1
486 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES 217 220 PROTEIN KINASE C IS INVOLVED IN THE DOWN-MODULATION OF ZYMOSAN-STIMULATED TUMOR NEGROSIS FACTOR alpha PRODUCTION BY HUNAN NoNocYTEs : EFFECT ON CR3XYTO8KELETAL INTERACTION RELATIONSHIP BETWEEN PROTEIN KINASE C AND INTERLEUKIN-1 IN HELPER T CELLS. M.R. Sierra-Honiama"". E. Rubalcaba. and P.A. Murohv. The Johns Hopkins Univ., Baltimore, MD 21205. M.V. SANGIJEDOLCE. C. CAPO, P. BONGRAND, JL HEGE Lab. Immunol. Hosp. Sainte Marguerite 270. Bd de Ste Yargue- rite 13277 MAR8EILLE CEDEK 9 The regulatory role of protein kinase C &KC) on tumor necrosis factor alpha (TRF) production by hums" monocytes in response to zynmsan, a particulate agonist wee investigated. The pretreatment of awnccytes with phorbol myristate acetate @MA) induced a dose-dependent inhibition of zymosan- stimulated TNF production. This inhibition was prevented by en inhibitor of PKC, sphingosine. Moreover, FUA elicited a profound dovn-modulation of zymosan binding to monocytes. The inhibition of zynmsen binding and TNF production displayed similar dose-dependence, suggesting that both events were closely related. In addition, pMA did no modify the expres- sion of CDllb/CD18 receptor which is involved in zymosan recognition. In view of these data, qualitative changes of CDllb/GDlB molecles might account for the inhibition of zymosan binding and TNF production. We also found that cytc- chalasin B partially prevented the inhibition of zymosen binding and that PMA increased the eesociation of GDllb/CD18 with the detergent-insoluble cytoskeleton. Hence, the inhibitory action of F?4A on TNF secretion seems to be mediated by a" increase in attachment of zymosan receptor to cytoskeleton. The regulatory activity of PKC might represent a first way of limiting cytokine CIV~P production. To investigate the mechanism of signal transduction elicited by Interleukin-1 (IL-l) in helper T cells, the role of protein kinase C activation upon Interleukin-2 nC3NA induction (IL-Z) was evaluated. For this purpose, murine EL4 thymoma cells were used as targets. These cells respond to low doses of photbol esters [TPA 16nM] plus IL-1 (0.1 nM) and display a ten-fold increase of mRNA for IL-2 at approximately 2 hr post-stimulation. When PKC antagonist H7 (10 uM) is added, the mRNA for IL-2 returns to its basal levels. To determine whether PKC activation occurred as a direct consequence of IL-l interaction with the cells, we then performed experiments using "P in viva labelling of both DlO.G4.1 murine Th2 clone (DlO) and EL4 cells. The auto- radiographic analysis of "on equilibrium two dimensional gel electrophoresis, showed that IL-1 alone failed to change the "P incorDoration oattern of total cell extracts. However. both TPA and 503 (ant<-TCR mAb to DlO cells) displayed changes in phosphorylation. To ascertain if IL-1 could influence the phosphorylation of intracellular substrates of PKC, a polyclonal antibody that recognizes all forms of the lipocortin proteins was used for immunoprecipitation. Although both TPA and 3D3 induced phosphorylation oi such proteins, IL-1 not only did not have any effect by itself but failed to enhance their relative phosphorylation. Also of interest is our observation that, when DlO cells are pretreated with IL-l, a refractory state develops within l-Zhrs which precludes the response to TCR (by 303); yet, IL-1 addition after 3D3 could be delayed up to 12hr without significant change in the proliferative response measured 48hr later. The evidence presented here suggest that the potentiating effects of IL-1 upon helper T cell activation are distal to the activation of TCR signal and of its induction of PKC activity. 218 221 TNFRECEPTORMEDIATEDACTIVATIONOFAPHOSPHATIDYL-CHOLINE CALPNOSTIN C, A PRC INHIBITOR, COUNTERACTS THE LPS- SPECIFIC PHOSPHOLIPASE C. Stefa" Schfitze'. Dinko Berkovi?. Clemens Unser2. and Martin Krenke', 'Inst.f.Med.Mikrobioloeie u.Hveie"e. TU Mii"che". FRG. and *Abt.HBmatoloeie u.Onkologie, Zenhum I&e"? h&Ii&, UniversihitG6ttinge", FRG. Protein kiuass C (PKC) represents one major pathway by which TNF receptors communicate signals from the membrane to tbe cell nucleus. The present study addressesthemechanisms ofTNFinducedPKCactivation.Followi"gTNFstimulation of U937 cells, a rapid and transient increase of 1'2'diacylglycerol (DAG) was observed which was associated with an increase of phosphorylcholine and a co"comitantreductic"ofphospbstidylcholine(FC). These results suggestoperationof aPC-specificphospholipaseC(PC-PLC). Sinceno changesi"phosphatidicacid(PA) and choline were observed and tbe production of DAG by TNF could "&b-s blocked by either propranclol, a PA-phosphohydrolassinhibitor, or ethanol, which substitutes for Hz0 in the trans-phospbstidylationreactio" catalyzed by phospholipaseD (PLD), a combined activation of PLD and PA-phosphohydrolase in DAG production appears unlikely. Phospholipass A, (PLA3 is also activated by TNF, however with a delayed time course when compared to PKC-activation, suggesting that PLA, is not involved in TNF mediated PKC stimulation. Inhibition of TNF-stimulated PC-PLC activation by p-bromphesacylbromid (BPB)also prevented TNF-mediated PKC activation. Since BPB did"otinhibitPKC directly stimulatedby phorbolester (PMA), the data strongly suggest that TNF activates PKC via DAG produced by PC-PLC. TNF stimulation of DAG production could be inhibitedby preincubatio" of cells with a monoclonal anti- TNF receptor @60)antibody,indicatingthatactivation of PC-PLC is aTNF receptor- mediated event. 