Interleukin-32α expression in human colonic subepithelial myofibroblasts.

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by induction of nuclear factor (NF)-κB activation. We studied IL-32α expression in human colonic subepithelial myofibroblasts (SEMFs). Colonic SEMFs were isolated from normal human colon tissue. IL-32α protein expression was evaluated by Western blot analyses, and IL-32α mRNA expression was analyzed by real-time PCR. IL-32α mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1ß and TNF-α. IL-1ß and TNF-α enhanced intracellular accumulation of IL-32α protein, but IL-32α was not detected in supernatants. Each cytokine dose- and time-dependently induced IL-32α mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1ß- and TNF-α-induced IL-32α mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blotting. Blockade of NF-κB activation by an adenovirus expressing a stable mutant form of IκBα markedly suppressed IL-1ß- and TNF-α-induced IL-32α mRNA expression. Human colonic SEMFs expressed IL-32α in response to IL-1ß and TNF-α. IL-32α mRNA expression depends on the phosphatidylinositol 3-kinase and the NF-κB system.

Interleukin-33 expression is specifically enhanced in inflamed mucosa of ulcerative colitis.

BACKGROUND: Interleukin (IL)-33 is a cytokine belonging to the IL-1 family. IL-33 has been shown to elicit a Th2-like cytokine response in immune cells. In this study, we investigated IL-33 expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for IL-33 expression in human colonic subepithelial myofibroblasts (SEMFs). METHODS: IL-33 mRNA expression was determined by real-time polymerase chain reaction (PCR). IL-33 expression in the IBD mucosa was evaluated by immunohistochemical methods. RESULTS: IL-33 mRNA expression was significantly elevated in active lesions from patients with ulcerative colitis (UC), but was not detected in inactive lesions from UC patients or in lesions from patients with either active or inactive Crohn's disease. Colonic SEMFs were identified as a major source of IL-33 in the mucosa. IL-1ß and tumor necrosis factor-α (TNF-α) significantly enhanced IL-33 mRNA and protein expression in isolated colonic SEMFs. IL-1ß and TNF-α did not affect IL-33 expression in intestinal epithelial cell lines (HT-29 and Caco-2 cells). This IL-1ß- and TNF-α-induced IL-33 mRNA expression was mediated by p42/44 mitogen activated protein kinase (MAPK) pathway-dependent activation of nuclear factor (NF)-κB and activator protein (AP)-1. CONCLUSIONS: IL-33, derived from colonic SEMFs, may play an important role in the pathophysiology of UC.

Feasibility of five days of consecutive leukocytapheresis for the treatment of ulcerative colitis: a preliminary study.

Leukocytapheresis (LCAP) has been applied for the treatment of steroid refractory ulcerative colitis (UC). A standard protocol employs one or two sessions of LCAP per week. Our aim was to determine whether five consecutive LCAP sessions can be performed safely and effectively for UC patients. Six patients with moderately active UC were enrolled. The patients received five days of consecutive LCAP in which the processing volume of blood was limited to 1500 mL per session. The hemoglobin levels in each patient gradually decreased, and the platelet count by the fifth session reached half of the value before the first session. The clinical activity index in two patients improved daily, and they went into remission with an improvement in the colonic endoscopic appearance after one week. This preliminary study showed that five consecutive LCAP sessions are safe and feasible for active UC patients. The therapeutic efficacy and suitable patients for this treatment protocol should be confirmed by further studies.

Inhibitory effects of short-chain fatty acids on matrix metalloproteinase secretion from human colonic subepithelial myofibroblasts.

BACKGROUND AND AIMS: Short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate, are the major by-product of bacterial fermentation of dietary fiber in the colon. In this report, we investigated how SCFAs modulate matrix metalloproteinase (MMP) secretion from human colonic subepithelial myofibroblasts (SEMFs). MATERIALS AND METHODS: SEMFs were identified by expression of alpha-smooth muscle actin and vimentin. Cytokine-induced MMP-1 and MMP-3 levels were determined by enzyme-linked immunosorbent assay. Cytokine-induced MMP mRNA expression was analyzed by RT-PCR and real-time PCR methods. RESULTS: Acetate had no effect on MMP secretion. Propionate and butyrate significantly attenuated IL-1 beta- and TNF-alpha-induced MMP-1 and MMP-3 secretion. Similar responses were also observed at the mRNA levels. Propionate and butyrate did not modulate IL-1 beta- and TNF-alpha-induced activation of mitogen-activated protein kinases (MAPKs), which play a crucial role in MMP induction. Trichostatin A, a histone-deacetylase inhibitor, reduced IL-1 beta-induced MMP-1 and MMP-3 mRNA expression, and suppressed TNF-alpha-induced MMP-3 mRNA expression. CONCLUSION: SCFAs play an anti-inflammatory role through suppression of MMP secretion in the colon. Inhibitory effects of SCFAs on MMP secretion might be associated with their action of histone hyperacetylation.

