IL-2-dependent proliferation of murine T cells requires expression of both the p55 and p70 subunits of the IL-2 receptor

An IL-4-dependent T cell clone (LD8) was isolated from the murine IL-2-dependent cytotoxic T cell line C30.1. This clone has lost the capacity to proliferate in response to IL-2 after long-term culture in IL-4. LD8 cells express the p70, but not the p55, subunit of the IL-2R on their cell surface. The number of p70 IL-2R molecules on LD8 cells is comparable with the number of high-affinity IL-2R on the parental C30.1 cell line. LD8 cells can efficiently internalize IL-2 through the p70 IL-2R subunit. Following stimulation by IL-2, LD8 cells up-regulate p70 IL-2R mRNA, but do not express p55 IL-2R mRNA. IL-2-dependent proliferation of LD8 cells was reconstituted after introduction and expression of a human p55 IL-2R cDNA. To further investigate the role of p70 IL-2R, we have measured IL-2-induced proliferation of C30.1 cells in the presence of three anti-p55 IL-2R mAb (5A2, PC61, and 7D4) that recognize different epitopes. Under the experimental conditions used, the combination of anti-p55 IL-2R mAb prevents the formation of high-affinity IL-2R, but does not affect the binding of IL-2 to p70 IL-2R or IL-2 internalization. However, these three mAb inhibit proliferation of C30.1 cells even in the presence of IL-2 concentrations sufficient to saturate p70 IL-2R. Together these results demonstrate that p70 IL-2R alone is not sufficient to transmit IL-2-induced growth signals and that formation of p55-p70 IL-2R complex is required for IL-2-dependent proliferation of murine T cells.     

Inhibition of interleukin 2 production by prostaglandin E2 is not absolute but depends on the strength of the stimulating signal

In view of the eminently important role of interleukin-2 (IL-2) in T-cell responses, and in view of reports about immune stimulatory effects of PGE2, we reinvestigated the question whether PGE2 inhibits IL-2 production. It was found that PGE2 does not inhibit IL-2 production in murine spleen cell cultures after optimal stimulation (5 micrograms/ml concanavalin A) but does inhibit at suboptimal stimulation conditions. The failure of PGE2 to inhibit IL-2 production at optimal concanavalin A concentration was demonstrated by two independent IL-2 assays namely by the co-stimulator assay and by the proliferation of IL-2-dependent T-cell clone W-2. Our observations indicated that the inhibitory effect of PGE2 depends on the strength of the stimulating signal. IL-2 production in cultures with 5 micrograms/ml concanavalin A was also not suppressed by PGE1, by prostaglandin D2, thromboxane B2 (T X B2), and prostaglandin F2.     

Interleukin-2 receptor beta chain as a possible target for low doses of mafosfamide

The 7-day cytotoxic lymphocytes (CTL) induced in mixed lymphocyte culture express only the chain of the interleukin-2 receptor (IL-2R). In the present study this fact has been confirmed in a murine semi-allogeneic system. The ability of low doses of mafosfamide (Mf) to affect IL-2-induced CTL proliferation has been demonstrated. It was also shown that IL-2 activated resting suppressor cells. The pretreatment of the suppressor cells with either monoclonal antibodies (mAbs) against the p75 chain of IL-2R, or with Mf abolished the suppressive effect of these cells. No restoration of the proliferative response occurred when the anti-IL-2Ralpha mAb had been used. Flow cytometry analysis of 7-day CTL was carried out with mAbs against the alpha and beta chains of IL-2R. CTL treatment with Mf inhibited anti-IL-2Rbeta mAb binding. It may be assumed that the anti-proliferative effects of Mf which have been demonstrated in this paper, were a result of blocking the IL-2R beta chain.     

