TGF-β1 down-regulates the expression of its receptors on rabbit articular chondrocytes (RAC)
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THIRD INTERNATIONAL WORKSHOP ON CYTOKINES / 477 163 EFFECT OF INTERLEUKIN-I AND NAPROXEN ON IL-1 RECEPTOR EXPRESSION IN PROLIFERATING HUMAN RHEUMATOID SYNOVIAL CELLS IN CULTURE. S. Prottost, F. Raini. D. Vivien. P. Gal&a, G. Loyau. and J.P. Pujol. Laboratoire de Biochimie du Tissu Conjonctif, C.H.U. Niveau 3, 14033 cAENcEDExFR4NcE To get insights into the mechanism whereby non-steroidal anti-inflammatory drugs (AINS) could modulate the action of interlcukin-1 (IL-l) on human rheumatoid synovial cells (HRSC), we determine whether Naproxen was capable of modulating the expression of IL-l receptors on these cells. When Naproxen (lo-4 M) was added to proliferating cultures of HRSC for 96h. a decrease of lz51-IL-I binding was observed (20 to 50% compared to control values). Scatchard analysis revealed that this effect results from a down-regulation of the IL-I receptors at the cell surface without apparent change in binding affinity. IL-l itself was shown to reduce the total binding of 1251.IL-1 on HRSC by 35%. Scatchard plots indicated that the effect was due to a decrease of the number of receptors at the surface with no change in binding affinity. These results suggest that IL-1 could exert a feed-back control on the expression of its receptors. In addition, the data demonstrate that non-steroidal anti-inflammatory drugs such as Naproxen may reduce the effects of IL-1 by down-regulating the expression of the monokine receptors on rheumatoid synovial cells. If it were the case in viva, Naproxen could be of some benefit in reducing the IL- l-induced deleterious effects associated with osteo-articular diseases. 164 REGULATED EXPRESSION OF TNF RECEPTORS ON HUMAN T CELLS: EFFECTS OF PHORBOL ESTER AND CYCLIC-AMP ANALOGUES. Gerald E. Ranges* Keith Kelly* and Terje Espevik. *Miles Research Institute, West Haven, CT. and Institute for Cancer Research, University of Trondheim, Norway. We have examined the expression of TNF receptors on resting and activated human T cells using monoclonal antibodies directed at p75 and p55 receptors. Resting T cells predominantly express p75 receptors and little or no detectable p55 receptor. Overnight incubation in the presence of PMA will markedly increase the expression of P75 TNF receptors while P55 remains undetectable. Treatments which increase the apparent Cyclic-AMP concentration (addition of 1OOuM cyclic-CPTAMP, a C-AMP analogue or treatment with the phosphodiesterase inhibitor IBMX) also result in enhanced receptor expression. Addition of calcium ionophore to PMA cultures prevents receptor upregulation while it enhances the C-AMP Effect. Calcium ionophore alone shows no apparent effect on receptor expression Cyclohexsmide inhibits receptor up regulation by both PMA and C-AMP. Neither Cyclosporin A or TGF-beta inhibit the activity by either inducer. These results suggest multiple pathways involved in TNF receptor regulation. 165 TRANSFORMING GROWTH FACTOR-8 (TGF-8) PREVENTS THE INTERLEUKIN-1 (IL-l)-INDUCED EFFECTS ON MATRIX METABOLISM IN CULTURED RABBIT ARTICULAR CHONDROCYTES (RAC): POSSIBLE DOWN-REGULATION OF IL- 1 RECEPTORS F. RCdini. A. Mauviel. S. Pronost. G. Loyau and J.-P. Pujol. Laboratoire de Biochimie du Tissu Conjonctif. CHU C&e de Nacre, Niv. 3, 14033 Caen Ctdex. France Incubation of RAC cultures with IL-10 for the last 3 days of experiment produced a huge decrease in collagen and proteoglycans (PC&) synthesis paralleled with an increase of collagenolytic activity. When RAC were treated with TGF-E (I-25 rig/ml) for 3 days and then with IL-l (500 pg/ml) for another 3- day period, the production of collagen and PGs overcame that of IL- l-treated cultures as well as control cells. In addition, the collagenolytic activity of chondrocytes was dose-dependently diminished when oretreated with TGF-8. resoective to cultures . incubated with IL-l alone. When IL-l was present during 6 days and TGF-6 added together with IL-1 for the last 3 days, TGF-8 at 5 and 25 nglml totally reversed the inhibiting effects of the cytokine on collagen and PGs synthesis and its stimulating effect on collagenase activity. In order to get insights into this phenomenon, we used affinity labeling techniques to characterize cell surface receptors for IL-IS on cultured cells. RAC exhibit a single class of high-affinity IL-IS binding sites (Kd =72 pM; n=3350 sites/cell). A pretreatment of the chondrocytes with TGF-8 for 3 days clearly shows a diminution of IL-l binding to its receptors. 