Helicobacter pylori-Induced IL-8 Production by Gastric Epithelial Cells Up-Regulates CD74 Expression1
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免疫学杂志 2005年第13期
Abstract
CD74, or the class II MHC-associated invariant chain, is best known for the regulation of Ag presentation. However, recent studies have suggested other important roles for this protein in inflammation and cancer studies. We have shown that CD74 is expressed on the surface of gastric cells, and Helicobacter pylori can use this receptor as a point of attachment to gastric epithelial cells, which lead to IL-8 production. This study investigates the ability of H. pylori to up-regulate one of its receptors in vivo and with a variety of gastric epithelial cell lines during infection with H. pylori. CD74 expression was increased dramatically on gastric biopsies from H. pylori-positive patients and gastric cell lines exposed to the bacteria. Gastric cells exposed to H. pylori-conditioned medium revealed that the host cell response was responsible for the up-regulation of CD74. IL-8 was found to up-regulate CD74 cell surface expression because blocking IL-8Rs or neutralizing IL-8 with Abs counteracted the increased expression of CD74 observed during infection with H. pylori. These studies demonstrate how H. pylori up-regulates one of its own receptors via an autocrine mechanism involving one of the products induced from host cells.
Introduction
Helicobacter pylori is a common human pathogen associated with chronic gastritis, duodenal ulcers, and at least 80% of gastric ulcers worldwide (1, 2). There is also considerable evidence linking this pathogen to gastric cancer (3, 4), which led to H. pylori becoming classified as a class I carcinogen by the World Health Organization. Pathogenesis of infection often includes inflammation, mucosal damage, or gastric atrophy. During infection, H. pylori maintain close association with gastric epithelium (5, 6). Clearly bacterial adhesion and colonization of the gastric mucosa are key events in pathogenesis (7). Although multiple bacterial adhesins and host cell receptors have been implicated in the attachment of the bacterium to the gastric epithelium (8, 9, 10, 11), the interactions that lead to host cell responses associated with inflammation are not well understood. We have shown recently that CD74 is a receptor used by H. pylori, and engagement of this receptor leads to the production of IL-8 (12).
CD74 is also known as the class II MHC-associated invariant chain. The development of immune responses is influenced by CD74 because it regulates the intracellular transport and functions of class II MHC in APCs (13, 14). Recently, we and others (12, 15, 16) have found that CD74 is expressed on the surface of a variety of cell types, including gastric epithelial cells. CD74 has a long cytoplasmic tail that has been implicated in signaling events. A variety of studies involving signaling leading to NF-B activation (17) and B cell maturation have been shown to be dependent on CD74 (18). Macrophage migration inhibitory factor (MIF)3, an important inflammatory cytokine, has been shown to bind to CD74 expressed on cell surfaces and initiate the MAPK activation (19). We recently showed that H. pylori binding to CD74 stimulates the NF-B-signaling cascade that leads to IL-8 production (12).
Given the importance of CD74 in H. pylori attachment and signaling leading to a proinflammatory response, this study examines the mechanism leading to increased expression of this receptor by gastric cells in vivo and with a variety of gastric cell lines during infection with H. pylori. We observed that surface expression of CD74 on epithelial cells is increased in biopsies from H. pylori-infected individuals. Gastric cell lines infected with H. pylori also had increased CD74 expression as determined by flow cytometry and quantitative real-time RT-PCR. Interestingly, IL-8, which is among the earliest cytokines produced by the epithelium in response to H. pylori infection (20, 21), was found to induce CD74 up-regulation. The IL-8Rs CXCR-1 and CXCR-2 were also up-regulated during infection. CD74 up-regulation was decreased upon blocking these receptors with mAbs or by the addition of IL-8-neutralizing Abs to cell cultures before H. pylori exposure. These studies offer insights into H. pylori interaction with the gastric epithelium leading to inflammation and how the bacterium up-regulates one of its receptors through an autocrine mechanism involving one of the products induced from host cells.
