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Infection and Immunity, May 2005, p. 2736-2743, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.2736-2743.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Helicobacter pylori Binds to CD74 on Gastric Epithelial Cells and Stimulates Interleukin-8 Production

Ellen J. Beswick,1 David A. Bland,2 Giovanni Suarez,1 Carlos A. Barrera,3 Xuejung Fan,1 and Victor E. Reyes1,2*

Departments of Pediatrics,1 Pathology,3 Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 775552

Received 13 October 2004/ Returned for modification 29 November 2004/ Accepted 21 December 2004


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ABSTRACT
 
The pathogenesis associated with Helicobacter pylori infection requires consistent contact with the gastric epithelium. Although several cell surface receptors have been suggested to play a role in adhesion, the bacterium-host interactions that elicit host responses are not well defined. This study investigated the interaction of H. pylori with the class II major histocompatibility complex (MHC)-associated invariant chain (Ii; CD74), which was found to be highly expressed by gastric epithelial cells. Bacterial binding was increased when CD74 surface expression was increased by gamma interferon (IFN-{gamma}) treatment or by fibroblast cells transfected with CD74, while binding was decreased by CD74 blocking antibodies, enzyme cleavage of CD74, and CD74-coated bacteria. H. pylori was also shown to bind directly to affinity-purified CD74 in the absence of class II MHC. Cross-linking of CD74 and the engagement of CD74 were verified to stimulate IL-8 production by unrelated cell lines expressing CD74 in the absence of class II MHC. Increased CD74 expression by cells increased IL-8 production in response to H. pylori, and agents that block CD74 decreased these responses. The binding of H. pylori to CD74 presents a novel insight into an initial interaction of H. pylori with the gastric epithelium that leads to upregulation of inflammatory responses.


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INTRODUCTION
 
Helicobacter pylori is a gram-negative, spiral-shaped bacterium that colonizes the gastric mucosa. Infection with this organism is a major cause of chronic gastritis, gastric and duodenal ulcers, and gastric carcinomas throughout the world (9, 21). The evidence linking this pathogen to gastric cancer led to H. pylori becoming classified as a class 1 carcinogen by the World Health Organization. Although infection of gastric epithelium with H. pylori elicits marked inflammatory responses and epithelial cell apoptosis (14, 22, 39), the events that promote these responses are not clearly defined. H. pylori maintains close association with gastric epithelial cells during infection (16, 36). Clearly bacterial adhesion and colonization of the gastric mucosa are key events in pathogenesis (38).

H. pylori interactions with gastric epithelial cells result in increased expression of class II major histocompatibility complex (MHC) (11), release of proinflammatory cytokines (18, 44), and increased apoptosis of the epithelium (8, 43). Although various epithelial receptors for H. pylori have been suggested (15, 33), it is not known how they are involved in the initiation of epithelial cell responses leading to inflammation and tissue damage (30). The prolonged inflammatory response during infection and the associated tissue damage, together with the complicated treatment, necessitate a further understanding of the early events in adhesion of the bacterium to gastric epithelial cells and how these events influence the ensuing host responses.

The expression of class II MHC by gastric epithelial cells together with the presence of activated CD4+ T cells adjacent to the epithelium suggests that gastric epithelial cells are more than a target of the infection and may act as antigen-presenting cells. A Th1 response to H. pylori has been well documented (24, 26), with production of gamma interferon (IFN-{gamma}) as an important early response by T cells. IFN-{gamma} leads to a marked increase in the expression of class II MHC on gastric epithelial cells (11). In addition to their traditional role of presenting antigen, class II MHC molecules, after their engagement by T cells or superantigens, may initiate signaling processes within the cells that express them, resulting in increased cytokine release and apoptosis. We have previously shown that H. pylori urease adheres to class II MHC molecules, leading to signaling that results in apoptosis of epithelial cells during infection (12). Other molecules reported to be used in attachment by H. pylori have not been shown to elicit signaling, but adherence of the bacterium is required for some key responses, such as interleukin-8 (IL-8) production (6, 35), and this response has been shown to be dependent upon NF-{kappa}B activation (20, 29).

