A Functional Role for CCR6 on Proallergic T Cells in the Gastrointestinal Tract

CCL20 is up-regulated 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 with 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 (percent of mice with visible diarrhea) after each feed was recorded. n = 33 (+/+) and 36 (−/−).

Allergen-induced jejunal mast cells and serum IgE are CCR6 independent. CCR6^+/+ and CCR6^−/− mice were sensitized and with OVA. Four mice were killed 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 mice killed after 4 (OVA/OVA) or 0 (OVA/PBS) feeds of OVA. **P < .01; ns = not significant.

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 hours, mice were killed, and ligated loops were prepared from small intestine. Wet/dry weights were calculated as in Materials and Methods section. n = 3/group. (B) CCR6^+/+ and CCR6^−/− mice were injected with 0.2 mg of anti-CD3 antibody or left uninjected as control. After 2 hours, ligated loops were prepared as above. n = 7/group. **P < .01; ***P < .001; ns = not significant.

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 killed within 1–2 hours after symptom onset. (n = 10 samples/group). Right panels: Mice were killed 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).

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 restimulated 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 hours, T-cell proliferation in the MLN was assessed by CFSE dilution (panel B).

T-cell reactivation occurs locally in the absence of homing from lymph nodes. Mice were treated with FTY720 daily beginning 1 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 2 independent experiments, with a total of 10 mice per group.

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 restimulated 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.

Flow cytometric detection of lamina propria mast cells. Lamina propria cells were isolated from CCR6^+/+ and CCR6^−/− mice with allergic diarrhea (OVA/OVA) or control (PBS/OVA) mice and stained with anti-FcεRI and c-kit antibodies. Mice with allergic diarrhea had an expansion of the FcεRI⁺c-kit⁺ population. This was also observed in CCR6^−/− mice that did not develop allergic diarrhea symptoms.

Lamina propria T cells in CCR6^+/+ and CCR6^−/− mice. Cells were isolated from the lamina propria of CCR6^+/+ and CCR6^−/− mice (4/group). Cells were stained with the pan-leukocyte marker CD45 and CD4. The proportion of CD4⁺ T cells was unchanged in CCR6^−/− mice (top left). In addition, the total yield of cells per small intestine (right) was also unchanged. Cells were stimulated with anti-CD2 and anti-CD28 for 72 hours in complete media, and cytokine release measured by ELISA (bottom panel).

Impact of CCR6 on lamina propria expression of Th1 and proinflammatory cytokines. CCR6^+/+ and CCR6^−/− mice were sensitized and fed with OVA 4 times (OVA/OVA) or were sensitized and left unfed as controls (OVA/PBS). Jejunum was removed, RNA was isolated, and RT-PCR was performed for IFN-γ, IL-17, TNF-α, and IL-18. Expression was normalized to the housekeeping gene GAPDH.

Induction of T-cell cytokine production by CCR6^+/+ or CCR6^−/− DCs from the lamina propria. Lamina propria cells were isolated from CCR6^+/+ and CCR6^−/− mice, followed by positive selection for CD11c⁺ DCs. CD4⁺ T cells were isolated from DO11.10 mice and cocultured with DCs plus OVA peptide. Cytokine secretion from CD3/CD28 restimulated T cells was measured by ELISA. Data are the mean + SEM from 3 individual isolations.

Impact of FTY720 treatment on resident lamina propria CD4⁺ T cells. CCR6^+/+ and CCR6^−/− mice were fed with FTY720 on 2 consecutive days prior to isolation of lamina propria cells from the small intestine. Cells were stained with the pan-leukocyte marker CD45 and CD4, and cells were acquired on a flow cytometer. Percent of CD4⁺ T cells among the total CD45⁺ population was calculated.