THIRD INTERNATIONAL WORKSHOP ON CYTOKINES / 479

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SPECIES SPECIFICITY OF IL-l RECEPTOR BINDING Klaus Vosbeck, Urs Joss, Albert Schmitz, and Jan van Oostrum, Ciba-Geigy Ltd., Pharmaceuticals Division, CH-4002 Basel, Switzerland

Single point mutants of human recombinant IL-lbeta (hrIL-lbe- ta) were obtained by site directed mutagenesis. They were tested for specific binding to the mouse thymoma cell line, EL-4.61, and to human foreskin fibroblasts using a competi- tion assay with radiolabelled human IL-lalpha or IL-lbeta. Their functional activity was assessed by measuring their ability to induce the formation of PGEE in mouse connective tissue fibroblasts, as well as in human foreskin fibroblasts. Two point mutants (Arg120 to Ser, and Glu221 to Lys) were identified that exhibited 40- and loo-fold reduced binding to the mouse, but only 5-fold reduced binding to the human receptor as compared to wild type hrIL-lbeta. PGE2 induction in human and mouse fibroblasts also exhibited a ten-fold difference that paralleled the decrease in receptor binding. Another mutant (24461~ to Lys) showed a 50-fold reduction in both human and mouse receptor binding and PGEE induction, whereas several other mutants were indistinguishable from wild type hrIL-lbeta in all four assays. These data suggest species differences in receptor recognition of the mouse and human type I (MW 80'000) IL-l receptor.

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EXPRESSION OF P55 AND P75 TNF RECEPTORS ON NEOPLAS- TIC CELLS FROM PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA. A. Waage, N. Liabakk, 3. Lamvik, T.Espevik Section of Haematology, and Institute of Cancer Research, University of Trondheim, N-7006 Trondheim, Norway.

Peripheral blood mononuclear cells (PBMC) separa- ted from 15 patients with chronic lymphocytic leuke- mia (CLL) were studied by flow cytometry and anti- bodies against ~55 and ~75 TNF receptors (htr-9 and utr-1, Hoffman-La Roche, Base11 and CD19 (Beckton Dickinson). The disease was staged from O-IV with duration from 0 to 12 years in individual patients. In patients with CLL ~55 was found in 31% of the cells (range O-77%) and ~75 in 44% of the cells (range O-81%). In healthy subjects the correspon-

ding percentages were 4 and 8%. CD19 positive cells were purified by positive selection with monodis- perse magnetic beads (Dynabeads). Both CD19 positive cells and CD19 depleted cells expressed TNF recep- tors. In in vitro cultures of CD19 positive cells, TNF stimulated to proliferation in all patients tested. Soluble TNF receptors were also studied in serum and urine from the patients. The results demonstrate that both types of TNF receptors are increased on cells from CLL patients and that TNF stimulates the cells to proliferate.

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STRUCI’URE AND FUNCTION OF MIJRINE IW AND GM-CSP RECEPTOR COMPLEXES. SYonehara. A.Ishii M.Yonehara. S.Kovasu and LYahara. The Tokyo Metro.Iwt.Med.Sci., Tokyo 113.

We have isolated the marine IL-3R gene, AIC2A, by using anti-Aic2 mAb. Aic2A protein, encoded by AIC2A gene, bids IL3 with low aftin- ity AIc2B gene has also been isolate+, which encodes a protein (Aic- ZB) 91% identical to Aic2A at amino acid IeveL Aic2B bids anti- AicY& but does not bid IL3. To examine functions of these proteins, we generated specitic mAbs agaiost Aic2A and Aic2B. Recombiiaot solu- ble form of Aic2.4 sod Aic2El were expressed in COS7 cells and purified from the supematants. Three mAbs (Hcd, Hdd and HDF) were obtained from annenian hamsters bmnudzed with the soluble A&A and Aic75. Hcd bids both Aic2A and Aic2B, and inhibits &3-biding to mouse mast cell progenitor cell line, IC2. Hdd binds Aic2.4 but not Ai&B, and inhibits IL3-biiding to ICZ. HDF bids AicZB but not Aic2.4 and inhibits GM-CSF-binding to IC2. Hcd and Hdd inhibit ILf-dependent proliferation of IC.2, but have no effed on GM-CSF-dependent prolif- eration of ICZ. In contrast, HDP inhibits GM-CSF-dependent prolifera- tion, but has no effect oo IL3-dependent proliferation. These results indicate that Aic2A and AicZB are the major biiding components of IL- 3R and GM-CSFR, respectively. Hdd immunoprezipitatea three cell sur- face proteins of lZOk @i&A), 95k and 63k from ICZ. HDF immonopre- cipitated a different set of proteins of 130k @i&B), 98k and 75k. The present study together with previous cross-linking studies suggest that 1ZOk @i&A) and 63k proteins are ligand-biding components of IL-3R, and l30k (Aic2.B) and 7.5kd proteins ax. those of GM-CSFR. The 95k and 98k proteins may be the third components of IL3R and GM-CSFR, respectively.