CD74, or the invariant chain (Ii), is closely associated with class II MHC molecules and is a determining factor in the development of immune responses. The intracellular transport and functions of class II MHC are regulated by CD74 (3, 10, 34). For instance, CD74 blocks the peptide-binding groove of class II MHC molecules to prevent premature binding of antigenic peptides or binding of self-peptides. Reports have shown that CD74 is expressed by a variety of cell types (31) and is suggested to play a role in signaling by activating through the long cytoplasmic tail (23, 27). Since we found CD74 to be expressed on gastric epithelial cell surfaces and since it is closely associated with class II MHC, we sought to determine its potential role in H. pylori interactions. H. pylori binding to CD74 was determined by studies blocking CD74 with specific antibodies, which resulted in reduced attachment, in addition to using H. pylori as "bait" to capture CD74 from a pool of gastric epithelial cell lysates. Further, we determined that this interaction has consequences for the epithelium, since H. pylori binding to CD74 results in IL-8 secretion, which is a well-documented response of H. pylori binding to gastric epithelium that is associated with pathogenesis (6, 35). Interestingly, this IL-8 response was found to be independent of the cag pathogenicity island (PAI), since cag PAI-deficient H. pylori stimulated IL-8 production.


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MATERIALS AND METHODS
 
Cell lines. N87 and Kato III human gastric carcinoma epithelial cells and Hs738.st/int human fetal gastric epithelial cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). P3HR1 B cells expressing CD74, but not class II MHC (40), were obtained from Robert E. Humphreys (Antigen Express, Worcester, MA). Cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics. M1 human fibroblast cells transfected with RSV.2 vector or RSV.2-p33 (M1-p33) (25) were obtained from Eric Long (National Institutes of Health, Rockville, MD) and cultured in Dulbecco's modified Eagle's medium (DMEM) selective medium with 10% fetal bovine serum, 2 mM L-glutamine, and 0.5 mg/ml Geneticin.

Bacterial cultures. H. pylori clinical isolate LC11 (1), 26695 lab strain (ATCC), 26695 cag pathogenicity island-deficient mutant (a kind gift from Yoshio Yamaoka at Baylor School of Medicine, Houston, TX), and Campylobacter jejuni 33291 (ATCC) were grown on blood agar plates (Becton Dickinson, San Jose, CA) at 37°C under microaerophilic conditions as previously described (11). Bacteria were transferred after 48 h into brucella broth containing 10% fetal bovine serum for 24 h. After centrifugation at 2,500 x g for 10 min, bacteria were resuspended in sterile phosphate-buffered saline (PBS). The concentration of bacteria per ml was determined by measuring the absorbance (at 530 nm) using a spectrophotometer (DU-65, Becton Dickinson Instruments, Inc., Fullerton, CA) and comparing the value to a standard curve generated by quantifying viable organisms from aliquots of bacteria at various concentrations that were also assessed by absorbance.

CD74 surface expression. Gastric epithelial cell lines were pretreated with IFN-{gamma} (100 U/ml) for 48 h to maximize CD74 expression. IFN-{gamma}-treated and untreated cells were incubated with anti-CD74 monoclonal immunoglobulin G1 (IgG1) clone BU-45 (Serotec, Raleigh, NC) or the isotype control. Fibroblast cells and P3HR1 cells were also stained with the anti-class II MHC antibody IVA-12 purified from culture supernatants of the HB145 hybridoma (ATCC) to verify lack of class II MHC. Cells were then washed with PBS and stained with secondary phycoerythrin-conjugated antibody (DAKO, Carpentaria, CA). Samples were analyzed on the FACScan Flow Cytometer (Becton Dickinson) using CellQuest software.

