THIRD INTERNATIONAL WORKSHOP ON CYTOKINES / 451

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DUAL CONTROL OF HUMAN INTERLEUKIN-2 AND INTERFERON-y GENE EXPRESSION BY HISTAMINE: ACTIVATION AND SUPPRESSION. Gila Arad. Mali Ketzinel, Rachel Nussinovich and Ravmond KaemDfer, Department of Molecular Virology, The Hebrew University-Hadassah Medical School, 91010 Jerusalem, Israel

Concomitant with induction of IL-2 and IFN--f gene expression in cultnred human PBMC or tonsil cells, mitogenic stimulation induces a tlansient activation of cells able to effectively suppress expression of these genes. Induction of IL-2 and 1FN-y genes largely precedes appearance of suppressor cell activity, allowing expression of both genes to occur before it is hnned off by the action of suppressor cells. Indeed, mitogen-induced expression of IL-2 and IFN-y RNA is superinduced extensively by treatments that affect primarily the activation of suppressor T cells: y-irradiation, cyclophosphamide, or the HZ histamine receptor antagonist, cimetidine. Conversely, histamine, a known activator of suppressor cells, elicits a strong inhibition of induced IL-2 and IFN? gene expression. However, early in the induction process, histamine exerts an unexpected and powerful stimulation of the expression of IL-2 and IFNr RNA. An immediate, 15 to 25fold stimulation by histamine is converted into an equally strong inhibitory response in the course of mitogenic induction. Activation and inhibition of IL-2 and 1FN-y gene expression by histamine are both exerted rapidly via the H2 receptor. These results support the concept that expression of IL-2 and IFNr genes is regulated by two distinct cell types. Histamine exerts its dual effect by rapidly activating cells that express IL-2 and IFNr genes and, on the other hand, suppressor cells that become responsive to histamine mom gradually but once activated, strongly inhibit expression of these genes.

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REQUIREMENT OF TWO CIS-ACTING DNA ELEMENTS FOR THE INDUCTION OF GM-CSF yGE,NE DURING T, CELL ACTIVATION. N. Arai. A. Tsubo Y. a aauchi-lwai. S. Mvaw

suda. J Shlomai and K. Arai’ DNAX Research I&titute Palo Alto, Ck 94304, ‘The Institute of Medical Science, Thi

PRIMITIVE CYTOKINES ARE INVOLVED IN INVERTEBRATE HOST DEFENSE. B Gail S. w Depallmenf of Pathology, SUNY at Stony Brook, Stony Brook, NY 117944691, USA

Cytokines are polypeptide mediators released by cells involved in vertebrate host defenses that are used to communicate with similat ot different cells. IL-l and TNF are major immunoregulatoty cytokines with many host defense related properties. The crucial need for and importance of cytokines to vertebrates suggests to us that they are proteins that have developed and evolved with the host defense systems of all animals. Since the host defense systems of invertebrates are diverse, we are interested in studying cytokine-like mediators that can be found throughout the animal kingdom. We have isolated and characterized IL-l and TNF from several invertebrate phyla using vertebrate assay systems. Invertebrate IL-1 shares many biological activities wirh vertebrate IL-l when assayed in several vertebrate systems (A375 cytotoxicity, fibroblast proliferation, and I’GEz production). TNF from invertebrates has been identified and characterized by using the L 929 cytotoxicity assay. In invertebrate systems, invertebrate IL-l stimulates coelomocyte phagocytosis and proliferation. By studying defense mechanisms of invertebrates and their regulatory cytokines, we may be able to unravel the complex web of interactions among cells and factors of vertebrate immune responses. This will help to demonstrate conclusively cytokine regulation of host defenses. Since phagocytosis is as old as animal life itself, it is likely that the cytokine armamentatium of the phagocyte has an ancient origin as well. (Supported by NSF Grant DCB 8810448)

University of Tokyo, Tokyo, Japan. Induction of lymphokine genes is coordinately regulated

upon antigen stimulation in T cells. Two critical cis-acting DNA regions in addition to TATA sequence are identified for the GM- CSF gene induction, i.e., CLEL(GM2)/GC box and CLEO elements. The GM2/GC box covering -96 to -72, is composed of two DNA binding motifs for inducible (NF-GML) and constitutive (Al, A2, 8) proteins’). NF-GM2, purified from activated Jurkat cells consists f 50 and 65 Kd polypeptides and is indistinguishable from NF-KB 29 Al seems to be identical with Spl and stimulates transcripti;yri$ vitro,, whereas A2 and B are inhibitory to Al action. requirements of two adjacent motifs suggests the importance of interaction between two proteins, NF-GM2 and Al in induction. CLEO, extending from -40 to -54, shows homology to the sequences found in the 5’ flanking region of IL-4, IL-5 and G-CSF. NF-CLEO consists of two complexes, NF-CLEOa and NF-CLEOb which act as a stimulator and inhibitor, respectively. The CLEO elements of the IL-4 and IL-5 genes, but not of G-CSF, are also recognized by NF-CLEOa and b, suggesting that NF-CLEOa and b protein plays an important role in the coordinate induction of these lymphokine genes in activated T cells. 1) Sugimoto, K. et al. (1990) Intl. Immunol. 2:787. 2) Tsuboi, A. et al. (1991) Intl. /mmuno/. in press.

