Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (P40), we have analyzed the interaction between OmpA and macrophages. We report that Alexa488-labeled P40 binds (at 4°C) to murine and human macrophages in a dose-dependent manner and is rapidly internalized (at 37°C). No binding or internalization of the Alexa488-labeled glycophorin A control protein is observed under the same conditions. Furthermore, P40 up-regulates the production of IL-1β, IL-8, IL-10, IL-12, and TNF-α by human macrophages and of NO by the RAW 264.7 murine macrophage cell line. P40 also synergizes with IFN-γ and suboptimal concentrations of LPS to up-regulate the production of these mediators. In conclusion, P40 binds to and activates macrophages. These data suggest that recognition of OmpA by macrophages may be an initiating event in the antibacterial host response.

The effective host defense against bacterial infection is dependent primarily upon the rapid clearance of bacteria by neutrophils and macrophages. On initial encounter with bacteria, macrophages recognize and phagocyte bacteria before killing them intracellularly and secreting proinflammatory mediators and chemokines that activate and recruit leukocytes into the bacterial foci (1, 2, 3, 4). This may result in a proinflammatory amplification loop between macrophages, neutrophils, and lymphocytes. In addition to an antibacterial activity, inflammatory mediators activate APCs and allow the initiation of a specific immune response (1, 5). Later, resident leukocytes produce a second wave of anti-inflammatory cytokines, such as IL-10, which down-regulate the inflammation (2).

Macrophages discriminate between infectious agents and self by using a restricted number of receptors that recognize structures shared by large groups of pathogens. Pathogen recognition by most of these receptors triggers macrophage activation. These receptors can be divided into the following three types: humoral proteins circulating in the plasma (i.e., soluble CD14), endocytic receptors expressed on the cell surface (mannose and scavenger receptors), and signaling receptors (i.e., Toll-like receptors) (4, 5). Bacterial components such as cell-surface structures (e.g., LPS and lipoproteins in Gram-negative bacteria; Refs. 1 and 6), heat shock proteins (7), and unmethylated CpG motif (8) stimulate macrophage functions. For most of these molecules, the nature of the receptor and the signaling pathways they use remain undefined. In addition to lipoproteins, Gram-negative bacteria express other outer membrane proteins (Omp)2 such as OmpA and porins (OmpC and F). The observations that the OmpC from Salmonella typhimurium mediates adherence to macrophages (9) and that peptides derived from the OmpF from Escherichia coli enhance macrophage cytotoxicity (10) suggest that these proteins may interact with macrophages.

OmpA is one of the major Omp that assembles into the outer membrane via an N-terminal eight-transmembrane amphipathic β-barrel region with the C-terminal region retained in the periplasm. Unlike other surface-exposed components of the bacterial cell envelope, OmpA is highly conserved among the Enterobacteriaceae family and throughout evolution (11). Functions attributed to OmpA include maintenance of structural cell integrity and a role in bacterial conjugation as well as bacteriophage binding. It also contributes to the ability of Gram-negative bacteria to invade mammalian cells; OmpA-deficient E. coli mutants grow normally but exhibit attenuated virulence, invasive capacity, and resistance to serum bactericidal activity (12). However, due to the difficulty of purifying this class of proteins without contamination by lipoprotein or endotoxin, their interactions and effects on cells involved in innate immunity remain unknown (1).

We analyzed here the interaction between macrophages and the OmpA from K. pneumoniae (an enterobacteria responsible for respiratory tract and urinary infections). We report that this recombinant 40-kDa OmpA (P40) (13) binds to, is internalized by, and activates macrophages.

P40 was expressed in E. coli and purified as described (13, 14) with the following final additional steps. After ethanol precipitation and solubilization in 7 M urea, P40 was submitted to size-exclusion chromatography in water, using Fractogel EMD BioSEC (Merck, Nogent sur Marne, France). Urea was removed by this final gel filtration step as shown by enzymatic urea assay (Boehringer Mannheim, Indianapolis, IN). Analytical size-exclusion chromatography of final P40 showed that the peak obtained was homogeneous without the presence of protein aggregates (Fig. 1). The two last purification steps were performed with water for injection and using depyrogenated vessels, gels, and columns; endotoxin levels determined by the Limulus assay were <0.25 EU/mg of P40. P40 batches contained <10 pg DNA/mg protein as observed by dot blot (data not shown), and were produced according to pharmaceutical quality standards intended for clinical trials.