H. pylori-CD74 binding assays. Gastric epithelial cells were treated with 100 U/ml IFN-{gamma} for 48 h, and 2 x 106 cells were seeded into each well of a 24-well plate. Medium was replaced with IFN-{gamma}-free medium for 24 h. Cells were removed with trypsin and pretreated with either isotype control antibodies or 1 µg of the anti-CD74 BU-45 for 1 h. Some bacteria were incubated with 10 µg of immunoprecipitated CD74 for 1 h. H. pylori cells were stained with the red fluorochrome PKH26 (Sigma) according to the manufacturer's instructions, washed multiple times with RPMI, and resuspended in RPMI. Bacteria were washed and added to pelleted untreated cells; IFN-{gamma}-treated cells were also treated with antibodies, as indicated, in a ratio of 30:1 bacteria to cells, and incubated for 90 min at 37°C. Cells were washed and resuspended in 2% paraformaldehyde and analyzed by flow cytometry. Negative control samples consisted of cells treated with the wash from stained H. pylori to assess background staining of cells. Some cells were treated with 2.5 µg each of a cocktail of cathepsin L (Sigma) and cathepsin S (Athens Research and Technology, Athens, GA) for 6 h at 37°C in 400 mM sodium acetate buffer containing 4 mM EDTA and 8 mM dithiothreitol, pH 5.5, in order to remove surface-expressed CD74. Cells were then stained with anti-CD74 antibodies to confirm CD74 removal, while other samples were exposed to PKH26-labeled H. pylori for binding studies.

H. pylori binding of immunoprecipitated CD74. P3HR-1 cells (2 x 107) were washed and incubated in methionine-free RPMI for 2 h at 37°C. Cells were pelleted, resuspended, and incubated with [35S]methionine (0.5 mCi/107 cells) for six hours. Cells were then washed three times with serum-free RPMI; lysed with buffer containing Tris-HCl, NaCl, NP-40, Na-deoxycholate, and protease inhibitor cocktail (Sigma); and centrifuged at 10,000 x g for 10 min to remove nuclei. CD74 was immunoprecipitated using protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA) that were preincubated with BU-45 monoclonal antibody for two hours at room temperature. After washing, beads were incubated with P3HR-1 lysates for 2 h at 4°C. Beads were then washed four times, and the bound CD74 was eluted with 0.1 M citrate buffer (pH 2.7) and neutralized immediately. The resulting CD74 was incubated with H. pylori or C. jejuni for 45 min at 20°C. The bound material was eluted, and samples were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and exposed for autoradiography.

IL-8 induction and detection. Gastric epithelial cells or fibroblast transfectants were incubated for 24 h with H. pylori (30:1 bacterium/cell ratio). M1 and M1-p33 were incubated with serum-free medium for 24 h before bacterial exposure because of high basal IL-8 production. Some samples were incubated with 1 µg anti-CD74 antibodies before exposure to H. pylori. Cross-linking studies were done with anti-CD74 antibodies that were biotinylated using the Fluoreporter Biotinylation kit (Molecular Probes, Eugene, Oreg.) according to the manufacturer's instructions. Streptavidin was added for cross-linking purposes. Negative controls consisted of primary biotinylated anti-CD74 antibodies with no streptavidin to cross-link or streptavidin alone. Supernatants were harvested at 24 h and analyzed using IL-8 enzyme-linked immunosorbent assays (ELISAs) (Becton Dickinson) according to the manufacturer's instructions to compare levels of IL-8 secretion from IFN-{gamma}-treated and untreated cells exposed to either H. pylori, CD74 cross-linking, or blocking anti-CD74 antibodies.

I{kappa}B degradation. Cells grown in 24-well plates (2 x 105) were incubated overnight with serum-free medium and stimulated with CD74 cross-linking antibodies or H. pylori for specified times. Cells were removed from plates by scraping and lysed as described by similar studies (29). Lysates were centrifuged at 14,000 x g for 10 min at 4°C. Ten µg of the resulting protein from each sample was loaded into wells of 10% SDS gels for electrophoresis and transferred to nitrocellulose membranes for Western blotting. The membranes were treated with anti-I{kappa}B{alpha} antibody (Santa Cruz Biotechnology) recognizing a 37-kDa protein (1:5,000), and antitubulin antibody (Sigma) recognizing a 50-kDa protein (1:3,000) as a loading control for protein concentration for two hours at room temperature. After washing four times for 15 min with Tris-buffered saline-Tween, donkey anti-mouse horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnologies) was used, diluted 1:10,000, and incubated with the membrane for one hour at room temperature. The membrane was again washed four times for 15 min. Immunoreactive proteins were detected using Super Signal West Pico Chemiluminescent Substrate (Pierce Biotechnologies, Rockford, IL).

