Abstract: Anesthesia and surgical trauma are considered major oxidative and nitrosative stress effectors resulting in the development of SIRS. In this study we evaluated the usefulness of early enteral nutrition after surgical trauma. Sixty male Wistar rats were subjected to midline laparotomy and feeding-gastrostomy. Twenty of these rats served as controls after recovering from the operation stress. The remaining rats received, through gastrostomy, enteral nutrition or placebo-feeding for 24 h. Oxidative stress markers and CC chemokine production were evaluated in rat serum and liver tissue. The operation itself was found to increase nitric oxide (NO) and malondialdehyde (MDA) and to decrease superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as liver tissue energy charge (EC) in relation to controls. The rats receiving enteral feeding exhibited statistically significantly lower levels of NO and MDA, and higher levels of SOD, GSH-Px, and liver EC, in relation to placebo feeding rats. The operation significantly increased the chemokines monocyte chemoattractant protein (MCP)-1 and regulated upon activation, normal T-cell expressed, and secreted (RANTES) in rat serum, while enteral nutrition caused a further significant increase in chemokine levels in serum. mRNA chemokine expression in liver was increased in a similar pattern. These findings indicate that early enteral feeding might play an important role after surgery ameliorating oxidative stress, affecting positively the hepatic EC and regulating, via chemokine production, cell trafficking, and healing process.
Abstract: Vascular endothelial growth factor (VEGF) is a major angiogenic factor and prime regulator of endothelial cell proliferation. During pregnancy, VEGF is essential for the proliferation of trophoblasts, the development of embryonic vasculature and the growth of maternal and fetal blood cells in utero. In cases of pre-eclampsia and in some circumstances of preterm labor-raised umbilical cord serum, VEGF levels might be correlated with the clinical development of the above pathological disorders. Genetic alteration as 936C/T VEGF gene polymorphism has a statistical significant correlation with the severity of pre-eclampsia. The same VEGF gene polymorphism, which has been associated with lower protein production, has an increased risk of spontaneous preterm delivery in a Greek-studied population. Homozygotes were found to carry the greatest risk with a lesser proportionate risk associated with heterozygosity, whereas women with the -1154 allele of the VEGF gene have an increased risk of recurrent pregnancy loss. In this review, we present evidence that demonstrates an implication of VEGF gene polymorphisms in the pathological disorders of pregnancy. However, further genetic studies are needed to confirm these data.
Abstract: 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.
Abstract: Mounting evidence suggests that stress is implicated in the development of inflammatory bowel disease (IBD), via initial nervous disturbance and subsequent immune dysfunction through brain-gut interactions. The corticotropin-releasing factor (CRF) system, being the principal neuroendocrine coordinator of stress responses, is involved in the inflammatory process within the gastrointestinal tract, via vagal and peripheral pathways, as implied by multiple reports reviewed here. Blocking of CRF receptors could theoretically exert beneficial anti-inflammatory effects in colonic tissues. The recently synthesised small-molecule CRF(1) antagonists or alternatively non-peptide CRF(2) antagonists when available, may become new reliable options in the treatment of IBD.
Abstract: BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs), conventional and selective cyclooxygenase-2 (COX-2) inhibitors, are among the most widely used medications for the treatment of various inflammatory conditions. There is strong evidence of a possible association between the use of these drugs and the relapse of inflammatory bowel diseases (IBD). OBJECTIVE: Our objective was to examine the literature regarding the exacerbation of IBD associated with the use of conventional NSAIDs and selective COX-2 inhibitors and the underlying pathogenetic mechanisms. STUDY DESIGN: We reviewed articles, including original papers, controlled trials, case reports, reviews, and editorials published in English at the PubMed, Scopus Database, and Science Direct database, searching with the following keywords: nonsteroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, Coxibs, inflammatory bowel diseases (IBD), ulcerative colitis (UC), Crohn's disease (CD). RESULTS: There is substantial evidence that exacerbation of IBD happens after treatment with NSAIDs, but the available data remain conflicting, and it is not clear whether selective COX-2 inhibitors are safer than traditional NSAIDs. However, there is some evidence that selective COX-2 inhibition and COX-1 inhibition (with low-dose aspirin) appear to be well-tolerated in the short term. Regarding the mechanisms of relapse, the reduction of prostaglandins appears to be the hallmark of the NSAIDs adverse effects. CONCLUSIONS: Further randomized, double-blind, controlled trials should be performed to address this issue, and more in vitro studies to identify the pathways involved are required.
Abstract: Nitric oxide (NO) and Monocyte Chemoattractant Protein (MCP)-1 co-regulation has been found in endotoxin-activated macrophages. Kupffer cells (KC) are a main source of soluble-mediators production in liver abnormalities. We investigated in vitro similar co-regulation of NO and MCP-1 production in rat activated KC. Isolated rat KC were cultured in the presence of 1 microg/ml LPS and various concentrations of Wortmannin (0-300 nM), L-NAME (0-500 microM) or MCP-1 (0-100 ng/ml). Production of MCP-1 and NO were measured in supernatants, by ELISA and a modification of the Griess reaction, respectively. Growth arrested KC, stimulated with vehicle, produced a basal amount of NO and MCP-1. In the presence of LPS, cultured KC secreted significantly (P < 0.01) increased amounts of MCP-1 and NO. Pre-treatment of KC with various concentrations of L-NAME significantly (P < 0.05) reduced the LPS-induced secretion of NO in a concentration dependent manner, but the MCP-1 production remained unaffected. Pre-treatment with Wortmannin significantly (P < 0.05) inhibited LPS-induced secretion of MCP-1 and NO in a concentration dependent manner. Linear regression analysis revealed a positive correlation between MCP-1 and NO in the LPS (r = 0.59171, P < 0.0001) and Wortmannin (r = 0.9215, P = 0.009) treated groups, but not in the L-NAME (r = -0.08513, P = 0.873). Incubation of KC with various concentrations of MCP-1 did not increase the NO production. These results indicate that KC might be the main source of NO and MCP-1 production in liver disorders, probably through the induction of PI3-kinase(s) and without any co-regulation between these molecules, which might represent two independent immunoregulatory pathways in the role of KC in hepatic disorders.