Materials and Methods
Gastric biopsy staining
Gastric antrum biopsy tissues were obtained from consenting donors undergoing gastro-esophageal-duodenoscopy at the University of Texas Medical Branch. The biopsies were obtained from grossly inflamed areas of the antrum, as well as from regions that appeared uninvolved. The tissue was acquired following informed consent from patients and volunteers and with prior approval of the Institutional Review Board. The immunohistochemical expression of CD74 was evaluated in 45 consecutive endoscopy gastric biopsy specimens previously determined to be H. pylori positive or negative; however, the specimens were obtained from our institutional gastrointestinal endoscopy suites, so there was no bias as to the diagnosis or the clinical status of the patients. The tissue samples were fixed in 10% neutral buffered solution and then embedded in paraffin for immunohistochemistry. Four-micrometer thick sections of the paraffin-embedded tissue blocks were mounted on plus (coated) glass slides and deparaffinized in xylene followed by rehydration in ethanol. Endogenous peroxidase was blocked by incubation in 3% methanol peroxide for 10 min. For CD74 immunohistochemical staining, Ag retrieval was done by pretreating the sections with citrate buffer (0.01 ml/L citric acid at pH 6.0) for 20 min at 99°C in a microwave oven and then allowed to cool for 20 min at room temperature. Nonspecific endogenous proteins were blocked using the avidin-biotin blocking kit (Vector Laboratories), followed by 30 min of incubation at room temperature with biotinylated, mouse, and anti-human CD74 Ab clone LN-2 (DakoCytomation) at 1/200 dilution. The sections were stained with a streptavidin-biotin peroxidase detection kit (DakoCytomation), incubated with 3,3'-diaminobenzidine, and counterstained with hematoxylin followed by coverslipping. The immunostaining reactions were examined by light microscopy.
Cell lines
N87 human gastric carcinoma epithelial cells and Hs738.st/int (CRL-7869) normal human fetal gastric/intestinal cells were obtained from the American Type Tissue Culture. Cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics. Antibiotics were removed from the medium before infection of the cells with H. pylori.
Bacterial cultures
H. pylori clinical isolate LC11 was described previously (22). The 26695 lab strain and its cag pathogenicity island (PAI) totally deleted mutants (cag PAI mutants) were also constructed. For constructing a cag mutant, regions upstream (hp0518-hp0519; 545,254-547,164 bp: hp number and location from H. pylori strain 26695: GenBank accession no. AE000511) and downstream (hp0549-hp0550; 584,570-586,563 bp) of the cag PAI were amplified from the H. pylori strain 26695 chromosome to delete the entire cag PAI from the H. pylori chromosome. These fragments, separated by a chloramphenicol resistance cassette (cat) (a gift from Dr. D. E. Taylor, University of Alberta, Edmonton, Canada), were cloned into the T7Blue vector (Novagen), resulting in the deletion mutant’s plasmid cag PAI::cat. The plasmids (1–2 μg) were used for inactivation of chromosomal genes by natural transformation as described previously (23). Inactivation of the genes was confirmed by PCR amplification followed by Southern blot hybridization.
H. pylori were grown on blood agar plates (BD Biosciences) at 37°C under microaerophilic conditions as described previously (22). Bacteria were transferred after 48 h into Brucella broth containing 10% FBS for 24 h. After centrifugation at 2500 x g for 10 min, bacteria were resuspended in sterile PBS. The concentration of bacteria per milliliter was determined by measuring the absorbance (at 530 nm) using a spectrophotometer (DU-65; BD Biosciences) and comparing the value to a standard curve generated by quantifying viable organisms from aliquots of bacteria at varying concentrations that were also assessed by absorbance.
Abs and reagents
The anti-CD74 mAb MB-741 was obtained from BD Biosciences along with isotype controls. Anti-CXCR-1 and anti-CXCR-2 mAbs were obtained from Sigma-Aldrich. For samples treated with human rIL-8 (Sigma-Aldrich), 100 pg/ml for N87 or 1000 pg/ml for HS-738 were added for 24 h. The IL-8 concentration that we typically see in each gastric cell line exposed to H. pylori was used (12). Monoclonal anti-IL-8-neutralizing Ab was obtained from Sigma-Aldrich and was used at a working concentration of 1 μg/ml.
CD74, CXCR1, and CXCR2 staining for flow cytometry
Gastric epithelial cells (2 x 105) were grown in medium alone or supplemented, when indicated, with IFN- (100 U/ml) for 48 h. Cells were incubated with anti-CD74 MB-74 mAb (1 μg) or anti-CXCR1 and anti-CXCR-2 mixture (1 μg each) in PBS buffer for 1 h on ice. The cells were washed with PBS and then incubated with PE-conjugated goat anti-mouse IgG (1 μg) for an additional 30 min on ice. Following incubation, the cells were washed and fixed with 1% paraformaldehyde in PBS. Then, 10,000 events/sample were collected for additional analysis. The flow cytometry analysis was done on a FACScan cytometer (BD Biosciences), and the data analysis was done using CellQuest software.
