496 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES

211 280

COMPLEMENT ACTIVATION DURING SUBCUTANEOUS kc.) IL-l RECEPTOR ANTAGONIST INHIBITS IL-l INDUCED CYTOKINE ADMINISTRATION OF RECOMBINANT INTERLEUKIN 2 llL2J IN PATIENTS SYNTHESIS AND THE BINDING OF IL-l TO THE TYPE II IL-1 RECEPTOR WITH SMALL CELL LUNG CANCER ISCLCI. Vipgiano V.. Coronelli S.. IN HUMAN MONOCYTES. EV Granowitz. BD Clark. E Vannier. MV Spinazze’ S., Rivoltini L. and Cicardi M. Dept. Clinical Medicine, University of Milan, 20142 Milan. and lstituto Nazionale Tumori, 20133 Milan (ItalyI.

Callahan. and CA Dinarello, New England Medical Center Hospitals and Tufts University School of Medicine, Boston, MA

In order to assess complement activation during IL2 administration, we analyzed the plasma levels of the complement fractions in 5 patients with SCLC who underwent to s.c. injections of IL2. All the patients previously achieved partial response after induction chemotherapy. The therapeutic schedule consisted of 9 x lo6 IU/m*/12 h for 2 days, followed by 3 x 10e IU/m*l12 h for 18 days (5 days/week of treatment). Every cycle was repeated in presence of response or stable disease. Plasma samples from these patients were obtained before therapy and 2-3 times weekly during the course of treatment. We measured the plasma levels of C3a. C3, C4, Cl INH (AQ and functional) and the functional activity of the alternative pathway IAP 50). All the patients had normal values at the beginning of the treatment and we observed a progressive increase of all the complement fractions analyzed. Peak levels were reached at the end of the first week of treatment. Furthermore, the levels of the complement fractions tended to remain above normal range even during the following weeks. The expression of complement receptor type 1 (CRl, CD351 on the surface of eosinophil granulocytes and lymphocytes was also enhanced, resembling the same panern observed for the complement fractions. None of the patients developed any of the side effects which are often observed during i.v. administration of IL2. These data suggest that the changes of the complement fractions observed could be related to an “acute phase proteins” response. No definitive signs of complement activation have been detected during this regimen of IL2 administration.

We investigated the ability of human recombinant IL-l receptor antagonist (IL-lra) to inhibit IL-l induced cytokine production in peripheral blood mononuclear cells (PBMC) and isolated monocytes. IL-lra alone at concentrations as high as 1 pg/ml did not induce IL-la, TNFcr, or IL-6 synthesis. A ten-fold molar excess of IL-lra over IL-lb reduced IL-l!3 induced IL-la by 95% (p=.Ol) and IL-lcx induced IL-l!3 by 73% (p<.OOl). The effect of IL-lra on IL-l!3 induced IL-la, TNFa, and IL-6 was dose-dependant with a two-fold molar excess of IL-lra inhibiting cytokine synthesis by SO%. A ten-fold molar excess of IL-lra added to PBMC 8 h after stimulation with IL-lp was still able to inhibit IL-la synthesis by 74% (p=.OOl). TNFa synthesis by 46% (p=.OOl), and IL-6 synthesis by 31% (p<.Ol). In PBMC treated with IL-lp alone, a similar reduction in IL-lp induced IL-la was obtained when IL-ID was removed from the media after 8 h of stimulation. In monocytes isolated by elutriation, a ten-fold molar excess of IL-lra over IL-ID reduced IL-lb induced IL-la by 82% (pc.05). TNFa by 64% (p=.OS), and IL-6 by 47% (pc.05). Monocytes were also incubated with 1251-IL-lb followed by cross-linking and immunoprecipation. SDS- PAGE demonstrated a band at 85 kD corresponding lo the 68 kD IL-1RtII. However, in the presence of excess unlabeled IL-ID or IL-lra, the 85 kD IL-l/IL-1RtII complex was not present. We conclude that IL-lra inhibits IL-l induced cytokine synthesis by blockine the bindine of IL-l to the IL-lRtI1 on human monocytes.

278

IL-3 MODULATES T AND B CELLS IN IRRADIATED MICE. A. Zingoni,

281

EXPRESSION OF INTRACELLULAR IL-1 RECEPTOR ANTAGONIST (icIL-lra) IN E. COLT. P. Ralph, V. Wittman. A. Sampson-Johannes, S. Fonq, I. Nakoinz, D. Liu, M. Breen, and R. Drummond Cetus CorDoration. Emervville. CA 94ficJR.

D. Frasca, M. Spano', G. Leter, G. Doria. --- ---- Division of Physics and Biomedical Sciences, ENEA C.R.E. Casaccia, Rome (Italy). Administration of murine recombinant IL-3 to sublethally irradiated mice induced in the thymus complete recovery of the cell count and mitotic responsiveness to Con A, as well as a decrease in CD4-CD& cells concomitant with an increase in CD4+CD8- cells. Also, in the spleen, the cell count and mitotic responsiveness to Con A and LPS as well as antibody response and helper activity were completely recovered by IL-3 treatment. These findings show that IL-3 induces differentiation and growth of thymocytes and full recovery of T and B cell functions in sublethally irradiated mice.

