Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts.

BACKGROUND & AIMS: Interleukin (IL)-22, a member of the IL-10 subfamily, is a recently identified T-cell-derived cytokine. We investigated IL-22 expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD) and analyzed its biologic activities in human colonic subepithelial myofibroblasts (SEMFs). METHODS: Mucosal IL-22 expression was evaluated by immunohistochemical procedures. The effects of IL-22 on colonic SEMFs were investigated by cDNA microarrays, Northern blots, enzyme-linked immunosorbent assay, and electrophoretic gel mobility shift assays (EMSAs). RESULTS: IL-22 was not detectable in normal colonic mucosa. In IBD mucosa, IL-22 expression was detectable in CD4-positive T cells. IL-22-positive cells were increased in ulcerative colitis and even more so in Crohn's disease. IL-22 receptor expression colocalized with a marker of SEMFs. IL-22 did not modulate SEMF proliferation and collagen synthesis. cDNA microarray analyses demonstrated that, in colonic SEMFs, IL-22 increased the messenger RNA (mRNA) expression of inflammatory cytokines (IL-6, IL-8, IL-11, and leukemia inhibitory factor [LIF]), chemokines, and matrix metalloproteinases. IL-22 induced an activation of nuclear factor (NF)-kappaB and activating protein (AP)-1 within 1 hour, and a blockade of NF-kappaB and AP-1 activation markedly reduced IL-22 induction of IL-6, IL-8, IL-11, and LIF mRNA. MAP-kinase inhibitors (PD98059, U0216, and SB202190) significantly reduced IL-22 induction of cytokine secretion. The combination of either IL-17 plus IL-22 or IL-19 plus IL-22 additively up-regulated cytokine secretion. CONCLUSIONS: IL-22 derived from activated T cells acts on SEMFs to elicit expression of proinflammatory cytokines and matrix-degrading molecules indicating proinflammatory/remodeling roles in IBD.

Suppression of interleukin-1beta- and tumor necrosis factor-alpha-induced inflammatory responses by leukocytapheresis therapy in patients with ulcerative colitis.

BACKGROUND: To elucidate the molecular mechanisms involved in the therapeutic effects of leukocytapheresis (LCAP), we investigated the alterations in the cytokine responses of peripheral blood mononuclear cells (PBMCs) before and after LCAP therapy in ulcerative colitis (UC) patients. METHODS: Twelve patients with UC who did not respond to steroid therapy were enrolled. Nine patients responded to LCAP therapy, but 3 patients did not show clinical improvement. PBMCs were isolated from peripheral venous blood obtained within 5 min before and after the first and second session of LCAP treatment. Cells were stimulated with interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha for 24 h, and the levels of secreted IL-8 and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: LCAP induced a significant decrease in peripheral lymphocyte, monocyte, and platelet counts. IL-1beta- and TNF-alpha-induced IL-8 and IL-6 secretion was significantly decreased after the first and second LCAP treatments. These responses were associated with inhibitory effects on nuclear factor (NF)-kappaB DNA-binding activity. CONCLUSIONS: LCAP downregulates the IL-1beta- and TNF-alpha-induced inflammatory responses in PBMCs isolated from UC patients. The induction of hyporesponsiveness to proinflammatory cytokines may be an important factor mediating the clinical effects of LCAP in UC patients.

Fibroblast growth factor-2 stimulates interleukin-6 secretion in human pancreatic periacinar myofibroblasts.

OBJECTIVES: Fibroblast growth factor-2 (FGF-2) plays an important role in the pathophysiology of acute and chronic pancreatitis. In the present study, to evaluate the proinflammatory nature of FGF-2, we investigated the effects of FGF-2 on IL-6 secretion in human pancreatic periacinar myofibroblasts. METHODS: IL-6 supernatant levels were determined by enzyme-linked immunosorbent assays (ELISA). IL-6 mRNA expression were determined by Northern blots and quantitative PCRs. Activated protein (AP)-1 DNA-binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). RESULTS: FGF-2 induced IL-6 release in a dose- and time-dependent manner. FGF-2 activity for IL-6 induction was the same as that of IL-17. The combination of FGF-2 and IL-17 exerted additive effects at mRNA and protein levels. FGF-2 induced AP-1 DNA-binding activity, but blockage of AP-1 signaling by adenovirus-mediated transfer of a dominant negative c-Jun gene did not affect FGF-2-induced IL-6 mRNA expression. FGF-2 rapidly induced activation of ERK1/2 and p38 MAP kinases, and specific inhibitors for these enzymes significantly reduced FGF-2-induced IL-6 release. CONCLUSION: In the pancreas, FGF-2 may not only play a role as a growth factor in tissue injury repair processes but also as an inducer of acute-phase response via stimulation of IL-6 release.

