As part of a study to identify novel genes associated with macrophage activation, we have cloned a new member of the transforming growth factor ␤ (TGF-␤) superfamily designated macrophage inhibitory cytokine 1 (MIC-1). MIC-1 is synthesized as a 62-kDa intracellular protein, which, after cleavage by a furin like protease, is secreted as a 25-kDa disulfide-linked dimeric protein. Sequence analysis indicates that it does not cluster within any existing TGF-␤ families, suggesting it may be the first member of a new grouping within the TGF-␤ superfamily. Tissue Northern blots show that MIC-1 transcripts are only found abundantly in placenta, although smaller amounts are seen in a limited number of other adult and fetal tissues. MIC-1 is not expressed in resting macrophages but is induced by a number of different activation agents, including phorbol myristate acetate, interleukin 1, tumor necrosis factor ␣, and macrophage colony-stimulating factor but not by lipopolysaccharide or interferon-␥. We have hypothesized that it may be an autocrine inhibitor of macrophage activation but its major biological role is still uncertain.J. Leukoc. Biol. 65: 2-5; 1999.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-␤ (TGF-␤) superfamily. While it is synthesized in a pre-pro form, it is unique among superfamily members because it does not require its propeptide for correct folding or secretion of the mature peptide. To investigate factors that enable these propeptide independent events to occur, we constructed MIC-1/TGF-␤1 chimeras, both with and without a propeptide. All chimeras without a propeptide secreted less efficiently compared with the corresponding constructs with propeptide. Folding and secretion were most affected after replacement of the predicted major ␣-helix in the mature protein, residues 56 -68. Exchanging the human propeptide in this chimera with either the murine MIC-1 or TGF-␤1 propeptide resulted in secretion of the unprocessed, monomeric chimera, suggesting a specific interaction between the human MIC-1 propeptide and mature peptide. Propeptide deletion mutants enabled identification of a region between residues 56 and 78, which is important for the interaction between the propeptide and the mature peptide. Cotransfection experiments demonstrated that the propeptide must be in cis with the mature peptide for this phenomenon to occur. These results suggest a model for TGF-␤ superfamily protein folding.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily whose increased expression is associated with macrophage activation and which is expressed highly in placenta as compared to other tissues. There are two known allelic forms of human MIC-1 due an amino acid substitution at position 6 of the mature protein. We have raised four monoclonal antibodies (MAbs) and one polyclonal antiserum to the mature protein region of human MIC-1 and have used an extensive panel of MIC-1 relatives, mutants, and chimeras to map their epitopes. None of the MAbs were able to cross-react with either the murine homologue of MIC-1 or with hTGF-beta1, and all of the MAb epitopes were conformation-dependent. A distinct cross-reactivity pattern with the various antigens was observed for each of the monoclonal and polyclonal antibodies suggesting the presence of at least five immunogenic regions on the MIC-1 surface. One of the MAbs is directed against the amino terminus of the protein and can distinguish between the two allelic forms of MIC-1. The epitopes for the other three MAbs were located near the tips of the so-called "fingers" of the protein and appeared to be partially overlapping as each involved amino acids in the region 24-37. In one case, it was possible to mutate murine MIC-1 so that it could be recognized by one of the MAbs. Finally, the use of another mutant in which Cys 77 was replaced by serine enabled confirmation of the location of the MIC-1 interchain disulfide bond.