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Post-transcriptional regulation of cytokine and cytokine antagonist expression
452 / THIRD INTERNATIONAL
WORKSHOP
ON CYTOKINES
13
16
CLONING OF ILl-INDUCIBLE GENES IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS: PRELIMINARY CHARACTERISATLON. F.Breviario. &Aniello. J. Goand M. lnirona lstiluto Ricerche Farmacologiche “Mario Negri”, via Eritrea 62. 20157 Milano, Italy In an attempt to understand more directly the molecular mechanisms involved in the cellular response lo Interleukin-1 (ILl), we have made several cDNA libraries from mRNA isolated from human umbilical vein endothelial cells (HUVEC), after 2 lo 6 passages, and stimulated for 1 hour with 20 rig/ml human recombinant ILl-8, in the presence of 10 pg/ml cycloheximide. The cDNA libraries were differentially screened with radiolabelled cDNA derived from mRNA isolated from either untreated or ILl- treated HUVEC. 8 different cDNA clones induced by IL1 were identified and partially sequenced. 5 out of 8 corresponded 10 genes which had previously been identified and characterised as ILl-inducible genes in human endothelial cells (PA&l, N-CAF, GRO-d GRO-8 and ELAM-1). The other 3 cDNA clones on the contrary represent novel genes. In all cases, Northern analysis confirmed that these novel genes are induced upon exposure of HUVEC cells to Ll-8 for 1 hour in the presence of cycloheximide. The 3 new genes are identified as clones 100, 133 and 170 and their mRNA size has been estimated 10 be of 3.5, 1.6 and 2.0 kb respectively. The characteristics 01 the cDNA clones will be discussed.
POST-TRANSCRIPTIONAL REGULATION OF CYTOKINE AND CYTOKINE ANTAGONIST EXPRESSION. M.J. Fentonl, J.A. Burns*, and R.P. Donnelly2, ‘Boston Univ. Sch. Med., Boston MA 02118 USA, and 2U.S. Food and Drug Administration, Bethesda MD 20892 USA. The T cell-derived cytokine IL-4 inhibits the production of IL-16 by LPS-stimulated normal human monocytes. We recently showed that IL-4 exerts this inhibitory effect by selectively reducing both the rate of IL-l 0 gene transcription and the stability of IL-10 mRNA in these cells (Donnelly et al., J. Immunol. 146: 3431, 1991). Our data further suggest that this inhibition is dependent on the synthesis of an IL-4-induced autoregulatory protein. We have extended these findings to now show that IL-4 inhibits IL-6 production through a similar post-transcriptional mechanism. In contrast, while the stability of IL-lb and IL-6 mRNA is selectively decreased by IL-4, the turnover of IL-lra message is not affected. In view of reports that IL-4 itself can upregulate IL-lra expression in monocytic cells, these data suggest that IL-4 has potent antiinflammatory properties based in part on its ability to differentially regulate cytokine and cytokine antagonist expression at the post-transcriptional level.
14
17
INDUCTION OF CYTOKINE SECRETION BY A HUMAN INSULINOMA CELL M G Cavallo, A Gearina. A Toto. T LINE BY "IPAL INFECTION. Forsev, P Pozzilli and R ThorDe. NIBSC, Blanche Lane, South Hiruns, Herts., EN3 640, U.K. and Cattedea di Endocrinologia, II Clinica Medica, Universita di ROma, La Sapienza, Roma, Italy. Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease characterieed by the T-cell mediated destruction of pancreatic Beta-cells. Viral infection has been implicated in the Dathooenesis of IDDM a* a trioaerins event, responsibie f&the activation of a cascade w&h "itimately leads to the cytotoxic process against Beta-2 cells. In a human insulinoma cell line, used as an in this studv. vitro mod;i for the study of Beta-cells, was infected with Culture supernatants were mumps and measles viruses. collected every 24 hours for 3 days and cytokine (IL-l, IL2, IL-6, IL-4, TNF, GM-CSF and yIFN) levels were estimated. Infection with both measles and mumps viruses induced the secretion of IL-1 and IL-6 by the cell line as detected by bioassays. The IL-l and IL-6 activity in the supernatants were completely neutralised by specific antisera against IL1 and IL-6. NO other cytokines were detected. These data show for the first time that IL-l and IL-6 secretion may occur after viral infection of human ineulinoma cells and suggest that cytokine secretion by Beta-cells may be relevant in the induction of the immune response towards B cells which occurs in IDDM.
