Oncostatin M Induces a Pro-inflammatory Phenotype in Intestinal Subepithelial Myofibroblasts.

BACKGROUND: Oncostatin-M (OSM) is associated with antitumor necrosis factor (anti-TNF)-α resistance in inflammatory bowel disease (IBD) and fibrosis in inflammatory diseases. We studied the expression of OSM and its receptors (OSMR, gp130) on intestinal subepithelial myofibroblasts (SEMFs) and the effect of OSM stimulation on SEMFs. METHODS: The mRNA and protein expression of OSM, OSMR, gp130, and several fibrotic and chemotactic factors were studied in mucosal biopsies and isolated human intestinal SEMFs of patients with IBD and healthy controls (HCs) and in a model of human intestinal organoids (HIOs). Subepithelial myofibroblasts and HIOs were stimulated with OSM and interleukin (IL)-1α/TNF-α. RNAseq data of mucosal biopsies were also analyzed. RESULTS: Oncostatin-M receptors and gp130 were overexpressed in mucosal biopsies of patients with IBD (P < .05), especially in inflamed segments (P < .05). The expression of OSM, OSMR, and gp130 in SEMFs from HCs was increased after stimulation with IL-1α/TNF-α (P < .001; P < .01; P < .01). The expression of CCL2, CXCL9, CXCL10, and CXCL11 was increased in SEMFs from patients with IBD and HCs after stimulation with OSM in a dose-dependent manner (P < .001; P < .05; P < .001; P < .001) and was further increased after prestimulation with IL-1α/TNF-α (P < .01 vs OSM-alone). Similar results were yielded after stimulation of HIOs (P < .01). Oncostatin-M did not induce the expression of collagen I, III, and fibronectin. Oncostatin-M receptor expression was positively correlated with CCL2, CXCL9, CXCL10, and CXCL11 expression in mucosal biopsies (P < .001; P < .001; P = .045; P = .033). CONCLUSIONS: Human SEMFs overexpress OSMR in an inflammatory microenvironment. Oncostatin-M may promote inflammation in IBD via its stimulatory effects on SEMFs, which primarily involve chemoattraction of immune cells to the intestinal mucosa.

Oncostatin-M/OSMR show elevated expression on intestinal fibroblasts that is regulated by IBD-relevant pro-inflammatory stimuli. In turn, OSM induces a pro-inflammatory phenotype on primary intestinal fibroblasts, with prominent overexpression of chemotactic factors, without demonstrating a substantial profibrotic effect.

Co-expression of fibrotic genes in inflammatory bowel disease; A localized event?

Introduction: Extracellular matrix turnover, a ubiquitous dynamic biological process, can be diverted to fibrosis. The latter can affect the intestine as a serious complication of Inflammatory Bowel Diseases (IBD) and is resistant to current pharmacological interventions. It embosses the need for out-of-the-box approaches to identify and target molecular mechanisms of fibrosis. Methods and results: In this study, a novel mRNA sequencing dataset of 22 pairs of intestinal biopsies from the terminal ileum (TI) and the sigmoid of 7 patients with Crohn's disease, 6 with ulcerative colitis and 9 control individuals (CI) served as a validation cohort of a core fibrotic transcriptomic signature (FIBSig), This signature, which was identified in publicly available data (839 samples from patients and healthy individuals) of 5 fibrotic disorders affecting different organs (GI tract, lung, skin, liver, kidney), encompasses 241 genes and the functional pathways which derive from their interactome. These genes were used in further bioinformatics co-expression analyses to elucidate the site-specific molecular background of intestinal fibrosis highlighting their involvement, particularly in the terminal ileum. We also confirmed different transcriptomic profiles of the sigmoid and terminal ileum in our validation cohort. Combining the results of these analyses we highlight 21 core hub genes within a larger single co-expression module, highly enriched in the terminal ileum of CD patients. Further pathway analysis revealed known and novel inflammation-regulated, fibrogenic pathways operating in the TI, such as IL-13 signaling and pyroptosis, respectively. Discussion: These findings provide a rationale for the increased incidence of fibrosis at the terminal ileum of CD patients and highlight operating pathways in intestinal fibrosis for future evaluation with mechanistic and translational studies.