166 TGF-El DOWN-REGULATES THE EXPRESSION OF ITS RECEPTORS ON RABBIT ARTICULAR CHONDROCYTES (RAC). F. Redini, D. Vivien. S. Pronost, G. Loyau and J.-P. Pujol. Laboratoire de Biochimie du Tissu Conjonctif. CHU C&e de Nacre. Niv. 3. 14033 Caen C&dex, France. We have previously demonstrated that expression of receptors for TGF-E on cultured RAC depends on the cell cycle distribution. We now investigate the effect of TGF-!3 on its receptor distribution in relation to the cell density. We have used affinity labeling techniques to characterize cell surface receptors for TGF-I3 in sparse and confluent cultures. Confluent RAC cultured for 7 days in the presence of 10% FCS (73% in GO/l phase) express two classes of high affinity binding sites (Kdl = 240 pM. nl = 3100 sites/cell; Kd2 = 1500 pM. n2 = 9300 sites/cell). Sparse RAC cultured for 3 days in the presence of 10% FCS (44% in GO/l phase) exhibit also two classes of binding sites (K’dl = 280 pM, n’l = 3100 sites/cell; K’d2 = 1057 pM, n’2 = 8210 sites/cell). In both experimental conditions, cells pretreated with TGF-6 (1 @ml) express one single class of high affinity binding sites to TGF-6 (confluent cells: Kd = 570 pM, n = 5470 sites/cell: sparse cells: K’d = 810 pM, n’ = 6660 sites/cell). All these results suggest that the distribution of TGF-E receptors is not dependent on the cell density conditions. Moreover, in both experimental conditions. we clearly demonstrate a down-regulation of TGF-6 receptors expression by itself. 167 STRUCTURE-FUNCTION STUDIES ON THE HUMAN HEPATIC INTERLEUKIN-BRECEPTOR S. RoseJohn, H. Schooltink, T. Stovan, D. Lenz, D. Zohlnhlifer, L. Graeve. P.C. Heinrich lnstfiut fiir Biochemie der RVvlH Aachen. PauweisstraEe 30,510O Aachen, FRG In previous studies with rat and human hepatocyte primary cultures as well as in the rat in vlvo we have identified IL-6 as the major inducer of acute phase protein synthesis. IL6 acts via a specific hepatic cell surfacs receptor. Using a cDNA cloning approach we could show that hepatocytes express an Mentical IL-&receptor as leukocytes. Affinity-crosslinking of ‘251-rhlLB to human hepatoma cells (HepGP) allowed the detection of three ILdcontaining complexes with mdecular masses of 100, 120 and 200 kDa. For the mRNA of the 80 kDa IL&eceptor subunit a time- and dose-dependent up-regulation was observed after treatment of HepGP cells with dexamethaaons. Under these conditions of IL+receptor up-regulation by dexamethasone 7-fibrinogen-mRNA induction by IL6 is stronger and occurs earlier than without dexamethasone. At present, there Is not much known about the signal transduction of IL-S As a first approach, we have stably expressed the cDNA coding for the human lL-6- receptor in HepG2 cells. We have shown that ‘251-rhlL-6 is internalized by these cells. A genetically engineered sduble human IL&receptor was found to act as an agonist on HepG2 cells In acute phase protein induction. Furthermore, the ability of the sduble IL&receptor to bind its ligand is exploited to study receptor binding of ILB analogs (agonists/antagonists),
Transcript of TGF-β1 down-regulates the expression of its receptors on rabbit articular chondrocytes (RAC)
THIRD INTERNATIONAL WORKSHOP ON CYTOKINES / 477
163
EFFECT OF INTERLEUKIN-I AND NAPROXEN ON IL-1 RECEPTOR EXPRESSION IN PROLIFERATING HUMAN RHEUMATOID SYNOVIAL CELLS IN CULTURE. S. Prottost, F. Raini. D. Vivien. P. Gal&a, G. Loyau. and J.P. Pujol. Laboratoire de Biochimie du Tissu Conjonctif, C.H.U. Niveau 3, 14033 cAENcEDExFR4NcE
