Effects of in vivo priming on the expression of IL-4 and IFN-γ in short-term murine T cell clones
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454 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES 25 TYROSINE KINASE PATHWAY IS INVOLVED IN THE PROTEIN KINASE C DEPENDENT INDUCTION OF IL-18 GENE EXPRESSION. M. Hurme, S. Matikainen and .I. Vakkila. Department of Bacteriology and Immunolow, University of Helsinki. Helsinki, Finland. The phbrbol ester 6MA is s wsll'chsrsctsrizsd activator of the protein kinsss C (PKC), a serine-threonine kinase. It has been reported that PMA can also induce tyrosine phosphor- ylation of different substrates in various cell types. As the expression of the IL-18 gene is readily induced by PMA in monocytic cells, we wanted to examine whether the induced tyrosine kinase activity has a role in this activation. In the monocytic leukemia cell line, THP-1, PMA induced IL-l6 mRNA expression and IL-16 protein production as well as ty- rosine-specific phosphorylation of several proteins. The sx- pression of IL-16 could be inhibited by preincubating the cells with the tyrosine kinase inhibitor, genistein. In order to exclude the possible effect of genistein on the PKC-medi- ated activities, THP-1 cells were transfscted with 5x AP-l-CAT reporter plasmid (i.e. containing 5 repeats of the AP-1 enhancer element). The same genistein concentration (301~ M) downregulating the IL-18 expression did not have any effect on the PMA-induced CAT activity in these cells. Thus we can conclude that the PMA-induced tyrosins kinase activity contributes to the activation of the IL-16 gene expression and that its effect is distal from the classical PMA-induced AP-1 element-dependent induction of transcription. 26 A MONOCYTIC CELL LINE IS TRIGGERED TO PRODUCE IL-1pBY CELL-CELL CONTACT WITH ACTIVATED T CELLS. P.Islar,J.-H.Zbarg,E. RoasnekandJ.-M. Dayar Div. of Immunology, Dept. of Medicine, H6pital Cantonal Universitaire, 1211 Geneva 4, SwRzerland. We have measured IL-lp production by a monocytic cell line after co-culture v&h either T cell line% M T cells isolated from peripheral blood. To address the importance of cell-cell contact required fw monocyte activation by T cells, we co-cultured both cell types in double chamber plates separated by a membrane. Our results show that T cells, when activated by PHA and PMA during 72 hours culture, acquire the capacity to induce IL-1 orelease by the monocyte. 12 hours after co-culture, IL-l p could be detected intracellulary, followed 24 hours later by cytokine release imo the culture medium. Under bath conditions, T cells were able to trigger the monocytic cell line , but IL-1 b production was increased 2 to 4 fold when cells were in close contact. Therefore, it is likely that both soluble factors and imermembrane signaIling play a role in monocyte activation by T cells. We were able to discriminate these two phenomenons further by showing that paraformaldehyde fixed T cells were still able to activate the monocytk cell line. Aftw fixation, PMA treated T cells remain still inducers for IL- 10 production, but the effects of PHA-activated T cells was abrogated. Eqwinents done with several T cell clones showed that activated CD4+ clones were more potent inducers of IL-1 0 production after cell-cell contact than CD8+ cells. 