by competition

with free wild-type NF-nB-consensus oligonucleotides (wt)as well as mutated NF-nB-consensus oligonucleotides (mt).

As shown, both protein DNA complexes, i.e., p65 and p50,

were specific because soluble oligonucleotides encoding for

the wild-type NF-nB-consensus binding site competed

completely with the binding activity of the titer-plate-

Fig. 3. Effect of RSV-WT and RSV-DsG on RANTES expression in A549

cells. The release of RANTES was determined by ELISA (A) and the

level of RANTES mRNA was quantified by Quantikine RANTES mRNA

ELISA (B). Cells were infected at a m.o.i. of 3 and cultured for 20 h. (A)

The release of RANTES from mock-infected cells (black bars) was

compared with the release from RSV-WT-infected cells (open bars) and

RSV-DsG-infected cells (shaded bars), respectively. Results are means FSEM (n = 6). (B) The amount of RANTES mRNA was quantified 20 h

postinfection. Results are means F SEM (n = 5). Significant differences

from mock infected cells (black bars) are indicated by *P b 0.01.

Significant differences from RSV-WT infected cells (open bars) are

indicated by #P b 0.01.

Fig. 4. Effect of RSV-WT and RSV-DsG on IL-8 expression in A549

cells. The release of IL-8 into the cell supernatant (A) and the amount of

cellular IL-8 mRNA (B) were quantified from mock-infected cells (black

bars), RSV-WT-infected cells (open bars), and RSV-DsG-infected cells

(shaded bars). (A) IL-8 was measured by ELISA 20 h postinfection in the

cell supernatants of mock-infected A549 cells and cells infected at a

m.o.i. of 1 and 3, respectively. Results are means F SEM (n = 5). (B)

The amount of IL-8 mRNA in mock-infected A549 cells and cells

infected with RSV (m.o.i. = 3) was determined by Taqman real-time RT-

PCR 20 h postinfection. Results are means F SEM (n = 7). *P b 0.01,

vs. mock infected cells (black bars); #P b 0.01, vs. RSV-WT-infected

cells (open bars).


bound DNA encoding for the NF-nB consensus binding

site (Figs. 5A,B: see control + wt). In contrast, soluble

oligonucleotides encoding for a mutated NF-nB-consensusbinding site did not compete with the immobilized DNA

for the binding of the activated transcription factors (Figs.

5A,B: see control + mt).

Replication of RSV-DsG and RSV-WT

It has been shown that replication of RSV is a

prerequisite for the RSV-induced activation of NF-nB(Bitko et al., 1997; Fiedler et al., 1996). We hypothesized

whether the observed increased binding activity of NF-nBin RSV-DsG-infected A549 cells might be a direct

consequence of a more increased replication rate of RSV-

DsG when compared to RSV-WT. We therefore analyzed the

growth of RSV-DsG and RSV-WT in A549 cells. First, we

determined the amount of infectious virus particles released

into the cell supernatants. As can be seen from Fig. 6A, the

virus strains RSV-DsG and RSV-WT showed a different

growth rate in A549 cells. During 96 h postinfection

(m.o.i. = 0.01), the mutant strain replicated less efficiently

in A549 cells. The growth was reduced nearly 10-fold when

compared to the growth rate of RSV-WT. To exclude that

RSV-DsG accumulated intracellularly instead of budding

from the cell, we analyzed the virus titer in the prepared cell

lysates and the corresponding supernatants 36 h post-

infection (m.o.i. = 1). As depicted in Fig. 6B, A549 cells

infected with the RSV-DsG strain possessed intracellularly

less infectious virus particles, suggesting that the mutant

RSV-DsG was not retained inside the cells to a higher

degree than the RSV-WT. Obviously, these data suggest that

the increased binding activity of NF-nB in RSV-DsG-

infected cells might not be explained by an increased

proliferation rate.

Fig. 5. NF-nB (p65, p50) binding activity in RSV-infected A549 cells. A549 cells were infected with RSV-WT and RSV-DsG, respectively at a m.o.i. of 3.

After 6 and 20 h, prepared cell lysates were assayed for p65 activity (A) and p50 activity (B). Jurkat nuclear extract provided by the manufacturer served as

positive control. To demonstrate specificity of the NF-nB binding assay, wild-type (wt) and mutant (mt) NF-nB oligonucleotides were used as competitors

during the binding reaction (shaded bars). Results are means F SEM (n = 8). Significant differences from mock-infected cells (black bars) are indicated by

*P b 0.01. Significant differences from RSV-WT infected cells (open bars) are indicated by #P b 0.01.


