Exposure of mononuclear phagocytes to certain soluble factors in vitro (eg. lymphokines, bacterial products, phorbol diesters) is known to stimulate a variety of cellular activities that include an increase in phagocytosis, oxidative metabolism, synthesis and secretion of monokines, and cytotoxicity of microbes and tumor cells. Many of the functional attributes of the """"""""activated state"""""""" are mediated at the cell membrane level and may therefore reflect alterations in the macromolecular composition of the plasma membrane. Through the use of hybridoma monoclonal technology, we have sought to identify membrane antigens that are uniquely or preferentially expressed by activated monocytes. We have succeeded in generating a series of 5 murine monoclonal reagents that bind to a surface antigen, Mo3, that is expressed by human monocytes that havce been exposed to culture to bacterial lipopolysacchyaride (LPS), phorbol myristate acetate (PMA), or muramyl dipeptide (MDP). Because Mo3 is barely detectable on the surface of unstimulated human monocytes, the expression of this marker by LPS-, PMA-, or MDP-stimulated cells represents an immunological marker for moncyte activation by these factors. In terms of monocyte function anti-Mo3 monoclonal antibody blocks monocyte responsiveness to migration inhibitory factor (MID), indication the possibility that Mo3 may represent the plasma membrane receptor for MIF whose expression is up-modulated by LPS, PMA, or MDP. The goal of this proposal is the further characterization of the Mo3 determinant including: (a) a determination of its biochemical features (composition, MW, mode of biosynthesis); (b) a precise quantitation of Mo3 antigenic sites expressed by monocytes and differentiated macrophages (resting and activated); (c) an investigation of the metabolic factors that requlate the biosynthesis and surface espression of Mo3; and (d) an assessment of the physiological significance of Mo3 as a ligand-binding receptor by determining whether anti-Mo3 blocking the monocyte MIF responsiveness is due to competitive binding to the MIF receptor, and whether other plasma membrane-mediated monocyte activites are affected by anti-Mo3 binding. Since the expression of Mo3 is restricted to cells exposed to some (LPS, PMA, MDP) but not all (IFNGamma) activating stimuli, we will attempt to generate additional monoclonal reagents that recognize other activation-related determinants whose expression may have physiological significance.
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