A cell-free Salmonella typhimurium extract modulates interleukin-2 (IL-2) receptor expression but not IL-2-stimulated responses of murine splenic lymphocytes

Abstract In a previous study, we observed that a cell-free Salmonella typhimurium extract induced suppression of mitogen-induced T-cell proliferation and this suppression involved non-responsiveness of T-cells to interleukin-2 (IL-2). In this study, we found that a cell-free S. typhimurium extract modulated IL-2 receptor (IL-2R) expression on phytohemagglutinin (PHA)-stimulated murine spleen cells and this was a mechanism of T-cell non-responsiveness to IL-2, but did not affect IL-2 binding to IL-2R and the consequent responses. Western blotting using anti-phosphotyrosine antibodies showed that IL-2R-mediated tyrosine phosphorylation of protein substrates in PHA-activated murine splenic T-cells, which express a high-affinity IL-2R (α- and β-chains), was not affected by treatment with the S. typhimurium cell-free extract. Furthermore, PHA-activated spleen T-cells responded to recombinant IL-2 and this was not inhibited by the extract. Surprisingly, IL-2R expression was augmented by treatment with the extract, although this was independent of IL-2 production. These results suggest that the suppression of T-cell proliferation induced by the Salmonella cell-free extract was associated with augmentation of IL-2R expression, rather than down-regulation of the IL-2 response. This may be a mechanism responsible for the Salmonella extract-evoked suppression of mitogen-induced T-cell proliferation.     

Agents that mimic antigen receptor signaling inhibit proliferation of cloned murine T lymphocytes induced by IL-2

We have shown previously that stimulation of cloned murine T lymphocytes via the TCR inhibits their responsiveness to rIL-2. Signaling via the TCR is believed to result in a variety of biochemical events that include a rise in intracellular free calcium and activation (translocation) of protein kinase C. These two signals also can be generated by calcium ionophores, such as ionomycin, and by activators of protein kinase C, such as PMA. We report here that treatment of cloned murine T lymphocytes with PMA, ionomycin, or the combination led to a dose-dependent inhibition of IL-2-dependent proliferation but did not inhibit lymphokine secretion. Concentrations of PMA and ionomycin that maximally inhibited proliferation stimulated maximal lymphokine secretion and increased mitochondrial activity as assessed by measurement of cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium-bromide. Furthermore, PMA, ionomycin, the combination, or immobilized anti-CD3 mAb added after 12 to 16 h of culture with IL-2 could inhibit proliferation. These results demonstrate that PMA and ionomycin mimic stimulation of the TCR by high concentrations of immobilized anti-TCR mAb in that proliferation is inhibited and lymphokine secretion is induced. In addition, PMA or ionomycin could independently inhibit proliferation of some cells. These findings suggest that alternative mechanisms exist to regulate proliferation. Either increased levels of intracellular calcium or the physiologic events corresponding to those induced by PMA can inhibit IL-2-dependent replication of T lymphocytes.     

Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites

Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman’s disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients’ blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL- 6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.     

Spontaneous proliferation of HTLV-II-infected peripheral blood lymphocytes: HLA-DR-driven, IL-2-dependent response.

Peripheral blood lymphocytes obtained from HTLV-II-infected persons (n = 13) and cultured in the absence of exogenous stimulator demonstrated augmented spontaneous proliferation (17,672 +/- 5,498 cpm) when compared with cells from healthy donors (1,921 +/- 1,306 cpm). Removal of non-T population did not abrogate the proliferative response of patients' PBMC, suggesting that the proliferation is not related to the autologous mixed lymphocyte reaction. Addition of recombinant interleukin-2 (rIL-2; 0.1 U/ml) to spontaneously proliferating cultures from HTLV-II-infected persons resulted in a 3- to 4-fold increase in proliferation (61,985 +/- 16,003); in contrast, PBMC from controls demonstrated 38- to 42-fold increase in their proliferative capacity in response to rIL-2 (77,256 +/- 13,044). Antibodies to both IL-2 receptor and HLA-DR were able to inhibit the spontaneous proliferation of PBMC from HTLV-II-infected persons in a dose-dependent manner. Furthermore, addition of cyclosporin A, which preferentially blocks accumulation of IL-2 mRNA, also inhibited spontaneous proliferation in a dose-dependent manner. These observations suggest that the spontaneous proliferation of HTLV-II-infected PBMC is at least in part an HLA-DR-driven, IL-2-dependent event, which is not analogous to the AMLR.     

Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18

We established two monoclonal antibodies (mAbs) which specifically recognize human 'functionally inactive' recombinant IL-18, and IL-18 protein polymorphism was examined using human monocytes and macrophages (M phi). In 6 day GM-CSF-treated M phi, an 'inactive' IL-18-recognizing mAb 21 detected the IL-18 proform (24 kDa) and a 48-kDa protein, which were gradually increased concomitant with maturation stage. Majority of the 24- and 48-kDa forms were barely detectable with other mAbs recognizing 'active' IL-18. No reagents including Toll stimulators up-regulated these IL-18 populations in M phi. The 21-recognizable IL-18 species were separated using an anion-exchanger column and their IFN gamma-inducing activity was assessed with human lymphocytes plus IL-12. Virtually no as yet known activity was detected with these IL-18 species. After processed with M phi proteases, an 18-kDa form was generated to express the IFN gamma-inducing activity, although the activity was far weaker than that of control 'active' IL-18. These observations suggested that large amounts of various IL-18 species are produced with monocyte-M phi differentiation and most of these IL-18 species are functionally 'inactive' in terms of the reported IL-18 function even after proteolytic 18-kDa conversion.     

Mapping of biologically relevant sites on human IL-1 beta using monoclonal antibodies

mAb have been raised that recognize human IL-1 beta. Using overlapping peptide fragments expressed in yeast and bacteria, we have mapped the regions of the protein to which these antibodies bind. To assess the relevance of the different regions of IL-1 beta for the expression of its biologic activity, the ability of the antibodies to block IL-1 activity was assayed. Antibodies recognizing the regions 133-148 and 251-269 of human IL-1 beta could inhibit the activity of IL-1 beta, but not of IL-1 alpha, in two different biologic assays, the murine thymocyte proliferation and PGE2 release from human fibroblasts. Conversely, antibodies that recognize the region 218-243 have only a moderate inhibitory effect on the IL-1 beta biologic activity in both assays. Finally, an antibody mapping to the region 148-192 did not inhibit IL-1 beta activity either on thymocytes or on fibroblasts. It is suggested that IL-1 beta-induced cell activation involves different regions of the protein and that both N-terminal and C-terminal fragments are involved in the correct functioning of the IL-1 beta molecule.     

Genetic construction and characterization of a fusion protein consisting of a chimeric F(ab') with specificity for carcinomas and human IL-2.

A genetic construct was created incorporating gene fragments encoding the H chain V region of the human carcinoma specific antibody L6, the CH1 domain of human IgG1, a linker region, and human IL-2. This construct was cotransfected with a chimeric L6 L chain construct into the murine myeloma cell line Ag8.653 for expression. First round clones produced the fusion protein at an estimated 5 to 10 micrograms/ml based on idiotypic reactivity. Dual binding activity was demonstrated through specific interaction with the L6 Ag on human tumor cells and the IL-2R on activated human T cells. The IL-2 portion of the molecule was shown to support the growth of the IL-2-dependent T cell line CTLL2, and the qualitative nature of the IL-2 signal was found to be the same as rIL-2 with respect to induction of tyrosine-phosphorylation of intracellular protein substrates. Tumor cells coated with the fusion protein were shown to cause T cell proliferation and the presence of the fusion protein was found to enhance cell-mediated destruction of human tumor cells.     

Sandwich enzyme immunoassay for murine IL-3

A reproducible, sensitive immunoassay for murine interleukin-3 (IL-3) has been developed using two preparations of polyclonal antipeptide antibodies. Rabbits were immunized with the N-terminal peptide 1-29 (IL-3) coupled to KLH and the antibodies were affinity purified on immobilized peptide 1-29 (IL-3). This antibody preparation showed good reactivity with native IL-3, and was used to coat polyvinyl microtiter trays. IL-3 captured by this first antibody was detected by the addition of anti-IL-3 serum (second antibody) raised in sheep against synthetic full length IL-3 (1-140). This test reliably detects IL-3 from every source tested (T cells, WEHI-3B cells, recombinant material from transfected COS 7 cells or murine myeloid FDC-P1 cells transfected with an IL-3 containing retrovirus) with a sensitivity to 2 to 4 U/ml of bioactive IL-3 or about 60 pg synthetic IL-3/ml. The test is performed within 5 to 6 h compared to 2 to 3 d of a standard bioassay.     