TL1A (TNFSF15) and DR3 (TNFRSF25): A Co-stimulatory System of Cytokines With Diverse Functions in Gut Mucosal Immunity.

TL1A and its functional receptor DR3 are members of the TNF/TNFR superfamilies of proteins. Binding of APC-derived TL1A to lymphocytic DR3 provides co-stimulatory signals for activated lymphocytes. DR3 signaling affects the proliferative activity of and cytokine production by effector lymphocytes, but also critically influences the development and suppressive function of regulatory T-cells. DR3 was also found to be highly expressed by innate lymphoid cells (ILCS), which respond to stimulation by TL1A. Several recent studies with transgenic and knockout mice as well as neutralizing or agonistic antibodies for these two proteins, have clearly shown that TL1A/DR3 are important mediators of several chronic immunological disorders, including Inflammatory Bowel Disease (IBD). TL1A and DR3 are abundantly localized at inflamed intestinal areas of patients with IBD and mice with experimental ileitis or colitis and actively participate in the immunological pathways that underlie mucosal homeostasis and intestinal inflammation. DR3 signaling has demonstrated a dichotomous role in mucosal immunity. On the one hand, during acute mucosal injury it exerts protective functions by ameliorating the severity of acute inflammatory responses and facilitating tissue repair. On the other hand, it critically participates in the pro-inflammatory pathways that underlie chronic inflammatory responses, such as those that take place in IBD. These effects are mediated through modulation of the relative mucosal abundance and function of Th1, Th2, Th17, Th9, and Treg lymphocytes, but also of all types of ILCs. Recently, an important role was demonstrated for TL1A/DR3 as potential mediators of intestinal fibrosis that is associated with the presence of gut inflammation. These accumulating data have raised the possibility that TL1A/DR3 pathways may represent a valid therapeutic target for chronic immunological diseases. Nevertheless, applicability of such a therapeutic approach will greatly rely on the net result of TL1A/DR3 manipulation on the various cell populations that will be affected by this approach.

Gut Microbial Signatures Underline Complicated Crohn's Disease but Vary Between Cohorts; An In Silico Approach.

Microflora dysbiosis is implicated in the pathophysiology of Crohn's disease. This work analyzes differences in microbial communities and relevant metabolic pathways among the nonstricturing nonpenetrating (B1), stricturing (B2), and penetrating (B3) subphenotypes of Crohn's disease vs healthy controls. We conducted a bioinformatics analysis using the QIIME pipeline and the Calypso, linear discriminant analysis effect size, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, and STAMP tools on publicly available 16S bacterial rRNA sequencing data from terminal ileum mucosal biopsies of healthy controls and the 3 subphenotypes of Crohn's disease. We analyzed differences in microbial diversity and taxonomy, inferred active metabolic pathways via relevant genes' abundance, and detected bacterial families that could serve as biomarkers. Microbiota α-diversity was decreased within all 3 Crohn's disease subphenotypes vs control samples, with more significant reductions in B2 and B3 compared with B1. ß-diversity analysis identified similar microbial patterns in B2 and B3 samples, different from those of B1 and from those of healthy controls. Abundance analysis of microbial families in cohorts, beyond altered abundances compared with healthy controls, highlighted significant differences between the B2 and B3 subphenotypes and the B1 subphenotype. A similar pattern was observed in the inference of microbial metabolomics: the B2 and B3 cohorts had different predicted metabolotypes from the B1 cohort, in addition to differences observed in Crohn's disease vs healthy controls. Our findings indicate distinct microbiome signatures in complicated Crohn's disease subphenotypes and provide the basis for further investigation into the role of gut microflora in the natural course of Crohn's disease.

The Length of Surgical Skin Incision in Postoperative Inflammatory Reaction.

