Ratmucosal mast cells: the cultured bone marrow-derived mast cell is biochemically and functionally analogous to its counterpartin vivo

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Abstract

Mast cells (MC) are biochemically and functionally heterogeneous and the mixture of MC phenotypes varies according to anatomical location. Intestinal mucosal MC (IMMC) have been used to study the mucosal MC subset in the rat, but they are difficult to isolate in sufficient numbers and with consistent purity and viability. Bone marrow-derived MC (BMMC), with an apparent mucosal MC phenotype, can be cultured in large numbers and with high purity from normal rat bone marrow using supernatants from mesenteric lymph node cells of rats infected with the nematode, Nippostrongylus brasiliensis. We have compared serine proteinase content, tumour necrosis factor-α (TNF-α) storage and secretion, and TNF-α-dependent cytotoxicity of IMMC and BMMC to assess the appropriateness of BMMC as in vitro models of mucosal MC. Two-dimensional gel electrophoretic analysis revealed that the overall protein constituents of BMMC and IMMC were highly homologous. Immunoblotting confirmed that both MC types expressed the MMC-associated enzyme, rat mast cell proteinase-2 (RMCP-2), but not RMCP-1, mast cell proteinase-5 (MCP-5) or carboxypeptidase A (CPA), which characterize the connective tissue MC in the rat and which were detected in a representative of this subset, namely, the peritoneal MC (PMC). BMMC demonstrated levels of TNF-α-dependent cytotoxicity that were equivalent to those of IMMC. Like IMMC, BMMC contained little stored TNF-α, in comparison with PMC, but both MC types generated substantial amounts of TNF-α 6 hr following IgEmediated activation. Pretreatment of PMC with recombinant rat interferon-γ (IFN-γ) for 20 hr inhibited anti-immuoglobulin E (anti-IgE)-mediated release of the granule-associated enzyme, β-hexosaminidase, whereas identically treated BMMC were unresponsive to this cytokine. Similar results have previously been reported for IMMC. Rat BMMC, unlike their more immature and less phenotypically committed counterparts in the mouse, appear therefore to be more appropriate models for studies on the mucosal MC.

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