To get insights into the mechanism whereby non-steroidal anti-inflammatory drugs (AINS) could modulate the action of interlcukin-1 (IL-l) on human rheumatoid synovial cells (HRSC), we determine whether Naproxen was capable of modulating the expression of IL-l receptors on these cells. When Naproxen (lo-4 M) was added to proliferating cultures of HRSC for 96h. a decrease of lz51-IL-I binding was observed (20 to 50% compared to control values). Scatchard analysis revealed that this effect results from a down-regulation of the IL-I receptors at the cell surface without apparent change in binding affinity. IL-l itself was shown to reduce the total binding of 1251.IL-1 on HRSC by 35%. Scatchard plots indicated that the effect was due to a decrease of the number of receptors at the surface with no change in binding affinity. These results suggest that IL-1 could exert a feed-back control on the expression of its receptors. In addition, the data demonstrate that non-steroidal anti-inflammatory drugs such as Naproxen may reduce the effects of IL-1 by down-regulating the expression of the monokine receptors on rheumatoid synovial cells. If it were the case in viva, Naproxen could be of some benefit in reducing the IL- l-induced deleterious effects associated with osteo-articular diseases.
164
REGULATED EXPRESSION OF TNF RECEPTORS ON HUMAN T CELLS: EFFECTS OF PHORBOL ESTER AND CYCLIC-AMP ANALOGUES. Gerald E. Ranges* Keith Kelly* and Terje Espevik. *Miles Research Institute, West Haven, CT. and Institute for Cancer Research, University of Trondheim, Norway.
We have examined the expression of TNF receptors on resting and activated human T cells using monoclonal antibodies directed at p75 and p55 receptors. Resting T cells predominantly express p75 receptors and little or no detectable p55 receptor. Overnight incubation in the presence of PMA will markedly increase the expression of P75 TNF receptors while P55 remains undetectable. Treatments which increase the apparent Cyclic-AMP concentration (addition of 1OOuM cyclic-CPTAMP, a C-AMP analogue or treatment with the phosphodiesterase inhibitor IBMX) also result in enhanced receptor expression. Addition of calcium ionophore to PMA cultures prevents receptor upregulation while it enhances the C-AMP Effect. Calcium ionophore alone shows no apparent effect on receptor expression Cyclohexsmide inhibits receptor up regulation by both PMA and C-AMP. Neither Cyclosporin A or TGF-beta inhibit the activity by either inducer. These results suggest multiple pathways involved in TNF receptor regulation.
165
TRANSFORMING GROWTH FACTOR-8 (TGF-8) PREVENTS THE INTERLEUKIN-1 (IL-l)-INDUCED EFFECTS ON MATRIX METABOLISM IN CULTURED RABBIT ARTICULAR CHONDROCYTES (RAC): POSSIBLE DOWN-REGULATION OF IL- 1 RECEPTORS F. RCdini. A. Mauviel. S. Pronost. G. Loyau and J.-P. Pujol. Laboratoire de Biochimie du Tissu Conjonctif. CHU C&e de Nacre, Niv. 3, 14033 Caen Ctdex. France
Incubation of RAC cultures with IL-10 for the last 3 days of experiment produced a huge decrease in collagen and proteoglycans (PC&) synthesis paralleled with an increase of collagenolytic activity. When RAC were treated with TGF-E (I-25 rig/ml) for 3 days and then with IL-l (500 pg/ml) for another 3- day period, the production of collagen and PGs overcame that of IL- l-treated cultures as well as control cells. In addition, the collagenolytic activity of chondrocytes was dose-dependently diminished when oretreated with TGF-8. resoective to cultures . incubated with IL-l alone. When IL-l was present during 6 days and TGF-6 added together with IL-1 for the last 3 days, TGF-8 at 5 and 25 nglml totally reversed the inhibiting effects of the cytokine on collagen and PGs synthesis and its stimulating effect on collagenase activity. In order to get insights into this phenomenon, we used affinity labeling techniques to characterize cell surface receptors for IL-IS on cultured cells. RAC exhibit a single class of high-affinity IL-IS binding sites (Kd =72 pM; n=3350 sites/cell). A pretreatment of the chondrocytes with TGF-8 for 3 days clearly shows a diminution of IL-l binding to its receptors.