BACKGROUND AND OBJECTIVES: Surgery provokes inflammatory and immune responses, so efforts have been made to reduce host response by using less invasive techniques. The purpose of this experimental study was to investigate the surgical stress induced by skin incision and the role of liver response in this process. METHODS: Seventy male anesthetized Wistar rats were subjected to a midline incision confined strictly to the skin (dermis) of either 1 cm long (n = 20), 10 cm long (n = 20), or no incision (n = 20) or served as controls (n = 10). Skin trauma was left open for a 20-minutes period, and then was meticulously sutured. At 3 and 24 hours later, laparotomy was performed on half the rats of each group, for blood and liver sampling. In serum and liver homogenates, cytokine-induced neutrophil chemoattractant (CINC)1/interleukin (IL)-8 and tumor necrosis factor (TNF)-α levels were measured with enzyme-linked immunosorbent assays and nitric oxide (NO) using a Griess reaction. RESULTS: Skin trauma was found to significantly (P < .01) increase all inflammatory mediators tested (CINC1/IL-8, TNF-α, NO) in serum of operated rats versus controls, the increase being proportionally dependent on the length of skin incision. In liver homogenates, CINC1/IL-8 was significantly (P < .01) increased in operated animals versus controls, similarly to serum levels. In contrast, liver TNF-α levels were inversely related to serum levels, and a significant (P < .01) decrease in TNF-α was observed in liver homogenates of operated animals compared with the controls, indicating that the increased TNF-α in blood reflects liver TNF-α secretion. CONCLUSION: Our findings suggest that inflammatory and immune reactions induced by skin-only surgical trauma are closely correlated to the length of skin incision.

Stromal and immune cells in gut fibrosis: the myofibroblast and the scarface.

Post-inflammatory scarring is the end-result of excessive extracellular matrix (ECM) accumulation and tissue architectural destruction. It represents a failure to effectively remodel ECM and achieve proper reinstitution and healing during chronic relapsing inflammatory processes. Scarring may affect the functionality of any organ, and in the case of inflammatory bowel disease (IBD)-associated fibrosis leads to stricture formation and often surgery to remove the affected bowel. The activated myofibroblast is the final effector cell that overproduces ECM under the influence of various mediators generated by an intense interplay of classic and non-classic immune cells. This review focuses on how proinflammatory mediators from various sources produced in different stages of intestinal inflammation can form profibrotic pathways that eventually lead to tissue scarring through sustained activation of myofibroblasts.

Intraperitoneal application of phospholipids for the prevention of postoperative adhesions: a possible role of myofibroblasts.

BACKGROUND: Peritoneal adhesions, organized as fibrous bands after abdominal surgery, are related with considerable morbidity and repeated hospitalization. Phospholipids, natural constituents of the peritoneal fluid, seem to display excellent antiadhesive properties. The aim of this study was to investigate whether intraperitoneal application of phospholipids is capable of reducing postoperative adhesions and the possible underlying mechanisms. MATERIALS AND METHODS: Twenty male Wistar rats were subjected to a midline laparotomy and a standard peritoneal and cecum abrasion trauma. Before laparotomy closure, a bolus of 3 mL of phospholipids (12 mg/mL) or NaCl (placebo) was given intraperitoneally. Seven days later, the quality and the quantity of adhesions, as well as serum proinflammatory and/or profibrotic mediators, were blindly assessed. Human colonic subepithelial myofibroblasts were isolated from normal controls and cultured with transforming growth factor-ß1 (TGFß1, 5 ng/mL) in the presence of phospholipids (30-300 µg/mL). Collagen production in culture supernatants and migratory activity of myofibroblasts were also assessed. RESULTS: Phospholipids reduced intra-abdominal adhesions (P < 0.001), with respect to their intensity and area, and serum levels of cytokines (interleukin 1ß, interleukin 6, platelet-derived growth factor-1, and TGFß1) compared with placebo-treated rats. Stimulation of myofibroblasts with TGFß1 significantly increased (P < 0.001) the basic collagen production. The presence of phospholipids significantly reduced (P < 0.001) both the TGFß1 induced and the basic collagen production. Using a wound healing assay, phospholipids were found to reduce the basic and the TGFß1-induced migration of myofibroblasts in a concentration-dependent manner. CONCLUSIONS: Intraperitoneal phospholipids might be involved in the prevention of postoperative adhesions formation via the reduction of proinflammatory and/or profibrotic mediators and by inhibiting fibrogenic properties of mesenchymal cells.