Real-time PCR
Real-time RT-PCR assays were used to determine gene expression using TaqMan technology on an Applied Biosystems 7000 sequence detection system. Applied Biosystems Assays-By-Design containing a 20x assay mix of primers and predeveloped 18S rRNA (VIC dye-labeled probe) TaqMan assay reagent (/N 4319413E) for internal control were used for real-time PCR measurements. These assays were designed using primers that span exon-exon junctions so as not to detect genomic DNA. All primer and probe sequences were searched against the Celera database to confirm specificity. Target gene CD74 primers were designed using accession no. NM_004355 (GenBank) and probe sequence 5'-CCCGGAGAGCAAGTGCAGCCGCGGA-3' (Applied Biosystems). HLA-DR target gene primers were designed using accession no. NM_019111 (GenBank) and probe sequence 5'-TGTAAGGCACATGGAGGTGATGATG-3' (Applied Biosystems). The efficiency of target amplification was validated using a reference amplification reaction. Absolute values of the slope of log input RNA amount vs CT were <0.1 in all experiments. One-step RT-PCR reactions were performed on 20 ng of input RNA for both target genes and endogenous controls using the TaqMan one-step RT-PCR master mix reagent kit (Applied Biosystems). The cycling parameters were as follows: reverse transcription, 48°C for 30 min; AmpliTaq activation, 95°C for 10 min; denaturation, 95°C for 15 s; and annealing/extension, 60°C for 1 min for 40 cycles. Duplicate cycle threshold values were analyzed in Microsoft Excel using the comparative CT(CT) method as described by the manufacturer (Applied Biosystems). The amount of target (2CT) was obtained by normalizing to an endogenous reference (18S mRNA) and relative to a calibrator.
Attachment assays
Cells were treated with 100 U/ml IFN- (Roche) or 100 or 1000 pg/ml IL-8 (Sigma-Aldrich) for 48 h to up-regulate CD74 expression. Media were replaced with IFN--free medium for 24 h. H. pylori was stained with the red fluorochrome PKH26 (Sigma-Aldrich), according to the manufacturer’s instructions, washed three times with RPMI 1640 medium, and resuspended in RPMI 1640 medium. Bacteria were added to pelleted cells that were treated with medium, IFN-, or IL-8 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.
Statistical analysis
Results are expressed as mean ± SEM. In vivo CD74 expression among groups were compared using a Fisher’s exact text and H. pylori binding, and IL-8 production results were compared using an ANOVA analysis of the variance and considered significant if p was <0.05.
Results
CD74 expression is increased on gastric epithelial cell biopsy samples during H. pylori infection
Because we have shown previously that cell surface CD74 plays a pivotal role in H. pylori attachment leading to the inflammatory response (12), we sought to determine the effect of H. pylori infection on CD74 expression. Gastric antrum biopsy tissues were obtained from consenting donors undergoing gastro-esophageal-duodenoscopy. Staining was done using anti-CD74 clone LN-2. As seen in Fig. 1, there is a marked increase in CD74 staining of epithelial cells during H. pylori infection. In this study, 45 patient biopsies were examined for CD74 expression. Of the 45 patients, 13 were positive for H. pylori infection (Table I), as determined by staining, and of the 13 biopsies, 11 (85%) were found to have high CD74 expression. High expression was considered >30% of epithelial cells staining positive for CD74. CD74 expression also correlated with an influx of inflammatory cells. There were nine samples from patients that did not have H. pylori infection or gastritis, and only two (22%) of those had high CD74 expression (p < 0.01 compared samples from patients with H. pylori-positive gastritis). These results indicate a correlation between H. pylori infection and CD74 expression. Of the 23 samples from patients with gastritis but no infection, 14 (61%) had high CD74 expression, suggesting there is also a link between inflammation, which is a hallmark response to H. pylori infection, and CD74 expression.
