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Abstract
Eosinophils are multifunctional leukocytes that reside in several tissues, most abundantly in the small intestinal lamina propria under the steady state. To date, the phenotypic and functional characteristics of small intestinal eosinophils have remained poorly understood. In this study, we found that proliferation of ovalbumin (OVA)-specific CD4+ T cells isolated from the mesenteric lymph nodes of eosinophil-deficient ΔdblGATA mice were decreased relative to wild-type mice after oral immunization with OVA and cholera toxin (CT), the typical mucosal adjuvant that induces CD4+ T cell-dependent responses. ΔdblGATA mice showed reduced mucosal secretion of OVA-specific IgA and IgG1 while maintaining a systemic level of anti-OVA IgG1 upon oral immunization with OVA and CT. These findings suggest that eosinophils might have a role in the modulation of T cell-mediated immune responses including mucosal antibody responses in the gastrointestinal tract following oral immunization.
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Figure 1.
Phenotypic characteristics of leukocytes isolated from the small intestinal lamina propria (LP) of wild-type (WT) and ΔdblGATA mice. (A) Small intestinal LP cells of WT and ΔdblGATA mice were stained with anti-CD11b and anti-CD172a, anti-SiglecF, anti-CCR3, or anti-IL-5Rα, then analyzed by flow cytometry. The R1 gate represents the medium to high side scatter (SSC) subset, and the R2 and R3 gates correspond to the mononuclear cell fractions of WT and ΔdblGATA mice, respectively. FSC indicates a forward light scatter. (B) The R1 and R2 of the LP cells of WT mice and the R3 of ΔdblGATA intestinal LP cells were analyzed for the expression of various cell-surface molecules. (C and D) Isolated blood (C) and bone marrow cells (D) were stained with anti-CCR3 and anti-SiglecF and analyzed by flow cytometry. A granulocyte population with medium to high SSC was gated and analyzed for their expression of CCR3 and SiglecF. Eosinophil populations (CCR3+ SiglecF+) were not observed in the blood or bone marrow of ΔdblGATA mice. The CCR3−SiglecF+ subset, observed only in the bone marrow of WT mice, might represent an immature type of eosinophil. The results shown are representative of three independent experiments.
Figure 2.
Reduced proliferation of CD4+ T cells isolated from the mesenteric lymph node (MLN) of ΔdblGATA mice after oral immunization with ovalbumin (OVA) and cholera toxin (CT). (A and B) CD4+ T cells isolated from the MLN of WT or ΔdblGATA mice after oral immunization with PBS (control), OVA or OVA+CT were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and co-cultured with OVA-loaded APC for 72 h. Representative flow cytometry analysis of CD4+ CFSE+ cells (A). (B) CD4+ T cell proliferation presented as percentage of CD4+CFSE+ cells. (C and D) CD4+ T cells isolated from the MLN of WT or ΔdblGATA mice after oral immunization with PBS, OVA or OVA+CT were labeled with CFSE and cultured for 72 h with anti-CD3 and anti-CD28 antibodies. Representative flow cytometry analysis of CD4+ CFSE+ cells (C). (D) CD4+ T cell proliferation presented as a percentage of CD4+ CFSE+ cells. Data are mean ± s.e.m. values. ∗ p < 0.05, ∗∗∗ p < 0.001 (Student's t-test).
Figure 3.
OVA-specific intestinal IgA and IgG1 are reduced in ΔdblGATA mice orally immunized with OVA plus CT. (A) The frequencies of IgA+ cells in the Peyer's patch (PP) and small intestinal LP of WT and ΔdblGATA mice after oral immunization with PBS (control) or OVA+CT. The results shown are representative of three independent experiments. (B) OVA-specific IgA and IgG1 titers in serum and intestinal wash of WT and ΔdblGATA mice after oral immunization with PBS (control), OVA or OVA+CT. Each group consists of at least three individual mice. The absorbance at 450 nm represents antibody titers. Data are mean ± s.e.m. values. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (Student's t-test).
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