TIP27: a novel repressor of the nuclear orphan receptor TAK1/TR4.

The nuclear orphan receptor TAK1/TR4 functions as a positive as well as a negative regulator of transcription; however, little is known about the factors regulating or mediating its activity. Yeast two-hybrid analysis using the ligand-binding domain (LBD) of TAK1 as bait identified a novel TAK1-interacting protein, referred to as TIP27, which functions as a repressor of TAK1-mediated transactivation. TIP27 is a 27 kDa protein containing two zinc finger motifs. Mammalian two-hybrid analysis showed that TIP27 interacts specifically with TAK1 and not with several other nuclear receptors tested. The region between Asp39 and Lys79 of TIP27, referred to as TAK1-interaction domain (TID), is critical for its interaction with TAK1 while the TAK1-LBD from helix 3 until the C-terminus is required for the optimal interaction with TIP27. Pull-down assays demonstrated that the TIP27 physically interacts with TAK1 and supported the critical importance of the TID. Confocal microscopy showed that in the nucleus, TIP27 and TAK1 co-localize. TIP27 acts as a strong repressor of DR1-dependent transcriptional activation by TAK1. This repression does not involve the inhibition of TAK1 homodimerization or DR1 binding but may be due to an effect on co-activator recruitment by TAK1. Our results indicate that TIP27 functions as a TAK1-selective repressor.

Epimorphin expression in human colonic myofibroblasts.

Epimorphin is a membrane-associated protein that has been postulated to regulate epithelial morphogenesis in several tissues. However, epimorphin expression in the human intestine has not been fully investigated. In this study, we investigated epimorphin expression in the inflamed mucosa of inflammatory bowel disease (IBD). Tissue samples were obtained surgically from patients with active ulcerative colitis (UC) (n=5) and active Crohn's disease (CD) (n=5). Epimorphin and alpha-smooth muscle actin (SMA) were stained immunohistochemically. Epimorphin expression in human intestinal subepithelial myofibroblasts (SEMFs) was analyzed by Western and Northern blotting. In the normal colon, epimorphin expression was detected partly in the alpha-SMA-positive cells under the epithelial cells. Epimorphin was also expressed in alpha-SMA-positive cells in the capillary wall. In the inflamed mucosa of UC and CD patients, epimorphin expression was not altered. In isolated human SEMFs, epimorphin was detected as a single band of molecular weight 34-kDa under reducing and non-reducing conditions. In intestinal SEMFs, epimorphin mRNA expression was not affected by inflammatory cytokines and growth factors. Epimorphin was constitutively expressed in the normal colonic mucosa, and this was not altered in the inflamed mucosa of IBD patients. The localization of epimorphin may indicate a potential role in maintaining normal tissue structure in normal and IBD mucosa.

Dietary bile acids inhibit potentially elemental diet-induced small intestinal atrophy in rats.

The mechanism responsible for elemental diet (ED)-induced small intestinal atrophy is still unknown. However, it is possible that bile acids in the gut lumen influence this process. The aim of this study was to evaluate the effects of oral bile acid administration during ED feeding. Specific pathogen-free male Sprague-Dawley rats, 10 weeks old, were fed an ED only, ED plus 0.1% (w/w) hyocholic acid, or ED plus 0.1% (w/w) hyodeoxycholic acid ad libitum for 4 weeks. The control rats were fed standard chow ad libitum for 4 weeks. After 4 weeks, the wet weight and whole length of the small intestine, and the mucosal diamine oxidase (DAO) and alkaline phosphatase (ALP) activities were measured. Microscopic histological observation was also performed. ED feeding induced atrophy and elevations in the mucosal DAO and ALP activities in the small intestine. Hyocholic acid and hyodeoxycholic acid administration both tended to inhibit these alterations. In conclusion, ED feeding induced atrophy and elevations in the mucosal DAO and ALP activities in the small intestine. Oral bile acid administration may prevent this atrophy and the elevations in mucosal DAO and ALP activities, which may lead to new therapeutic strategies in patients managed with ED.

IL-17 selectively down-regulates TNF-alpha-induced RANTES gene expression in human colonic subepithelial myofibroblasts.