Discussion
CD74 has been thought traditionally to only play a role intracellularly in regulating the transport and functions of class II MHC molecules (13, 14). Reports have shown that CD74 is present on a variety of cell surfaces (16), whereas we have shown it is present on gastric epithelial cell surfaces (12). We have also shown that it plays an important role in mucosal immunology (24). A variety of roles have been suggested recently for CD74 independent of its function in the regulation of Ag presentation. CD74 has been shown to be a receptor for MIF, which is an important regulator of innate and acquired immune responses (19). MIF binding to CD74 has been shown to lead to signaling events such as ERK activation, cell proliferation, and PGE2 production (25). Other studies have indicated that CD74 is required for the signaling that induces B cell maturation (17, 18). One study indicated that the cytoplasmic domain of CD74 activates a pathway leading to up-regulation of several transcription factors that allow B cells to differentiate into mature cells capable of participating in immune responses.
We have previously shown that CD74 is among the receptors H. pylori uses to attach to gastric epithelial cells (12). Upon attachment to CD74, NF-B is activated, and the inflammatory response is initiated. This study demonstrating that signaling through the attachment to CD74 was the first to demonstrate a H. pylori receptor directly initiating signaling. This study illustrates that H. pylori up-regulates one of its receptors on gastric cells through an autocrine response system. Increased expression of CD74 in the presence of H. pylori was demonstrated on the biopsies of infected patients. This observation was confirmed in vitro by increased CD74 mRNA levels and surface protein expression on two different gastric cell lines. Additional investigation using the H. pylori CM of previously exposed gastric cells, filtered to remove bacteria, or a cag PAI mutant revealed that a gastric cell product or a bacterial product could be responsible for the up-regulation of CD74. The recent discovery of the presence of IL-8Rs on gastric epithelial cells (26), along with IL-8 being an important early response after H. pylori attachment to gastric epithelial cells and being induced from cross-linking CD74 (12), led us to consider IL-8 as a candidate responsible for the up-regulation of CD74 expression by gastric cells. Concentrations of rIL-8, equivalent to those produced by the cells in response to H. pylori, were found to up-regulate CD74 surface expression. The addition of anti-IL-8-neutralizing Abs or blocking IL-8Rs on cultures of gastric cells exposed to H. pylori prevented the up-regulation of CD74. Although a previous study showed that H. pylori infection induces an increased expression of the IL-8R CXCR2 by gastric epithelial cells and had a minimal effect on the expression of the CXCR1 IL-8R (26), in our studies to determine the effect of IL-8 on CD74 expression by gastric epethelial cells, we found that blocking either receptor reduced the level of CD74 induction by H. pylori or IL-8; however, the effect of blocking CXCR2 was slightly more pronounced and perhaps reflecting the level of that receptor that is induced by H. pylori (data not shown). To confirm the role of IL-8 in the noted increase of CD74 expression by gastric epethelial cells, we blocked both receptors with the corresponding Abs.
These results suggest that IL-8 induced by H. pylori or by general inflammatory conditions when IL-8 is induced is a mechanism for up-regulation of CD74 surface expression.
Adherence to the gastric epithelium is undoubtedly a primordial event in bacterial colonization of the gastric mucosa (7). We have shown that CD74 is an essential receptor for H. pylori on gastric epithelial cells, and this bacterium is capable of up-regulating one of its receptors. When CD74 is up-regulated on the surface of gastric cells, more receptors are present for increased bacterial attachment, leading to enhanced attachment. These results uncover relevant information pertaining to lengthy infections and chronic gastritis often seen with H. pylori infection. IL-8 is a central player in this system, and the up-regulation of CD74 by IL-8 is an important immunological finding that extends beyond H. pylori studies and should be further studied for its role in the inflammatory response in gastritis unrelated to H. pylori infection.
Acknowledgments
We thank Dr. D. E. Taylor (University of Alberta, Edmonton, Alberta, Canada) for the kind gift of the chloramphenicol resistance cassette.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the National Institutes of Health Grants DK50669 and DK56338. E.J.B. was a recipient of a fellowship under National Institutes of Health T32 AI007536-06 Training Grant.
2 Address correspondence and reprint requests to Dr. Victor E. Reyes, Children’s Hospital, Room 2.300, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555. E-mail address: vreyes@utmb.edu
3 Abbreviations used in this paper: MIF, macrophage migration inhibitory factor; PAI, pathogenicity island; CM, conditioned medium.
Received for publication February 16, 2005. Accepted for publication April 27, 2005.
