Partial protection against Helicobacter pylori in the absence of mast cells in mice

Abstract

The goal of this study is to evaluate the contribution of mast cells to Helicobacter pylori immunity in a model of vaccine-induced protection. Mast cell-deficient Kitl(Sl)/Kitl(Sl-d) and control mice were immunized with H. pylori sonicate plus cholera toxin and challenged with H. pylori, and the bacterial loads, inflammatory infiltrates, and cytokine responses were evaluated and compared at 1, 2, and 4 weeks postchallenge. In vitro stimulation assays were performed using bone marrow-derived mast cells, and recall assays were performed with spleen cells of immunized mast cell-deficient and wild-type mice. Bacterial clearance was observed by 2 weeks postchallenge in mast cell-deficient mice. The bacterial load was reduced by 4.0 log CFU in wild-type mice and by 1.5 log CFU in mast cell-deficient mice. Neutrophil numbers in the gastric mucosa of immune Kitl(Sl)/Kitl(Sl-d) mice were lower than those for immune wild-type mice (P < 0.05). Levels of gastric interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-alpha) were also significantly lower in immune Kitl(Sl)/Kitl(Sl-d) mice than in wild-type mice (P < 0.001). Immunized mast cell-deficient and wild-type mouse spleen cells produced IFN-gamma and IL-17 in response to H. pylori antigen stimulation. TNF-alpha and CXC chemokines were detected in mast cell supernatants after 24 h of stimulation with H. pylori antigen. The results indicate that mast cells are not essential for but do contribute to vaccine-induced immunity and that mast cells contribute to neutrophil recruitment and inflammation in response to H. pylori.

FIG. 1.

Mast cell-deficient mice exhibit compromised immunity to vaccine-induced H. pylori-specific immunity. Immunized and unimmunized wild-type (WT) and mast cell-deficient (Kit) mice were challenged with H. pylori, and subgroups of mice were assessed for bacterial load at 1, 2, and 4 weeks by CFU determination. Each group contained 6 to 11 mice. Error bars indicate standard deviations from the means. **, P < 0.01; ***, P < 0.001 (compared to U/C control mice).

FIG. 2.

Immunized mast cell-deficient mice have reduced neutrophil counts in the gastric mucosa postchallenge compared to the counts for immunized wild-type mice. Immunized and unimmunized wild-type (WT) and mast cell-deficient (Kit) mice were challenged with H. pylori, and the gastric mucosa was assessed at 2 (a and b) and 4 (c and d) weeks postchallenge for granulocyte recruitment. Tissue sections were Giemsa stained, and the numbers of mast cells (a and c) and neutrophils (b and d) were quantified in a blinded fashion. Each group contained 6 to 11 mice. Error bars indicate standard deviations from the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to the wild-type I/C group).

FIG. 3.

BMMC produce neutrophil recruitment and activate cytokines in response to stimulation with H. pylori antigens. Mast cells were grown from mouse bone marrow cells for 4 weeks and then stimulated with decreasing concentrations of H. pylori lysate antigens (Hp Ag) or E. coli lipopolysaccharide (LPS) in vitro for 24 h. TNF-α (a), CXCL1/KC (b), and MIP-2 (c) concentrations were determined by quantitative ELISA. The data are representative of two independent experiments. Assays were performed in quadruplicate. Error bars indicate standard deviations from the means. **, P < 0.01; ***, P < 0.001 (compared to unstimulated cells [PBS]).

FIG. 4.

Helicobacter-induced cytokine production by BMMC is not strain dependent. Mast cells were grown from mouse bone marrow cells for 4 weeks, and then 2 × 10⁵ cells were stimulated with 100 μg/ml of bacterial lysate prepared from one of seven H. pylori strains of various genotypes and phenotypes or from H. felis. Cell culture supernatants were assessed for TNF-α and KC at 24 h by ELISA. Assays were performed in quadruplicate. Error bars indicate standard deviations from the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to stimulation with H. pylori SS1). LPS, lipopolysaccharide.

FIG. 5.

Immunized mast cell-deficient mice have reduced levels of IL-17 and TNF-α in the gastric mucosa 2 weeks after challenge. Immunized and unimmunized wild-type (WT) and mast cell-deficient (Kit) mice were challenged with H. pylori. In one experiment, real-time PCR analysis was performed with the mice that were evaluated at 2 weeks postchallenge for the presence of IL-17 (a) and TNF-α (b) expression in the gastric mucosa. Values obtained for challenged mice are expressed as relative units indicating the increase (n-fold) over the level for naïve control samples. Each group contained 10 mice. Error bars indicate standard deviations from the means. ***, P < 0.001 (compared to all other groups).

FIG. 6.

The T-cell cytokine recall response of immunized mast cell-deficient mice is not reduced compared to that for immunized wild-type mice. Wild-type C57BL/6 mice (W), Kitl^Sl/Kitl^Sl-d mast cell-deficient mice (K), and Kitl^Sl/Kitl^Sl-d wild-type littermates (L) were immunized, and the spleen cells were removed 2 weeks after the final booster. Spleen cells (1 × 10⁶ cells per well) were stimulated with either 10 or 100 μg/ml H. pylori lysate for 48 h. Supernatants were assessed by ELISA for IFN-γ (a) and IL-17 (b). Assays were performed in triplicate. I, immunized (two mice in each group); N, naïve (two mice in each group). Error bars indicate standard deviations from the means. ***, P < 0.001 (compared to I/W mice).