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Fas-mediated apoptosis in human intestinal primary epithelial cell (HIPEC) cultures
A360 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
1923 INDUCIBLE NO SYNTHASE AND INTERFERON-r EXPRESSION IN DUODENAL EPITHELIUM FROM PATIENTS WITH ANKYLOSING SPONDYLITIS. Dominique Lamarque, Pascal Claudepierre, Zoltan Szepes, Christophe Bernardeau, Jeanne Tran Vannhieu, Nadine Martin-Garcia, Maxime Breban, Jean Charles Delchier, Brendan Jr Whittle, INSERM U99, Creteil, France; CHU Henri Mondor, Creteil, France; INSERM U477, Paris, France. Inflammatory processes have been detected in the ileal mucosa from patients with ankylosing spondylitis (AS). The inducible isoform of NO synthase, (iNOS), may be expressed early in the inflammatory process. The aim of this study was to determine iNOS activity and expression of mRNA for the pro-inflammatory cytokine, interferon-v (IFN-y) in the duodenal epithelium from patients with AS, rheumatoid arthritis (RA) and in controls. Methods. Gastroscopy with duodenal biopsies was conducted in 22 patients with AS who had not been treated by NSAID for at least 5 days, in 6 patients with RA and 28 controls, 5 of whom were treated by NSAID. H pylori status was determined by histology and culture from antral biopsies. Lymphocytic infiltration in lamina propria were semi-quantified by a histologic score ranging from 0 to 3. The iNOS expression was assessed by immunohistochemistry with monoclonal antibodies (Transduction Laboratories®) and the extent of the immunoreactivity was quantified by a score ranging from 0 to 3 in epithelium and in lamina propria. The percentage of patients with lymphocytic infiltration and positive immunofluoresence for iNOS in mucosa was compared between the different groups. Mucosal samples from duodenum were assayed for iNOS activity, measured as the conversion of L_'4C arginine to '4C-citrulline, in the presence of EGTA (l mM), and expressed in pmol/min.mg of protein (M ::t:SEM). Expression of mucosal IFN-y was estimated by mRNA detection with RT-PCR semi-quantified by comparison with actin mRNA expression. Results. Endoscopic examination of the gastro-duodenal mucosa did not find any lesion except in one patient with peptic ulcer in AS group. Conclusion: iNOS and IFN-y induction were detected in duodenal mucosa from patients with AS, which was independent of Hp status. This induction, which was not found in patients with RA or in controls, may reflect a specific inflammatory process in the duodenal epithelium in AS.
Control (n=28) AS (n=22) RA(n=6)
Hpstatus +
Infiltration
MucosaiiNOS
15 13 4
10 14 3
14 19· 2
INOS activity IFN·g mRNA
0,02 ± 0.04 0,29 ± ca0,06 ± 0.04.
O±O 0.7±0,3·
.: significant difference with control group; P<0.02
1924 HUMAN INTESTINAL MAST CELLS EXPRESS DIFFERENT CY· TOKINE PROFILES. Axel Lorentz, Gernot Sellge, Michael P. Manns, Stephan C. Bischoff, Med Sch of Hannover, Hannover, Germany. The recognition of mast cells as cytokine-producing cells has extended their potential functions from proinflammatory effector cells to regulatory components of the immune system. So far, little is known about cytokine profiles of mucosa-derived human mast cells. In this study, we examined the expression of cytokines by human intestinal mast cells triggered with different stimuli. Mast cells were purified up to 99% by magnetic cell separation and subsequently cultured with stem cell factor. Cytokine expression was analyzed by RT-PCR, immunocytochemistry and ELISA. Proinflammatory cytokines like TNF-a, ILl /3, IL-6, IL8, IL-16 and IL-18 were expressed without further stimulation. Activation by both IgE-receptor crosslinking and IgE-independent agonists such as grarnnegative bacteria enhanced the expression of TNF-a. Another set of cytokines, namely the Th2 type cytokines IL-3, IL5, IL-9 and ILl3, was expressed in response to stimulation by IgE receptor-crosslinking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and ILl3 was increased up to 4-fold compared to mast cells cultured with SCF alone. By contrast, IL-6 expression was totally blocked following culture with IL-4. Conclusion: Human intestinal mast cells produce constitutively proinflammatory cytokines which may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor-crosslinking results in the expression of Th2 type cytokines. IL-4 enhances the expression of Th2 type cytokines but does not affect or even down-regulates proinflammatory cytokines.
