SECOND INTERNATIONAL WORKSHOP ON CYTOKINES / 95

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LECTIN BINDING SUGGEST DIFFERENT GLYCOSYLATION PATTERNS OF IL-1 RECEPTORS EXPRESSED ON DIFFERENT CELLS. J. MancilIa, T. Ikejima, B.D. Clark, S.F. Orencole, S. Sirko, and C.A. Dinarello. Tufts University School of Medicine and New England Medical Center, Boston, MA 02111.

The wide variety of cellular responses to IL-l are mediated via specific membrane receptors (IL-lR), and these may possibly be modified by specific cell-recognition characteristics. To determine whether different types or degrees of membrane glycosylation patterns play a role in the IL-l/IL-IR interactions. we have done lectin-bindine assavs on the EL.4-6.1 T-cell line’(EL.4). and on an IL-I dep&dent ‘subclone (DIOS) of DlO.Ci4.1 cells. These assays used human recombinant 1251-IL-la and four lectins of different sugar specificities were studied: concanavalin A (Con A), peanut agglutinin (PNA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA). We observed that Con A blocked 70% of the binding of IL-l to both cell lines; PNA had no effect, whereas WGA blocked more than 90% of the binding of IL-1 to EL.4 and only 60% on DlOS (p < 0.05). and PHA blocked 70% on EL.4 and 30% on DIOS (p < 0.05). Additionally, we have observed that these lectins differentially alter, in a dose- dependent manner. the IL-la stimulated proliferation of DlOS cells. WGA produced an anti-mitogenic effect, whereas the other three lectins induced different degrees of mitogenesis. These results suggest different glycosylation patterns on IL-IRS are expressed on different cells.

134 EXPRESSION AND FUNmIONAL NIXXJIATION OF CD3- L%XF’NOCYTES BY THE IL-P NXXPTOR fi CH&IN. .I. Ortaldo. .I. Frey-Vasconcells, W. Farrar. T. Takeshita and K. Sueamura. LEI h LHI, NCI- FCRF, Frederick, MD 21701 and Tohoku University School of Medicine. Sendai. Jaoan.

The monoclonai antibody (MAb) TU27 recognizes the IL-2Ro chain ~75 orotein. We are studvine the abilitv of this MAb s . , I 2

to block the activation of CD3. lymphocytes by IL-2 and to directly activate the cells by binding to the IL-2R,9 chain. The IL-2R@ chain, based on binding of TU27 MAb and 12'I-IL-2 cross-linking, was found to be expressed primarily on CD3., Leul9' lymphocytes, with a small [and donor dependent] expression on CD3+ cells. TU27 blocked both augmentation of NK activity and induction of L4K activity. Interestingly. treatment of CD3- lymphocytes by MAb alone was able to augment NK activity and induce 1FN-j production, but was unable to induce L4K activity or cell growth. Subsequently we examined the ability of other cytokines to regulate expression of the IL-2R,9 chain on CD3- lymphocytes. Most cvtokines tested (IL-l. IL-3. IL-4. IL-6. IFN a/-i. TNFa) did

. , . . I

not directly modulate expression of the IL-2Rj3 chain based on reactivity with TU27. However, TCFB was able to diminish both the reactivity to TU27 and totally abrogate rhe IL-2- induced expression of ~55. Collectively, these results indicate that TU27 reacts with the p75 IL-2v, augments NK activity and IFNy production, and inhibits IL-2 binding and subsequent activation events in CD3* lymphocytes.

135

Specific Binding of lZSI-Labelled Human Recombinant IL-3 to Human Bone Marrow and Chr5Tic MpYelogenous Lqukemia Cells. . E.W. Palaszvnskl. Falk. sari alazxnsk’. R hard Schulo

and Paul ZQC&Y George WashiigtorUniversity f,

Medical Center, Washington, Dd 20037 and BRMP. NCI-FCRF. Frederick, MD 21701.

Interleukin-3 is a major hematopwitic factor of early stem cell progenitors in both human and murine systems. However, the human receptor has been difficult to characterize due to low expression of receptors, few cell lines available and human IL-3 appears to be sensitive to radiolabelling via conventional methods.

We have radiolabelled human recombinant IL-3 (HR-IL-31 with 1251 Na to sufficient specific activities (50 uCi/ug) which rendered the molecule biologically active as compared to unlabeled HR-IL-3 and assayed for growth of human bone marrow cells in soft agar. Minimal loss of bioactivity was observed after radiolabelling. The 1251-HR-IL-3 can be immune precipitated with a commercial anti-human IL-3 antibody (Genzyme). yielding titers of 1: 4500. We have also been successful in using this I251- HR-IL-3 as a receptor probe for the human IL-3 receptor using either freshly isolated human bone marrow cells or peripheral blood cells from patients diagnosed as having chronic myelogenous leukemia. Using conventional radioreceptor techniques, the radiolabelled IL-3 binds to these cells in a specific, time dependent and saturable manner.