Measurement and partial characterization of an interleukin-2 inhibitor (IL-2-IN) in human urine

We observed a human urine-derived protein complex (IL-2-IN) which competitively inhibits interleukin-2 (IL-2) dependent murine lymphocyte proliferation. Measurements of urinary IL-2-IN have been used to stratify the immune response of patients to bacteria in the bladder. Partial characterization of IL-2-IN indicates that it is a heat-stable, 75 kDa complex comprised of interleukin-2 bound to another protein(s). Although the IL-2-IN complex is stable in physiologic buffers, the complex can be disrupted using acidic or low-ionic strength buffers, thereby liberating IL-2. IL-2-IN activity is susceptible to bacterial and endogenous urinary proteolysis. The IL-2 bound in the IL-2-IN complex cannot be detected using a double monoclonal antibody radioimmunoassay for IL-2. Unlike other IL-2 binding proteins, the IL-2 binding protein of the IL-2-IN complex is not a soluble interleukin-2 receptor. A modification of the bioassay for interleukin-2 activity is the method of choice for the detection and quantification of urinary IL-2-IN.     

Modulation of IL-1 inflammatory and immunomodulatory properties by IL-6.

Among the major cytokines present in inflammatory lesions interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) share many biological activities. Since IL-1 alpha, IL-1 beta and TNF alpha have been previously demonstrated to play an important role in connective tissue destruction by stimulating the production of prostaglandin E2 (PGE2) and collagenase, these functions were investigated in the presence or absence of natural human IL-6 (nhIL-6) or recombinant human IL-6 (rhIL-6). IL-6 was found 1 degree to stimulate immunoglobulin A production by the CESS B cell line up to 19 fold without being affected by the presence of IL-1 beta and 2 degrees to stimulate murine thymocytes proliferation up to 2-4 fold, with an increase up to 60-fold in costimulation with either IL-1 alpha or beta. IL-6 alone, even at very high concentrations (up to 200 U/ml and 50 ng/ml), did not induce PGE2 production by fibroblasts and synovial cells. However, IL-1 alpha or beta induced PGE2 production by human dermal fibroblasts and by human synovial cells was inhibited (in 5/8 experiments) up to 62% by addition of IL-6. On the contrary in 2/4 experiments TNF alpha-induced PGE2 production was increased (approximately 2 fold) by the addition of IL-6. IL-1 and TNF alpha-induced collagenase production in synovial cells remained unchanged in the presence of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)     

The BCL1 B lymphoma responds to IL-4, IL-5, and GM-CSF

Proliferation in vitro of the in vivo passaged murine B cell tumor line BCL1 has been used as a standard assay for mouse interleukin-5 (IL-5) for a number of years. We demonstrate that this line will also respond to human IL-5. The response to murine IL-5 is abrogated by transforming growth factor-beta and to a lesser extent by interferon-gamma. This suggests a possible regulatory role for these lymphokines in the proliferation of B cells induced by IL-5. Other purified recombinant lymphokines were also tested for their ability to induce BCL1 proliferation. The lymphokines IL-1, IL-2, IL-3, and IL-6 had no effect on the growth of BCL1. In contrast, IL-4 and more surprisingly granulocyte-macrophage colony-stimulating factor (GM-CSF) also induced proliferation of this cell. These effects could be inhibited by specific antibodies directed against the respective lymphokines. These data suggest that GM-CSF, as well as IL-4 and IL-5, may be yet another regulator of neoplastic and possibly even normal B-cell growth and differentiation.     

A sensitive sandwich enzyme immunoassay for calcitonin gene-related peptide (CGRP): characterization and application.