1925 RESISTANCE OF ANTIGEN·SPECIFIC T CELLS TO FAS-MEDIATED APOPTOSIS INDUCED BY IL-12 ANTAGONISTS IS MEDIATED BY BCL-2 AND BCL-XL. Thomas Marth, Sabine Ring, Dirk Schulte, Martin Zeitz, Internal Medicine II, Homburg/Saar, Germany. 1440720 Background: Feeding or injection of antigen to ovalbumin (OVA) T cell receptor (TCR) transgenic mice has been shown to induce systemic tolerance. This tolerance is augmented when antibodies to interleukin-12 (anti-IL-12) are applied concomitantly with antigen. The effect of antiIL-12 is brought over, at least in part, by induction of a Fas-mediated
apoptosis as also shown in a model of chronic murine colitis. The aim of the present study was to analyze the role of members of the bcl-2 family in this system. Methods: OVA-TCR transgenic mice were treated three times with injections of OVA protein with or without simultaneous anti-IL-12 injections; mice in the control group were not treated. On day four, mRNA and proteins were extracted from splenocytes and Peyers patches by standard methods and were analyzed by PCR and western blot, respectively. Results: OVA plus anti-IL-12 treated mice showed higher levels of Fas-mediated T cell apoptosis as compared to OVA alone treated and control mice in both peripheral and mucosal lymphoid tissues. The remaining, Fas-resistent and TGF-beta producing cells in the OVA plus anti-IL-12 group showed a significantly higher levels of FasL, bcl-2 and bcl-xl protein and mRNA expression as determined by optical densitometry measurements. Bcl-2 expression was also elevated in paralell studies using flow cytometry. Conclusions: Thus, the treatment with anti-IL-12 and antigen results in an induction of Fas-mediated apoptosis. Fas-resistant cells may escape clonal deletion because of upregulated anti-apoptotic molecules such as bcl-2 and bcl-xl, Our studies contribute to the understanding of the anti-inflammatory effects of anti-IL-12 which represent a promising therapeutic approach in autoimmune diseases.
1926 FAS-MEDIATED APOPTOSIS IN HUMAN INTESTINAL PRIMARY EPITHELIAL CELL (HIPEC) CULTURES. Carla A. Martin, Asit Panja, Winthrop-University Hosp, Mineola, NY. The mechanisms of regulation for Fas-mediated apoptosis in intestinal epithelial cells (IEC) is not well understood, in part due to an inability to grow non-transformed IEC in culture. Using a recently developed model of HIPEC lines, we determined the susceptibility and mechanism of Fasmediated apoptosis in the presence, and absence of inflammatory cytokines. Untreated, IFNy- and TNFa-treated HIPEC were incubated with agonist anti-Fas antibody, CHII (20 hrs) and apoptosis was measured by AnnexinV staining, TUNEL and immunoblotting for activated caspase 3 (csp3). Jurkat cells were used as controls in all experiments. HIPEC consistently expressed high levels of Fas. However, incubation of HIPEC with IFN'Y or TNFa did not alter the level of Fas expression. Despite ample surface Fas, incubation of HIPEC with CHI I, induced less than a 25% increase in apoptosis above background. Pretreatment (20 hrs) of HIPEC with IFN'Y, but not TNFa, increased the number of CHll-induced apoptotic cells detected (jejunum»colon). Whole celllysates from CHllstimulated Jurkat contained csp3 proenzyme, partially processed p25 subunit and fully activated, p22 and p20 subunits. In contrast, HIPEC treated with CHll alone, contained only procsp3 and p25 subunit, while IFN'Ytreated HIPEC contained proenzyme and all three subunits. While IFN'Y primed HIPEC for the generation of activated csp3, it did not alter expression of the proenzyme. The onset of Pas-mediated apoptosis in HIPEC was delayed as compared to Jurkat. In IFNy- treated HIPEC, apoptotic cells were detected after 8 hours incubation with CHI 1 while the onset of apoptosis in Jurkat cells was seen by 2 hours. The generation of csp3 p22 and p20 subunits correlated with the onset of cell death. Experiments using caspase specific peptide inhibitors, revealed that, like Jurkat, HIPEC required csp9 as well as csp8 and csp3. Furthermore, HIPEC expressed significantly less procsp8 and 9 than Jurkat, but comparable levels of adapter protein FADD, and csp8 inhibitor, I-FLICE. IFN'Y treatment did not induce significant changes in the expression of any of these proteins. In conclusion, consistent with previous reports, we found that IFN'Y regulated the susceptibility of IEC to Fas-mediated apoptosis. However, the increased susceptibility of IFNv-treated HIPEC could not be fully accounted for by changes in expression of surface Fas, procaspases, FADD or I-FLICE. Also, our data show that HIPEC utilize caspases 8, 9 and 3 in Pas-mediated apoptosis.