MEMBRANES OF MYCOPLASMA AND SPIROPLASMA AS POTENT INDUCERS FOR TNFa AND IL-I SECRETION BY MACROPHAGES IN VITRO: R. Gallilv. N. Bronstein . T. Sher and S. Rottem The Lautenbera Ctr. for General and Tumor Immunoloov. The Hebrew U.- Haiassah Med. Sch., Jerusalem, Israel. --. We found that membranes of Mvcoplasma capricolum and Spiroplasma sp strain MQ-1, although not containing LPS, activate pronouncedly murine macrophages (M#) to secret TNFa and IL-l. Following M. caoricolu activation, human monocytes also secrete TNFo. We further observed that Mcaoricolum and MQ-1 membranes indcced high mitogenic response in splenic lymphocytes derived from LPSresponsive and non-responsive strains of mice. The mechanism of MO activation by Mycoplasma/Spiroplasma seems to differ from that of LPS based on the following: 1. LPS-Unresponsive C3H/HeJ MQ secrete TNFcr following activation by Mycoplasmal Spiroplasma. 2. Mycoplasma or Spiroplasma membranes synergize with LPS to augment TNFu oroduction. Biochemical analvsis of the active component is) of M. caoricolun and Ma-i membranes showed that it is in part, protein.
15
18
GENETIC VARIATION OF INTERLEUKIN-1 BETA. Section of F.S. da Glowne. F Tam. F Sim. Molecular Medicine, Dept. Med., Univ. Sheffield, Royal Hallamshire Hospflal, Sheffield, SlO 2JF. U.K.
INDUCED FACTOR THAT CHARACTERIZATION OF AN IFN-a BINDS TO THE INTERFERON RESPONSE SEQUENCE OF THE 202 GENE IN MURINE LEUKEMIA CELLS. ~an1co ( G. Cavallo , 2. Dembic t-l. Garjglio , S. arad $. . Landolfn . Microbiology, Llnlversity of Torina, 1n5t1 tute of Base1 , Italy, and Hoffmann-La Roche, Inc., Switzerland. We have investigated events following treatment of L1210 murine leukemia cells with IFN-8x that lead to the immediate transcriptional activation of the 202. The SF flanking region of IFN--inducible gene, the gene contains a 46-base-pare IFN-stimulatable called GA box, homoloQous response element (ISRE), several IFN-inducible genee. The to the ISKEs of ISRE was shown to be necessary and sufficient for IFN-8~. In band 202 transcriptional induction by with nuclear shi,ft aE.says the GA box yielded extracts of L1210 cell.5 treated with IFN-ox a DNAprotein cample:x. This complex was ma:ximally induced 30 ’ after level5 IFN-treatment, remained at high for the ne::t 90’ and gradually decreased. Treatment of cells with cycloheximlde decreased of abnut 50% WE are now the levels of the Inducible complex. DNA blndiny factor which attxnpting to clone the the 202 GA box by screening an binds tcj gene expression l.ambda gtll cDNA library made from Liz10 ceils treated with IFN-8~.
There is some evidence of polymorphism of the lnterleukinl p gene,a cyiokine whose over-production has been implicated in several autoimnwne diseases, including Rheumatoid Arthritis and other chronic inflammatory diseases. We have studied the 5' region of this gene (1231-2536), which contains several consensus potential binding sites for regulatory proteins and seems to rule transcriptional activation and down-regulation of IL-1 p gene. We used PCR to isolate this region of DNAfrom RA patients (n=5) and normal controls (n=4). We have directly sequenced these products from two of the normal individuals and three RA patients. In different individuals, we found variability within potential transcriptional factor-binding sites (one of the two TATA boxes in this region, CTF/NFl, OCT l/2, possibly AP-2). We could not identify a pattern of linkage in any individual genome between these mutations. These consistent sites of variability are the first clear evidence for genetic variation in the IL-1 p gene. Further studies are in progress to assess if these structural differences are reflected in changes in gene regulation, and if they changethe susceptibility to chronic inflammatory diseases.
