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Outer Membrane Protein A from Klebsiella pneumoniae Activates Bronchial Epithelial Cells: Implication in Neutrophil Recruitment¹

Muriel Pichavant^*, Yves Delneste, Pascale Jeannin, Catherine Fourneau^*, Anne Brichet, André-Bernard Tonnel^*, and Philippe Gosset^2,*

^* Institut National de la Santé et de la Recherche Médicale, Unité 416, Institut Pasteur, Lille, France; Centre d’Immunologie Pierre Fabre, St. Julien en Genèvois, France; and Clinique des Maladies Respiratoires, Centre Hospitalier Régional Universitaire, Lille, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Epithelium represents the primary interface between the external and internal surroundings of the airways. Aside from its mechanical barrier function, bronchial epithelium 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)³ express various types of cell adhesion molecules, such as ICAM-1, conjugated with cytokine and chemokine secretion, which both orchestrate inflammatory cell migration toward the airway 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 chemoattractant protein-1), and GM-CSF, after a variety of mucosal injuries (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 recruitment (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 activation, 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 development of immunity: LPS, membrane proteoglycans, and outer membrane 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 described (reviewed in Refs.7 and 9), only few studies evaluated Omp properties, mainly focused on its immunomodulatory function (8, 10, 11, 12). It has been reported that the recombinant OmpA of Klebsiella pneumoniae (kpOmpA) is a potent carrier protein (13, 14, 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 between kpOmpA and immunological cells may represent an initiating event in acquired host defense mechanisms.

In contrast, OmpA involvement in lung inflammation, and particularly 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 neutrophil influx.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 epithelial cell growth and basal medium (Promocell, Heidelberg, Germany), antibiotics (penicillin G sodium, 10,000 U/ml; streptomycin sulfate, 10,000 mg/ml; and amphotericin B, 25 µg/ml); and 2 mM L-glutamine (Invitrogen), 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, Fontenay sous Bois, France) and isopropanol (Carlo Erba, Milan, Italy), gelstar (FMC bioproducts, Rockland, ME); and recombinant human TNF-, IFN- (R&D Systems, Abingdon, U.K.); SB203580, RO318220, 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 eBioscience (San Diego, CA).

Cell culture

Human bronchial epithelial biopsies were obtained by fiberoptic bronchoscopy from patients who were being investigated for bronchopulmonary carcinoma. Biopsies were taken largely at a distance from the tumor. Histologic 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 included in the study.

BEC were cultured as previously described (17). Briefly, one explant (0.5 x 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 Collection (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-cm² culture flasks, until passage 20, and maintained in BEC growth medium, containing 1% antibiotic solution. After seeding, 5 x 10⁵ cells were plated on six-well cluster plates (Costar, Cambridge, 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 µg/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 were expressed as a ratio: gene of interest/GAPDH or -actin mRNA for the human or the mouse genes, respectively.

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 µg/ml) or -tetanus toxin C (20 µg/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 µg/ml) were preincubated with BEAS-2B cells for 30 min before the addition of biotinylated kpOmpA.

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 µg/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 analyzed on 10,000 events using a flow cytometer (FACSCalibur; BD Biosciences). 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 intensity (MFI) with specific Ab minus the isotype control MFI (MFI).

Cytokine measurement

Concentrations of human IL-6, CCL2, CCL5, CXCL1, CXCL8, and CXCL10 in BEC culture supernatants were determined by sandwich enzyme 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.

EMSA

BEAS-2B cells were cultured in medium alone, or in presence of TNF- (200 U/ml) (positive control) or kpOmpA (5, 20, and 40 µg/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' end ³²P-labeling of the double-stranded oligonucleotide 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 image-analysis system.

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 expression GL3 plasmids containing or not a tandem repeat of an NF-B site and luciferase promoter. Luciferase assays were performed 24 h after transfection 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 µg) or kpOmpA solution (20 or 100 µg) 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 Immunohistowax (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 incubation 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 obtained with a Fast-Red solution (Sigma-Aldrich). Slides were washed three times between each step. Gill’s hematoxylin was used to counterstain. Leukocyte infiltrate was shown by May-Grünwald Giemsa staining (Sigma-Aldrich) on lung sections.

