Transforming growth factor β alters the cytoskeleton of rat lung fibroblasts, in vitro
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120 / SECOND INTERNATIONAL WORKSHOP ON CYTOKINES 283 ABNORMAL EFFECT OF TGF-8 ON FIBRONECTIN AND COLLAGEN GENE EXPRESSION IN SCLERODERMA FIBROBIASTS. M. Troianowska. 0, Ishikawa. E. Smith. E.C. LeRov. Med Univ of South Carolina, Charleston, SC 29425. We compared the effect of TGF-fl on extracellular matrix gene expression and proliferation in scleroderma and control fibroblasts. Confluent cells were starved for 24 hours in serum free medium prior to TGF-fl (0.25 "g/ml) addition. Analysis of the steady state levels of collagens type I, type III and fibronectin mRNAs in control cells showed increases in message levels 1 hour after TGF-,9 addition with maximum stimulation after 8-12 hours. Scleroderma fibroblasts exhibited higher uninduced levels of collagen and fibronectin mRNAs, reduced induction of these messages by TGF-j3 and different kinetics of induction as compared to normal cells. While TGF-fl is not mitogenic for human adult fibroblasts, simultaneous addition of TGF-p and PDGF significantly increased DNA replication in these cells in comparison to PDGF alone. Both scleroderma and normal fibroblasts responded similarly to this combination of growth factors. The abnormal effect of TGF-fl on extracellular matrix gene expression in scleroderma fibroblasts suggests a possible role for this cytokine in the pathogenesis of scleroderma. 284 SELECTIVE IIWIBITION OF CYTOKINB RELEASE AND FUNCTION BY TGFB. M. Turner, D. Chantry and M. Feldman". Charing Cross Sunley Research Centre, London, W6 8LW. Transforming growth factor B (TGFB) has potent inhibitory effects on a number of immune cell effector functions. The inhibitory effect of TGFE may be due to the inhibition of cytokine release. We have shown TGFt? is able to induce mRNA for IL-la, IL-lt?, TNFa and IL-6 in peripheral blood cells. Analysis of culture supernatants and cell lysates shows that only IL-6 mRNA is translated. When stimulants such as LPS and TNF are used IL-l production is inhibited by TGFB but IL-6 is slightly enhanced. The inhibitory effects of TGFB are dependent on the nature of the inducing stimulus and the activation state of the cells. Thus ohorbol ester induction of IL-l and TNF is resistant to TGFB'inhibition and cells preactivated with LPS are not inhibited in their ability to induce IL-l. TGFB selectively inhibits IL-6 biological activities but enhances others. IgG, thymocyte proliferation and fibrinogen induction were inhibited while IgM production was enhanced. 285 ROLE OF CELLULAR INTERACTIONS AND CYTOKINE PRODUCTION IN EBV-ASSOCIATED NASO-PHARYNGEAL CARCINOMA (NPC). 1. Tursz, G. Rousselet, Q. Zhang, L. Ferradini, H. Wakasugi and P. Busson. Laboratoire d’hnmunobiolo ie des Tumeurs, UA 1156 CNRS, Institut Gustave Roussy, Vll qulf, France. .f.. 286 TRANSFOliMINGWulIHFAEmRBPLTERSlHECYmSKELmr( OFPdTATFIRBIIDBLASTS, IN VITFlX W. Ussler III, V. Kaltg and A.R. Etrozy, Nat'l. Inst. of Environ. Hlth. Sci.. Research Triamle park. NC 277Kl Early p&sage rat llng fitroblasts (RLFs) cultured in IEEM + 10% fetal tovine sentn m glass cov~lips ard ezqo& to T@& and TGFBt exhibit dose dependent changes in cytoskeletaloqanizatim. MJswzre plated 3 hrs tefcreaddingtigrcwth factcr,andwffe slrbsecprentlyincubated fcr 4Bhrs befcre staining the cells with rhodamne-@alloidin fa- e&nation of actin filaments with fluorescence micruscopy. Cmcentratims ranged ftwn 0.001 rig/ml to 1.0 rg/ml, tiich is an interval over tiich siplificant grcwth inhi- bitim has been observed in a unpanicn study. Cells plated at 1~ density (1.3~10~ cells/ti) or at hi@ density (2.6~10’ cells/mn') sho& little dif- fgenceintheirrespansetotreatmentwithTGFB,a~6,. -tit0 untreated cmtruls,both factcrs induced cell spr&ingtiichwasmifested byanincreaseincrcss-sectionalareaardatrendtocsrdsamrecircular cell artline. At 1~ cell density, there was a neasureable increase in the nu&er of focal contacts per cel1,wit.h a correspading reorganizatim of the actin cytoskeletm. Fcr cells treated with 0.5 rig/ml TGFLJ,, the mean nu&r of focal contacts (i.e., temdnatim sites of cancmtrated actin filarr?nts) per cell v.as 5.m.3 caqzared to a ccntrul value of4.4K1.2 (pal.025). The effect of 0.5 rig/ml TGFB, was similar, with a mBan nurber of focal cc&acts per cell of 5.4 mrpared to 4.0 fcr the cmtruls (p<o.C05). Accmpanying this increase in then* offocalcmtacts,wasan apparent increase in the axunt of filanentcus actin and a dramtic polarization of son? cells. This was most pmarnced at 1.0 rig/ml at-d distinguished by actin filament- deficient contact regims. It is not clear at this time &tker these changes in cyt.oskeletalorganizaticn areadirect effect ofTGF6orare secmdary effects of gr&h inhibition. 