Next, we asked whether the increased binding activity of

NF-nB might be due to an increased cellular virus protein

load. Therefore, we assayed the cell surface expression of

mG and mF protein on A549 cells infected with RSV-WT

and RSV-DsG, respectively. The reduced production of

infectious RSV-DsG particles correlated with a diminished

expression of mG- and mF protein on A549 cells 24 h

Fig. 6. Growth of RSV-WT and RSV-DsG in A549 cells. (A) Cells were infected

and analyzed by plaque assay on HEp-2 cells. Results are means from two exp

(m.o.i. = 1) and 36 h postinfection aliquots of cell supernatants and cell lysates wer

SEM (n = 5). *P b 0.01 vs. RSV-WT infected cells (open bars).

postinfection with RSV-DsG (Fig. 7A). Furthermore, a

reduced expression of mF and mG protein on A549 cells

infected with the mutant strain was still observed after 36 h

postinfection (data not shown). Additionally, like mG and

mF protein, the cell surface staining of the mSH protein

verified a reduced expression of this protein on RSV-DsG-

infected cells (data not shown). Again, to exclude that cells

at a m.o.i. of 0.01. Supernatants were harvested at the indicated time points

eriments performed in duplicate. (B) A549 cells were infected with RSV

e assayed for the presence of infectious virus particles. Results are meansF

Fig. 7. Comparison of the expression of mF and mG protein and fusion

activity of RSV-WT and RSV-DsG in A549 cells. (A) The amount of cell

surface expression of mG and mF protein was determined by FACS

analysis. Results are means F SEM (n = 6). *P b 0.01, vs. RSV-WT

infected cells (open bars). (B) The percentage of cells that stained positive

for mG and mF protein was determined by FACS analysis. Results are

means F SEM (n = 6). *P b 0.01, vs. RSV-WT infected cells (open

bars). (C) Relief of self-quenching of aminofluorescein-labeled virus

bound to A549 cells. A representative experiment performed in triplicate

is shown (n = 3).


infected with RSV-DsG do not retain synthesized G- and F

protein to a higher degree than RSV-WT-infected cells, we

stained permeabilized cells for these proteins. Our data

showed that these virus proteins were not stored within cells

either infected with RSV-DsG and RSV-WT, respectively

(data not shown). But when cells were stained for mG and

mF protein, we obtained less positive cells in case of

infection with RSV-DsG, suggesting that the mutant either

infects their target cell less efficiently or replicates more

slowly (Fig. 7B). In this regard, we analyzed whether

different fusion rates between the virus envelope and the

target cell membrane may occur. The fusion of RSV labeled

with aminofluorescein was measured by means of a

fluorescence dequenching assay. As depicted in Fig. 7C,

the fluorescence dequenching curves obtained with RSV-

DsG and RSV-WT were identical. Therefore, we concluded

that different fusion kinetics do not account for the different

replication rates of both virus strains.

Discussion

It has been shown that infection of human lung epithelial

cells with RSV induces expression of ICAM-1, RANTES,

and IL-8. In this study, we supply evidence that the

expression of these inflammatory mediators were even

more up-regulated in A549 cells when cells were infected

with a mutant strain RSV-DsG, lacking the production of sG

protein. This different expression pattern was observed 20 h

postinfection and correlated with an increased binding

activity of the transcription factor NF-nB and a diminished

growth rate in A549 cells. However, these different cell

responses were time constricted since with prolonged

culture time, i.e., 48 h postinfection, both RSV-DsG and

RSV-WT induced the same expression of ICAM-1,

RANTES, and IL-8, respectively.

The RSV-induced bronchiolitis is characterized by

peribronchial infiltration of leukocytes and epithelial

necrosis (Faden et al., 1984) as well as prominent

accumulation of PMN in the airway lumen (Everard et al.,

1994). It has been shown that the enhanced expression of

ICAM-1 on RSV-infected human epithelial cells is respon-

sible for an increased adhesion of neutrophils (Stark et al.,

1996) leading consequently to epithelial cell damage and

death (Wang et al., 1998). Therefore, in primary RSV

infection, lung epithelial cells infected with RSV-DsG,

expressing a further increased level of cell surface ICAM-

1, seem to be more susceptible to cell damage than RSV-

WT-infected cells. Whether the increased ICAM-1 cell

surface expression on RSV-DsG-infected cells might be

partly a consequence of an altered cell surface shedding is

still not known. However, the increase in the cellular mRNA

level encoding for ICAM-1 together with the enhanced

binding activity of NF-nB (as discussed below) suggest that

the enhanced cell surface expression is primarily mediated

by de novo protein synthesis.

