Macrophage (mphi) activation, the acquisition of enhanced cytotoxic capacity, has been studied intensively and induced clinically, with therapeutic success. Recently there has been increasing recognition of the potential importance of the induced loss of mphi cytotoxic capacity, or mphi deactivation. Mphi deactivation takes place both in physiologic settings and in response to products of tumor cells and microbes. Only a few purified proteins have been identified that are potent mphi deactivators. These include mphi deactivation factor (MDF) and transforming growth factor-beta types 1-3 (TGF-beta). MDF is a 13-kDa protein discovered during this grant and recently purified from medium conditioned by mouse tumor cells. MDF selectively, profoundly and nontoxically suppresses the ability of mphi to secrete at least 2 major classes of cytotoxic anti-tumor and anti-microbial effector molecules; reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). MDF shares these bioactivities with TGF-beta's, and TGF-beta1 reportedly suppresses a third major class of mphi cytotoxins represented by tumor necrosis factor-alpha (TNFalpha). MDF shares at least 3 other bioactivities with TGF-beta's--suppression of concanavalin A-driven T lymphocyte proliferation, promotion of proliferation of rat kidney fibroblasts, and inhibition of proliferation of mink lung epithelial cells- -but does not appear to bind to TGF-beta receptors. The purpose of this grant is to further characterize MDF, a presumptive new cytokine displaying TGF-beta-like properties, to define its biologic role, and to analyze the mechanism by which it deactivates mphi. Goal 1 is to clone mouse MDF cDNA and produce monoclonal and polyclonal antibodies to natural MDF, synthetic MDF-based peptides, and/or fusion proteins containing recombinant MDF. Goal II is to compare the bioactivities of MDF and TGF-betas in vitro. We will analyze the mechanism of suppression of production of ROI and RNI and determine whether MDF also suppresses TNFalpha release. We will test if MDF regulates the cell surface expression of interferon-gamma (IFNgamma) receptors, TNFalpha receptors, or CD11/CD18 cell adhesion molecules, each of which can transduce signals that activate mphi. We will complete the comparison of MDF and TGF-betas for their actions on cells other than mphi, notably neutrophils, T cells, mesenchymal and epithelial cells. Goal III is to characterize MDF receptors on mphi and other cells, to determine if MDF competes with the binding of the mphi activating factors IFNgamma and TNFalpha, and to characterize and isolate the MDF binding proteins of mphi. Goal IV is to determine the range of cell types that produce MDF in vitro and in vivo. We will determine how much of the weak ROI-suppressing bioactivities released by some nontransformed cells are due to MDF or to TGF-beta. MDF in tissues of the fetal mouse and in the normal and tumor- bearing adult mouse will be characterized immunohistochemically, and MDF will be quantitated during tumor growth. Goal V is to test whether MDF has predicted effects in vivo, namely, the ability to suppress host defenses against tumors and against the opportunistic pathogens listeria and legionella. This will be tested both by neutralizing MDF in vivo with antibodies, and by inducing constitutive transgenic mice expressing MDF under the control of the mammary tumor virus promoter, and mating them with previously described transgenic mice expressing c-myc under the same promoter.
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