One project in the laboratory is focused on the structure of the receptor for interferongamma (IFN-gamma). We have been able to isolate the receptor on polyacrylamide gel electrophoresis (PAGE) using antibodies specific to the receptor. With cells biosynthetically labelled with 35S methionine, we have solubilized, immunoprecipitated, and separated out the receptor on PAGE. After studying the effects of several detergents such as CHAPS, octylglucoside, TX100, and digitonin, we have used TX100 most frequently since this detergent allows us to separate the receptor into various subcomponents. In addition to the previously described and cloned binding protein of 80-90 kD, we have observed an additional protein of 200kD. This protein is poorly labelled with 125I by the lacto- peroxidase system, and with 3H mannose, which implies that it is not a glyco-protein and supports the contention that it may be associated with the binding protein as an intracellular protein. We are further characterizing the relationship between this 200kD protein and the binding protein of 80-kD. Another project in the laboratory focuses on the inhibition by IL-4 of the effects of IFN gamma on monocytes. We have characterized the binding of IL-4 on the human monocyte. The data revealed that monocytes have very few receptors, 150-300 per cell with a high affinity. We have also shown that IFN gamma will induce the expression of the IL-4 receptor on the murine macrophage cell line, J774.16. This occurs rapidly (1-2 hr) after addition of IFN gamma to the cells, and protein synthesis is necessary for the induction. We also have shown using a RNAse protection assay that the MRNA for the receptor is induced within 15-30 min following exposure to IFN gamma. This model will explore the mechanism whereby IFN-gamma activates the macrophage to induce this receptor. In addition, we are studying the effects of IL-4 on the induction of the MRNA for IP-10 by IFN-gamma in human monocytes. We have shown that IL-4 can inhibit the induction of IP-10 by IFN-gamma and are beginning to explore the mechanisms involved.