1927 ENDOTHELIAL CELLS SUPPORT SURVIVAL OF HUMAN INTESTINAL MAST CELLS BY A DIRECT CELL-CELL INTERAC· TION. Claudia T. Mierke, Mathias Ballmaier, Michael P. Manns, Karl Welte, Stephan C. Bischoff, Med Sch of Hannover, Hannover, Germany. The regulation of human intestinal mast cell (MC) function is largely unknown. Recently, we found that human MC isolated from intestinal tissue specimen and purified up to 95 % maintain in culture for several weeks if stem cell factor (SCF) is added to the culture medium. Without addition of SCF, all purified MC die within 7 days. We examined if human umbilical cord vein endothelial cells (HUVEC) are capable of providing signals for MC survival in culture and if they are able to interact through adhesion molecules with endothelial cells. HUVEC (l st to 3rd passage) were layered into 24 well plates to adhere before the purified MC were added. The total MC culture time was 21 days, but once a week MC were transferred onto new HUVEC mono-layers to avoid confluence of the HUVEC. We could show that MC co-cultured with HUVEC could be maintained in culture for up to 21 days without addition of SCF to the culture medium. The MC number increased tol52 ::t: 19 % (mean ::t: SD, n = 13) as analyzed by MC counting and measurement of total histamine after cell lysis. In contrast, MC recovery was only 46 ::t: 27 % (n = 13), if MC were cultured with SCF. Addition of neutralizing anti-SCF antibody to the co-cultures as well as separation of MC and HUVEC using two chamber wells caused a marked reduction of MC survival. After 4 h of co-culture 82 ::t: 9 % of purified MC adhere to HUVEC. Addition of
GASTROENTEROLOGY Vol. 118, No.4
1923 INDUCIBLE NO SYNTHASE AND INTERFERON-r EXPRESSION IN DUODENAL EPITHELIUM FROM PATIENTS WITH ANKYLOSING SPONDYLITIS. Dominique Lamarque, Pascal Claudepierre, Zoltan Szepes, Christophe Bernardeau, Jeanne Tran Vannhieu, Nadine Martin-Garcia, Maxime Breban, Jean Charles Delchier, Brendan Jr Whittle, INSERM U99, Creteil, France; CHU Henri Mondor, Creteil, France; INSERM U477, Paris, France. Inflammatory processes have been detected in the ileal mucosa from patients with ankylosing spondylitis (AS). The inducible isoform of NO synthase, (iNOS), may be expressed early in the inflammatory process. The aim of this study was to determine iNOS activity and expression of mRNA for the pro-inflammatory cytokine, interferon-v (IFN-y) in the duodenal epithelium from patients with AS, rheumatoid arthritis (RA) and in controls. Methods. Gastroscopy with duodenal biopsies was conducted in 22 patients with AS who had not been treated by NSAID for at least 5 days, in 6 patients with RA and 28 controls, 5 of whom were treated by NSAID. H pylori status was determined by histology and culture from antral biopsies. Lymphocytic infiltration in lamina propria were semi-quantified by a histologic score ranging from 0 to 3. The iNOS expression was assessed by immunohistochemistry with monoclonal antibodies (Transduction Laboratories®) and the extent of the immunoreactivity was quantified by a score ranging from 0 to 3 in epithelium and in lamina propria. The percentage of patients with lymphocytic infiltration and positive immunofluoresence for iNOS in mucosa was compared between the different groups. Mucosal samples from duodenum were assayed for iNOS activity, measured as the conversion of L_'4C arginine to '4C-citrulline, in the presence of EGTA (l mM), and expressed in pmol/min.mg of protein (M ::t:SEM). Expression of mucosal IFN-y was estimated by mRNA detection with RT-PCR semi-quantified by comparison with actin mRNA expression. Results. Endoscopic examination of the gastro-duodenal mucosa did not find any lesion except in one patient with peptic ulcer in AS group. Conclusion: iNOS and IFN-y induction were detected in duodenal mucosa from patients with AS, which was independent of Hp status. This induction, which was not found in patients with RA or in controls, may reflect a specific inflammatory process in the duodenal epithelium in AS.
