1
478 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES 169 172 CHIMERIC TNF RECEPTORS OF VIRAL AND CELLULAR ORIGIN. CraiP A.. L. Lauffer*. T, &rvmond G. GoodwinJmmunex Corp., Seattle, WA 98101; *Behringwerke AG, FRG. The oligomeric nature of TN’F provides a rationale for the multiple affinity classes this cytokine usually exhibits for its receptors. We therefore inferred that while soluble, monomeric forms of these receptors should inhibit ligand binding to surface receptors, dimeric constructs should be considerably more potent (higher affmity). We therefore engineered soluble monomeric and dimeric forms containing extracellular portions of the human ~80 TNF receptor. Several dimeric forms were constructed as Fc fusion proteins containing truncated Ig heavy chains. These dimers bound TNF with a KI similar to high affinity sites of surface-bound receptors, and 20X higher than monomers. Dimers were also a much more effective inhibitor of TNF cytolysis in vitro and LPS-induced shock in vim. The generation of higher ligand affinity by oligomeric forms of soluble TNF receptors is illuminated by the T2 protein encoded within at least five Poxvirus genomes: by chemical cross-linking and gel filtration, this soluble form of the type I TNF receptor appears to be a dimer, underscoring the significance of multiphasic binding in the TNF system. 170 LNJKOTRIENE B4 UPREGULATES THE EXPRJQSION OF IL 2RP ON HUMAN LYMPHOCYTES AND ENHANCES THEIR RESPONSES TO IL 2. J. Stankova. N. Gammon and M. Rola- Pleszczvnski, Immunology Division, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QC, Canada, JlH 5N4. The 5 chain of the IL 2 receptor (IL 2RP) is expressed constitutively on lymphocytes and is responsible for transduction of some IL a-mediated effects, including NK cell activation by IL 2. Since LTB4 can enhance NK cell activity, we studied the modulation of IL 2R5 expression and function by LTB4. Cell surface expression of IL 2R9 on lymphocytes exposed to LTB4 increased two-fold, both in terms of % positive cells and intensity of expression, at 24 h. Peak effects were seen at lo-lo-lo-sM LTB4 and were preferentially observed in CD56+ and CD8+ cells. IL 2R5 mRNA was upregulated by LTB4 within 3 h, with peak effects at lo-lo - 10~sM LTB4. LTB4-pretreated lymphocytes also responded with enhanced cytotoxic activity against NK-sensitive K562 target cells when treated with IL 2 during the cytotoxicity assay, suggesting that LTBd-induced augmentation of IL 2R5 expression was functionally relevant to IL a-mediated effects. Our studies indicate a potential participation of LTB4 in modulation of lymphocyte cytotoxic functions through regulation of cytokine receptor expression. Supported by NCI Canada 171 IL1 INDUCES NF-KB THROUGH ITS TYPE I AND NOT TYPE II RECEPTOR ‘E. Stylianou, *L.A..J.O’Neil, ‘M. Edbrooke, and ‘J L Saklatvala Strangeways Research Laboratory, Warts Causeway Cambridge; ZBiochemistry Department, Trinity college, Dublin 2, Ireland; 3Glaxo Group Research, Greenford, UK. NF-KB is a DNA binding protein known to activate transcription of several IL1 responsive genes. The induction of NF-KB activity by IL1 was studied in the murine lines, EL4.NOB.l which expresses the Type I (8OKD), and the 702/3 pre-B cell line which bears Type II (67KD) IL1 receptor. Nuclear extracts were analysed using an electrophoretic mobility shift assay. NF-KB was induced by 5 mins and maximum at 15 mins in response to IL1 in both cell types. Pre-incubation with the IL1 receotor antaeonist (ILlR.4) inhibited IL1 induction of NF-KB in a dose-dependent manner in both cell types. Direct binding of ILlRA to the Type I and not the Type II receptor was demonstrated in these cells. Forskolin did not activate NF-KB and staurosporine did not inhibit IL1 induced NF-KB. These results suggest that the activation of NF-KB by IL1 is mediated through the Type I and not Type II receptor and does not involve protein kinases A Of c. PURIFICATION AND CHARACTERISATION OF A NATURAL SOLUBLE RECEPTOR FOR INTERLEUKIN-1. n W Duff, Section of Molecular Medicine. Dept. Med., Univ. Sheffield, Royal Hallamshire Hospital, Sheffield, U.K. Affinity chromatography and Reverse-Phase HPLC was used to purify a soluble IL-1 receptor (sIL-1 R) from the supernatant of a human B cell line, Raji. The purified protein specifically bound 1 251 IL-1 p forming a 60kD comolex in non-reducina conditions and a 70kD comolex