The chemokine RANTES, a member of the C–C

chemokine family, is a chemotactic and activation factor

for monocytes, basophils, and eosinophils, with no activity

on neutrophils (Rot et al., 1992). With regard to our data,

showing an enhanced release of RANTES from RSV-DsG-

infected A549 cells during the first day postinfection, one

may hypothesize whether the above-mentioned cells may

accumulate earlier and/or to a more increased amount in the

RSV-DsG-infected lung.

The C–X–C chemokine IL-8 is mainly a chemotactic

factor and activator for neutrophils (Baggiolini et al., 1989).

The high amount of IL-8 released from lung epithelial cells


may be primarily responsible for the prominent accumu-

lation of PMN in the alveolar space of the infected child

(Everard et al., 1994). Similar to the increased release of

RANTES, one is tempted to speculate whether the enhanced

release of IL-8 after infection with RSV-DsG may contribute

to a further increased recruitment of neutrophils into the

infected lung.

Several recently published studies have shown that

chemokines, such as IL-8, RANTES, and MIP-1a are

induced in respiratory secretions collected from RSV-

infected patients, and that their amount correlated with

disease severity (Noah et al., 2002; Smyth et al., 2002;

Teran et al., 1999). Furthermore, instead of T helper

cytokines, the chemokines MIP-1a and RANTES seem to

be better predictive markers for severe bronchiolitis and

recurrent wheezing (Chung and Kim, 2002; Garofalo et al.,

2001). Quite recently, Schwarze and Schauer (2004)

analyzed this mutant RSV-DsG strain in a murine model

of primary infection. Following infection with RSV-DsG, a

more impaired lung function, an enhanced airway inflam-

mation, and increased lung virus titers were observed when

compared with infection of RSV-WT. Also, the numbers of

pulmonary eosinophils, macrophages, and neutrophils were

more increased in mice infected with RSV-DsG. Therefore,

future studies in this in vivo infection model have to address

the question whether lung epithelial cells infected with

RSV-DsG are mainly responsible for this impaired infection

course.

The transcription factor NF-nB is one of the pivotal

regulators of proinflammatory gene expression, i.e., it

induces the transcription of proinflammatory cytokines,

chemokines, and adhesion molecules (Tak and Firestein,

2001). NF-nB is a collective name for a family of dimeric

transcription factors consisting of five members: RelA

(p65), RelB, c-Rel, NF-nB1 (p50), and NF-nB2 (p52).

They form various hetero- and homodimers. The RelA/NF-

nB1 (p65/p50) heterodimer is the most commonly found in

activated cells. It has been shown that the RSV-induced

expression of RANTES and IL-8 is highly dependent on the

activation of NF-nB (Casola et al., 2001; Garofalo et al.,

1996; Thomas et al., 1998). We observed that infection with

RSV-DsG as well as RSV-WT led to a more pronounced

binding activity of RelA and NF-nB1, respectively. Thissuggests that NF-nB heterodimers consisting of p65/p50

were formed regardless of the used RSV strain. That p65/

p50 complexes are primarily formed in RSV-infected cells is

in agreement with recently published results (Bitko et al.,

1997; Garofalo et al., 1996). The observed enhanced

binding activity of NF-nB after infection with RSV-DsG

correlated with a further increase of the cellular mRNA level

encoding for ICAM-1, IL-8, and RANTES, respectively.

Since all three genes under investigation have been shown

to be induced by RSV in a NF-nB-dependent manner, our

data suggest that RSV-DsG might lead to an increased

ICAM-1, IL-8, and RANTES gene expression via the

observed increased binding activity of NF-nB. Obviously,

the transcription rates of these genes are regulated by

additional transcription factors (Casola et al., 2000; Chini et

al., 1998, 2001), and the expression of the RANTES- and

IL-8 gene is further regulated by modulating mRNA

stability (Hoffmann et al., 2002; Koga et al., 1999).

