A functional role for CCR6 on proallergic T cells in the gastrointestinal tract - PubMed (original) (raw)

A functional role for CCR6 on proallergic T cells in the gastrointestinal tract

Ana Belén Blázquez et al. Gastroenterology. 2010 Jan.

Abstract

Background & aims: CCL20 is a chemokine that regulates the homeostatic and inflammatory trafficking of leukocytes to the small intestine and regulates the development of the gastrointestinal lymphoid architecture. T cells expressing T helper cell (Th) 2 cytokines are critical for experimental food allergy, and we hypothesized that CCL20 is involved in the localization of these cells to the gut.

Methods: We evaluated the role of CCR6 in allergic diarrhea induced by sensitization and oral challenge with ovalbumin (OVA) using CCR6(+/+) and CCR6(-/-) mice.

Results: CCR6(-/-) mice were protected from OVA-induced diarrhea but surprisingly were not impaired in mastocytosis or allergen-specific immunoglobulin E. CCR6(-/-) mice were also protected from T cell-mediated diarrhea induced by anti-CD3 antibody. Allergic diarrhea was associated with an increased expression of Th2 cytokines within the intestinal mucosa that was significantly reduced in CCR6(-/-) mice. Inhibition of lymphocyte homing by treatment with FTY720 did not impair allergic diarrhea, indicating that reactivation of T cells could occur locally within the small intestine. Finally, T-cell transfer studies demonstrated that CCR6 was required both on the transferred T cells and in the recipient mouse to manifest allergic disease in the gastrointestinal tract.

Conclusions: These studies highlight a mast cell- and immunoglobulin E-independent role for CCR6-bearing T cells in the pathogenesis of gastrointestinal allergic disease.

Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

The authors have no financial disclosures. The authors have no conflicts of interest to disclose.

Figures

Figure 1

Figure 1. CCL20 is upregulated during allergic diarrhea and CCR6 is required for diarrhea symptoms

CCR6+/+ and CCR6−/− mice were sensitized and orally challenged with OVA. A. CCL20 mRNA expression in the jejunum of CCR6+/+ and CCR6−/− mice. Data are expressed as fold-changed compared to unchallenged controls. B. Immunostaining for CCL20 in the small intestine (bottom panel shows anti-CCL20 staining, top panel is isotype control). Note the dense immunoreactivity in the follicle-associated epithelium. C. Onset of symptoms (% of mice with visible diarrhea) after each feed was recorded. n = 33 (+/+) and 36 (−/−).

Figure 2

Figure 2. Allergen-induced Jejunal Mast Cells and serum IgE are CCR6-independent

CCR6+/+ and CCR6−/− mice were sensitized and with OVA. 4 mice were sacrificed after each feed of OVA (from 0 to 4). Top panel: Serum OVA-specific IgE. Middle panel: Jejunum mast cell counts per high power field (HPF). Bottom panel: MMCP-1 mRNA expression in jejunum from sacrificed after 4 (OVA/OVA) or 0 (OVA/PBS) feeds of OVA. ** p < 0.01, NS = not significant.

Figure 3

Figure 3. CCR6 is required for T cell-mediated, but not toxin-induced diarrhea

A: CCR6+/+ and CCR6−/− mice were fed 50 μg of cholera toxin or PBS as control. After 3 h, mice were euthanized and ligated loops were prepared from small intestine. Wet/dry weights were calculated as in methods. n = 3/group. B: CCR6+/+ and CCR6−/− mice were injected with 0.2 mg of anti-CD3 antibody or left un-injected as control. After 2 h, ligated loops were prepared as above. n = 7/group. ** p < 0.01; *** p < 0.001; ns = not significant.

Figure 4

Figure 4. Allergen-induced Th2 cytokine expression in the small intestine is impaired in CCR6 −/− mice

Left panels: CCR6+/+ and CCR6−/− mice were sensitized to OVA and challenged with OVA (OVA/OVA) or PBS (OVA/PBS) as control. Mice were euthanized within 1–2 h after symptom onset. (n= 10 samples/group) Right panels: Mice were sacrificed after each OVA feeding (4 mice/group). RNA was isolated from jejunum, and RT-PCR for IL-4 (top panel), IL-13 (middle panel) and IL-10 (bottom panel) was performed on individual samples (left panels) or pooled samples from the time course (right panels).

Figure 5

Figure 5. Th2 responses in the mesenteric lymph node are normal in CCR6 −/− mice

CCR6+/+ and CCR6−/− mice were sensitized to OVA and challenged with OVA (OVA/OVA) or PBS (OVA/PBS) as control. MLN cells were re-stimulated with OVA and IL-4, IL-5, IL-13, and IL-10 were measured in culture supernatants (Panel A). CFSE-labeled OVA-specific DO11.10 cells were transferred to naïve CCR6+/+ and CCR6−/− mice. Mice were fed 50 mg OVA (+OVA) or remained unfed as control (−OVA). After 96 h, T cell proliferation in the MLN was assessed by CFSE dilution (Panel B).

Figure 6

Figure 6. T cell reactivation occurs locally in the absence of homing from lymph nodes

Mice were treated with FTY720 daily beginning one day prior to beginning the OVA feeds. Efficacy of the FTY720 treatment was checked by flow cytometry of peripheral blood (A), which showed a near abolishment of circulating CD4 and CD8 T cells. FTY720 treatment had no effect on onset of OVA-induced diarrhea (B), jejunal mastocytosis (C), or jejunal IL-4 (D) or IL-13 (E) expression. Data are representative of two independent experiments, with a total of 10 mice per group.

Figure 7

Figure 7. CCR6 on T cells is necessary, but not sufficient, for allergic diarrhea

MLN cells from OVA-sensitized and fed (AD) CCR6+/+ or CCR6−/− mice were re-stimulated with OVA in vitro before transferring to naïve Balb/c mice. Mice were then fed with OVA every second day and diarrhea symptoms recorded. (A) The cytokine output of the transferred cells prior to transfer. (B) Symptoms of wild-type mice receiving primed CCR6+/+ or CCR6−/− T cells as above. (C) Donor cells were CCR6+/+, and recipients were CCR6+/+ or CCR6−/− as indicated.

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