in reducina conditions. We have pre&usly identified a similar molecule in healthy se&? and supernatants from activated PBMNC. Binding was found to be displaceable by mature human and murine IL-1 6, and human 31 kD IL-1 6 propeptide, but not displaceable by human and murine IL-1 a or human IL-1 receotor antaaonist. Liaand blottina revealed a 47kD molecule that spectically bo&d IL-1 6. -Microseque&ing of this 47kD band revealed that the NH2terminal was blocked and we are currently sequencing tryptic fragements to generate internal peptide sequence. Measurement of binding affinity of the cell surface Raji IL-1R (Kd=2.2nM) and the soluble Raji IL-1R (Kd=2.7nM) demonstrated a similar affinity lor 1251 IL-1 fi. Purified sIL-1 R inhibited binding of IL-1 6 to cell lines with both Type I (60kD) and Type II (66kD) IL-1 receptors, but did not interfere with IL-la binding. A serine protease inhibitor prevented release of the slL-1R indicating that solubilization occurs via proteolytic cleavage at the cell surface. This natural slL-1 R may function as an important regulatory molecule of IL-1 6 invire 173 DOES TNFa BINDING TO THE TWO RECEPTOR TYPES PRODUCE AN IDENTICAL CELLULAR RESPONSE? DJ Taylor Univ.Dept of Medicine, University Hospital of South Manchester, UK. Two Cellular receptors for TNFa have recently been cloned with approx. Mwt of 55-and 75-KDa which are recognised by the McAbs htr-9 and utr-1, respectively. Preincubation with the antibody utr-1 (lOug/ml) inhibited (approx.50%) the TNFa- stimulation of PO: production by rheumatoid synovial fibro- lasts (RSF) in vitro. However within the same cells, the TNF a-stimulation of fructose 2,6-bisphosphate (Fru(‘2,6)PZ) was only marginally inhibited (approx.lS%), achieving significance in only 3 out of 5 experiments. Increasing the antibody concentration or the preincubation time did not increase the level of inhibition. The antibody htr-9 (lOug/ml) had TNFa like activity on RSF, stimulating both PGE production and Fru (2,6)P2, with the latter being preferentially increased. The agonistic effect of htr-9 was inhibited by a second antibsdy (htr-5) raised against the 55-KDa receptor, demonstrating the agonistic effect was not the result of endotoxin contamination Taken together, these data suggest the TNFa-stimulation of PGE production involves both receptor types in RSF, but that the stimulation of cellular Fru(2,6)P2, and thus glycolysis, may be mediated largely via the 55-KDa receptor. This is the first evidence suggesting that TNFu binding to the two receptor types may not produce identical cellular responses. Dr. M. Brockhaus of Hoffman-La Roche generously provided the McAbs and the work was supported by the Arthritis Fi Rheumatism Council, UK. 174 E ULTURED RABBIT ARTICULAR CHONDROCYTES (RAC) XPRESS DISTINCT TRANSFORMING GROWTH FACTOR-B ECEPTOR PHENOTYPES AS A FUNCTION OF CELL CYCLE bIS+RIBUTION. D.Vivien, F. R6dini. SRonost, P.Galtra, G.Loyaa. J- p.Pujol. Laboratoire de Biochimie du Tissu Conjonctif, CHU Caen C&e de Nacre. 14033. France. I” culture, TGF-El is a bifunctional regulator of RAC proliferation depending on the serum concentrations. In low level of semm (2% FCS). cycling cells are growth-inhibited by the peptide, whereas in high level of serum (10% FCS). TGF-5 transiently potentiates their growth. Cycling RAC in 10% and 2% FCS exhibited two distinct classes of high-affinity TGF-6 binding sites. However, in 2% FCS we show a significant increase (60%) of sites per cell for the highest affinity receptors (in 10% FCS: Kdl=280 pM, 3.100 sites/cell; Kd2=1,057 pM. 8,200 sites/cell: In 2% FCS: Kdl=346 pM. 4,900 sites/cell; Kd2=900 pM. 9,990 sites/cell). Cytofluorometric analyses of DNA content show that 55% of cells are in GO/l in 10% FCS versus 70% in 2% FCS. To get more insight into the receptor distribution in relation with the cell cycle phases, we have performed similar experiments in cells synchronized in late Gl or early S phase. Synchronized cells in 10% FCS (>80% of cells in S phase). exhibited a single class of TGF-5 high affinity receptor (Kdl=l.OEO pM, 5,772 sites/cell). In contrast, RAC cultured in the presence of 2% FCS (41% of cells in GO/l) expressed two distinct classes of binding sites (Kdl=314 pM, 2,664 sites/cell; Kd2=884 pM. 5,500 sites/cell). In addition, we have oberved no modification of the distribution of TGF-O receptors between sparse cycling cells and confluent cells.