219 CHEMOTAKIS IS MEDIATED BY &-RECEPTORS BUT NOT a-RECEPTORS FOR PLATELET-DERIVED GROWTH FACTOR. A. Siepbahn'. A. ErikssonJ. B. WestermarkZ. C-H. Heldin and L. Claesson-Welsha. Department of Clinical Chemistry1 and Pathologya, University Hospital, S- 751 85 Uppsala, and Ludwig Institute for Cancer Researcha, Biomedical Center, Box 595, S-751 24 Uppsala. Platelet-derived growth factor (PDGF) is a dimeric molecule composed of disulphide bonded polypeptid chains and exists as three isoforms, PDGF-AA, PDGF-AB and PDGF-BB. The PDGF u-receptor binds all three isoforms whereas the PDGF R- receptor binds PDGF-BB with high affinity, PDGF-AB with lower affinity and does not bind PDGF-AA. In a previous study we have shown that the R-receptors mediate a chemotactic response. Furthermore, the motility response required a functional receptor protein-tyrosine kinase activity. In this study we have expressed human u- and I-receptors in aortic endothelial cells which lack endoeenous recerrtors. PDGF-BB induced increased [JH]thymidi"eY incorpora&" in four different clones expressing a-receptors and four clones with K-receptors, whereas PDGF-AA induced proliferation only in a- receptor bearing cells. PDGF-BB induced a strong, dose- dependent chemotactic response in the four different Is- receptor expressing cell-clo"es. In contrast to these results, the ol-receptor bearing PAE cell-clones did not migrate towards a concentration gradient of either PDGF-BB or PDGF-AA. Our results suggest that different signal transduc- tion pathways are mediated by PDGF rx- and R-receptors. INDUCED TCROR NECROBIS FACTOR NODLILRTION BY CRRRO- TACTIC PEPTIDE IN ELICITED PERITONRRL NACROPEAQES. P Tremblav. G Nercier, 8 Gauthier, G Sauvd, R Turcotte, and D 0th. Centres de recherche en immunologic et en microbiologic appligude, Institut Rrmand-Frappier, Laval-des-Rapides, Qudbec, Canada, H7N 423. Formyl-methionyl-leuoyl-phenylalanine (PRLP), a potent chemotaotic peptide, modulates LPS-induced tumor necrosis factor secretion (TNP-8) (L929 cells test) and gene expression (TRP-0) (Northern blot) in thioglycollate-elicited murine peritoneal maoro- phages. when added 30 mist before LPS, FRLP eu- hances, in a dose-dependent manner, TNP-8 (up to 3- fold) and TRP-G (1.2-fold). when added either & multaneously or 30 min after LPS, it inhibits TRP-S by 30% and TNP-G by 10%. Calphostin C (0.5 F&I), a potent PKC inhibitor, prevents the inhibitory effect of FRLP, as opposed to H-7, another PKC inhibitor, which results in the inhibition of TRP in all si- tuations. The discrepancy in the effects of these two inhibitors could be explained by their different target sites on the PKC molecule. These results give new insights into the mechanisms implicated in the modulatory action of FRLP on TNP production. Supported by NSERC, Canada. 222 TGF-B-INDUCED G2lM DELAY IN PROLIFERATING RABBIT ARTICULAR CHONDROCYTES IS ASSOCIATED WITH AN! E NHANCEMENT OF REPLICATION RATE AND A CAMP ECREASE : POSSIBLE INVOLVEMENT OF PERTUSSIS TOXIN- ENSITIVE PATHWAY. b.Vivien. P.GalCra, G.Loyau and Jean-Pierre Pujol. Lab. de Biochimie pu Tissu Conjonctif. CHU C6te de Nacm. 14033 Caen Cedex, France Using proliferating chondrocytes in fetal calf serum-containing pedium, we have previously shown that TGF-8 induced a recruitment of cells at the end of the S phase (GZ/M) observed 24 h after addition. The delayed cells may then be released. producing a proliferative effect in 10% FCS-or an inhibition of growth in 2% FCS-containing medium. In chondrocytes synchronized in S phase by a thymidine block. we investigated the rate of [3H]-thymidine incorporation, the cell cycle traverse, the expression of PCNA (Proliferating Cell Nuclear Antigen) and CAMP levels. The data demonstrate that TGF-6 caused a decrease of CAMP content (0.5 - 1 h) followed by an enhancement of the DNA synthesis rate (4 h). In contrast, addition of CAMP analognes to the cultures resulted in an inhibition of the replication rate. We also showed that penussis toxin produced a decrease of DNA synthesis rate. in a transient manner and only in the presence of TGF-0. All these results suggest that TGF-8 may accelerate the replication process of cyclized chondrocytes. making then accumulate at the G2/h4 boundary, via a: mechanism that could involve the adenylate cyclase activity and a i protein. This finding contributes to the multifunctional of TGF-6 and may have general physiological in tissue repair and cancer.

