of June 13, 2013.
This information is current as
Cells: Implication in Neutrophil Recruitment
pneumoniae Activates Bronchial Epithelial
Outer Membrane Protein A from Klebsiella
PhilippeFourneau, Anne Brichet, André-Bernard Tonnel and
Muriel Pichavant, Yves Delneste, Pascale Jeannin, Catherine
2003; 171:6697-6705; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
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The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 13, 2013
Outer Membrane Protein A from Klebsiella pneumoniae
Activates Bronchial Epithelial Cells: Implication in Neutrophil
Muriel Pichavant,* Yves Delneste,†Pascale Jeannin,†Catherine Fourneau,* Anne Brichet,‡
Andre ´-Bernard Tonnel,*‡and Philippe Gosset2*
Aside from its mechanical barrier function, bronchial epithelium plays an important role both in the host defense and in the
pathogenesis of inflammatory airway disorders. To investigate its role in lung defense, the effect of a bacterial cell wall protein,
the outer membrane protein A from Klebsiella pneumoniae (kpOmpA) on bronchial epithelial cells (BEC) was evaluated on
adhesion molecule expression and cytokine production. Moreover, the potential implication of this mechanism in kpOmpA-
induced lung inflammation was also determined. Our in vitro studies demonstrated that kpOmpA strongly bound to BEAS-2B
cells, a human BEC line, and to BEC primary cultures, resulting in NF-?B signaling pathway activation. Exposure to kpOmpA
increased ICAM-1 mRNA and cell surface expression, as well as the secretion of IL-6, CXC chemokine ligand (CXCL)1, CXCL8,
C-C chemokine ligand 2, CXCL10 by BEAS-2B cells, and BEC primary cultures (p < 0.005). We analyzed in vivo the consequences
of intratracheal injection of kpOmpA to BALB/c mice. In kpOmpA-treated mice, a transient neutrophilia (with a maximum at
24 h) was observed in bronchoalveolar lavage and lung sections. In vivo kpOmpA priming induced bronchial epithelium activation
as evaluated by ICAM-1 and CXCL1 expression, associated with the secretion of CXCL1 and CXCL5 in bronchoalveolar lavage
fluids. In the lung, an increased level of the IL-6, CXCL1, CXCL5, CXCL10 mRNA was observed with a maximum at 6 h. These
data showed that kpOmpA is involved in host defense mechanism by its ability to activate not only APC but also BEC, resulting
in a lung neutrophilia. The Journal of Immunology, 2003, 171: 6697–6705.
is involved both in normal host defense and in the pathogenesis of
inflammatory airway disorders by its implication in inflammatory
cell recruitment (1, 2). For this purpose, bronchial epithelial cells
(BEC)3express various types of cell adhesion molecules, such as
ICAM-1, conjugated with cytokine and chemokine secretion,
which both orchestrate inflammatory cell migration toward the air-
way wall. BEC are able to produce cytokines and growth factors,
particularly IL-6, CXC chemokine ligand (CXCL)1 (macrophage
inflammatory protein-2), CXCL8 (IL-8), CXCL10 (IFN-?-induced
protein-10), C-C chemokine ligand (CCL)2 (monocyte chemoat-
pithelium represents the primary interface between the
external and internal surroundings of the airways. Aside
from its mechanical barrier function, bronchial epithelium
tractant protein-1), and GM-CSF, after a variety of mucosal inju-
ries (3, 4). In addition to their chemotactic activity for neutrophils,
CXCL8 and other CXC chemokines with an ELR amino acid motif
activate these cells. CXCL10 is implicated in Th1 lymphocyte re-
cruitment (5). Conversely, CCL2 is a potent chemoattractant and
activator of monocytes, macrophages, and T lymphocytes. So,
BEC activation may be implicated in the lung defense mechanism
by its ability to recruit granulocytes and mononuclear cells.