Abstract: The intestinal microflora is a large bacterial community that colonizes the gut, with a metabolic activity equal to an organ and various functions that affect the physiology and pathology of the host's mucosal immune system. Intestinal bacteria are useful in promotion of human health, but certain components of microflora, in genetically susceptible individuals, contribute to various pathological disorders, including inflammatory bowel disease. Clinical and experimental observations indicate an imbalance in protective and harmful microflora components in these disorders. Manipulation of gut flora to enhance its protective and beneficial role represents a promising field of new therapeutic strategies of inflammatory bowel disease. In this review, we discuss the implication of gut flora in the intestinal inflammation that justifies the role of probiotics and prebiotics in the prevention and treatment of inflammatory bowel disease and we address the evidence for therapeutic benefits from their use in experimental models of colitis and clinical trials.
Abstract: BACKGROUND AND AIM: T-lymphocyte migration is implicated in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). CXC chemokines MIG, IP-10, and I-TAC act by binding to CXCR3 receptor on T-lymphocytes. We investigated the role of these chemokines and their receptor in patients with UC, CD, and normal controls (NC). METHODS: Chemokine expression and serum levels were examined in colonic biopsies from patients and NC using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. HT-29 and Caco2 colonic epithelial cells were studied following in vitro stimulation with proinflammatory (Th1) and Th2-derived cytokines. CXCR3 receptor expression was assessed in CD3+ peripheral blood lymphocytes (PBL) from patients and NC and in stimulated Jurkat leukaemia cells, using RT-PCR and flow cytometry. RESULTS: Full size CXCR3 mRNA (FS) expression was found in CD3+ PBL from controls and UC, but not from CD patients. In contrast, CD3+ PBL from CD patients showed a marked mRNA expression of the spliced variant CXCR3 (TV). This finding explains the high expression of CXCR3 on CD3+ PBL from CD patients in flow cytometry. Increased chemokine expression and production was found in colonic biopsies and serum from CD compared to UC patients and controls. Stimulation of epithelial cells with proinflammatory cytokines significantly induced chemokine production. The addition of Th2 cytokines had an inhibitory effect. Stimulation of Jurkat cells with cytokines and supernatant conditioned media from epithelial cells induced CXCR3TV expression. CONCLUSIONS: These data demonstrate that PBL from CD patients express a spliced variant of the CXCR3 receptor and suggest a role for the colonic epithelial cells in T-lymphocyte migration in intestinal inflammation.
Abstract: The present study investigated the presence of somatostatin receptor subtypes (ssts) and the endogenous peptides somatostatin and cortistatin in rat Kupffer cells, since modulation of these cells by somatostatin may be important for the beneficial effect of somatostatin analogues in a selected group of hepatocellular carcinoma patients. Kupffer cells were isolated from rat liver in agreement with national and EU guidelines. RT-PCR was employed to assess the expression of somatostatin, cortistatin and ssts in Kupffer cells. Western blot analysis and immunocytochemistry were employed to assess the expression and the localization of the receptors, respectively. Quiescent Kupffer cells were found to express sst(1-4) mRNA, while immunocytochemical studies supported the presence of only the sst(3) and sst(4) receptors, which were found to be internalized. However, sst1 and sst(2A) receptors were detected by western blotting. RT-PCR and RIA measurements support the presence of both somatostatin and cortistatin. Stimulation of the cells with LPS activated the expression of the sst(2), sst(3) and sst(4) receptors. The present data provide evidence to support the presence of ssts and the endogenous neuropeptides somatostatin and CST in rat Kupffer cells. Both peptides may act in an autocrine manner to regulate sst receptor distribution. Studies are in progress in order to further characterize the role of ssts in Kupffer cells and in hepatic therapeutics.
Abstract: OBJECTIVES: Sepsis is a common complication in the early postoperative period, leading to the augmentation of oxidative and nitrosative stresses. The present study investigated the role of enteral nutrition on nitric oxide (NO) production after a lipopolysaccharide (LPS)-induced endotoxemia as an index of nitrosative stress. METHODS: Fifty rats were subjected to midline laparotomy and feeding gastrostomy. Ten rats served as controls after recovering from operative stress. The remaining rats received, through gastrostomy, enteral nutrition or placebo feeding for 24 h, after which they were injected intraperitoneally with LPS or equal volume of saline. Two hours later blood and liver tissue were collected. NO production was quantified in serum samples and homogenates of liver tissue by a modification of Griess's reaction. NO synthase (NOS) mRNA expression was examined in homogenate of liver tissue by reverse transcription-polymerase chain reaction. RESULTS: The operation significantly increased basal NO production in rat serum. LPS induced a further significant increase of NO levels. Enteral feeding of rats significantly decreased NO levels in both groups. In contrast, enteral nutrition was found to increase significantly NO levels in liver homogenates from rats treated with LPS. A constitutive endothelial NOS mRNA expression was found in liver tissue, whereas LPS administration induced inducible NOS mRNA expression in liver tissue regardless of enteral feeding. CONCLUSION: These findings indicate that early enteral feeding leads to a reduction in circulating NO levels induced by operation and endotoxemia, but increases hepatic NO levels in endotoxemia probably by the effect of LPS-induced inducible NOS on the increased L-arginine uptake.
