- Email: [email protected]
Accessory cell functions of fibroblasis
108 / SECOND
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
211
214
EARLY PRODUCTION OF IL-1 BY DRAINING LYMPH NODE CELLS FOLLOWING CONTACT SENSITIZATION. s. J. Ho"kins. M, d I Ki University of Ii!-mQ.>A.& Manchester Rheumatic Diseases Centre, Hope Hospital, Salford, M6 BHD and Central Toxicology Laboratories, ICI, Cheshire,, SK10 4TJ. Since interleukin-1 (IL-l) can potentiate the activation of lymphoid cells, we wished to determin whether IL-1 was produced by the draining lymph node cells of contact sensitized mice during the early stages of activation. Lymph node cells were collected at various times after sensitization of mice with oxasolone and cultured for 24hr. Supernatants from these cultures were assesssed for the presence of IL-1 using a sensitive, IL-2-saturated, bioassay with the DlO(N4)M sub-line of the DlO.G4.1 murine T cell clone. IL-l-like activity was detected in supernatants from cells taken as early as 4hr after sensitization and increased in a dose-related fashion with increasing sensitizing dose of oxasolone. The activity was detected in assays additionally saturated with IL-4 and was antagonized by an anti-murine IL-1 serum. Depletion of dendritic cells from the sensitized lymph node population resulted in a marked reduction or loss in the capacity to produce IL-l. The data suggest that production of IL-1 may be a" important event in the activation of draining lymph node cells following application of contact sensitizing agents.
EFFECT OF V-SIS TRANSFECTION ON PROLIFERATION AND MATRIX SYNTHESIS OF ADULT HUMAN FIBROBLASTS. 0. Ishikawa. E.C, LeRov. and M, Troianowska. Medical University of South Carolina, Charleston, SC 29425. Adult human diploid fibroblasts grow" on plastic surfaces were transfected with v-sis oncogene devoid of regulatory sequences. V-sis transformed fibroblasts grew as welldefined foci which facilitated clonal expansion. Two clones (designated sis-1 and sis-2) were studied. Transfected cells synthesized PDGF, most of which was membrane bound, were completely unresponsive to exogenous PDGF, and showed anchorage independence and growth to higher cell density in the presence of serum, but did not proliferate in serum-free medium. In addition, the PDGF-inducible, growth-related genes c-myc and c-fos were constitutively expressed at high lSVSlS, while the expression of collagen type I and fibronectin m9NA were dramatically decreased. The properties of v-sis-transformed foreskin and adult human fibroblasts were compared. Our data indicate that adult human diploid fibroblasts are suitable cell strains for studying the effect of v-sis on in vitro cell transformation.
212
215
Fibroblasts actively participate in wound healing and chrmic inflasrnations in connfftive tissues. Studies wxa pxfonnsdto assess inlnu"oregulat.ory fuxtimsof fibxhlasta. l"&?easbryo flbrchlaatacr densal fibroblasts-~tizedwith~ehs~toind~inviho 2, G/K-C& and lTN-~)or cytokinea (&I 'IR" and 13&&l as&l) followed by a aacrnd sigmlprovidedbybxtnr%aledotcnin (LPS). IL-1 was shamto~l~~saWyintraoellularlyorassaiatedto~sl1 sxambrane, howevermtdetectedinths qematants.nRNAtralscriptsd TL-lasndDwered~intlnactivan?dflbrcblast. Itismtewcutl~, that for the expression of full biological activity both signals (lymphokine/cytokine and LPS) are needed, howaver each of them is sufficient for mRNA expression. ILrl-like biological activity of fibroblast originvas mu&$&ad withanti-IL&xmt%xdies. Pnhuxad prostaglandin generation by -~"a~ mrcblasts vas 4w.a to ~v~yregulate~prod~ti~~IL1aswellas~cytddnes.Q1 the cmbary, baqdetic ,znlmy sdmikhng factds (Ghl end MGF) were show"tohaszra&dccnstitutiv~ybysrh fibrcblastpoIxd.&.iuts,~ their mRNA was cmstitutivelyaqressed.Plbrcblasts~eatedtithgwsra interferon also -asafficimtmtigsnqesanti"gcallsto~ic helper T cells. Thus, fibrc&sts nmifest vmuroregulatciy activities, and their role in controlling cellular issnm reqnses in cmtive t.issuesi~~ncnimlar~plthologicalatates&.xldbe&demd.