FIGURE 1.

Analytical size-exclusion chromatography of P40. P40 was submitted to analytical size-exclusion chromatography on 1.6 × 60 cm Fractogel EMD BioSEC column. P40 is detected by UV absorption at 279 nm.

FIGURE 1.

Analytical size-exclusion chromatography of P40. P40 was submitted to analytical size-exclusion chromatography on 1.6 × 60 cm Fractogel EMD BioSEC column. P40 is detected by UV absorption at 279 nm.

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Monocytes were purified from PBMC by positive selection using a magnetic cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany). Macrophages were generated by culturing monocytes for 5 days with 106 cells/ml in culture medium consisting of complete RPMI 1640 medium supplemented with 10% FCS, 2 mM l-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 10 mM HEPES, and 0.1 mM nonessential amino acids (all from Life Technologies, Cergy Pontoise, France) in the presence of 20 ng/ml GM-CSF (R&D Systems, Abingdon, U.K.). Macrophages were stimulated with P40 in the presence or absence of 0.2 ng/ml LPS (from E. coli isotype 0111:B4; Sigma, Saint Louis, MO). In some experiments, macrophages were stimulated overnight with 10 ng/ml IFN-γ (R&D Systems) before stimulation with P40. As a positive control, cells were stimulated with 10 ng/ml LPS. In some experiments P40 was preincubated with 10 μg/ml polymixin B sulfate (Sigma). Cell lines were obtained from the American Type Culture Collection (Manassas, VA). RAW 264.7 cells cultured at 0.5 × 106/ml in complete RPMI 1640 were primed or not 6 h with 5 μg/ml murine IFN-γ (R&D Systems), and stimulated with different concentrations of P40.

P40 was labeled with Alexa488 (Molecular Probes, Eugene, OR). Flow cytometric analysis was performed using a FACSvantage cytofluorometer (Becton Dickinson, San Jose, CA). P40 (2 × 105 cells/well in a 96-V-bottom wells plate) were incubated for 20 min at 4°C in FACS buffer (RPMI 1640 medium-0.1% BSA) with Alexa488-labeled P40 or glycophorin A. In other experiments, macrophages were incubated with 0.5 μM P40, washed in FACS buffer, incubated with 5 μg/ml purified IgG1 anti-P40 mAb or IgG1 control mAb (Becton Dickinson), and revealed by FITC-labeled anti-mouse IgG Ab (Silenus, Hauworth, Australia). Results are expressed either as mean fluorescence intensity (MFI) or as a relative level of MFI values. In neutralization experiments, macrophages were preincubated for 10 min in FACS buffer with different concentrations of P40 or glycophorin A before the addition of 0.2 μM Alexa488-labeled P40. Results are expressed as a percentage of inhibition defined as follows: A-B/A × 100, where A and B are the MFI obtained in the absence or presence of the unlabeled protein, respectively. For confocal microscopy, human macrophages were incubated 20 min at 4°C in FACS buffer with 0.5 μM Alexa488-labeled P40, Alexa488-labeled glycophorin A, or with 0.5 μM Alexa488-labeled P40 plus an anti-HLADR Ab coupled to Cy3 using the Cy3 Ab labeling kit (Amersham, Arlington Heights, IL), washed in FACS buffer, incubated or not 10 min at 37°C, cytospun, and examined using a LSM510 Zeiss (Oberkochen, Germany) inverted microscope with a ×63 plan apochromat objective. In some experiments, 150 μM dimethyl amiloride (Sigma) was added in the FACS buffer. Alexa488 fluorescence was measured with a 530-nm filter after excitation with a 488-nm argon ion laser. Cy3 fluorescence was measured at 565 nm after excitation with a 543-nm HeNe laser.

