Human monocytes induced with adherent IgG secrete an interleukin-1 receptor antagonist which could be important for the in vivo regulation of IL-1 activity. A complementary DNA for this molecule has been isolated from a human monocyte library. Analysis of monocyte RNA indicates that the gene is transcriptionally regulated. The sequence of the receptor antagonist indicates that it is structurally similar to IL-1 beta. Expression of the cDNA in Escherichia coli yields IL-1 receptor antagonist activity.

Tumor necrosis factor-␣ (TNF) is initially expressed as a 26-kDa membrane-bound precusor protein (pro-TNF) that is shed proteolytically from the cell surface, releasing soluble 17-kDa TNF. We have identified human ADAM 10 (HuAD10) from THP-1 membrane extracts as a metalloprotease that specifically clips a peptide substrate spanning the authentic cleavage site between Ala 76 and Val 77 in pro-TNF. To confirm that HuAD10 has TNF processing activity, we cloned, expressed, and purified an active, truncated form of HuAD10. Characterization of recombinant HuAD10 (rHuAD10) suggests that this enzyme has many of the properties (i.e. substrate specificity, metalloprotease activity, cellular location) expected for a physiologically relevant TNF-processing enzyme. Tumor necrosis factor-␣ (TNF)1 is a cytokine that is produced primarily by activated monocytes and macrophages in response to a variety of physiological stresses such as infection or injury (1). Clinical and experimental evidence has also identified TNF as a mediator of chronic autoimmune diseases such as rheumatoid arthritis (2) and Crohn's disease (3), as well as being involved in the pathology associated with sepsis (1). Accordingly, TNF has become a primary target for therapeutic intervention of several inflammatory diseases.TNF is initially synthesized as a 26-kDa membrane-bound protein (pro-TNF) that is subsequently cleaved to release soluble 17-kDa TNF with an NH 2 terminus of Val 77 (4). The identity of the protease(s) responsible for TNF processing remains controversial. Robache-Gallea et al. (5) detected a serine protease activity (PR3) in monocyte membrane preparations which was able to generate a 17-kDa active TNF with an NH 2 terminus of Arg 78 . In 1994, the partial isolation and characterization of a membrane-bound activity capable of generating the 17-kDa form of TNF were reported (6). The TNF-processing enzyme was thought to be a non-matrix metalloprotease since it was not inhibited by TIMP-1,2 or phosphoramidon, and no calcium requirement was detected. More recently, two members of the ADAM family (TNF-␣ converting enzyme (TACE) and bovine ADAM 10 (BoAD10)) have been shown to possess pro-TNF processing activity (7-9).In this report we describe the isolation, cloning, and characterization of a TNF-processing enzyme from the human monocytic cell line THP-1. The purified recombinant enzyme, rHuAD10, specifically recognizes the authentic cleavage site in pro-TNF and is sensitive to metalloprotease inhibitors that block soluble TNF production (6). MATERIALS AND METHODSReagents-Dinitrophenol-labeled polypeptides were synthesized by the Fmoc (N-(9-fluorenyl)methoxycarbonyl)/t-butyl-based solid phase peptide chemistry method using an Applied Biosystems, Inc. 431A peptide synthesizer (10). All peptides were purified by reversed phase HPLC, and molecular weights were verified by mass spectrometry.HPLC Peptide Assay-TNF processing activity was measured as the ability to cleave a 12-residue peptide spanning the Ala 76 -Val 77 site in pro-TNF. The chromatopho...

