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Effect of Anapsos (Polypodium leucotomos extract) on cytokines production
Cytokines in in$hmmation
25 June 1997 - Poster presentations
studied with respect to their DNA binding properties in the presence and absence of PGE2 (AP-1, NFAT, NF-~6). We observed that PGEp only inhibited the binding capacity of NF-~8, whereas AP-1 and NFAT were not affected. In addiion, the effect of PGE2 was also reflected in the amount of secreted IL-5 protein in anti-CD3 plus anti-CD26 or Con A stimulated T lymphocytes. In contrast to these observations, Con A plus PMA induced IL-5 mRNA accumulation was slightly upregulated (+20X) by PGEp (1O-5 M). In conclusion, these data demonstrate that the ultimate effect of intracellular cAMP elevation can be inhibitory as well as stimulatory on the expression of 11-5,and depends on the mode of stimulation.
1 P.3.10.41
) Characterization of P-adrenoceptor subtype(s)
415
well as IFN-y and TNF-(r induced RANTES production was fully inhibited by TGF-6. The presence of IL-4 in combination with IL-la and IFN-y, IFN-y and TNFa or IL-la and TNFa augmented RANTES production. 11-2, IL-10 and IL-15 had no effect on IL-la and IFN-y or IFN-y and TNFa induced RANTES production by PTEC. Conolurlons: The production of RANTES by PTEC is induced in the presence of both T cell- and macrophagederived cytokines. Therefore we hypothesize that during transplant rejection RANTES produced by PTEC plays a more important role in the amplification phase of the immune response rather than in the initiation phase.
1P3.10.43
1 IL-17 and CD40-L act synerglstlcaliy in the activation of human renal epithdial cells and can both be detected during renal allograft rejection
involved in the regulation of cytokine gene expression in activated human T lymphocytes Peter Borger, Yke Hoekstra, Dirkje S. Posbna, Johan Zaagsma, Edo Vellenga, Henk F. Kauffman. Divisions ofA//efgo/ogy, Pu/mono/~, Mofecu/ar Pharmscology and Hemato/ogg Depertment of Internal Medicine and Universify Centre br Pharmacy, University of Groningen, The Netherlands
C. van Kooten ‘, J.G. Boonstra ‘, M.E. Paape ‘, F. Fossiez2, J. Banchereau 2, S. Lebecque 2, L.A. van Es’, M.R. Daha' ’ Department of Nephrology; Leiden University Hospital, Leiden, the Netherlands, zLaboratory for lmmunobgical Research, Schering Plough, Dardilly, France
Cytokine gene expression in T lymphocytes is a stringently regulated process, involving both stimulatory and inhibitory signals. j3-Adrenoceptor (B-AR) agonists are able to induce an inhibitory signal on immunological responses after binding to their specific receptors. In the present paper the characterization of the B-AR subtypes (pi, 82, and 83) involved in the regulation of the expression of IL-3, 11-4, GM-CSF and IFN-y mRNA was studied by using various B-AR agonists and antagonists. Northern analvsis revealed that concanavalin A (Con A) induced IFN-V. GM-CSF and IL-i mRNA are dose-dependently inhibited by’the nonselect& ,!?-ARagonist isoproterenol (ISO; lO-4 M-10-* M) and by the selective BP-AR &nistenoterol: IL-4 mRNA accumulation was less susceptable to B-AR stimulation. The obsen/ed inhibition of IS0 on IFN-y, GM-CSF. and IL-3 mRNA was blocked by the 82 specific antagonist ICI 118,551 (1O-6 M). In contrast, the selective PI-AR antagonist atenolol(0.3 x 10-s M) did not have any effect. The biological relevance ofthemRNAfindings was investigated by measuring GM-CSF protein secretion. GM-CSF protein followed a similar pattern as observed for GM-CSF mRNA. In addition it was shown that the B-AR mediated inhibltion on the expression of IFN-y, GM-CSF. and IL-3 mRNA coincided with an accumulation of Intracellular cAMP levels. Finally, we were able to demonstrate B 3-AR transctfpts in Con A activated T lymphocytes by using the FIT-PCRtechnique. However, no functional activity of the ,3 3-AR in the regulation of cvtokine expression was shown: the selective 6 3-Ak agonist BRL 37344 had no effect on the accumulation of the studied cytokine mRNAs, though at higher concentrations BRL 37344 (10m4M) moderately increased cell&r cyclic-AMP levels (30% of the amount generated by lO-5 M ISO). In conclusion, these data demonstrate that the B-agonist-induced inhibition of IFN-y, GM-CSF and IL-3 mRNA expression and GM-CSF protein secretion is solely mediated by BP-adrenoceptors.