287 CYTOKINES CONTROLLING PITUITARY FUNCTION. H. Vankelecom*, P. Carmeliett, A. Billiau* and C. Deneft. Laboratory of Immuno- biology (The Rega Institute)* and Laboratory of Cell Pharma- cology+, University of Leuven School of Medicine, B-3000 Leuven, Belgium. The immune and endocrine systems are interconnected at the level of the anterior pituitary. Evidence is already avail- able that cytokines, e.g. IL-l, affect secretion of specific hormones by the pituitary. In reverse, anterior pituitary hormones affect immune and inflammatory responses, e.g. by stimulation of glucocorticoid release. Release of anterior pituitary hormones (ACTH, prolactin, growth hormone) is pri- marily controlled by hypothalamic peptides (e.g. corticotro- pin releasing factor). Previous work from our laboratory has indicated that paracrine factors secreted by the anterior pi- tuitary itself constitute an additional level of regulation and that a specific cell type, the folliculostellate cell, produces cytokines s.a. IL-6. In order to define the rela- tionship between these paracrine factors and cytokines, we have studied the production and modulatory action of IL-l, IFN-cr/B, IFN-Y and IL-6 in freshly isolated rat pituitary cell populations that were cultured as dense monolayers and induced to release specific pituitary hormones. The results of these studies suggest that cytokines do have a role in the local paracrine regulation of the anterior pituitary. 288 TUMOR NECROSIS FACTOR-ALPHA CAN PARTIALLY REVERSE CYCLOSPORIN A MEDIATED IMMUNOSUPPRESSION. s. Wallick. I. Fieari. R. Morris+ and M. Palladino. Genentech, Inc., So. San Francisco, CA, 94080 and +Stanford University, Palo Alto, CA, 94304. immunosuppressive effects of CsA on mouse s lenoc tes. We reDort that in a dose-deuendent manner r K UT X-u can siinificantly reverse CsA &p ression of BALB/c spleen cell proliferation to~$tohemag&inin-P. In support of earlier observations, r u1 -4 and r uIL-2, both mitogenic for mouse the immuno-
Transcript of Transforming growth factor β alters the cytoskeleton of rat lung fibroblasts, in vitro
120 / SECOND INTERNATIONAL WORKSHOP ON CYTOKINES
283
ABNORMAL EFFECT OF TGF-8 ON FIBRONECTIN AND COLLAGEN GENE EXPRESSION IN SCLERODERMA FIBROBIASTS. M. Troianowska. 0, Ishikawa. E. Smith. E.C. LeRov. Med Univ of South Carolina, Charleston, SC 29425.
We compared the effect of TGF-fl on extracellular matrix gene expression and proliferation in scleroderma and control fibroblasts. Confluent cells were starved for 24 hours in serum free medium prior to TGF-fl (0.25 "g/ml) addition. Analysis of the steady state levels of collagens type I, type III and fibronectin mRNAs in control cells showed increases in message levels 1 hour after TGF-,9 addition with maximum stimulation after 8-12 hours. Scleroderma fibroblasts exhibited higher uninduced levels of collagen and fibronectin mRNAs, reduced induction of these messages by TGF-j3 and different kinetics of induction as compared to normal cells. While TGF-fl is not mitogenic for human adult fibroblasts, simultaneous addition of TGF-p and PDGF significantly increased DNA replication in these cells in comparison to PDGF alone. Both scleroderma and normal fibroblasts responded similarly to this combination of growth factors. The abnormal effect of TGF-fl on extracellular matrix gene expression in scleroderma fibroblasts suggests a possible role for this cytokine in the pathogenesis of scleroderma.
284
SELECTIVE IIWIBITION OF CYTOKINB RELEASE AND FUNCTION BY TGFB. M. Turner, D. Chantry and M. Feldman". Charing Cross Sunley Research Centre, London, W6 8LW.
Transforming growth factor B (TGFB) has potent inhibitory effects on a number of immune cell effector functions. The inhibitory effect of TGFE may be due to the inhibition of cytokine release. We have shown TGFt? is able to induce mRNA for IL-la, IL-lt?, TNFa and IL-6 in peripheral blood cells. Analysis of culture supernatants and cell lysates shows that only IL-6 mRNA is translated. When stimulants such as LPS and TNF are used IL-l production is inhibited by TGFB but IL-6 is slightly enhanced.
The inhibitory effects of TGFB are dependent on the nature of the inducing stimulus and the activation state of the cells. Thus ohorbol ester induction of IL-l and TNF is resistant to TGFB'inhibition and cells preactivated with LPS are not inhibited in their ability to induce IL-l. TGFB selectively inhibits IL-6 biological activities but enhances others. IgG, thymocyte proliferation and fibrinogen induction were inhibited while IgM production was enhanced.
