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Human astrocyte response to tumor necrosis factor (TNF): Enhancement by interleukin 1 (IL-1) and platelet-derived growth factor (PDGF)
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154
GAMMA INTERFERON SYNERGIZES WITH IL-1 AND IL-6 IN REVERSING DEXAMETBASONE (DEX)-MEDIATED SUPPRESSIVE EFFECTS. W.Y. Aha"i K.L. Sewell, E.T. Hadro, B. Zanker, and T.B. Strom.(Spon: T.B. Strom). Dept. Nephrol. and Internal Med., Beth Israel Hosp. and Harvard Med. Sch., Boston, MA 02215. Although DEX has been employed clinically in combating allograft rejection and autoimmune disorders, the exact mechanism by which it mediates its suppressive effects remains evasive. Using mitogen-induced proliferation as a biological read-out, we previously showed that DEX inhibits T cell proliferation by a mechanism which %res not include 1) Induction of Lipocortin, 2) Blockade of Ca flux and, 3) Interference with PK-C function. We proposed that DEX directly suppresses cytokine gene expression. The purpose of this study was to test the effects of DEX on cytokine gene expression, as well as the effects of cytokines on DEX-induced suppression. OUT data show that DEX, in a concentration-dependent fashion, suppressed IL-6 gene induction. In addition, rIL-1, rIL-2, rIL-4 and rIL-6 did not alter DEX-induced suppression. G.Wl!Jl.3 IFN, in a concentration-dependent fashion, partially reversed DEX-mediated immu”osuppresslon. However, gamma IFN (50 U/ml) when added to IL-1 (10 U/ml) and IL-6 (10 U/ml), completely reverses DEX-mediated effects. Other cytokine combinations were not as effective as gamma IFN+IL-l+IL-6 in reversing DEX mediated effects. These, and other results, further indicate that DEX acts at the level of cytokine gene expression in suppressing mitogen& antigen-induced cellular proliferation.
HUMAN ASTROCYTE RESPONSE ~0 TUMOR NECROSIS FACTOR (TNF): ENHANCEMENT BY INTERLEUKIN I (IL-i) AND PLATELET-DERIVED GROWTH FACTOR (PDGF). B.P. Barna, S.M. Chou, B.S. Jacobs, M.L. Estes and R.M. Ransohoff The Cleveland Clinic Foundation, Cleveland, OH 44i95. Current evidence suggests that the astrocyte hypertrophy and proliferation characteristic of central nervous system (CNS) injury may be in part associated with inflamnatory We recently reported that TNF enhanced DNA cytokine action. synthesis and proliferation of cultured adult human Here, we demonstrate that IL-1 non-neoplastic astrocytes. and PDGF augment the TNF effect. Two non-neoplastic astrocyte cell lines were established previously from epilepsy surgery specimens: Glial fibrillary acidic protein was detectable in both by immunostaining. DNA synthesis was comparably enhanced by TNF or IL-1 in both cell lines: at 10 rig/ml TNF or 0.5 U/ml IL-l, 1.7 to 2.0-fold enhancement occurred over medium alone (n = 2 experiments). An additive effect (3.6-fold) was seen with IL-1 + TNF. PDGF (10 rig/ml) enhanced DNA synthesis 8.4-fold but with PDGF + TNF, 18.6-fold stimulation was observed, suggesting synergism. These results indicate that combinations of cytokines can markedly alter growth characteristics of human astrocytes in vitro. Data support the concept of astrocyte responsiveness to cytokines in CNS injury.