IL-1β, IL-8, and biologically active IL-12 (p75) were quantified by ELISA using commercial kits (R&D Systems; sensitivity of 1, 10, and 0.5 pg/ml, respectively). TNF-α and IL-10 were quantified by ELISA using specific capture and detection Abs from R&D Systems and PharMingen (San Diego, CA), respectively (sensitivity of 1 ng/ml and 10 pg/ml, respectively). Results are expressed in pg/ml or in ng/ml. Nitrite production was determined using the Griess reaction with NaNO2 as standard (sensitivity of 2 μM).

The expression of IL-1β, IL-8, IL-10, IL-12 p35, IL-12 p40, and TNF-α mRNA was evaluated by RT-PCR. The sequences of the primers have been previously reported (15). RNA integrity and cDNA synthesis were verified by amplifying GAPDH cDNA. Amplified fragments were size separated by electrophoresis and visualized by ethidium bromide.

We analyzed the binding at 4°C of P40 to human macrophages. Results from FACS analysis show that Alexa488-labeled P40 binds to macrophages (Fig. 2,A). No binding of Alexa488-labeled glycophorin A, a transmembrane protein used as a negative control, is observed (Fig. 2,A). Similar results are obtained using unlabeled P40 revealed by a specific mAb (Fig. 2,B). The binding of Alexa488-labeled P40 is dose-dependent, significant at 50 nM P40 and saturable at 0.5 μM P40 (the highest concentration tested; Fig. 2,C), and partly competed by unlabeled P40 but not by a control protein (Fig. 2,D). Alexa488-labeled P40 also binds to thioglycolate-elicited murine macrophages, to the RAW 264.7 cell line, and to the U937 human monocytic cell line (Fig. 2,E). In contrast, no binding is detectable on the murine P815 and human HL60 myeloid cell lines (Fig. 2 E). The partial saturability of P40 binding to macrophages and the absence of binding to some myeloid cells suggest the existence of P40-binding element(s) on macrophages. Similar observations were made using the OmpA purified from E. coli (data not shown), thereby suggesting that macrophages may recognize this class of protein. The conservation of OmpA throughout evolution is in agreement with this hypothesis (5).

FIGURE 2.

P40 binds to macrophages. A, Human monocyte-derived macrophages were incubated (white histograms) or not (gray histograms) for 20 min at 4°C with 0.5 μM Alexa488-labeled P40 or glycophorin A, washed, and analyzed by FACS. B, Macrophages were incubated (right histogram) or not (left histograms) for 20 min at 4°C with 0.5 μM Alexa488-labeled P40, washed, and incubated (white histograms) or not (gray histogram) with an IgG1 anti-P40 mAb revealed by FITC-labeled anti-mouse Ig analyzed by FACS. A and B, Results are representative of one of ten experiments. C, Human macrophages were incubated with different concentrations of Alexa488-labeled P40 (□) or glycophorin A (•) and analyzed by FACS; results are expressed in MFI values (mean ± SD; n = 3). D, Human macrophages were incubated with different concentrations of P40 or glycophorin A for 10 min at 4°C before addition of 0.2 μM Alexa488-labeled P40. Results are expressed as a percentage of inhibition as described in Materials and Methods, and are representative of one of three experiments. E, Human monocytes, human macrophages, murine thioglycolate-elicited macrophages, and the human HL-60, U937, and THP-1, and murine RAW 264.7 and P815 myeloid cell lines were incubated with 0.5 μM Alexa488-labeled P40 and analyzed by FACS. Results are presented as a relative level of MFI values.

FIGURE 2.

