T-bet upregulation and subsequent interleukin 12 stimulation are essential for induction of Th1 mediated immunopathology in Crohn's disease

Abstract

Background and aims: Many lines of evidence suggest that T helper cell type 1 (Th1) immune responses predominate in Crohn's disease (CD). Recently, a novel transcription factor T-box expressed in T cells (T-bet) has been reported as the master regulator of Th1 development. This study was designed to investigate the role of T-bet and proinflammatory cytokines in Th1 mediated immunopathology in CD.

Materials: CD4+ lamina propria mononuclear cells (LPMCs) were isolated from surgically resected specimens (CD, n = 10; ulcerative colitis (UC), n = 10; normal controls (NL), n = 5).

Methods: (1) T-bet expression of CD4+ LPMCs was examined by quantitative real time polymerase chain reaction and western blotting. (2) T-bet expression of LPMCs stimulated by interleukin (IL)-12/IL-18 was analysed by western blotting. (3) Interferon gamma (IFN-gamma) production and T-bet expression of CD4+ peripheral blood mononuclear cells (PBMCs) were examined with or without stimulation by anti-CD3/CD28 monoclonal antibodies and/or IL-12.

Results: (1) T-bet expression of CD4+ LPMCs was increased in CD compared with UC and NL. (2) Synergistically, augmentation of IFN-gamma production by IL-12/IL-18 was independent of T-bet expression in LPMCs. (3) T-bet was induced by T cell receptor stimulation in CD4+ PBMCs. T-bet induction correlated with IFN-gamma production and with augmentation of surface expressed IL-12 receptor beta2.

Conclusions: T-bet induction by antigenic stimulation and subsequent stimulation by macrophage derived IL-12/IL-18 are important for establishing Th1 mediated immunopathology in CD.

Figure 1

T-box expressed in T cells (T-bet) expression of CD4+ lamina propria mononuclear cells (LPMCs) was increased in Crohn’s disease (CD). Total RNA was extracted from CD4+ LPMCs (CD, n = 10; ulcerative colitis (UC), n = 10; normal controls (NL), n = 5). (A) Levels of T-bet mRNA transcripts were measured by quantitative real time polymerase chain reaction and adjusted to β-actin mRNA transcripts. **p<0.01. (B) T-bet protein expression was assessed by western blotting in CD and UC patients and in NL. AU, arbitrary units. (C) LPMCs were cultured in medium alone for 48 hours (CD, n = 6; UC, n = 6; NL, n = 6). Production of interferon γ (IFN-γ) was measured by ELISA. **p<0.01.

Figure 2

T-box expressed in T cells (T-bet) did not contribute to synergistic augmentation of interferon γ (IFN-γ) production by interleukin (IL)-12 and IL-18 stimulation. (A) Lamina propria mononuclear cell (LPMCs) were cultured in medium alone for 48 hours. Production of IL-12 was measured by ELISA in Crohn’s disease (CD) and ulcerative colitis (UC) patients, and in normal controls (NL). **p<0.01. (B) LPMCs from CD patients were stimulated by IL-12 (1 ng/ml) and/or IL-18 (1 ng/ml) for 48 hours. Production of IFN-γ was measured by ELISA (n = 6). (C) T-bet protein expression was assessed by western blotting. Results are representative of three independent experiments.

Figure 3

Induction of T-box expressed in T cells (T-bet) via the T cell receptor (TCR) signalling pathway is necessary for interferon γ (IFN-γ) production before interleukin (IL)-12 stimulation. CD4+ PBMCs were stimulated with anti-CD3 (10 μg/ml)/CD28 (5 μg/ml) and/or IL-12 (10 ng/ml) for 48 hours. (A) Production of IFN-γ was measured by ELISA (n = 3). (B) T-bet mRNA expression was analysed by quantitative real time polymerase chain reaction after stimulation for 12 hours in patients with Crohn’s disease (CD) and ulcerative colitis (UC), and in normal controls (NL). (C) T-bet protein expression was assessed by western blotting. (D) Surface expression of IL-12Rβ2 was analysed by FACS.