Therefore, with regard to increased gene expression after

infection with RSV-DsG, additional mechanisms have to be

taken into account.

The mechanisms leading to the activation of NF-nB in

RSV-infected lung epithelial cells are still not known. In

A549 cells, infected with RSV-DsG, we observed no

intracellular accumulation of mG protein due to the lack

of secreted G protein (data not shown). Moreover, the

mutant strain replicated less efficiently when compared to

the wild-type strain. In this regard, one may exclude an

increased activation of NF-nB in RSV-DsG-infected A549

cells due to an enhanced cellular virus load and/or

replication rate (Fiedler et al., 1996). Similar to UV-

inactivated wild-type strain, UV-inactivated RSV-DsG did

not induce any expression of the proinflammatory mediators

under investigation (data not shown). Together with our data

showing identical fusion kinetics of both virus strains, we

conclude that adherence/fusion phenomena by themselves

cannot be responsible for the increased activation of NF-nBobserved in A549 cells infected with the mutant strain. In

this regard, we also exclude that the two point mutations in

the F gene of the mutant strain do not influence the infection

behavior of RSV-DsG.

Quite recently, Bose et al. (2003) demonstrated that the

activation state of NF-nB, at the beginning of a RSV

infection, controls the replication of RSV. When A549 cells

were preactivated with TNF-a and IL-1h, respectively, andsubsequently infected with RSV, the replication of the virus

was significantly inhibited due to the enhanced activation

state of NF-nB. One may hypothesize whether the reduced

replication rate of RSV-DsG in A549 cells might be at least

in part a direct consequence of the more increased activation

of NF-nB. The observed increased activation of NF-nB in

A549 cells 6 h postinfection with RSV-DsG supports this

thesis.

Recently, Teng et al. (2001) used reverse genetics to

construct a RSV-mutant deficient in the release of sG

protein. When compared with the wild-type strain, this

mutant did not differ markedly with regard to replication in

HEp-2 and Vero cells. However, when virus growth was

analyzed in the lower and upper respiratory tract of infected

BALB/c mice, a slightly reduced replication rate of the

mutant strain was observed. We obtained similar results

showing that the replication of RSV-DsG and RSV-WT did

not differ significantly in HEp-2 cells (data not shown). The

mechanisms for these cell-type-dependent replication rates

of RSV-DsG are still unknown. Both A549 cells and HEp-2

cells secrete a variety of cytokines following infection with

RSV (Arnold et al., 1994; Harrison et al., 1999). Whether

differences in the amount or cytokine profile secreted by the

infected epithelial cell type are responsible for the different


growth rates of RSV-DsG in A549 cells and HEp-2 cells

remains to be determined (Merolla et al., 1995).

The soluble G protein is capable of binding to epithelial

cell surfaces, and can substitute for the mG protein in the

course of infection (Escribano-Romero et al., 2004; Teng et

al., 2001). To analyze whether the sG protein might

influence the expression pattern of ICAM-1 in an autocrine

manner, we incubated RSV-DsG-infected A549 cells in the

presence of conditioned HEp-2 cell supernatant (1:2 diluted)

that was harvested from confluent cell monolayers (75 cm2,

10 ml supernatant) 20 h postinfection with RSV-WT

(m.o.i. = 3). The A549 cells were cultured for 20 h and

expression of ICAM-1 was determined thereafter. These

experiments clearly showed that ICAM-1 expression was

not diminished due to the added cell conditioned medium.

Also, purified sG protein (1 Ag/ml) or fractalkine (20 ng/ml)

added to the media of RSV-DsG-infected A549 cells did not

reduce the RSV-DsG-induced ICAM-1 expression pattern

(data not shown). Therefore, we concluded that sG protein

may not modulate the cell surface expression of ICAM-1 in

an autocrine manner as it was shown for IL-1a with regard

to IL-8 and ICAM-1 (Patel et al., 1995, 1998). Whether

RSV-DsG might be able to induce a substantial amount of

IL-1a earlier than RSV-WT, mediating ICAM-1 and IL-8

expression, remains to be determined.

Evidence accumulated that detrimental G-protein-

specific immune responses play a central role in RSV-

induced lung disease and eosinophilia (Openshaw, 1995).