P40 binds to macrophages. A, Human monocyte-derived macrophages were incubated (white histograms) or not (gray histograms) for 20 min at 4°C with 0.5 μM Alexa488-labeled P40 or glycophorin A, washed, and analyzed by FACS. B, Macrophages were incubated (right histogram) or not (left histograms) for 20 min at 4°C with 0.5 μM Alexa488-labeled P40, washed, and incubated (white histograms) or not (gray histogram) with an IgG1 anti-P40 mAb revealed by FITC-labeled anti-mouse Ig analyzed by FACS. A and B, Results are representative of one of ten experiments. C, Human macrophages were incubated with different concentrations of Alexa488-labeled P40 (□) or glycophorin A (•) and analyzed by FACS; results are expressed in MFI values (mean ± SD; n = 3). D, Human macrophages were incubated with different concentrations of P40 or glycophorin A for 10 min at 4°C before addition of 0.2 μM Alexa488-labeled P40. Results are expressed as a percentage of inhibition as described in Materials and Methods, and are representative of one of three experiments. E, Human monocytes, human macrophages, murine thioglycolate-elicited macrophages, and the human HL-60, U937, and THP-1, and murine RAW 264.7 and P815 myeloid cell lines were incubated with 0.5 μM Alexa488-labeled P40 and analyzed by FACS. Results are presented as a relative level of MFI values.

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We then analyzed by confocal microscopy whether P40 binding to macrophages is followed by its internalization. After incubation at 4°C, fluorescence is located on the surface of the macrophages (Fig. 3,A). A further incubation at 37°C leads to the appearance of fluorescent endosomes the sizes of which vary from 0.8 to 2 μm in the cytosol as early as 5 min after incubation (Fig. 3,B). Dimethyl amiloride, an inhibitor of macropinocytosis (16), prevents the formation of these P40-containing endosomes (Fig. 3,C), thereby suggesting a macropinocytic uptake. In agreement with previous data showing that P40 is a potent carrier protein (14, 17), we also report that P40 colocalizes intracellularly with MHC class II molecules (Fig. 3,D). In contrast, no binding or internalization of Alexa488-labeled glycophorin A is observed (Fig. 3, E and F). These data show that P40 binds to and is internalized by macrophages.

FIGURE 3.

P40 is endocytosed by macrophages. Human macrophages were incubated for 20 min at 4°C with 0.5 μM Alexa488-labeled P40, washed, and analyzed by confocal microscopy before (A) or after a further incubation for 10 min at 37°C in the absence (B and D) or presence (C) of dimethyl amiloride. D, Macrophages were incubated for 20 min at 4°C with 0.5 μM Alexa488-labeled P40 and Cy3-labeled anti-MHC class II mAb, washed, and incubated for 10 min at 37°C before analysis. E and F, Macrophages were incubated for 20 min at 4°C with 0.5 μM Alexa488-labeled glycophorin A, washed, and analyzed by confocal microscopy before (E) or after (F) a further incubation for 10 min at 37°C.

FIGURE 3.

P40 is endocytosed by macrophages. Human macrophages were incubated for 20 min at 4°C with 0.5 μM Alexa488-labeled P40, washed, and analyzed by confocal microscopy before (A) or after a further incubation for 10 min at 37°C in the absence (B and D) or presence (C) of dimethyl amiloride. D, Macrophages were incubated for 20 min at 4°C with 0.5 μM Alexa488-labeled P40 and Cy3-labeled anti-MHC class II mAb, washed, and incubated for 10 min at 37°C before analysis. E and F, Macrophages were incubated for 20 min at 4°C with 0.5 μM Alexa488-labeled glycophorin A, washed, and analyzed by confocal microscopy before (E) or after (F) a further incubation for 10 min at 37°C.

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Macrophages phagocyte opsonized pathogens through Fc and complement receptors (1). Because our experiments were performed in a medium free of Ig and complement, and macrophages from SCID mice bind P40 (data not shown), we have excluded a binding of P40 through these receptors. To recognize pathogens, macrophages have evolved a restricted number of phagocytic receptors like mannose receptors or scavenger receptors (1, 5). Using a neutralizing anti-mannan mAb (3.29 IgG1 mAb; generous gift from M. Cella, Basel Institute for Immunology, Basel, Switzerland), we failed to prevent P40 binding. Currently, the identification of OmpA-binding elements is under investigation.