Introduction: Renal allograft rejection is charactetfzed by local inflammatory processes and the infiltration of both T cells and monocytes. T cells are considered a central cell type in the local regulatory processes, as well as in the final target destruction. In previous studies we have demonstrated that proximal tubular epithelial cells (PTEC), a cellular component involved in local renal inflammatory responses, express the CD40 receptor and respond to several cytokines. In the present study we investigated a possible role of IL-17 and CD40L, two specific T cell products, in kidney allograft rejectim and renal inflammation. MaterIsIs and Methods: Primary cultures of human PTEC were established from normal kidney tissue specimens. PTEC were activated by recombinant 11-17, CWO-llgand or combinations, and the activation products IL-6, 11-6, MCP-1 and RANTES were measured by specific ELISA. Expression of both IL-17 and CD4OL in renal biopsy material was investigated by RT-PCR as well as by immunofluorescence stainings. Reeutts: Activation of PTEC by IL-17 resulted in an enhanced production of IL-6, IL-6 and MCP-1, but not of RANTES. Simultaneous actfvation of PTEC by IL-17 and CD40L resulted in strong synergistic effects on the production of 11-6, 11-6, MCP-1 and RANTES. Up to 50-fold increase in cytokine production was observed after this combined activation. lmmunofluorescence staining showed a strong expression of IL-17 in biopsies of patients with diagnosis of rejection (n = 6). No expression of IL-17 was observed in pre-transplant biopsies. Expression of IL-17 was confirmed at the mRNA level by RT-PCR analysis of whole kidney isolate, as well as in in vitro cultured graft infiltrating T cells. In the same biopsies expression of CD4OL,a marker of activated T cells for which the counter-receptor CD40 is expressed on PTEC, could be demonstrated. Conclusion: These results represent the first demonstration of IL-17 expression in pathological conditions and suggest that IL-17 might be important for the regulation of the local inflammatory response during renal allograft rejection. Furthermore, the presence of CD4OL during rejection and the synergistic signalling between IL-17 and CD40, suggest an important role for T cells in the regulation of this local inflammatory response.
( P.3.10.42 1 RANTES production by human proximal tubular epithelial cells (pTEC) In vffru J.G.M. Decker& S.W. van Der Kooij, F.J. van Der Woude, M.R. Daha. Dept. of Nephrol~, Leiden Univetsify Hospital, Leiden, The Netherlands Introduction: In biopsies from renal transplant patients with rejection, RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) is detected in both infiltrating mononuclear cells and renal tubular epithelium. The mechanisms involved in upregulation of RANTES in tubular epithelium have not yet been resolved. Th&f&e, we studied the effect of vahous monocyte- and T cell-derived cytokines on RANTES production by PTEC in vitro. Yaterlals~andMethods:Hum& primary PTEC lines were cultured in DMEMMAM F12 under serum free conditions. PTEC tissue-culture supernatants were harvested after 72 hr stimulation with eRher IL-la, 11-2, 11-4, IL-lo, 11-15, IFN-y, TNFa or TGF-p and assessed for RANTES by ELISA (lower detection limit 3 pg/ml). Reeulta: No detectable RANTES was found in 72 hr culture suoematants of 7 different PTEC lines when cultured in medium alone. The pres&ce of IL-la, IL-2,lL-4, IL-lo, IL-15, IFN-y, TNFa or TGF-,?Ialone did not result in significant induction of RANTES. FtANTES production was not detectable after stimulation with either 11-2, 11-4, IL-IO, IL-15 or TGF-@in combination with IL-la, IFN-y or TNFa, respectively. However, combinations of IFN-y together with IL-la or with TNF-a resulted in strong induction of RANTES production (2046 f 617 and 2595 f 525 w/ml, respectively) in all PTEC lines. RANTES production after stimulation with a combination of IL-la and TNF-(r was less prominent (352 f 532 pg/ml) and only detectable in 517 PTEC lines. The cytokine induced RANTES production was inhibited by cycioheximide indicating that de now protein synthesis is required for RANTES production. Both IL-la and IFN-y as
IP.3.10.44
Effect of Anapsos (Po/ypocf/um leucotomos extract) on cytokines production
*.