Statistical analysis. Results are expressed as the mean ± standard error of the mean. H. pylori binding, and IL-8 production results were compared using a two-tailed Student's t test and considered significant if P is <0.05.


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RESULTS
 
CD74 expression on the surface of gastric epithelial cells correlates with H. pylori binding. Three gastric epithelial cell lines that express basal levels of CD74 (Fig. 1A) were examined for CD74 expression before and after treatment with IFN-{gamma}, which is produced by T cells in the gastric mucosa during H. pylori infection (1, 19). Cells were stained with anti-CD74 monoclonal antibodies and analyzed by flow cytometry. All three cell lines were found to have some CD74 surface expression that was increased by approximately 40% in N87 and HS-738 cells and 60% in Kato III cells as a result of the IFN-{gamma} treatment. Additionally, M1 fibroblast cells transfected with CD74 in an RSV.2 vector (RSV.2-p33) or with the vector alone (25) were also analyzed for CD74 expression. The RSV.2-p33-transfected cells were found to express CD74 on the surface, while the cells with the vector control did not (Fig. 1B). Fibroblast transfectants were also stained with anti-class II MHC antibodies to verify the lack of class II MHC expression by these cell lines.



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  		FIG. 1. CD74 expression on the surface of gastric epithelial cells. (A) Flow cytometric analysis of the gastric epithelial cells lines N87, HS-738, and Kato III untreated and IFN-{gamma} treated for 48 h. Cells were stained with the BU-45 anti-CD74 antibody followed by phycoerythrin-conjugated anti-mouse IgG. The percent positive cells above background staining with isotype control antibody is shown. (B) Cells of the M1 fibroblast line transfected with the vector alone (RSV.2) or with the vector carrying the p33 form of CD74 (RSV.2-p33) were similarly stained. The means are shown as the results of duplicates in four experiments (n = 8).

The ability of gastric epithelial cells and fibroblast transfectants to bind PHK26-labeled H. pylori was examined. Flow cytometric analysis of IFN-{gamma}-treated cells revealed increased binding of H. pylori compared to untreated cells (Fig. 2A). Minimal binding of H. pylori to M1 fibroblasts was observed unless CD74 was present (Fig. 2B). The mean fluorescence intensities of each cell line for CD74 expression and H. pylori attachment were compared, and a correlation was found: as CD74 expression increased, H. pylori attachment increased proportionally (Fig. 2C).



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  		FIG. 2. The level of CD74 expression by gastric epithelial cells correlates with H. pylori binding. PKH26-labeled H. pylori cells were incubated with the indicated cells for 90 min, and unbound bacteria were removed by washing. Binding was determined by flow cytometry. IFN-{gamma}-treated N87 gastric epithelial cells (A) exhibit increased H. pylori binding compared to untreated cells, and CD74 p33 fibroblast transfectants (B) show increased binding of M1-p33-transfected cells over vector (RSV.2) transfectants (M1), as shown in representative histograms. (C) Correlation of increasing mean fluorescence intensities of CD74 expression and H. pylori (Hp) binding in all cell lines.

CD74 plays a role in H. pylori attachment to gastric epithelial cells. Since CD74 is closely associated with class II MHC, which was shown to bind H. pylori (11), the role of CD74 in H. pylori binding to gastric epithelial cells was considered. In order to investigate the role of CD74 in attachment, cathepsins L and S, endosomal proteases that proteolytically cleave CD74 from class II MHC to allow for class II MHC to bind foreign peptides (17) were used to cleave CD74 from the surface of gastric epithelial cells. Attachment of PKH26-stained H. pylori was measured after enzymatic treatment. As shown in Fig. 3A, cathepsin treatment dramatically decreased surface CD74 of N87 cells, as it did with other gastric epithelial cells, and led to a substantial decrease in bacterial binding (Fig. 3B).