The value of experimental models of colitis in predicting efficacy of biological therapies for inflammatory bowel diseases.

During the last decade, biological therapies have an increasing share in the modern therapeutics of various diseases including inflammatory bowel diseases (IBD). Animal models of IBD have often been used to identify the targets of biological therapies, to test their relevance to disease pathogenesis, to assess their therapeutic efficacy in vivo, and to check for drug toxicity. In the field of inflammatory diseases the majority of biologics under development have failed to reach the clinic. This review examines the ability of preclinical data from animal models of IBD to predict success or failure of biologics in human IBD. Specifically, it describes the murine models of IBD, the mechanism of disease induction, the phenotype of the disease, its relevance to human IBD, and the specific immunological features of disease pathogenesis in each model and mainly compares the results of the phase II and III trials of biologics in IBD with preclinical data obtained from studies in animal models. Finally, it examines the possible reasons for low success in translation from bench to bedside and offers some suggestions to improve translation rates.

Proinflammatory cytokines induce crosstalk between colonic epithelial cells and subepithelial myofibroblasts: implication in intestinal fibrosis.

BACKGROUND AND AIMS: Colonic epithelial cells and adjacent subepithelial myofibroblasts are important counterparts in the pathogenesis of intestinal inflammation and fibrosis. We investigated the possible crosstalk between them, whilst focusing on the mucosal inflammation pathways that potentially trigger intestinal fibrosis. METHODS: We studied the effects of proinflammatory cytokines (IL-1α, TNF-α, IFN-γ) on human colonic epithelial cell lines and the effects of epithelial cell-conditioned media on primary human colonic subepithelial myofibroblasts isolated from normal controls or patients with inflammatory Crohn's disease along with the corresponding 18CO cell line. Readouts included production of TGF-ß and TIMP-1, total collagen synthesis, matrix metalloproteinases MMP-2 and MMP-9 and myofibroblast migration/mobility. RESULTS: Proinflammatory cytokines upregulated TGF-ß and TIMP-1 in colonic epithelial cells. Conditioned medium from these epithelial cell cultures induced production of MMP-9 and collagen and inhibited the migration/mobility of subepithelial myofibroblasts. MMP-9 production depended on endothelin receptor A signalling on responding myofibroblasts. Collagen up-regulation was independent of TGF-ß, CTGF, TF and endothelin. Subepithelial myofibroblasts isolated from Crohn's disease patients had similar responses to those isolated from normal controls, with the exception of higher basal collagen production. CONCLUSIONS: Our study indicates that colonic epithelial cells may respond to an inflammatory milieu by inducing myofibroblast functions similar to those observed during intestinal fibrosis.

Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon.

Dendritic cells (DCs) and macrophages (MPs) are important for immunological homeostasis in the colon. We found that F4/80(hi)CX3CR1(hi) (CD11b(+)CD103(-)) cells account for 80% of mouse colonic lamina propria MHC-II(hi) cells. Both CD11c(+) and CD11c(-) cells within this population were identified as MPs based on multiple criteria, including an MP transcriptome revealed by microarray analysis. These MPs constitutively released high levels of IL-10 at least partially in response to the microbiota via an MyD88-independent mechanism. In contrast, cells expressing low to intermediate levels of F4/80 and CX3CR1 were identified as DCs based on phenotypic and functional analysis and comprise three separate CD11c(hi) cell populations: CD103(+)CX3CR1(-)CD11b(-) DCs, CD103(+)CX3CR1(-)CD11b(+) DCs, and CD103(-)CX3CR1(int)CD11b(+) DCs. In noninflammatory conditions, Ly6C(hi) monocytes (MOs) differentiated primarily into CD11c(+) but not CD11c(-) MPs. In contrast, during colitis, Ly6C(hi) MOs massively invaded the colon and differentiated into proinflammatory CD103(-)CX3CR1(int)CD11b(+) DCs, which produced high levels of IL-12, IL-23, iNOS, and TNF. These findings demonstrate the dual capacity of Ly6C(hi) blood MOs to differentiate into either regulatory MPs or inflammatory DCs in the colon and that the balance of these immunologically antagonistic cell types is dictated by microenvironmental conditions.