TGF-β-induced G2/M delay in proliferating rabbit articular chondrocytes is associated with an enhancement of replication rate and A cAMP decrease: possible involvement of pertussis

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486 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES

217 220

PROTEIN KINASE C IS INVOLVED IN THE DOWN-MODULATION OF ZYMOSAN-STIMULATED TUMOR NEGROSIS FACTOR alpha PRODUCTION BY HUNAN NoNocYTEs : EFFECT ON CR3XYTO8KELETAL INTERACTION

RELATIONSHIP BETWEEN PROTEIN KINASE C AND INTERLEUKIN-1 IN HELPER T CELLS. M.R. Sierra-Honiama"". E. Rubalcaba. and P.A. Murohv. The Johns Hopkins Univ., Baltimore, MD 21205.

M.V. SANGIJEDOLCE. C. CAPO, P. BONGRAND, JL HEGE Lab. Immunol. Hosp. Sainte Marguerite 270. Bd de Ste Yargue- rite 13277 MAR8EILLE CEDEK 9

The regulatory role of protein kinase C &KC) on tumor necrosis factor alpha (TRF) production by hums" monocytes in response to zynmsan, a particulate agonist wee investigated. The pretreatment of awnccytes with phorbol myristate acetate @MA) induced a dose-dependent inhibition of zymosan- stimulated TNF production. This inhibition was prevented by en inhibitor of PKC, sphingosine. Moreover, FUA elicited a profound dovn-modulation of zymosan binding to monocytes. The inhibition of zynmsen binding and TNF production displayed similar dose-dependence, suggesting that both events were closely related. In addition, pMA did no modify the expres- sion of CDllb/CD18 receptor which is involved in zymosan recognition. In view of these data, qualitative changes of CDllb/GDlB molecles might account for the inhibition of zymosan binding and TNF production. We also found that cytc- chalasin B partially prevented the inhibition of zymosen binding and that PMA increased the eesociation of GDllb/CD18 with the detergent-insoluble cytoskeleton. Hence, the inhibitory action of F?4A on TNF secretion seems to be mediated by a" increase in attachment of zymosan receptor to cytoskeleton. The regulatory activity of PKC might represent a first way of limiting cytokine CIV~P production.