Thirty mouse monoclonal antibodies (mAbs) directed against rat calcitonin gene-related peptide-alpha (CGRP-alpha) have been obtained. These mAbs are classified in 2 groups, one recognizing the peptide N-terminus and the other binding the C-terminus. A two-site immunometric assay was developed using mAb CGRP-83 as capture antibody, whereas mAb CGRP-72 acts as tracer, covalently labeled with enzyme acetylcholinesterase. This assay appeared sensitive (limit of detection: 2 pg/ml) and precise, allowing quantitative measurement of all human and murine CGRP isoforms. The assay was used to determine specific concentrations of CGRP in different rat, mice and guinea pig samples. The validity of the test was demonstrated by HPLC fractionation experiments.     

Identification of a novel protein capable of interacting with the IL-3 receptor

IL-3 has numerous functions in hematopoiesis yet its receptor has not been fully characterized. We have developed two mAb, 4G8 and 2F2, that markedly inhibited IL-3-dependent proliferation whereas only marginally affecting IL-2 or IL-4-induced proliferation. On Western blots, both antibodies identified the same protein, which varied in size from 115 to 145 kDa in six cell lines tested. The 4G8/2F2 Ag was detected at moderate density, on a wide variety of cells including IL-3-dependent cell lines and T lymphocytes. Radioligand binding studies revealed that 4G8, but not 2F2, could inhibit the binding of 125I-IL-3 to the high affinity IL-3R. These data suggest that the mAb 4G8 and 2F2 recognize different epitopes on the same Ag, and suggest furthermore that the inhibition of IL-3-dependent proliferation mediated by 2F2, in particular, does not occur via inhibition of ligand binding. Neither antibody showed an enhanced level of fluorescent staining of Cos 7 cells transfected with the low affinity IL-3R cDNA. In addition, 4G8 did not inhibit IL-3 binding to L cells transfected with the cloned IL-3R or IL-4R despite the fact that 4G8 was expressed on these cells. These data suggest that the 4G8/2F2 Ag is a unique cell surface protein that can interact with the endogenous functional IL-3R.     

Inhibition of IL 2-dependent proliferation of rat T lymphoblasts by the monoclonal antibody ART62 which reacts with MHC class 1 antigens

During the course of studies designed to obtain monoclonal antibodies (mAb) that recognize the rat interleukin 2 receptor, a mouse IgG1 mAb (ART62) was identified which inhibits the interleukin 2 (IL 2)-dependent proliferation of rat T lymphoblasts without affecting the binding of IL 2 to such cells. In order to characterize the cell surface components that react with the mAb ART62, T lymphoblasts were surface-labeled with 125I, and the radioactive molecules were immunoprecipitated by the antibody analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The mAb ART62 precipitated two major components of 48,000 m.w. and 12,000 m.w., respectively, which were different from those which react with the anti-IL 2-receptor antibody ART18, a molecule of 50,000 to 55,000 m.w. Sequential immunoprecipitation studies revealed that the mAb ART62 reacts with the MHC class 1 antigen that reacts with the classical anti-rat MHC class 1 mAb OX18, and vice versa. In contrast to the mAb ART62, OX18 that does not affect and several other mAbs known to inhibit the rat MLR failed to inhibit IL 2-dependent proliferation of rat T lymphoblasts. In contrast to the anti-IL 2 receptor antibody ART18, ART62 effectively inhibited IL 2-driven proliferation even when added to cells already committed to proliferate by IL 2-IL 2 receptor interaction. These data raise the possibility that MHC class 1 antigens could be involved in the chain of reactions mediating the signals required for cell proliferation.     

A conformation-sensitive monoclonal antibody against the A2 domain of von Willebrand factor reduces its proteolysis by ADAMTS13

