Research Project
3180665
The overall goal of this proposal is to increase our understanding of the mechanisms of lymphokine action in reactions of cellular immunity and hypersensitivity. Lymphokines are elaborated by antigen or mitogen stimulation of sensitized lymph node cells and are thought to play a critical role in cellular immune events. This proposal is aimed at the dissection of the interaction of one lymphokine, migration inhibitory factor (MIF), with its target cell the macrophage. The immediate objective of the proposed study is to describe the molecular mechanisms by which the human monocyte/macrophage responds to MIF and, in particular, the role of two macrophage molecules, a glycoprotein and a glycolipid, in this response. We will use purified homogeneous components. We will first study the protein target of Mo3Ab, a monoclonal antibody directed against the monocyte surface which we showed to specifically block the MIF response, and will determine if Mo3Ab recognizes the receptor for MIF. The glycoprotein component will be purified from the monocyte-like cell line, U937, and will be chemically characterized with respect to amino acid composition and sequence and carbohydrate composition. We will then make a cDNA library from U937 mRNA and attempt to get expression of the Mo3 antigen with recombinant DNA technology. The glycolipid component, which enhances and converts the MIF response, will be purified from the U937 cell line, and will be chemically characterized with respect to the ceramide and carbohydrate compositions, the sequence, anomeric configurations and glycosidic bond linkages. We will then examine the binding of MIF to mynocytes and to the isolated glycoprotein and glycolipid components including an examination of the kinetics of binding, and a determination of whether internalization and recycling are characteristic of this receptor-ligand interaction. Furthermore, we will utilize HL-60 cells, a promyelocytic leukemia line, as a model for the macrophage response to MIF. The molecular events that parallel the conversion of HL-60 cells into MIF-responsive cells by treatment with phorbol diester will be analyzed. This study will contribute insights into the biochemical mechanisms of lymphokine action. This will be relevant for the human clinical situation where an increase in certain macrophage functions mitigates the severity of infections and the proliferation of tumors or where a decrease in macrophage functions alleviates adverse reactions of hypersensitivity.