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THE EFFECT OF INTERLEUKIN-1 AND TNF ON CHLAMYDIA TRACHflIATTS GROWTH: REVERSION BY TRYPTOPHAN AND PRO-F PROSTACLANDIN D. Wallach, I. Sarov, BGU, Ez. Y. Shemer-Avni, --H. Holtman, Beer aheva, Israel: Hannover acedi&lSchool, FRRr;;zmann Institute of Scien&, Rehovot, Israel. Infection with Chlamydia trachomatis, an obligate intracellular bacterium, can become persistent and was found to be associated with arthritis. TNF (tumor necrosis factor) and IL-1 (interleukin l), recently recognized as mediators in the pathogenesis of inflmmation, were shown to stimulate prostaglandin E2 (PGE2) and collagenase nroduction in synovial cells. In the present study we demonstrate that IL-l and TNF inhibit the growth of C. trachomatis in HEp-2 cells in a dose-dependent manner. Firrthermore , III ’ chlamydia infected cells treated with TNF, PGE2 formation was strikingly augmented, while in chlamydia infected cells or in uninfected cells treated with TNF, only a small increase of PGE was observed. The production of PGE2 and the inhibition o..3 chlamydial growth by IL-1 or TNF could effectively be reversed by addition of dexamethasone (10e6M) or by elevation of tryptophan concentration in the medium (above 100 pg/ml). Degradation of tryptophan could be observed in cultures treated with TNF. These results demonstrate that the interaction between TNF or IL-1 and the intracellular parasite, chlamydia, result not only in chlamydial inhibition but also in enhanced PGE2 production which might be associated with the pathogenesis of rheumatic diseases.
153 MODULATION OF TYPE IV COLLAGEN AND GELATINASE PRODUCTION BY HUMAN GINGIVAL KERATINOCYTES BY IL-1 8, TGF 8, TNF a AND B AND IFN ‘I. J. Bamuton S. Fishburn. J Reynolds and M Wilton. London and Strangeways Research MRC Dental Research Unit, Laboratories, Cambridge. UK. In inflammatory periodontitis, attachment of epithelium to the tooth, via a basement membrane(BM),is lost.We studied the role of some cytokinos on breakdown/repair of BM proteins by keratinocytes(KC) using a) changes in Type IV collagen (TIVC) synthesis and b) production of gelatinase. Gingival KC were cultured in vitro and stimulated with cytokines and PMA as control. TIVC was quantitated by immunoblotting and scanning by laser densitometry. To ensure the results were not due to changes in cell number, this was assessed colourimetricafiy. Gelatinase production was measured by degradati.on of Clabelled gelatin. PMA inhibited production of TIVC at 10e7 M but this appeared to be due to and at higher concentrations, decreased cell numbers. Gelatinase was also produced at these concentrations. IL-18 (150 pq/ml), IFNI (0.1 pM) and TGFB (2 rig/ml) increased TIVC production, unrelated to any changes in cell numbers. IL-18 and IFNy had no effect on qelatinase production but TGF@ stimulated latent qelatinase production. TNFa and B (10 “g/ml) had no effect on TIVC production but stimulated production of latent qelatinaso. These results show that these cytokines, probably produced at the disease site, may contribute to the regulation of BM inteqrity, and thus play a role in the pathoqenesis of periodontal diseases.
REGULATION
OF IL-1 EXPRESSION
BY 11-3.
David I. Bollrr, Matth.w.J. Fenton, Gyorgy Frendl, Boston Unhwrity Medical Center, Evans Medical Foundation, Borbn, MA 02118
In our accomDanyino abstract we reDorl that IL-3 (multi-CSF) can enhance macrophage$%+) properties related to anti&n presentation. Therefore ii was of interest to studv. in oarlicular. whether IL-3 was involved in the regulation of MB IL-1 e!&e&iin. We f&md that IL-3 has a limited potential to induce IL-1 protein expression, while showing a marked synergy with suboptimal concentrations of LPS. This phenomenon can be observed on normal murine Peritoneal exudate MB as well as on the P388Dl cell line. Further analysis of this interaction indicates that LPS-inducible MB cytokines (IL-l, IL-6. or TNF-a) can not provide the necessary complementary signal for IL-3 to effiiiently induce IL-1 protein expression. In our hands GM-CSF behaves similarly to IL-3. Our -studies oh the induction of IL-l in response lo IL-3 al&e or in combination with subootimal doses of LPS indicate that IL-3 is able to induce an increse in 'IL-1 mRNA as well as synergyze with LPS in production of both IL-I mRNA and bioactivity. Experiments examining total cellular and the cvtoplasmic RNA after induction of P388Dl cells with IL-3+LPS indicate ihat the increase of IL-I message indeed occurs in the translatable IL-I mRNA DOOI. Northern blot analvsis of the same RNA demonstrated that the pot&tial of IL-3 lo induce IL:1 mRNA as well as its pronounced synergy with LPS were reflected in changes occuring in the 1.9 kB cvtoolasmic IL-1 mRNA. Furthermore. interactions of IL-3 and LPS are $sd manifested in changes in the kinetics of both IL-1 mRNA as well as protein production. Moreover our preliminary results indicate the interactions of the lymphokine-mediated and -independent pathways of T cell induction of IL-l, since GM-CSF and IL-3 can reconstitute the IL-1 induction induced by fixed T cells lo the level of induction provided by fife T cells.
