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Expression and functional activity of tumor necrosis factor (TNF)-α converting enzyme (TACE) in human colonic epithelial cells
the production of cytokines by macrophagesfrom IL-lO-deficient mice to the level of those by macropbagesfrom control mice. Conclusions: Deficiency in autocrine inhibition by IL-IO potently upregulates the activation of macrophages induced by LPS or CD40 stimulation. Enhancedactivation of macrophagesfrom IL-10-deficient mice may be involved in the progression of the enterocolitis.
1629 IL-18 Is Produced by Human Intestinal Epithelial Cells and Enhances Introopitbellal Lymphocyte Proliferation Synergistically with IL-2, IL-7, and IL-15 Takanori Kanai, GraduateSch, Tokyo Medical and Dental Unlv, Tokyo Japan; Akira Okazawa, Kouichi Nakamaru, Sch of Medicine, Keio Unlv, Tokyo Japan; Masashi Kudmoto, Hayashibara Biochemical Ctr, OkayamaJapan; Tosbitumi Hibi, Keio cancer Ctr, Tokyo Japan; Mikiko Sakae, Teruharu Totsuka, Takahiro Ishikurs, Tetsuya Nomura, Kenichi Ishii, Motomi Yamazaki, Mamoru Watanabe, Graduate Sch, Tokyo Medical and Dental Unlv, Tokyo Japan BACKGROUND: Growth and functional differentiation of intestinal mucosal lymphocytes is regulated by a network of soluble factors produced by mononuclearcells and epithelial cells. Several cytokines including SCF, IL-7, and IL-15 are known to be required for development of -y&TCR intestinal intraepitheliallymphocytes(IELs). Recently,intestinal epithelialcells were found to produce a novel cytokine, IL-18 in humans and mice. Here, we show the effects of epithelial cell-derived IL-18 on the proliferation of human IELs. METHODS:Expressionof IL18 and IL-18 receptor in normal human intestinal mucosa was determined using RT-PCR, immunohistochemistry, ELISA, and flow ¢ytometry. The functional ~ of IL-f8 was assessed by the utility of recombinant 11.-18 to stimulate the growth of intestinal IEt.s. RESULTS: IL-18 transcripts were detected in isolated human colonic epithelial cells and colonic epithelial cell lines, iL-18 protein expression was demonstrated in colonic epithelial cells by immunohistochemistry. IL-18 activity was also detected by IL-18-specitic ELISA in cell lysates of these ceils. IL-18 protein was also detected in colonic epithelial cell lines using flow cytometric analysis. Colonic epithelial cells expressedmRNA for the IL-18 receptor ¢=and AcPL (IL-18 receptor p), indicating that these cells were supposed to be responsive to IL18. IELs constitutively expressed IL-18 receptor. Significant proliferative responses of IELs were not observed after stimulation with various concentrations of iL-18. In contrast, IL-2, IL-7 and IL-15 induced the proliferation of IELs. However, the combination of IL-18+IL-2, IL-18+IL-7, or IL-18+ IL-15 was far more effective in inducing significant proliteratb/e responses. In the presenceof low dose of anfl-CO3 mAb, significaltly increased proliferative responses of IELs were observed in a dose-dependentmanner after stimulabon with various concentrations of IL-18. CONCLUSION:These results suggest that intestinal epithelial celF derived IL-18 plays a role in the proliferation of intestinal IELs and the maintenanceof intestinal immune responses.