TNF receptors in Kupffer cells.

INTRODUCTION: Somatostatin is a mediator of immune functions and has been used as an antineoplastic agent in animal models and human neoplasias. We have demonstrated that Octreotide inhibits only LPS induced secretion of proinflammatory cytokines including TNFa by Kupffer cells (KC). We, therefore, tested the hypothesis that somatostatin modulates the expression of tumor necrosis factor alpha (TNF?) receptors and apoptosis of KC. METHODS: Rat KC were isolated by centrifugal elutriation. TNFR1 and TNFR2 expression was studied by RT-PCR, quantitative PCR, Western Blot and immunofluorescence before and after Octreotide pre-incubation. Apoptosis was assessed by quantitative measurement of cytoplasmic histone-associated DNA fragments. TNFa mRNA expression was assessed by semiquantitative PCR and TNFa was measured in cell supernatants by ELISA. RESULTS: TNFR1 and TNFR2 mRNA are constitutively expressed in KC. Octreotide incubation increased both receptors expression with a peak at 6?h and return to basal levels at 24?h. TNFR1 was mostly influenced. However, only increase in TNFR2 protein was identified, whereas a band at 90 kD was present instead of a band at 55 kD as expected for TNFR1. TNF? mRNA expression was inhibited by Octreotide and a significant inhibition was observed at 48?h. TNF had no effect on KC apoptosis, whereas Octreotide significantly increased their apoptosis, and this effect was not influenced by co-incubation with TNFa. CONCLUSION: TNFR1 and TNFR2 are constitutively expressed in KC and their expression is strongly increased by somatostatin. Moreover, somatostatin increases KC apoptosis. These findings may in part explain the antineoplasmatic effect of somatostatin.

Endoscopic treatment for high-risk bleeding peptic ulcers: a comparison of epinephrine alone with epinephrine plus ethanolamine.

BACKGROUND: Among the various methods of combined endoscopic therapy for high-risk bleeding peptic ulcers the use of adrenaline followed by injection of ethanolamine is minimally demanding in terms of the endoscopic skills and instrumentation but has not been adequately studied. The aim of the present study is to determine whether the injection of ethanolamine in combination with epinephrine compared to injection of epinephrine alone reduces rebleeding rates, need for surgery and overall mortality of patients with bleeding ulcers. METHODS: Patients with ulcers and endoscopic features indicative of a high risk for spontaneous recurrent bleeding were included. High risk was defined by the Forrest classification. Patients were assigned to injection of epinephrine alone (n = 284) or epinephrine plus ethanolamine (n = 131). RESULTS: Initial hemostasis was achieved in 96% of patients in both groups. We detected significant difference in rates of recurrent bleeding, 16.4% vs. 8.7%, for epinephrine and epinephrine plus ethanolamine respectively (P<0.05). When patients were stratified according to Forrest criteria, no significant difference could be found, although there was a trend towards less recurrent bleeding in the case of dual injection therapy in all patient subgroups. There was no significant difference in the proportions of patients who required surgery, 7.7% vs. 7.6% respectively. Mortality was equal (3.2 vs. 3.1%) in the two groups. No major complications from endoscopic treatment were observed in either group. CONCLUSION: Adding ethanolamine to epinephrine for injection treatment of bleeding peptic ulcers decreases bleeding recurrence rates and represents a safe endoscopic treatment for high-risk bleeding ulcers.

Ciprofloxacin decreases survival in HT-29 cells via the induction of TGF-beta1 secretion and enhances the anti-proliferative effect of 5-fluorouracil.

