The Role Of Dendritic Cells And Toll-like Receptor 2 In The Pathogenesis Of Ulcerative Colitis

Ulcerative colitis (UC) is characterized by chronic inflammation of the rectum and colon. But its pathogenesis has not been fully clarified. In general, the abnormal immune response and imbalance immune regulation are thought to be an important segment in this process.Dendritic cells (DCs) are specialized antigen presenting cells (APC) that have a dominant role in initiating naive T cells. There are a number of cell surface markers on DCs, including S-100 protein which is thought to be a marker of DCs in local tissue. The capacity of DCs to activate T cells depends on their mature states, CD83 is a surface marker with relative specificity expressed by mature DCs. DCs might play a pivotal role in the development of UC, however, the pathogenesis is still unknown.Toll like receptors (TLRs) are recent discovered transmembrance protein and receptors of the innate immune system, which are abundant in macrophage and DCs. Lipopolysaccharide (LPS) and microbial lipopeptides can stimulate DCs maturation via TLR2.Janus kinase (JAK)/signal transducer and activator of transcription (STAT) is an important cell signaling pathway, which is responsible for the development of UC. The expression of STAT3 and phosphorylated STAT3 in colonic mucosa of patients with UC were higher than that in normal colonic mucosa. Some studies showed that there was a binding site of STAT3 in TLR2 promoter. Causative agent may directly regulate the expression of TLR2 gene.ObjectiveThe aim of our study was to investigate the disposition of DCs and the expression of TLR2 in colonic mucosa of patents with UC and their correlations with clinical activity and endoscopic classification. We also analyzed their potential role in pathogenesis of UC.MethodsColonic biopsy specimens were obtained from patients with UC and normal controls. The cases were divided into classⅠgroup, classⅡgroup and classⅢgroup, according to endoscopic classification. And according to clinic activity the cases were divided into mild group, moderate group and severe group.Immunohistochemistry (IHC) was employed to study the disposition of DCs with markers of S-100 and CD83 in colonic mucosa. Protein was extracted from colonic mucosa of patients with UC and normal controls to detect the expression of TLR2 and phosphorylated STAT3 by Western Blot. TLR2 mRNA expression in mucosa was determined by performing semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results1 Clinical and pathological performance of UC patients 26 of 47 UC patients mainly suffered from bloody purulent stool, 14 from hematochezia and 7 only from abdominal pain. Fever occurred in 8 patients.Coloscopy showed that there were hyperemia, edema, erosion or ulcer in the mucosa with cover of bloody purulent secretion in moderate and severe patients.In mild patients, grave edema and hyperemia were the main appearances which made the mucosa to be grainy, fragile and more bleeding.[0] Optical microscope observation revealed that in normal mucosa the colon glands were well-arranged complete epithelial cells and crypt, and there were small amounts of inflammatory cells in lamina proia, but in the colonic mucosa of patients with UC, the tissue presented hyperemia, edema, erosion, ulcer and the loss or decrease of goblet cells with considerable inflammatory cells infiltration in lamina propria. We also detected analosis of glands, thickening of colon paries and formation of crypt abscess. Atypical hyperplasia and formation of fibrous connective tissue also could be found in the mucosa of patients with polypi.2 The expressions of S-100 in colonic mucosa of patients with UCUnder light microscope, there was mild expression of S-100 in lamina propria of normal colonic mucosa[0], and the cells with S-100 had brown and yellow kytoplasm and round nuclear.In normal control few S-100 positive cells were detected, in comparison to classⅠg roup, classⅡgroup and classⅢgroup; There were no differences between classⅠgroup and the normal control (23.31 vs 16.00), P>0.05; Compared with the normal control, the level of S-100 protein in colonic mucosa of classⅡgroup and classⅢgroup had obviously increased (23.31 vs 16.00, 32.61 vs 16.00), P<0.01; the expression of S-100 was significantly enhanced in classⅢgroup than that in classⅠgroup (23.31 vs 42.24), P<0.01; There were no significant differences between classⅠgroup and classⅡgroup and between classⅡgroup and classⅢgroup (23.31 vs 32.61, 32.61 vs 42.24), P>0.05.Compared with the normal control, the expression of S-100 in the colonic mucosa of mild group, moderate group and severe group were significantly increased (30.13 vs 16.00, 34.43 vs 16.00, 42.00 vs 16.00), P<0.01; But there were no differences among mild group, moderate group and severe group, P>0.05. 