J.M. Sempere 1,2,A. Campos 3,2,C. Rodrfgo ’ ASAC Pharmaceutical Intemati~al, Spain, 2Division of Immunobg~ University of A&Me, Spain, 3Blood Sank Centre of Alicante, Spain Introduction: Poiypodium leucotomos is a typical fern of Central America. The extract of its rhizomes (Anapsos) is used to treat skin diseases. Early studies of this product in vitro, evidenced an antiiumoral effect. Other atiors also showed an immunomodulating effect in several diseases. Matedaland Methods:after dehydration, immersion and maceration with polar solvents, the extract obtained (Anapsos) was filtered, freeze-dried and granted by ASAC for the present research work. Mononuclear cells from healthv sonon w&e cultured v&h LPS and/or PHA and/or Anapsos, and complete kinetics for different cytokines (IL-l& TNFa, 11-2, INF-y, IL-4, IL-lo) were measured in supematants (ELISA). Results: A significant reduction in IL-18 levels was observed with LPS + Anapsos vs. LPS (p < 0.025). Addition of Anapsos to LPS + PHA, consistently vielded a sianificant rise in 11-2. INF-v and IL-10 levels. Correlation between iL-1s and 11-2with LPS + PHA.(r = 0160; p = 0.004). reversed when Anapsos was added to LPS + PHA (r = -0.60; p = 0.005). The same happened with the correlation between INF-y and IL-IO (with LPS was r = -0.70-p = 0.002; with LPS + ANP was r = 0.65 p = 0.001). Conclusion: The present study suggest that Anapsos has a pleiotropic effect in vitro for the different cytokines analysed, which may be due to a different mode of acting on different cell populations in the immune system.
25 June 1997 - Poster presentations
studied with respect to their DNA binding properties in the presence and absence of PGE2 (AP-1, NFAT, NF-~6). We observed that PGEp only inhibited the binding capacity of NF-~8, whereas AP-1 and NFAT were not affected. In addiion, the effect of PGE2 was also reflected in the amount of secreted IL-5 protein in anti-CD3 plus anti-CD26 or Con A stimulated T lymphocytes. In contrast to these observations, Con A plus PMA induced IL-5 mRNA accumulation was slightly upregulated (+20X) by PGEp (1O-5 M). In conclusion, these data demonstrate that the ultimate effect of intracellular cAMP elevation can be inhibitory as well as stimulatory on the expression of 11-5,and depends on the mode of stimulation.
1 P.3.10.41
) Characterization of P-adrenoceptor subtype(s)
415
well as IFN-y and TNF-(r induced RANTES production was fully inhibited by TGF-6. The presence of IL-4 in combination with IL-la and IFN-y, IFN-y and TNFa or IL-la and TNFa augmented RANTES production. 11-2, IL-10 and IL-15 had no effect on IL-la and IFN-y or IFN-y and TNFa induced RANTES production by PTEC. Conolurlons: The production of RANTES by PTEC is induced in the presence of both T cell- and macrophagederived cytokines. Therefore we hypothesize that during transplant rejection RANTES produced by PTEC plays a more important role in the amplification phase of the immune response rather than in the initiation phase.