We are currently screening additional cell sources to find a consistent high expressor for the human IL-3 receptor.

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This work was supported by NIH grant GM35483.

EVIDENCE FOR THE HUMAN B CELL GROWTH FACTOR RECPTOR AS A HETERO-TRIMER. C.G. Sahasrabuddhe, M. Gara and K. Furukawa. The University of Texas M.D.Anderson Cancer Center, Houston, TX 77030.

An intracellular 60kDa protein induces growth in factor dependent normal and neoplastic long term B cells. This growth factor dependent modulation was achieved through a cell surface receptor on the B cells. Presence of such a receptor was demonstrated by double antibody staining procedure. In this report, we present biochemical characteristics of this IC-BCGF receptor. Our results show that putative IC- BCGF receptor is a hetero-trimeric protein complex. The molecular masses of the three subunits, estimated by SDS-PAGE, are approximately 80 kDa, 50 kDa and 30 kDa. Further experimentation indicated that protein kinase activity associated with this trimeric complex phosphorylated 30 kDa subunit. This 30 kDa phosphorylated protein was specifically detected in B cell membrane proteins and was absent from activated T cell membrane proteins. These results suggested a possible role for protein-kinase as a second messenger in BCGF- receptor mediated growth signal transduction.

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EVIDENCE FOR ADDITIONAL SUBUNITS ASSOCIATED WITH THE MOUSE INTERLEUKIN-2 RECEPTOR / COMPLEX. Horatio Saragovi and Thomas R. Malek. Univ. of Miami School of Medicine, Miami, FI 7710 - . 1 - - - - _ .

High affinity interleukin-2 receptors (IL-2R) are composed of at least 2 distinct IL-Z binding subunits, the well known p55 (d) and the p75 (B). Recent cloning and transfection studies of p75 suggest that additional subunits may be required for competent expression and IL-Z binding capacity of ~75. In order to biochemically analyze murine IL-2 binding subunits we have used affinity precipitations with immobilized IL-2 to isolate IL-2R complexes. We now report that the murine IL-2R p75 exists as a disulfide-linked heterodimer with a novel subunit of Mr 22,000 (~22 or IL-2RY). These findings suggest that functional high affinity murine IL-2R are comprised of at least three distinct subunits. The possibility of an even more complex subunit structure is suggested by the co-precipitation of an additional protein of Mr 4D,OOO-45,000 (~40) that may represent the IL-LRc? subunit. The ~55, p75/p22 and p4D are non-covalently associated and were shown to be distinct by 2-Dimensional analysis and Cleveland mapping.

138

HOMOMGOUS AND HETEROLOGOUS DOWN-REGULATION OF THE IL-6 RECEPTOR. M. Schwabe. A.T. Brini and C.R. Faltvnek, LBP, LMI and BCDP-PRI. Biological Response Modifiers Proeram. NCI- Frederick Cancer Rerearch Facility, Frederick MD' 21jOl.

We characterized the human IL-6 receptor on a monocytic (U 937) and a B-cell line (U 266). U 937 cells expressed 6880 low affinity sites with a Kd of 480 pM. On U 266 cells we detected 7100 low affinity sites (Kd-210 PM) es well as high affinity sites with a Kd of 10 pM. Affinity-crosslinking experiments performed with iodinated IL-6 and subsequent analysis of the receptor complexes by SDS-PAGE under reducing conditions revealed a single band of 100 kD on U 937 cells. U 266 cells showed a doublet of 100 and 130 kD. IL-6 down- regulated its own receptor on both lines in a time and concentration dependent fashion. However, IFN-7 (100 U/ml) or TNF-o (100 U/ml) but not IL-1 (100 U/ml) led to a 40% (24 h) and 75% (48 h), decrease in IL-6 binding on U 937 cells. This phenomenon was due to loss of receptor number leaving the affinity unchanged. The heterologous down-regulation of the IL-6 receptor was specific since neither cytokine had an effect of the IFN-u receptor after 48 h. On the contrary, TPA treatment of U 266 cells for 48 h led to a selective loss of the 130 kD band with little effect on the 100 kD band as determined by affinity-crosslinking. We conclude that IFN-.I and TNF-a down-regulate the IL-6 receptor on monocytes whereas TPA selectively abrogates the high MW IL-6 receptor on B- cells.