MOUSE BONE-MARROW-DERIVED MAST-CELLS UNDERGO EXOCYTOSIS, PROSTANOID GENERATION, AND CYTOKINE EXPRESSION IN RESPONSE TO G-PROTEIN-ACTIVATINGPOLYBASIC COMPOUNDS AFTER COCULTURE WITH FIBROBLASTS IN THE PRESENCE OF C-KIT LIGAND

Polycationic mast cell activators, such as compound 48/80 and substanc e P, have been reported to activate connective tissue-type mast cells specifically by interacting directly with the G(i) family of trimeric GTP-binding protein. We now demonstrate that mouse bone marrow-derived mast cells (BMMC) developed in IL-3, an immature mast cell population lacking responsiveness to the G(i)-coupled polycationic mast cell act ivators, underwent maturation toward a connective tissue-type mast cel ls-like phenotype that responded to polycationic compounds after only 4 to 6 days of coculture with Swiss 3T3 fibroblasts in concert with re combinant soluble c-kit ligand (KL), whereas 3T3 or KL alone was insuf ficient to mediate this process. Under optimal conditions, cocultured BMMC released similar to 30% beta-hexosaminidase and generated similar to 1 ng of PGD(2)/10(6) cells within a few minutes in response to com pound 48/80 or substance P. Furthermore, these cells expressed cytokin es, such as IL-1 beta and IL-6, and PG endoperoxide synthase-2 1 to 4 h after stimulation with compound 48/80 or substance P. All these resp onses were suppressed effectively by pertussis toxin, implicating func tional G(i) coupling. Regardless of the remarkable change in polycatio nic compound sensitivity, there was only a minimal change in the const itutive expression of G(i3)alpha after coculture. These results togeth er with the observation that before coculture BMMC responded to thromb in through its G(i)-coupled receptor suggest that the alteration in a certain step(s) distinct from the level of C(i3)alpha protein expressi on is important for the acquisition of responsiveness to the polycatio nic compounds by the synergistic action of KL and 3T3 fibroblast-deriv ed factor. Several lines of evidence have revealed that 3T3-derived fa ctor appears to differ from the known cytokines, prostanoids, and adhe sion molecules and is a labile soluble substance.