3 The expressions of CD83 in colonic mucosa of patients with UCUnder light microscope, there was mild expression of S-100 in lamina propria of normal colonic mucosa[0], and the cells with CD83 had brown and yellow kytoplasm and round nuclear.In normal control few CD83 positive cells were detected, in comparison to classⅠg roup, classⅡgroup and classⅢgroup; There were no differences between classⅠgroup and the normal control (19.00 vs 16.00), P>0.05; Compared with the normal control, the level of CD83 protein in colonic mucosa of classⅡgroup and classⅢgroup had increased obviously (34.14 vs 16.00, 42.29 vs 16.00), P<0.01; In comparison to classⅠgroup, the level of CD83 protein in colonic mucosa of classⅡgroup and classⅢgroup had statistic differences (34.14 vs 19.00, 42.29 vs 19.00), P<0.01; There were no statistic differences between classⅡgroup and classⅢgroup (34.14 vs 42.29), P>0.05.Compared with the normal control, the expression of CD83 protein had an increasing tendency in mild group, moderate group and severe group (30.10 vs 16.00, 33.76 vs 16.00, 43.78 vs 16.00), P<0.01. But no significant differences were found among mild group, moderate group and severe groups, P>0.05. 4 The expressions of TLR2 protein in colonic mucosa of patients with UCThe TLR2 band of 90 kD was showed by Western Blot. In comparison to the normal control, the level of TLR2 protein by Western blot in classⅠgroup, classⅡgroup and classⅢgroup, was obviously increased; No differences were found between classⅠgroup and the normal control (15.77 vs 15.94), P>0.05; Compared with the normal control, the level of TLR2 protein in classⅡgroup and classⅢgroup was obviously increased (29.48 vs 15.77, 49.94 vs 15.77), P<0.01; There were statistic differences among classⅠgroup, classⅡgroup and classⅢgroup, P<0.01.Compared with the normal control, the level of TLR2 protein by Western blot in mild group, moderate group and severe group was obviously increased; There were no differences between mild group and the normal control (17.07 vs 15.77), P>0.05; In comparison to the normal control, the level of TLR2 protein in moderate group and severe group was obviously increased (39.26 vs 15.77, 51.98 vs 15.77), P<0.01; There were statistic differences among mild group, moderate group and severe groups, P<0.01.5 The expressions of TLR2 mRNA in colonic mucosa of patients with UCThe expression of TLR2 andβ-actin gene were examined by RT-PCR, it showed that there were the TLR2 band of 223 bp and theβ-actin band of 500 bp in UC group and normal control.Compared with the normal control, the expression of TLR2 mRNA in classⅠgroup, classⅡgroup and classⅢgroup, was obviously increased; There were no differences between classⅠgroup and the normal control (16.75 vs 11.38), P>0.05; In comparison to the normal control, the expression of TLR2 mRNA in classⅡgroup and classⅢgroup was obviously increased (32.61 vs 11.38, 48.85 vs 11.38), P<0.01; There were significant differences among classⅠgroup, classⅡgroup and classⅢgroup, P<0.01.Compared with the normal control, the expression of TLR2 mRNA in mild group, moderate group and severe group had an increasing tendency (28.93 vs 11.38, 32.37 vs 11.38, 55.94 vs 11.38), P<0.01; There were no differences between mild group and moderate group (28.93 vs 32.37), P>0.05; Compared with the severe group, the expression of TLR2 mRNA in mild group, moderate group were obviously decreased (28.93 vs 55.94, 32.37 vs 55.94), P<0.01.6 The expressions of pSTAT3 protein in colonic mucosa of patients with UCThe pSTAT3 band of 90 kD was showed by Western Blot. In comparison to normal control, the expressions of pSTAT3 protein in classⅠgroup, classⅡgroup and classⅢgroup had an increasing tendency (17.75 vs 7.00, 33.61 vs 7.00, 50.44 vs 7.00), P<0.01; There were statistic differences among classⅠgroup, classⅡgroup and classⅢgroup, P<0.01.Compared with the normal control, the expression of pSTAT3 in mild group, moderate group and severe group had an increasing tendency (21.70 vs 7.00, 42.33 vs 7.00, 48.89 vs 7.00), P<0.01; There were significant differences between mild group and moderate group and between mild group and severe group reapectively, P<0.01; There were no differences between moderate group and severe group, P>0.05.7 The correlation analysis between pSTAT3 and TLR2 suggested that the relationship between pSTAT3 and TLR2 is positive correlation, r=0.716, P<0.01.ConclusionsCompared with the normal control, the expression of S-100 and CD83 in colonic mucosa of patients with UC was up-regulated, which to some extend reflected the degree of clinical activity and endoscopic classification. At the same time, the expression of TLR2 protein, TLR2 mRNA and pSTAT3 in colonic mucosa of patients with UC were increased, which to some extend associated with the degree of clinical activity and endoscopic classification too. The correlation analysis between pSTAT3 and TLR2 suggested that the relationship between pSTAT3 and TLR2 is positive correlation. Thereby the increase in DCs expression of S-100 and CD83 might contribute to occurrence and development of UC.