Recombinant human interleukin 1 receptor antagonist (IL-1ra) and 35F5, a neutralizing monoclonal antibody (mAb) to the type I mouse IL-1 receptor, were examined for their ability to bind to IL-1 receptors (IL- 1Rs) on various types of mouse cells and to block immune and inflammatory responses to IL-1 in vitro and in mice. IL-1ra competed for binding of 125I-IL-1 alpha to type I IL-1R present on EL-4 thymoma cells, 3T3 fibroblasts, hepatocytes, and Chinese hamster ovary cells expressing recombinant mouse type I IL-1R. The IC50 values for IL-1ra binding (ranging from 2 to 4 ng/ml) were similar to those of IL-1 alpha. In contrast, IL-1ra bound with very low affinity (IC50 values ranging from 10 to 200 micrograms/ml) to cells expressing type II IL- 1R, i.e., 70Z/3 pre-B cell line and polymorphonuclear leukocytes (PMN) derived from bone marrow and acute inflammatory exudates. The mAb 35F5 bound specifically to type I IL-1R; no inhibition of 125I-IL-1 alpha binding to cells having type II IL-1R was observed with very high concentrations of antibody. While neither IL-1ra nor 35F5 had intrinsic activity in bioassays using T helper D10.G4.1 cells and mouse thymocytes, both agents blocked the ability of IL-1 to stimulate proliferation of these cells. The effects of IL-1ra and 35F5 on acute inflammatory responses in mice were also evaluated. IL-1ra and 35F5 blocked the local accumulation of PMN after intraperitoneal injection of rIL-1 alpha. The response to IL-1 was inhibited when IL-1ra or 35F5 was administered simultaneously with or before administration of IL-1. IL-1ra and 35F5 also blocked PMN accumulation after intraperitoneal injection of lipopolysaccharide or proteose peptone, suggesting IL-1 is important in mediating responses to these agents. In addition, IL-1ra and 35F5 significantly blocked the ability of IL-1 to stimulate egress of PMN from bone marrow, to induce a transient neutrophilia, and to elevate serum levels of hepatic acute phase proteins, IL-6, and corticosterone. Thus, IL-1ra and 35F5 competitively inhibit the binding of IL-1 to the IL-1R on certain cell types. These two IL-1 receptor antagonists act to inhibit biological responses induced by IL-1 and other inflammatory agents.

Interleukin 1 (IL-1) is a polypeptide hormone that mediates a broad range ofbiological activities and interacts with surface receptors on numerous cell types. Equilibrium binding studies have identified a class of IL-1 receptors on T cells, fibroblasts, and epithelial cells that have 2-to 5-fold higher affinity than the receptors on bone marrow cells, pre-B cells, and macrophage cell lines. Affinity cross-linking with human 125I-labeled IL-la (125I-IL-1a) labels an .100-kDa protein on T cells and fibroblasts and an "80-kDa protein on pre-B cells and macrophage cell lines. Monoclonal and polyclonal antibodies specific for the IL-1 receptor on T cells and fibroblasts block human 125I-IL-la binding to T cells, fibroblasts, and epithelial cells but cannot block IL-i binding to bone marrow cells, pre-B cells, and macrophages. These antibodies immunoprecipitate the IL-1 receptor-human 125I-IL-la complex from T cells and fibroblasts but not from pre-B cells and macrophage cell lines. An S1 nuclease protection assay demonstrated that T cells and fibroblasts contain identical IL-1 receptor mRNA but that pre-B cells and macrophages do not contain this receptor mRNA. Taken together, the data demonstrate that mouse T cells, fibroblasts, and epithelial cells express an identical IL-1 receptor, whereas the IL-i receptor on pre-B cells, macrophages, and bone marrow cells represents a different gene product.Interleukin 1 proteins (IL-la and IL-13) act on a variety of cell types and have multiple biological activities (1-12). The diversity of IL-1 action is mediated by specific receptors on membranes of mouse (3, 13-17), human (17-21), rat (13), and porcine (21) cells. The binding of IL-1 is specific and saturable and occurs with high affinity (5-50 x 10-11 M) on many cell types, including T cells (3, 15-17), B cells (17, 18), fibroblasts (3, 16, 17), macrophages (22), and neutrophils (23). Both IL-la and IL-1p bind to the same receptor site on mouse (15, 24) and human (18) cells.Analysis of the structure of the IL-1 receptor by affinity cross-linking techniques identified an :80-kDa cell membrane protein on both mouse (3, 13, 17) and human (17,18) cells. An -'80-kDa IL-1 binding protein has been purified to homogeneity from the mouse EL4 thymoma cell line (25, 26), and a cDNA that encodes this protein has been isolated by expression cloning techniques from mRNA from these cells (27). An identical mRNA has been detected in 3T3-Swiss cells (27), which indicates that both fibroblasts and T cells express the same IL-1 receptor. The purified natural 80-kDa receptor from mouse EL4 cells and the recombinant EL4 type IL-1 receptor expressed in COS cells bind radiolabeled IL-1 with an affinity equal to the affinity of the cell-bound IL-1 receptor (ref. 27; R.C., P.L.K., and U.G., unpublished observations). These results indicate that a single recombinant polypeptide can duplicate the high-affinity binding of the natural membrane-bound EL4 IL-1 receptor.However, it is not known whether this 80-kDa receptor protein mediates all th...

