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

Based on biochemical and functional characteristics, mast cells are broadly classified into mucosal and connective tissue phenotypes. Both types may be effector cells in the pathogenesis of allergic diseases, but intestinal mucosal mast cells (MMCs) are also involved in the immune response against intestinal nematodes. 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 IgEdependent stimulation without directly causing mediator release. Furthermore, SCF enhanced the total percentage release of RMCP-II and (3-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 phenoiype 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 -28.5 mV and a mean whole-cell capacitance of 4.8 pF. With the amphotericin B perforated-patch technique, both inwardly rectifying (IR) and 11 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 CI" concentration and by the CI" channel blocker DIDS, indicating a CI" conductance. The IRK current could also be detected in the majority of BMMCs using the conventional whole-cell recording technique at room temperature. In contrast, the activity of the ORu current was dependent on temperature and the maintenance of cytoplasmic integrity. The ORc, current may be involved in voltage control of the cell during degranulation. However, in addition, the ORa current was influenced by changes in extracellular osmolality, suggesting a possible role in volume control.

These studies provide further insight into the regulation of secretion in rat BMMCs, and therefore suggest mechanisms that may be involved in the modulation of gastrointestinal immune responses.