BACKGROUND AND PURPOSE: Fluoroquinolones are potent anti-microbial agents with multiple effects on host cells and tissues. Previous studies have highlighted their pro-apoptotic effect on human cancer cells and an immunoregulatory role in animal models of inflammatory bowel disease. We examined the effect of ciprofloxacin on proliferation, cell cycle and apoptosis of HT-29 cells, a human colonic epithelial cell line sensitive to transforming growth factor (TGF)-beta1-mediated growth inhibition and its role in TGF-beta1 production. We also examined the effect of ciprofloxacin on proliferation of HT-29 cells in combination with 5-fluorouracil (5-FU), a well-established pro-apoptotic agent. EXPERIMENTAL APPROACH: Using subconfluent cultures of HT-29 and Caco-2 cells, we studied the effect of ciprofloxacin, TGF-beta1 and 5-FU on proliferation, apoptosis, necrosis and cell cycle. The effect of ciprofloxacin on TGF-beta1 mRNA expression and production was studied in RNA extracts and cell culture supernatants respectively, using confluent cultures. KEY RESULTS: Ciprofloxacin decreased proliferation of HT-29 cells in a concentration- and time-dependent manner. This was mediated by accumulation of HT-29 cells into the S-phase but without any effect on apoptosis or necrosis. Additionally, ciprofloxacin enhanced the antiproliferative effect of 5-FU. Interestingly, ciprofloxacin was found to up-regulate TGF-beta1 production by HT-29 cells and its anti-proliferative effect was abolished when TGF-beta1 was blocked. Confirming this mechanism further, ciprofloxacin had no effect on Caco-2, a human colonic epithelial cell line that lacks functional TGF-beta1 receptors. CONCLUSIONS AND IMPLICATIONS: We demonstrate a novel anti-proliferative and immunoregulatory effect of ciprofloxacin on human intestinal epithelial cells mediated via TGF-beta1.

Role of Kupffer cells in the pathogenesis of liver disease.

Kupffer cells, the resident liver macrophages have long been considered as mostly scavenger cells responsible for removing particulate material from the portal circulation. However, evidence derived mostly from animal models, indicates that Kupffer cells may be implicated in the pathogenesis of various liver diseases including viral hepatitis, steatohepatitis, alcoholic liver disease, intrahepatic cholostasis, activation or rejection of the liver during liver transplantation and liver fibrosis. There is accumulating evidence, reviewed in this paper, suggesting that Kupffer cells may act both as effector cells in the destruction of hepatocytes by producing harmful soluble mediators as well as antigen presenting cells during viral infections of the liver. Moreover they may represent a significant source of chemoattractant molecules for cytotoxic CD8 and regulatory T cells. Their role in fibrosis is well established as they are one of the main sources of TGFbeta1 production, which leads to the transformation of stellate cells into myofibroblasts. Whether all these variable functions in the liver are mediated by different Kupffer cell subpopulations remains to be evaluated. In this review we propose a model that demonstrates the role of Kupffer cells in the pathogenesis of liver disease.

Immunomodulatory cytokines suppress epithelial nitric oxide production in inflammatory bowel disease by acting on mononuclear cells.

Inducible nitric oxide synthase (iNOS) activity in colonic epithelial HT-29 cells is modulated by the T-cell-derived cytokines IL-4 and IL-13, but is not affected by IL-10 despite its effect in models of colitis. We studied the effects of these cytokines on nitric oxide (NO) production by colonic tissue. IL-10 and IL-4 but not IL-13 suppressed the NO production and iNOS expression by inflamed tissue and cytokine-stimulated noninflamed tissue from patients with ulcerative colitis, whereas the three cytokines suppressed NO production in cytokine-stimulated biopsies from controls. To examine why colonic biopsies and HT-29 cells respond differently to immunomodulatory cytokines, a coculture of mixed mononuclear monocytes (MMC) and HT-29 cells was studied. Treatment of HT-29 cells with conditioned medium from IFN-gamma/LPS-stimulated MMC produced significant amounts of NO, which suggested the presence of an MMC-derived soluble factor modifying epithelial NO production. Pretreatment of IFN-gamma/LPS-stimulated MMC with IL-10 and IL-4 but not IL-13 suppressed NO production by HT-29 cells. Interestingly, pretreatment of HT-29 cells with IL-1 receptor antagonist suppressed the IFN-gamma/LPS-stimulated MMC-induced NO production. These results suggest that immunomodulatory cytokines might exert an inhibitory effect on NO up-regulation by colonic epithelium via the inhibition of MMC-derived soluble mediators, such as IL-1.