An inhibitor of tumor necrosis factor (TNF) has been isolated from the human histiocytic tymphoma cell line U-937 that is capable of inhibiting both TNF-a and TNF-j3. Protein sequencing has verified that it is distinct from a previously described TNF inhibitor that is a soluble fragment of a TNF receptor molecule (TNFrI). The cDNA sequence of this second TNF inhibitor clone suggests that it is also a soluble fragment of a TNF receptor. Expression of this cDNA sequence in COS-7 cells verified that it encodes a receptor for TNF-a (TNFrII) that can give rise to a soluble inhibitor of TNF-a, presumably through proteolytic cleavage. The extracellular

Interleukin 1 receptor antagonist (IL-lra) is a protein that binds to the IL-1 receptor and blocks the binding of both IL-la and -.8 without inducing a signal of its own.Human IL-lra has some sequence identity to human IL-1(3, but the evolutionary relationship between these proteins has been unclear. We show that the genes for human, mouse, and rat IL-lra are similar to the genes for IL-la and IL-1(3 in intron-exon organization, indicating that gene duplication events were important in the creation of this gene family. Furthermore, an analysis of sequence comparisons and mutation rates for IL-la, IL-1pj, and IL-lra suggests that the duplication giving rise to the IL-lra gene was an early event in the evolution of the gene family. Comparisons between the mature sequences for IL-lra, IL-la, and IL-1(3 suggest that IL-lra has a P-stranded structure like to IL-la and IL-1iB, consistent with the three proteins being related. The N-terminal sequences of IL-lra appear to be derived from a region of the genome different than those of IL-la and IL-1p3, thus explaining their different modes of biosynthesis and suggesting an explanation for their different biological activities.Interleukin 1 (IL-1) is believed to be important in mediating inflammatory and immune responses (1, 2). IL-1 proteins have been cloned from several species, and in each case two proteins have been identified (3). These two proteins, IL-la and IL-1,3, have the same biological activities and bind to the same cell-surface receptors (4-6).Inhibitors of IL-1 have been reported in the literature over the past several years (7,8). We recently reported the isolation, cloning, and characterization of a cDNA for a protein from human monocytes, IL-lra, that acts as a receptor antagonist on the 80-kDa IL-1 receptor (9, 10). Subsequently, Carter et al. (11) isolated and cloned an IL-1 inhibitor from the human monocyte-like cell line U937 and showed on the basis of its sequence that this molecule is IL-lra. IL-lra appears to be related to the IL-1 family based on its homology and similar hydropathy profile to IL-1,, but unlike the IL-1 proteins, IL-lra has a classical secretory leader peptide and is glycosylated at a consensus N-linked glycosylation site (9, 10).To determine whether IL-1 and IL-lra evolved from a common precursor or whether their similarities are the result of convergent evolution of a structure that can bind to the IL-1 receptor, we isolated genomic clones for IL-lra from human, mouse, and rat and compared their coding sequences with those of IL-la and IL-1,8 from the same and other species.* Our findings support the view that these three proteins have a common ancestor and that the gene leading to IL-lra diverged early in the evolutionary history of the IL-1 family. The results also argue for an important role ofthe sequence around the N terminus of IL-1ra in the unique properties of this protein.

The secretory leukoprotease inhibitor (SLPI) gene codes for a 12-kD protein that within the lung protects the airway epithelium from neutrophil elastase. Screening of 228 alleles in 114 individuals for sequence differences by RNase protection of genomic DNA revealed no detectable polymorphisms in SLPI gene exons II-IV. SLPI gene expression in the lung was demonstrated by identifying SLPI mRNA transcripts in bronchial epithelial cells freshly isolated from normals. Cell lines derived from mucosal surfaces (HS-24 bronchial squamous cell carcinoma, HeLa cervical carcinoma) actively transcribe the SLPI gene and contain SLPI mRNA transcripts, while lung fibroblasts demonstrate no evidence of SLPI gene expression. SLPI mRNA transcripts appear to be relatively stable, with mRNA levels only mildly affected by inhibition of RNA synthesis. Chromatin DNA of HS-24 cells demonstrates two DNase I hypersensitivity sites within the 5' flanking region of exon I of the SLPI gene, whereas fibroblast chromatin has no DNase I accessible sites in the same region. Further analysis of the 5' flanking region demonstrated two contiguous transcription start sites, CAAT and TATA boxes, and several potential regions of known DNA binding proteins. Overall, the SLPI gene appears to be a relatively nonpolymorphic, stable gene that is constitutively expressed at specific tissue sites, but has the potential to be modulated at both the transcriptional and posttranscriptional levels. (J. Clin. Invest. 1991. 87:2207-2215