Interleukin-12 drives the Th1 signaling pathway in Helicobacter pylori-infected human gastric mucosa

Abstract

In this study we examined mechanisms that regulate T-helper lymphocyte 1 (Th1) commitment in Helicobacter pylori-infected human gastric mucosa. The levels of gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-12 in total extracts of gastric biopsies taken from H. pylori-infected and uninfected patients were determined by an enzyme-linked immunosorbent assay. The levels of signal transducer and activator of transcription 4 (STAT4), STAT6, and T-box expressed in T cells (T-bet) in total proteins extracted from gastric biopsies were determined by Western blotting. Finally, the effect of a neutralizing IL-12 antibody on expression of Th1 transcription factors and the levels of IFN-gamma in organ cultures of H. pylori-infected biopsies was examined. Increased levels of IFN-gamma and IL-12 were found in gastric biopsy samples of H. pylori-infected patients compared to the levels in uninfected patients. In addition, H. pylori-infected biopsies exhibited high levels of expression of phosphorylated STAT4 and T-bet. Higher levels of IFN-gamma and expression of Th1 transcription factors were associated with greater infiltration of mononuclear cells in the gastric mucosa. By contrast, production of IL-4 and expression of phosphorylated STAT6 were not associated with the intensity of mononuclear cell infiltration. In ex vivo organ cultures of H. pylori-infected biopsies, neutralization of endogenous IL-12 down-regulated the expression of phosphorylated STAT4 and T-bet and reduced IFN-gamma production. Our data indicated that IL-12 contributes to the Th1 cell commitment in H. pylori-infected human gastric mucosa.

FIG. 1.

Levels of the IFN-γ (A) and IL-4 (B) proteins were determined by ELISA using 100-μg portions of total protein extracts obtained from fresh gastric biopsies from H. pylori-infected patients (n = 10; median inflammation score, 2.5 ± 0.26) and from uninfected patients with NSAID-related gastritis (n = 6; median inflammation score, 1.5 ± 0.22) (w gastritis) and without gastritis (n = 4; inflammation score, 0) (w/o gastritis). Statistical significance was assessed by ANOVA and Tukey's multiple-comparison tests. ns, not significant.

FIG. 2.

Activation of Th1 and Th2 transcription factors in gastric mucosa in H. pylori-infected patients (n = 10; median inflammation score, 2.5 ± 0.26) and in uninfected patients with NSAIDS-related gastritis (n = 6; median inflammation score, 1.5 ± 0.22) (w gastritis) and without gastritis (n = 4; inflammation score, 0) (w/o gastritis). Western blot analyses of phosphorylated STAT4 and STAT4 (A), T-bet and β-actin (B), and pSTAT6 and STAT6 (C) were performed by using total proteins extracted from gastric biopsies from 20 patients. In each panel a polyacrylamide gel shows the results of one representative experiment for 10 (lane 1), 6 (lane 2), and 4 (lane 3) patients. The results are expressed in arbitrary units (a.u.). Statistical significance was assessed by ANOVA and Tukey's multiple-comparison tests. NS, not significant.

FIG. 3.

Levels of IL-12 protein as determined by ELISA in 100-μg portions of total protein extracts obtained from freshly gastric biopsies from H. pylori-infected patients (n = 10; median inflammation score, 2.5 ± 0.26) and from uninfected patients with NSAID-related gastritis (n = 6; median inflammation score, 1.5 ± 0.22) (w gastritis) and without gastritis (n = 4; inflammation score, 0) (w/o gastritis). Statistical significance was assessed by ANOVA and Tukey's multiple-comparison tests. ns, not significant.

FIG. 4.

Neutralization of endogenous IL-12 results in decreased activation of Th1 transcription factors. Western blot analyses of STAT4 (A), T-bet (B), and STAT6 (C) were performed by using total protein extracts from gastric biopsy cultures from 10 H. pylori-positive patients treated without or with a control IgG antibody (1 μg/ml) or anti-IL-12 antibody (5 μg/ml). In each panel a polyacrylamide gel shows the results of one representative experiment for the 10 patients. The results are expressed in arbitrary units (a.u.). Statistical significance was assessed by ANOVA and Tukey's multiple-comparison tests. Ab, antibody; ns, not significant.

FIG. 5.

Neutralization of endogenous IL-12 results in decreased IFN-γ production but not decreased IL-4 production. IFN-γ (A) and IL-4 (B) levels were determined by ELISA by using 100 μg of total protein from the supernatant after 24 h of culture of gastric biopsies from the 10 H. pylori-infected patients treated without or with a control IgG antibody (1 μg/ml) or an anti-IL-12 antibody (5 μg/ml). Statistical significance was assessed by ANOVA and Tukey's multiple-comparison tests. Ab, antibody; ns, not significant.