156 INHIBITORS OF SERINE/THREONINE KINASES INHIBIT IL-l-INDUCTION OF C-MYC mRNA. A. Berger and S. Davidson. Cell Biology Unit, The Upjohn Co., Kalamazoo, MI 49008. Interleukin-1 increases the steady state level of c-myc mRNA in human lymphocytes (J.I. 137:3649, 1986). This response is rapid (2 hr), occurs in human T cells in the absence of other stimuli, and, therefore, can be used as the basis of an assay for inhibitors of the intracellular action of IL-l. Northern analyses of mRNA from a human T cell hybridoma, 1169, incubated with or without human IL-l@ or IL-&, and with or without drugs were performed. Drugs which increased intracellular CAMP, inhibited cyclooxygenase or lipoxygenase, inhibited protein synthesis, inhibited lymphokine release, blocked calcium channels or inhibited calmodulin, inhibited phospholipases, or inhibited EGF-receptor tyrosine kinase failed to decrease basal or IL-l-induced c-m c steady state mRNA levels. K252a ( ICF,O, 100 nM), 20 uM) and H9 (IC60, 100 pM), all H7 -7&o, inhibitors of serine/threonine kinases, inhibited both basal and IL-l-induced c-myc. Treatment with PM4 and IL-1 led to an additive increase in c-myc mRNA levels, suggesting that IL-1 was not acting through protein kinase C alone. The lack of effect of analogs or inducers of CAMP indicated that CAMP-dependent kinase was not involved in the effect of IL-1 on c-myc mRNA. These data support the conclusion that the action of IL-1 on steady state mRNA levels of c-myc may be mediated by a serine/threonine protein kinase other than PKA or PKC.
/ SECOND
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
151
154
GAMMA INTERFERON SYNERGIZES WITH IL-1 AND IL-6 IN REVERSING DEXAMETBASONE (DEX)-MEDIATED SUPPRESSIVE EFFECTS. W.Y. Aha"i K.L. Sewell, E.T. Hadro, B. Zanker, and T.B. Strom.(Spon: T.B. Strom). Dept. Nephrol. and Internal Med., Beth Israel Hosp. and Harvard Med. Sch., Boston, MA 02215. Although DEX has been employed clinically in combating allograft rejection and autoimmune disorders, the exact mechanism by which it mediates its suppressive effects remains evasive. Using mitogen-induced proliferation as a biological read-out, we previously showed that DEX inhibits T cell proliferation by a mechanism which %res not include 1) Induction of Lipocortin, 2) Blockade of Ca flux and, 3) Interference with PK-C function. We proposed that DEX directly suppresses cytokine gene expression. The purpose of this study was to test the effects of DEX on cytokine gene expression, as well as the effects of cytokines on DEX-induced suppression. OUT data show that DEX, in a concentration-dependent fashion, suppressed IL-6 gene induction. In addition, rIL-1, rIL-2, rIL-4 and rIL-6 did not alter DEX-induced suppression. G.Wl!Jl.3 IFN, in a concentration-dependent fashion, partially reversed DEX-mediated immu”osuppresslon. However, gamma IFN (50 U/ml) when added to IL-1 (10 U/ml) and IL-6 (10 U/ml), completely reverses DEX-mediated effects. Other cytokine combinations were not as effective as gamma IFN+IL-l+IL-6 in reversing DEX mediated effects. These, and other results, further indicate that DEX acts at the level of cytokine gene expression in suppressing mitogen& antigen-induced cellular proliferation.