27 CONTROL OF HUMAN IL-la, IL-lt3 AND TN&a GENE EXPRESSION AT PROCESSING OF PRE-mRNA. Navef larrous and Ravmond Kaemufer. Department of Molecular Virology, The Hebrew University-Hndassah Medical School, 91010 Jerusalem, Israel Re!inoic acid (RA), phorbol ester and LPS each induce, with distinct kinetics, a transient wave of mRNA encoded by IL-la, IL-lp and TNFa genes. Appearance of mRNA in PBMC depends on de noun tmntiption. When resting PBMC are exposed to the translation inhibitor cycloheximide (CHX) alone, in the absence of other inducers, a far greater induction is observed, of 48%fold for IL-l@ and ILla RNA, and of ZO-fold for TNF-a RNA, accompanied by a greatly increased rate of transcription. The transient, CHX-induced waves of RNA de&e promptly to basal level. ndine out stabilization of mRNA. Maximal induction of IL-16 mRNA -s at i04 M of RA, in good correlation with the &on of active protein. Expression of IL-la, IL-lp and TNFa mRNA is superinduced extensively (by 50-, 50-, and Z-fold, respectively] when CHX is added at any time after tie onset of induction by RA. This superinduction is not accompanied by any increase in primary transcription rate-In the absence or presence of CHX,.equ& halflives of mRNA are observed, showing that stabilization of mRNA also rannot account for suoerinduction. Yet. suoerinduction of the three eenes bv CHX is blocked bv i&ibitors of tmnsc&tio~ and thus depends critic&y on s&thesis of new RNA molecules. Simultaneous qua&it&ion of pre-mRNA and mRNA by RNase protection analysis, using genomic probes covering an adjoining exon and intron, shows that CHX does not stabilize pre-mRNA and supports the conclusion that a labile protein acts post-transcriptionally to inhibit the flow of newly synthesized, short-lived pre-mRNA molecules into IL-la, IL-l@ and TNF-a mRNA. 28 EFFECTS OF IN VW0 PRIMING ON THE EXPRESSION OF IL-4 AND IFN-yIN SHORT-TERM MURINE T CELL CLONES Anne K&o. Michael If. Pech and Lynn Morris The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia. We have previously reported that, while many short-term alloreactive T cell clones express IFN-y without IL-4, most IL-4-expressing clones coexpress IL-2 and IFN-y and therefore do not conform to the Thlm classification. To test the effects of in viva priming on lymphokine production patterns, mice were immunized subcutaneously with alum-precipitated KLH and killed Bordetella o-xtussis, or infected with Leisbmania major promastigotes. CDl+ T cells from draining lymph nodes were cloned at limiting dilution with antigen and IL-2 then assayed after 14 days for lymphokine secretion. In the KLH response, 80-904 of clones secreted IL-4 without IF%y. The remaining clones included both IFNr+ IL4 and IFN-r+ IL-4+ clones, of which many double-producers retained the ability to secrete both lymphokines on single-cell recloning. In the response to mmaior, antigen-specific clones from 4 wk-infected mice of the resistant strain C57BL/6 were mainly IFN-y+ IL& whereas those from the susceptible strain BALB/c were mainly IL-4+ IFN-y-. However, CD4+ cells harvested early in the infection of C57BL/6 mice gave rise to many IL-4+ clones, some of which coexpressed IFN=y. It is concluded that in viva priming markedly affects the distribution of lymphokine profiles but that even in these circumstances many clones coexpress ThI and Th2 lymphokines. 29 MACROPHAGES CONSTITUTIVELY EXPRESS A NUCLEAR FAIZOR WHICH BINDS TO THE PUTATIVE SILENCER ELEMENT OF THE HUMAN INTERFERON7 GENE. E.J. Kovacs~. S. VanStedum’, P. Ghosh+, and H.A. &a& DeDt. of Cell. Biol.. Nwmbio. & Anat. and ‘Moles. Biol. Pmn.. Lovola Univ. Stritch-Sch. Med., May&d, IL 60153 and +Lab of Expt. Imm&l:, B&P, NCI-FCRDC, Frederick, MD 21701. The expression of interfemny (lFN-y) is restricted to two normal cell types. T lymphocytes and huge granular IymPhccytes (LGL.a). These cells do not spontmeousIy express IFN-y, but can be induced to express it following stimulation with various combinations of cytokines, mitogens, and phorbol eaters. In contrsst to T cells and LGLs. mscrophages do not express IFNy spontaneously and CaOnOthe. induced to express the gene. Since the 5’ flsnking region of the human IFNr gene. contains negative as well as positive regulatory elements, we hypothesized thst the lack of expression of IFNy in macmpbages may be a consequence of the constihltive production of nuclear biidiig proteins which interact with the putative silencer element at -251 to -215. Herein, we sport that the humsn THP-I monocytic leukemia and murine ANA-I macrophage cell lines produce nuclear factors which specitically bids to the silencer region of the IFNy gene. The nuclear factors, which appear as a doublet of approximately 75-85 kd, are constitutively produced snd cannot be upregulated by treatment of cells with phorbol myristste a&ate, interleukin-2, IFN-7, or lipopolyssccharide. Similar proteins are present in nuclear extracts of T cells and LGLs, but not non-lymphoid cells. Tlx,se data demonstrate that cells of my&id lineages produce nuclear factors which specifically bids to a negative regulatory element of the 1FNv-r gene and suggest thst the lack of induciblity of IFNy in macrophages may he the result of the high level of coastihltive production of these proteins. (Supported by ONR #NC0014-89-J-1130 and The Elss U. Pardee Foundation.) 30 ACOPAFANT (RP 55778) SELECTIVELY INHIBITS IN YIYD ENDOTOXIN-INDUCED TNFol PRODUCTION BY MURINE PERITONEAL MACROPHAGES AT THE mRNA LEVEL. 8. Lionne, A. Naussac. A. Floch. M. Lemaitre. A. Bousseau RhBne-Poulenc Rarer Central Research. CRVA, 94403 Vitrv sur Seine. FRANCE RP 55778 is a PAF-receptor antagonist which has been shown to inhibit in vitro endotoxin-induced TNFu production by murine peritoneal macrophages. In the presence of RP 55778, the TNFu mRNA level is stronrzlv hepressed, whereas Ii, p mRNA remains unaffected. - - To assess the efficacy of the drug in viva, 6. to 8. week-old female OFI mice were treated orally 3 days with 5 to 100 mg/kg RP 55778. Animals were then challenged with endotoxin (100 rig/mouse) administered either intravenously or intraperitoneally. Circulatina TNFcl and IL. levels were measured in plasma 30 min to 2 h afeer intravenous endotoxin challenge. TNFu, IL1 and IL f; mRNA in peritoneal macrophages was measured 3 ours after intraperitoneal challenge. RP 55778 selectively blocked TNFa mRNA expression and protein synthesis in viva without affecting IL6 or IL1 production. These results indicate that RP 55778 may have therapeutic potential for the treatment of TNF-related toxic manifestations characteristic of certain acute and chronic infectious diseases.
Transcript of Effects of in vivo priming on the expression of IL-4 and IFN-γ in short-term murine T cell clones
454 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES
25
TYROSINE KINASE PATHWAY IS INVOLVED IN THE PROTEIN KINASE C DEPENDENT INDUCTION OF IL-18 GENE EXPRESSION. M. Hurme, S. Matikainen and .I. Vakkila. Department of Bacteriology and Immunolow, University of Helsinki. Helsinki, Finland.
The phbrbol ester 6MA is s wsll'chsrsctsrizsd activator of the protein kinsss C (PKC), a serine-threonine kinase. It has been reported that PMA can also induce tyrosine phosphor- ylation of different substrates in various cell types. As the expression of the IL-18 gene is readily induced by PMA in monocytic cells, we wanted to examine whether the induced tyrosine kinase activity has a role in this activation. In the monocytic leukemia cell line, THP-1, PMA induced IL-l6 mRNA expression and IL-16 protein production as well as ty- rosine-specific phosphorylation of several proteins. The sx- pression of IL-16 could be inhibited by preincubating the cells with the tyrosine kinase inhibitor, genistein. In order to exclude the possible effect of genistein on the PKC-medi- ated activities, THP-1 cells were transfscted with 5x AP-l-CAT reporter plasmid (i.e. containing 5 repeats of the AP-1 enhancer element). The same genistein concentration (301~ M) downregulating the IL-18 expression did not have any effect on the PMA-induced CAT activity in these cells. Thus we can conclude that the PMA-induced tyrosins kinase activity contributes to the activation of the IL-16 gene expression and that its effect is distal from the classical PMA-induced AP-1 element-dependent induction of transcription.