The priming of mice with vaccinia viruses that expressed

mG protein led to a release of IL-5 into lung supernatants

and lung eosinophilia when subsequently challenged with

RSV. Moreover, mice primed with vaccinia viruses express-

ing sG protein and challenged with RSV revealed an even

more enhanced amount of IL-5 in the cell supernatant

combined with greatest lung eosinophilia (Johnson et al.,

1998). An established TH2-like response might be advanta-

geous for the survival of the virus. Therefore, the ability of

RSV to produce sG protein may represent a viral strategy

for immunomodulation in the course of adaptive immunity.

Cane (2001) proposed that sG protein might also act as a

decoy receptor in the course of a RSV-specific antibody

immune response. But to date, no data are available

supporting this concept. Also, Ray et al. (2001) did not

find any direct influence of purified sG protein on RSV-

specific T cell responses.

On the contrary, a lot of data accumulated that support

the thesis that sG protein seems to play an important

regulatory role in the course of innate immunity. Recently,

Tripp et al. (2000) published that a RSV mutant, deficient in

the G- and SH protein, induced an increased expression of

chemokines in the lung of infected BALB/c mice. However,

this mutant also contained amino acid changes in the F and

L protein. Therefore, further experiments are necessary to

determine exactly the role of the sG protein in this mouse

infection model. With respect to our observed two amino

acid changes in the F protein of the mutant RSV-DsG, we

hypothesize that they are not responsible for the observed

mutant phenotype since RSV-WT and RSV-DsG showed

identical fusion rates with their target cell membrane. The

data published by Tripp et al. (2000) are in line with recently

published results showing an increased lung infiltration of

immune effector cells in mice infected with RSV lacking the

secretion of sG protein (Schwarze and Schauer, 2004).

Furthermore, evidence accumulated that sG protein might

act as a chemokine by itself via binding through its con-

served CX3C motif to the fractalkine receptor (CX3CR1)

expressed on immune effector cells (Tripp et al., 2001). In

contrast, whether sG protein might be able to complex

chemokines and thereby blocking their chemotactic activity

as it was shown for a variety of viral soluble glycoproteins

remains to be determined (Seet and McFadden, 2002).

With respect to our data represented herein, one may

suggest that RSV is able to reduce the inflammatory

response by the synthesis of sG protein. This regulatory

activity seems to be time constricted. To control the

inflammatory response established by the infected lung

epithelial cell should be advantageous for the virus.

Materials and methods

Cell culture

Human A549 pulmonary type II epithelial cells and

HEp-2 cells [American Type Culture Collection (ATCC),

Rockville, USA] were cultured in DMEM supplemented

with 5% (v/v) FBS, streptomycin (100 Ag/ml), and

penicillin (100 IU/ml) (Gibco BRL, Karlsruhe, Germany).

All cell cultures and prepared virus stocks were free of

chlamydia and mycoplasmic contamination.

Virus preparation and titration

The human long strain of RSV (ATCC) and a mutant

thereof deficient in the production of sG protein both

designated as RSV-WT and RSV-DsG, respectively, were

propagated and titrated in HEp-2 cells. Cells were cultured

in DMEM containing 0.5% (v/v) FBS, streptomycin

(100 Ag/ml), and penicillin (100 U/ml). Confluent mono-

layers of HEp-2 cells were infected with RSV (WT or DsG)

for 3 h (m.o.i. = 0.1) in DMEM. The monolayers were

washed, overlayed with DMEM (0.5% FBS), and incubated

at 37 8C in 5% CO2 atmosphere until cytopathic effect

reached ~80%. Thereafter, the supernatants were harvested

and cellular debris was removed by centrifugation (5000 �g, 20 min). RSV was concentrated by polyethylene glycol

precipitation (final 10%, 60 min, 4 8C) and purified by

means of discontinuous sucrose gradient centrifugation

(Ueba, 1978). To stabilize the purified virus particles, they

were resolved in 20% sucrose/NT-buffer [150 mM NaCl,

50 mM Tris–HCl (pH 7.5)] and stored at �80 8C. The virustiter was determined using the microplaque immunoperox-


idase method (Cannon, 1987). Briefly, RSV stocks as well

as cell supernatants and cell lysates (prepared from 24-well

plates in a volume of 1 ml) were serially 10-fold diluted

onto confluent HEp-2 monolayers in microtiter plates. After

24 h, the monolayers were fixed with paraformaldehyde

(2%) and stained with mouse anti-G protein RSV polyclonal

antibody (Ab) (Biotrend, Kfln, Germany) followed by

HRP-conjugated rabbit anti-mouse immunoglobulins

(Dako, Hamburg, Germany). The RSV plaques were

identified by colorimetric staining and counted microscopi-

cally. Stock titer of the used virus stocks were adjusted to

108 plaque forming units (pfu)/ml.