Abstract: BACKGROUND: The fluoroquinolone ciprofloxacin is a broad-spectrum antibiotic that has been used in the treatment of inflammatory bowel diseases. There is evidence that quinolones have immunomodulating activities via the regulation of cytokine production. MATERIALS AND METHODS: We investigated the effect of ciprofloxacin on the nitric oxide (NO) production by colonic epithelium. HT-29 cells and colonic biopsies from patients (n = 4) with ulcerative colitis (UC) and normal controls (n = 4) were cultured with various concentrations of ciprofloxacin (10-100 microg mL(-1)) in the presence and absence of pro-inflammatory cytokines. The production of NO was measured in culture supernatants with a spectrophotometric method and inducible nitric oxide synthase (iNOS) mRNA expression was examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Ciprofloxacin did not have any effect on the basal NO production by HT-29 cells. In contrast, ciprofloxacin significantly (P < 0.001) inhibited the pro-inflammatory cytokines (interleukin-1alpha + tumour necrosis factor-alpha + interferon-gamma)-induced NO production in HT-29, in a concentration-dependent manner, via the inhibition of the cytokine-induced iNOS mRNA expression. Wortmannin produced a concentration related reversal of the inhibitory effect of ciprofloxacin at both iNOS mRNA expression and NO production in HT-29 cells. A similar inhibitory effect of ciprofloxacin on the cytokine-induced NO production and iNOS mRNA expression was detected in vitro in cultures of normal colonic tissue. In addition, ciprofloxacin significantly inhibited the NO production and iNOS mRNA expression in cultures of colonic tissue from ulcerative colitis patients, in a concentration-dependent manner. CONCLUSIONS: These data suggest that ciprofloxacin, in addition to its antimicrobial role, might have an immunoregulatory effect on intestinal inflammation, via the modulation of inflammatory mediators.
Abstract: 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.
Abstract: The stress neuropeptides, corticotropin-releasing hormone (CRH) and urocortin (UCN), modulate the inflammatory response via the hypothalamus-pituitary-adrenal axis and locally, in a paracrine manner, act on mast and macrophage cells. Kupffer cells (KCs) are the resident macrophages of the liver. They represent the bulk of tissue macrophages in the body and they are the first to face invading noxious agents reaching the body via the portal circulation. The aim of the present report was to study the expression of the CRH system in rat KC and test its functionality. Our findings are as follows: (1) In highly purified KCs the transcripts of UCN, of its receptors CRHR1, CRHR2 and that of the pseudoreceptor CRH-binding protein (CRHBP) were present while that of CRH was not detectable. (2) Similarly, immunoreactive UCN, CRHR1, CRHR2 and CRHBP were easily detectable by immunohistochemistry and immunofluorescence in sections of whole rat liver (localized in KC) as well as in purified KC while CRH was again not detectable. (3) Exposure of purified KC to CRH or UCN suppressed lipopolysaccharide-induced tumor necrosis factor alpha production, an effect completely prevented by the CRHR1 and CRHR2 receptor antagonist astressin. Our data demonstrate the presence of UCN and its receptors in rat KC, the absence of CRH, and the functionality of these receptors. We propose that a UCN-based system may affect local inflammatory phenomena in the liver acting in a paracrine manner.
Abstract: BACKGROUND: Endothelins and nitric oxide regulate sinusoidal blood flow and the perfusion of the peribiliary vascular plexus. AIMS: To study the serum and hepatic vein concentration of ET-1, ET-2, ET-3 and nitric oxide in patients with primary biliary cirrhosis and the effect of ursodeoxycholic acid treatment. METHODS: Endothelins and nitrites/nitrates were measured in serum and hepatic vein blood in primary biliary cirrhosis and viral cirrhotic patients prior and after ursodeoxycholic acid therapy and in serum in controls. Endothelins were measured with commercial enzyme-linked immunosorbent assays and nitrites/nitrates with a modification of Griess reaction. RESULTS: The ET-1 and ET-3 levels were similar in patients and controls. Primary biliary cirrhosis patients had the highest serum ET-2 (P < 0.001) compared with other groups. Nitrites/nitrates was increased in primary biliary cirrhosis (P < 0.05) compared with normal. ET-2 and nitric oxide were similar in all primary biliary cirrhosis stages. Ursodeoxycholic acid significantly decreased ET-2 in all stages (I and II: P < 0.05 and III and IV: P < 0.01) and increased nitric oxide (P < 0.05) in early primary biliary cirrhosis. Hepatic vein ET-1 and ET-3 were higher in viral cirrhosis patients, but only in primary biliary cirrhosis a significant difference for ET-1 and ET-3 between hepatic and peripheral veins was found. CONCLUSIONS: Increased ET-2 is an early defect in primary biliary cirrhosis that is significantly reduced by the ursodeoxycholic acid treatment. The possibility of a more generalized endothelial cell dysfunction in primary biliary cirrhosis requires further investigation.
Abstract: Kupffer cells may be involved in liver fibrogenesis through production of TGF-beta1. Their role in fibrinolysis is less clear. Octreotide, a synthetic analogue of somatostatin, is often used in cirrhotic patients. Its effect on Kupffer cells was studied. Isolated rat Kupffer cells were cultured in the presence of lipopolysaccharide and/or octreotide. TGF-beta1, leptin, collagenase (MMP-1), and urokinase-type plasminogen activator (uPA) were assessed in supernatants by ELISA, and MMP-2 and MMP-9 by zymography. Kupffer cells produced large amounts of MMP-1 and lipopolysaccharide induced a significant (P < 0.02) early increase. Octreotide and lipopolysaccharide caused a synergistic effect on MMP-1 secretion. By contrast, MMP-9 production stimulated by lipopolysaccharide was suppressed by octreotide. Kupffer cells produced a basal amount of uPA, significantly increased after lipopolysaccharide or octreotide incubation (P < 0.001). Large amounts of TGF-beta1 were produced in a time-dependent manner by unstimulated Kupffer cells. Lipopolysaccharide and octreotide, alone or in combination, induced a significant inhibition of this production (P < 0.01). Kupffer cells did not produce leptin, a recently identified mediator of liver fibrosis, or MMP-2. Kupffer cells may play a significant role in liver fibrinolysis. Octreotide, acting on TGF-beta1, uPA, and MMP-1 production, may be a useful agent for fibrosis resolution.