Among the agents responsible for resident airway cell activa-
tion, many different stimuli such as fungi, viruses, pollutants, and
bacteria are present in inhaled air. Klebsiella pneumoniae is a
Gram-negative aerobic organism, which is a major pathogen for
nosocomial pneumonia (6). The clearance of this bacteria from the
lungs requires effective host defense mechanisms, to which the
bacterial surface takes part. Three components of the outer wall of
Gram-negative bacteria are suspected to be involved in develop-
ment of immunity: LPS, membrane proteoglycans, and outer mem-
brane proteins (Omp) (7, 8). OmpA is one of the major proteins of
the outer membrane of Gram-negative bacteria. This protein is
highly conserved among the enterobacteriacae family and is
thought to consist of two domains. Whereas the effects of LPS and
membrane proteoglycans on immune cells have been largely de-
scribed (reviewed in Refs. 7 and 9), only few studies evaluated
Omp properties, mainly focused on its immunomodulatory func-
tion (8, 10–12). It has been reported that the recombinant OmpA
of Klebsiella pneumoniae (kpOmpA) is a potent carrier protein
(13–15) that binds to, is internalized, and activates macrophages
(16) and professional APCs such as dendritic cells (DC) (14).
kpOmpA also favors the cross-presentation of exogenous Ags and
the induction of cytotoxic responses (14). These interactions be-
tween kpOmpA and immunological cells may represent an initi-
ating event in acquired host defense mechanisms.
*Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 416, Institut Pasteur,
Lille, France;†Centre d’Immunologie Pierre Fabre, St. Julien en Gene `vois, France;
and‡Clinique des Maladies Respiratoires, Centre Hospitalier Re ´gional Universitaire,
Received for publication February 24, 2003. Accepted for publication October
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Institut National de la Sante ´ et de la Recherche
Me ´dicale and the Pasteur Institute of Lille. M.P. is the recipient of doctoral fellow-
ships from the Ministe `re de l’Education Nationale. P.G. is a member of the Institut
National de la Sante ´ et de la Recherche Me ´dicale.
2Address correspondence and reprint requests to Dr. Philippe Gosset, Institut Na-
tional de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 416, Institut Pasteur de Lille, 1,
rue du Professeur Calmette, BP 245, 59019 Lille cedex, France. E-mail address:
3Abbreviations used in this paper: BEC, bronchial epithelial cell; CXCL, CXC che-
mokine ligand; CCL, C-C chemokine ligand; Omp, outer membrane protein;
kpOmpA, OmpA of Klebsiella pneumoniae; DC, dendritic cell; TLR, Toll-like receptor;
MFI, mean fluorescence intensity; BAL, bronchoalveolar lavage; MAPK, mitogen-acti-
vated protein kinase; PKC, protein kinase C; PI3-K, phosphatidylinositol 3-kinase.
The Journal of Immunology
Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00
by guest on June 13, 2013
In contrast, OmpA involvement in lung inflammation, and par-
ticularly its action on BEC, is not documented. The purpose of this
work was to identify potential interactions between kpOmpA and
BEC in vitro and in vivo and secondarily the development of lung
inflammation. Our results demonstrated that kpOmpA binds to
BEC and induces adhesion molecule and cytokine expression by
these cells. In vivo, kpOmpA triggers BEC activation and a neu-
Materials and Methods
Cytokines, Abs, and other reagents
kpOmpA was expressed in Escherichia coli and purified as described as an
endotoxin-free preparation (14). The following materials were purchased:
DMEM/F12 medium (Invitrogen, Cergy Pontoise, France), airway epithe-
lial cell growth and basal medium (Promocell, Heidelberg, Germany), an-
tibiotics (penicillin G sodium, 10,000 U/ml; streptomycin sulfate, 10,000
mg/ml; and amphotericin B, 25 ?g/ml); and 2 mM L-glutamine (Invitro-
gen), collagen G (3 mg/ml in 12 mM HCl; Biochrom, Berlin, Germany),
FCS, Ultroser G, trypsin (containing 1 mM EDTA); agarose, PBS, TRIzol
reagent (Life Technologies, Grand Island, NY), chloroform (Merck, Fon-
tenay sous Bois, France) and isopropanol (Carlo Erba, Milan, Italy), gelstar
(FMC bioproducts, Rockland, ME); and recombinant human TNF-?,
PD98059, genistein (Calbiochem, San Diego, CA), LY294002 (Sigma-
Aldrich, St. Louis, MO). The following mouse mAb were used: anti-
cytokeratin 5/6/18 Ab (NeoMarkers, Fremont, CA); anti-ICAM-1 mAb
(IgG1), clone B159 and the isotype control (IgG1), clone MOPC 21 (BD
Biosciences, Erembodegem, Belgium), as well as the secondary Abs FITC
or PE-labeled streptavidin (Sigma-Aldrich). Neutralizing anti-human Toll-
like receptor (TLR)-2 and TLR-4 mouse mAb were purchased by eBio-
science (San Diego, CA).