References
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Utt, M., T. Wadstrom. 1997. Identification of heparan sulphate binding surface proteins of Helicobacter pylori: inhibition of heparan sulphate binding with sulphated carbohydrate polymers. J. Med. Microbiol. 46: 541-546.(Ellen J. Beswick*, Soumit)
CD74, or the class II MHC-associated invariant chain, is best known for the regulation of Ag presentation. However, recent studies have suggested other important roles for this protein in inflammation and cancer studies. We have shown that CD74 is expressed on the surface of gastric cells, and Helicobacter pylori can use this receptor as a point of attachment to gastric epithelial cells, which lead to IL-8 production. This study investigates the ability of H. pylori to up-regulate one of its receptors in vivo and with a variety of gastric epithelial cell lines during infection with H. pylori. CD74 expression was increased dramatically on gastric biopsies from H. pylori-positive patients and gastric cell lines exposed to the bacteria. Gastric cells exposed to H. pylori-conditioned medium revealed that the host cell response was responsible for the up-regulation of CD74. IL-8 was found to up-regulate CD74 cell surface expression because blocking IL-8Rs or neutralizing IL-8 with Abs counteracted the increased expression of CD74 observed during infection with H. pylori. These studies demonstrate how H. pylori up-regulates one of its own receptors via an autocrine mechanism involving one of the products induced from host cells.
Introduction
Helicobacter pylori is a common human pathogen associated with chronic gastritis, duodenal ulcers, and at least 80% of gastric ulcers worldwide (1, 2). There is also considerable evidence linking this pathogen to gastric cancer (3, 4), which led to H. pylori becoming classified as a class I carcinogen by the World Health Organization. Pathogenesis of infection often includes inflammation, mucosal damage, or gastric atrophy. During infection, H. pylori maintain close association with gastric epithelium (5, 6). Clearly bacterial adhesion and colonization of the gastric mucosa are key events in pathogenesis (7). Although multiple bacterial adhesins and host cell receptors have been implicated in the attachment of the bacterium to the gastric epithelium (8, 9, 10, 11), the interactions that lead to host cell responses associated with inflammation are not well understood. We have shown recently that CD74 is a receptor used by H. pylori, and engagement of this receptor leads to the production of IL-8 (12).
CD74 is also known as the class II MHC-associated invariant chain. The development of immune responses is influenced by CD74 because it regulates the intracellular transport and functions of class II MHC in APCs (13, 14). Recently, we and others (12, 15, 16) have found that CD74 is expressed on the surface of a variety of cell types, including gastric epithelial cells. CD74 has a long cytoplasmic tail that has been implicated in signaling events. A variety of studies involving signaling leading to NF-B activation (17) and B cell maturation have been shown to be dependent on CD74 (18). Macrophage migration inhibitory factor (MIF)3, an important inflammatory cytokine, has been shown to bind to CD74 expressed on cell surfaces and initiate the MAPK activation (19). We recently showed that H. pylori binding to CD74 stimulates the NF-B-signaling cascade that leads to IL-8 production (12).
Given the importance of CD74 in H. pylori attachment and signaling leading to a proinflammatory response, this study examines the mechanism leading to increased expression of this receptor by gastric cells in vivo and with a variety of gastric cell lines during infection with H. pylori. We observed that surface expression of CD74 on epithelial cells is increased in biopsies from H. pylori-infected individuals. Gastric cell lines infected with H. pylori also had increased CD74 expression as determined by flow cytometry and quantitative real-time RT-PCR. Interestingly, IL-8, which is among the earliest cytokines produced by the epithelium in response to H. pylori infection (20, 21), was found to induce CD74 up-regulation. The IL-8Rs CXCR-1 and CXCR-2 were also up-regulated during infection. CD74 up-regulation was decreased upon blocking these receptors with mAbs or by the addition of IL-8-neutralizing Abs to cell cultures before H. pylori exposure. These studies offer insights into H. pylori interaction with the gastric epithelium leading to inflammation and how the bacterium up-regulates one of its receptors through an autocrine mechanism involving one of the products induced from host cells.