'rhe IL-i receptor antagonist is a secreted protein with 26% homology to IL-lb that binds to IL-1 receptors and blocks IL-1 signaling. The intracellular form is expressed mainly in epithelial cells. We introduced the PL promoter, crystal protein terminator, an ampicillin selectable marker and a temperature sensitive origin of replication. Induction of recombinant protein expression was accomplished by heat shock at 42' followed by 5 hr at 39'. icIL-lra accounted for about 20% of total cell protein and was in the soluble fraction of a cell sonicate. The purified protein specifically blocked IL-1 induced IL-2 production. IL-1 induced proliferation of murine T line DlO.G4, binding of monocytes to IL-1 stimulated endothelial cells, and IL-1 toxicity assayed on melanoma A375-S2 cells. Clinical grade icIL-lra showed partial protection of mice from lethal LPS challenge.

279

CONSTRUCTIONOFANINTERLEUKIN-QANTAGONIST J.PJ. Brakenhoff, F.D. de Han, V. Fontaine*, M. Hart, E.R. de Groat, J. Content* and LA Amden. Central Laboratory of the Netherlands Red Cmss Blood Transfusion Service, PO box 9190,lOOd AD, Amsterdam, The Netherlands; *Pasteur Institute, Brussels, Belgium. IL-6 is involved in the pathogen&s of various discascs. Antagonists of IL-6 might therefore be of therapeutic value. In previous studies we have identified two regions on the IL6 molecule important for bioactivity. recognized by two groups (I and II) of neutralizing monoclonal antibodies (mAb). When we compared the e&t of the group I and II mAb on bindine of ?+IL-6 to IL-6 recemors that are oresem on CESS cells (EBV transformed hum& B cells), f a lC@fold higher concent&ion of group II mAb 16 was required for 50% inhibition as compared fo group I mAb Bl. In inhibiting bioactivity however, mAb 16 was f four-fold more potent then mAb Bl. This suggested that site I, recognized by group I antibodies, is involved in binding m the 80 kDa chain of the IL-6 receptor, whereas site II is somehow involved in the interaction of the IL-6/80 kDa complex with the signal transducing chain gp130. These observations led to the hypothesis that site II mutants, that retain binding m the 80 kDa chain, but fail 10 induce the interaction of the IL-6/80 kDa complex with gp130, can act as IL-6 receptor antagonists. Group II mAb bind around IL6 residue Trp158. Random mutagenesis in the region around Trpl58 resulted in the isolation of a number of mutants, binding to group I-, but not to group II antibodies, that were biologically active as measured in the murine B9 hybridoma growth factor assay. Surprisingly, when tested on human CESS cells, two of the mutants failed lo induce IgGl production. One of these was capable of inhibiting the &Cl inducing activity of wild type IL6. Furthermore, this mutant inhibited IL-6 induced tibrinogen and Cl inhibitor synthesis by human HepG2 cells, whereas IFNy induced Cl inhibitor synthesis was unaffected.

282

MODULATION OF CELL PROLIFERATION AND CYTOKINE PRODUCTION IN ACUTE MYELOBLASTIC LEUKEMIA BY RECOMBINANT IL-1 RECEPTOR ANTAGONIST AND LACK OF ITS EXPRESSION BY LEUKEMIC CELLS, &Rambaldi’. M.Torcia’. S.BertpaL . . .F Vannier’,~~.Dinarello’ and F.Cozzolino’. Division9 di Ematologia Ospedali Riuniti e lstituto Mario Negri Bergamo. Department of Medicine Tufts University Boston. Clinica Medics General9 IV Universita di Firenze. An IL-1 inhibitor has been recently puriiied and cloned. This molecule bmds to the IL-1

receptor but has no IL-1 like activity fulfilling the characteristics of a pure Interleukin-1 receptor antagonist (IL-l ra). In this paper we studied the in vitro effect of human recombinant IL-lra on proliferationon of Acute Myelogenous Leukemia (AML) cells. Spontaneous as well as IL-1 stimulated AML proliferation was inhibited by values ranging from 30% to 95% by the addition of 50 nglml of recombinant IL-lra A dose dependent activity of IL-1 ra on leukemic growth was observed in all the cases studied with significant effects at concentrations as low as 1 nglml. fnteraction of IL-1 ra with high affinity IL-I receptors on AML cells was analyzed by radioligand binding studies. As expected. IL-1 ra could wmpete with IL-1 in bindlng experiments on AML cells. In the same cases, leukemic cell production of IL-la and GM-CSF was assessed, by radiolmmuno assays, in basal conditions or in the presence of exogenous recombinant IL-l-a or IL-In Culture supernatanis of unstimulated leukemic samples contained IL-lb and GM-CSF activity. The Incubation of the same leukemic blasts with IL-I ra was followed by reduction or dtsappearance of GM-CSF I” the cufture supernatants whereas the IL-l!3 productwn was only patlially modulated. Expression of IL-lra and IL-lb gene was investigated by Northern hybridization on 23 cases of freshly isolated, uncultured AML. Interestingly. constI1utws expression of the IL-lp gene was detected in 19 of 23 AML cases analayzed whereas only three of these express the IL-I RA mRNA. These results suggest that imbalanced secretion of IL-1 and its natural receptor antagonist could contribute 10 the unrestricted growth of AML cells.