1630 IGF-1 Protects Colonic Epithelial Cells From Cytokine Induced Apoptocts By Maintaining Cellular Phospho-Tyrosine Content And Preventing Cytochrome C Release Kenneth Nally, National Univ of Ireland, Cork, Cork Ireland; Joe O'Connell, John Morgan, John K. Collins, Gerald C. O'Sullivan, Fergus Sbaoahan,National Univ of Ireland, Cork Ireland Background: IFN~and TNF~re prominent cytokines associatedwith epithelial cell apoptosis in inflammatory bowel diseases (IBD). IFN-ypfimesTNF~esistant cells for a death inducing signal. Several growth factors have been identified as regulators of cell survival, and among them IGF-1 has been shown to have a potent ability to protect a wide range of cells from various proapoptoticchallenges.Therefore,we investigatedthe potential for IGF-1 to protect a colonic epithelialcell line from IFN-t+ TNFo~inducedapoptosis.To identify possible molecular mechanisms underlying this protective effect we monitored NFKBfunction, cellular phosphotyrosine content and mitochondrial cytochrome C status. Methods: Human colonic HT29 epithelialcells were co-treatedwith IFN~/TNF~z,IGF-1/IFN.y/TNFa~ndZVAD-FMK(casp.1inhibitor)/IFN~//1NFotoverseveral time points up to 24hr. Induction of apoptosis was measured using MIT assay,DNAladderinggels and M30 cytndeathantibody. Thetransactivationfunction of NF,/B was assessedat 4hr and 8hr post treatment using a luciferase-basedreporter gene assay. Cellular phosphotyrosine content and cytochrome C distribution were estimated at 4, 8, 12 and 16hr by immunofluorescant staining. Results: Co-treatment of HT29 with IFN-y/ TNF~xresultedin > 60% apoptosis by 16hr. This was associatedwith a significant decrease in NF~Btransactivation function by 8hr, a decreasein and re-distribution of phospho-tyrosine content at 8 and 12hr and release of mitochondrial cytochrome C at 12hr. IGF-1 treatment blocked cytokine-inducedapoptnsis completely. This was associatedwith retention of cellular phosphotyrosine content, N~B transactivation function and mitochondrial cytochrome C localization. ZVAD-FMK treatment (casp,1 inhibitor) also blocked induction of apoptosis by the IFN,y/'l'NF~ombination. In this case, however, the decrease in phosphotyrosine content and release of cytochrome C were not blocked. Conclusion:Thesedata demonstratethat IGF1 is a potent survival factor for colonic epithelialcells under conditions of inflammatory stress. Furthermore, the molecular mechanisms underlying this effect involve the retention of total cellular phosphotyrosine content, NF-yBactivity and mitochondrlal cytochrome C content.
1631 Expression and Functional Activity of Tumor Necrosis Factor (TNF)-~ Converting Enzyme ('fACE) in Human Colonic Epithelial Ceils. Tove Kirkegaard,Gitte Pedersen,Torben Saermark, Jorn Brynskov, Herlev Univ Hosp, Herlev Denmark Background:TACEis A DisintagrinAnd Metalloproteinase(ADAM 17) that cleavesmembranebound pro-TNF-~to solubleTNF-~, but other zinc-dependlngmatrix metalloproteinases(MMP) may havea similar effect. We have previously shown that TACEis responsiblefor processing of soluble TNF-c=in human colonic mucosa, but little is known about the cellular origin (1,2).
A-316
It is now recognizedthat epithelial cells are actively involved in immune signalling in the gut, which raises the question whether these cells also participate in TNF-a processing in colonic mucosa. Objectives:To investigateTACEexpressionand functional activity in human colonic epithelial cells. Methods: HT-29 and DLD-1 cell cultures were grown to semiconfluence and RT-PCR was carried out on extracted total RNA using specific primers for TACE. RT-PCR products were quantified by densitometry and adjustedto GAPDHexpression. In other experiments, isolatedcell membraneswere dissolved in 1% Nonidet P-40 and the resulting detergent extracts were collected. Functional TACE activity was quantified by a HPLC-basedcleavage assay using a synthetic