Figure 1.
CXCL16 and CXCR6 are upregulated in colonic mucosa of CD patients.
(A, B) mRNA expression of CXCL16 (A) and CXCR6 (B) was evaluated by Q-PCR. The expression of CXCL16 and CXCR6 is higher in colonic mucosa of patients with Crohn’s disease (CD) compared with patients with ulcerative colitis (UC) and healthy controls. Data were normalized to expression of GAPDH mRNA. (n = 5–10; mean and s.e.m.). *, P < 0.05. (C) Correlation between CXCL16 and CXCR6 mRNA expression in the colonic mucosa of CD patients. Statistical analysis was performed by Spearman’s correlation; correlation coefficient = 0.76, P = 0.024. (D, E) Immunohistochemistry of CXCL16 (D) and CXCL16 (E) was performed on colonic mucosa of patients with CD and healthy controls. CXCL16 positive staining was observed on epithelial cells and a subset of colonic LP cells in CD patients (D). CXCR6 was strongly expressed by small round cells in CD mucosa (E). Scale bars, 50 µm, IgG indicates a control antibody. Representative photomicrographs obtained from the analysis of five or six specimens per group are shown.
Figure 2.
Expression of CXCL16 increased in the inflamed mucosa of CD45RBhigh transfer colitis.
(A) Cxcl16 mRNA levels in colonic epithelium (CEC) and distal colon tissues were analyzed by Q-PCR 8 weeks after transfer. The expression of Cxcl16 mRNA increased in both epithelium and colon tissues of the transfer model compared with healthy Rag1−/− mice. Data were normalized to expression of Gapdh mRNA. (n = 5; mean and s.d.). **, P < 0.01. (B) CXCL16 immunostaining of the distal colon in colitic and healthy Rag1−/− mice. Scale bars, 100 µm. Data are representative of two independent experiments. (C) CXCR6 expression on CD4+ T cells was analyzed by flow cytometry using a mouse CXCL16-human IgG-Fc fusion protein or control human IgG-Fcγ at 8-week post transfer. CXCR6 was expressed at high levels by the majority of colonic LP CD4+ T cells in the colitic mice, by about half of the SP and MLN CD4+ T cells, and by ∼20% of BM cells. (D) Absolute numbers of CXCR6+ and CXCR6− CD4+ T cells in each of tissues were calculated based on the flow cytometric analysis described in (C). Data are representative of three independent experiments (mean and s.d.). **, P < 0.01.
Figure 3.
CXCR6 is expressed both by the effector and effector memory CD4+ T cells in the inflamed colon.
LP CXCR6− and CXCR6+ cells were analyzed for expression of activation and memory markers at week 8 post-transfer of naïve CD4+ T cells. (A–C) CD4+ T cells were gated as CD127−CD62L−CD27−CD43+CD44+ to measure the proportion of effector T cells (a). (D–F) The effector memory population (CD44+CD127+) was subdivided using CD62L and CD27 to measure early effector memory cells (CD62L−CD27+CD43+, b) and late effector memory cells (CD62L−CD27−CD43+, c). Data are representative of three independent experiments. (G) The relative percentages of effector, early effector memory and late effector memory in each subset are shown in a pie chart.
Figure 4.
CXCR6 expression is related to the production of Th1 and Th17 cytokines.
Intracellular staining for cytokine and transcription factors in CD4+ T cells was performed 8 weeks after naïve CD4+ T-cell transfer. (A, B) The frequency of IL-2+ cells and IFN-γ+ cells was analyzed in the CXCR6− (upper) and CXCR6+ (lower) subsets (A) and the absolute number of T cells were graphed on the basis of the flow cytometric analysis (B). (C, D) The frequency of IL-17A+ cells and TNF-α+ cells was analyzed in CXCR6− (upper) and CXCR6+ (lower) subset (C) and the numbers were graphed on the basis of flow cytometric analysis (D). (E) The frequency of IFN-γ+ cells and IL-17A+ cells was analyzed. (F) The frequency of T-bet+ cells and RORγt+ cells was analyzed. All data are representative from four independent experiments (mean and s.d.). *, P < 0.05 **, P < 0.01 ***, P < 0.001.
Figure 5.
CXCR6 expression is not required for development of transfer colitis.
(A) Body weight of Rag1−/− recipients of i.v. injected purified CD45RBhighCD4+ T cells form Cxcr6+/Egfp or Cxcr6Egfp/Egfp (CXCR6-deficient) mice on day 0, presented as percent of original weight. (B) Colon weight of the mice in (A) on week 7. Data are representative of two independent experiments (mean and s.d.). (C) Histology of colon tissues from the mice in B. (D) CXCR6 expression by LP CD4+ T cells was analyzed by flow cytometry using CXCL16-hFc at 7-week post transfer. (E) Expression levels of indicated cytokines in distal colon were analyzed by Q-PCR at 7 weeks after the transfer. Data were normalized to expression of Gapdh. (n = 4 or 5; mean and s.d.).
Figure 6.
LP CXCR6− but not CXCR6+ cells can transfer wasting and colitis upon retransfer into Rag1−/− recipients.
(A) Schematic transfer protocol. An equal number of CD25−CXCR6− cells or CD25−CXCR6+CD4+ T cell isolated from colon LP of colitic mice was retransferred into Rag1−/− mice. (B) Time course of changes in body weight after retransfer of the two subsets or transfer of CD45RBhigh naïve T cells. CXCR6–transferred Rag1−/− mice manifested progressive body weight loss to a similar extent to CD45RBhigh-transferred Rag1−/− mice, whereas the cohort that received CXCR6+cells did not show wasting. Data are expressed as the mean and s.e.m of two independent experiments. *, P < 0.05 ***, P < 0.001 versus CXCR6+-transferred animals. (C) Histopathological analysis of distal colon tissue at 8 weeks post transfer. The transfer of CXCR6−CD4+ T cells alone induced intestinal inflammation. Scale bars, 50 µm. (D) Number of CD4+ T cells in each group were calculated based on the flow cytometric analysis. (E) CXCR6 expression on CD4+ T cells in each group was analyzed by flow cytometry using CXCL16-hFc at 8-week post transfer. CXCR6−CD4+ T cells express CXCR6 upon retransfer. (F) Intracellular cytokine staining was performed in LP CD4+ T cells in recipients of CXCR6−CD4+ T cells at 8 weeks after transfer. The CXCR6− subset produced IFN-γ and IL-17A coincident with the expression of CXCR6 upon retransfer. (G) LP CXCR6− and CXCR6+ subsets were purified from CD45RBhigh-transferred Rag1−/− mice at 8-week post transfer, labeled with CFSE and cultured with LP MHC class II+CD11c+ cells at a 5∶1 ratio in the presence of 5 µg/ml anti-CD3ε Abs and 20 ng/ml IL-23 for 3 days. Proliferation was measured by CFSE dilution. Proliferation by CXCR6− and CXCR6+ cells is depicted in the right columns. All data are representative from four independent experiments (mean and s.d.).