The long-term objectives of this research proposal are to develop and utilize immunological mutants of the mouse as genetic tools to elucidate the etiology of autoimmune and immunodeficiency diseases. This research proposal focuses on the genetic and cellular mechanisms underlying the profound dysregulation of the immune system in mice homozygous for the motheaten (me) and viable motheaten (mev) mutations. It is hypothesized that the primary genetic defect in me/me and mev/mev mice is expressed in Mac-1+ monomyelocytic cells. This defect results in the abnormal expression of transforming growth factor-beta(TGF-beta), a potent regulator of cell growth, thereby causing severe immunological dysfunction. This hypothesis will be tested by the following four specific aims.
The first aim i s to construct a high resolution genetic map of the region encompassing the motheaten locus on Chromosome 6. This will be accomplished by a Mus musculus castaneus intersubspecific backcross. A detailed recombinational map encompassing the me locus will provide a basis for a physical map for future studies on the isolation of the me gene and will test the possibility that me and mev are allelic with any of four candidate loci.
The second aim i s to determine the role of TGF-beta` in defective hematopoiesis in mev/mev mutant mice. Mechanisms underlying abnormal myelopoiesis and lymphopoiesis in these mice will be investigated in vitro. Such investigations will include measurements of TGF-beta functional and immunoreactive protein as well as determination of levels of TGF-beta mRNA. The ability of antagonists to TGF-beta to correct the defective hematopoiesis will also be determined.
The third aim i s to assess the role of Mac-1+ monomyelocytic cells in the development of immunopathologic changes in me/me and mev/mev mice. Mobilization and recruitment of such cells will be blocked in vivo by the administration of monoclonal anti-Mac-1 antibody.
The fourth aim i s to evaluate the effects of macrophage and B cell depletion on phenotypic expression of the mev mutation using other mutations or disrupted transgenes as genetic tools to deplete these cell populations. Macrophages will be depleted by development of mice doubly homozygous for the mev mutation and the osteopetrosis (op) mutation. The op mutation is within the structural gene (Csfm) for the macrophage colony stimulating factor. B cells will be depleted by development of mice doubly homozygous for the mev mutation and a disrupted immunoglobulin mu chain constant region gene (muMT). Homozygosity for muMT results in a selective block in B cell development. Findings from these studies will contribute to an understanding of complex immunological diseases and increase the knowledge of mechanisms controlling immunological function in normal and pathological states.
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