Distinct patterns of mucosal apoptosis in H pylori-associated gastric ulcer are associated with altered FasL and perforin cytotoxic pathways

Abstract

Aim: To analyze the level of apoptosis in different mucosal compartments and the differential expression of Fas/Fas-ligand and perforin in H pylori-associated gastric ulcer.

Methods: Antral specimens from patients with H pylori-related active gastric ulcer (GU), H pylori-related gastritis, and non-infected controls were analysed for densities and distribution of apoptotic cells determined by the TdT-mediated dUDP-biotin nick-end-labelling method. GU patients were submitted to eradication therapy with follow-up biopsy after 60 d. Fas, FasL, and perforin-expressing cells were assessed by immunoperoxidase, and with anti-CD3, anti-CD20 and anti-CD68 by double immunofluorescence and confocal microscopy. Quantitative analysis was performed using a computer-assisted image analyser.

Results: H pylori-infected antrum showed greater surface epithelial apoptosis which decreased after eradication therapy. In the lamina propria, higher rates of mononuclear cell apoptosis were observed in H pylori-gastritis. Co-expression of Fas with T-cell and macrophage markers was reduced in GU. FasL- and perforin-expressing cells were increased in H pylori-infection and correlated with epithelial apoptosis. Perforin-expressing cells were also increased in GU compared with H pylori-gastritis.

Conclusion: Epithelial apoptosis is increased in H pylori-infection and correlates to FasL- and perforin-expression by T cells. Expression of perforin is correlated with the tissue damage, and may represent the enhancement of a distinct cytotoxic pathway in GU. Increased expression of FasL not paralleled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells in H pylori-infection.

Figure 1

Terminal deoxynucleotidyltransferase (TdT)-mediated dUDP-biotin nick end labelling (TUNEL)-positive apoptotic cells. Photomicrographs show tissues obtained from the antral mucosa of: a non-infected control patient (A), a patient with H pylori-associated gastritis (B), a patient with active gastric ulcer (C), at the ulcer margin of the same patient (D), and after eradication therapy (E), and a negative control, without TdT enzyme (F), respectively. Arrowheads show positive cells in the epithelium and in the lamina propria (Original magnification × 400).

Figure 2

Percentages of apoptotic cells in the surface and crypt epithelium, and in the lamina propria of patients with gastric ulcer (GU) [at the ulcer margin, and the antrum before and after eradication therapy (ET)], H pylori-associated gastritis, and non-infected controls, respectively. Horizontal bars represent medians, boxes represent the 25th and 75th percentiles, and vertical bars represent ranges. In the surface epithelium values were significantly different compared to GU after ET (P = 0.018; P = 0.003; P = 0.027); gastritis (P = 0.003; P = 0.002), and the control group (P < 0.001; P = 0.001). In the lamina propria values were significantly different compared to GU after ET (P = 0.001), and the control group (P = 0.001). In the crypt epithelium values were not significantly different among groups (n: GU = 12, H pylori-associated gastritis = 11, control = 10). Differences were analysed using one-way ANOVA with the Dunnett’s test for multiple comparisons. The Wilcoxon signed rank test was used for comparisons between GU patients.

Figure 3

Quantitative analysis of apoptosis-related proteins in the gastric lamina propria of patients with gastric ulcer (GU) [at the ulcer margin, and the antrum before and after eradication therapy (ET)], H pylori-associated gastritis, and non-infected controls, respectively. Horizontal bars represent medians, boxes represent the 25th and 75th percentiles, and vertical bars represent ranges. Values of Fas were significantly different compared with the antral mucosa after ET (^aP = 0.005, ^bP = 0.008), and the normal group (^aP < 0.001, ^bP = 0.031). Values of FasL were significantly different compared to GU after ET (^cP = 0.003, ^dP = 0.008), gastritis (^cP < 0.001), and the control group (^cP < 0.001, ^dP = 0.018). Values of perforin were significantly different compared to GU after ET (^eP = 0.005, ^fP = 0.008), gastritis (^eP = 0.004, ^fP = 0.033), and the control group (^eP = 0.001, ^fP = 0.006) (n: GU = 12, H pylori-associated gastritis = 11, control = 10). Differences were analysed using one-way ANOVA with the Dunnett’s test for multiple comparisons. The Wilcoxon signed rank test was used for comparisons between GU patients.

Figure 4

Double immunofluorescence analysis. Expression of CD3 (FITC) and Fas (R-PE) in the ulcer margin of a patient with gastric ulcer (A) and a non-infected control (B). Expression of CD68 (FITC) and Fas (R-PE) in the antrum of a patient with gastric ulcer (C) and a patient with H pylori-related gastritis (D). Images obtained by confocal microscopy. Arrows show CD3 or CD68 (FITC)-single-positive cells (green) without correspondent Fas (R-PE)-positive cells in the lamina propria of the same tissue section.

Figure 5

Percentages of the expression of Fas by CD3, CD20, and CD68 positive cells in the antral lamina propria of patients with gastric ulcer (GU) [at the ulcer margin, and at the antrum before and after eradication therapy (ET)], patients with H pylori-associated gastritis, and from non-infected controls, respectively. Horizontal bars represent medians, boxes represent the 25th and 75th percentiles, vertical bars represent ranges. In regard of the expression of Fas with CD3 values were significantly different compared to GU after ET (^a,bP = 0.043), gastritis (^aP = 0.021); and the control group (^aP < 0.001; ^bP = 0.012). For Fas expression with CD20 values were not significantly different among groups. For Fas expression with CD68 values were significantly different compared to GU after ET (^c,dP = 0.043), gastritis (^cP = 0.007; ^dP = 0.034); and the control group (^cP = 0.012; ^dP = 0.027) (n = 6, in each group). Differences were analysed using one-way ANOVA with the Dunnett’s test for multiple comparisons. The Wilcoxon signed rank test was used for comparisons between GU patients.

Figure 6

Apoptosis-related proteins in the gastric mucosa stained with immunoperoxidase. Photomicrographs show the antral mucosa stained for FasL in a non-infected control patient (A), at the ulcer margin (B), and after eradication therapy of the same patient (C). Immunostaining for perforin is shown in a patient with H pylori-associated gastritis (D), and in a patient with gastric ulcer before (E), and after eradication therapy (F), respectively. Arrowheads show immunoreactive cells (Original magnification × 400).