dnp-labaledoli9opeptide substrate (dnp-acSPLAOAVRSSSRTPS-NH2, 10/zg/ml) spanning the known TACE-cleavegesite (Ala76-Va177)in pro-TNF-tx. TACE/MMP inhibitors were added in certain experiments. Results: TACEmRNA was equally expressedin HT-29 (1.3 - 0.2 arbitrary units (AU)) and DLD-1 cells (1.3 ± 0.2 AU). Functional TACElike activity, as measured by cleavageof the oligopeptide substrate, was present in detergent extracts of call membranesfrom both HT-29 (AU: 2100 _+ 190) and DLD-1 cells (AU: 3400 _+ 540). This activity was inhibited by EDTA (5 mM) (a metal-chelating agent), CH4474 (1 /.,.g/ml) (a known TACE/MMP inhibitor), but not Trocade (5 p.M) (a broadspectrum MMP inhibitor with low inhibitory activity against TACE). Conclusion: This study shows that an enzyme with properties similar to those of TACE is present in human colonic epithelial cell lines. The results suggest that the colonic epithelium also representsa therapeutictarget for drugs designed to inhibit the final stage of TNF-~zprocessing. 1) Foegh et al. Ann NY Acad Sci 1999;878:692-5 2) Brynskov et al. Submitted 2000
1632 Pynofldia Olthimmdiamate Abrogates Spontaneous NF-KB Activation And IL-8 Troosedption in Crohn's Disease Intestinal Smooth Muscle Cells Flamesh Natarajan, Bernard J. Fisher, Shobha Ghosh, Martin F. Graham, Robert F. Diegelmann, Alpha A. Fowler III, Virginia Commonwealth Univ, Richmond, VA
BACKGROUND:Crohn's disease is typically manifested by infiltration of the intestinal wall with inflammatory cells. Interieukin-8 (IL-8) is central to inflammation and its expression is regulated primarily by the oxidant-sensitivetranscription nuclear factor kappa B (NF-KB). IL8 secretedby cells at inlury-sites promotes neutrophil migration and activationfor production of cytoldnesand reactiveoxygen species.Pethogenesisof enhancedspontaneoustranscription of IL-8 by the bowel in patients with Crohn s disease is undefined. However, the outcome of exuberant secretion of IL-8 and other pro-inflammatory peptides is chronic, persistent tissue inflammation. HYPOTHESIS:Altered intrinsic cellular redox balance in Cmhn s disease intestinalsmooth musclecells causesspontaneousIL-8 secretion.METHODS:Humanintestinal smooth muscle cells were isolated from surgically resected human ileum of normal (NHISM) and Crohn s disease (CDISM) subjects by collagenasedigestion and grown in Dulbecco's modified eagles medium plus 10% serum. RESULTS: Electmphoretic mobility shift assays for NF-KB showed that it is 7-fold more active in resting CDISM compared to resting NHISM and may be responsiblefor the spontaneous IL-8 transcription observed in CDISM. We also chemically enhanced reducing equivalents (i.e., antioxidant molecules) in CDISM cells with pyrrolidins dithiocarbamate(25 IO0/AVl) and found that it potently suppressed NF-KB-drlven IL-8 promoter activity by transient transfection assays(see figure). CONCLUSIONS:Intestinal smooth muscle cells in Crohn s disease patients are associated with significantly altered redox balance.Treatment with agents that restore the redox balance could have therapeutic implications.
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In Vitro Evelootloo of IL-18 Effects in Crohn's Disease Philippe Maerten, Stetaan Colpaert, Zhanju Liu, Karel Geboes,Jan Ceuppens, Paul Rutgeerts, Unlv Hoop Gasthuisberg, Leuven Belgium; Guido Vanbam, Tropical Institute, Antwerp Belgium BACKGROUND:An imbalanceof immunorsgulatory factors contributes to uncontrolled mucosal Thl cell activation in Crohn's disease (CO). It has been reported that the bio-actlve form of IL-18, a macrophagederived cytokine, is enhancedin CO. We wanted to examinethe effect of 11_-18on Thl cytokines produced by lamina proprla mononuclear cells (LPMC) and on membranousCD4Oligand(CD4OL)expressionon laminapropria T cells (LPT) in CD. METHODS: Ileal LPMC were isolated from involved and non-involved surgical specimens of 9 CD and 4 controls. Ileal