Secretion of inflammatory mediators by isolated rat Kupffer cells: the effect of octreotide.

AIMS: We studied the production of inflammatory mediators by rat KC and the possible in vitro effect of the somatostatin analogue octreotide. METHODS: Primary KC cultures were incubated with LPS added alone or with different concentrations of octreotide. The production of TNFalpha, IL-6, IL-10, IL-12 and IL-13 was assessed in culture supernatants by ELISA and that of nitric oxide (NO) by a modification of the Griess reaction. RESULTS: Isolated KC produced a basal amount of TNFalpha, IL-6, IL-12, IL-13, and NO but not IL-10. LPS-stimulated KC secreted significantly increased amounts of TNFalpha (P < 0.001), IL-6 (P < 0.01), IL-10 (P < 0.001), IL-12 (P < 0.01), and NO (P < 0.001) whereas IL-13 production remained constant. Octreotide reduced IL-12 (P < 0.05) and increased IL-13 (P < 0.05) production by unstimulated KC. Furthermore, octreotide suppressed TNFalpha production (P < 0.05), without modifying TNFalpha mRNA expression and decreased iNOS expression and NO (P approximately 0.05) production by LPS-activated KC. These effects were reversed with Wortmannin pre-treatment suggesting that octreotide may act via interference with phosphatidylinositol 3-kinase pathways. CONCLUSIONS: These data demonstrate that KC is a source of multiple inflammatory mediators, indicating a critical role in liver inflammatory disorders. Octreotide modulates inflammatory mediator production by isolated KC, suggesting that it might have immunoregulatory and anti-inflammatory effects in liver diseases.

Effect of octreotide on apoptosis-related proteins in rat Kupffer cells: a possible anti-tumour mechanism.

BACKGROUND: Octreotide may prolong survival in patients with hepatocellular carcinoma, through an as yet unidentified mechanism. Kupffer cells play a key role in antitumour activity. Kupffer cell apoptosis is of major importance for the maintenance of this antitumour activity. MATERIALS AND METHODS: We studied the in vitro effects of octreotide in the RNA expression of apoptotic and antiapoptotic proteins in isolated rat Kupffer cells, before and after exposure to lipopolysaccharide (LPS). Apoptotic and antiapoptotic gene expression was assessed using a semi-quantitative multiplex RT-PCR measuring bax, bcl-xS, bcl-2 and bcl-xL. LICE (caspase-3) mRNA was used as a measure of apoptosis. RESULTS: Unstimulated Kupffer cells exhibited increased proapoptotic gene expression in a time-dependent manner, paralleled by a similar increase of LICE. LPS stimulation decreased the expression of proapoptotic bax and bcl-xS mRNA without effecting the antiapoptotic proteins. A decrease of LICE expression became significant at 48 hours. Octreotide showed a reduction of proapoptotic proteins, accompanied by an early increase and a late reduction of antiapoptotic proteins and a net decrease of LICE expression. A combination of LPS and octreotide produced a similar effect with the exception of a late increase of LICE expression, probably caused by a late increase of bax and bcl-xS. CONCLUSION: LPS and octreotide reverse the observed increased apoptosis of cultured Kupffer cells by influencing both proapoptotic and antiapoptotic proteins. Therefore, the antitumour effect of octreotide in hepatocellular carcinoma may, in part, be explained by its antiapoptotic effect on Kupffer cells.