The pathogenesis and progression of rheumatoid arthritis involves the production of biologically active lymphokines and monokines. Of these, interleukin 1 (IL-1) has been somewhat of a controversial molecule because it seems to evoke various biological responses in several different tissues. In these studies we demonstrate that three biological properties of human monocyte-derived IL-i (T-lymphocyte activation and human synovial cell prostaglandin E2 and collagenase production) co-purify. The complementary DNA for the prominent pI 7 form of human IL-1 was expressed, purified, and tested. Any controversy now appears resolved since homogeneous recombinant human IL-1 stimulates prostaglandin E2 and collagenase from human synovial cells as well as activates T cells in vitro.

The high-level expression of human interleukin-la in Escherichiu coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion-and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N-and C-terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vifro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1 /LAF) assay. The specific activities determined with the IL-1/MCF and IL-l/ LAF assays, are 2 x lo7 and 4 x lo7 units mg-', respectively.The term interleukin-1 (IL-1) applies to at least two cytokines which play a role in immunity and in the pathophysiology of inflammation [I -31. Preparations of authentic IL-1 tend to be contaminated with other cytokine mediators which can complicate the interpretation of sensitive IL-1 bioassays (see, for example [4]). The availability of IL-1 produced by recombinant DNA technology would eliminate the problem of potential contamination and provide a reliable source of well characterized cytokine.Recently, the coding sequences for human IL-la and have been cloned from peripheral blood monocytes and their expressed products shown to possess IL-1 activities [5, 61. Using published sequence data we have isolated clones of IL-lP cDNA from a library constructed from mRNA expressed in human histiocytic lymphoma U937 cells. Here we report the expression of the mature IL-la protein in Escherichiu coli, a method for its purification, and the physiochemical and biological properties of the purified protein. MATERIALS AND METHODS Bacterial hosts and plusmids

A better method was developed for analysis and identification of protein and DNA components of gel-shift assays. The protein-DNA complexes, separated in polyacrylamide gels, were transferred onto stacked nitroceDulose and anion-exchange membranes. The proteins, bound to nitrocellulose, were identified by immunoblotting, while the DNA, which bound only to the anion-exchange membrane, was detected by autoradiography. The technique readily identified thyroid hormone receptors interacting with response elements representing inverted or direct repeats of the consensus halfsite AGGTCA. In addition, specific antisera identified both the thyroid hormone and the retinoic acid receptors in het-

The effects of thyroid hormone agonists on thyroid hormone receptor (TR)/DNA complex formation was investigated to elucidate the mechanism by which TRs transactivate genes in response to ligand. The data, obtained from gel shift experiments, indicate that thyroid hormones alter the conformation of TRs bound to DNA, irrespective of if the element is occupied by monomeric TR, homodimeric TR/TR, or heterodimeric complexes with the retinoid receptors RAR or RXR. Furthermore, triiodo-thyronine (T3) prevents 2 TR molecules from binding to oligonucleotides containing direct repeats or inverted palindromes of the consensus AGGTCA motif, an effect that was not detected with palindromic elements. Heterodimers bound to direct repeats were less affected: RXR/TR were fully and RAR/TR complexes partially resistant to thyroid hormone. The data suggest that a ligand-induced conformational change in TR prevents double TR occupancy of a response element containing 2 direct repeats of the consensus binding motif, possibly by steric hindrance, whereas such an event does not prevent TR/RXR heterodimers from binding to DNA. Finally, our data show that a monomeric, liganded TR bound preferentially to the second half site in a AGGTCActcaAGGTCA element, and therefore indicate that nucleotides adjacent to the consensus half site contribute to binding specificity.

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