Statistical analysis

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.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. kpOmpA activates the NF-B signaling pathway in BEAS-2B cells. A, BEAS-2B cells were incubated with medium alone (negative control), TNF- (positive control), or kpOmpA (5, 20, and 40 µg/ml). After 2 h of incubation, nuclear protein extracts were prepared and EMSA for NF-B were performed as described in Materials and Methods. Results are representative of one of three experiments. B, BEAS-2B cells were transiently transfected with GL3 reporter plasmids containing (), or not (), a tandem repeat of an NF-B site. Cells were incubated with medium alone (control), kpOmpA (20 µg/ml), or TNF- (200 UI/ml) for 24 h. Epithelial cells were harvested, and luciferase activity in protein equivalent cell lysates was assessed as described in Materials and Methods. Results are representative of one of three experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. kpOmpA up-regulates CXCL1, CXCL5, CCL2, CXCL10, IL-6, and ICAM-1 mRNA levels in the lung parenchyma after local administration of kpOmpA. Mice were primed with a single intratracheal administration of BSA (thin) or kpOmpA (bold) (100 µg). The right lung was processed to assess cytokine mRNA expression by semiquantitative RT-PCR, at 6, 24, and 48 h after instillation in comparison with control mice (t0). mRNA expression for CXCL1, CXCL5, CCL2, CXCL10, IL-6, and ICAM-1 and -actin was analyzed. Data are expressed as the mean of the ratio "gene of interest/-actin" for three mice and are representative of one of three separate experiments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

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 µg/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 carrier 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 observed, 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).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. kpOmpA binds to BECs. BEAS-2B cells (A) and BEC primary cultures (B) were incubated, for 30 min, at 37°C, with medium (thin), biotinylated-tetanic toxin (20 µg/ml) (bold), or biotinylated-kpOmpA (20 µg/ml) (shaded), washed, and analyzed by FACS. The binding was revealed by PE-labeled streptavidin. Results are representative of one of six experiments.

Studies in BEAS-2B cells were conducted to determine whether kpOmpA may affect ICAM-1 expression. ICAM-1 mRNA expression in response to kpOmpA (8 or 20 µg/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 µg/ml kpOmpA, at 6 h, whereas this level was close to baseline levels at 24 h.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. kpOmpA up-regulates ICAM-1 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 µg/ml kpOmpA. One representative of three separate experiments is shown. B, Membrane ICAM-1 expression was measured after a 24-h incubation by flow cytometry on BEAS-2B epithelial cells, cultured with medium alone () or activated with 8–40 µg/ml kpOmpA (). Results are expressed as MFI. Data are mean ± SEM (n = 6). **, p < 0.01 compared with unstimulated cells.

kpOmpA induces cytokine mRNA expression and secretion in BECs

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 transcripts 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 µg/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).

View larger version (39K): [in this window] [in a new window]   FIGURE 3. kpOmpA induces cytokine mRNA expression 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 µg/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 production was measured after a 24-h incubation by ELISA in supernatants of BEAS-2B cells (B) cultured with medium alone (), 8 (), and 20 µg/ml kpOmpA () and BEC primary culture (C) exposed to 20 µg/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 activates intracellular signaling pathway in BEAS-2B cells

To investigate transduction pathway involved in kpOmpA activation, different inhibitors of protein kinases were tested on chemokine production (Table II). After kpOmpA stimulation, P38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) reduced CCL2, CXCL8, and CXCL10 secretion by 39, 40, and 34%, respectively, whereas the extracellular signal-regulated kinase 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%) production, but not CXCL8 (20%).

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 µg/ml) or TNF- induced NF-B activation, as evidenced by the increase in luciferase activity (10- and 13-fold, respectively). No luciferase activity was observed when cells were transfected with plasmid without NF-B site.

kpOmpA potentiates TNF- and TNF- + IFN- effects on CXCL8 and CCL2 production by BEAS-2B cells

To investigate the potential synergy between inflammatory cytokines and kpOmpA, BEAS-2B cells were activated by TNF- and TNF- + IFN-. CXCL8 and CCL2 production was up-regulated in the presence of these cytokines and kpOmpA alone (Fig. 5). BEC costimulation with kpOmpA and TNF-, or TNF- + IFN- had a synergistic effect on CXCL8 and CCL2 production. The dose-dependent effect of kpOmpA was also observed in the presence of TNF- and TNF- + IFN-. These data demonstrated that kpOmpA potentiates TNF- and TNF- + IFN- effects on CXCL8 and CCL2 production by BEAS-2B cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Synergistic effect of TNF- or TNF- + IFN- on kpOmpA-induced cytokine production by BEAS-2B cells. CXCL-8 and CCL-2 levels were measured in 24-h supernatants, after addition of 2, 8, and 20 µg/ml kpOmpA on BEAS-2B cells cultured with medium alone (•), or activated with TNF- (200 UI/ml) () or TNF- + IFN- (100 UI//ml) (). Results are expressed as mean ± SEM of three experiments.