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A MONOCYTIC CELL LINE IS TRIGGERED TO PRODUCE IL-1pBY CELL-CELL CONTACT WITH ACTIVATED T CELLS. P.Islar,J.-H.Zbarg,E. RoasnekandJ.-M. Dayar Div. of Immunology, Dept. of Medicine, H6pital Cantonal Universitaire, 1211 Geneva 4, SwRzerland. We have measured IL-lp production by a monocytic cell line after co-culture v&h either T cell line% M T cells isolated from peripheral blood. To address the importance of cell-cell contact required fw monocyte activation by T cells, we co-cultured both cell types in double chamber plates separated by a membrane. Our results show that T cells, when activated by PHA and PMA during 72 hours culture, acquire the capacity to induce IL-1 orelease by the monocyte. 12 hours after co-culture, IL-l p could be detected intracellulary, followed 24 hours later by cytokine release imo the culture medium. Under bath conditions, T cells were able to trigger the monocytic cell line , but IL-1 b production was increased 2 to 4 fold when cells were in close contact. Therefore, it is likely that both soluble factors and imermembrane signaIling play a role in monocyte activation by T cells. We were able to discriminate these two phenomenons further by showing that paraformaldehyde fixed T cells were still able to activate the monocytk cell line. Aftw fixation, PMA treated T cells remain still inducers for IL- 10 production, but the effects of PHA-activated T cells was abrogated. Eqwinents done with several T cell clones showed that activated CD4+ clones were more potent inducers of IL-1 0 production after cell-cell contact than CD8+ cells.
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CONTROL OF HUMAN IL-la, IL-lt3 AND TN&a GENE EXPRESSION AT PROCESSING OF PRE-mRNA. Navef larrous and Ravmond Kaemufer. Department of Molecular Virology, The Hebrew University-Hndassah Medical School, 91010 Jerusalem, Israel
Re!inoic acid (RA), phorbol ester and LPS each induce, with distinct kinetics, a transient wave of mRNA encoded by IL-la, IL-lp and TNFa genes. Appearance of mRNA in PBMC depends on de noun tmntiption. When resting PBMC are exposed to the translation inhibitor cycloheximide (CHX) alone, in the absence of other inducers, a far greater induction is observed, of 48%fold for IL-l@ and ILla RNA, and of ZO-fold for TNF-a RNA, accompanied by a greatly increased rate of transcription. The transient, CHX-induced waves of RNA de&e promptly to basal level. ndine out stabilization of mRNA. Maximal induction of IL-16 mRNA -s at i04 M of RA, in good correlation with the &on of active protein. Expression of IL-la, IL-lp and TNFa mRNA is superinduced extensively (by 50-, 50-, and Z-fold, respectively] when CHX is added at any time after tie onset of induction by RA. This superinduction is not accompanied by any increase in primary transcription rate-In the absence or presence of CHX,.equ& halflives of mRNA are observed, showing that stabilization of mRNA also rannot account for suoerinduction. Yet. suoerinduction of the three eenes bv CHX is blocked bv i&ibitors of tmnsc&tio~ and thus depends critic&y on s&thesis of new RNA molecules. Simultaneous qua&it&ion of pre-mRNA and mRNA by RNase protection analysis, using genomic probes covering an adjoining exon and intron, shows that CHX does not stabilize pre-mRNA and supports the conclusion that a labile protein acts post-transcriptionally to inhibit the flow of newly synthesized, short-lived pre-mRNA molecules into IL-la, IL-l@ and TNF-a mRNA.
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EFFECTS OF IN VW0 PRIMING ON THE EXPRESSION OF IL-4 AND IFN-yIN SHORT-TERM MURINE T CELL CLONES
Anne K&o. Michael If. Pech and Lynn Morris The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia.
We have previously reported that, while many short-term alloreactive T cell clones express IFN-y without IL-4, most IL-4-expressing clones coexpress IL-2 and IFN-y and therefore do not conform to the Thlm classification. To test the effects of in viva priming on lymphokine production patterns, mice were immunized subcutaneously with alum-precipitated KLH and killed Bordetella o-xtussis, or infected with Leisbmania major promastigotes. CDl+ T cells from draining lymph nodes were cloned at limiting dilution with antigen and IL-2 then assayed after 14 days for lymphokine secretion. In the KLH response, 80-904 of clones secreted IL-4 without IF%y. The remaining clones included both IFNr+ IL4 and IFN-r+ IL-4+ clones, of which many double-producers retained the ability to secrete both lymphokines on single-cell recloning. In the response to mmaior, antigen-specific clones from 4 wk-infected mice of the resistant strain C57BL/6 were mainly IFN-y+ IL& whereas those from the susceptible strain BALB/c were mainly IL-4+ IFN-y-. However, CD4+ cells harvested early in the infection of C57BL/6 mice gave rise to many IL-4+ clones, some of which coexpressed IFN=y. It is concluded that in viva priming markedly affects the distribution of lymphokine profiles but that even in these circumstances many clones coexpress ThI and Th2 lymphokines.