Characterization of the spontaneous RSV-DsG mutant

The mutant of RSV (long strain) only deficient in the

production of sG protein (RSV-DsG) was isolated in the

Department of Medical Microbiology and Virology at the

Ruhr-University Bochum. The G gene possesses the greatest

genetic variability (Johnson et al., 1987). This inherent

quality was exploited for isolating a spontaneous mutant

deficient in the production of sG protein. For this purpose,

the RSV-WT was serially passaged at high multiplicity of

infection (undiluted) in HEp-2 cells. Twenty hours after

each passage, the cell supernatants were screened for the

presence of sG protein and the cells refed with fresh

medium. At the 7th passage, no sG protein could be

detected. The amount of sG protein was assessed by ELISA

(Roder et al., 2000). The isolated mutant (7th passage)

possess a point mutation in the G gene at position 157 (A to

G), which led to an exchange at amino acid 48 from

methionine to valine and therefore in an inactivation of the

second start codon in the ORF of the G gene. To exclude

any other mutations detrimental for the replication of the

mutant RSV-DsG, we performed sequence analysis of the

genes, known to be important for viral replication, i.e.,

NS1-, NS2-, M2- N-, P-, L-, and F gene. Gene sequences

obtained from our wild-type RSV (RSV-WT) strain were

directly compared with sequences of the isolated mutant

RSV-DsG. Virus RNA was isolated from supernatants from

infected HEp-2 cells using the RNeasy isolating kit

according to the instructions of the manufacturer (Qiagen,

Hilden, Germany). Primers for the NS1-, NS2-, M2-, N-, P-,

L-, F-, and the G gene of RSV (Metabion, Planegg-

Martinsried, Germany) were designed based on sequences

published previously (accession number M74568) and are

available upon request from the authors. PCR fragments

were constructed with sufficient overlap with adjacent

fragments such that there were no primer-masked regions.

Virus RNA was reverse transcribed at 50 8C for 30 min

using Superscript reverse transcriptase, oligo(dT)12–18 as

primer, and reaction conditions according to the protocols

supplied by the manufacturer (Invitrogen, Karlsruhe, Ger-

many). The resulting cDNA was used as template in the

PCR procedure to generate a series of overlapping PCR

products, spanning the desired RSV genes. The PCR

mixture (50 Al) consisted of cDNA, 2 Al of the respective

primers (50 pmol) each, 10 Al of 10 mM deoxyribonucleo-

side triphosphate (dNTPs), and 2 units of Taq polymerase

(Invitrogen). The cDNA was amplified during 40 cycles of

95 8C for 1 min, 53 8C for 2 min, 72 8C for 3 min, with a

final extension at 72 8C for 10 min. After examination on a

2% agarose gel, the RT-PCR products were purified using a

QIAquick purification kit (Qiagen) and sequenced directly

using the ABI Prism BigDyek Terminator Cycle Sequenc-

ing Ready Reaction kit [PE Applied Biosystems (PE),

Weiterstadt, Germany] on an ABI PRISM 3100 automatic

DNA sequencer according to the instructions of the

manufacturer. Sequence alignments were made using the

Sequencerk software V.4.0.5 from Gene Codes Coopera-

tion (Ann Arbor, MI, USA). When compared to RSV-WT,

the genes encoding for L-, NS1-, NS2-, M2-, P, and N

protein were unchanged in RSV-DsG. We observed two

point mutations in the F gene at position 227 (T to A) and

635 (G to A) leading to amino acid changes Val to Glu and

Ser to Asn, respectively. In addition to the point mutation at

position 157, the G gene of the mutant strain accumulated

27 point mutations leading to 18 amino acid changes.

However, the four cysteine residues within the conserved

gene sequence of the G protein, which is known to be

responsible for enhanced lung eosinophilia, were unchanged

(Sparer et al., 1998; Tebbey et al., 1998). Since both RSV-

DsG and RSV-WT showed identical fusion rates (see

Fig. 7C), we concluded that no additional mutations, except

the loss of the sG protein production, might influence the

biological behavior of the RSV-DsG.

