Down-regulation of p38 mitogen-activated protein kinase activation and proinflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease.

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)-α, a known agonist of the mitogen-activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho-p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex- and age-matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF-α, interleukin (IL)-1ß and IL-6 were measured in the organ and cell culture supernatants by enzyme-linked immunosorbent assay. We found higher levels of phospho-p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF-α, IL-1ß and IL-6 from IBD LPMCs and biopsies. Activated p38α MAPK is up-regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down-regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines.

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

BACKGROUND AND AIMS: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor beta (TGFbeta) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFbeta signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn's disease (CD). METHODS: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFbeta blocking antibody or TGF beta 1. TGFbeta transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. RESULTS: TGFbeta transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFbeta transcripts, a greater pSmad2-3 response to TGFbeta, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFbeta blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. CONCLUSIONS: Changes in TGF-beta signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Blockade of transforming growth factor beta upregulates T-box transcription factor T-bet, and increases T helper cell type 1 cytokine and matrix metalloproteinase-3 production in the human gut mucosa.

BACKGROUND AND AIMS: The role of transforming growth factor beta (TGFbeta) in inhibiting T cell function in the normal gut has been studied in animal models. However, the impact of TGFbeta inhibition on T cells in the normal human gut remains poorly understood. The effect of TGFbeta blockade in normal intestinal biopsies grown ex vivo and lamina propria mononuclear cells (LPMCs) on T-bet, a T-box transcription factor required for T helper cell type (Th)1 differentiation, interferon gamma (IFN gamma) production, T cell apoptosis and matrix metalloproteinase (MMP)-3 production has therefore been tested. METHODS: TGFbeta transcripts were determined by quantitative reverse transcription-PCR in laser-captured gut epithelium and lamina propria. Biopsies and LPMCs were cultured with anti-TGFbeta neutralising antibody. After 24 h culture, T-bet was determined by immunoblotting, and T cell apoptosis was assessed by flow cytometry. IFN gamma, tumour necrosis factor alpha (TNFalpha), interleukin (IL) 2, IL6, IL8, IL10, IL12p70 and IL17 were measured by ELISA. MMP-3 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were assessed by immunoblotting. RESULTS: A higher number of TGFbeta transcripts was found in the lamina propria than in the epithelium in normal gut. T-bet expression was significantly higher in biopsies and LPMCs cultured with anti-TGFbeta antibody than in those cultured with control antibody. TGFbeta blockade downregulated T cell apoptosis, and induced a significant increase in IFN gamma, TNFalpha, IL2, IL6, IL8 and IL17 production. A higher expression of MMP-3, but not TIMP-1, was observed in the tissue and supernatant of biopsies treated with anti-TGFbeta antibody. CONCLUSIONS: The findings support a crucial role for TGFbeta in dampening T cell-mediated tissue-damaging responses in the human gut.