Chimeric TNF receptors of viral and cellular origin

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Page 1: Chimeric TNF receptors of viral and cellular origin

478 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES

169 172

CHIMERIC TNF RECEPTORS OF VIRAL AND CELLULAR ORIGIN. CraiP A.. L. Lauffer*. T,

&rvmond G. GoodwinJmmunex Corp., Seattle, WA 98101; *Behringwerke AG, FRG.

The oligomeric nature of TN’F provides a rationale for the multiple affinity classes this cytokine usually exhibits for its receptors. We therefore inferred that while soluble, monomeric forms of these receptors should inhibit ligand binding to surface receptors, dimeric constructs should be considerably more potent (higher affmity). We therefore engineered soluble monomeric and dimeric forms containing extracellular portions of the human ~80 TNF receptor. Several dimeric forms were constructed as Fc fusion proteins containing truncated Ig heavy chains. These dimers bound TNF with a KI similar to high affinity sites of surface-bound receptors, and 20X higher than monomers. Dimers were also a much more effective inhibitor of TNF cytolysis in vitro and LPS-induced shock in vim. The generation of higher ligand affinity by oligomeric forms of soluble TNF receptors is illuminated by the T2 protein encoded within at least five Poxvirus genomes: by chemical cross-linking and gel filtration, this soluble form of the type I TNF receptor appears to be a dimer, underscoring the significance of multiphasic binding in the TNF system.

170

LNJKOTRIENE B4 UPREGULATES THE EXPRJQSION OF IL

2RP ON HUMAN LYMPHOCYTES AND ENHANCES THEIR RESPONSES TO IL 2. J. Stankova. N. Gammon and M. Rola- Pleszczvnski, Immunology Division, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QC, Canada, JlH 5N4.

The 5 chain of the IL 2 receptor (IL 2RP) is expressed constitutively on lymphocytes and is responsible for transduction of some IL a-mediated effects, including NK cell activation by IL 2. Since LTB4 can enhance NK cell activity, we studied the modulation of IL 2R5 expression and function by LTB4. Cell surface expression of IL 2R9 on lymphocytes exposed to LTB4 increased two-fold, both in terms of % positive cells and intensity of expression, at 24 h. Peak effects were seen at lo-lo-lo-sM LTB4 and were preferentially observed in CD56+ and CD8+ cells. IL 2R5 mRNA was upregulated by LTB4 within 3 h, with peak effects at lo-lo - 10~sM LTB4. LTB4-pretreated lymphocytes also responded with enhanced cytotoxic activity against NK-sensitive K562 target cells when treated with IL 2 during the cytotoxicity assay, suggesting that LTBd-induced augmentation of IL 2R5 expression was functionally relevant to IL a-mediated effects. Our studies indicate a potential participation of LTB4 in modulation of lymphocyte cytotoxic functions through regulation of cytokine receptor expression. Supported by NCI Canada

171

IL1 INDUCES NF-KB THROUGH ITS TYPE I AND NOT TYPE II RECEPTOR

‘E. Stylianou, *L.A..J.O’Neil, ‘M. Edbrooke, and ‘J L Saklatvala Strangeways Research Laboratory, Warts Causeway Cambridge; ZBiochemistry Department, Trinity college, Dublin 2, Ireland; 3Glaxo Group Research, Greenford, UK.

NF-KB is a DNA binding protein known to activate transcription of several IL1 responsive genes. The induction of NF-KB activity by IL1 was studied in the murine lines, EL4.NOB.l which expresses the Type I (8OKD), and the 702/3 pre-B cell line which bears Type II (67KD) IL1 receptor. Nuclear extracts were analysed using an electrophoretic mobility shift assay. NF-KB was induced by 5 mins and maximum at 15 mins in response to IL1 in both cell types. Pre-incubation with the IL1 receotor antaeonist (ILlR.4) inhibited IL1 induction of NF-KB in a dose-dependent manner in both cell types. Direct binding of ILlRA to the Type I and not the Type II receptor was demonstrated in these cells. Forskolin did not activate NF-KB and staurosporine did not inhibit IL1 induced NF-KB. These results suggest that the activation of NF-KB by IL1 is mediated through the Type I and not Type II receptor and does not involve protein kinases A Of c.

PURIFICATION AND CHARACTERISATION OF A NATURAL SOLUBLE RECEPTOR FOR INTERLEUKIN-1.

n W Duff, Section of Molecular Medicine. Dept. Med., Univ. Sheffield, Royal Hallamshire Hospital, Sheffield, U.K.