Abstract: Nitric oxide (NO) production is increased in the human colonic mucosa in intestinal inflammation. We examined the effect of corticosteroids and the role of mononuclear cells in this production. Colonic biopsies from patients with ulcerative colitis and normal controls were cultured with either budesonide or prednisolone in the presence of proinflammatory cytokines. Human mixed mononuclear cells (MMCs) were cocultured with HT-29 cells stimulated with IFN-gamma and LPS in the presence or absence of corticosteroids. Nitrite production was measured in supernatants by a modification of the Griess reaction, and inducible NO synthase (iNOS) mRNA expression was studied in colonic tissue by RT-PCR. Both steroids significantly suppressed the nitrite production and iNOS mRNA expression in inflamed colonic biopsies from ulcerative colitis patients and in cytokine-stimulated normal colonic biopsies but not in cytokine-stimulated HT-29 cells. Nitrite production by HT-29 cells was significantly increased (P < 0.01) in cocultures with MMCs stimulated with IFN-gamma and LPS. The presence of either prednisolone or budesonide significantly (P < 0.01) suppressed nitrite production from cocultures of HT-29 cells and MMCs but not from cultures of HT-29 cells stimulated with conditioned media from activated MMCs. Interestingly, stimulation of HT-29 with conditioned media from MMCs pretreated with steroids before stimulation with LPS and IFN-gamma induced a significantly (P < 0.01) lower nitrite production. These results suggest that the inhibitory effect of corticosteroids on the NO production in the intestinal inflammation might be via the inhibition of MMC-produced mediators responsible for NO production by colonic epithelial cells.
Abstract: 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.
Abstract: Intestinal myofibroblasts have been implicated in the pathogenesis of chronic inflammatory conditions such as Crohn's disease via interactions with an elaborate network of cytokines, growth factors, and other inflammatory mediators. CXCR3 is a Galpha(i) protein-coupled receptor that binds the proinflammatory chemokines CXCL9, CXCL10, and CXCL11, which are released from the intestinal epithelium. The three CXCR3 ligands shared the ability to activate biochemical (e.g., PI3K and MAPK activation) and functional events (actin reorganization) in intestinal myofibroblasts. However, CXCL11 is unique in its ability to elevate intracellular calcium. Surprisingly, although CXCR3 mRNA is detectable in these myofibroblasts, there is no detectable surface expression of CXCR3. Furthermore, the biochemical responses and actin reorganization stimulated by the CXCR3 ligands in intestinal myofibroblasts are insensitive to the Galpha(i) inhibitor, pertussis toxin. This suggests either the existence of differential receptor coupling mechanisms in myofibroblasts for CXCR3 that are distinct from those observed in PBLs and/or that these cells express a modified or variant CXCR3 compared with the CXCR3 expressed on PBLs.
Abstract: BACKGROUND/AIMS: Recently, trials of octreotide have shown a significant survival benefit in the treatment of advanced hepatocellular carcinoma but new data are controversial. We, therefore, examined the production of somatostatin and cortistatin, the expression and distribution of somatostatin receptors (sst) in HepG2 human hepatocellular carcinoma cells, and the possible antiproliferative effect of octreotide on these cells. METHODS: Radioimmunoassay and RT-PCR studies were performed for the detection of somatostatin and cortistatin. RT-PCR, radioligand binding and immunocytochemistry assays were employed for the detection of the ssts. Growth and viability of cells were measured by the tetrazolium salt assay. RESULTS: HepG2 cells were found to express sst(2), sst(3) and sst(5) receptors. Immunocytochemistry revealed a mainly intracellular distribution of all ssts with unique patterns for each of them. Membrane binding sites for somatostatin were mainly of the sst(3) (39+/-8%) and sst(5) (59+/-5%) types, while only minor sst(2) binding could be detected (5+/-12%). Octreotide was found to inhibit the proliferation of HepG2 cells (IC(50) 1.25 x 10(-9)M) via protein tyrosine phosphatases. HepG2 cells produced cortistatin while somatostatin expression was not detected. CONCLUSIONS: In conclusion, HepG2 cells express cortistatin, which regulates somatostatin receptors. Cell proliferation was reduced by octreotide via a protein tyrosine phosphatase dependent mechanism.
Abstract: 1. Cyclooxygenase (COX)-2 expression and activity in response to pro-inflammatory cytokines TNF alpha and IFN gamma was evaluated in the colonic epithelial cell line HT29 and the airway epithelial cell line A549. 2. TNF alpha induced concentration- and time-dependent upregulation of COX-2 mRNA, protein and prostaglandin (PG)E(2) synthesis. 3. Co-stimulation of TNF alpha with IFN gamma resulted in reduced COX-2 mRNA and protein expression. 4. IFN gamma had no effect on the stability of TNF alpha-induced COX-2 mRNA. 5. TNF alpha-induced PGE(2) biosynthesis was significantly enhanced by the simultaneous addition of IFN gamma and was COX-2 dependent. 6. The combination of IFN gamma and TNF alpha induced the microsomal prostaglandin E synthase (mPGES), comensurate with the enhanced PGE(2) synthesis. 7. These results suggest that, in terms of PGE(2) biosynthesis, IFN gamma plays a negative regulatory role at the level of COX-2 expression and a positive regulatory role at the level of mPGES expression. This may have important implications for the clinical use of IFN gamma in inflammatory diseases.