LPT were isolated from 4 involved CO. LPMC were stimulated with mouse mastocytoma cells (5x105/ml) transfected with human CD8Oand soluble anti-CD3 (UCTH-1; 1/zg/ml) in the absence and presenceof IL-18 (2.5, 5, 10, 20, 40 ng/ml) and IL-12 (1 no/ ml). After 48 hours of culture, supematans were collected and assayedfor cytoldnes ( IFN-1, IL-IO and TNF) by ELISA.CD40Lexpressionwas analysedon a FACScanon LPT, incubated in anti-CD3 coated 96 wells plates, in the absence or presence of IL-18 (40 ng/ml) and IL12 (1 ng/ml). RESULTS: In involved CD, a dose-dependentincrease of IFN--yproductiooby IL-18 was observed only in the presence of IL-12. Significant up-regulation could however only be achievedat the dose of 40 ng/ml IL-18, as comparedwith absenceof IL-18 stimulation. IL-IO was found to be significantly decreasedin non-involved but not in involved CD tissue in a dose-dependentway, again only if IL-12 was present. TNF levels were not influenced by IL-18. Furthermore IL-18 could not upregulate membranous CD40L expression. Comparing the functional results in CDwith control ileal LPMCprovidedevidencefor diminished sensitivity for IL-18 in CD versus control. CONCLUSIONS:Our results indicate that IL-18 is synergistic
1629 IL-18 Is Produced by Human Intestinal Epithelial Cells and Enhances Introopitbellal Lymphocyte Proliferation Synergistically with IL-2, IL-7, and IL-15 Takanori Kanai, GraduateSch, Tokyo Medical and Dental Unlv, Tokyo Japan; Akira Okazawa, Kouichi Nakamaru, Sch of Medicine, Keio Unlv, Tokyo Japan; Masashi Kudmoto, Hayashibara Biochemical Ctr, OkayamaJapan; Tosbitumi Hibi, Keio cancer Ctr, Tokyo Japan; Mikiko Sakae, Teruharu Totsuka, Takahiro Ishikurs, Tetsuya Nomura, Kenichi Ishii, Motomi Yamazaki, Mamoru Watanabe, Graduate Sch, Tokyo Medical and Dental Unlv, Tokyo Japan BACKGROUND: Growth and functional differentiation of intestinal mucosal lymphocytes is regulated by a network of soluble factors produced by mononuclearcells and epithelial cells. Several cytokines including SCF, IL-7, and IL-15 are known to be required for development of -y&TCR intestinal intraepitheliallymphocytes(IELs). Recently,intestinal epithelialcells were found to produce a novel cytokine, IL-18 in humans and mice. Here, we show the effects of epithelial cell-derived IL-18 on the proliferation of human IELs. METHODS:Expressionof IL18 and IL-18 receptor in normal human intestinal mucosa was determined using RT-PCR, immunohistochemistry, ELISA, and flow ¢ytometry. The functional ~ of IL-f8 was assessed by the utility of recombinant 11.-18 to stimulate the growth of intestinal IEt.s. RESULTS: IL-18 transcripts were detected in isolated human colonic epithelial cells and colonic epithelial cell lines, iL-18 protein expression was demonstrated in colonic epithelial cells by immunohistochemistry. IL-18 activity was also detected by IL-18-specitic ELISA in cell lysates of these ceils. IL-18 protein was also detected in colonic epithelial cell lines using flow cytometric analysis. Colonic epithelial cells expressedmRNA for the IL-18 receptor ¢=and AcPL (IL-18 receptor p), indicating that these cells were supposed to be responsive to IL18. IELs constitutively expressed IL-18 receptor. Significant proliferative responses of IELs were not observed after stimulation with various concentrations of iL-18. In contrast, IL-2, IL-7 and IL-15 induced the proliferation of IELs. However, the combination of IL-18+IL-2, IL-18+IL-7, or IL-18+ IL-15 was far more effective in inducing significant proliteratb/e responses. In the presenceof low dose of anfl-CO3 mAb, significaltly increased proliferative responses of IELs were observed in a dose-dependentmanner after stimulabon with various concentrations of IL-18. CONCLUSION:These results suggest that intestinal epithelial celF derived IL-18 plays a role in the proliferation of intestinal IELs and the maintenanceof intestinal immune responses.
