Helicobacter pylori binds to CD74 on gastric epithelial cells and stimulates interleukin-8 production

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.

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-γ 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).

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-γ-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.

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.

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.

FIG. 5.

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

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-γ 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.

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.