We then evaluated whether P40 recognition is coupled to macrophage activation. TNF-α is a major activator of macrophages that also mediates lung antibacterial host defense in murine K. pneumoniae (18, 19). We show that P40 induces TNF-α production by macrophages in a dose-dependent manner; P40-induced TNF-α production is significant at 0.04 μM P40, maximal at 1 μM (Figs. 4,A and 5), and detectable as early as 3 h after stimulation (data not shown). Macrophages contain a pool of TNF-α mRNA (18), which expression is enhanced by P40, thereby suggesting that P40 acts at the transcriptional level (Fig. 4,B). Polymixin B inhibits LPS- but not P40-induced TNF-α (Fig. 4 A). Moreover, compared with macrophages from C3H/HeN mice, macrophages from LPS-resistant C3H/HeJ mice (carrying a mutant of Toll-like receptor 4) produce comparable levels of murine TNF-α in response to P40 but lower levels in response to LPS (Ref. 20 ; data not shown). Both of these observations show that the effect of P40 is not mediated by contaminating endotoxin, and that P40 does not act through Toll-like receptor 4. Moreover, the observation that P40 binds to but does not induce cytokine production by the U937 cell line (data not shown) suggests that the molecules involved in P40 binding and signaling may differ (21)

FIGURE 4.

P40 induces cytokine production by human macrophages. A, Human macrophages were incubated without or with 0.2 or 1 μM P40 or with 10 ng/ml LPS in the absence or presence (∗) of polymixin B. The cytokines were quantified in the cell-free supernatants by ELISA. IL-1β, IL-8, and TNF-α were quantified after 24-h incubation, IL-12 after 48 h, and IL-10 after 72 h. Results (mean ± SD of four separate experiments) are expressed in ng/ml, except IL-12 in pg/ml; <, Undetectable. B, Macrophages were incubated without or with 1 μM P40 or 10 ng/ml LPS. After 12 h, total RNA was isolated and the mRNA encoding for IL-1β, IL-8, IL-10, IL-12, TNF-α, and GAPDH was analyzed by RT-PCR.

FIGURE 4.

P40 induces cytokine production by human macrophages. A, Human macrophages were incubated without or with 0.2 or 1 μM P40 or with 10 ng/ml LPS in the absence or presence (∗) of polymixin B. The cytokines were quantified in the cell-free supernatants by ELISA. IL-1β, IL-8, and TNF-α were quantified after 24-h incubation, IL-12 after 48 h, and IL-10 after 72 h. Results (mean ± SD of four separate experiments) are expressed in ng/ml, except IL-12 in pg/ml; <, Undetectable. B, Macrophages were incubated without or with 1 μM P40 or 10 ng/ml LPS. After 12 h, total RNA was isolated and the mRNA encoding for IL-1β, IL-8, IL-10, IL-12, TNF-α, and GAPDH was analyzed by RT-PCR.

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When stimulated, macrophages produce other mediators such as IL-1β, IL-8, IL-12, NO, and IL-10 that are involved in the antibacterial response. IL-1 is an inflammatory cytokine that stimulates numerous cell types (22). IL-8 controls neutrophil and lymphocyte infiltration and protects mice challenged with K. pneumoniae intraperitoneally (2, 23). IL-12 induces IFN-γ production and favors cytotoxic responses (24). NO up-regulates macrophage phagocytosis and killing, especially in K. pneumoniae infection (25). IL-10, produced at a later stage, down-regulates the inflammatory response (3). We show that P40 up-regulates the production of human IL-1β and IL-8 in a manner that is dose-dependent and significant at 0.04 μM, induces low but detectable levels of bioactive IL-12 (<5 pg/ml at 1 μM), and induces the production of IL-10 that is detectable 3 days after stimulation (Figs. 4,A and 5). P40 enhances the expression of the mRNA encoding for all these cytokines (Fig. 4,B). Finally, P40 also induces NO production by the RAW macrophage cell line (Fig. 5). As expected, LPS used as a positive control induces the expression of all these mediators (Fig. 4 A). These data show that P40 triggers a signal leading to macrophage activation.

FIGURE 5.

