Neutral proteases of rodent mast cells

Detta är en avhandling från Uppsala : Acta Universitatis Upsaliensis

Sammanfattning: Mast cells are important effector cells in general as well as in allergic inflammation andin the defence against helminthic parasites and bacteria. Two major populations, withdifferences in tissue distribution, have been identified; connective tissue mast cells andmucosal mast cells. Neutral proteases are the major granule constituents of mast cells.This investigation focuses on the characterization of rodent mast cells using serineproteases as markers to study their tissue distribution, heterogeneity and differentiation.The expression of recently identified rat mast cell proteases (RMCPs) was studied in Nippostrongylus brasiliensis -infected rats. mRNA for five of them, RMCP-3, -4, -8, -9, and -10, was detected together with the classical mucosal mast cell protease RMCP-2during the parasite-induced mast cell hyperplasia. This finding shows that these five novel proteases can be classified as true mucosal mast cell proteases in the rat. RMCP-8, -9 and -10, together with their mouse homologue, MMCP-8, form a new subfamily ofmast cell/basophil-specific proteases that are clearly distinct from the classical chymasesand tryptases. The gene encoding MMCP-8 was isolated and its structure was determined.The gene and its encoded protein show higher similarity to the T-cell granzymes andcathepsin G than to the classical mast cell proteases. MMCP-8 has been identified as thefirst basophil-specific marker in mouse. To study early events in mast cell and basophildevelopment, the phenotype of a panel of murine cell lines was determined.Interestingly, one of the cell lines, IC-2, showed characteristics of both early mast cellsand basophils. This indicates that the cell line is arrested at a stage of development wherethe cells can still differentiate into either mast cells or basophils. To study functional andbiochemical characteristics of the various members of the MMCP-8 subfamily, thecorresponding recombinant proteins were produced in a mammalian expression system.The proteins were highly glycosylated and had different affinities for heparin. Theabsolute necessity of heparin for storage of granule mediators was shown in micedeficient in NDST-2, an enzyme essential for heparin synthesis. The mast cells of thesemice have empty granules, but there are no differences in levels of mRNA encoding theMMCPs, indicating that absence of heparin does not influence transcription of theprotease genes.

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