Cell infection and stimulation

Confluent monolayers of A549 cells were washed with

DMEM, exposed to RSV for 2 h, washed and incubated

with fresh medium DMEM (2% FBS) for 20 and 48 h,

respectively. Thereafter, cells were harvested and immedi-

ately stained for FACS analysis. Corresponding cell super-

natants were collected and stored at �70 8C until cytokine

determination.

Preparation of soluble and membrane-anchored G protein

HEp-2 monolayers were infected with RSV-WT or

RSV-DsG (m.o.i. = 3), washed and cultured for 20 and

36 h, respectively. After 20 h, cell supernatants were

harvested, cleared by centrifugation (100000 � g, 30

min) and sG protein was prepared by ConA-sepharose 4B

affinity chromatography (Amersham Biosciences, Frei-

burg, Germany) (Johnson et al., 1998). Cellular proteins

were isolated from cells by suspension in RIPA buffer

(1� PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate,

0,1% SDS) containing 100 Ag/ml PMSF, 60 Ag/ml

aprotinin, and 1 mM sodium orthovanadate. Cell lysates

were centrifuged and the supernatants collected. Mem-

brane-bound G protein from RSV-infected HEp-2 cells


was prepared as previously described (Roder et al.,

2000).

Fluorescence dequenching assay

The virus envelope was labeled with the fluorescent

aminofluorescein (Molecular Probes, Leiden, Netherlands).

An aliquot (10 Al) of the aminofluorescein solution (50

mg/ml DMSO) was added to a RSV solution containing

5 � 106 pfu/500 Al HBSS (Miller and Hutt-Fletcher,

1992). Following incubation for 60 min at RT, the

unincorporated fluorescent dye was removed by gel

filtration on Sephadex G-75 columns using HBSS as the

eluting solution. The labeled virus was used for experi-

ments at the same day. A549 cells (5 � 105) were

incubated with RSV (5 � 104) in a volume of 100 AlHBSS for 2 h. Fluorescence intensity was measured on a

SpectraFluorPlus Reader (TECAN, Crailsheim, Germany)

with an excitation wavelength of 485 nm and an emission

wavelength of 535 nm. Each experiment was run in

triplicate. At the end of each experiment, Triton X-100

was added to 1% (v/v) into the reaction mixture, and the

maximum of fluorescence was obtained, which was

considered to be equivalent to 100% fusion of RSV with

target cells. The percentage of fusion (F) was determined

as follows: %F = 100 � (Fd � F0)/(Ft � F0), where

F0 = Em 535 nm of aminofluorescein labeled virus, Fd =

Em 535 nm after incubation with target cells, and Ft = Em

535 nm after addition of Triton X-100.

Western immunoblot

SDS-PAGE, semidry electroblotting, and detection of

bound sheep anti-mouse HRP-conjugated secondary Ab

(Dako) by ECL Plusk Western blotting detection system

(Amersham Biosciences) was performed as already

described (Roder et al., 2000). The expression of several

virus proteins was analyzed by means of HRP-labeled

polyclonal goat anti-RSV Ab (Biotrend). Protein molecular

weight markers were obtained from Amersham Bioscien-

ces. The chemiluminescence signal was measured by

means of Lumi-Imagerk F1 workstation with Lumi-

Analystk 3.x software from Roche Diagnostics (Mann-

heim, Germany).

Flow cytometry

The expression of ICAM-1 on cell surfaces of A549 cells

was measured with a FACSCalibur (BD Biosciences,

Heidelberg, Germany). Detachment and staining of cells

was performed as previously described (Arnold and Konig,

1996). PE-labeled mouse anti-human ICAM-1 mAb and

PE-labeled IgG isotype control Abs were obtained from BD

Biosciences. Monoclonal mouse anti-G protein- and anti-F

protein Abs were obtained from Biotrend. The binding of

primary Abs was detected by secondary staining with Cy3-

labeled AffiniPure Goat anti-mouse IgG (H+L) (Dianova,

Hamburg, Germany). By using the Fix and Perm kit for

intracellular staining (BD Biosciences), the cells were fixed

and permeabilized according to the manufacturer’s instruc-

tions. Data were analyzed by means of CellQuest software

(BD). The mean fluorescence intensity (MFI) of 10000 cells

was determined and corrected by subtraction of background

fluorescence of isotype control.