P40 synergizes with IFN-γ and/or LPS to up-regulate NO and cytokine production by macrophages. Human monocyte-derived macrophages were (□) or were not (▪, ▦) primed for 6 h with IFN-γ and were incubated or not with different concentrations of P40 in the absence (▪, □) or presence (▦) of 0.2 ng/ml LPS. IL-1β, IL-8, and TNF-α were quantified by ELISA in the supernatants after 24 h, IL-12 after 48 h, and IL-10 after 72 h. Results (mean ± SD of three separate experiments) are expressed in ng/ml, except IL-12 in pg/ml. The murine RAW 264.7 cell line was (□) or was not primed for 6 h with IFN-γ (▪, ▦) and was incubated or not with different concentrations of P40 in the absence (▪, □) or presence (▦) of 0.2 ng/ml LPS. NO was measured in the 24-h supernatants. Results are expressed in μM as mean ± SD of three separate experiments.

FIGURE 5.

P40 synergizes with IFN-γ and/or LPS to up-regulate NO and cytokine production by macrophages. Human monocyte-derived macrophages were (□) or were not (▪, ▦) primed for 6 h with IFN-γ and were incubated or not with different concentrations of P40 in the absence (▪, □) or presence (▦) of 0.2 ng/ml LPS. IL-1β, IL-8, and TNF-α were quantified by ELISA in the supernatants after 24 h, IL-12 after 48 h, and IL-10 after 72 h. Results (mean ± SD of three separate experiments) are expressed in ng/ml, except IL-12 in pg/ml. The murine RAW 264.7 cell line was (□) or was not primed for 6 h with IFN-γ (▪, ▦) and was incubated or not with different concentrations of P40 in the absence (▪, □) or presence (▦) of 0.2 ng/ml LPS. NO was measured in the 24-h supernatants. Results are expressed in μM as mean ± SD of three separate experiments.

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Because Gram-negative bacteria express LPS, we analyzed the combined effect of P40 and LPS on macrophage activation. P40 synergizes with LPS to induce TNF-α production with a maximal effect seen at a concentration of 0.2 ng/ml LPS (Fig. 5). In the presence of 0.2 ng/ml LPS, P40 up-regulates the production of IL-1β, IL-8, IL-10, and NO (with a significant effect at 8 nM P40 for IL-1β, IL-8, and TNF-α and at 40 nM P40 for IL-10 and IL-12) (Fig. 5).

IFN-γ produced locally by T cells also exerts antibacterial immune effects. Although it induces a limited production of cytokines by itself, it primes macrophages to produce cytokines in response to a second stimuli (26). When added to IFN-γ-primed macrophages, the effect of P40 on TNF-α, IL-1β, IL-8, IL-10, and IL-12 is highly enhanced (a significant effect of P40 on IL-1β, IL-8, IL-10, and TNF-α being observed with 8 nM P40 and on IL-12 with 40 nM P40) (Fig. 5). Taken together, these data show that P40 synergizes with LPS to induce IL-12 and TNF-α (two cytokines that induce IFN-γ production; Refs. 18 and 24) and with IFN-γ to induce TNF-α and IL-12 production, and thereby suggest a paracrine positive-feedback cycle with local T cells.

We show that P40 binds to macrophages, is endocytosed, and triggers macrophage activation. These data, in accordance with the crucial role played by macrophages in the elimination of K. pneumoniae in the lung (27), suggest that upon contact with an enterobacteria, OmpA may participate in the activation of resident macrophages. The observation that LPS and IFN-γ potentiate the production of cytokines induced by P40 suggests that a limited number of Gram-negative bacteria could be sufficient to efficiently stimulate macrophages. Furthermore, as macrophages process and present Ag to T cells, these properties of P40 may contribute to explain why OmpA is highly immunogenic in the absence of adjuvant (14, 17).

Because OmpA is highly represented in bacterial cell walls and conserved among the Enterobacteriaceae, one could speculate that the immune system has acquired the ability to recognize and to be activated by this class of protein. The identification of the phagocytic cell surface receptor(s) and of the molecular pathway involved in P40-induced macrophage activation is currently under investigation. Finally, OmpA appears as a new class of molecules expressed by bacteria that is recognized by and activates macrophages.

2

Abbreviations used in this paper: Omp, outer membrane protein; MFI, mean fluorescence intensity.

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