Abstract: Kupffer cells (KC) and lipopolysaccharide (LPS) interaction is the initial event leading to hepatic inflammation and fibrosis in many types of liver injury. We studied chemokine secretion by KC activated with LPS and the possible effect of the somatostatin analogue octreotide, in the regulation of this process. KC isolated from Sprague-Dawley rats were cultured in the presence of LPS added alone or with different concentrations of octreotide for 24 and 48 h, and chemokine production was assessed in culture supernatants by ELISA. CC chemokine mRNA expression was assessed by semiquantitative RT-PCR. Vehicle-stimulated KC produced a basal amount of CC and CXC chemokines. LPS-stimulated KC secreted significantly increased amounts of IL-8 (GRO/CINC-1) (P<0.001), MIP-2 (P<0.001), MCP-1 (P<0.001), and RANTES (P<0.01). Octreotide inhibited LPS-induced secretion of the CC chemokines MCP-1 (P<0.05) and RANTES (P<0.05), but not the CXC chemokines IL-8 (GRO/CINC-1) and MIP-2, in a concentration-dependent manner. Downregulation of basal and LPS-induced mRNA expression of the CC chemokines was also observed in the presence of octreotide. Pretreatment with phosphatidylinositol 3 (PI3)-kinase inhibitors reduced chemokine production by LPS-treated KC in both the mRNA and protein level. Furthermore, it prevented the octreotide inhibitory effect on LPS-induced chemokine secretion, indicating a possible involvement of the PI3-kinase pathway. In conclusion, these data demonstrate that chemokine secretion by KC can be differentially regulated by octreotide, and suggest that this somatostatin analogue may have immunoregulatory effects on resident liver macrophages. British Journal of Pharmacology (2004) 141, 477-487. doi:10.1038/sj.bjp.0705633
Abstract: PURPOSE: To investigate the presence of somatostatin and its receptors (sst(1-5) receptors) and their possible involvement in the regulation of nitric oxide (NO) production in human RPE cell cultures. METHODS: Human RPE cells (D407) were used for all studies performed. Somatostatin levels were detected by radioimmunoassay, and RT-PCR and immunocytochemistry studies were performed to identify the somatostatin receptors (sst1-sst5). Radioligand binding assays were also performed examining the ability of certain somatostatin ligands (sst1, sst2, sst5) to compete for [125I]Tyr11 somatostatin binding. The presence of NO synthase in the cultures was assayed with NADPH-diaphorase cytochemistry, and RT-PCR, and NO levels were assessed by examining the production of its stable metabolites NO2- and NO3- (NOx-). RESULTS: SRIF was detected in a concentration of 0.56 +/- 0.13 picomoles/mg protein. sst1, sst2, and sst5 mRNAs were detected, yet only sst2B and sst5 immunoreactivity was observed in human RPE cell cultures. sst1- and sst5- but not sst2-selective ligands displaced the specific [125I]Tyr11 somatostatin binding to RPE cell membranes. NADPH-diaphorase stain and iNOS mRNA were detected. SRIF and the sst2-selective analogue MK678 increased the levels of NOx- in a concentration-dependent manner. This increase was blocked by the sst2 antagonist CYN-154806 (Ac-4NO2-Phe-c(dCys-Tyr-dTrp-Lys-Thr-Cys)-dTyr-NH2). CONCLUSIONS: These results demonstrate the presence of somatostatin, and its receptors sst1, sst2B, and sst5 in human RPE cells and suggest an autocrine or paracrine role for somatostatin. Somatostatin's ability to regulate NO production, by activating sst2 receptors, provides a functional role of somatostatin in the RPE.
Abstract: 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.
Abstract: In recent years, nitric oxide (NO), a gas previously considered to be a potentially toxic chemical, has been established as a diffusible universal messenger that mediates cell-cell communication throughout the body. Constitutive and inducible NO production regulate numerous essential functions of the gastrointestinal mucosa, such as maintenance of adequate perfusion, regulation of microvascular and epithelial permeability, and regulation of the immune response. Up-regulation of the production of NO via expression of inducible nitric oxide synthase (iNOS) represents part of a prompt intestinal antibacterial response; however, NO has also been associated with the initiation and maintenance of inflammation in human inflammatory bowel disease (IBD). Recent studies on animal models of experimental IBD have shown that constitutive and inducible NO production seems to be beneficial during acute colitis, but sustained up-regulation of NO is detrimental. This fact is also supported by studies on mice genetically deficient in various NOS isoforms. However, the mechanism by which NO proceeds from being an indispensable homeostatic regulator to a harmful destructor remains unknown. Furthermore, extrapolation of data from animal colitis models to human IBD is questionable. The purpose of this review is to update our knowledge about the role of this universal mediator and the enzymes that generate it in the pathogenesis of IBD.
Abstract: BACKGROUND: Inflammatory bowel disease is associated with an increased incidence of thromboembolic complications. The aim of this study was to investigate the role of the soluble CD40 ligand (sCD40L), which displays prothrombotic properties, in patients with ulcerative colitis (UC) and Crohn's disease (CD) in comparison with inflammatory and healthy controls. METHODS: Plasma levels of sCD40L, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin (TAT) complex and soluble P-selectin were measured in 104 inflammatory bowel disease patients (54 ulcerative colitis and 50 Crohn's disease), in 18 cases with other causes of intestinal inflammation and in 80 healthy controls using commercially available enzyme-linked immunosorbent assays. Plasma levels of sCD40L were correlated with disease activity, type, localization and treatment as well as with the measured thrombophilic parameters. RESULTS: CD patients had significantly higher sCD40L levels than both groups of controls (CD vs HC P < 0.001; CD vs non-IBD P < 0.05). UC patients had higher but not significantly different sCD40L levels compared with the controls. Both UC and CD patients with active disease had significantly higher sCD40L levels in comparison with patients with inactive disease. Plasma levels of sCD40L were correlated with platelet count (r = 0.27, P = 0.001). They also showed a correlation with prothrombin F1+2 (r = 0.16, r = 0.03) and TAT (r = 0.15, r = 0.04) as well as with P-selectin (r = 0.19, P = 0.01). CONCLUSIONS: The increased sCD40L plasma levels may represent, at least in some degree, a molecular link between inflammatory bowel disease and the procoagualant state.
Abstract: 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.
Abstract: We report a four-step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED-2 immunophenotyping and non-specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80-100 x 10(6)KCs per liver, over 95% positive for ED-2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time-dependent LPS-induced TNF-alpha and nitric oxide production.