1P3.10.43
1 IL-17 and CD40-L act synerglstlcaliy in the activation of human renal epithdial cells and can both be detected during renal allograft rejection
involved in the regulation of cytokine gene expression in activated human T lymphocytes Peter Borger, Yke Hoekstra, Dirkje S. Posbna, Johan Zaagsma, Edo Vellenga, Henk F. Kauffman. Divisions ofA//efgo/ogy, Pu/mono/~, Mofecu/ar Pharmscology and Hemato/ogg Depertment of Internal Medicine and Universify Centre br Pharmacy, University of Groningen, The Netherlands
C. van Kooten ‘, J.G. Boonstra ‘, M.E. Paape ‘, F. Fossiez2, J. Banchereau 2, S. Lebecque 2, L.A. van Es’, M.R. Daha' ’ Department of Nephrology; Leiden University Hospital, Leiden, the Netherlands, zLaboratory for lmmunobgical Research, Schering Plough, Dardilly, France
Cytokine gene expression in T lymphocytes is a stringently regulated process, involving both stimulatory and inhibitory signals. j3-Adrenoceptor (B-AR) agonists are able to induce an inhibitory signal on immunological responses after binding to their specific receptors. In the present paper the characterization of the B-AR subtypes (pi, 82, and 83) involved in the regulation of the expression of IL-3, 11-4, GM-CSF and IFN-y mRNA was studied by using various B-AR agonists and antagonists. Northern analvsis revealed that concanavalin A (Con A) induced IFN-V. GM-CSF and IL-i mRNA are dose-dependently inhibited by’the nonselect& ,!?-ARagonist isoproterenol (ISO; lO-4 M-10-* M) and by the selective BP-AR &nistenoterol: IL-4 mRNA accumulation was less susceptable to B-AR stimulation. The obsen/ed inhibition of IS0 on IFN-y, GM-CSF. and IL-3 mRNA was blocked by the 82 specific antagonist ICI 118,551 (1O-6 M). In contrast, the selective PI-AR antagonist atenolol(0.3 x 10-s M) did not have any effect. The biological relevance ofthemRNAfindings was investigated by measuring GM-CSF protein secretion. GM-CSF protein followed a similar pattern as observed for GM-CSF mRNA. In addition it was shown that the B-AR mediated inhibltion on the expression of IFN-y, GM-CSF. and IL-3 mRNA coincided with an accumulation of Intracellular cAMP levels. Finally, we were able to demonstrate B 3-AR transctfpts in Con A activated T lymphocytes by using the FIT-PCRtechnique. However, no functional activity of the ,3 3-AR in the regulation of cvtokine expression was shown: the selective 6 3-Ak agonist BRL 37344 had no effect on the accumulation of the studied cytokine mRNAs, though at higher concentrations BRL 37344 (10m4M) moderately increased cell&r cyclic-AMP levels (30% of the amount generated by lO-5 M ISO). In conclusion, these data demonstrate that the B-agonist-induced inhibition of IFN-y, GM-CSF and IL-3 mRNA expression and GM-CSF protein secretion is solely mediated by BP-adrenoceptors.
Introduction: Renal allograft rejection is charactetfzed by local inflammatory processes and the infiltration of both T cells and monocytes. T cells are considered a central cell type in the local regulatory processes, as well as in the final target destruction. In previous studies we have demonstrated that proximal tubular epithelial cells (PTEC), a cellular component involved in local renal inflammatory responses, express the CD40 receptor and respond to several cytokines. In the present study we investigated a possible role of IL-17 and CD40L, two specific T cell products, in kidney allograft rejectim and renal inflammation. MaterIsIs and Methods: Primary cultures of human PTEC were established from normal kidney tissue specimens. PTEC were activated by recombinant 11-17, CWO-llgand or combinations, and the activation products IL-6, 11-6, MCP-1 and RANTES were measured by specific ELISA. Expression of both IL-17 and CD4OL in renal biopsy material was investigated by RT-PCR as well as by immunofluorescence stainings. Reeutts: Activation of PTEC by IL-17 resulted in an enhanced production of IL-6, IL-6 and MCP-1, but not of RANTES. Simultaneous actfvation of PTEC by IL-17 and CD40L resulted in strong synergistic effects on the production of 11-6, 11-6, MCP-1 and RANTES. Up to 50-fold increase in cytokine production was observed after this combined activation. lmmunofluorescence staining showed a strong expression of IL-17 in biopsies of patients with diagnosis of rejection (n = 6). No expression of IL-17 was observed in pre-transplant biopsies. Expression of IL-17 was confirmed at the mRNA level by RT-PCR analysis of whole kidney isolate, as well as in in vitro cultured graft infiltrating T cells. In the same biopsies expression of CD4OL,a marker of activated T cells for which the counter-receptor CD40 is expressed on PTEC, could be demonstrated. Conclusion: These results represent the first demonstration of IL-17 expression in pathological conditions and suggest that IL-17 might be important for the regulation of the local inflammatory response during renal allograft rejection. Furthermore, the presence of CD4OL during rejection and the synergistic signalling between IL-17 and CD40, suggest an important role for T cells in the regulation of this local inflammatory response.