Control (n=28) AS (n=22) RA(n=6)
Hpstatus +
Infiltration
MucosaiiNOS
15 13 4
10 14 3
14 19· 2
INOS activity IFN·g mRNA
0,02 ± 0.04 0,29 ± ca0,06 ± 0.04.
O±O 0.7±0,3·
.: significant difference with control group; P<0.02
1924 HUMAN INTESTINAL MAST CELLS EXPRESS DIFFERENT CY· TOKINE PROFILES. Axel Lorentz, Gernot Sellge, Michael P. Manns, Stephan C. Bischoff, Med Sch of Hannover, Hannover, Germany. The recognition of mast cells as cytokine-producing cells has extended their potential functions from proinflammatory effector cells to regulatory components of the immune system. So far, little is known about cytokine profiles of mucosa-derived human mast cells. In this study, we examined the expression of cytokines by human intestinal mast cells triggered with different stimuli. Mast cells were purified up to 99% by magnetic cell separation and subsequently cultured with stem cell factor. Cytokine expression was analyzed by RT-PCR, immunocytochemistry and ELISA. Proinflammatory cytokines like TNF-a, ILl /3, IL-6, IL8, IL-16 and IL-18 were expressed without further stimulation. Activation by both IgE-receptor crosslinking and IgE-independent agonists such as grarnnegative bacteria enhanced the expression of TNF-a. Another set of cytokines, namely the Th2 type cytokines IL-3, IL5, IL-9 and ILl3, was expressed in response to stimulation by IgE receptor-crosslinking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and ILl3 was increased up to 4-fold compared to mast cells cultured with SCF alone. By contrast, IL-6 expression was totally blocked following culture with IL-4. Conclusion: Human intestinal mast cells produce constitutively proinflammatory cytokines which may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor-crosslinking results in the expression of Th2 type cytokines. IL-4 enhances the expression of Th2 type cytokines but does not affect or even down-regulates proinflammatory cytokines.
1925 RESISTANCE OF ANTIGEN·SPECIFIC T CELLS TO FAS-MEDIATED APOPTOSIS INDUCED BY IL-12 ANTAGONISTS IS MEDIATED BY BCL-2 AND BCL-XL. Thomas Marth, Sabine Ring, Dirk Schulte, Martin Zeitz, Internal Medicine II, Homburg/Saar, Germany. 1440720 Background: Feeding or injection of antigen to ovalbumin (OVA) T cell receptor (TCR) transgenic mice has been shown to induce systemic tolerance. This tolerance is augmented when antibodies to interleukin-12 (anti-IL-12) are applied concomitantly with antigen. The effect of antiIL-12 is brought over, at least in part, by induction of a Fas-mediated
apoptosis as also shown in a model of chronic murine colitis. The aim of the present study was to analyze the role of members of the bcl-2 family in this system. Methods: OVA-TCR transgenic mice were treated three times with injections of OVA protein with or without simultaneous anti-IL-12 injections; mice in the control group were not treated. On day four, mRNA and proteins were extracted from splenocytes and Peyers patches by standard methods and were analyzed by PCR and western blot, respectively. Results: OVA plus anti-IL-12 treated mice showed higher levels of Fas-mediated T cell apoptosis as compared to OVA alone treated and control mice in both peripheral and mucosal lymphoid tissues. The remaining, Fas-resistent and TGF-beta producing cells in the OVA plus anti-IL-12 group showed a significantly higher levels of FasL, bcl-2 and bcl-xl protein and mRNA expression as determined by optical densitometry measurements. Bcl-2 expression was also elevated in paralell studies using flow cytometry. Conclusions: Thus, the treatment with anti-IL-12 and antigen results in an induction of Fas-mediated apoptosis. Fas-resistant cells may escape clonal deletion because of upregulated anti-apoptotic molecules such as bcl-2 and bcl-xl, Our studies contribute to the understanding of the anti-inflammatory effects of anti-IL-12 which represent a promising therapeutic approach in autoimmune diseases.