A pilot study of leukocytapheresis efficacy with 1.5 liter blood processing volume in patients with ulcerative colitis.

Standard leukocytapheresis (LCAP) protocols recommend the processing of a 3 L blood volume. In this study, we evaluated the clinical effects of LCAP with 1.5 L of blood processing (1.5L-LCAP) in patients with active ulcerative colitis (UC). Ten patients with moderate to severe UC were enrolled. Their clinical and endoscopic responses, the kinetics of the peripheral blood counts and cytokine responses were evaluated. Clinical and endoscopic effects were assessed using the clinical activity index described by Rachmilewitz, and by Matts' endoscopic classification, respectively. The 1.5L-LCAP induced clinical remission in 8 out of 10 patients (80%). Endoscopic improvement was noted in 6 out of 7 patients (85.7%). Prednisolone (PSL) was used in 8 patients; the PSL dose could be reduced in 6 patients, and weaning was possible in one patient. Adverse effects were not observed during 1.5L-LCAP therapy. During the 1.5L-LCAP session, the leukocyte count reached the minimum at 1.0 L of blood processing, but promptly increased after completion of the session, and reached a maximum after 30 min. Interleukin (IL)-1beta-induced IL-8 and IL-6 secretion by peripheral blood mononuclear cells were both significantly reduced by 1.5L-LCAP therapy. 1.5L-LCAP was clinically effective for active UC patients. Cellular responses induced by 1.5L-LCAP were similar to those induced by a standard LCAP session.

Mucosal cytokine network in inflammatory bowel disease.

Inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD) are characterized by ongoing mucosal inflammation in which dysfunction of the host immunologic response against dietary factors and commensal bacteria is involved. The chronic inflammatory process leads to disruption of the epithelial barrier, and the formation of epithelial ulceration. This permits easy access for the luminal microbiota and dietary antigens to cells resident in the lamina propria, and stimulates further pathological immune cell responses. Cytokines are essential mediators of the interactions between activated immune cells and non-immune cells, including epithelial and mesenchymal cells. The clinical efficacy of targeting TNF-alpha clearly indicates that cytokines are the therapeutic targets in IBD patients. In this manuscript, we focus on the biological activities of recently-reported cytokines [Interleukin (IL)-17 cytokine family, IL-31 and IL-32], which might play a role through interaction with TNF-alpha in the pathophysiology of IBD.

Amphiregulin and epiregulin expression in neoplastic and inflammatory lesions in the colon.

Amphiregulin and epiregulin belong to the epidermal growth factor family and mediate the biological functions of epithelial and mesenchymal cells through epidermal growth factor receptors. In this study, we evaluated the amphiregulin and epiregulin expression in neoplastic and inflammatory lesions from the human colon. Surgically-obtained specimens were stained using standard immunohistochemical procedures. Amphiregulin and epiregulin were not expressed in the normal colonic mucosa, but were clearly detectable in adenomas and carcinomas. Weak immunostaining was also detected in mesenchymal cells from the tumor tissues. In the active mucosa of patients with ulcerative colitis and Crohn's disease, amphiregulin was mainly expressed by the epithelial cells. In addition, positive immunostaining was also detectable in the surrounding mesenchymal cells. In conclusion, amphiregulin and epiregulin may play important roles in colonic tumor growth and mucosal repair in the inflamed mucosa of inflammatory bowel disease.

Inflammatory responses induced by interleukin-17 family members in human colonic subepithelial myofibroblasts.

BACKGROUND: We investigated the potential role of interleukin (IL)-17 family members (IL-17A to IL-17F) in the induction of inflammatory responses in human colonic subepithelial myofibroblasts (SEMFs). METHODS: The expression of the inflammatory cytokines IL-6, IL-8, leukemia inhibitory factor (LIF), and matrix metalloproteinases (MMP)-1 and MMP-3 were evaluated by enzyme-linked immunosorbent assay and Northern blotting. Activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. RESULTS: IL-17A and IL-17F significantly enhanced IL-6, IL-8, LIF, MMP-1, and MMP-3 secretion. The effects of IL-17A were relatively stronger than those induced by IL-17F. The effects of IL-17B, IL-17C, IL-17D, and IL-17E were modest as compared with those induced by IL-17A and IL-17F. Both IL-17A and IL-17F augmented IL-1beta-induced secretion of IL-6, IL-8, LIF, MMP-1, and MMP-3. A similar augmentation was also observed in tumor necrosis factor (TNF)-alpha-induced cytokine and MMP secretion. IL-17A and IL-17F rapidly induced phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, p38 MAPKs, and c-Jun-NH(2)-terminal kinase (JNK) as early as 15 min after stimulation. Inhibitors for ERK (PD98059 and U0216) and p38 MAPK (SB203580) significantly reduced the IL-17F-induced IL-6, IL-8, LIF, MMP-1, and MMP-3 secretion. CONCLUSIONS: Among IL-17 family members, IL-17A and IL-17F strongly stimulate human colonic SEMFs, inducing inflammatory responses.

