Monocyte chemoattractant protein 1 (MCP-1) released from Helicobacter pylori stimulated gastric epithelial cells induces cyclooxygenase 2 expression and activation in T cells

Abstract

Background: and aims: To clarify the interaction between gastric epithelial and mucosal T cells, we examined the role of cytokines released from epithelial cells in response to Helicobacter pylori water extract protein (HPWEP) in regulating T cell cyclooxygenase 2 (COX-2) expression and activation.

Methods: Media from MKN-28 cells incubated with HPWEP for 48 hours were added to Jurkat T cells and human peripheral T cells. C-C and CXC chemokine concentrations in MKN-28 cell media, and COX-2 expression, interferon gamma (IFN-gamma), and interleukin (IL)-4 secretions in T cells were determined by western blot analysis and ELISA methods. Distributions of COX-2 positive T cells and monocyte chemoattractant protein 1 (MCP-1) in tissue specimens with H pylori associated gastritis were determined as single or double labelling by immunohistochemistry.

Results: MCP-1, IL-7, IL-8, and RANTES were detected in media from MKN-28 cells incubated with HPWEP. Media as a whole, and MCP-1 alone, stimulated COX-2 expression and peripheral T cell proliferation. Anti-MCP-1 antibody inhibited media stimulated COX-2 mRNA expression in Jurkat T cells. Media stimulated IFN-gamma but not IL-4 secretion from peripheral T cells, while MCP-1 stimulated IL-4 but not IFN-gamma secretion. Both stimulated cytokine release, and peripheral T cell proliferation was partially inhibited by NS-398, a specific COX-2 inhibitor. In mucosa with gastritis, COX-2 was expressed in T cells and MCP-1 was localised mainly in epithelial and mononuclear cells. MCP-1 levels and the intensity of COX-2 expression in tissue samples were closely related.

Conclusions: Cytokines such as MCP-1, released from gastric epithelial cells in response to HPWEP, seem to modulate T cell immune responses, at least in part via COX-2 expression.

Figure 1

Cyclooxygenase (COX) protein expression in peripheral T cells and Jurkat T cells stimulated by MKN-28 cell media. (A) COX-2 protein expression in T cells stimulated by MKN-28 cell media. COX-2 expression in peripheral T cells incubated with media from Helicobacter pylori water extract protein (HPWEP) exposed MKN-28 cells (lane c), HPWEP (lane d), and unstimulated MKN-28 cell media (lane e). Lanes f–h: as in lanes a–e except that Jurkat T cells instead of peripheral T cells are represented. Lanes a and b indicate COX-2 positive control (70 kDa) and COX-1 positive control (68 kDa), respectively. (B) COX-1 protein expression in T cells stimulated by media from MKN-28 cells. Lane a, COX-1 positive control; lane b, COX-2 positive control; lanes c–h, as in (A) except that COX-1 instead of COX-2 expression is represented. Each panel is representative of four separate experiments.

Figure 2

Comparison of cyclooxygenase (COX) activity in Jurkat T cells. COX activity in stimulated and unstimulated Jurkat T cells was determined as described in materials and methods. Jurkat T cells were stimulated with Helicobacter pylori water extract protein (HPWEP+Jurkat T cells), media from HPWEP exposed MKN-28 cells (HPWEP/MKN-28 medium+Jurkat T cells), and 20 ng/ml PMA (Jurkat T cells+PMA), respectively. Prostaglandin E₂ generated in each sample was determined in duplicate. Each value represents the mean (SEM) of four separate experiments. *p<0.05.

Figure 3

mRNA and protein expression of cytokines in MKN-28 cells and their release into the media. (A) Monocyte chemoattractant protein 1 (MCP-1), interleukin (IL)-7, RANTES, and IL-8 mRNAs were found in Helicobacter pylori water extract protein (HPWEP) exposed MKN-28 cells. In contrast, interferon γ (IFN-γ), macrophage inflammatory protein (MIP)-1α, MIP-1β, and IP-10 mRNAs were not expressed. Each panel is representative of four separate experiments. (B) Cytokine levels were determined as described in materials and methods. Each cytokine was determined in duplicate for each sample. Values are mean (SEM) of four separate experiments.

