Vassilis Valatas

Nitric oxide in inflammatory bowel disease: a universal messenger in an unsolved puzzle

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.

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.

Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures.

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 80–100×106KCs 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‐α and nitric oxide production.

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.

Octreotide regulates CC but not CXC LPS-induced chemokine secretion in rat Kupffer cells

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.

Apoptosis and apoptosis related proteins in chronic viral liver disease

AbstractBackground: Apoptosis may be an important mechanism of hepatocyte death in chronic viral liver disease. Methods: We studied apoptosis in liver biopsies from 30 patients with chronic viral hepatitis and 8 patients with viral cirrhosis by the TUNEL method. 12 cases of non-alcoholic steatohepatitis and 12 cases of primary biliary cirrhosis were used as non-viral disease controls. Immunohistochemical expression of p53, p21/waf1, bcl-2 and mdm-2 proteins was also studied in the same patients. Results: A statistically significant increase of apoptotic liver cells was found in severe chronic viral hepatitis (5.3 ± 0.3%), cirrhosis (3.4 ± 0.5%) and PBC (4.4 ± 0.4%) cases compared to patients with non-alcoholic steatohepatitis (0.8 ± 0.3%). The expression of p53 protein was increased in the cases of viral cirrhosis and in chronic severe viral hepatitis whereas in the cases of chronic mild hepatitis, PBC and non-alcoholic steatohepatitis we found no expression of p53. P21/waf1 expression was increased in severe chronic hepatitis, cirrhosis and PBC cases compared to mild hepatitis and non-alcoholic steatohepatitis cases. However no induction of mdm-2 was observed in the subgroups of chronic liver disease. Bcl-2 was expressed only in epithelium of bile ducts and mononuclear cells of the portal tracts and liver lobules. A weaker Bcl-2 expression was noted in the epithelium of bile ducts of 7/12 PBC cases. Conclusion: Our results provide evidence of increased apoptosis in severe chronic viral liver disease, suggesting that apoptotic cell death might be involved in the pathogenesis of hepatocellular damage of viral hepatitis and cirrhosis. Furthermore we analysed part of the apoptotic pathways implicated in the above process and found an increased expression of p21/waf1, probably p53 mediated, without overexpression of the apoptosis inhibiting bcl-2 and mdm-2 proteins. By contrast p21/waf1 overexpression in PBC seems to be propagated by a p53 independent mechanism.

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 Crohns disease along with the corresponding 18CO cell line. Readouts included production of TGF-β and TIMP-1, total collagen synthesis, matrix metalloproteinases MMP-2 and MMP-9 and myofibroblast migration/mobility. RESULTS Proinflammatory cytokines upregulated TGF-β 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-β, CTGF, TF and endothelin. Subepithelial myofibroblasts isolated from Crohns 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.

Production of pro- and anti-fibrotic agents by rat Kupffer cells; the effect of octreotide.

Kupffer cells may be involved in liver fibrogenesis through production of TGF-β1. 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-β1, 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-β1 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-β1, uPA, and MMP-1 production, may be a useful agent for fibrosis resolution.

Increased expression of chemokine receptor CCR3 and its ligands in ulcerative colitis: the role of colonic epithelial cells in in vitro studies

Human colonic epithelial cells express T helper type 1 (Th1)‐associated chemoattractants, yet little is known about the production of Th2‐associated chemoattractants. CCL11/eotaxin‐1, CCL24/eotaxin‐2 and CCL26/eotaxin‐3 are known to attract CCR3‐expressing, Th2‐polarized lymphocytes. We studied constitutive and inflammation‐induced expression and production of CCR3 together with its ligands in the colon and peripheral blood of patients with inflammatory bowel disease (IBD) by flow cytometry, reverse transcription–polymerase chain reaction (RT–PCR) and enzyme‐linked immunosorbent assay (ELISA). We further defined the regulated expression of these chemokines by RT–PCR and ELISA using cultured human epithelial cell lines. A higher fraction of peripheral T lymphocytes were found to be positive for CCR3 in patients with ulcerative colitis (UC) compared to Crohns disease (CD), while almost no CCR3+ T cells were found in normal controls (NC). Similarly, higher and more frequent expression of CCR3 was observed in colonic biopsies from patients with UC, regardless of the disease activity, when compared to CD or NCs. Serum CCL11/eotaxin‐1 was increased significantly in UC (306 ± 87 pg/ml) and less so in CD (257 ± 43 pg/ml), whereas CCL24/eotaxin‐2, and CCL26/eotaxin‐3 were increased only in UC. Colonic expression of the three chemokines was minimal in NCs but high in inflammatory bowel diseases (especially UC) and was independent of disease activity. Th2, and to a lesser extent Th1, cytokines were able to induce expression and production of all three eotaxins from colonic epithelial cells in culture. CCR3 and ligands over‐expression would appear to be a characteristic of UC. The production of CCR3 ligands by human colonic epithelial cells suggests further that epithelium can play a role in modulating pathological T cell‐mediated mucosal inflammation.

Experimental colitis models: Insights into the pathogenesis of inflammatory bowel disease and translational issues.

Inflammatory bowel diseases, ulcerative colitis and Crohn׳s disease are characterized by chronic relapsing inflammation of the gastrointestinal tract of unknown etiology that seems to be the consequence of a genetically driven dysregulated immune response against various local and environmental triggers through a defective epithelial barrier. During the last decades, a large number of animal experimental models of intestinal inflammation have been generated and provided valuable insights into the mechanisms that either maintain mucosal homeostasis or drive intestinal inflammation. Their study enabled the identification of various treatment targets and the development a large pipeline of new drugs, mostly biologics. Safety and therapeutic efficacy of these agents have been evaluated in a large number of clinical trials but only a minority has reached the clinic so far. Translational successes but mostly translational failures have prompted to re-evaluate results of efficacy and safety generated by pre-clinical testing and to re-examine the way to interpret experimental in vivo data. This review examines the contribution of the most popular experimental colitis models to our understanding of the pathogenesis of human inflammatory bowel diseases and their translational input in drug development and discusses ways to improve translational outcome.