Immune reactions against elongation factor 2 kinase: specific pathogenesis of gastric ulcer from Helicobacter pylori infection

Abstract

Helicobacter pylori (H. pylori) infection is a definite causative factor for gastric ulcers (GUs). In the present study we detected a specific antigen of gastric epithelial cells (HGC-27) using cell ELISA, which was recognized by the sera of GU patients (n = 20) but not in patients with chronic gastritis (CG; n = 20) or in healthy volunteers (HC; n = 10). This antigen was over-expressed by a stressful (heat-stressed) environment, and was identified as elongation factor 2 kinase (EF-2K) by western blotting. The GU patients' lymphocytes stimulated by H. pylori specifically disrupted heat-stressed HGC-27 cells in a cytotoxic assay. In flow cytometry, the effector cells (lymphocytes) from GU patients were significantly differentiated to T helper type 1 lymphocyte (Th1) and cytotoxic T lymphocyte (CTL) as opposed to those from CG patients. The target cells (HGC-27) expressed EF-2K and MHC-class I together with costimulatory molecules from heat stress. This antigen specific immune mechanism could have a prominent role in the pathogenesis of GU.

Figure 1

Autoantibody to heat-stressed HGC-27 cells in GU patients. (a) The titers of anti-HGC27 antibodies in patients with H. pylori infection and healthy volunteers are shown. The open column indicates the antibody titer in GU patients, the striped is CG patients, and the solid is HC. The left column displays the titers of anti-nonheat-stressed HGC27 antibody and the right antiheat-stressed HGC27 antibody in each group. The titers of anti-HGC-27 antibody were significantly elevated by heat stress in GU and CG patients (*P < .01).

Figure 2

The putative antigen in heat-stressed HGC-27 cells was detected as EF-2K. (a) Western blotting of HGC-27 cells and heat-stressed HGC-27 cells by the sera of GU and CG. Lane 1; molecular weight marker, lanes 2–4; the proteins from heat-stressed HGC-27 cells reacted by GU sera, lanes 5–7; the proteins from heat-stressed HGC-27 cells reacted by CG sera, lanes 8–10; the proteins from nonstressed HGC-27 cells reacted by GU sera, lanes 11–13; the proteins from nonstressed HGC-27 cells reacted by CG sera. 90 kDa protein from heat-stressed HGC-27 cells reacted by GU sera specifically. (b) The expression and purification of EF-2K recombinant protein. About 100 kDa protein was purified. (c) Antibody to EF-2K recombinant protein in patients with GU and CG. The titer of anti-EF-2K recombinant protein antibodies in GU was significantly higher than that in CG (*P < .05). (d) Quantification of EF-2K expressions in HGC-27 infected by H. pylori. The EF-2K expressions in HGC-27 were enhanced by H. pylori infection as by heat stress.

Figure 3

Specific cytotoxicity induced by lymphocytes from GU patients. 4 target cells were prepared. (a) HGC-27 cells treated with heat stress and patients' sera, (b) HGC-27 cells treated with heat stress, (c) HGC-27 cells treated with patients' sera, (d) nontreated HGC-27 cells. PBMCs were taken from GU patients (n = 8). PBMCs stimulated with conA and H. pylori (■) in addition to IL-12 (∆) or IL-4 (●) were used as effector cells. Nonstimulated PBMCs (◊) were also used as a control. Both cells reacted with various E/T ratios. An E/T ratio dependent cytotoxicity was observed only against heat-stressed HGC-27 cells or heat-stressed and sera-treated HGC-27 cells. The cytotoxicities of effector cells stimulated by H. pylori lysate were significantly enhanced, regardless of additional cytokine stimulations (*; P < .05). (e) The comparison of cytotoxicities among lymphocytes from CG, GU, and GU posteradication patients. Target cells were HGC-27 cells treated with heat stress and patients' sera. Effector cells were PBMCs stimulated with conA, H. pylori, and IL-12. The E/T ratio was 4. Specific cytotoxicity was only observed in GU patients' lymphocytes.

Figure 4

Flow cytometric analyses of effector cells. Typical subsets analysis of CD4⁺ (a) and CD8⁺ (b) lymphocytes from GU patients, and CD4⁺ (c) and CD8⁺ (d) lymphocytes from CG patients are shown. H. pylori lysate induced the expression of IFN-γ in CD4⁺ cells regardless of additional cytokine stimulations, while CG indicated the expression of IFN-γ in CD4⁺ cells only by IL-12 stimulation. The same tendency was observed in CD8⁺ cells.

Figure 5

Flow cytometric analyses of target cells. (a) Typical analysis of EF-2K expression on HGC-27 cells is shown. The shadowed histogram indicates the EF-2K detected by GU sera in nontreated HGC-27 cells. And the bold histogram indicates the EF-2K detected by GU sera in heat-stressed HGC-27 cells. (b) Typical analysis of MHC class I molecule expression on HGC-27 cells is shown. The shadowed histogram indicates MHC class I molecules detected by PE-Cy5 conjugated mouse antihuman HLA-A, B, C antibody in nontreated HGC-27 cells and the bold histogram indicates MHC class I molecules detected by PE-Cy5 conjugated mouse antihuman HLA-A, B, C antibody in heat-stressed HGC-27 cells. The costimulatory molecules detected by FITC conjugated mouse antihuman CD80 antibody and PE conjugated mouse antihuman CD86 antibody on nontreated HGC-27 cells are shown in (c) and those of heat-stressed HGC-27 cells in (d). The heat stress enhanced the expression of EF-2K, MHC class I molecules, and costimulatory molecules on HGC-27 cells (target cells).