Affinity chromatography and Reverse-Phase HPLC was used to purify a soluble IL-1 receptor (sIL-1 R) from the supernatant of a human B cell line, Raji. The purified protein specifically bound 1 251 IL-1 p forming a 60kD comolex in non-reducina conditions and a 70kD comolex in reducina conditions. We have pre&usly identified a similar molecule in healthy se&? and supernatants from activated PBMNC. Binding was found to be displaceable by mature human and murine IL-1 6, and human 31 kD IL-1 6 propeptide, but not displaceable by human and murine IL-1 a or human IL-1 receotor antaaonist. Liaand blottina revealed a 47kD molecule that spectically bo&d IL-1 6. -Microseque&ing of this 47kD band revealed that the NH2terminal was blocked and we are currently sequencing tryptic fragements to generate internal peptide sequence. Measurement of binding affinity of the cell surface Raji IL-1R (Kd=2.2nM) and the soluble Raji IL-1R (Kd=2.7nM) demonstrated a similar affinity lor 1251 IL-1 fi. Purified sIL-1 R inhibited binding of IL-1 6 to cell lines with both Type I (60kD) and Type II (66kD) IL-1 receptors, but did not interfere with IL-la binding. A serine protease inhibitor prevented release of the slL-1R indicating that solubilization occurs via proteolytic cleavage at the cell surface. This natural slL-1 R may function as an important regulatory molecule of IL-1 6 invire

173

DOES TNFa BINDING TO THE TWO RECEPTOR TYPES PRODUCE AN IDENTICAL CELLULAR RESPONSE? DJ Taylor Univ.Dept of Medicine, University Hospital of South Manchester, UK.

Two Cellular receptors for TNFa have recently been cloned with approx. Mwt of 55-and 75-KDa which are recognised by the McAbs htr-9 and utr-1, respectively. Preincubation with the antibody utr-1 (lOug/ml) inhibited (approx.50%) the TNFa- stimulation of PO: production by rheumatoid synovial fibro- lasts (RSF) in vitro. However within the same cells, the TNF a-stimulation of fructose 2,6-bisphosphate (Fru(‘2,6)PZ) was only marginally inhibited (approx.lS%), achieving significance in only 3 out of 5 experiments. Increasing the antibody concentration or the preincubation time did not increase the level of inhibition. The antibody htr-9 (lOug/ml) had TNFa like activity on RSF, stimulating both PGE production and Fru (2,6)P2, with the latter being preferentially increased. The agonistic effect of htr-9 was inhibited by a second antibsdy (htr-5) raised against the 55-KDa receptor, demonstrating the agonistic effect was not the result of endotoxin contamination Taken together, these data suggest the TNFa-stimulation of PGE production involves both receptor types in RSF, but that the stimulation of cellular Fru(2,6)P2, and thus glycolysis, may be mediated largely via the 55-KDa receptor. This is the first evidence suggesting that TNFu binding to the two receptor types may not produce identical cellular responses. Dr. M. Brockhaus of Hoffman-La Roche generously provided the McAbs and the work was supported by the Arthritis Fi Rheumatism Council, UK.

174

E

ULTURED RABBIT ARTICULAR CHONDROCYTES (RAC) XPRESS DISTINCT TRANSFORMING GROWTH FACTOR-B ECEPTOR PHENOTYPES AS A FUNCTION OF CELL CYCLE

bIS+RIBUTION. D.Vivien, F. R6dini. SRonost, P.Galtra, G.Loyaa. J- p.Pujol. Laboratoire de Biochimie du Tissu Conjonctif, CHU Caen C&e de Nacre. 14033. France.

I” culture, TGF-El is a bifunctional regulator of RAC proliferation depending on the serum concentrations. In low level of semm (2% FCS). cycling cells are growth-inhibited by the peptide, whereas in high level of serum (10% FCS). TGF-5 transiently potentiates their growth. Cycling RAC in 10% and 2% FCS exhibited two distinct classes of high-affinity TGF-6 binding sites. However, in 2% FCS we show a significant increase (60%) of sites per cell for the highest affinity receptors (in 10% FCS: Kdl=280 pM, 3.100 sites/cell; Kd2=1,057 pM. 8,200 sites/cell: In 2% FCS: Kdl=346 pM. 4,900 sites/cell; Kd2=900 pM. 9,990 sites/cell). Cytofluorometric analyses of DNA content show that 55% of cells are in GO/l in 10% FCS versus 70% in 2% FCS. To get more insight into the receptor distribution in relation with the cell cycle phases, we have performed similar experiments in cells synchronized in late Gl or early S phase. Synchronized cells in 10% FCS (>80% of cells in S phase). exhibited a single class of TGF-5 high affinity receptor (Kdl=l.OEO pM, 5,772 sites/cell). In contrast, RAC cultured in the presence of 2% FCS (41% of cells in GO/l) expressed two distinct classes of binding sites (Kdl=314 pM, 2,664 sites/cell; Kd2=884 pM. 5,500 sites/cell). In addition, we have oberved no modification of the distribution of TGF-O receptors between sparse cycling cells and confluent cells.