1630 IGF-1 Protects Colonic Epithelial Cells From Cytokine Induced Apoptocts By Maintaining Cellular Phospho-Tyrosine Content And Preventing Cytochrome C Release Kenneth Nally, National Univ of Ireland, Cork, Cork Ireland; Joe O'Connell, John Morgan, John K. Collins, Gerald C. O'Sullivan, Fergus Sbaoahan,National Univ of Ireland, Cork Ireland Background: IFN~and TNF~re prominent cytokines associatedwith epithelial cell apoptosis in inflammatory bowel diseases (IBD). IFN-ypfimesTNF~esistant cells for a death inducing signal. Several growth factors have been identified as regulators of cell survival, and among them IGF-1 has been shown to have a potent ability to protect a wide range of cells from various proapoptoticchallenges.Therefore,we investigatedthe potential for IGF-1 to protect a colonic epithelialcell line from IFN-t+ TNFo~inducedapoptosis.To identify possible molecular mechanisms underlying this protective effect we monitored NFKBfunction, cellular phosphotyrosine content and mitochondrial cytochrome C status. Methods: Human colonic HT29 epithelialcells were co-treatedwith IFN~/TNF~z,IGF-1/IFN.y/TNFa~ndZVAD-FMK(casp.1inhibitor)/IFN~//1NFotoverseveral time points up to 24hr. Induction of apoptosis was measured using MIT assay,DNAladderinggels and M30 cytndeathantibody. Thetransactivationfunction of NF,/B was assessedat 4hr and 8hr post treatment using a luciferase-basedreporter gene assay. Cellular phosphotyrosine content and cytochrome C distribution were estimated at 4, 8, 12 and 16hr by immunofluorescant staining. Results: Co-treatment of HT29 with IFN-y/ TNF~xresultedin > 60% apoptosis by 16hr. This was associatedwith a significant decrease in NF~Btransactivation function by 8hr, a decreasein and re-distribution of phospho-tyrosine content at 8 and 12hr and release of mitochondrial cytochrome C at 12hr. IGF-1 treatment blocked cytokine-inducedapoptnsis completely. This was associatedwith retention of cellular phosphotyrosine content, N~B transactivation function and mitochondrial cytochrome C localization. ZVAD-FMK treatment (casp,1 inhibitor) also blocked induction of apoptosis by the IFN,y/'l'NF~ombination. In this case, however, the decrease in phosphotyrosine content and release of cytochrome C were not blocked. Conclusion:Thesedata demonstratethat IGF1 is a potent survival factor for colonic epithelialcells under conditions of inflammatory stress. Furthermore, the molecular mechanisms underlying this effect involve the retention of total cellular phosphotyrosine content, NF-yBactivity and mitochondrlal cytochrome C content.
1631 Expression and Functional Activity of Tumor Necrosis Factor (TNF)-~ Converting Enzyme ('fACE) in Human Colonic Epithelial Ceils. Tove Kirkegaard,Gitte Pedersen,Torben Saermark, Jorn Brynskov, Herlev Univ Hosp, Herlev Denmark Background:TACEis A DisintagrinAnd Metalloproteinase(ADAM 17) that cleavesmembranebound pro-TNF-~to solubleTNF-~, but other zinc-dependlngmatrix metalloproteinases(MMP) may havea similar effect. We have previously shown that TACEis responsiblefor processing of soluble TNF-c=in human colonic mucosa, but little is known about the cellular origin (1,2).
A-316
It is now recognizedthat epithelial cells are actively involved in immune signalling in the gut, which raises the question whether these cells also participate in TNF-a processing in colonic mucosa. Objectives:To investigateTACEexpressionand functional activity in human colonic epithelial cells. Methods: HT-29 and DLD-1 cell cultures were grown to semiconfluence and RT-PCR was carried out on extracted total RNA using specific primers for TACE. RT-PCR products were quantified by densitometry and adjustedto GAPDHexpression. In other experiments, isolatedcell membraneswere dissolved in 1% Nonidet P-40 and the resulting detergent extracts were collected. Functional TACE activity was quantified by a HPLC-basedcleavage assay using a synthetic dnp-labaledoli9opeptide substrate (dnp-acSPLAOAVRSSSRTPS-NH2, 10/zg/ml) spanning the known TACE-cleavegesite (Ala76-Va177)in pro-TNF-tx. TACE/MMP inhibitors were added in certain experiments. Results: TACEmRNA was equally expressedin HT-29 (1.3 - 0.2 arbitrary units (AU)) and DLD-1 cells (1.3 ± 0.2 AU). Functional TACElike activity, as measured by cleavageof the oligopeptide substrate, was present in detergent extracts of call membranesfrom both HT-29 (AU: 2100 _+ 190) and DLD-1 cells (AU: 3400 _+ 540). This activity was inhibited by EDTA (5 mM) (a metal-chelating agent), CH4474 (1 /.,.g/ml) (a known TACE/MMP inhibitor), but not Trocade (5 p.M) (a broadspectrum MMP inhibitor with low inhibitory activity against TACE). Conclusion: This study shows that an enzyme with properties similar to those of TACE is present in human colonic epithelial cell lines. The results suggest that the colonic epithelium also representsa therapeutictarget for drugs designed to inhibit the final stage of TNF-~zprocessing. 1) Foegh et al. Ann NY Acad Sci 1999;878:692-5 2) Brynskov et al. Submitted 2000