Human bronchial epithelial biopsies were obtained by fiberoptic bronchos-
copy from patients who were being investigated for bronchopulmonary
carcinoma. Biopsies were taken largely at a distance from the tumor. His-
tologic features of the bronchial mucosa were normal in all specimens. All
procedures were reviewed and approved by Hospital Institutional Review
Board and written informed consent was obtained from all subjects in-
cluded in the study.
BEC were cultured as previously described (17). Briefly, one explant
(?0.5 ? 0.5 mm in size) was placed on sterile plastic dishes coated with
collagen G matrix (type I and III collagen). After an adherence phase,
explants were cultured in DMEM/F12 medium containing 2% Ultroser G,
1% antibiotics; and 2 mM L-glutamine. Culture medium was changed every
3–4 days. Explants were cultured until BEC were confluent. Then, explants
were transferred three times to new dishes to initiate new BEC primary
cultures. After transfer, confluent epithelial cells were incubated for 24 h
with medium alone (negative control) and then incubated with kpOmpA in
fresh medium. Epithelial phenotype (?96%) was confirmed by staining
with anti-cytokeratin 5/6/18 Ab (data not shown).
BEAS-2B cells were obtained from the American Type Culture Collec-
tion (Manassas, VA). This cell line was derived from human bronchial
epithelium transformed by an adenovirus SV-40 hybrid virus. BEAS-2B
cells were cultured in 75-cm2culture flasks, until passage 20, and main-
tained in BEC growth medium, containing 1% antibiotic solution. After
seeding, 5 ? 105cells were plated on six-well cluster plates (Costar, Cam-
bridge, MA) with collagen G coating. Confluent cells were then cultured in
BEC basal medium for 24 h.
Cell activation was achieved by addition of endotoxin-free recombinant
kpOmpA (2–40 ?g/ml) or a control glycoprotein such as BSA. In some
experiments, TNF-? (200 U/ml) with or without IFN-? (100 U/ml) were
added in the presence or not of kpOmpA. Activation by LPS (serotype
055B5; 100 ng/ml) and kpOmpA in the presence or not of polymyxin B (50
U/ml) (Sigma-Aldrich) was also performed as a control. Supernatants were
collected after 6 or 24 h incubation and cells were lysed in TRIzol reagent
to prepare RNA.
Quantification of mRNA expression
Total RNA were purified and reverse transcripted using oligo-dT primers
and Superscript reverse transcriptase (Invitrogen). In BEAS-2B cells,
mRNA expression for human IL-6, IL-18, CCL2, CCL5, CXCL8,
CXCL10, and ICAM-1 was evaluated by a two-step semiquantitative RT-
PCR using GAPDH mRNA as a reference. In mouse lungs, murine IL-6,
CCL2, CXCL1, CXCL5, CXCL10, and ICAM-1 mRNA expression was
determined by the same method using ?-actin mRNA as a reference. The
primer sequences and the size of the product are reported in Table I. After
gel electrophoresis and staining with gelstar, the intensity of each band was
measured with Gel Analyst system (Claravision, Orsay, France). Results
Table I. Primers for RT-PCR analysis of cytokine, chemokine, and adhesion molecule expressiona
Target Product (bp)Sequences
5?-GTC TTC ACC ACC ATG GAG A-3?
5?-CCA AAG TTG TCA TGG ATG ACC-3?
5?-GTC GGG GCG CCC CAG GCA CCA-3?
5?-CTC CTT AAT GTC ACG CAC GAT TTC-3?
5?-TCA ATG AGG AGA CTT GCC TG-3?
5?-GAT GAG TTG TCA TGT CCT GC-3?
5?-GGC ACA GGT CAT GAC AGA CGT GAG CCG AAA GAT TCG A-3?
5?-GCC GGA TCC CGC GCC CGC GGT TGC CGC TCC GCC-3?
5?-TCC AGC ATG AAA GTC TCT GC-3?
5?-TGG AAT CCT GAA CCC ACT TC-3?
5?-TTG GCA GCC TTC CTG ATT-3?
5?-AAC TTC TCC ACA ACC CTC TG-3?
5?-CGA TTC TGA TTT GCT GCC TTA T-3?
5?-GAC ATC TCT TCT CAC CCT TCT TTT T-3?
5?-AGC TGG CAC ATT GGA GTC TG-3?
5?-CCA ACG TCT CGT CCT TCT TC-3?
5?-GTG ACA ACC ACG GCC TTC CCT ACT-3?
5?-GGT AGC TAT GGT ACT CCA-3?
5?-TCA GCC AGA TGC AGT TAA CG-3?
