The proposed investigations on the cells, mast cells and eosinophils, and the mediators, preformed secretory granule constituents and newly derived membrane lipid metabolites, of immediate hypersensitivity reactions continue a long term commitment. With the culture/coculture of mast cells and eosinophils, the regulation of phenotype of non-transformed cells by fibroblast factors operating in concert with defined hemopoietic cytokines can be examined so that possible tissue effects on the chemical composition and/or function of these effector cell types can be identified. Kirsten sarcoma virus immortalized murine mast cells (KiSV-MC) provide a homogeneous and substantial source of connective tissue-like murine mast cells (CTMC) for isolation of specific proteins and cloning of their specific cDNA from the KiSV-MC cDNA library; the cDNA will be used to examine transcription of these proteins during phenotypic changes in interleukin 3 (IL-3)-dependent bone marrow- derived murine mast cells (BMMC) elicited by fibroblasts or during regulation of proliferation and/or phenotype of KiSV-MC by IL-3 and/or IL-4. These same cDNA probes will be used for genomic cloning of mouse secretory granule carboxypeptidase A and to initiate cloning of the cDNA that encodes the analogous human mast cell neutral proteases. With regard to the analysis of the 5-lipoxygenase pathway to leukotriene C4 generation (LTC4), the techniques for culturing eosinophils ex vivo and for retrovirus immortalization of murine mast cells, KiSV-MC, provide new approaches. The ability to culture peripheral blood human eosinophils ex vivo will permit the mechanism and regulation of the distinct step for LTC4 export to be fully characterized. The KiSV-MC provide a homogeneous source of cells with adequate amounts of LTC4 synthase for isolation and amino acid sequence analysis of selected portions so as to allow synthesis of oligonucleotides and initiation of cloning from the KiSV-MC library. Taken together, these studies will yield new insights into the following: the extracellular regulation of mast cell and eosinophil phenotypes; transcription, translation, and degradation of secretory granule constituents of mast cells; the protein and/or nucleotide sequence for microsomal LTC4 synthase and mechanistic information on LTC4 export; and the presence in selected human cDNA libraries of nucleotide sequences analogous to those that encode the murine mast cell proteases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37AI022531-06
Application #
3481190
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1984-12-01
Project End
1994-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Lam, Bing K (2003) Leukotriene C(4) synthase. Prostaglandins Leukot Essent Fatty Acids 69:111-6
Lam, Bing K; Austen, K Frank (2002) Leukotriene C4 synthase: a pivotal enzyme in cellular biosynthesis of the cysteinyl leukotrienes. Prostaglandins Other Lipid Mediat 68-69:511-20
Boyce, Joshua A; Mellor, Elizabeth A; Perkins, Brandy et al. (2002) Human mast cell progenitors use alpha4-integrin, VCAM-1, and PSGL-1 E-selectin for adhesive interactions with human vascular endothelium under flow conditions. Blood 99:2890-6
Castells, M C; Klickstein, L B; Hassani, K et al. (2001) gp49B1-alpha(v)beta3 interaction inhibits antigen-induced mast cell activation. Nat Immunol 2:436-42
Hsieh, F H; Lam, B K; Penrose, J F et al. (2001) T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C(4) synthase expression by interleukin 4. J Exp Med 193:123-33
Bannert, N; Farzan, M; Friend, D S et al. (2001) Human Mast cell progenitors can be infected by macrophagetropic human immunodeficiency virus type 1 and retain virus with maturation in vitro. J Virol 75:10808-14
Mellor, E A; Maekawa, A; Austen, K F et al. (2001) Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells. Proc Natl Acad Sci U S A 98:7964-9
Daheshia, M; Friend, D S; Grusby, M J et al. (2001) Increased severity of local and systemic anaphylactic reactions in gp49B1-deficient mice. J Exp Med 194:227-34
Friend, D S; Gurish, M F; Austen, K F et al. (2000) Senescent jejunal mast cells and eosinophils in the mouse preferentially translocate to the spleen and draining lymph node, respectively, during the recovery phase of helminth infection. J Immunol 165:344-52
Lam, B K; Frank Austen, K (2000) Leukotriene C4 synthase. A pivotal enzyme in the biosynthesis of the cysteinyl leukotrienes. Am J Respir Crit Care Med 161:S16-9

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