To investigate the mechanism of signal transduction elicited by Interleukin-1 (IL-l) in helper T cells, the role of protein kinase C activation upon Interleukin-2 nC3NA induction (IL-Z) was evaluated. For this purpose, murine EL4 thymoma cells were used as targets. These cells respond to low doses of photbol esters [TPA 16nM] plus IL-1 (0.1 nM) and display a ten-fold increase of mRNA for IL-2 at approximately 2 hr post-stimulation. When PKC antagonist H7 (10 uM) is added, the mRNA for IL-2 returns to its basal levels. To determine whether PKC activation occurred as a direct consequence of IL-l interaction with the cells, we then performed experiments using "P in viva labelling of both DlO.G4.1 murine Th2 clone (DlO) and EL4 cells. The auto- radiographic analysis of "on equilibrium two dimensional gel electrophoresis, showed that IL-1 alone failed to change the "P incorDoration oattern of total cell extracts. However. both TPA and 503 (ant<-TCR mAb to DlO cells) displayed changes in phosphorylation. To ascertain if IL-1 could influence the phosphorylation of intracellular substrates of PKC, a polyclonal antibody that recognizes all forms of the lipocortin proteins was used for immunoprecipitation. Although both TPA and 3D3 induced phosphorylation oi such proteins, IL-1 not only did not have any effect by itself but failed to enhance their relative phosphorylation. Also of interest is our observation that, when DlO cells are pretreated with IL-l, a refractory state develops within l-Zhrs which precludes the response to TCR (by 303); yet, IL-1 addition after 3D3 could be delayed up to 12hr without significant change in the proliferative response measured 48hr later. The evidence presented here suggest that the potentiating effects of IL-1 upon helper T cell activation are distal to the activation of TCR signal and of its induction of PKC activity.

218 221

TNFRECEPTORMEDIATEDACTIVATIONOFAPHOSPHATIDYL-CHOLINE CALPNOSTIN C, A PRC INHIBITOR, COUNTERACTS THE LPS- SPECIFIC PHOSPHOLIPASE C. Stefa" Schfitze'. Dinko Berkovi?. Clemens Unser2. and Martin Krenke', 'Inst.f.Med.Mikrobioloeie u.Hveie"e. TU Mii"che". FRG. and *Abt.HBmatoloeie u.Onkologie, Zenhum I&e"? h&Ii&, UniversihitG6ttinge", FRG. Protein kiuass C (PKC) represents one major pathway by which TNF receptors communicate signals from the membrane to tbe cell nucleus. The present study addressesthemechanisms ofTNFinducedPKCactivation.Followi"gTNFstimulation of U937 cells, a rapid and transient increase of 1'2'diacylglycerol (DAG) was observed which was associated with an increase of phosphorylcholine and a co"comitantreductic"ofphospbstidylcholine(FC). These results suggestoperationof aPC-specificphospholipaseC(PC-PLC). Sinceno changesi"phosphatidicacid(PA) and choline were observed and tbe production of DAG by TNF could "&b-s blocked by either propranclol, a PA-phosphohydrolassinhibitor, or ethanol, which substitutes for Hz0 in the trans-phospbstidylationreactio" catalyzed by phospholipaseD (PLD), a combined activation of PLD and PA-phosphohydrolase in DAG production appears unlikely. Phospholipass A, (PLA3 is also activated by TNF, however with a delayed time course when compared to PKC-activation, suggesting that PLA, is not involved in TNF mediated PKC stimulation. Inhibition of TNF-stimulated PC-PLC activation by p-bromphesacylbromid (BPB)also prevented TNF-mediated PKC activation. Since BPB did"otinhibitPKC directly stimulatedby phorbolester (PMA), the data strongly suggest that TNF activates PKC via DAG produced by PC-PLC. TNF stimulation of DAG production could be inhibitedby preincubatio" of cells with a monoclonal anti- TNF receptor @60)antibody,indicatingthatactivation of PC-PLC is aTNF receptor- mediated event.

219

CHEMOTAKIS IS MEDIATED BY &-RECEPTORS BUT NOT a-RECEPTORS FOR PLATELET-DERIVED GROWTH FACTOR. A. Siepbahn'. A. ErikssonJ. B. WestermarkZ. C-H. Heldin and L. Claesson-Welsha. Department of Clinical Chemistry1 and Pathologya, University Hospital, S- 751 85 Uppsala, and Ludwig Institute for Cancer Researcha, Biomedical Center, Box 595, S-751 24 Uppsala.