To investigate in vivo kpOmpA effects, mice were injected intratracheally. BAL and lung fragments were collected to analyze lung inflammation.

In mice primed with kpOmpA, there was a trend to an increase in total BAL cell number compared with the control BSA (104 x 10³ and 82 x 10³ cells in comparison with 60 x 10³ cells). The absolute number of macrophages is not significantly modified 6 and 24 h after intratracheal injection of 100 µg of kpOmpA (49 x 10³ cells and 36 x 10³ cells, respectively) compared with the number obtained after BSA instillation (42 x 10³ cells). In contrast, the absolute number of neutrophils was significantly increased 6 and 24 h after instillation of 100 µg of kpOmpA (34 x 10³ cells and 42 x 10³ cells, respectively) in comparison with naive mice (6 x 10³ cells) (p < 0.05). This was confirmed by the analysis of BAL cell differential count in mice receiving kpOmpA (Fig. 6). Whereas BAL cells from control mice (receiving BSA) consisted predominantly of macrophages, a pronounced influx of neutrophils was found in BAL of mice primed with 100 µg of kpOmpA, with a maximum at day 1 (85% total cells). kpOmpA exhibited an in vivo dose-dependent effect as intratracheal injection of 20 µg induced a lower neutrophil influx on day 1 (55% total cells). Neutrophil percentage progressively returned to baseline level at day 7. Lymphocyte, eosinophil, and epithelial cell counts were not markedly modified after kpOmpA injection (data not shown).

View larger version (33K): [in this window] [in a new window]   FIGURE 6. kpOmpA intratracheal injection in mice induces neutrophil influx in BAL fluids. Mice received an intratracheal injection of BSA (100 µg) or kpOmpA (20 or 100 µg). BAL cells were spun on slides and stained with May-Grünwald-Giemsa. These data showed percentages of macrophages and neutrophils in BAL fluids collected at 6 h, 1, 2, 3, and 7 days after instillation. Results are expressed as mean ± SEM for three mice and are representative of three experiments.

View larger version (144K): [in this window] [in a new window]   FIGURE 7. Histologic evaluation of lung inflammation in mice primed with BSA or kpOmpA. Lung sections from mice sacrificed 24 h after intratracheal injection of kpOmpA (100 µg) were stained by eosin-hematoxylin and observed at a magnification of x500 (A) or x1250 (B). Neutrophils (arrows) were present in bronchial lumen (BL), bronchial epithelium, and alveolar spaces (AS). BAL immunohistochemistry was performed for ICAM-1 on lung sections from mice treated for 6 h with BSA (D) or exposed to kpOmpA for 6 h (E) or 24 h (F), in comparison with an isotype control (C). Same experiments were done for CXCL1: mice treated for 6 h with BSA (H) or exposed to kpOmpA for 6 h (I) or 24 h (J), compared with an isotype control (G). BECs (arrow) and macrophages (arrow head) were positive for both ICAM-1 and CXCL1. The magnifications were x500.

View larger version (20K): [in this window] [in a new window]   FIGURE 9. Intratracheal injection of kpOmpA in mice increases IL-10, IFN-, TNF- CXCL1, and CXCL5 levels in BAL fluids. Mice were primed by a single intratracheal administration of kpOmpA (100 µg). IL-12, IL-10, IFN-, TNF-, CXCL1, and CXCL5 levels in BAL fluids were assessed by ELISA, 6 h () or 24 h ( after instillation, in comparison with naive mice (). Data shown are representative of one of three separate experiments, and are expressed as the means ± SEM of three mice. *, p < 0.05 and **, p < 0.01 compared with BSA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