Cytokine measurements

Cytokines were measured by ELISA. RANTES was

determined according to the manufacturer’s instructions

(R&D Systems, Wiesbaden-Nordenstadt, Germany), and

IL-8 was determined as previously described (Arnold et al.,

1999).

NF-jB binding activity

The binding activity of NF-nB subunits Rel A (p65) and

NF-nB1 (p50) was measured using the Trans-AMk tran-

scription factor assay kit (Active Motif Europe, Rixensart,

Europe) according to the manufacturer’s recommendations

(Renard et al., 2001). Briefly, DNA encoding for the NF-nBconsensus binding site was bound to microtiter plates. The

binding of active forms of p65 or p50 to this immobilized

DNA was revealed by incubation with NF-nB specific

antibodies using ELISA technology. Whole cell lysates were

assayed for p65 and p50 binding activity. Optical density

was measured at 450 nm.

Quantikine mRNA ELISA and quantitative real-time

RT-PCR

The amount of RANTES mRNA was determined by

means of the Quantikine mRNA detection kit in accordance

with the manufacturer’s instructions (R&D Systems). The

amount of ICAM-1 and IL-8 mRNA was quantified by

Taqman real-time RT-PCR. The total cellular RNA from

uninfected and RSV-infected epithelial cells (5 � 105) was

extracted using the QiAmp 96 viral RNA Biorobotk kit on

the Biorobot 3000 System fromQiagen. Using the Gene Amp

5700 Sequence Detector (PE Applied Biosystems) the RNA

(1 Ag) was reverse transcribed to 2.5 Al cDNA and amplified

in a volume of 25 Al by means of TaqMan Universal PCR

Master Mix (PE Applied Biosystems) containing specific

primers for IL-8 (10 pmol) and a fluorescently labeled probe

(7 pmol) that were selected by means of Primer Express

Software version 2.0 [IL-8 primers: forward, 5V-CTTA-GATGTCAGT GCATAAAGACATACTCC-3V/reverse,5V-CTCAGCCCTCTTCAAAAACTTCTCCACA-3V/probe,5V-6-carboxy-fluorescein-TTGAGAGTGGACCACACTGC-6-carboxytetramethyl-rhodamine-3V (Metabion)]. The reac-

tions were incubated at 50 8C for 2 min followed by 95 8C for

10 min. The amplification profile was 95 8C for 15 s and 60

8C for 1 min for 45 cycles. For quantitative determination, a


control plasmid encoding for IL-8 was simultaneously

amplified. For this purpose, amplified IL-8 cDNA,

prepared from LPS-stimulated PBMC by use of the

above-mentioned IL-8 primers, was eluted from an agarose

gel (2%) with QIAquick gel extraction kit (Qiagen). By

using Zero Blunt TOPO PCR cloning kit for sequencing

(Invitrogen), the extracted IL-8 cDNA was cloned in a

pCR 4Blunt-TOPO vector and multiplied in transformed E.

coli. Purification of plasmids was performed by using

Qiaprep Miniprep kit (Qiagen). Plasmids used in the range

of 107 to 2 � 1010 copies of IL-8 cDNA/2.5 Al were in

parallel amplified with the samples. Data were analyzed

with PE Biosystems 5700 Sequence detection system

software and obtained fluorescence signals, in a linear

range, were used as standard curve for determination of the

amount of IL-8 cDNA/2.5 Al cDNA. The primer/probe sets

for human ICAM-1 and the housekeeping gene glycer-

aldehyde-3-phosphate dehydrogenase (GAPDH) were pur-

chased as predeveloped kits from Qiagen. The comparative

CT method was used to determine relative quantities of

ICAM-1 in cell samples. The relative RNA amount was

analyzed by using the following equation: 2�DDCT were

DDCT = DCT,q � CT,cb. CT is defined as the CT value for

ICAM-1 minus the CT value for GAPDH for a given

sample; q is the unknown sample, and cb is the calibrator

(noninfected sample).

Statistics

All data were expressed as the mean F SEM. Statistical

significance analysis of data was performed using Student’s

t test (two-sided). A value of P b 0.05 was considered

significant.

Acknowledgments

This work was partially supported by grants from

Bundesministerium fqr Arbeit und Forschung (BMBF-

NBL3). The authors wish to thank Ms. Bettina Polte for

her excellent technical assistance in all the performed

experiments.

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respiratory syncytial virus deficient in soluble g protein induced an increased proinflammatory...

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