1926 FAS-MEDIATED APOPTOSIS IN HUMAN INTESTINAL PRIMARY EPITHELIAL CELL (HIPEC) CULTURES. Carla A. Martin, Asit Panja, Winthrop-University Hosp, Mineola, NY. The mechanisms of regulation for Fas-mediated apoptosis in intestinal epithelial cells (IEC) is not well understood, in part due to an inability to grow non-transformed IEC in culture. Using a recently developed model of HIPEC lines, we determined the susceptibility and mechanism of Fasmediated apoptosis in the presence, and absence of inflammatory cytokines. Untreated, IFNy- and TNFa-treated HIPEC were incubated with agonist anti-Fas antibody, CHII (20 hrs) and apoptosis was measured by AnnexinV staining, TUNEL and immunoblotting for activated caspase 3 (csp3). Jurkat cells were used as controls in all experiments. HIPEC consistently expressed high levels of Fas. However, incubation of HIPEC with IFN'Y or TNFa did not alter the level of Fas expression. Despite ample surface Fas, incubation of HIPEC with CHI I, induced less than a 25% increase in apoptosis above background. Pretreatment (20 hrs) of HIPEC with IFN'Y, but not TNFa, increased the number of CHll-induced apoptotic cells detected (jejunum»colon). Whole celllysates from CHllstimulated Jurkat contained csp3 proenzyme, partially processed p25 subunit and fully activated, p22 and p20 subunits. In contrast, HIPEC treated with CHll alone, contained only procsp3 and p25 subunit, while IFN'Ytreated HIPEC contained proenzyme and all three subunits. While IFN'Y primed HIPEC for the generation of activated csp3, it did not alter expression of the proenzyme. The onset of Pas-mediated apoptosis in HIPEC was delayed as compared to Jurkat. In IFNy- treated HIPEC, apoptotic cells were detected after 8 hours incubation with CHI 1 while the onset of apoptosis in Jurkat cells was seen by 2 hours. The generation of csp3 p22 and p20 subunits correlated with the onset of cell death. Experiments using caspase specific peptide inhibitors, revealed that, like Jurkat, HIPEC required csp9 as well as csp8 and csp3. Furthermore, HIPEC expressed significantly less procsp8 and 9 than Jurkat, but comparable levels of adapter protein FADD, and csp8 inhibitor, I-FLICE. IFN'Y treatment did not induce significant changes in the expression of any of these proteins. In conclusion, consistent with previous reports, we found that IFN'Y regulated the susceptibility of IEC to Fas-mediated apoptosis. However, the increased susceptibility of IFNv-treated HIPEC could not be fully accounted for by changes in expression of surface Fas, procaspases, FADD or I-FLICE. Also, our data show that HIPEC utilize caspases 8, 9 and 3 in Pas-mediated apoptosis.
1927 ENDOTHELIAL CELLS SUPPORT SURVIVAL OF HUMAN INTESTINAL MAST CELLS BY A DIRECT CELL-CELL INTERAC· TION. Claudia T. Mierke, Mathias Ballmaier, Michael P. Manns, Karl Welte, Stephan C. Bischoff, Med Sch of Hannover, Hannover, Germany. The regulation of human intestinal mast cell (MC) function is largely unknown. Recently, we found that human MC isolated from intestinal tissue specimen and purified up to 95 % maintain in culture for several weeks if stem cell factor (SCF) is added to the culture medium. Without addition of SCF, all purified MC die within 7 days. We examined if human umbilical cord vein endothelial cells (HUVEC) are capable of providing signals for MC survival in culture and if they are able to interact through adhesion molecules with endothelial cells. HUVEC (l st to 3rd passage) were layered into 24 well plates to adhere before the purified MC were added. The total MC culture time was 21 days, but once a week MC were transferred onto new HUVEC mono-layers to avoid confluence of the HUVEC. We could show that MC co-cultured with HUVEC could be maintained in culture for up to 21 days without addition of SCF to the culture medium. The MC number increased tol52 ::t: 19 % (mean ::t: SD, n = 13) as analyzed by MC counting and measurement of total histamine after cell lysis. In contrast, MC recovery was only 46 ::t: 27 % (n = 13), if MC were cultured with SCF. Addition of neutralizing anti-SCF antibody to the co-cultures as well as separation of MC and HUVEC using two chamber wells caused a marked reduction of MC survival. After 4 h of co-culture 82 ::t: 9 % of purified MC adhere to HUVEC. Addition of