TNF AND IL-1 GENE ACTIVATION IN HUNAN NDNDCYTES TREATED WITH KRESTIN (PSK). Anahid Jewett and Benjamin Bonavida. Department of Microbioloav and Immunoloav,-UCLA School of Medicine. Los Angeles, CA. %D24 The biological response modifier PSK has been used in cancer therapy. PSK potentiates several immunological functions and its effects on peripheral human blood monocytes (PBM) were assessed by direct cell mediated cytotoxicity (CMC) and secretion of both TNF and IL-l. Coculture of PBM with PSK overnight resulted in significant augmentation of CMC and secretion of cytotoxic factors and TNF into the supernatants. The effects were dose dependent and PSK was significantly more potent activator than IFN-T. Further, the levels of IL-1 produced by PSK were several logs higher than that produced by IFN-T. Significant synergy was obtained when suboptimal concentrations of PSK and IFN-7 were used. Unlike IFNT monocytes treated with PSK resulted in minor changes in the expression of class II MHC on the cell surface. These results demonstrate that PSK is a potent activator of PBM and the mechanism of activation is different than that mediated by IFN-7. Current studies are comparing mRNA levels of TNF and IL-l in monocytes treated with IFN-T and PSK. (Supported in part by a gift from Kureha Chemical Company, Japan.)
213
216
APOPROTEIN E mRNA EXPRESSION IN HUMAN MACROPHAGES: ITS REGULATION BY LYMPHOKINE-CONTAINING MEDIUM. E.Hurt and G.Bondiers. Wallenberg Laboratory. Univ. of Gothenburg. Sahlgren's Hospital. S 413-45 Gothenburg, Sweden.
INTERFERON TREATMENT ENHANCED THE EXPRESSION OF LAMININ AND FIBR0NECTIN.V. Kedar, II. Coon, D. Bhartiya, and R. Maheshwari, Unif. Serv. Univ. of the Health Sciences, Bethesda, MD 20814. USA Lamini" (LMN) and fibronectin (FN) have been show" to be involved in various aspects of wound healing. Plasma concentration of FN has bee" show" to be reduced in most At wound types of surgical trauma and in many infections. site FN is synthesized by different cells (macrophage, endothelial, fibroblast and certain epithelial) involved in wound healing. Migration of epithelial cells has been shown to be increased in the presence of exogenous LMii and this may lead to rapid healing of bum, traumatic or infectious injuries. Mouse LB, 3T3 and macrophage cells were treated with mouse IFN o/B: human 1""s enithelial (A5491 and foreskin fibrob&; (FS4) celis were treated with human IFNa and y for varying periods. Using specific antibodies, FN and IMN were analyze2 by immunofluorescence and by metabolic labelling with 3 S-methionine, immunoprecipitatio" and analysis by SDS-PAGE. Immunofluorescense showed a more intense staining of LMN and FN in cells treated with IFN. Metabolic labelling showed a" increase in LMN and FN svnthesis and release from cells treated with IFN. To d&mi"e whether the increased synthesis is reflected at mBNA levels, poly (A)+ RNA was isolated and probed with specific cDNAs. IFN treatment increased the mBNA level of INN and FN. These results suggested that IFN regulated the biosynthesis of LMN and FN at the mBNA level.
Apoprotein E is an immunoregulatory monokine which has potent suppressive activity for lymphocyte proliferation. An apoE cDNA probe was used for apoE mRNA detection on northern blots of total cell RNA isolated by C&l gradient. Allogeneic activated human macrophages express less or even undetectable amounts of a"oE mRNA. cornoared with non-activated single donor cultures of HMDM. Medium from early culture of allogeneic activated macrophages, as well as PHA-lymphocyte conditioned medium, have a suppressive effect and are concentration dependent and fully reversible (upon both medium withdrawal and boiling) on apoE mRNA expression. The cell lysosomal enzyme activities and beta-actin mRNA levels did not change. ApoE mRNA expression was not suppressed in HepG2 cells. Secretion ex"eriments usine 35S-L-methio"i"e. followed bv showed intracellular accumulation, 6ut immunoprecipitation, not secretion, of apoE in the presence of lymphokinecontaining medium. Human recombinant IL-Z showed some suppressor effect on apoE mRNA. IL-l beta, Gamma-interferon and TGF-beta did not down-regulate apoE mRNA expression. Other cytokines may be involved in the regulation of apoE mRNA, or a combination of different cytokines. Experiments are currently being done in order to elucidate these possibilities.