5?-AGG TGC TGA AGA CCT TAG GG-3?
5?-AAC GGA GAA AGA AGA CAG ACT G-3?
5?-GAC GAG ACC AGG AGA AAC AG-3?
5?-AGC TCG CCA TTC ATG CGG ATG-3?
5?-CTA TTG AAC ACT GGC CGT TCT-3?
5?-CCT ATC CTG CCC ACG TGT TG-3?
5?-CGC ACC TCC ACA TAG CTT ACA-3?
5?-TCG GAG GAT CAC AAA CGA AGC-3?
5?-AAC ATA AGA GGC TGC CAT CAC G-3?
ah, human; m, mouse.
6698kpOmpA ACTIVATES BECs
by guest on June 13, 2013
were expressed as a ratio: gene of interest/GAPDH or ?-actin mRNA for
the human or the mouse genes, respectively.
Flow cytometric analysis
BEC were briefly treated with trypsin solution and, after neutralization of
proteinase activity, removed from plates by repeated pipetting. The binding
of kpOmpA on BEC was evaluated by their incubation for 30 min at 37°C
with biotinylated-kpOmpA (20 ?g/ml) or -tetanus toxin C (20 ?g/ml) as a
control. After washings, cells were incubated with PE-labeled streptavidin
for another 30 min and then washed twice. To block the binding of
kpOmpA, neutralizing anti-TLR-2 and anti-TLR-4 mAbs (20 ?g/ml) were
preincubated with BEAS-2B cells for 30 min before the addition of bio-
To assess the modulation of ICAM-1 expression on BEAS-2B cells,
cells were stimulated in presence or not of kpOmpA (8, 20, or 40 ?g/ml)
for 24 h. FITC-labeled anti-ICAM-1 mAb and the isotype control were
added for 30 min to cells, and then washed twice. Cells were resuspended
and fixed in PBS with 0.25% paraformaldehyde. Fluorescence was ana-
lyzed on 10,000 events using a flow cytometer (FACSCalibur; BD Bio-
sciences). Previous experiments showed that BEC treatment with trypsin
did not affect ICAM-1 expression compared with cells detached with PBS
plus EDTA 2 mM (data not shown).
Results were expressed as difference between mean fluorescence inten-
sity (MFI) with specific Ab minus the isotype control MFI (?MFI).
Concentrations of human IL-6, CCL2, CCL5, CXCL1, CXCL8, and
CXCL10 in BEC culture supernatants were determined by sandwich en-
zyme immunoassay (R&D Systems). Levels of murine IL-10, IL-12,
IFN-?, TNF-?, CXCL1, and CXCL5 (R&D Systems) were determined by
ELISA in bronchoalveolar lavage (BAL) fluids.
BEAS-2B cells were cultured in medium alone, or in presence of TNF-?
(200 U/ml) (positive control) or kpOmpA (5, 20, and 40 ?g/ml) for 2 h.
After washing, nuclear proteins were extracted by standard procedures
(18). The probes used for the gel retardation assay contained the consensus
?B (5?-CAG CGG CAG GGG AAT TCC CCT CTC CTT AGG TT-3?)
binding site. The 5? end32P-labeling of the double-stranded oligonucleo-
tide and EMSA were performed by standard procedures. Membranes were
exposed to a PhosphoImager screen (Molecular Dynamics, Sunnyvale,
CA) and the intensity of the bands were quantified by using a computer
Transient transfection assays
BEAS-2B cells, grown to 70–80% confluence in BEC growth medium and
starved during 24 h in BEC basal medium, were transiently transfected by
lipofection using lipofectamin (Invitrogen) technique with reporter and ex-
pression GL3 plasmids containing or not a tandem repeat of an NF-?B site
and luciferase promoter. Luciferase assays were performed 24 h after trans-
fection as previously described (19).
kpOmpA intratracheal injection in mice
Six- to 10-wk female BALB/c mice were anesthetized by i.p. injection of
avertin (Sigma-Aldrich) (2.5% v/v in PBS). Fifty microliters of BSA (100
?g) or kpOmpA solution (20 or 100 ?g) were administrated intratracheally
under direct vision through the opening vocal cords using a 23G metal
catheter connected to the outlet of a micropipette. Mice were sacrificed
after 6 h, 1, 2, 3, and 7 days, and BAL fluids were performed by PBS
instillation (1 ml).