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  		FIG. 3. Removal or blocking of CD74 on the surface of gastric epithelial and control cells reduces H. pylori binding. (A) Cathepsin L and S digestion of CD74 on N87 cell surfaces leads to a decrease in CD74 being detected by flow cytometry using an anti-CD74 antibody (BU-45). (B) This treatment also reduces the attachment of H. pylori, as shown in a representative histogram of N87 cells. (C) H. pylori (Hp) LC11 attachment was significantly decreased by blocking CD74 with monoclonal antibodies by all cells expressing CD74. Results are expressed as the percentage of cells staining positive for bacterial attachment. The means of duplicates from four experiments (n = 8) are shown here.

To further determine the role of CD74 in H. pylori adherence to gastric epithelial cells, PKH26-labeled H. pylori LC11 and 26695 were incubated with gastric epithelial cells or transfected fibroblast cells in the presence of anti-CD74 blocking antibodies. M1-p33 cells that were shown to express high levels of CD74, but not class II MHC, were utilized to study binding in the absence of class II MHC. IFN-{gamma}-treated cells or fibroblast cells were incubated with anti-CD74 monoclonal antibodies, and PKH26-labeled H. pylori binding to the cells was analyzed by flow cytometry. Anti-CD74 antibodies led to approximately 40% decreased cells positive for H. pylori LC11 binding compared to cells incubated with isotype control antibody (Fig. 3C). Binding to CD74+ M1-p33 fibroblast transfectants was decreased by 80% in the presence of anti-CD74 antibodies, and minimal attachment to M1 fibroblasts was observed in the absence of CD74 in the previous experiment. These results were further confirmed using H. pylori strain 26695, which led to proportional results, but with approximately 15% less binding (not shown). Another approach to examine the role of CD74 on the binding of H. pylori to the gastric epithelium was to use affinity-purified CD74 in order to block CD74 binding proteins on the bacterial surface prior to incubation of the bacteria with the gastric epithelial cells. The coating of H. pylori with CD74 caused a decrease in bacterial attachment to gastric epithelial cells by approximately 50% compared to controls and 90% for M1-p33 fibroblast transfectants. These results indicate an important role for CD74 in the binding of H. pylori to gastric epithelial cells.

H. pylori binds CD74 in the absence of class II MHC. In order to confirm the interaction between H. pylori and CD74, binding of H. pylori to CD74 was determined by incubating bacteria with metabolically labeled, affinity-purified CD74 from P3HR1 cells to ensure that class II MHC was absent. After incubation for 45 min, unbound material was removed by washing with PBS. H. pylori-associated CD74 was eluted and run on SDS-PAGE. After autoradiography, binding of CD74 to H. pylori was determined, but binding did not occur to a related bacterial control, C. jejuni (Fig. 4). Affinity-purified CD74 was run as a control to reveal its size, showing the 33-kDa and 41-kDa isoforms of CD74 (2, 37). The most abundant isoform of CD74, p33, was shown to bind to H. pylori, with a faint band for the p41 isoform. The direct binding of CD74 from a cell line deficient in class II MHC to H. pylori further demonstrates a role for CD74 in H. pylori binding to gastric epithelial cells.



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  		FIG. 4. H. pylori precipitates soluble CD74. Radiolabeled affinity-purified CD74 from P3HR1 cells that express CD74, but not class II MHC bound to H. pylori (Hp) but not C. jejuni (Cj), a related bacterial control. The most common isoform of CD74, 33 kDa, is shown by a strong band, while the 41-kDa isoform is present as a lighter band. Affinity-purified CD74 was run as a control to verify molecular masses. MWS, molecular mass (kDa) standards.