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
211
214
EARLY PRODUCTION OF IL-1 BY DRAINING LYMPH NODE CELLS FOLLOWING CONTACT SENSITIZATION. s. J. Ho"kins. M, d I Ki University of Ii!-mQ.>A.& Manchester Rheumatic Diseases Centre, Hope Hospital, Salford, M6 BHD and Central Toxicology Laboratories, ICI, Cheshire,, SK10 4TJ. Since interleukin-1 (IL-l) can potentiate the activation of lymphoid cells, we wished to determin whether IL-1 was produced by the draining lymph node cells of contact sensitized mice during the early stages of activation. Lymph node cells were collected at various times after sensitization of mice with oxasolone and cultured for 24hr. Supernatants from these cultures were assesssed for the presence of IL-1 using a sensitive, IL-2-saturated, bioassay with the DlO(N4)M sub-line of the DlO.G4.1 murine T cell clone. IL-l-like activity was detected in supernatants from cells taken as early as 4hr after sensitization and increased in a dose-related fashion with increasing sensitizing dose of oxasolone. The activity was detected in assays additionally saturated with IL-4 and was antagonized by an anti-murine IL-1 serum. Depletion of dendritic cells from the sensitized lymph node population resulted in a marked reduction or loss in the capacity to produce IL-l. The data suggest that production of IL-1 may be a" important event in the activation of draining lymph node cells following application of contact sensitizing agents.
EFFECT OF V-SIS TRANSFECTION ON PROLIFERATION AND MATRIX SYNTHESIS OF ADULT HUMAN FIBROBLASTS. 0. Ishikawa. E.C, LeRov. and M, Troianowska. Medical University of South Carolina, Charleston, SC 29425. Adult human diploid fibroblasts grow" on plastic surfaces were transfected with v-sis oncogene devoid of regulatory sequences. V-sis transformed fibroblasts grew as welldefined foci which facilitated clonal expansion. Two clones (designated sis-1 and sis-2) were studied. Transfected cells synthesized PDGF, most of which was membrane bound, were completely unresponsive to exogenous PDGF, and showed anchorage independence and growth to higher cell density in the presence of serum, but did not proliferate in serum-free medium. In addition, the PDGF-inducible, growth-related genes c-myc and c-fos were constitutively expressed at high lSVSlS, while the expression of collagen type I and fibronectin m9NA were dramatically decreased. The properties of v-sis-transformed foreskin and adult human fibroblasts were compared. Our data indicate that adult human diploid fibroblasts are suitable cell strains for studying the effect of v-sis on in vitro cell transformation.
212
215
Fibroblasts actively participate in wound healing and chrmic inflasrnations in connfftive tissues. Studies wxa pxfonnsdto assess inlnu"oregulat.ory fuxtimsof fibxhlasta. l"&?easbryo flbrchlaatacr densal fibroblasts-~tizedwith~ehs~toind~inviho 2, G/K-C& and lTN-~)or cytokinea (&I 'IR" and 13&&l as&l) followed by a aacrnd sigmlprovidedbybxtnr%aledotcnin (LPS). IL-1 was shamto~l~~saWyintraoellularlyorassaiatedto~sl1 sxambrane, howevermtdetectedinths qematants.nRNAtralscriptsd TL-lasndDwered~intlnactivan?dflbrcblast. Itismtewcutl~, that for the expression of full biological activity both signals (lymphokine/cytokine and LPS) are needed, howaver each of them is sufficient for mRNA expression. ILrl-like biological activity of fibroblast originvas mu&$&ad withanti-IL&xmt%xdies. Pnhuxad prostaglandin generation by -~"a~ mrcblasts vas 4w.a to ~v~yregulate~prod~ti~~IL1aswellas~cytddnes.Q1 the cmbary, baqdetic ,znlmy sdmikhng factds (Ghl end MGF) were show"tohaszra&dccnstitutiv~ybysrh fibrcblastpoIxd.&.iuts,~ their mRNA was cmstitutivelyaqressed.Plbrcblasts~eatedtithgwsra interferon also -asafficimtmtigsnqesanti"gcallsto~ic helper T cells. Thus, fibrc&sts nmifest vmuroregulatciy activities, and their role in controlling cellular issnm reqnses in cmtive t.issuesi~~ncnimlar~plthologicalatates&.xldbe&demd.