Immunohistochemistry for ICAM-1 and CXCL1 expression in
mice lung tissue
Lung specimens were fixed with Immunohistofix and embedded in Immu-
nohistowax (Aphase, Mormont, Belgium). After permeabilization, sections
were overlaid overnight with rabbit anti-mouse ICAM-1 (BD Biosciences)
or CXCL1 Abs (R&D Systems). Ab binding was detected after a 2-h in-
cubation with biotin-conjugated goat anti-rabbit IgG Ab (dilution, 1/400) at
room temperature, followed by extravidin-alkaline phosphatase incubation
(dilution, 1/200; Sigma-Aldrich) for 30 min. Color development was ob-
tained with a Fast-Red solution (Sigma-Aldrich). Slides were washed three
times between each step. Gill’s hematoxylin was used to counterstain. Leu-
kocyte infiltrate was shown by May-Gru ¨nwald Giemsa staining (Sigma-
Aldrich) on lung sections.
Excepting Figs. 4 and 8, results were expressed as mean ? SEM. Statistical
analysis was performed by the use of nonparametric tests: the Wilcoxon
test for paired data or the Mann-Whitney U test for unpaired data. A value
of p ? 0.05 was considered as significant.
kpOmpA binds to BEC
Binding of biotinylated-kpOmpA was analyzed by flow cytometry
to the human BEC line BEAS-2B and BEC primary cultures. Fig.
1 shows that kpOmpA (20 ?g/ml) strongly bound to BEAS-2B
cells (Fig. 1A) and BEC primary cultures (Fig. 1B). In contrast, no
binding of biotinylated-tetanic toxin, a bacterial protein with car-
rier properties, was detected. Biotinylated kpOmpA binding was
inhibited by addition of a 10-fold excess of unconjugated kpOmpA
(data not shown). Previous studies reported that TLR-2 is involved
in kpOmpA signaling in macrophage and DC (14, 20). Therefore,
we tested the effects of anti-TLR-2 and -TLR-4 blocking Abs on
kpOmpA binding to BEAS-2B cells. No modification was ob-
served, suggesting, in agreement with others (14, 20), that TLR-2
and TLR-4 are not involved in kpOmpA binding to BEC but rather
than in cell activation (data not shown).
kpOmpA induces ICAM-1 mRNA and cell surface protein
expression in BEAS-2B cells
Studies in BEAS-2B cells were conducted to determine whether
kpOmpA may affect ICAM-1 expression. ICAM-1 mRNA expres-
sion in response to kpOmpA (8 or 20 ?g/ml) was studied by RT-
PCR at 6 and 24 h stimulation. BEAS-2B cells incubated with
medium alone expressed low levels of ICAM-1 mRNA (Fig. 2A).
ICAM-1 mRNA was strongly increased with 8 and 20 ?g/ml
kpOmpA, at 6 h, whereas this level was close to baseline levels at
kpOmpA effects on ICAM-1 cell surface protein expression
were tested after a 24-h incubation. Fig. 2B shows that BEAS-2B
cells constitutively express ICAM-1, as previously reported (17).
kpOmpA increased dose-dependently ICAM-1 expression (p ?
0.01 compared with unstimulated cells). MFI was 3-, 4-, and 5-fold
mary cultures (B) were incubated, for 30 min, at 37°C, with medium (thin),
biotinylated-tetanic toxin (20 ?g/ml) (bold), or biotinylated-kpOmpA (20
?g/ml) (shaded), washed, and analyzed by FACS. The binding was re-
vealed by PE-labeled streptavidin. Results are representative of one of six
kpOmpA binds to BECs. BEAS-2B cells (A) and BEC pri-
6699The Journal of Immunology
by guest on June 13, 2013
higher than in medium alone, in the presence of 8, 20, and 40
?g/ml kpOmpA, respectively.
kpOmpA induces cytokine mRNA expression and secretion in
Effect of kpOmpA on cytokine production by BEAS-2B cells and
BEC primary cultures was analyzed. kpOmpA effects on mRNA
expression for CCL2, CXCL8, CXCL10, IL-6, and IL-18 were
quantified in BEAS-2B cells by RT-PCR. Cytokine mRNA tran-
scripts were undetectable in resting BEAS-2B cells, except for
IL-18, as shown in Fig. 3A. However, CCL2, CXCL8, CXCL10,
and IL-6 mRNA levels increased in a dose-dependent manner after
treatment with 8 and 20 ?g/ml kpOmpA. Cytokine mRNA levels
reached a maximum at 6 h postexposure, and persisted at 24 h.
