Ourania Sfakianaki

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.

Octreotide modulates the effects on fibrosis of TNF-α, TGF-β and PDGF in activated rat hepatic stellate cells ☆

BACKGROUND AND AIMS Somatostatin and its analogs may influence hepatic fibrosis interfering through several mechanisms. The aim of this study was to investigate the effect of octreotide on cytokine activated hepatic stellate cells (HSC). METHODS Primary HSCs were isolated from rats and were cultured on plastic for activation. Expression of somatostatin receptors (SSTR) was investigated in cultured HSCs by immunofluorescence and western blot. The effect of octreotide on cellular proliferation was studied with the MTT assay and western blot for α1-procollagen (α1-PROC) production in TNFα, TGF-β1 or PDGF treated HSCs. Phosphotyrosine (PTP) and phosphoserine-phosphothreonine (STP) phosphatases inhibition was performed with sodium orthovanadate and okadaic acid respectively. RESULTS Activated HSC express SSTR subtypes 1, 2A, 2B, 3 and 4 and their expression is enhanced by further HSC activation. Octreotide did not have an effect on HSC proliferation but inhibited plastic induced α1-PROC production. Interestingly, it enhanced PDGF-induced HSC proliferation but inhibited PDGF and TGFβ1 dependent expression of α1-PROC, while an opposite effect was observed in TNFα-induced cell proliferation and collagen production. PTP inhibition reversed the inhibitory effect of octreotide on α1-PROC, but potentiated its effect on PDGF and TGFβ1 dependent α1-PROC production. Finally, STP inhibition profoundly inhibited α1-PROC expression in all cases suggesting that both STP and PTP phosphatases are important regulators of pro-fibrotic mechanisms. CONCLUSIONS The net effect of octreotide on HSCs and therefore liver fibrosis is subject to the cytokine microenvironment of these cells. This effect is modulated by PTPs and STPs inhibition. Especially in the case of STPs their profibrotic effects could be an interesting new therapeutic target in liver fibrosis.

Serum surrogate markers of liver fibrosis in primary biliary cirrhosis

BACKGROUND Hyaluronan, leptin, laminin and collagen IV have been used extensively for the assessment of liver fibrosis. The aim of this study was to assay these markers in the peripheral and hepatic vein blood of primary biliary cirrhosis (PBC) patients and to study their ability to discriminate early from advanced disease. METHODS Sera from 62 PBC patients were compared to 60 controls, 44 chronic Hepatitis C, 38 hepatocellular carcinoma and 34 viral cirrhosis patients. Serum from the hepatic vein of 15 cirrhotic PBC patients and 17 patients with viral cirrhosis was also assayed. RESULTS All disease groups had significantly increased levels of hyaluronan and collagen IV, compared to controls, while laminin was significantly increased only in viral cirrhosis. Hyaluronan levels were statistically different between early (54.5 ng/ml; 95%CI 27.3-426.9) and late PBC (154.5 ng/ml; 95%CI 55.3-764.4, p<0.05). The area under the curve (AUC) for the identification of late PBC was 0.74 for hyaluronan, 0.63 for leptin, 0.59 for laminin and 0.70 for collagen IV. Hyaluronan had high sensitivity and NPV in identifying late stages of PBC (96% and 90%, respectively). Short term UDCA had no effect on these markers. CONCLUSION No single measurement can differentiate between advanced and early fibrosis in PBC. However serum hyaluronan is a promising single serum marker for longitudinal studies in PBC.

Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis.

AIM To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis (PBC). METHODS Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B (HBV) virus, 3 patients with hepatitis C virus (HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis (AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targeted by autoantibodies present in the patients sera was performed by indirect immunofluorescence (IIF) analysis using paraffin-embedded liver sections as a substrate. Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy. RESULTS In total, 18/21 (85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21 (47.6%) in lymphocytes, 8/21 (38%) in cholangiocytes and 7/21 (33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G (IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining (20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody, positive staining was observed in 75% of lymphocytes, 62.5% of cholangiocytes, 37.5% of hepatocytes and 50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections, weak positive staining of cholangiocytes was observed in 3/21 (14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients. CONCLUSION In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.

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 (TNFα) 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. TNFα 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.