Abstract: BACKGROUND: Non specific esterases (NSE) are a group of cellular carboxylesterases, enzyme markers of monocytes/macrophages, whose tissue distribution in the human body and changes in various disease states have not been adequately studied. We investigate the presence and localization of NSE, in the normal and inflamed human colonic mucosa. DESIGN: NSE were studied histochemically and biochemically using alpha-naphthyl acetate as the substrate, in the colonic mucosa from 67 patients with colitis of various aetiologies and 10 normal controls. In addition, esterase activity was studied biochemically in serum from colitic patients and normal controls. RESULTS: Histochemical study of the colonic tissue demonstrated that NSE were localised in the epithelial brush border, the goblet cells of the glands and a macrophage population of the lamina propria in the colonic mucosa of normal controls and patients with non specific colitis. In active ulcerative colitis, esterase depletion and esterase negative macrophages were identified in parallel with goblet cell disappearance. Gradual reappearance of esterase activity was found after successful treatment. Biochemical study of NSE activity showed that serum and colonic tissue esterase levels were greatly (P < 0.001) reduced in active ulcerative colitis compared to the normal controls or non specific colitis patients and they were increased after successful treatment. Despite this increase, the esterase activity in the colonic tissue from ulcerative colitis patients after treatment was significantly reduced compared to the normal controls. Interestingly, the enzyme levels from non-inflamed areas of the bowel of patients with ulcerative colitis were also significantly (P < 0.01) decreased compared to the normal controls. CONCLUSIONS: These data suggest that esterase reduction in ulcerative colitis is not a simple result of the inflammatory process but rather it precedes its development. This enzyme depletion might have an important pathogenetic implication in the inflammatory process.
Abstract: BACKGROUND AND AIMS: Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells. METHODS: In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography. RESULTS: The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly. CONCLUSIONS: COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.
Abstract: BACKGROUND/AIMS: Nitric oxide synthesis is increased in rectal biopsies from patients with ulcerative colitis and colonic epithelial cells are considered to be a major source of nitric oxide in intestinal inflammation. METHODOLOGY: Human colonic biopsies from normal bowel mucosa and colonic epithelial cell line HT-29 were cultured in the presence of the inflammatory cytokines IL-1 alpha + TNF-alpha + IFN-alpha added after 1 hour pretreatment with vehicle or Interleukin-13. Nitrite levels were determined at 30 hours in culture supernatants by a fluorometric assay. RESULTS: Unstimulated human colonic biopsies and HT-29 cells produced a basal amount of nitrite. Stimulation with IL-1 alpha + TNF-alpha + IFN-alpha induced a significant (P < 0.001) increase of nitrite generation by both human colonic biopsies and HT-29 cells. The presence of Interleukin-13 produced a significant (P < 0.001) suppression of the cytokine-induced nitrite generation from both colonic biopsies and HT-29 cells. CONCLUSIONS: Nitric oxide generation in human colonic mucosa is susceptible to manipulation by proinflammatory cytokines. Interleukin-13 has an inhibitory effect on cytokine induced nitrite production in colonic mucosa and could play an anti-inflammatory role in intestinal inflammation.
Abstract: Differential chemokine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected population of leukocytes cells in inflammatory bowel disease. We have previously demonstrated that IL-13 enhances IL-1alpha-induced IL-8 secretion by the colonic epithelial cell line HT-29. We have now explored the C-C chemokine expression and modulation in this system. The combination of TNF-alpha and IFN-gamma was the minimal stimulation required for regulated on activation, normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein (MCP-1) mRNA expression and secretion by HT-29 cells. The same stimulation induced a stronger IL-8 mRNA expression and secretion. Pretreatment with IL-13 or IL-4, reduced significantly the RANTES, and MCP-1, but not IL-8 mRNA expression and secretion. In contrast, IL-10 had no effect on either MCP-1, or RANTES, or IL-8 generation. Pretreatment of HT-29 cells with wortmannin suggested that the IL-13-induced inhibition of C-C chemokine expression is via activation of a wortmannin-sensitive phosphatidylinositol 3-kinase. These data demonstrate that colonic epithelial cell chemokine production can be differentially regulated by T cell-derived cytokines and suggest an interplay between epithelial cells and T lymphocytes potentially important in the intestinal inflammation.
Abstract: In addition to their role as regulators of leukocyte migration and activation, chemokines and their receptors also function in angiogenesis, growth regulation, and HIV-1 pathogenesis--effects that involve the action of chemokines on nonhematopoietic cells. To determine whether chemokine receptors are expressed in human colonic epithelium, HT-29 cells were examined by RT-PCR for the expression of the chemokine receptors for lymphotactin, fractalkine, CCR1-10, and CXCR1-5. The only receptor consistently detected was CXCR4 (fusin/LESTR), although HT-29 cells did not express mRNA for its ligand, stromal cell-derived factor (SDF-1alpha). Flow cytometric analysis with anti-CXCR4 antibody indicated that the CXCR4 protein was expressed on the surface of roughly half of HT-29 cells. CXCR4 was also expressed in colonic epithelial cells in vivo as shown by immunohistochemistry on biopsies from normal and inflamed human colonic mucosa. The mRNA for SDF-1alpha and other CC and CXC chemokines was present in normal colonic biopsies. The CXCR4 receptor in HT-29 cells was functionally coupled, as demonstrated by the elevation in [Ca2+]i, which occurred in response to 25 nM SDF-1alpha and by the SDF-1alpha-induced upregulation of ICAM-1 mRNA. Sodium butyrate downregulated CXCR4 expression and induced differentiation of HT-29 cells, suggesting a role for CXCR4 in maintenance and renewal of the colonic epithelium. This receptor, which also serves as a coreceptor for HIV, may mediate viral infection of colonic epithelial cells.