NF-{kappa}B is activated by cross-linking CD74 and H. pylori exposure. Since other studies have shown that CD74 has the ability to signal due to its long cytoplasmic tail (23, 27), it was important to determine whether binding of H. pylori to CD74 on the surface of gastric epithelial cells was implicated in signaling within these cells. In order to verify the ability of CD74 to initiate signaling events, gastric epithelial cells were exposed to CD74 cross-linking antibodies for 15-min time intervals and I{kappa}B-{alpha} degradation was assessed in order to confirm NF-{kappa}B activation. Tubulin-{alpha} was used as a loading control to ensure equal amounts of protein were loaded into each lane. As shown in Fig. 5A, cross-linking CD74 on HS-738 cells led to I{kappa}B-{alpha} degradation at 45 min and began to recover at 75 min. Similar results were seen with N87 and Kato III cells (not shown). The ability of H. pylori to activate NF-{kappa}B was also investigated by exposing cells to bacteria for 15-min time periods. Similar to CD74 cross-linking, H. pylori binding led to I{kappa}B-{alpha} degradation at 45 min and recovery began at 75 min (Fig. 5B). The role of CD74 in NF-{kappa}B activation by H. pylori was then examined by exposing cells to CD74 blocking antibodies prior to exposure to H. pylori. The results indicate that I{kappa}B-{alpha} degradation was decreased with blocking antibodies compared to unblocked cells with H. pylori (Fig. 5C), indicating a role for CD74 in H. pylori-induced NF-{kappa}B activation within gastric epithelial cells.



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  		FIG. 5. Engagement of CD74 on gastric epithelial cells causes I{kappa}B degradation. Cross-linking CD74 with biotinylated monoclonal antibodies to CD74 and streptavidin resulted in (A) I{kappa}B-{alpha} degradation at 45 min, with HS-738 shown in a representative figure with a tubulin-{alpha} control. Exposure to H. pylori led to (B) I{kappa}B-{alpha} degradation at 45 min, with HS-738 shown in a representative figure with a tubulin-{alpha} control. Blocking CD74 with monoclonal antibodies decreased (C) I{kappa}B-{alpha} degradation compared to unblocked cells above, as shown in a representative figure with HS-738 cells with a tubulin-{alpha} control.

CD74 plays an important role in the IL-8 response to H. pylori. IL-8 is one of the first inflammatory mediators reported to be produced by the epithelium in response to H. pylori (4, 7). Because we have shown that CD74 plays a role in signaling, leading to I{kappa}B-{alpha} degradation, we further examined IL-8 production after engagement of CD74. N87, HS-738, Kato III, and p33-transfected fibroblast (M1-p33) cells were also exposed to CD74 cross-linking antibodies to assess IL-8 responses. IL-8 ELISAs were used to quantify the amount of IL-8 produced by the various cells. As shown in Fig. 6A and B, HS-738 and Kato III gastric epithelial cells treated with anti-CD74 antibodies under cross-linking conditions released 20 times more IL-8 than untreated cells, while treated N87 cells had a lower basal level of IL-8 that increased 12-fold upon cross-linking CD74. P33 transfectants had an increase in IL-8 production of about 10-fold upon cross-linking CD74, demonstrating that CD74 engagement results in the stimulation of IL-8 release.



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  		FIG. 6. Engagement of CD74 on multiple cell types causes IL-8 production. Cross-linking CD74 with biotinylated monoclonal antibodies to CD74 and streptavidin resulted in (A and B) IL-8 production by all cells expressing CD74 after 24 h in culture. Results are expressed as the ratio of IL-8 produced by cells with cross-linked CD74 compared to untreated cells. (C) IL-8 production by gastric epithelial cells is increased by exposure to H. pylori. IFN-{gamma} treatment of cells to upregulate CD74 drastically increased IL-8 production. (D) IL-8 production by M1 cell transfectants was minimal with H. pylori exposure, while M1 transfectants with CD74 p33 drastically increased IL-8 production upon exposure to H. pylori. (E and F) IL-8 production in response to H. pylori was significantly decreased by blocking CD74 with monoclonal antibodies by all cells expressing CD74. The means of duplicates from four experiments (n = 8) are shown here.

The contribution of CD74 in the IL-8 response of gastric epithelial to H. pylori was investigated by exposing cells to H. pylori for 24 h. Since CD74 expression increases with IFN-{gamma}, the IL-8 response due to H. pylori binding was examined with IFN-{gamma}-treated and untreated cells exposed to H. pylori for 24 h. IL-8 produced by gastric epithelial cells was increased 10 times upon H. pylori exposure of both treated and untreated cells, and the levels of IL-8 produced by IFN-{gamma}-pretreated cells were 2.5 times higher than those of untreated cells (Fig. 6C), suggesting that IL-8 production is higher when there is a higher expression of CD74. HS-738 and Kato III are shown here as representative cell lines, but studies with N87 cells led to similar results (not shown). IL-8 production was also examined with fibroblast transfectants. Cells without CD74 produced minimal IL-8 upon exposure to H. pylori, while CD74 p33 transfectants (M1-p33) had a robust IL-8 response following H. pylori exposure (Fig. 6D). These results indicate that cells that normally lack an IL-8 response to H. pylori become responsive upon expression of CD74.