1632 Pynofldia Olthimmdiamate Abrogates Spontaneous NF-KB Activation And IL-8 Troosedption in Crohn's Disease Intestinal Smooth Muscle Cells Flamesh Natarajan, Bernard J. Fisher, Shobha Ghosh, Martin F. Graham, Robert F. Diegelmann, Alpha A. Fowler III, Virginia Commonwealth Univ, Richmond, VA
BACKGROUND:Crohn's disease is typically manifested by infiltration of the intestinal wall with inflammatory cells. Interieukin-8 (IL-8) is central to inflammation and its expression is regulated primarily by the oxidant-sensitivetranscription nuclear factor kappa B (NF-KB). IL8 secretedby cells at inlury-sites promotes neutrophil migration and activationfor production of cytoldnesand reactiveoxygen species.Pethogenesisof enhancedspontaneoustranscription of IL-8 by the bowel in patients with Crohn s disease is undefined. However, the outcome of exuberant secretion of IL-8 and other pro-inflammatory peptides is chronic, persistent tissue inflammation. HYPOTHESIS:Altered intrinsic cellular redox balance in Cmhn s disease intestinalsmooth musclecells causesspontaneousIL-8 secretion.METHODS:Humanintestinal smooth muscle cells were isolated from surgically resected human ileum of normal (NHISM) and Crohn s disease (CDISM) subjects by collagenasedigestion and grown in Dulbecco's modified eagles medium plus 10% serum. RESULTS: Electmphoretic mobility shift assays for NF-KB showed that it is 7-fold more active in resting CDISM compared to resting NHISM and may be responsiblefor the spontaneous IL-8 transcription observed in CDISM. We also chemically enhanced reducing equivalents (i.e., antioxidant molecules) in CDISM cells with pyrrolidins dithiocarbamate(25 IO0/AVl) and found that it potently suppressed NF-KB-drlven IL-8 promoter activity by transient transfection assays(see figure). CONCLUSIONS:Intestinal smooth muscle cells in Crohn s disease patients are associated with significantly altered redox balance.Treatment with agents that restore the redox balance could have therapeutic implications.
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In Vitro Evelootloo of IL-18 Effects in Crohn's Disease Philippe Maerten, Stetaan Colpaert, Zhanju Liu, Karel Geboes,Jan Ceuppens, Paul Rutgeerts, Unlv Hoop Gasthuisberg, Leuven Belgium; Guido Vanbam, Tropical Institute, Antwerp Belgium BACKGROUND:An imbalanceof immunorsgulatory factors contributes to uncontrolled mucosal Thl cell activation in Crohn's disease (CO). It has been reported that the bio-actlve form of IL-18, a macrophagederived cytokine, is enhancedin CO. We wanted to examinethe effect of 11_-18on Thl cytokines produced by lamina proprla mononuclear cells (LPMC) and on membranousCD4Oligand(CD4OL)expressionon laminapropria T cells (LPT) in CD. METHODS: Ileal LPMC were isolated from involved and non-involved surgical specimens of 9 CD and 4 controls. Ileal LPT were isolated from 4 involved CO. LPMC were stimulated with mouse mastocytoma cells (5x105/ml) transfected with human CD8Oand soluble anti-CD3 (UCTH-1; 1/zg/ml) in the absence and presenceof IL-18 (2.5, 5, 10, 20, 40 ng/ml) and IL-12 (1 no/ ml). After 48 hours of culture, supematans were collected and assayedfor cytoldnes ( IFN-1, IL-IO and TNF) by ELISA.CD40Lexpressionwas analysedon a FACScanon LPT, incubated in anti-CD3 coated 96 wells plates, in the absence or presence of IL-18 (40 ng/ml) and IL12 (1 ng/ml). RESULTS: In involved CD, a dose-dependentincrease of IFN--yproductiooby IL-18 was observed only in the presence of IL-12. Significant up-regulation could however only be achievedat the dose of 40 ng/ml IL-18, as comparedwith absenceof IL-18 stimulation. IL-IO was found to be significantly decreasedin non-involved but not in involved CD tissue in a dose-dependentway, again only if IL-12 was present. TNF levels were not influenced by IL-18. Furthermore IL-18 could not upregulate membranous CD40L expression. Comparing the functional results in CDwith control ileal LPMCprovidedevidencefor diminished sensitivity for IL-18 in CD versus control. CONCLUSIONS:Our results indicate that IL-18 is synergistic