Abstract: A combination of the pro-inflammatory cytokines interleukin (IL)-1alpha, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the human colon carcinoma cell line HT-29. This can be inhibited by pretreatment with IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wright, K., Ward, S. G., Kolios, G., and Westwick, J. (1997) J. Biol. Chem. 272, 12626-12633). Since NO has been implicated in regulating mechanisms leading to cell death, while activation of PI 3-kinase-dependent signaling cascades are thought to be involved with promoting cell survival events, we have investigated the outcome of these cytokine treatments on apoptosis and cell survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-gamma/TNF-alpha, IFN-gamma/CD95, IL-1alpha/IFN-gamma, and IL-1alpha/IFN-gamma/TNF-alpha to varying extents. Induction of apoptotic markers by HT-29 cells in response to cytokine treatment is not dependent on NO production. Pretreatment with IL-13 protects against IL-1alpha/IFN-gamma/TNF-alpha- and IFN-gamma/TNF-alpha- as well as IFN-gamma/CD95-induced (but not IL-1alpha/IFN-gamma-induced) cell death. In addition, IFN-gamma/TNF-alpha and IL-1alpha/IFN-gamma/TNF-alpha stimulate activation of caspase-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effector, protein kinase B. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit IL-13 stimulation of protein kinase B as well as the cell survival effects of IL-13. These data demonstrate that cytokine-induced apoptosis of HT-29 cells is NO-independent and that the activation of a PI 3-kinase-dependent signaling cascade by IL-13 is a key signal responsible for the inhibition of apoptosis.
Abstract: BACKGROUND: Cholangitis and septicaemia are serious complications of endoscopic retrograde cholangiopancreatography (ERCP). They occur mainly following therapeutic ERCP in the setting of an obstructed biliary system. The optimum prophylactic antibiotic regimen in such patients is not yet defined but usually depends on intravenous agents. AIM: To compare the efficacy of oral ciprofloxacin with intravenous cephazolin. METHODS: One hundred and fifty patients at high risk from septic complications were randomized prospectively to either oral ciprofloxacin (750 mg b.d.) or intravenous cephazolin (1 g b.d.), commenced at least 90 min prior to the ERCP and continued for 3 days. Bacteriological cultures were taken from bile during the procedure and from blood both immediately and at 24 h post-procedure. RESULTS: There were no significant differences between the two treatment groups in the pre-ERCP clinical or radiological findings or in the types of procedure performed. One patient did not undergo an ERCP and was excluded from the final analysis. Of the 77 patients in the ciprofloxacin group there were no positive blood cultures and one positive culture from a nasobiliary drain. Two out of the 72 cephazolin patients had positive blood cultures immediately post-ERCP; one of these two patients and one other cephazolin patient had positive bile cultures. There were no cases of cholangitis or septicaemia in the ciprofloxacin group and three cases in the cephazolin group. One patient from each treatment group died within the 7-day study. Adverse drug reactions were minimal and none of the different clinical outcomes in the two groups reached statistical significance. CONCLUSION: Oral ciprofloxacin is a cost-effective prophylactic agent for high-risk ERCP.
Abstract: Ulcerative colitis and Crohn's disease, regardless of the initiating events, share common immunologically mediated pathways of tissue injury and repair. Although their etiology remains unknown, increasing evidence suggests that activated immunological effector mechanisms within the intestinal mucosa are responsible for the pathogenesis of the diseases. Activation of immune, mesenchymal and epithelial cells; transmigration of leukocytes from the circulation to the sites of inflammation; tissue damage; and healing phase are mediated by a number of soluble mediators released by activated intestinal cells. These mediators are involved in a network of cell communication, affecting immune response, synthesis and release of enzymes, and cell proliferation. In the last decades, the identification of potential mediators in intestinal inflammation has expanded to include eicosanoids, platelet activating factor, biogenic amines, kinins, proteases, reactive oxygen species, complement components, cytokines, chemokines, nitric oxide, and neuropeptides. An increasing understanding suggests that in inflammatory bowel disease, regardless of the predisposing and trigger factors, a disruption of certain regulatory mechanisms, mediated by these soluble molecules, results in pathological immune responses to antigens and in chronic inflammation.
Abstract: BACKGROUND: Nitric oxide (NO) synthesis and inducible nitric oxide synthase (iNOS) expression are increased in colonic biopsy specimens from patients with ulcerative colitis, but the cellular source of NO production is not known. AIMS: To examine the distribution of iNOS in human colonic mucosa and to explore the ability of T lymphocyte derived cytokines to regulate iNOS expression and activity in human colonic epithelial cells. METHODS: iNOS expression was examined using immunohistochemistry in colonic biopsy samples from 12 patients with ulcerative colitis and three with infectious colitis and compared with 10 normal controls. In vitro iNOS expression and activity were determined in HT-29 cell cultures; nitrite levels were measured using a fluorescent substrate, iNOS mRNA expression by northern blot analysis, and iNOS protein expression by western blot analysis. RESULTS: No iNOS expression was detected (10 of 10) in non-inflamed mucosa derived from normal controls. In 11 of 12 cases of newly diagnosed ulcerative colitis, iNOS protein was expressed in the epithelial cells, while no other positive cells were found in the lamina propria. Similar iNOS labelling was found in colonic biopsy samples from patients with infectious colitis in the acute phase, but when re-examined in samples from patients in total remission, no iNOS staining was observed. Both interleukin (IL)-13 and IL-4, but not IL-10, are potent inhibitors of iNOS expression and activity induced by an optimal combination of cytokines, namely IL-1 alpha, tumour necrosis factor alpha and interferon gamma. CONCLUSIONS: The data suggest that the epithelium is the major source of iNOS activity in ulcerative colitis and that IL-13 and IL-4 may act as intrinsic regulators of NO generation in intestinal inflammation.