Platelet-derived growth factor (PDGF) is a dimeric molecule composed of disulphide bonded polypeptid chains and exists as three isoforms, PDGF-AA, PDGF-AB and PDGF-BB. The PDGF u-receptor binds all three isoforms whereas the PDGF R- receptor binds PDGF-BB with high affinity, PDGF-AB with lower affinity and does not bind PDGF-AA. In a previous study we have shown that the R-receptors mediate a chemotactic response. Furthermore, the motility response required a functional receptor protein-tyrosine kinase activity. In this study we have expressed human u- and I-receptors in aortic endothelial cells which lack endoeenous recerrtors. PDGF-BB induced increased [JH]thymidi"eY incorpora&" in four different clones expressing a-receptors and four clones with K-receptors, whereas PDGF-AA induced proliferation only in a- receptor bearing cells. PDGF-BB induced a strong, dose- dependent chemotactic response in the four different Is- receptor expressing cell-clo"es. In contrast to these results, the ol-receptor bearing PAE cell-clones did not migrate towards a concentration gradient of either PDGF-BB or PDGF-AA. Our results suggest that different signal transduc- tion pathways are mediated by PDGF rx- and R-receptors.

INDUCED TCROR NECROBIS FACTOR NODLILRTION BY CRRRO- TACTIC PEPTIDE IN ELICITED PERITONRRL NACROPEAQES. P Tremblav. G Nercier, 8 Gauthier, G Sauvd, R Turcotte, and D 0th. Centres de recherche en immunologic et en microbiologic appligude, Institut Rrmand-Frappier, Laval-des-Rapides, Qudbec, Canada, H7N 423.

Formyl-methionyl-leuoyl-phenylalanine (PRLP), a potent chemotaotic peptide, modulates LPS-induced tumor necrosis factor secretion (TNP-8) (L929 cells test) and gene expression (TRP-0) (Northern blot) in thioglycollate-elicited murine peritoneal maoro- phages. when added 30 mist before LPS, FRLP eu- hances, in a dose-dependent manner, TNP-8 (up to 3- fold) and TRP-G (1.2-fold). when added either & multaneously or 30 min after LPS, it inhibits TRP-S by 30% and TNP-G by 10%. Calphostin C (0.5 F&I), a potent PKC inhibitor, prevents the inhibitory effect of FRLP, as opposed to H-7, another PKC inhibitor, which results in the inhibition of TRP in all si- tuations. The discrepancy in the effects of these two inhibitors could be explained by their different target sites on the PKC molecule. These results give new insights into the mechanisms implicated in the modulatory action of FRLP on TNP production. Supported by NSERC, Canada.

222

TGF-B-INDUCED G2lM DELAY IN PROLIFERATING RABBIT ARTICULAR CHONDROCYTES IS ASSOCIATED WITH AN!

E

NHANCEMENT OF REPLICATION RATE AND A CAMP ECREASE : POSSIBLE INVOLVEMENT OF PERTUSSIS TOXIN- ENSITIVE PATHWAY.

b.Vivien. P.GalCra, G.Loyau and Jean-Pierre Pujol. Lab. de Biochimie pu Tissu Conjonctif. CHU C6te de Nacm. 14033 Caen Cedex, France

Using proliferating chondrocytes in fetal calf serum-containing pedium, we have previously shown that TGF-8 induced a recruitment of cells at the end of the S phase (GZ/M) observed 24 h after addition. The delayed cells may then be released. producing a proliferative effect in 10% FCS-or an inhibition of growth in 2% FCS-containing medium. In chondrocytes synchronized in S phase by a thymidine block. we investigated the rate of [3H]-thymidine incorporation, the cell cycle traverse, the expression of PCNA (Proliferating Cell Nuclear Antigen) and CAMP levels. The data demonstrate that TGF-6 caused a decrease of CAMP content (0.5 - 1 h) followed by an enhancement of the DNA synthesis rate (4 h). In contrast, addition of CAMP analognes to the cultures resulted in an inhibition of the replication rate. We also showed that penussis toxin produced a decrease of DNA synthesis rate. in a transient manner and only in the presence of TGF-0. All these results suggest that TGF-8 may accelerate the replication process of cyclized chondrocytes. making then accumulate at the G2/h4 boundary, via a: mechanism that could involve the adenylate cyclase activity and a

i protein. This finding contributes to the multifunctional of TGF-6 and may have general physiological in tissue repair and cancer.