HUMAN ASTROCYTE RESPONSE ~0 TUMOR NECROSIS FACTOR (TNF): ENHANCEMENT BY INTERLEUKIN I (IL-i) AND PLATELET-DERIVED GROWTH FACTOR (PDGF). B.P. Barna, S.M. Chou, B.S. Jacobs, M.L. Estes and R.M. Ransohoff The Cleveland Clinic Foundation, Cleveland, OH 44i95. Current evidence suggests that the astrocyte hypertrophy and proliferation characteristic of central nervous system (CNS) injury may be in part associated with inflamnatory We recently reported that TNF enhanced DNA cytokine action. synthesis and proliferation of cultured adult human Here, we demonstrate that IL-1 non-neoplastic astrocytes. and PDGF augment the TNF effect. Two non-neoplastic astrocyte cell lines were established previously from epilepsy surgery specimens: Glial fibrillary acidic protein was detectable in both by immunostaining. DNA synthesis was comparably enhanced by TNF or IL-1 in both cell lines: at 10 rig/ml TNF or 0.5 U/ml IL-l, 1.7 to 2.0-fold enhancement occurred over medium alone (n = 2 experiments). An additive effect (3.6-fold) was seen with IL-1 + TNF. PDGF (10 rig/ml) enhanced DNA synthesis 8.4-fold but with PDGF + TNF, 18.6-fold stimulation was observed, suggesting synergism. These results indicate that combinations of cytokines can markedly alter growth characteristics of human astrocytes in vitro. Data support the concept of astrocyte responsiveness to cytokines in CNS injury.
152
155
THE EFFECT OF INTERLEUKIN-1 AND TNF ON CHLAMYDIA TRACHflIATTS GROWTH: REVERSION BY TRYPTOPHAN AND PRO-F PROSTACLANDIN D. Wallach, I. Sarov, BGU, Ez. Y. Shemer-Avni, --H. Holtman, Beer aheva, Israel: Hannover acedi&lSchool, FRRr;;zmann Institute of Scien&, Rehovot, Israel. Infection with Chlamydia trachomatis, an obligate intracellular bacterium, can become persistent and was found to be associated with arthritis. TNF (tumor necrosis factor) and IL-1 (interleukin l), recently recognized as mediators in the pathogenesis of inflmmation, were shown to stimulate prostaglandin E2 (PGE2) and collagenase nroduction in synovial cells. In the present study we demonstrate that IL-l and TNF inhibit the growth of C. trachomatis in HEp-2 cells in a dose-dependent manner. Firrthermore , III ’ chlamydia infected cells treated with TNF, PGE2 formation was strikingly augmented, while in chlamydia infected cells or in uninfected cells treated with TNF, only a small increase of PGE was observed. The production of PGE2 and the inhibition o..3 chlamydial growth by IL-1 or TNF could effectively be reversed by addition of dexamethasone (10e6M) or by elevation of tryptophan concentration in the medium (above 100 pg/ml). Degradation of tryptophan could be observed in cultures treated with TNF. These results demonstrate that the interaction between TNF or IL-1 and the intracellular parasite, chlamydia, result not only in chlamydial inhibition but also in enhanced PGE2 production which might be associated with the pathogenesis of rheumatic diseases.
153 MODULATION OF TYPE IV COLLAGEN AND GELATINASE PRODUCTION BY HUMAN GINGIVAL KERATINOCYTES BY IL-1 8, TGF 8, TNF a AND B AND IFN ‘I. J. Bamuton S. Fishburn. J Reynolds and M Wilton. London and Strangeways Research MRC Dental Research Unit, Laboratories, Cambridge. UK. In inflammatory periodontitis, attachment of epithelium to the tooth, via a basement membrane(BM),is lost.We studied the role of some cytokinos on breakdown/repair of BM proteins by keratinocytes(KC) using a) changes in Type IV collagen (TIVC) synthesis and b) production of gelatinase. Gingival KC were cultured in vitro and stimulated with cytokines and PMA as control. TIVC was quantitated by immunoblotting and scanning by laser densitometry. To ensure the results were not due to changes in cell number, this was assessed colourimetricafiy. Gelatinase production was measured by degradati.on of Clabelled gelatin. PMA inhibited production of TIVC at 10e7 M but this appeared to be due to and at higher concentrations, decreased cell numbers. Gelatinase was also produced at these concentrations. IL-18 (150 pq/ml), IFNI (0.1 pM) and TGFB (2 rig/ml) increased TIVC production, unrelated to any changes in cell numbers. IL-18 and IFNy had no effect on qelatinase production but TGF@ stimulated latent qelatinase production. TNFa and B (10 “g/ml) had no effect on TIVC production but stimulated production of latent qelatinaso. These results show that these cytokines, probably produced at the disease site, may contribute to the regulation of BM inteqrity, and thus play a role in the pathoqenesis of periodontal diseases.