The size of von Willebrand factor (VWF), controlled by ADAMTS13-dependent proteolysis, is associated with its hemostatic activity. Many factors regulate ADAMTS13-dependent VWF proteolysis through their interaction with VWF. These include coagulation factor VIII, platelet glycoprotein 1bα, and heparin sulfate, which accelerate the cleavage of VWF. Conversely, thrombospondin-1 decreases the rate of VWF proteolysis by ADAMTS13 by competing with ADAMTS13 for the A3 domain of VWF. To investigate whether murine monoclonal antibodies (mAbs) against human VWF affect the susceptibility of VWF to proteolysis by ADAMTS13 in vitro, eight mAbs to different domains of human VWF were used to evaluate the effects on VWF cleavage by ADAMTS13 under fluid shear stress and static/denaturing conditions. Additionally, the epitope of anti-VWF mAb (SZ34) was mapped using recombinant proteins in combination with enzyme-linked immunosorbent assay and Western blot analysis. The results indicate that mAb SZ34 inhibited proteolytic cleavage of VWF by ADAMTS13 in a concentration-dependent manner under fluid shear stress, but not under static/denaturing conditions. The binding epitope of SZ34 mAb is located between A1555 and G1595 in the central A2 domain of VWF. These data show that an anti-VWF mAb against the VWF-A2 domain (A1555-G1595) reduces the proteolytic cleavage of VWF by ADAMTS13 under shear stress, suggesting the role of this region in interaction with ADAMTS13.     

Functional comparison of recombinant interleukin 2 (IL-2) with IL-2-containing preparations derived from cultured cells

Due to its purity and potential availability in large amounts, human recombinant interleukin 2 (IL-2) expressed in Escherichia coli is an important source of IL-2 for experimentation and possible therapy. To date, very few comparisons between the activity of recombinant IL-2 and conventional cell-derived preparations of IL-2 have been made. This is particularly important since the use of recombinant IL-2 may have some specific limitations. For example, recombinant IL-2 is not post-translationally modified as are cell-derived preparations. Lack of modifications such as glycosylation of threonine 3 may alter efficacy or stability. Comparative studies are necessary to demonstrate the efficacy, species specificity, and stability of recombinant IL-2. By comparing IL-2 activity of recombinant IL-2 to that of cell-derived IL-2, we have demonstrated that each of the preparations are equally active in several murine and human IL-2 proliferation assays and that IL-2 is the active moiety in these assays. In contrast to previous reports, we also show that recombinant IL-2 is sufficient to establish and maintain long-term cell lines. Additionally, by using "synthetic" recombinant IL-2 of defined protein sequence, we have demonstrated that this amino acid-defined structure is indeed responsible for the functions attributed to IL-2.     

A monoclonal antibody recognizing 68- to 75-kilodalton protein(s) associated with the human IL-1 receptor

We produced an IgM mAb termed 4.9 against an EBV-containing lymphoblastoid cell line, termed 3B6. This mAb reacted with both various B and T cell lines such as HSB2 cells, with an NK-like cell line YT-C3 cells, and with human fibroblast MCR-5 cells. It also reacted with normal resting peripheral B lymphocytes, monocytes, and anti-CD2- or anti-CD3-activated T lymphocytes. The 4.9 mAb immunoprecipitated two bands estimated to be of Mr 68 and 75 kDa from iodinated 3B6 cells. The 4.9 mAb inhibited the proliferation of peripheral T lymphocytes induced either by anti-CD3 mAb or anti-CD2 mAb. The 4.9 mAb inhibited also the proliferation of murine thymocytes both in the presence of PHA and IL-1 and the proliferation of human fibroblasts in the presence of IL-1. Radiolabeled IL-1 binding on 3B6 cells revealed two types of IL-1 binding sites with high and low affinity for IL-1 (300 sites/cell with a Kd of 6 x 10(-11)M and 6000 sites/cell with a Kd of 3 x 10(-9)M). On both 3B6 and YT-C3 cells, mAb 4.9 inhibited specifically the binding of 125I-labeled rIL-1, alpha or beta, whereas the irrelevant IgM mAb did not. Conversely, rIL-1, alpha or beta, could inhibit specifically the binding of radioiodinated 4.9 mAb to 3B6 or YT-C3 cells, whereas rIL-2, rIFN, or the irrelevant IgM mAb were ineffective. 125I-4.9 mAb bound 3B6 cells with an association constant (Ka) of 2 x 10(8)/M and demonstrated 6000 binding sites/cell. We thus conclude that mAb 4.9 recognizes a protein complex (68 to 75 kDa) closely associated with the IL-1R.