WORKSHOP
ON CYTOKINES
13
16
CLONING OF ILl-INDUCIBLE GENES IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS: PRELIMINARY CHARACTERISATLON. F.Breviario. &Aniello. J. Goand M. lnirona lstiluto Ricerche Farmacologiche “Mario Negri”, via Eritrea 62. 20157 Milano, Italy In an attempt to understand more directly the molecular mechanisms involved in the cellular response lo Interleukin-1 (ILl), we have made several cDNA libraries from mRNA isolated from human umbilical vein endothelial cells (HUVEC), after 2 lo 6 passages, and stimulated for 1 hour with 20 rig/ml human recombinant ILl-8, in the presence of 10 pg/ml cycloheximide. The cDNA libraries were differentially screened with radiolabelled cDNA derived from mRNA isolated from either untreated or ILl- treated HUVEC. 8 different cDNA clones induced by IL1 were identified and partially sequenced. 5 out of 8 corresponded 10 genes which had previously been identified and characterised as ILl-inducible genes in human endothelial cells (PA&l, N-CAF, GRO-d GRO-8 and ELAM-1). The other 3 cDNA clones on the contrary represent novel genes. In all cases, Northern analysis confirmed that these novel genes are induced upon exposure of HUVEC cells to Ll-8 for 1 hour in the presence of cycloheximide. The 3 new genes are identified as clones 100, 133 and 170 and their mRNA size has been estimated 10 be of 3.5, 1.6 and 2.0 kb respectively. The characteristics 01 the cDNA clones will be discussed.
POST-TRANSCRIPTIONAL REGULATION OF CYTOKINE AND CYTOKINE ANTAGONIST EXPRESSION. M.J. Fentonl, J.A. Burns*, and R.P. Donnelly2, ‘Boston Univ. Sch. Med., Boston MA 02118 USA, and 2U.S. Food and Drug Administration, Bethesda MD 20892 USA. The T cell-derived cytokine IL-4 inhibits the production of IL-16 by LPS-stimulated normal human monocytes. We recently showed that IL-4 exerts this inhibitory effect by selectively reducing both the rate of IL-l 0 gene transcription and the stability of IL-10 mRNA in these cells (Donnelly et al., J. Immunol. 146: 3431, 1991). Our data further suggest that this inhibition is dependent on the synthesis of an IL-4-induced autoregulatory protein. We have extended these findings to now show that IL-4 inhibits IL-6 production through a similar post-transcriptional mechanism. In contrast, while the stability of IL-lb and IL-6 mRNA is selectively decreased by IL-4, the turnover of IL-lra message is not affected. In view of reports that IL-4 itself can upregulate IL-lra expression in monocytic cells, these data suggest that IL-4 has potent antiinflammatory properties based in part on its ability to differentially regulate cytokine and cytokine antagonist expression at the post-transcriptional level.
14
17
INDUCTION OF CYTOKINE SECRETION BY A HUMAN INSULINOMA CELL M G Cavallo, A Gearina. A Toto. T LINE BY "IPAL INFECTION. Forsev, P Pozzilli and R ThorDe. NIBSC, Blanche Lane, South Hiruns, Herts., EN3 640, U.K. and Cattedea di Endocrinologia, II Clinica Medica, Universita di ROma, La Sapienza, Roma, Italy. Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease characterieed by the T-cell mediated destruction of pancreatic Beta-cells. Viral infection has been implicated in the Dathooenesis of IDDM a* a trioaerins event, responsibie f&the activation of a cascade w&h "itimately leads to the cytotoxic process against Beta-2 cells. In a human insulinoma cell line, used as an in this studv. vitro mod;i for the study of Beta-cells, was infected with Culture supernatants were mumps and measles viruses. collected every 24 hours for 3 days and cytokine (IL-l, IL2, IL-6, IL-4, TNF, GM-CSF and yIFN) levels were estimated. Infection with both measles and mumps viruses induced the secretion of IL-1 and IL-6 by the cell line as detected by bioassays. The IL-l and IL-6 activity in the supernatants were completely neutralised by specific antisera against IL1 and IL-6. NO other cytokines were detected. These data show for the first time that IL-l and IL-6 secretion may occur after viral infection of human ineulinoma cells and suggest that cytokine secretion by Beta-cells may be relevant in the induction of the immune response towards B cells which occurs in IDDM.