Microarray analysis of leukocytapheresis-induced changes in gene expression patterns of peripheral blood mononuclear cells in patients with ulcerative colitis.

To elucidate the molecular mechanisms involved in the therapeutic effects of leukocytapheresis (LCAP), we performed microarray analysis for gene expression patterns in peripheral blood mononuclear cells (PBMCs) before and after LCAP therapy in patients with ulcerative colitis (UC). Four patients with UC were enrolled. PBMCs were isolated from peripheral venous blood obtained within 5 min before and after the first session of LCAP therapy. Cells were stimulated with IL-1beta for 12 h, and gene expression patterns were analyzed by an IntelliGene HS Human Expression Chip. The LCAP session reduced various genes, such as proinflammatory cytokines (IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha, and IFN-gamma), cytokine receptors (IL-1R and IL-2Ralpha), chemokines, chemokine receptors, and intracellular signal transduction molecules. Genes which had increased after the LCAP session included those regulating anti-inflammatory cytokines and proteins (TGF-beta1 and IL-R antagonist), receptors for anti-inflammatory cytokines (IL-10R and IL-4R), growth factor receptors (IGF-R1, R2) and antioxidant proteins. Total changes in gene expression patterns after LCAP session were a combination of a decrease in pro-inflammatory genes and an enhancement of anti-inflammatory genes. These changes may explain some parts of the mechanisms by which LCAP improves clinical symptoms of UC patients.

Interleukin-31 stimulates production of inflammatory mediators from human colonic subepithelial myofibroblasts.

Interleukin (IL)-31 is mainly produced by CD4+ T cells, in particular T cells skewed toward a Th2 phenotype. Here we report for the first time that IL-31 stimulates secretion of proinflammatory cytokines, chemokines and matrix metalloproteinases (MMPs) from human colonic subepithelial myofibroblasts (SEMFs). The effects of IL-31 were investigated by cDNA microarrays, enzyme-linked immunosorbent assay, and real-time PCR. IL-31 effectively induced chemokines [IL-8, GRO-alpha (growth-related oncogene-alpha), MCP-3 (monocyte chemoattractant protein-3), CXCL3, CCL13 and CCL15], proinflammatory cytokines (IL-6, IL-16 and IL-32) and matrix metalloproteinases (MMP-1, MMP-3, MMP-25 and MMP-7). IL-31 dose-dependently induced secretion of IL-6, IL-8, GRO-alpha, MCP-3, MMP-1 and MMP-3. The effects of IL-31 were comparable to the effects of IL-17A. IL-31 and IL-17A showed additive effects on IL-6, IL-8, GRO-alpha, MCP-3, MMP-1 and MMP-3 secretion. In conclusion, we demonstrated that IL-31 is a potent inducer of proinflammatory mediators in human colonic SEMFs. IL-31 may function as a proinflammatory cytokine derived from Th2 cells.

Curcumin prevents the development of dextran sulfate Sodium (DSS)-induced experimental colitis.

Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric). We evaluated the effects of curcumin on the development of dextran sulfate sodium (DSS)-induced experimental colitis. BALB/c mice were fed a chow containing either 3.5% (wt/wt) DSS or 3.5% DSS + 2.0% (wt/wt) curcumin. The body weight loss was more apparent in DSS-treated mice than in DSS + curcumin-treated mice. The disease activity index, histological colitis score, and MPO activity were all significantly higher in DSS-treated mice than in DSS plus curcumin-treated mice. Microscopically, mucosal edema, cellular infiltration, and epithelial disruption were much more severe in DSS-treated mice than in DSS + curcumin-treated mice. In DSS + curcumin-treated mice, NF-kappaB activation was blocked in the mucosa. In conclusion, the development of DSS-induced colitis was significantly attenuated by curcumin. Being a nontoxic natural dietary product, curcumin could be useful in treatment of IBD patients.

Matrix metalloproteinase-3 secretion from human pancreatic periacinar myofibroblasts in response to inflammatory mediators.