Interleukin 1 (IL-1) receptor antagonist (IL-

Human interleukin 113 (IL-113) exerts its diverse biological effects by binding to specific receptors on target cells. Two ypes of IL-1 receptor (IL-1R) have been identified: the type I IL-1R (p80) and the type II IL-1R (p68). Using site-specific mutagenesis, we have identified the binding site on IL-11 for the murine type I IL-1R. Analogs of the IL-1fL protein containing defined amino acid substitutions were produced and tested for competitive binding to the two IL-1Rs. Substitutions of the amino acids at seven positions resulted in analogs that had .100-fold reductions in competitive binding to the type I IL-1R, while maintaining substantial binding to the type II IL-1R. These seven amino acids (Arg-4, Leu-6, Phe-46, Ile-56, are clustered in the IL-13 molecule, forming a discontinuous binding site. The side chains of all seven residues are exposed on the surface of IL-14. The cumulative binding energies contributed by each of the residues predict a binding affinity that is consistent with the observed Kd of the wild-type protein for the type I IL-1R.Human interleukins la and 1,B (IL-la and IL-1*3) are polypeptide hormones that share limited identity in amino acid sequence but are functionally similar and exhibit a multiplicity of biological activities. Their diverse biological effects indicate that IL-1 is one of the major mediators in the regulation of immunologic and inflammatory responses (1-3). Both IL-la and IL-1p8 bind to the same receptors on different cell types with high affinity (4-6). Two types of IL-1 receptor (IL-lR) have been identified. The IL-lR that is present on T cells is an 80-kDa glycoprotein that has been molecularly cloned from mouse and human cells (7,8 MATERIALS AND METHODSBacterial Strains and Plasmids. The Escherichia coli expression plasmid for human IL-1f, pEV1-IL-lbeta, has been described (27). Oligonucleotide-directed mutagenesis was performed as described (27). For each mutation, a specific restriction site was included in the synthetic oligonucleotide to monitor the incorporation of the mutated sequence at the correct location in the IL-1p8 coding region. Mutations were confirmed by nucleotide sequence analysis as described (28).Preparation of IL-1(3 Analog Proteins. Crude extracts. Bacterial cells containing the wild-type or mutated IL-1p3 expression plasmids were grown at 300C to early logarithmic phase (OD6w = 0.5) in M9 medium. The cultures were then induced for protein synthesis by transfer to 420C for --2 h, until OD600 = 1.0 ± 0.1. Samples of the cultures were then pelleted by centrifugation at 12,000 x g for 20 min at 40C. Extracts were made from the cell pellets by vigorous Vortex mixing and heating at 1000C in Laemmli sample buffer (29). The extracted proteins were analyzed by SDS/PAGE to assess the size and levels of expression of the IL-1.3 analog proteins, relative to that of wild-type IL-1,3 induced in parallel cultures.Purified proteins. Pellets of E. coli cells induced for 8 synthesis were disrupted by sonication and then adjusted to pH 4 with 6 M HCL....

Previous studies suggest that prostaglandin E2 (PGE2) is important in normal endometrial function and that it may be involved in certain uterine dysfunctions. In this study, the ability of the purified E. coli-derived recombinant interleukin-1 alpha (rIL-1 alpha) to regulate the production of PGE2 by the endometrial epithelium is investigated. PGE2 levels determined by the RIA consistently increase upon incubation of cultured glandular epithelial cells with rIL-1 alpha. A significant increase is obtained with 17 and 170 ng/L rIL-1 alpha. A maximal effect is obtained within 24 h of incubation with rIL-1 alpha. In the seven endometria evaluated, rIL-1 alpha increases PGE2 synthesis in all cases, but the maximal increase relative to the basal levels varies between 2- to 10-fold for a given preparation. Vibratome sections retaining the integrity of the endometrial tissue also show an increased PGE2 synthesis in response to rIL-1 alpha. rIL-1 alpha-stimulated PGE2 production is blocked by the addition of a neutralizing antibody to rIL-1 alpha. In addition, indomethacin, a cyclooxygenase inhibitor, suppresses both rIL-1 alpha-induced and basal PGE2 synthesis. Experiments using radioiodinated rIL-1 alpha reveal that the membranes prepared from human endometrial epithelium possess high affinity receptors for IL-1, suggesting that IL-1 regulates PGE2 synthesis by binding to this receptor site. The expression of IL-1 receptors and the ability to modulate PGE2 production by IL-1 in endometrial epithelium suggest that IL-1 may play a significant role in human uterine function via modulation of PGE2 production.