MEMBRANES OF MYCOPLASMA AND SPIROPLASMA AS POTENT INDUCERS FOR TNFa AND IL-I SECRETION BY MACROPHAGES IN VITRO: R. Gallilv. N. Bronstein . T. Sher and S. Rottem The Lautenbera Ctr. for General and Tumor Immunoloov. The Hebrew U.- Haiassah Med. Sch., Jerusalem, Israel. --. We found that membranes of Mvcoplasma capricolum and Spiroplasma sp strain MQ-1, although not containing LPS, activate pronouncedly murine macrophages (M#) to secret TNFa and IL-l. Following M. caoricolu activation, human monocytes also secrete TNFo. We further observed that Mcaoricolum and MQ-1 membranes indcced high mitogenic response in splenic lymphocytes derived from LPSresponsive and non-responsive strains of mice. The mechanism of MO activation by Mycoplasma/Spiroplasma seems to differ from that of LPS based on the following: 1. LPS-Unresponsive C3H/HeJ MQ secrete TNFcr following activation by Mycoplasmal Spiroplasma. 2. Mycoplasma or Spiroplasma membranes synergize with LPS to augment TNFu oroduction. Biochemical analvsis of the active component is) of M. caoricolun and Ma-i membranes showed that it is in part, protein.
15
18
GENETIC VARIATION OF INTERLEUKIN-1 BETA. Section of F.S. da Glowne. F Tam. F Sim. Molecular Medicine, Dept. Med., Univ. Sheffield, Royal Hallamshire Hospflal, Sheffield, SlO 2JF. U.K.
INDUCED FACTOR THAT CHARACTERIZATION OF AN IFN-a BINDS TO THE INTERFERON RESPONSE SEQUENCE OF THE 202 GENE IN MURINE LEUKEMIA CELLS. ~an1co ( G. Cavallo , 2. Dembic t-l. Garjglio , S. arad $. . Landolfn . Microbiology, Llnlversity of Torina, 1n5t1 tute of Base1 , Italy, and Hoffmann-La Roche, Inc., Switzerland. We have investigated events following treatment of L1210 murine leukemia cells with IFN-8x that lead to the immediate transcriptional activation of the 202. The SF flanking region of IFN--inducible gene, the gene contains a 46-base-pare IFN-stimulatable called GA box, homoloQous response element (ISRE), several IFN-inducible genee. The to the ISKEs of ISRE was shown to be necessary and sufficient for IFN-8~. In band 202 transcriptional induction by with nuclear shi,ft aE.says the GA box yielded extracts of L1210 cell.5 treated with IFN-ox a DNAprotein cample:x. This complex was ma:ximally induced 30 ’ after level5 IFN-treatment, remained at high for the ne::t 90’ and gradually decreased. Treatment of cells with cycloheximlde decreased of abnut 50% WE are now the levels of the Inducible complex. DNA blndiny factor which attxnpting to clone the the 202 GA box by screening an binds tcj gene expression l.ambda gtll cDNA library made from Liz10 ceils treated with IFN-8~.
There is some evidence of polymorphism of the lnterleukinl p gene,a cyiokine whose over-production has been implicated in several autoimnwne diseases, including Rheumatoid Arthritis and other chronic inflammatory diseases. We have studied the 5' region of this gene (1231-2536), which contains several consensus potential binding sites for regulatory proteins and seems to rule transcriptional activation and down-regulation of IL-1 p gene. We used PCR to isolate this region of DNAfrom RA patients (n=5) and normal controls (n=4). We have directly sequenced these products from two of the normal individuals and three RA patients. In different individuals, we found variability within potential transcriptional factor-binding sites (one of the two TATA boxes in this region, CTF/NFl, OCT l/2, possibly AP-2). We could not identify a pattern of linkage in any individual genome between these mutations. These consistent sites of variability are the first clear evidence for genetic variation in the IL-1 p gene. Further studies are in progress to assess if these structural differences are reflected in changes in gene regulation, and if they changethe susceptibility to chronic inflammatory diseases.