Treatment of Helicobacter gastritis with IL-4 requires somatostatin

Abstract

Fifty percent of the world's population is infected with Helicobacter pylori; however, treatment has been insufficient to eradicate the organisms due to rising antibiotic resistance. Helicobacter infection is characterized by induction of a T helper 1 lymphocyte (Th1) immune response, hypergastrinemia, and suppressed tissue somatostatin (SOM) levels. However, the mechanism by which the immune response regulates acid secretion is not known. We show here that treatment with IFN-gamma, a Th1 cytokine, was sufficient to induce gastritis, increase gastrin, and decrease SOM levels within 7 days. In contrast, the T helper 2 lymphocyte cytokine IL-4 increased SOM levels and effectively suppressed gastrin expression and secretion. This result demonstrated reciprocal regulation of acid regulatory peptides by immune modulators. IL-4 pretreatment prevented gastritis in infected wild-type but not in SOM null mice. Thus, the ability of IL-4 to oppose a Th1-mediated infection required SOM. Immunofluorescence was used to document the presence of IL-4 receptors on the gastric SOM-secreting cell (D cell). Moreover, IL-4 stimulated SOM release from primary D cell cultures. Treatment of mice chronically infected with Helicobacter felis for 2 mo with the SOM analogue octreotide resolved the inflammation. Thus, a mechanism by which IL-4 resolves inflammation in the stomach is by stimulating the release of SOM from gastric D cells.

Fig. 1.

Reciprocal changes in gastrin and SOM by IFN-γ and IL-4. (a) Immunohistochemical staining of G cells after 7 days of IFN-γ or IL-4 infusion. (b) The G cells were quantified by morphometry, and changes in plasma gastrin were determined by RIA. (c) Tissue SOM was quantified by RIA. IFN-γ inhibited, whereas IL-4 stimulated, the expression of tissue SOM. Shown is the mean ± SEM for eight mice from two independent experiments. *, P < 0.05 relative to PBS-treated mice (unpaired t test). (d) Normal gastric glands in control mice (Top). Recruitment of lymphocytes to the gastric mucosa after 7 days of IFN-γ treatment is shown (arrow, Middle). Mucous gland metaplasia is indicated (open arrow, Bottom). Periodic acid Schiff/alcian blue stains of sections from IFN-γ-infused mice showing mucous gland metaplasia are shown (open arrow, Bottom)(×400).

Fig. 2.

IL-4 prevention of H. felis gastritis is mediated by SOM. Shown are hematoxylin/eosin stains of SOM^+/+ (a) or SOM^-/- (b) mice treated with PBS, H. felis, or IL-4 plus H. felis. Quantitative RT-PCR was performed on total stomach RNA. Shown is the ratio of IFN-γ to GAPDH mRNA in SOM^+/+ (c) or SOM^-/- (d) mice treated with PBS, H. felis, or IL-4 plus H. felis. Data are the mean ± SEM for n = 8 mice, *, P < 0.05 vs. PBS-treated mice (unpaired t test). IL-4 reduces H. felis colonization. Shown is quantitation by qRT-PCR and morphometry of the amount of H. felis colonizing SOM^+/+ (e) or SOM^-/- (f) mice. The log fluorescent emission measured continuously during PCR per cycle was determined. Early amplification showed increased “starting” fluorescent emission and was used to compute the number of Helicobacter per gram of tissue for each mouse by using a standard curve generated from known quantities of H. felis. Scatter plots showing the bacterial count for each mouse are shown. The mean indicated by a horizontal bar for n = 8 mice is shown. Bacteria per crypt were counted on the basis of the presence of spiral-shaped organisms characteristic of Helicobacter. The mean ± SEM for n = 8 mice is shown in parentheses. *, P < 0.05 vs. PBS mice; #, P < 0.05 vs. H. felis mice (unpaired t test). N/D, nondetectable.

Fig. 3.

Inhibition of IFN-γ by IL-4 in CD3⁺ cells is mediated by SOM. FACS of CD3⁺ cells expressing IFN-γ was performed on primary cells isolated from SOM^+/+ (a and b) and SOM^-/- (c and d) mouse stomachs. Primary cultures were exposed to PBS, H. felis, IL-4, or OCT. In addition, cultures were also exposed to H. felis plus IL-4 or OCT with or without SOM antagonist (ANT). Results are expressed as the mean ± SEM for n = 6 experiments performed in triplicate incubations. *, P < 0.05 vs. PBS-treated cultures (unpaired t test).

Fig. 4.

SOM-secreting D cells express IL-4 receptors. (a Left) Immunofluorescence of isolated canine fundic SOM-secreting D cells (Texas red) that colocalizes with IL-4 receptors (FITC). A cytoplasmic process characteristic of the D cell is shown by the arrow. (b) Summary of FACS of the percent of isolated canine fundic D cells expressing IL-4 receptors after 2, 12, and 24 h of treatment with 100 nM IL-4. (c) SOM release in response to100 nM IL-4 for 2, 12, and 24 h. The results are expressed as the percent of initial cell SOM content (700–1,000 pmol/liter) for three independent experiments. *, P < 0.05 vs. unstimulated (unpaired t test).

Fig. 5.

OCT resolves H. felis gastritis. The number of T cells (CD3⁺) after a 2-mo H. felis infection was analyzed by FACS. Shown is the mean ± SEM for eight mice. *, P < 0.05 vs. OCT alone-treated mice; #, P < 0.05 vs. H. felis-treated mice (unpaired t test).