Such modulation was not observed for IL-18 (Fig. 3A).
kpOmpA-induced mRNA expression was associated with the
dose-dependent production of CCL2, CXCL1, CXCL8, CXCL10,
and IL-6 release, in BEAS-2B cell supernatants after 24 h of stim-
ulation (p ? 0.01 for these cytokines) (Fig. 3B). CXCL1 and
CXCL8 production was strongly induced by 480- and 110-fold,
respectively, in the presence of 20 ?g/ml kpOmpA compared with
medium alone. Moreover, treatment with polymixin B (an inhib-
itor of endotoxin activation) did not affect CCL2 and CXCL8 pro-
duction, whereas it neutralized the effect of 10 ng/ml LPS (data not
shown). Similar results were obtained with BEC primary cultures:
kpOmpA (20 ?g/ml) increased significantly the production of
CCL2, CXCL1, CXCL10, and IL-6 (p ? 0.05), and CXCL8 (p ?
0.01) compared with unstimulated cells after 24 h (Fig. 3C). Taken
together, these data show that kpOmpA triggers CCL2, CXCL8,
CXCL10, and IL-6 mRNA expression and secretion in BEC.
kpOmpA activates intracellular signaling pathway in BEAS-2B
To investigate transduction pathway involved in kpOmpA activa-
tion, different inhibitors of protein kinases were tested on chemo-
kine production (Table II). After kpOmpA stimulation, P38 mito-
gen-activated protein kinase (MAPK) inhibitor (SB203580)
reduced CCL2, CXCL8, and CXCL10 secretion by 39, 40, and
34%, respectively, whereas the extracellular signal-regulated ki-
nase 1/2 MAPK inhibitor PD98059 had no effect. The inhibitor of
phosphatidylinositol 3-kinase (PI3-K) LY294002 inhibited in a
higher manner CCL2, CXCL8, and CXCL10 secretion (62, 49, and
60%, respectively). Protein kinase C (PKC) inhibitor, referred to as
RO318220, also decreased CCL2 (45%) and CXCL10 (70%) pro-
duction, but not CXCL8 (20%).
NF-?B nuclear translocation was also investigated in BEAS-2B
cells by EMSA. In these experiments, NF-?B was activated in a
dose-dependent manner with kpOmpA (Fig. 4A). TNF-?-induced
translocation was used as a positive control.
To evaluate NF-?B promoter activity, the luciferase activity was
measured in lysates of BEAS-2B cells transfected with the reporter
plasmid pGL3, containing or not a tandem repeat of an NF-?B site
(Fig. 4B). The data showed that kpOmpA (20 ?g/ml) or TNF-?
mRNA expression and cell surface expression on
BEAS-2B cells. A, ICAM-1 and GAPDH mRNA
expression were analyzed by RT-PCR on
BEAS-2B epithelial cells cultured for 6 or 24 h,
with medium alone (control) or 8 and 20 ?g/ml
kpOmpA. One representative of three separate
experiments is shown. B, Membrane ICAM-1 ex-
pression was measured after a 24-h incubation by
flow cytometry on BEAS-2B epithelial cells, cul-
tured with medium alone (?) or activated with
8–40 ?g/ml kpOmpA (u). Results are expressed
as ?MFI. Data are mean ? SEM (n ? 6). ??, p ?
0.01 compared with unstimulated cells.
kpOmpA up-regulates ICAM-1
pression and production by BEAS-2B cells and BEC
primary cultures. A, Cytokine mRNA expression was
quantified in BEAS-2B cells, cultured for 6 and 24 h
with medium alone, or kpOmpA (8 and 20 ?g/ml).
CCL2, CXCL8, CXCL10, IL-6, IL-18, and GAPDH
mRNA expression were evaluated by RT-PCR. One
representative of six experiments is shown. CCL2,
CXCL1, CXCL8, CXCL10, and IL-6 cytokine produc-
tion was measured after a 24-h incubation by ELISA in
supernatants of BEAS-2B cells (B) cultured with me-
dium alone (?), 8 (u), and 20 ?g/ml kpOmpA (f) and
BEC primary culture (C) exposed to 20 ?g/ml
kpOmpA (right column) or not (left column). B and C,
Results were expressed as the mean ? SEM of 6–10
experiments. ?, p ? 0.05 and ??, p ? 0.01 compared
with unstimulated cells.
kpOmpA induces cytokine mRNA ex-
6700kpOmpA ACTIVATES BECs
by guest on June 13, 2013