First, we report that kpOmpA binds to BEC in a saturable fashion to similar levels as observed with monocyte-derived DC (data not shown), suggesting at least that one of its receptors is expressed on epithelial cells. The innate immune system recognizes diverse pathogen-associated molecular patterns by members of the TLR family, leading to an inflammatory reaction (21). TLR-2 and TLR-4 are expressed by numerous cell types such as monocytes, macrophages, DCs, endothelial cells, and mucosal epithelial cells. TLR-2 is involved in the recognition of diverse bacteria and their products, including Gram-positive bacteria and peptidoglycans, whereas TLR-4 has been identified as the receptor for LPS, an endotoxin of Gram-negative bacteria (22). Blocking anti-TLR-4 Abs did not inhibit kpOmpA binding to BEC. kpOmpA-mediated activation is not due to LPS because endotoxin is undetectable in kpOmpA preparation and polymyxin B does not neutralize kpOmpA effects on bronchial epithelium. Using TLR-2-transfected cells and DC from TLR-2-deficient mice, Jeannin et al. (14). demonstrated that TLR-2 is involved in kpOmpA-mediated signaling but is not required in kpOmpA binding to and endocytosis by DC (14, 20). We also show here that blocking anti-TLR-2 Abs do not affect kpOmpA binding to BEC. These observations suggest that endocytic receptor(s) might be responsible for the capture of kpOmpA and for the subsequent mobilization of TLR-2 to activate the signaling cascade (23). Nevertheless, activation of airway epithelial cells by TLR-2 ligands does not necessarily require TLR-2 signaling because lipoteichoic acid, another constituant of bacteria wall, activates platelet-activating factor receptor in these cells, resulting in the transactivation of the epidermal growth factor receptor (24). In our study, BEC exposure to kpOmpA activates the NF-B pathway, a signaling pathway involved in TLR-2-mediated cell activation. This transcription factor has been identified as an inducer for the transcription of cytokine, chemokine, and adhesion molecule genes such as ICAM-1 (25). The present study also demonstrates that kpOmpA induces ICAM-1 expression and cytokine secretion by BEC through the activation of NF-B and protein kinases such as P38 MAPK, PI₃-K, and PKC.

Mobilization of innate defense mechanisms involves the recruitment of effector cells, and particularly neutrophils and macrophages. This process requires the coordinate activity of proinflammatory cytokines, chemokines, and adhesion molecules. We showed that in vitro, kpOmpA induces proinflammatory cytokine production by BEC which implicate, at least in part, NF-B signaling pathway activation. This included the production of the cytokine IL-6 and chemokines such as CCL2, CXCL1, CXCL8, or CXCL10. IL-6 was initially considered as a proinflammatory cytokine, but more recent data showed that IL-6 could participate to the resolution of inflammatory reactions (26). It induces the secretion of cytokine inhibitors such as IL-1 receptor antagonist and soluble TNFR by monocytes, and of protease inhibitors such as tissue inhibitor of metalloproteinase 1 or 2-macroglobulin. Among chemokines, CXCL1, CXCL5, and CXCL8, as well as other chemokines containing an AA motif ELR, are chemotactic factors for neutrophils through the interaction with CXCR1 and 2 (5). CCL2 and CXCL10 are implicated in the recruitment of mononuclear cells through the interaction with CCR2 and CXCR3. Both chemokines are involved in the recruitment of monocytes/macrophages and they are also chemotactic for different T cell subsets including memory T and Th1 cells, respectively (27).

In our in vivo model, intratracheal kpOmpA administration induced mRNA expression of several chemokines in the lung, including CCL3 (macrophage inflammatory protein-1), CXCL1, CXCL5, and CXCL10. In the murine model, kpOmpA instillation caused an important and transient accumulation of neutrophils in lung and BAL fluids, probably mediated by the production of chemoattractant factors such as CXCL1 and CXCL5. Interestingly, CXCL5 production seems to be delayed (maximal at 24 h) compared with CXCL1 secretion (at 6 h), suggesting that these chemokines act sequentially in this model. Both CXCL1 and CXCL5 potently activate neutrophils via the murine CXCL8 receptor homologue, resulting in increased Mac-1 expression and in respiratory burst activity (28, 29, 30). The recruitment and the subsequent activation of neutrophils play a key role in lung bacterial clearance and improved survival (31, 32). CXCL5 is predominantly produced by fibroblasts and lung epithelial cells. Macrophages are reported to be the main source of CXCL1 (33, 34). However, immunochemistry analysis showed that both macrophages and BEC are strongly positive for CXCL1 expression after exposure to kpOmpA, suggesting that both cell types are involved in this process. The adhesion molecule ICAM-1 participates in leukocyte diapedesis by mediating firm adhesion to endothelial or epithelial cells and by this way is involved in host defense (35). We also reported that ICAM-1 expression is up-regulated after kpOmpA exposure in alveolar and bronchial epithelium and in endothelium, witness of the local inflammation. In human BEC, there is some specificity in the kpOmpA-derived adhesion molecule expression, because kpOmpA alone or in presence of TNF- does not modulate VCAM-1 expression (data not shown) in contrast to ICAM-1. Therefore, these data demonstrated that chemokine secretion and adhesion molecule expression, in response to kpOmpA, specifically direct neutrophil migration across the epithelial barrier at infected mucosal sites.