( P.3.10.42 1 RANTES production by human proximal tubular epithelial cells (pTEC) In vffru J.G.M. Decker& S.W. van Der Kooij, F.J. van Der Woude, M.R. Daha. Dept. of Nephrol~, Leiden Univetsify Hospital, Leiden, The Netherlands Introduction: In biopsies from renal transplant patients with rejection, RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) is detected in both infiltrating mononuclear cells and renal tubular epithelium. The mechanisms involved in upregulation of RANTES in tubular epithelium have not yet been resolved. Th&f&e, we studied the effect of vahous monocyte- and T cell-derived cytokines on RANTES production by PTEC in vitro. Yaterlals~andMethods:Hum& primary PTEC lines were cultured in DMEMMAM F12 under serum free conditions. PTEC tissue-culture supernatants were harvested after 72 hr stimulation with eRher IL-la, 11-2, 11-4, IL-lo, 11-15, IFN-y, TNFa or TGF-p and assessed for RANTES by ELISA (lower detection limit 3 pg/ml). Reeulta: No detectable RANTES was found in 72 hr culture suoematants of 7 different PTEC lines when cultured in medium alone. The pres&ce of IL-la, IL-2,lL-4, IL-lo, IL-15, IFN-y, TNFa or TGF-,?Ialone did not result in significant induction of RANTES. FtANTES production was not detectable after stimulation with either 11-2, 11-4, IL-IO, IL-15 or TGF-@in combination with IL-la, IFN-y or TNFa, respectively. However, combinations of IFN-y together with IL-la or with TNF-a resulted in strong induction of RANTES production (2046 f 617 and 2595 f 525 w/ml, respectively) in all PTEC lines. RANTES production after stimulation with a combination of IL-la and TNF-(r was less prominent (352 f 532 pg/ml) and only detectable in 517 PTEC lines. The cytokine induced RANTES production was inhibited by cycioheximide indicating that de now protein synthesis is required for RANTES production. Both IL-la and IFN-y as
IP.3.10.44
Effect of Anapsos (Po/ypocf/um leucotomos extract) on cytokines production
*.
J.M. Sempere 1,2,A. Campos 3,2,C. Rodrfgo ’ ASAC Pharmaceutical Intemati~al, Spain, 2Division of Immunobg~ University of A&Me, Spain, 3Blood Sank Centre of Alicante, Spain Introduction: Poiypodium leucotomos is a typical fern of Central America. The extract of its rhizomes (Anapsos) is used to treat skin diseases. Early studies of this product in vitro, evidenced an antiiumoral effect. Other atiors also showed an immunomodulating effect in several diseases. Matedaland Methods:after dehydration, immersion and maceration with polar solvents, the extract obtained (Anapsos) was filtered, freeze-dried and granted by ASAC for the present research work. Mononuclear cells from healthv sonon w&e cultured v&h LPS and/or PHA and/or Anapsos, and complete kinetics for different cytokines (IL-l& TNFa, 11-2, INF-y, IL-4, IL-lo) were measured in supematants (ELISA). Results: A significant reduction in IL-18 levels was observed with LPS + Anapsos vs. LPS (p < 0.025). Addition of Anapsos to LPS + PHA, consistently vielded a sianificant rise in 11-2. INF-v and IL-10 levels. Correlation between iL-1s and 11-2with LPS + PHA.(r = 0160; p = 0.004). reversed when Anapsos was added to LPS + PHA (r = -0.60; p = 0.005). The same happened with the correlation between INF-y and IL-IO (with LPS was r = -0.70-p = 0.002; with LPS + ANP was r = 0.65 p = 0.001). Conclusion: The present study suggest that Anapsos has a pleiotropic effect in vitro for the different cytokines analysed, which may be due to a different mode of acting on different cell populations in the immune system.