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PRODUCTION OF LEUKEMIA INHIBITORY FACTOR (LIF) AND INTER- LEUKIN 6 (116) BY MACROPHAGES AND HEPATOMA CELL UNES.

Institute, La Jolla, CA 92037. G.H. Fey The Scripps Research

Leukemia inhibitory factor (LIF) and interleukin 6 (IL6) are produced by a similar spectrum of cell types and share several common functions. For example, both induce differentiation of myeloid leukemia cells and induce the expression of liver acute phase genes. We have recently demonstrated the autocrine action of IL6 in rat hepatoma cells. The expression of IL6 and LIF in both macrophage and hepatoma cells was stimulated by treatment with lipopolysaccharides (LPS) and this induction was inhibited by treatment with glucocorticoids. Secretion of newly synthesized LIF protein by hepatoma cells was also demonstrated by metabolic labeling with 36Smethionine and immunoprecipitation. Both IL6 and LIF mRNA synthesis in hepatoma cells was also induced by treatment with IL1 and thus both genes are controlled by similar mechanisms. We have recently cloned the rat IL6 and LIF cDNAs and the corresponding genes. The transcriptional start sites has been mapped and we have initiated a comparative investigation of their control elements for inducible expression.

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INDEPENDENT INDUCTION OF IL-la AND IL-16. 0. Bakouche nd L.B. Lachman, Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030.

When human monocytes were activated by LPS, both IL-l. and IL- ll3 mRNAs were detected along with intracellular production and secretion of these two cytokines. When human monocytes were activated by LPS presented in liposomes (MLV-LPS), IL-la mRNA was detected, but no IL-18 mRNA. When human monocytes were activated by LPS, mRNA for IL-16 was in a greater amount than the IL-l. messenger (5:l ratio). However, monocytes activated by LPS, 24 hours after plating displayed more IL-la than IL-18 messenger (ratio:3:1); Activation of human monocytes 5 to 7 days after plating led to the induction of the IL-l. messenger only and no IL-18 mRNA was found. Pretreatment of cells with dexamethasone or prednisolone and activation by LPS 7 days after plating, restored a ratio of 3:l respectively for IL-l. and IL-18 messenger. Finally, a pretreatment of cell with IL- ll3 in the presence of prednisolone and activation by LPS 7 days after treatment led to the presence of both IL-l. and IL-1B with an equal ratio. These findings indicate that IL-la and IL-18 can be independently induced and that aging monocytes are unable to produce IL-18 upon activation by LPS.

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CYCLOSPORINE A (CSA) INHIBITS IL-6 PRODUCTION BY MURINE SPLENOCYTES IN VITRO. Blot, C., Lebrec, H.. Bohuon, C., and Pallardv, M.. Laboratoire de Toxicologic, Faculte de Pharmacie de Paris XI, rue J.B Wment, 92296 Chatenay- Malabry & Dept. Biologic Clinique, Institut C. Roussy, 94805 Villejuif (France). IL-6 is involved in hematopoiesis, immune defense and in the acute phase response. IL-6 production was measured in supernatants from concanavaline-A (ConA), and lipopolysaccharide (LPS) - activated B6C3Fl mice splenocytes following exposure for 24 hours to CSA. IL-6 production was also assessed in supernatants from ConA, and LPS-stimulated peritoneal macrophages exposed to CSA in the same conditions than splenocytes. IL-6 measurements were performed using the hybridoma 8.9. bioassay. The results shown that CSA inhibits in a dose dependent manner the production of IL-6 by ConA- activated-splenocytes (-84% at 10-6 M, CI50 = 2,33 lo-8M) but not by LPS-activated splenocytes. However, the addition of CSA to ConA and LPS-stimulated peritoneal macrophages showed a significative augmentation of production of IL-6 at the highest doses (respectively +20% and +25% at 10-5 M). The results obtained in this study suggest that the action of CSA on IL-6 production is mainly affecting the T lymphocyte but not the macrophage synthesis of this cytokine. We are currently evaluating the in vitro production of IL-6 by anti-CD3-activated lymphocytes exposed to CSA using DlOS, a Th2 T cell clone.