The in vivo production of inflammatory and immunostimulatory cytokines, such as TNF- and IL-10, was investigated in the BAL. In lung, TNF- is predominantly produced by macrophages, but BEC may also secrete it (1). This cytokine improves lung bacterial clearance through an up-regulation of adhesion molecule expression and secretion of chemokines including CXCL1 (36). In our study, kpOmpA induces a weak production of TNF- by BEC, whereas IFN- and IL-10 remain undetectable in BEC supernatants (data not shown). In addition, kpOmpA potentiates the effects of TNF-, and TNF- + IFN- on CXCL8 and CCL2 production by BEC. kpOmpA activates macrophages, which produce high amounts of TNF- (16). In turn, these mediators may expand macrophage activation. So, in vivo, this may result in a proinflammatory amplification loop between macrophages and epithelial cells. In contrast, IL-10 is detrimental to innate and cell-mediated immunity in the lung, particularly by blocking the synthesis of proinflammatory cytokines, including IFN-, IL-1, TNF-, IL-12, and chemokine production (37). In vivo, neutralization by anti-IL-10 Ab results in an enhanced bacterial clearance (38) whereas IL-10 secretion serves to limit the production of proinflammatory cytokines in this model (38, 39). During infection, IL-10 seems to have a dual effect: detrimental on the bacterial clearance and beneficial on the inflammation as shown in septic peritonis (40). In our study, IL-10 production reaches its maximum at 6 h and persists at 24 h, whereas the production of chemokines and proinflammatory cytokines returned to baseline. These demonstrated that kpOmpA induced inflammatory burst was associated with the production of anti-inflammatory mediators (IL-6 and IL-10).

The different cell populations in the mucosal barrier are equipped to sense and respond to the molecular contents in the lumen and to translate this molecular information into signals that can reach local or distant sites within the body. In addition to its effect on BEC, kpOmpA interacts and activates DC, inducing their maturation and delivering Ag into the MHC class I presentation pathway (14). However, the IL-12 production induced by kpOmpA in macrophages and DC is modest compared with that obtained after LPS- or CD40-dependent activation (16). In this context, the lack of IL-12 secretion in BAL fluids, after kpOmpA treatment may be due to the capture of this cytokine by IL-12R-positive cells. In vivo IL-12 secretion by kpOmpA-activated APC may also require the action of a coactivator. Nevertheless, IFN- levels, whose production can be induced by IL-12, are increased after kpOmpA instillation. As IFN- production is observed after 6 h, kpOmpA presumably directly induces its production by immunocompetent cell activation. Further studies are required to elucidate this point. Because IFN- plays an essential role in the development of adaptative immunity (41), kpOmpA may orchestrate the transition from innate to adaptive immunity, and the polarization of this immune response to promote cell-mediated immunity.

In conclusion, the present data identify a new mechanism by which the bronchial epithelium is involved in anti-infectious defense. BEC exposed to kpOmpA show an increased expression of adhesion molecules and an up-regulation of chemokine secretion, which are implicated in vivo in inflammatory cell recruitment toward the lung. In addition to endotoxin and glycoprotein, OmpA is also an important component of the bacterial wall implicated in the development of a defense mechanism.

	Acknowledgments

 
We greatly thank Philippe Marquillies for his helpful technical assistance, Dr. Fabrice Bureau (Liège, Belgium) for the EMSA, and Dr. Catherine Duez for the critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by the Institut National de la Santé et de la Recherche Médicale and the Pasteur Institute of Lille. M.P. is the recipient of doctoral fellowships from the Ministère de l’Education Nationale. P.G. is a member of the Institut National de la Santé et de la Recherche Médicale.

² Address correspondence and reprint requests to Dr. Philippe Gosset, Institut National de la Santé et de la Recherche Médicale, Unité 416, Institut Pasteur de Lille, 1, rue du Professeur Calmette, BP 245, 59019 Lille cedex, France. E-mail address: Philippe.Gosset{at}pasteur-lille.fr

³ Abbreviations used in this paper: BEC, bronchial epithelial cell; CXCL, CXC chemokine 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-activated protein kinase; PKC, protein kinase C; PI3-K, phosphatidylinositol 3-kinase.

Received for publication February 24, 2003. Accepted for publication October 8, 2003.

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