Differential effects of somatostatin on tnf receptors and apoptosis in hepatocellular carcinoma cell lines

Introduction Somatostatin has antitumour activity in animal models of hepatocellular carcinoma. In human studies both favourable and unfavourable results have been reported. A 40% fraction of treated patients are benefited. Since its action may be through apoptosis, the authors tested the hypothesis that somatostatin may act differently in different hepatocellular cell lines. Methods The somatostatin synthetic analogue octreotide was tested for its effect on the expression of TNF receptors (RT-PCR and Western blot) and the TNFα-induced apoptosis (NF-kB nuclear translocation, P65 phosphorylation, DNA fragmentation and TUNEL cytochemistry) The HepG2 human hepatoblastoma cells and the Hep3B human hepatocellular carcinoma cells were used. Results TNFR1 but no TNFR2 receptor was constitutively expressed in both cell lines. Octreotide caused an early (within 1 h) reduction of both mRNA and TNFR1 protein in HepG2 and a late (at 6 and 12 h) reduction in Hep3B cells. TNFα induced NF-kB nuclear translocation, stronger in Hep3B which was not influenced by octreotide. Octreotide significantly increased P65Ser536 and P65Ser468 phosphorylation, more pronounced in Hep3B cells. Octreotide significantly decreased TNFα-induced Ser536 phosphorylation only in HepG2 cells. Octreotide or TNFα alone had no effect on apoptosis in HepG2 cells but TNFα greatly increased apoptosis in Hep3B cells. Co-incubation with Octreotide and TNFα increased apoptosis in HepG2 cells and decreased TNFα induced apoptosis in Hep3B cells, Conclusion (1) The differential effect of somatostatin on apoptosis may be due to its different effect on TNFR1 expression in the two cell lines. (2) Somatostatin may have no direct effect on apoptosis but it may indirectly act through TNF induced apoptosis. (3) This differential effect may in part explain the conflicting results of human HCC studies.

Prevalence of thiopurine S-methyltransferase gene polymorphisms in patients with inflammatory bowel disease from the island of Crete, Greece

Background There is evidence that genotyping for the thiopurine S-methyltransferase (TPMT) gene variants is useful for the prediction of response to thiopurine analogs (azathioprine and 6-mercaptopurine) in patients with inflammatory bowel disease (IBD). The aim of the present study was to determine the prevalence of TPMT gene polymorphisms in a genetic homogenous population of IBD patients in Crete and to correlate the results with adverse reactions to thiopurine drugs. Patients and methods Genotyping for the most common TPMT variants TPMT*2, TPMT*3A, TPMT3*C, and TPMT*3B was performed using the PCR-restriction fragment length polymorphism method in 223 consecutive IBD patients and 119 age-matched and sex-matched healthy controls. The hospital medical records were reviewed for thiopurine use in these patients and related adverse events. Results The prevalence of TPMT variants TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3C was 1.8, 2.7, 1.3, and 1.8%, respectively. The G238C mutation was detected in four (1.8%) out of 223 patients, three (1.3%) patients were carriers of the G460A mutation, four (1.8%) of the A719G mutation, and six (2.7%) of both G460A and A719G mutations. In healthy controls, only one (0.8%) carried both the G460A and the A719G mutation, whereas TPMT*2, TPMT*3C, and TPMT*3B were not detected. None of the genotypes was homozygous. A statistically significant correlation between the presence of the G460A mutation and the development of leucopenia after the administration of thiopurines was observed (P=0.048). Conclusion This study showed a lower frequency of total TPMT variants and a higher frequency of TPMT*3B in Cretan IBD patients compared with other Caucasian populations. The presence of the G460A mutation is associated with the development of leukopenia.

PTU-079 The Tpmt And Abcb1 Polymorphisms In Ibd Patients In Crete: Impact On Disease And Response To Treatment

