Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis - PubMed (original) (raw)

. 2004 May;113(10):1490-7.

doi: 10.1172/JCI19836.

Frank Heller, Monica Boirivant, Francisco Leon, Masaru Yoshida, Stefan Fichtner-Feigl, Zhiqiong Yang, Mark Exley, Atsushi Kitani, Richard S Blumberg, Peter Mannon, Warren Strober

Affiliations

Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis

Ivan J Fuss et al. J Clin Invest. 2004 May.

Abstract

While Crohn disease (CD) has been clearly identified as a Th1 inflammation, the immunopathogenesis of its counterpart inflammatory bowel disease, ulcerative colitis (UC), remains enigmatic. Here we show that lamina propria T (LPT) cells from UC patients produce significantly greater amounts of IL-13 (and IL-5) than control cells and little IFN-gamma, whereas comparable cells from CD patients produce large amounts of IFN-gamma and small amounts of IL-13. We then show that stimulation of UC LPT cells bearing an NK marker (CD161) with anti-CD2/anti-CD28 or with B cells expressing transfected CD1d induces substantial IL-13 production. While this provided firm evidence that the IL-13-producing cell is an NK T (NKT) cell, it became clear that this cell does not express invariant NKT cell receptors characteristic of most NKT cells since there was no increase in cells binding alpha-galactosylceramide-loaded tetramers, and alpha-galactosylceramide did not induce IL-13 secretion. Finally, we show that both human NKT cell lines as well as UC CD161(+) LPT cells are cytotoxic for HT-29 epithelial cells and that this cytotoxicity is augmented by IL-13. These studies show that UC is associated with an atypical Th2 response mediated by nonclassical NKT cells producing IL-13 and having cytotoxic potential for epithelial cells.

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Figures

Figure 1

Figure 1

Cytokine secretion by control, CD, and UC LPMCs. (A) IL-13, (B) IFN-γ, (C) IL-5, and (D) IL-4 secretion by LPMCs obtained from control (n = 6), involved CD (n = 8), and involved UC (n = 12) tissues induced by anti-CD2/anti-CD28. In all experiments error bars represent SEMs. Similar secretion patterns for all cytokine analyzed were found with anti-CD3/anti-CD28 stimulation of LPMCs.

Figure 2

Figure 2

Intracellular cytokine expression of CD161+ LPMC. (A) CD161 expression on unstimulated CD3+ LPMCs. LPMCs from control (n = 5), involved CD (n = 6), and involved UC (n = 8) tissues were stained with fluorochrome-labeled Ab’s recognizing various surface antigens (CyChrome-labeled anti-CD3, PE-labeled anti-CD4, and FITC-labeled anti-CD161) and then were analyzed for the expression of CD4+ versus CD161+ cells on CD3-gated cells. (B) Intracellular IL-13 cytokine production by stimulated LPMCs. LPMCs from control (n = 5), involved CD (n = 6), and involved UC (n = 8) tissues were cultured for 48 hours with anti-CD2/anti-CD28 with addition of monensin for the last 6 hours of culture. The resultant cells were stained with fluorochrome-labeled Ab’s recognizing various surface antigens (CyChrome-labeled anti-CD3, APC-labeled anti-CD8, and FITC-labeled anti-CD161) and then were fixed/permeabilized prior to intracellular cytokine staining with PE-labeled anti_IL-13. Cells were gated on CD3 and analyzed for the expression of CD8_ (CD4+) cells and CD161+ cells versus IL-13+ cells. (C) Intracellular IFN-γ cytokine production by stimulated LPMCs was the same as in B, except LPMCs were cultured for 6 hours with PMA and ionomycin with the addition of monensin for the last 5 hours of culture, and fixed/permeabilized cells were stained with APC-labeled anti_IFN-γ. (D) Intracellular IL-13 cytokine production by unstimulated LPMCs. LPMCs from involved CD (n = 4) and involved UC (n = 4) tissues were stained with fluorochrome-labeled Ab’s recognizing various surface antigens (CyChrome-labeled anti-CD3, APC-labeled anti-CD8, and FITC-labeled anti-CD161) and then fixed/permeabilized prior to intracellular cytokine staining with PE-labeled anti_IL-13. Cells were gated on CD3 and analyzed for the expression of CD4+ cells and CD161+ cells versus IL-13+ cells.

Figure 3

Figure 3

Stimulation of cytokine production in LPMCs by EBV-transformed B cells expressing surface CD1d. (A) LPMCs isolated from involved UC lamina propria were cultured for 48 hours with PMA/IL-2 (black line) (see Methods) in the presence or absence of EBV-transformed B cells transfected with CD1d (721 cells) (green line) with or without anti-CD1d mAb (51.1.3) (red line) or untransfected 721 (WT) B cells (blue line). (B and C) LPMCs isolated from involved CD lamina propria were treated as in A. In all cultures monensin was added 6 hours prior to cell harvest. Cells were initially surface stained with fluorochrome-labeled Ab’s (CyChrome-labeled anti-CD3 and APC- or PE-labeled anti-CD8) and then fixed/permeabilized prior to intracellular staining with PE-labeled anti_IL-13 or APC-labeled anti_IFN-γ. CD3-gated cells were then analyzed for CD4+ versus IL-13+ or IFN-γ cells. Histogram shown is representative of three for each patient population.

Figure 4

Figure 4

IL-13 secretion of UC LPMCs stimulated with EBV-transformed B cells transfected with CD1d. LPMCs extracted from involved UC tissue were cultured for 48 hours with PMA/IL-2 (see Methods) in the presence or absence of EBV-transformed B cells transfected with CD1d (721 cells) or untransfected 721 WT cells in the presence and absence of anti-CD1d mAb (51.1.3). Culture supernatants were assayed by ELISA for the secretion of IL-13. Error bars represent SEMs.

Figure 5

Figure 5

Expression of invariant chain TCR in LPMC CD3+ subpopulations. (A) LPMCs isolated from control, involved CD and UC tissues, as well as a control NKT cell line (see Methods), were stained with various fluorochrome-labeled Ab’s (CyChrome-labeled anti-CD3, FITC-labeled anti-Vα24, and PE-labeled anti-Vβ11). The resultant cells were gated on CD3 and analyzed for the expression of Vα24 versus Vβ11. (B) LPMCs isolated from control, involved CD and UC tissues, as well as a control NKT cell line, were stained with FITC-labeled anti-CD3 and PE-labeled α-GalCer_loaded CD1d tetramer. The resultant cells were then analyzed for surface expression of CD3 versus α-GalCer_loaded tetramer. The dot plots in both A and B are representative of results from three experiments.

Figure 6

Figure 6

Cytotoxicity of NKT cells. In these studies HT-29 epithelial cells were prestimulated with LPS (see Methods) to induce increased expression of CD1d (data not shown). (A) Invariant NKT cells were cultured with target HT-29 cells in the presence or absence of IL-13 Ab (see Methods), and the percentage of cytotoxicity was measured by the CytoTox 96 System (n = 4 experiments). Error bars represent SEMs. (B) Purified UC lamina propria (LP) CD4+CD161+T cells were cultured as in A. (C) Purified CD LP CD4+CD161+ T cells were cultured as in A. For both B and C, n = 2 experiments. Error bars represent SDs.

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