Immunological and electrophysiological studies of rat bone marrow-derived mast cells

Rat bone marrow-derived mast cells (BMMCs), cultured in the presence of a T lymphocyte conditioned medium, are analogous to MMCs as defined by the granule content of the soluble chymase, rat mast cell protease-II (RMCP-II); by the granule proteoglycan chondroitin sulphate; and by their secretory characteristics. To investigate the secretory response of BMMCs to IgE-dependent stimulation, a sensitive, specific and repeatable enzyme-linked immunospot (ELISPOT) assay was developed to detect the release of RMCP-II from individual cells. Within populations of BMMCs, only 6-24% of the cells responded to challenge with either anti-IgE or specific antigen, leaving a large residual refractory population. Pre-incubation of mature BMMCs with the multi-functional cytokine, stem cell factor (SCF), significantly increased (≈ 2-fold) the proportion of cells responding to IgE-dependent stimulation without directly causing mediator release. Furthermore, SCF enhanced the total percentage release of RMCP-II and β-hexosaminidase from populations of mature BMMCs in association with an increased proportion of cells secreting RMCP-II as detected by ELISPOT. These results suggest that SCF augments IgE-dependent secretion from rat BMMCs primarily by activating previously unresponsive cells. To further characterise the functional phenotype of rat BMMCs, the electrophysiological properties of the cells were investigated using the whole-cell configuration of the patch-clamp technique. Rat BMMCs had a mean membrane potential of -25.9 mV and a mean whole-cell capacitance of 4.8pF. With the amphotericin B perforated-patch technique, both inwardly rectifying (IR) and outwardly rectifying (OR) currents were observed in rat BMMCs. The reversal potential and conductance of the IR current depended on the extracellular K⁺ concentration, indicating that the channel was K⁺ selective. The OR current was reversibly decreased both by lowering the extracellular Cl^- concentration and by the Cl^- channel blocker DIDS, indicating a Cl^- conductance.