Analysis of cytokines in the early development of gastric secondary lymphoid follicles in Helicobacter pylori-infected BALB/c mice with neonatal thymectomy

Abstract

Immunological interaction between the host and Helicobacter pylori seems to play a critical role in follicular formation in gastric mucosa. We reported H. pylori-induced follicular gastritis model using neonatally thymectomized mice. In this study, we investigated the involvement of various cytokines in this model. BALB/c mice were thymectomized on the third day after birth (nTx). At 6 weeks old, these mice were orally infected with H. pylori. Histological studies showed that follicular formation occurred from 8 weeks after the infection and that most of the infiltrating lymphocytes were CD4(+) and B cells. Neutrophils increased transiently at 1 week after the infection. Gamma interferon, interleukin-7 (IL-7), and IL-7 receptor were expressed in the stomach of the nTx mice irrespective of the infection. In contrast, expressions of the tumor necrosis factor alpha, IL-4 and lymphotoxin-alpha genes were remarkably upregulated by the infection. Our findings suggest that follicular formation may require cooperative involvement of a Th2-type immune response, tumor necrosis factor alpha and lymphotoxin-alpha in addition to the Th1-type immune response in H. pylori-induced gastritis in nTx mice.

FIG. 1

Histologic findings of the gastric mucosa in neonatally thymectomized (nTx) BALB/c mice with H. pylori infection. Neonatal thymectomy was performed on the 3rd day after birth. nTx mice at 6 weeks old were orally infected with H. pylori and sacrificed 1, 2, 4, 8, and 12 weeks later. (a) At the age of 18 weeks, noninfected nTx mice showed infiltration of mononuclear cells and gland atrophy characterized by loss of parietal and chief cells, with replacement by proliferating epithelial cells. Magnification, ×50. Histologic findings of nTx mice without H. pylori infection were not changed throughout the experiment. (e and f) In infected nTx mice, follicle formation was observed at 8 weeks after infection in 7 of 10 mice (P < 0.05 versus noninfected nTx mice) (e), and the body mucosa clearly showed germinal center formation at 12 weeks after infection in 6 of 10 mice (P < 0.05 versus noninfected nTx mice) (f). The photographs in panels b, c, and d show gastric histology at 1, 2, and 4 weeks after H. pylori infection in nTx mice, respectively.

FIG. 2

Flow cytometric analysis of mononuclear cells infiltrating the gastric mucosa of nTx mice after H. pylori infection. Infected nTx mice were sacrificed 1, 2, and 4 weeks later. Infiltrating mononuclear cells were collected from the whole stomachs of three mice at each time point. (a) The panel shows that neutrophils were transiently increased at 1 week after H. pylori infection (P < 0.05 versus noninfected nTx mice; 2.7, 8.6, 2.9, and 0.3% at 0, 1, 2, and 4 weeks, respectively). Panels b and c show that the ratios of both macrophages (3.4, 4.0, 4.1, and 4.0% at 0, 1, 2, and 4 weeks) and γδ T cells (3.5, 1.8, 4.7, and 2.9% at 0, 1, 2, and 4 weeks) were not significantly changed by H. pylori infection. The data are representative of three separate experiments and are expressed as the percentage of all of the infiltrating cells.

FIG. 3

RT-PCR of various cytokine mRNAs in the gastric mucosa after H. pylori infection. IFN-γ mRNA was present in nTx mice, but its expression was not altered by H. pylori infection. LT-α, TNF-α, and IL-4 gene expressions were upregulated in all infected nTx-mice at 1, 1, and 2 weeks after infection, respectively. There were no differences in the gene expression of the IL-7 and IL-7R genes between infected and noninfected nTx mice. N, noninfected normal (non-nTx) mice; nTx, noninfected nTx mice at 18 weeks old. The designations 1w, 2w, 4w, 8w, and 12w refer to 1, 2, 4, 8, and 12 weeks, respectively, after infection in nTx mice.