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FUNCTIONAL AND COMPARATIVE ANALYSIS OF THE CYTOPIASMIC DOMAIN OF THE HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR RECEPI’OR. Steve” F. Zieeler. Ten-~ L. fianklin.

WCraie Immunex Corp.,Seattle, WA98101. Two cDNAs encoding human GCSF receptor differing only in the

cytoplasmic domain have previously been isolated from a human placental cDNA library (Larsen et al., 1990. J. Exp. Med. 172:1559). An analysis of the human GCSF-R gene showed that the difference was due to differential RNA splicing of a cryptic intro” within the coding region of the gene. Transfection of either cDNA in the murine IL-B-dependent line B9 or the mutine IL&dependent line BAF/BOB resulted in the expression of high-affinity GCSF receptors and conferred G-CSF-responsiveness. Deletional analysis of the cytoplasmic domain of the human G-CSF-R has revealed a region required for signal transduction. Within the hematopoietin receptor family the GCSF-Rismostcloselyrelatedtothe IL-6-R subunitgp130 and the recently cloned LIF-R (Gearing et al., submitted). We have constructed a number of cbimeric receptors using the extracellular and cytoplasmic domains of these three molecules. The chimeric receptors will be expressed in BAF/BOB cells in order to study the relationships between the signal transduction pathways utilized by each receptor.

THE BIOLCGICRL MECHANISMS OF TGF+ INHIBITION OF CYTOKINE INDUCED PROLIFERATION ARE STRICTLY TIME DEPENDENT, INVOLVE THE SLOWING OF CELL-CYCLE PROGRESSION AND THE DOWN REGULATION OF CYTOKINE RECEPTORS. , T Huckle, C H Wadhwa Bird, R Thorpe and A R Mire-Sluis. Div. of Immunobiology, NIBS?, Blanche Lane, South Mimms, Heats, EN6 3pC, UK. Transforming growth factor R, (TGF-O,) is a widely distributed 2SK homodimer that has been shown to inhibit the cytokine induced proliferation and activation of many haemopoetic cell lines although the mechanisms by which this process occurs remain obscure. Using the leukemic cell lines TFl and NO7e we show that the inhibitory effects of TGF-OI are strictly time dependent, whereby preincubation of cells with TGF-D, prior to addition of cytokine enhances the level of inhibition of "roliferatio". If cells are washed immediately after con&t with TGF-R,, the subsequent inhibitory response is only slightly reduced. This data stronolv suooeets that TGF-R. bindina to its receptors at a" early- stag;- is all that 'is required to initiate its inhibitory effects and that it is not required throughout to maintain inhibition. Therefore TGF-R, can act on the early signalling events that control cytokine action and we have evidence t" show that one such mechanism is the down regulation of cytokine receptors from the cell surface. Addition of TGF-B, up t" 44 hours after addition of cytokine can still result in inhibition of cytokine activity suggesting that TGF-R, can act both on very early and very late events involved in cytokine activity. Flow cytometric analysis together with 3[H)thymidine incorporation show that TGF-R does not alter the compartmentalisation of the cell lines in any one phase of the cell cycle, but slows the progression of cells through all phases of the cell cycle.

THE PNELIMOTOXICANT PARAQUAT INDUCES IL-8 GENE EXPRESSION IN HUMAN MONOCYTES AND PULMONARY EPITHELIAL CELLS. M. Bianchi, R. Bertini, G. Fantuzzi, L. Perin. M. -Salmona and P. Gbezzi. "Mario Negri" Institute for Pharmacological Research, 20157 Milan, Italy.

Paraquat (PQ) is an herbicide markedly pneumotoxic, indu- cing pulmonary edema and fibrosis via a free radical mechanism. Previous studies had shown that a" oxidative stress (e.g. is- chemia/reperfusion) induces IL-8. We therefore studied the ef- fect of PQ on IL-8 gene expression in human peripheral blood mononuclear cells in vitro. PQ (100 uM) induced high levels of IL-8 mRNA with a maximum at 24h. PQ, while not inducing IL-6 bioactivity (neutrophil chemotaxis) per se, potentiated LPS- induced production of chemotactic activity. PQ-induced IL-8 mRNA was not inhibited by cicloheximide, suggesting a direct transcriptional effect of PQ. IL-4 and gamma-IFN inhibited this effect of PQ, while indomethacin was uneffective. PQ also induced IL-~ mRNA in the human pulmonary epithelial cell line A549. Stimulated production of neutrophil chemotactic factors like IL-8 might be important in the fibrogenic and pneumotoxic action of PQ.