Introduction It is well known that polymorphisms of the TPMT gene (coding for thiopourine methyl-transferase), influence response to treatment with azathioprine. Polymorphisms of the ABCB1 gene (coding for p-glycoprotein 170) has been associated with IBD and resistance to treatment but results are conflicting. The aim of this study was to determine the frequencies of TPMT and ABCB1 gene polymorphisms in IBD patients from Crete, a population genetically homogeneous, and how these polymorphisms might influence response to treatment and disease behaviour. Methods A total of 222 IBD patients records were reviewed for intake of azathioprine, possible adverse reactions, response to treatment and need for colectomy. All patients were genotyped for TPMT gene polymorphisms, that have been related to intolerance to azathioprine (G238C, G460A and A719C) as well as ABCB1 gene polymorphisms (G2677T/A and C3435T), using a PCR-RFLP method. The same polymorphisms were also determined in 119 age and sex healthy controls. Results Allele frequencies of TPMT gene in our study population were found to be in concordance with those reported in other Caucasian populations. 76 IBD patients were identified receiving azathioprine, of whom 16 were discontinued (10 CD, 6 UC) due to adverse reaction. 2 of them were found to carry the G460A and A719G alleles (TPMT 3A genotype) (12.5%). For the ABCB1 gene, G2677T/A allele frequencies were found to be similar to those reported in the literature. There was no association of G2677T/A or C3435T with clinical phenotype, or resistance to treatment. However, 77.3% of 22/222 patients who did not respond to therapy and required surgery, where found to carry both the C3434T and the G2677T mutation. Conclusion Our study was conducted in a genetically homogenous population in the island of Crete. No correlation of any single SNP was found with either clinical activity or response to treatment. However, most patients who carried both the G2677T and C3435T mutations were refractory to treatment, a finding which implies that resistance to treatment in IBD patients is a more complex issue, which requires the presence of a genetic locus rather than a single SNP. Disclosure of Interest None Declared.

Differential effect of somatostatinergic signaling on collagen type i production and the proliferation of cytokine primed rat hepatic stellate cells

Introduction Somatostatin may influence hepatic fibrosis with mediators produced by Kupffer cells. The aim of this study is to investigate the role of somatostatinergic and cytokine signalling in hepatic stellate cells (HSC), the effector cells of hepatic fibrosis. Methods The production of a1-procollagen (a1-PROC) by rat HSCs treated with TNFα (100 ng/ml), TGFβ1 (5 ng/ml) or PDGF (32 ng/ml) and their cellular proliferation with or without octreotide was investigated. a1-PROC and aSMA were analysed by Western blotting and cellular proliferation was assayed by MTT. The role of the phosphotyrosine (PTP) and phosphoserine-phosphothreonine (STP) phosphatases on somatostatin signalling, was investigated by using the PTP inhibitor sodium orthovanadate (0.1 μM) and the STP inhibitor okadaic acid (0.1 μM). Results TGFβ1 and PDGF enhanced, whereas TNFα inhibited the expression of a1-PROC. Octreotide dose dependently inhibited the expression of a1-PROC in cells treated with TGFβ1, PDGF and increased the production of a1-PROC in TNF treated cells. Sodium orthovanadate significantly augmented the inhibition of a1-PROC production caused by octreotide only in TGFβ1 or PDGF treated cells. Okadaic acid uniformly inhibited the expression of a1-PROC. The expression of aSMA remained constant in all experiments. HSC proliferation increased by TGFβ1 and PDGF and was inhibited by TNFα Octreotide potentiated the effect of TGFβ1 and PDGF, and reversed TNFα inhibition. Orthovanadate and okadaic acid did not have any effect on the proliferation of cells. However, okadaic acid profoundly inhibited HSC proliferation when combined with octreotide, in TGFβ1 and PDGF treated cells. Conclusion Somatostatin differentially influences HSC a1 procollagen production according to cytokine microenvironment and this effect is modulated by tyrosine and threonin phosphatases. Proliferation of HSCs is similarly influenced by Somatostatin by a phosphatase dependent mechanism.

Somatostatin effect on TNFA 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-a somatostatin analogue- inhibits only LPS-induced secretion of pro-inflammatory cytokines including tumour necrosis factor (TNF) α by Kupffer cells.1 2 We therefore tested the hypothesis that somatostatin modulates the expression of TNFα receptors and apoptosis of Kupffer cells. Methods Rat Kupffer cells 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 (Roche). TNFα mRNa expression was assessed by a semiquantitative PCR and TNFα was measured in cell supernatants by ELISA. Results TNFR1 and TNFR2 m RNA are constitutively expressed in Kupffer cells. 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 TNFR2 protein increase was identified by Western blot, while a band at 90 kD was present instead of a band at 55 kD as expected for TNFR1. TNFa mRNA expression and protein secretion was not influenced by octreotide at 24 h. However a significant inhibition was observed at 48 h. TNF had no effect on Kupffer cell apoptosis while octreotide significantly increased their apoptosis and this effect was not influenced by co-incubation with TNFα. Conclusion TNFR1 and TNFR2 are constitutively expressed in Kupffer cells and their expression is strongly increased by somatostatin. Moreover somatostatin increases Kupffer cell apoptosis. These findings may in part explain the antineoplasmatic effect of somatostatin.