REGULATION
OF IL-1 EXPRESSION
BY 11-3.
David I. Bollrr, Matth.w.J. Fenton, Gyorgy Frendl, Boston Unhwrity Medical Center, Evans Medical Foundation, Borbn, MA 02118
In our accomDanyino abstract we reDorl that IL-3 (multi-CSF) can enhance macrophage$%+) properties related to anti&n presentation. Therefore ii was of interest to studv. in oarlicular. whether IL-3 was involved in the regulation of MB IL-1 e!&e&iin. We f&md that IL-3 has a limited potential to induce IL-1 protein expression, while showing a marked synergy with suboptimal concentrations of LPS. This phenomenon can be observed on normal murine Peritoneal exudate MB as well as on the P388Dl cell line. Further analysis of this interaction indicates that LPS-inducible MB cytokines (IL-l, IL-6. or TNF-a) can not provide the necessary complementary signal for IL-3 to effiiiently induce IL-1 protein expression. In our hands GM-CSF behaves similarly to IL-3. Our -studies oh the induction of IL-l in response lo IL-3 al&e or in combination with subootimal doses of LPS indicate that IL-3 is able to induce an increse in 'IL-1 mRNA as well as synergyze with LPS in production of both IL-I mRNA and bioactivity. Experiments examining total cellular and the cvtoplasmic RNA after induction of P388Dl cells with IL-3+LPS indicate ihat the increase of IL-I message indeed occurs in the translatable IL-I mRNA DOOI. Northern blot analvsis of the same RNA demonstrated that the pot&tial of IL-3 lo induce IL:1 mRNA as well as its pronounced synergy with LPS were reflected in changes occuring in the 1.9 kB cvtoolasmic IL-1 mRNA. Furthermore. interactions of IL-3 and LPS are $sd manifested in changes in the kinetics of both IL-1 mRNA as well as protein production. Moreover our preliminary results indicate the interactions of the lymphokine-mediated and -independent pathways of T cell induction of IL-l, since GM-CSF and IL-3 can reconstitute the IL-1 induction induced by fixed T cells lo the level of induction provided by fife T cells.
156 INHIBITORS OF SERINE/THREONINE KINASES INHIBIT IL-l-INDUCTION OF C-MYC mRNA. A. Berger and S. Davidson. Cell Biology Unit, The Upjohn Co., Kalamazoo, MI 49008. Interleukin-1 increases the steady state level of c-myc mRNA in human lymphocytes (J.I. 137:3649, 1986). This response is rapid (2 hr), occurs in human T cells in the absence of other stimuli, and, therefore, can be used as the basis of an assay for inhibitors of the intracellular action of IL-l. Northern analyses of mRNA from a human T cell hybridoma, 1169, incubated with or without human IL-l@ or IL-&, and with or without drugs were performed. Drugs which increased intracellular CAMP, inhibited cyclooxygenase or lipoxygenase, inhibited protein synthesis, inhibited lymphokine release, blocked calcium channels or inhibited calmodulin, inhibited phospholipases, or inhibited EGF-receptor tyrosine kinase failed to decrease basal or IL-l-induced c-m c steady state mRNA levels. K252a ( ICF,O, 100 nM), 20 uM) and H9 (IC60, 100 pM), all H7 -7&o, inhibitors of serine/threonine kinases, inhibited both basal and IL-l-induced c-myc. Treatment with PM4 and IL-1 led to an additive increase in c-myc mRNA levels, suggesting that IL-1 was not acting through protein kinase C alone. The lack of effect of analogs or inducers of CAMP indicated that CAMP-dependent kinase was not involved in the effect of IL-1 on c-myc mRNA. These data support the conclusion that the action of IL-1 on steady state mRNA levels of c-myc may be mediated by a serine/threonine protein kinase other than PKA or PKC.