Characterization of leukoregulin, a cytokine first described in this project in 1983 with the unique ability to increase tumor cell plasma membrane permeability, is being pursued at both the structural and functional levels. Isolation of the gene encoding for the protein with plasma membrane permeability activity is being carried out by direct expression cloning in mammalian cells using clones from a cDNA library prepared from leukoregulin secreting normal human peripheral blood mononuclear leukocytes. More than 200,000 clones in pools of 250 clones have been examined for leukoregulin synthesis. Approximately 1.5% of the clone pools are able to express membrane permeability activity following plasmid transfection into COS7 cells. The actively expressing pools are being subdivided to isolate the leukoregulin cDNA clones to obtain the oligonucleotide sequence encoding leukoregulin and to produce recombinant human leukoregulin. Leukoregulin while increasing membrane permeability also protects tumor cells from complement mediated lysis. Leukoregulin induction of complement lysis is blocked by both inhibitors of protein synthesis and inhibitors of protein kinase C activity suggesting that complement resistance may be mediated through leukoregulin induction of protein kinase C stimulated complement resistance proteins. Leukoregulin also induces glycosylaminoglycan, collagenase and other protease synthesis and extracellular secretion which are important components in the extracellular matrix and may indicate a role for leukoregulin in immunological inflammatory connective tissue disorders. Production of recombinant leukoregulin will allow critical examination of whether the diverse functional properties of this cytokine are resident in a single molecule and further delineation of the intracellular pathways responsible for the functional activities of this cytokine.