Materials and Methods
Gastric biopsy staining
Gastric antrum biopsy tissues were obtained from consenting donors undergoing gastro-esophageal-duodenoscopy at the University of Texas Medical Branch. The biopsies were obtained from grossly inflamed areas of the antrum, as well as from regions that appeared uninvolved. The tissue was acquired following informed consent from patients and volunteers and with prior approval of the Institutional Review Board. The immunohistochemical expression of CD74 was evaluated in 45 consecutive endoscopy gastric biopsy specimens previously determined to be H. pylori positive or negative; however, the specimens were obtained from our institutional gastrointestinal endoscopy suites, so there was no bias as to the diagnosis or the clinical status of the patients. The tissue samples were fixed in 10% neutral buffered solution and then embedded in paraffin for immunohistochemistry. Four-micrometer thick sections of the paraffin-embedded tissue blocks were mounted on plus (coated) glass slides and deparaffinized in xylene followed by rehydration in ethanol. Endogenous peroxidase was blocked by incubation in 3% methanol peroxide for 10 min. For CD74 immunohistochemical staining, Ag retrieval was done by pretreating the sections with citrate buffer (0.01 ml/L citric acid at pH 6.0) for 20 min at 99°C in a microwave oven and then allowed to cool for 20 min at room temperature. Nonspecific endogenous proteins were blocked using the avidin-biotin blocking kit (Vector Laboratories), followed by 30 min of incubation at room temperature with biotinylated, mouse, and anti-human CD74 Ab clone LN-2 (DakoCytomation) at 1/200 dilution. The sections were stained with a streptavidin-biotin peroxidase detection kit (DakoCytomation), incubated with 3,3'-diaminobenzidine, and counterstained with hematoxylin followed by coverslipping. The immunostaining reactions were examined by light microscopy.
Cell lines
N87 human gastric carcinoma epithelial cells and Hs738.st/int (CRL-7869) normal human fetal gastric/intestinal cells were obtained from the American Type Tissue Culture. Cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics. Antibiotics were removed from the medium before infection of the cells with H. pylori.
Bacterial cultures
H. pylori clinical isolate LC11 was described previously (22). The 26695 lab strain and its cag pathogenicity island (PAI) totally deleted mutants (cag PAI mutants) were also constructed. For constructing a cag mutant, regions upstream (hp0518-hp0519; 545,254-547,164 bp: hp number and location from H. pylori strain 26695: GenBank accession no. AE000511) and downstream (hp0549-hp0550; 584,570-586,563 bp) of the cag PAI were amplified from the H. pylori strain 26695 chromosome to delete the entire cag PAI from the H. pylori chromosome. These fragments, separated by a chloramphenicol resistance cassette (cat) (a gift from Dr. D. E. Taylor, University of Alberta, Edmonton, Canada), were cloned into the T7Blue vector (Novagen), resulting in the deletion mutant’s plasmid cag PAI::cat. The plasmids (1–2 μg) were used for inactivation of chromosomal genes by natural transformation as described previously (23). Inactivation of the genes was confirmed by PCR amplification followed by Southern blot hybridization.
H. pylori were grown on blood agar plates (BD Biosciences) at 37°C under microaerophilic conditions as described previously (22). Bacteria were transferred after 48 h into Brucella broth containing 10% FBS for 24 h. After centrifugation at 2500 x g for 10 min, bacteria were resuspended in sterile PBS. The concentration of bacteria per milliliter was determined by measuring the absorbance (at 530 nm) using a spectrophotometer (DU-65; BD Biosciences) and comparing the value to a standard curve generated by quantifying viable organisms from aliquots of bacteria at varying concentrations that were also assessed by absorbance.
Abs and reagents
The anti-CD74 mAb MB-741 was obtained from BD Biosciences along with isotype controls. Anti-CXCR-1 and anti-CXCR-2 mAbs were obtained from Sigma-Aldrich. For samples treated with human rIL-8 (Sigma-Aldrich), 100 pg/ml for N87 or 1000 pg/ml for HS-738 were added for 24 h. The IL-8 concentration that we typically see in each gastric cell line exposed to H. pylori was used (12). Monoclonal anti-IL-8-neutralizing Ab was obtained from Sigma-Aldrich and was used at a working concentration of 1 μg/ml.
CD74, CXCR1, and CXCR2 staining for flow cytometry
Gastric epithelial cells (2 x 105) were grown in medium alone or supplemented, when indicated, with IFN- (100 U/ml) for 48 h. Cells were incubated with anti-CD74 MB-74 mAb (1 μg) or anti-CXCR1 and anti-CXCR-2 mixture (1 μg each) in PBS buffer for 1 h on ice. The cells were washed with PBS and then incubated with PE-conjugated goat anti-mouse IgG (1 μg) for an additional 30 min on ice. Following incubation, the cells were washed and fixed with 1% paraformaldehyde in PBS. Then, 10,000 events/sample were collected for additional analysis. The flow cytometry analysis was done on a FACScan cytometer (BD Biosciences), and the data analysis was done using CellQuest software.