OBJECTIVES: Matrix metalloproteinases (MMPs) play roles in the pathophysiology of pancreatic disorders. However, the regulation of MMP-3 secretion in the pancreas remains unclear. In this study, we assessed the expression of MMP-3 in human pancreatic periacinar myofibroblasts. METHODS: MMP-3 secretion and MMP-3 mRNA expression were determined by enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. RESULTS: In human pancreatic myofibroblasts, MMP-3 secretion and mRNA expression were induced by interleukin (IL)-17, IL-1beta, and tumor necrosis factor (TNF) -alpha, respectively. The effects of IL-17 were detected as similar in extent to those induced by IL-1beta or TNF-alpha. Costimulation by IL-17 plus IL-1beta and/or IL-17 plus TNF-alpha induced a synergistic increase in MMP-3 secretion, although the costimulatory effects of these combinations were not detected in tissue inhibitor of matrix metalloproteinase-1 secretion. Adenovirus-mediated transfer of a stable form of IkappaBalpha markedly inhibited the effects of IL-17, IL-1beta, and TNF-alpha. Mitogen-activated protein kinase inhibitors (U0126, PD098059, and SB203580) also blocked MMP-3 secretion. These findings indicate a role for nuclear factor-kappaB and mitogen-activated protein kinases in cytokine-induced MMP-3 secretion. CONCLUSIONS: Pancreatic periacinar myofibroblasts actively secrete MMP-3 in response to IL-17, IL-1beta, and TNF-alpha. Pancreatic myofibroblasts may play an important role in extracellular matrix turnover via MMP-3 secretion in the pancreas.

Regulation of amphiregulin and epiregulin expression in human colonic subepithelial myofibroblasts.

Amphiregulin and epiregulin belong to the epidermal growth factor (EGF) family, and act as mitogenic stimulators via binding to EGF receptors (EGFRs). Amphiregulin and epiregulin are thought to play a role in regenerative responses in the gastrointestinal tract. In this study, we investigated secretion of amphiregulin and epiregulin in human colonic subepithelial myofibroblasts (SEMFs). The mRNA expression and protein secretion of amphiregulin and epiregulin were evaluated by Northern blotting and Western blotting, respectively. The trophic effects of amphiregulin and epiregulin on SEMFs were analyzed by MTT assays. Amphiregulin and epiregulin mRNAs were not detected in unstimulated SEMFs. Among the various cytokines and growth factors, interleukin-1beta, tumor necrosis factor-alpha, and EGF strongly induced amphiregulin and epiregulin mRNA expression. These responses were markedly reduced by AG1478, a specific inhibitor of EGF receptor tyrosine kinases. Amphiregulin and epiregulin secretion were also detected at the protein level. MTT assays demonstrated that amphiregulin and epiregulin stimulate the proliferation of SEMFs. We demonstrated expression of amphiregulin and epiregulin in SEMFs. Amphiregulin and epiregulin may play an important role in the mechanism underlying wound healing in damaged colonic mucosa.

Modulation of platelet aggregation responses by leukocytapheresis therapy in patients with active ulcerative colitis.

BACKGROUND: Recent studies suggest that platelet activation plays an important role in the pathophysiology of inflammatory bowel disease. In this study, we evaluated the effects of leukocytapheresis (LCAP) on platelet functions in patients with ulcerative colitis (UC). METHODS: Thirteen patients with active UC (five women and eight men) were treated with LCAP therapy. Platelet-rich plasma (PRP) was prepared, and platelet aggregation in response to agonist solution (epinephrine, collagen, and ADP) was measured with a platelet aggregometer. Platelet-derived microparticle (PDMP) plasma levels were determined by enzyme-linked immunosorbent assay. RESULTS: Nine patients responded to LCAP therapy, but no clinical responses were observed in four patients. The aggregation response to 0.1 microg/ml epinephrine was enhanced in all patients. In all responders, enhanced epinephrine aggregation was normalized after the LCAP session. However, in the four nonresponders, enhanced epinephrine aggregation was maintained after the LCAP session. In responders, the mean maximum aggregation induced by 0.1 microg/ml epinephrine was 76.8 +/- 5.0% before and 15.4 +/- 3.8% after LCAP, respectively (P < 0.05). Increased aggregation responses to both 0.2 microg/ml collagen and 1.0 microM ADP were observed, and LCAP also normalized these enhanced responses. LCAP significantly reduced circulating PDMP levels (56.8 +/- 28.3 U/ml before and 46.3 +/- 30.4 U/ml after LCAP, P < 0.05). CONCLUSIONS: LCAP reduced enhanced platelet aggregation responses in active UC patients. Because platelets play an important role in inflammatory and immune responses, therapeutic effects of LCAP may be partially mediated by reduction of increased platelet aggregation activities.