Abstract: The human colonic epithelial cell line HT-29 can be induced by a combination of the cytokines interleukin (IL)-1alpha, tumor necrosis factor alpha, and interferon-gamma to express the inducible form of nitric-oxide synthase (iNOS; Kolios, G., Brown, Z., Robson, R., Robertson, D. A. F., & Westwick, J. (1995) Br. J. Pharmacol. 116, 2866-2872). IL-13 is a potent inhibitor of cytokine-induced iNOS mRNA expression and nitric oxide generation in HT-29 cells via an unknown mechanism. We report here that in HT-29 cells, IL-13 induces a concentration and time-dependent increase in the formation of the lipid products of phosphatidylinositol (PtdIns) 3-kinase, namely phosphatidylinositol (3,4)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate. IL-13 also induces a parallel concentration and time-dependent increase in the in vitro lipid kinase activity present in immunoprecipitates of the p85 regulatory subunit of PtdIns 3-kinase. In addition, we also demonstrate that IL-13 stimulates the tyrosine phosphorylation of the adaptor molecule insulin receptor substrate 1, which may facilitate receptor coupling to PtdIns 3-kinase. Both the increases in D-3 phosphatidylinositol lipids and the increased in vitro lipid kinase activity of p85 immunoprecipitates were inhibited by wortmannin and LY294002. Inhibition of the PtdIns 3-kinase activity was paralleled by a reversal of the ability of IL-13 to inhibit iNOS mRNA expression and nitrite generation in HT-29 cells. These data demonstrate that the activation of PtdIns 3-kinase by IL-13 is a key signal that is responsible for the inhibition of iNOS transcription in activated epithelial cells.
Abstract: 1. We have determined which cytokines induce and modulate the production of the chemokine interleukin-8 (IL-8) by the human colonic epithelial cell line HT-29. 2. Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin-1 alpha (IL-1 alpha), tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-13 (IL-13), interleukin-10 (IL-10) or vehicle added alone or in combination. The production of IL-8 was determined by enzyme-linked immunosorbent assay (ELISA) and IL-8 messenger RNA expression by Northern blot analysis. 3. The production of IL-8 in unstimulated cells was undetectable by both ELISA and Northern blot analysis. 4. HT-29 cells produced IL-8 following stimulation with IL-1 alpha or TNF-alpha in a time- and a concentration-dependent manner, while IFN-gamma, IL-10 and IL-13 did not induce IL-8 production by HT-29 cells. 5. IL-13 was found to up-regulate significantly (P < 0.01) the IL-1 alpha but not the TNF-alpha-induced IL-8 generation by HT-29 cells. In contrast, IL-10 had no effect on either IL-1 alpha or TNF-alpha-induced IL-8 production. 6. Experiments using cycloheximide demonstrated that this synergistic effect of IL-13 and IL-1 alpha on IL-8 secretion was not through de novo protein synthesis. Using actinomycin-D, we demonstrated that the IL-13 up-regulation was at the level of transcription rather than messenger RNA stability. 7. These findings suggest that colonic epithelial cells have a functional IL-13 receptor, which is coupled to an up-regulation of IL-1 alpha, but not TNF-alpha induced IL-8 generation.
Abstract: The frequency and degree of gastrointestinal involvement in patients with Mediterranean Kaposi's sarcoma (non-AIDS), a newly recognized form of Kaposi's sarcoma, is unknown. Eighty-seven patients with Mediterranean Kaposi's sarcoma proven by skin and/or nodal biopsy underwent endoscopic study of the upper gastrointestinal tract. Of these, 71 (81.6%) had gastrointestinal lesions. All these patients had lesions in the stomach. Additional lesions were detected in the esophagus in 19 patients and in the proximal duodenum in 8 patients, whereas additional lesions in both the esophagus and duodenum were identified in 2 patients. The lesions were classified into 4 types according to their size, shape, and color. Most types of lesions showed characteristic discoloration, but lesions with the appearance and color of normal mucosa that histologically were shown to be Kaposi's sarcoma were also identified. The high prevalence of gastrointestinal involvement in patients with Mediterranean Kaposi's sarcoma (non-AIDS) suggests that an endoscopic examination of the upper gastrointestinal tract may be useful in non-AIDS-related forms of Kaposi's sarcoma.
Abstract: 1 We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic-epithelial cell line HT-29. 2 Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin-1 alpha (IL-1 alpha), tumour necrosisfactor-alpha (TNF-alpha), interferon gamma (IFN-gamma) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay. 3 Unstimulated cells produced a small time-dependent increase in nitrite generation of 50 +/- 4, 75 +/- 8, and 103 +/- 8 nM per 10(6) cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 micrograms ml-1) pretreatment and iNOS mRNA was not detected. 4 None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL-1 alpha/IFN-gamma produced a highly significant (P < 0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective. 5 Time course studies with IL-1 alpha/IFN-gamma combination revealed significant (P < 0.001) increases in nitrite at 24 h (153 +/- 7), 48 h (306 +/- 24), and 72 h (384 +/- 15) compared to basal values of 50 +/- 4, 75 +/- 8, and 103 +/- 8 nM per 10(6) cells respectively. 6 Studies with IL-1 alpha/IFN-gamma combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL-1 alpha (0.3-10 ng ml-1) and IFN-gamma (10-300 u ml-1) in combination induced a concentration-dependent expression of iNOS mRNA at 24 h. 7 Pretreatment with cycloheximide before IL-1 alpha/IFN-gamma stimulation reduced nitrite levels to basal values. These data suggest that the IL-1 alpha/IFN-gamma-induced nitrite production by HT-29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme. 8 The addition of TNF-alpha produced a significant (P < 0.001) 3 fold increase of IL-1 alpha/IFN-gamma-induced nitrite generation. In marked contrast the presence of TNF-alpha had no effect on IL-1 alpha/IFN-gamma-induced iNOS mRNA expression by HT-29 cells. These findings suggest that the up-regulation by TNF-alpha of IL-1 alpha/IFN-gamma-induced nitrite generation is at the post-transcriptional level. 9 These data suggest that pro-inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.