TNF AND IL-1 GENE ACTIVATION IN HUNAN NDNDCYTES TREATED WITH KRESTIN (PSK). Anahid Jewett and Benjamin Bonavida. Department of Microbioloav and Immunoloav,-UCLA School of Medicine. Los Angeles, CA. %D24 The biological response modifier PSK has been used in cancer therapy. PSK potentiates several immunological functions and its effects on peripheral human blood monocytes (PBM) were assessed by direct cell mediated cytotoxicity (CMC) and secretion of both TNF and IL-l. Coculture of PBM with PSK overnight resulted in significant augmentation of CMC and secretion of cytotoxic factors and TNF into the supernatants. The effects were dose dependent and PSK was significantly more potent activator than IFN-T. Further, the levels of IL-1 produced by PSK were several logs higher than that produced by IFN-T. Significant synergy was obtained when suboptimal concentrations of PSK and IFN-7 were used. Unlike IFNT monocytes treated with PSK resulted in minor changes in the expression of class II MHC on the cell surface. These results demonstrate that PSK is a potent activator of PBM and the mechanism of activation is different than that mediated by IFN-7. Current studies are comparing mRNA levels of TNF and IL-l in monocytes treated with IFN-T and PSK. (Supported in part by a gift from Kureha Chemical Company, Japan.)
213
216
APOPROTEIN E mRNA EXPRESSION IN HUMAN MACROPHAGES: ITS REGULATION BY LYMPHOKINE-CONTAINING MEDIUM. E.Hurt and G.Bondiers. Wallenberg Laboratory. Univ. of Gothenburg. Sahlgren's Hospital. S 413-45 Gothenburg, Sweden.
INTERFERON TREATMENT ENHANCED THE EXPRESSION OF LAMININ AND FIBR0NECTIN.V. Kedar, II. Coon, D. Bhartiya, and R. Maheshwari, Unif. Serv. Univ. of the Health Sciences, Bethesda, MD 20814. USA Lamini" (LMN) and fibronectin (FN) have been show" to be involved in various aspects of wound healing. Plasma concentration of FN has bee" show" to be reduced in most At wound types of surgical trauma and in many infections. site FN is synthesized by different cells (macrophage, endothelial, fibroblast and certain epithelial) involved in wound healing. Migration of epithelial cells has been shown to be increased in the presence of exogenous LMii and this may lead to rapid healing of bum, traumatic or infectious injuries. Mouse LB, 3T3 and macrophage cells were treated with mouse IFN o/B: human 1""s enithelial (A5491 and foreskin fibrob&; (FS4) celis were treated with human IFNa and y for varying periods. Using specific antibodies, FN and IMN were analyze2 by immunofluorescence and by metabolic labelling with 3 S-methionine, immunoprecipitatio" and analysis by SDS-PAGE. Immunofluorescense showed a more intense staining of LMN and FN in cells treated with IFN. Metabolic labelling showed a" increase in LMN and FN svnthesis and release from cells treated with IFN. To d&mi"e whether the increased synthesis is reflected at mBNA levels, poly (A)+ RNA was isolated and probed with specific cDNAs. IFN treatment increased the mBNA level of INN and FN. These results suggested that IFN regulated the biosynthesis of LMN and FN at the mBNA level.
Apoprotein E is an immunoregulatory monokine which has potent suppressive activity for lymphocyte proliferation. An apoE cDNA probe was used for apoE mRNA detection on northern blots of total cell RNA isolated by C&l gradient. Allogeneic activated human macrophages express less or even undetectable amounts of a"oE mRNA. cornoared with non-activated single donor cultures of HMDM. Medium from early culture of allogeneic activated macrophages, as well as PHA-lymphocyte conditioned medium, have a suppressive effect and are concentration dependent and fully reversible (upon both medium withdrawal and boiling) on apoE mRNA expression. The cell lysosomal enzyme activities and beta-actin mRNA levels did not change. ApoE mRNA expression was not suppressed in HepG2 cells. Secretion ex"eriments usine 35S-L-methio"i"e. followed bv showed intracellular accumulation, 6ut immunoprecipitation, not secretion, of apoE in the presence of lymphokinecontaining medium. Human recombinant IL-Z showed some suppressor effect on apoE mRNA. IL-l beta, Gamma-interferon and TGF-beta did not down-regulate apoE mRNA expression. Other cytokines may be involved in the regulation of apoE mRNA, or a combination of different cytokines. Experiments are currently being done in order to elucidate these possibilities.