To further examine the role of CD74 in the gastric epithelial response to H. pylori, monoclonal antibodies were used to block CD74 before H. pylori exposure. IL-8 secretion by cells pretreated with anti-CD74 blocking antibodies and exposed to H. pylori was compared to that of unblocked cells or cells treated with isotype control antibody. Gastric epithelial cells treated with H. pylori in the absence of anti-CD74 antibodies produced more than double the IL-8 levels of cells pretreated with anti-CD74 (Fig. 6E and F). Fibroblast CD74 p33 transfectants showed the most dramatic decrease in IL-8 expression upon blocking CD74, very close to basal levels. CD74 appears to be the sole molecule responsible for the IL-8 response in these transfected cells, whereas it is likely there are other molecules on gastric epithelial cells or mechanisms responsible for the H. pylori-induced IL-8 response in addition to CD74.

The role of CD74 in IL-8 production is independent of the cag pathogenicity island. Since the H. pylori cag PAI has been implicated in the induction of IL-8 production by gastric epithelial cells, it was important to investigate the role of the cag PAI in the CD74-induced IL-8 production. To that end, a cag-deficient mutant of the H. pylori strain 26695 was utilized. Attachment of PKH26-stained 26695 and the cag-deficient 26695 mutant to gastric epithelial cell lines and M1-p33 cells was measured by flow cytometry. No significant decrease in attachment of the cag-deficient mutant to any of the cell lines was observed, as compared to the 26695 wild type (Fig. 7A). IL-8 production by these cells after 24 h of exposure to 26695 and the cag-deficient mutant was also measured by ELISA. Kato III and HS-738 cells had decreased production of IL-8 by approximately 40% in response to the cag-deficient mutant compared to the wild type (Fig. 7B). In contrast, M1-p33 cells showed no decrease in IL-8 production in response to the cag-deficient mutant. These results suggest a role for the CD74-mediated IL-8 response in the overall IL-8 production by cells in response to H. pylori exposure, which is independent of the cag PAI contribution to IL-8 induction.



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  		FIG. 7. Engagement of CD74 and the resulting IL-8 production are independent of the cag pathogenicity island. Binding of PKH26-labeled H. pylori 26695 and its cag-deficient mutant was determined by flow cytometry. The cag-deficient bacteria (A) showed no decrease in attachment to all cell lines compared to the wild type, while (B) IL-8 production was decreased by approximately 40% by gastric epithelial cells, but no decrease was seen with M1-p33 cells. The means of duplicates from four experiments (n = 8) are shown here.


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DISCUSSION
 
The host and microbial factors that determine the outcome of colonization of gastric epithelium by H. pylori remain elusive. H. pylori adherence to the gastric epithelium is undoubtedly a primordial event for bacterial colonization of the gastric mucosa (38). he lack of definite answers is in part due to variation among H. pylori strains and expression of receptors by host cells. A variety of host cell receptors have been suggested to be involved in H. pylori attachment, including heparan sulfate (15, 42), Lewis B antigen (32, 33), and as we have previously suggested, class II MHC (11, 12). This study expands upon the previous observations and brings forth a novel receptor that may be used both in adhesion of the bacteria and in the initiation of the inflammatory process associated with H. pylori infection. We have shown that CD74 alone binds to H. pylori and that upon blocking or removing CD74 from gastric epithelial cells, the overall attachment of H. pylori to gastric epithelial cells is reduced. Small differences in attachment and IL-8 production were seen between the LC11 and 26695 strains of H. pylori. The lower levels of both processes seen with strain 26695 may be due to differences in the density of outer membrane proteins. A clinical strain and a lab strain were used because of probable differences to illustrate that decreases in attachment and cell responses were seen with two different strains.