166
TGF-El DOWN-REGULATES THE EXPRESSION OF ITS RECEPTORS ON RABBIT ARTICULAR CHONDROCYTES (RAC). F. Redini, D. Vivien. S. Pronost, G. Loyau and J.-P. Pujol. Laboratoire de Biochimie du Tissu Conjonctif. CHU C&e de Nacre. Niv. 3. 14033 Caen C&dex, France.
We have previously demonstrated that expression of receptors for TGF-E on cultured RAC depends on the cell cycle distribution. We now investigate the effect of TGF-!3 on its receptor distribution in relation to the cell density. We have used affinity labeling techniques to characterize cell surface receptors for TGF-I3 in sparse and confluent cultures. Confluent RAC cultured for 7 days in the presence of 10% FCS (73% in GO/l phase) express two classes of high affinity binding sites (Kdl = 240 pM. nl = 3100 sites/cell; Kd2 = 1500 pM. n2 = 9300 sites/cell). Sparse RAC cultured for 3 days in the presence of 10% FCS (44% in GO/l phase) exhibit also two classes of binding sites (K’dl = 280 pM, n’l = 3100 sites/cell; K’d2 = 1057 pM, n’2 = 8210 sites/cell). In both experimental conditions, cells pretreated with TGF-6 (1 @ml) express one single class of high affinity binding sites to TGF-6 (confluent cells: Kd = 570 pM, n = 5470 sites/cell: sparse cells: K’d = 810 pM, n’ = 6660 sites/cell).
All these results suggest that the distribution of TGF-E receptors is not dependent on the cell density conditions. Moreover, in both experimental conditions. we clearly demonstrate a down-regulation of TGF-6 receptors expression by itself.
167
STRUCTURE-FUNCTION STUDIES ON THE HUMAN HEPATIC INTERLEUKIN-BRECEPTOR S. RoseJohn, H. Schooltink, T. Stovan, D. Lenz, D. Zohlnhlifer, L. Graeve. P.C. Heinrich
lnstfiut fiir Biochemie der RVvlH Aachen. PauweisstraEe 30,510O Aachen, FRG
In previous studies with rat and human hepatocyte primary cultures as well as in the rat in vlvo we have identified IL-6 as the major inducer of acute phase protein synthesis. IL6 acts via a specific hepatic cell surfacs receptor. Using a cDNA cloning approach we could show that hepatocytes express an Mentical IL-&receptor as leukocytes. Affinity-crosslinking of ‘251-rhlLB to human hepatoma cells (HepGP) allowed the detection of three ILdcontaining complexes with mdecular masses of 100, 120 and 200 kDa. For the mRNA of the 80 kDa IL&eceptor subunit a time- and dose-dependent up-regulation was observed after treatment of HepGP cells with dexamethaaons. Under these conditions of IL+receptor up-regulation by dexamethasone 7-fibrinogen-mRNA induction by IL6 is stronger and occurs earlier than without dexamethasone. At present, there Is not much known about the signal transduction of IL-S As a first approach, we have stably expressed the cDNA coding for the human lL-6- receptor in HepG2 cells. We have shown that ‘251-rhlL-6 is internalized by these cells. A genetically engineered sduble human IL&receptor was found to act as an agonist on HepG2 cells In acute phase protein induction. Furthermore, the ability of the sduble IL&receptor to bind its ligand is exploited to study receptor binding of ILB analogs (agonists/antagonists),