285
ROLE OF CELLULAR INTERACTIONS AND CYTOKINE PRODUCTION IN EBV-ASSOCIATED NASO-PHARYNGEAL CARCINOMA (NPC). 1. Tursz, G. Rousselet, Q. Zhang, L. Ferradini, H. Wakasugi and P. Busson. Laboratoire d’hnmunobiolo ie des Tumeurs, UA 1156 CNRS, Institut Gustave Roussy, Vll qulf, France. .f..
286
TRANSFOliMINGWulIHFAEmRBPLTERSlHECYmSKELmr( OFPdTATFIRBIIDBLASTS, IN VITFlX W. Ussler III, V. Kaltg and A.R. Etrozy, Nat'l. Inst. of Environ. Hlth. Sci.. Research Triamle park. NC 277Kl
Early p&sage rat llng fitroblasts (RLFs) cultured in IEEM + 10% fetal tovine sentn m glass cov~lips ard ezqo& to T@& and TGFBt exhibit dose dependent changes in cytoskeletaloqanizatim. MJswzre plated 3 hrs tefcreaddingtigrcwth factcr,andwffe slrbsecprentlyincubated fcr 4Bhrs befcre staining the cells with rhodamne-@alloidin fa- e&nation of actin filaments with fluorescence micruscopy. Cmcentratims ranged ftwn 0.001 rig/ml to 1.0 rg/ml, tiich is an interval over tiich siplificant grcwth inhi- bitim has been observed in a unpanicn study. Cells plated at 1~ density (1.3~10~ cells/ti) or at hi@ density (2.6~10’ cells/mn') sho& little dif- fgenceintheirrespansetotreatmentwithTGFB,a~6,. -tit0 untreated cmtruls,both factcrs induced cell spr&ingtiichwasmifested byanincreaseincrcss-sectionalareaardatrendtocsrdsamrecircular cell artline. At 1~ cell density, there was a neasureable increase in the nu&er of focal contacts per cel1,wit.h a correspading reorganizatim of the actin cytoskeletm. Fcr cells treated with 0.5 rig/ml TGFLJ,, the mean nu&r of focal contacts (i.e., temdnatim sites of cancmtrated actin filarr?nts) per cell v.as 5.m.3 caqzared to a ccntrul value of4.4K1.2 (pal.025). The effect of 0.5 rig/ml TGFB, was similar, with a mBan nurber of focal cc&acts per cell of 5.4 mrpared to 4.0 fcr the cmtruls (p<o.C05). Accmpanying this increase in then* offocalcmtacts,wasan apparent increase in the axunt of filanentcus actin and a dramtic polarization of son? cells. This was most pmarnced at 1.0 rig/ml at-d distinguished by actin filament- deficient contact regims. It is not clear at this time &tker these changes in cyt.oskeletalorganizaticn areadirect effect ofTGF6orare secmdary effects of gr&h inhibition.
287
CYTOKINES CONTROLLING PITUITARY FUNCTION. H. Vankelecom*, P. Carmeliett, A. Billiau* and C. Deneft. Laboratory of Immuno- biology (The Rega Institute)* and Laboratory of Cell Pharma- cology+, University of Leuven School of Medicine, B-3000 Leuven, Belgium.
The immune and endocrine systems are interconnected at the level of the anterior pituitary. Evidence is already avail- able that cytokines, e.g. IL-l, affect secretion of specific hormones by the pituitary. In reverse, anterior pituitary hormones affect immune and inflammatory responses, e.g. by stimulation of glucocorticoid release. Release of anterior pituitary hormones (ACTH, prolactin, growth hormone) is pri- marily controlled by hypothalamic peptides (e.g. corticotro- pin releasing factor). Previous work from our laboratory has indicated that paracrine factors secreted by the anterior pi- tuitary itself constitute an additional level of regulation and that a specific cell type, the folliculostellate cell, produces cytokines s.a. IL-6. In order to define the rela- tionship between these paracrine factors and cytokines, we have studied the production and modulatory action of IL-l, IFN-cr/B, IFN-Y and IL-6 in freshly isolated rat pituitary cell populations that were cultured as dense monolayers and induced to release specific pituitary hormones. The results of these studies suggest that cytokines do have a role in the local paracrine regulation of the anterior pituitary.
288
TUMOR NECROSIS FACTOR-ALPHA CAN PARTIALLY REVERSE CYCLOSPORIN A MEDIATED IMMUNOSUPPRESSION. s. Wallick. I. Fieari. R. Morris+ and M. Palladino. Genentech, Inc., So. San Francisco, CA, 94080 and +Stanford University, Palo Alto, CA, 94304.
immunosuppressive effects of CsA on mouse s lenoc tes. We reDort that in a dose-deuendent manner r K UT X-u can siinificantly reverse CsA &p ression of BALB/c spleen cell proliferation to~$tohemag&inin-P. In support of earlier observations, r u1 -4 and r uIL-2, both mitogenic for mouse
the immuno-
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