IL-17 enhances the TNF-alpha-induced IL-6 and IL-8 secretion in human colonic subepithelial myofibroblasts. In this study, we investigated how IL-17 modulates RANTES secretion in these cells. TNF-alpha potently induced RANTES secretion, but IL-17 dose-dependently inhibited the TNF-alpha-induced RANTES secretion. This was also observed at the mRNA level. Even after pretreatment with TNF-alpha for 12 h, the inhibitory effect of IL-17 was detectable. IL-17 did not affect the TNF-alpha-induced stability of the RANTES gene. IL-17 significantly decreased the TNF-alpha-induced increase in RANTES promoter activity, and IL-17 actually blocked the TNF-alpha-induced RANTES gene transcription. EMSAs demonstrated that IL-17 did not modulate the TNF-alpha-induced NF-kappaB DNA-binding activity, but markedly decreased TNF-alpha-induced IFN regulatory factor-1 (IRF-1) DNA-binding activity. Because cooperation between NF-kappaB and IRF-1 is important in the TNF-alpha-induced RANTES gene expression, the major mechanism mediating the inhibitory effect of IL-17 may be achieved by the inhibition of IRF-1 DNA-binding activity.

Inhibitory effects of somatostatin on tumor necrosis factor-alpha-induced interleukin-6 secretion in human pancreatic periacinar myofibroblasts.

Pancreatic periacinar myofibroblasts are considered to be therapeutic targets for the suppression of acute pancreatitis. To elucidate the mechanisms mediating the therapeutic actions of somatostatin on acute pancreatitis, we investigated how somatostatin affects the tumor necrosis factor (TNF)-alpha-induced interleukin (IL)-6 and IL-8 secretion from pancreatic myofibroblasts. Cytokine secretion was determined by enzyme-linked immunosorbent assay (ELISA) and Northern blotting. Nuclear factor (NF)-kappaB DNA-binding activity was evaluated by electrophoretic mobility shift assay (EMSAs). The expression of somatostatin receptor (SSTR) mRNA was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Somatostatin dose-dependently inhibited the TNF-alpha-induced IL-6 secretion. In comparison, the effects on IL-8 secretion were modest. Northern blot analysis demonstrated that somatostatin decreased the TNF-alpha-induced IL-6 mRNA expression, and that this effect was completely blocked by the somatostatin antagonist cyclo-somatostatin. Furthermore, somatostatin suppressed TNF-alpha-induced NF-kappaB activation. These cells bear SSTR subtypes 1 and 2. Somatostatin down-regulated the TNF-alpha-induced IL-6 secretion in human pancreatic periacinar myofibroblasts. These findings suggest that some of the therapeutic actions of somatostatin on acute pancreatitis might be mediated by reducing local IL-6 secretion in the pancreas.

IL-17 stimulates inflammatory responses via NF-kappaB and MAP kinase pathways in human colonic myofibroblasts.

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-kappaB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-kappaB activation within 45 min after stimulation. A blockade of NF-kappaB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1beta or IL-17 + tumor necrosis factor (TNF)-alpha enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-alpha on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.

Intestinal subepithelial myofibroblasts in inflammatory bowel diseases.

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the regulation of a number of epithelial cell functions and in the mucosal repair process. In this study, we evaluated the changes in alpha-smooth muscle actin (SMA)- and vimentin-positive SEMFs in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Tissue samples were surgically obtained from patients with active ulcerative colitis (UC) (n = 5) and active Crohn's disease (CD) (n = 5). Normal intestinal tissues were also obtained (n = 5). The SMA and vimentin expression was evaluated by standard immunohistochemical procedures. In normal intestinal mucosa, SMA- and vimentin-positive SEMFs were located immediately subjacent to the basement membrane, juxtaposed against the bottom site of the epithelial cells. In the inflamed mucosa of active UC patients, there were relatively more SMA-positive cells compared with normal mucosa. In particular, the increase in SMA-positive cells was greatest at the marginal area of deep ulcers of UC patients. In active CD mucosa, SMA-positive cells were increased in all samples, and a marked increase was observed in two samples. The number of SMA-positive SEMFs was relatively higher in CD mucosa than in UC mucosa. An [3H]thymidine incorporation study demonstrated that platelet-derived growth factor (PDGF)-BB, basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF)-I significantly increased the uptake of [3H]thymidine into isolated SEMFs. In particular, PDGF had a strong stimulatory effect. We concluded that colonic SEMFs may play an important role in the repair process of IBD.