We have identified a cDNA from a human phytohemagglutinin-activated lymphoblast library encoding a protein that binds 125

Cytotoxic lymphocyte maturation factor (CLMF) is a disulfide-bonded heterodimeric lymphokine that (i) acts as a growth factor for activated T cells independent of interleukin 2 and (ii) synergizes with suboptimal concentrations of interleukin 2 to induce lymphokine-activated killer cells. We now report the cloning and expression of both human CLMF subunit cDNAs from a lymphoblastoid B-cell line, NC-37. The two subunits represent two distinct and unrelated gene products whose mRNAs are coordinately induced upon activation of NC-37 cells. Coexpression of the two subunit cDNAs in COS cells is necessary for the secretion of biologically active CLMF; COS cells transfected with either subunit cDNA alone do not secrete bioactive CLMF. Recombinant CLMF expressed in mammalian cells displays biologic activities essentially identical to natural CLMF, and its activities can be neutralized by monoclonal antibodies prepared against natural CLMF. Since this heterodimeric protein displays the properties of an interleukin, we propose that CLMF be given the designation interleukin 12.The molecular cloning and expression of recombinant cytokines has made possible both significant advances in our understanding of the molecular basis of immune responses and the development of new approaches to the treatment of disease states. As an example, recombinant interleukin 2 (recombinant IL-2) has been shown to be capable of causing regression of established tumors in both experimental animals (1) and in man (2); however, its clinical use has been associated with significant toxicity (2). One potential approach to improving the therapeutic utility of recombinant cytokines is to use them in combination (3,4 MATERIALS AND METHODScDNA Cloning. A subline of NC-37 cells selected for its ability to produce high levels of CLMF (7), NC-37.98, was induced with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 for 16 hr. Poly(A)+ RNA was isolated, and random hexamer-primed cDNA libraries were established in phage AgtlO by standard procedures. Mixedprimer polymerase chain reaction (PCR) using controlled ramp times (8) was performed as follows. PCR primers contained all possible codons and were 14 or 15 nucleotides long ( Fig. 1) with a 5' extension of 9 nucleotides containing an EcoRI site for subcloning. Degeneracies varied from 1 in 32 to 1 in 4096; 0.5-4 pmol per permutation of forward and reverse primer was used in a 50-to 100-,lI PCR mixture with 40 ng of cDNA made from NC-37.98 cells that had been activated by culture with 10 ng of PMA and 25 ng of calcium ionophore A23187 per ml for 16 hr (40-kDa subunit) or with 3 ,tg of human genomic DNA (35-kDa subunit). PCR cycling parameters were as follows. Initial denaturation was at 95°C for 7 min. Low-stringency annealing was performed by cooling to 37°C over 2 min, incubating 2 min at 37°C, heating to 72°C over 2.5 min, extending at 72°C for 1.5 min, heating to 95°C over 1 min, and denaturing at 95°C for 1 min. This cycle was repeated once. Thirty standard cycles (40-kDa subun...

Interleukin-1 (IL-1), a peptide hormone produced by activated macrophages, possesses the ability to modulate the proliferation, maturation and functional activation of a broad spectrum of cell types and may play a major role in the initiation and amplification of immune and inflammatory responses through its action on these diverse cell populations. IL-1 exhibits microheterogeneity in terms of its relative molecular mass (Mr, 13,000-19,000) and charge properties, and although murine IL-1 has been purified and some of its basic structure-function relationships have been elucidated, it has proved difficult to prepare sufficient amounts of IL-1 for direct and detailed sequence and structural studies. Here we report the cloning, sequence analysis and expression of murine IL-1 cDNA in Escherichia coli. The IL-1 cDNA codes for a polypeptide precursor of 270 amino acids. Biologically active IL-1 was produced in E. coli by expressing the carboxy-terminal 156 amino acids of the IL-1 precursor.

Interleukin 1 (IL-1) is a family of polypeptides initially found to be produced by activated monocytes and macrophages that mediate a wide variety of cellular responses to injury and infection. Epidermal epithelial cells (keratinocytes) produce "epidermal cell-derived thymocyte activating factor" or ETAF, which has been recently shown to be identical to IL-1. Human epidermis is normally exposed to significant amounts of solar ultraviolet radiation. Certain ultraviolet wavelengths (UVB, 290-320 nm) are thought to be responsible for most of the immediate and long-term pathological consequences of excessive exposure to sunlight. In this study, we asked whether exposure to UVB irradiation induced IL-1 gene expression in cultured human keratinocytes. Cultured human keratinocytes contain detectable amounts of IL-i a and ft mRNA and protein in the absence of apparent stimulation; these levels could be significantly enhanced 6 h after exposure to 10 ng/ml of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Exposure to UVB irradiation with an emission spectrum comparable to that of sunlight (as opposed to that of an unfiltered artificial UV light source) significantly increased the steady state levels IL-la and , mRNA in identical populations of human keratinocytes. This was reflected in the production of increased IL-i activity by these cultures in vitro. In the same cell population, exposures to UVB irradiation did not alter the level of actin mRNA; therefore, the effect of UV irradiation on IL-1 represents a specific enhancement of IL-1 gene expression. Local increases of IL-1 may mediate the inflammation and vasodilation characteristic of acute UVB inured skin, and systemic release ofthis epidermal IL-1 may account for fever, leukocytosis, and the acute phase response seen after excessive sun exposure.

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...