Real-time PCR
Real-time RT-PCR assays were used to determine gene expression using TaqMan technology on an Applied Biosystems 7000 sequence detection system. Applied Biosystems Assays-By-Design containing a 20x assay mix of primers and predeveloped 18S rRNA (VIC dye-labeled probe) TaqMan assay reagent (/N 4319413E) for internal control were used for real-time PCR measurements. These assays were designed using primers that span exon-exon junctions so as not to detect genomic DNA. All primer and probe sequences were searched against the Celera database to confirm specificity. Target gene CD74 primers were designed using accession no. NM_004355 (GenBank) and probe sequence 5'-CCCGGAGAGCAAGTGCAGCCGCGGA-3' (Applied Biosystems). HLA-DR target gene primers were designed using accession no. NM_019111 (GenBank) and probe sequence 5'-TGTAAGGCACATGGAGGTGATGATG-3' (Applied Biosystems). The efficiency of target amplification was validated using a reference amplification reaction. Absolute values of the slope of log input RNA amount vs CT were <0.1 in all experiments. One-step RT-PCR reactions were performed on 20 ng of input RNA for both target genes and endogenous controls using the TaqMan one-step RT-PCR master mix reagent kit (Applied Biosystems). The cycling parameters were as follows: reverse transcription, 48°C for 30 min; AmpliTaq activation, 95°C for 10 min; denaturation, 95°C for 15 s; and annealing/extension, 60°C for 1 min for 40 cycles. Duplicate cycle threshold values were analyzed in Microsoft Excel using the comparative CT(CT) method as described by the manufacturer (Applied Biosystems). The amount of target (2CT) was obtained by normalizing to an endogenous reference (18S mRNA) and relative to a calibrator.
Attachment assays
Cells were treated with 100 U/ml IFN- (Roche) or 100 or 1000 pg/ml IL-8 (Sigma-Aldrich) for 48 h to up-regulate CD74 expression. Media were replaced with IFN--free medium for 24 h. H. pylori was stained with the red fluorochrome PKH26 (Sigma-Aldrich), according to the manufacturer’s instructions, washed three times with RPMI 1640 medium, and resuspended in RPMI 1640 medium. Bacteria were added to pelleted cells that were treated with medium, IFN-, or IL-8 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.
Statistical analysis
Results are expressed as mean ± SEM. In vivo CD74 expression among groups were compared using a Fisher’s exact text and H. pylori binding, and IL-8 production results were compared using an ANOVA analysis of the variance and considered significant if p was <0.05.
Results
CD74 expression is increased on gastric epithelial cell biopsy samples during H. pylori infection
Because we have shown previously that cell surface CD74 plays a pivotal role in H. pylori attachment leading to the inflammatory response (12), we sought to determine the effect of H. pylori infection on CD74 expression. Gastric antrum biopsy tissues were obtained from consenting donors undergoing gastro-esophageal-duodenoscopy. Staining was done using anti-CD74 clone LN-2. As seen in Fig. 1, there is a marked increase in CD74 staining of epithelial cells during H. pylori infection. In this study, 45 patient biopsies were examined for CD74 expression. Of the 45 patients, 13 were positive for H. pylori infection (Table I), as determined by staining, and of the 13 biopsies, 11 (85%) were found to have high CD74 expression. High expression was considered >30% of epithelial cells staining positive for CD74. CD74 expression also correlated with an influx of inflammatory cells. There were nine samples from patients that did not have H. pylori infection or gastritis, and only two (22%) of those had high CD74 expression (p < 0.01 compared samples from patients with H. pylori-positive gastritis). These results indicate a correlation between H. pylori infection and CD74 expression. Of the 23 samples from patients with gastritis but no infection, 14 (61%) had high CD74 expression, suggesting there is also a link between inflammation, which is a hallmark response to H. pylori infection, and CD74 expression.