Although the traditional role of CD74 is regulating the intracellular biology of class II MHC molecules, CD74 has recently been suggested to have functions independent of the class II MHC. CD74 has been shown to be a receptor for macrophage migration inhibitory factor (MIF), an important regulator of innate and acquired immune responses (23). MIF binding to CD74 was shown to lead to signaling events such as extracelluar signal-regulated kinase activation, cell proliferation, and prostaglandin E2 production (41). Other studies have indicated that CD74 is required for signaling that induces B-cell maturation (27, 28). Those studies showed that CD74 activates a pathway leading to upregulation of several transcription factors that allow B cells to differentiate into mature cells capable of participating in immune responses. Among these transcription factors is NF-{kappa}B, which is an important control in immune and inflammatory responses (5, 29), especially IL-8. The variety of functions and signaling leading to immune responses that have recently been implicated in CD74 suggest that there is much more to be revealed about the functions of this molecule. Consequently, this study suggests a role for CD74 in gastric epithelial cell interaction with H. pylori leading to NF-{kappa}B signaling that results in IL-8 secretion.

CD74 engagement led to IL-8 production by gastric epithelial cells and CD74-transfected fibroblasts, while nontransfected control cells did not produce increased levels of IL-8. IL-8 has long been recognized as an early response to H. pylori by the gastric epithelium. While several studies have indicated that attachment of the bacteria to the gastric epithelium is crucial to the IL-8 response (6, 35), we have found that CD74 plays an important role in these events. Using specific antibodies to cross-link CD74 on the surface of gastric epithelial cells led to I{kappa}B degradation as well as a robust IL-8 production by the cells, suggesting a direct role for CD74 in signaling and IL-8 production by these cells. Furthermore, blocking CD74 during H. pylori exposure caused a decrease in NF-{kappa}B activation and in the IL-8 response by approximately 75%. Although some of this decrease may be due to reduced attachment that was also seen with CD74 blocking, attachment was only decreased by 40%. H. pylori undoubtedly uses other receptors for attachment, leading to IL-8 production evident by the response remaining upon blocking CD74. However, the overall contribution of this interaction obviously plays a significant role in the inflammatory response.

Since the cag pathogenicity island has been shown to play an important role in the H. pylori-induced IL-8 response (7, 30), we sought to investigate its role in the observed CD74-induced IL-8 production. The studies showed that the cag-deficient mutant was not impaired in its binding to any of the cell lines examined. IL-8 production, however, was decreased by 40% by gastric epithelial cells when comparing the mutant to the wild-type bacteria, whereas the IL-8 response of the CD74-transfected fibroblasts to H. pylori was unaffected by the cag deletion in H. pylori. These results suggest a system independent of the cag pathogenicity island for IL-8 induction via CD74 engagement. In one study, IL-8 production by AGS cells was not increased upon incubation with the cag-deficient mutant (13), suggesting that the IL-8 response to H. pylori with these cells is completely cag dependent. We have found this cell line to be deficient in class II MHC (11) and CD74 expression, thus explaining the difference between this cell line and the cell lines used in this study.

The novel finding that H. pylori interacts with CD74 is an important step in understanding the interaction between H. pylori and gastric epithelial cells, leading to characteristic epithelial cell responses. Other studies have suggested a variety of host and bacterial factors involved in H. pylori attachment, but little is known about the steps leading to pathogenesis (30). The overall role of CD74 in interaction of H. pylori with the host epithelium and the ensuing pathogenic processes must be further defined, and the bacterial factors binding to CD74 and upregulating the crucial responses must be determined.


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ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health grants DK50669 and DK56338. E.J.B. was a recipient of a fellowship under National Institutes of Health training grant T32 AI007536-06.


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FOOTNOTES
 
* Corresponding author. Mailing address: Children's Hospital, Room 2.300, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555. Phone: (409) 772-3897. Fax: (409) 772-1761. E-mail: vreyes{at}utmb.edu. Back

Editor: J. B. Bliska


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Infection and Immunity, May 2005, p. 2736-2743, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.2736-2743.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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