29
MACROPHAGES CONSTITUTIVELY EXPRESS A NUCLEAR FAIZOR WHICH BINDS TO THE PUTATIVE SILENCER ELEMENT OF THE HUMAN INTERFERON7 GENE. E.J. Kovacs~. S. VanStedum’, P. Ghosh+, and H.A. &a& DeDt. of Cell. Biol.. Nwmbio. & Anat. and ‘Moles. Biol. Pmn.. Lovola Univ. Stritch-Sch. Med., May&d, IL 60153 and +Lab of Expt. Imm&l:, B&P, NCI-FCRDC, Frederick, MD 21701.
The expression of interfemny (lFN-y) is restricted to two normal cell types. T lymphocytes and huge granular IymPhccytes (LGL.a). These cells do not spontmeousIy express IFN-y, but can be induced to express it following stimulation with various combinations of cytokines, mitogens, and phorbol eaters. In contrsst to T cells and LGLs. mscrophages do not express IFNy spontaneously and CaOnOt he. induced to express the gene. Since the 5’ flsnking region of the human IFNr gene. contains negative as well as positive regulatory elements, we hypothesized thst the lack of expression of IFNy in macmpbages may be a consequence of the constihltive production of nuclear biidiig proteins which interact with the putative silencer element at -251 to -215. Herein, we sport that the humsn THP-I monocytic leukemia and murine ANA-I macrophage cell lines produce nuclear factors which specitically bids to the silencer region of the IFNy gene. The nuclear factors, which appear as a doublet of approximately 75-85 kd, are constitutively produced snd cannot be upregulated by treatment of cells with phorbol myristste a&ate, interleukin-2, IFN-7, or lipopolyssccharide. Similar proteins are present in nuclear extracts of T cells and LGLs, but not non-lymphoid cells. Tlx,se data demonstrate that cells of my&id lineages produce nuclear factors which specifically bids to a negative regulatory element of the 1FNv-r gene and suggest thst the lack of induciblity of IFNy in macrophages may he the result of the high level of coastihltive production of these proteins. (Supported by ONR #NC0014-89-J-1130 and The Elss U. Pardee Foundation.)
30
ACOPAFANT (RP 55778) SELECTIVELY INHIBITS IN YIYD ENDOTOXIN-INDUCED TNFol PRODUCTION BY MURINE PERITONEAL MACROPHAGES AT THE mRNA LEVEL. 8. Lionne, A. Naussac. A. Floch. M. Lemaitre. A. Bousseau RhBne-Poulenc Rarer Central Research. CRVA, 94403 Vitrv sur Seine. FRANCE
RP 55778 is a PAF-receptor antagonist which has been shown to inhibit in vitro endotoxin-induced TNFu production by murine peritoneal macrophages. In the presence of RP 55778, the TNFu mRNA level is stronrzlv hepressed, whereas Ii, p mRNA remains unaffected. - - To assess the efficacy of the drug in viva, 6. to 8. week-old female OFI mice were treated orally 3 days with 5 to 100 mg/kg RP 55778. Animals were then challenged with endotoxin (100 rig/mouse) administered either intravenously or intraperitoneally. Circulatina TNFcl and IL. levels were measured in plasma 30 min to 2 h afeer intravenous endotoxin challenge. TNFu, IL1 and IL
f; mRNA in peritoneal
macrophages was measured 3 ours after intraperitoneal challenge. RP 55778 selectively blocked TNFa mRNA expression and protein synthesis in viva without affecting IL6 or IL1 production. These results indicate that RP 55778 may have therapeutic potential for the treatment of TNF-related toxic manifestations characteristic of certain acute and chronic infectious diseases.