Discussion
CD74 has been thought traditionally to only play a role intracellularly in regulating the transport and functions of class II MHC molecules (13, 14). Reports have shown that CD74 is present on a variety of cell surfaces (16), whereas we have shown it is present on gastric epithelial cell surfaces (12). We have also shown that it plays an important role in mucosal immunology (24). A variety of roles have been suggested recently for CD74 independent of its function in the regulation of Ag presentation. CD74 has been shown to be a receptor for MIF, which is an important regulator of innate and acquired immune responses (19). MIF binding to CD74 has been shown to lead to signaling events such as ERK activation, cell proliferation, and PGE2 production (25). Other studies have indicated that CD74 is required for the signaling that induces B cell maturation (17, 18). One study indicated that the cytoplasmic domain of CD74 activates a pathway leading to up-regulation of several transcription factors that allow B cells to differentiate into mature cells capable of participating in immune responses.
We have previously shown that CD74 is among the receptors H. pylori uses to attach to gastric epithelial cells (12). Upon attachment to CD74, NF-B is activated, and the inflammatory response is initiated. This study demonstrating that signaling through the attachment to CD74 was the first to demonstrate a H. pylori receptor directly initiating signaling. This study illustrates that H. pylori up-regulates one of its receptors on gastric cells through an autocrine response system. Increased expression of CD74 in the presence of H. pylori was demonstrated on the biopsies of infected patients. This observation was confirmed in vitro by increased CD74 mRNA levels and surface protein expression on two different gastric cell lines. Additional investigation using the H. pylori CM of previously exposed gastric cells, filtered to remove bacteria, or a cag PAI mutant revealed that a gastric cell product or a bacterial product could be responsible for the up-regulation of CD74. The recent discovery of the presence of IL-8Rs on gastric epithelial cells (26), along with IL-8 being an important early response after H. pylori attachment to gastric epithelial cells and being induced from cross-linking CD74 (12), led us to consider IL-8 as a candidate responsible for the up-regulation of CD74 expression by gastric cells. Concentrations of rIL-8, equivalent to those produced by the cells in response to H. pylori, were found to up-regulate CD74 surface expression. The addition of anti-IL-8-neutralizing Abs or blocking IL-8Rs on cultures of gastric cells exposed to H. pylori prevented the up-regulation of CD74. Although a previous study showed that H. pylori infection induces an increased expression of the IL-8R CXCR2 by gastric epithelial cells and had a minimal effect on the expression of the CXCR1 IL-8R (26), in our studies to determine the effect of IL-8 on CD74 expression by gastric epethelial cells, we found that blocking either receptor reduced the level of CD74 induction by H. pylori or IL-8; however, the effect of blocking CXCR2 was slightly more pronounced and perhaps reflecting the level of that receptor that is induced by H. pylori (data not shown). To confirm the role of IL-8 in the noted increase of CD74 expression by gastric epethelial cells, we blocked both receptors with the corresponding Abs.
These results suggest that IL-8 induced by H. pylori or by general inflammatory conditions when IL-8 is induced is a mechanism for up-regulation of CD74 surface expression.
Adherence to the gastric epithelium is undoubtedly a primordial event in bacterial colonization of the gastric mucosa (7). We have shown that CD74 is an essential receptor for H. pylori on gastric epithelial cells, and this bacterium is capable of up-regulating one of its receptors. When CD74 is up-regulated on the surface of gastric cells, more receptors are present for increased bacterial attachment, leading to enhanced attachment. These results uncover relevant information pertaining to lengthy infections and chronic gastritis often seen with H. pylori infection. IL-8 is a central player in this system, and the up-regulation of CD74 by IL-8 is an important immunological finding that extends beyond H. pylori studies and should be further studied for its role in the inflammatory response in gastritis unrelated to H. pylori infection.
Acknowledgments
We thank Dr. D. E. Taylor (University of Alberta, Edmonton, Alberta, Canada) for the kind gift of the chloramphenicol resistance cassette.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the National Institutes of Health Grants DK50669 and DK56338. E.J.B. was a recipient of a fellowship under National Institutes of Health T32 AI007536-06 Training Grant.
2 Address correspondence and reprint requests to Dr. Victor E. Reyes, Children’s Hospital, Room 2.300, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555. E-mail address: vreyes@utmb.edu
3 Abbreviations used in this paper: MIF, macrophage migration inhibitory factor; PAI, pathogenicity island; CM, conditioned medium.
Received for publication February 16, 2005. Accepted for publication April 27, 2005.
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