Figure 4

Effects of cytokines and MG-132 on Jurkat T cell cyclooxygenase 2 (COX-2) protein expression. Lane A, COX-2 positive control; lane B, COX-1 positive control; lane C, COX-2 protein expression in Jurkat T cells incubated with media from Helicobacter pylori water extract protein (HPWEP) exposed MKN-28 cells; lane D, COX-2 protein expression in Jurkat T cells pretreated with MG-132 and then incubated with media from HPWEP stimulated MKN-28 cells; lane E, COX-2 protein expression in Jurkat T cells stimulated with monocyte chemoattractant protein 1 at 100 pg/ml; lanes F–H, lack of COX-2 protein expression in Jurkat T cells stimulated with human recombinant IL-8 40 pg/ml (lane F), IL-7 5 pg/ml (lane G), and RANTES 600 pg/ml (lane H). Experiments were repeated four times and the panel shown is a representative experiment.

Figure 5

Comparison of cyclooxygenase 2 (COX-2) mRNA production in Jurkat T cells. COX-2/β-actin mRNA levels for stimulated Jurkat T cells were determined by real time polymerase chain reaction, as described in materials and methods. COX-2/β-actin mRNA levels of Jurkat T cells increased following incubation with Helicobacter pylori water extract protein (HPWEP) or HPWEP/MKN-28 medium (as in fig 2 ▶) and were suppressed by preincubation with anti-monocyte chemoattractant protein 1 (MCP-1) neutralising antibody (HPWEP/MKN-28 medium+Jurkat T cells+anti-MCP-1Ab). Each value represents the mean (SEM) of four separate experiments. *p<0.05.

Figure 6

T cell cytokine production and proliferation. (A) Proliferation of activated peripheral T cells was determined as described in materials and methods. Peripheral T cells were stimulated by anti-CD3 antibody (0.3 μg/ml), Helicobacter pylori water extract protein (HPWEP)/MKN-28 medium, HPWEP/MKN-28 medium+NS-398 (HPWEP/MKN-28 medium in the presence of 10 μM NS-398), HPWEP/MKN-28 medium+SC-560 (0.03 μM), 100 pg/ml monocyte chemoattractant protein 1 (MCP-1), 100 pg/ml MCP-1+NS-398 (10 μM), and 100 pg/ml MCP-1+SC-560 (0.03 μM). MKN-28 medium, peripheral T cells treated with unstimulated MKN-28 media. For each MTT assay, samples were determined in triplicate. *p<0.05. (B) Interferon γ (IFN-γ) production was determined as described in materials and methods. (C) Interleukin 4 (IL-4) production was determined as described in material and methods. Each value represents the mean (SEM) of 11 separate experiments.

Figure 7

Localisation of cyclooxygenase 2 (COX-2) positive mucosal T cells and monocyte chemoattractant protein 1 (MCP-1) positive cells in the gastric mucosa. (A) COX-2 positive cells in the lamina propria of Helicobacter pylori infected gastritis (original magnification, ×150). (B) CD3 positive cells in the lamina propria of H pylori infected gastritis (original magnification, ×150). (C) Double positive staining (yellow cells) reveals COX-2 positive mucosal T cells in H pylori infected gastritis (original magnification, ×150). (D) Double staining of H pylori uninfected gastritis mucosa with anti-COX-2 antibody and anti-CD3 antibody (original magnification, ×90). (E) MCP-1 positive epithelial cells and mononuclear cells in the lamina propria, as seen in H pylori infected gastritis (original magnification, ×200).

Figure 8

Correlation between monocyte chemoattractant protein 1 (MCP-1) levels and intensity of cyclooxygenase 2 (COX-2) expression in gastric mucosa. Pearson’s correlation analysis was used to assess the correlation between MCP-1 levels and intensity of COX-2 expression in Helicobacter pylori infected gastritis.