ACTIVATION OF PROTEIN KINASE C STIMULATES PRODUCTION OF IL-6 BY MAST CELLS AND ADHESION TO LAMININ

Abstract

Purpose: Mast cells are found in large numbers in the GI tract and may be in intimate contact with the basement membrane of the intestinal epithelium. We examined the ability of mast cells to adhere to laminin, a component of the basement membrane, and the role of protein kinase C (PKC) in this interaction. Methods: Bone marrow cultured mast cells (BMCMC) were derived from BALB/c mice and labeled with 3H-thymidine, then incubated in laminin-coated or uncoated plates with or without phorbol myristate acetate (PMA; 50 ng/ml). Non-adhered cells (wash) and adhered cells (lysed with Triton-X) were collected. Radioactive counts were measured and the percent adherent cells calculated. In some experiments, PKC inhibitors calphostin C (CC: 1 μM) or bisindolylmaleimide I (BIM; 10 μM) were incubated with cells prior to stimulation with PMA. In separate experiments, IL-6 production was measured by ELISA. Results: PMA induced significant adhesion of BMCMC on laminin-coated plates compared with medium alone at 150 min (20± 1% vs. 0.7±0.2%, respectively, p < 0.001), while no significant adhesion was seen with PMA or medium on uncoated plates. Either PKC inhibitor significantly diminished PMA-induced adhesion (CC by 27%; BIM by 99%). PMA also induced significant IL-6 release on either laminin- or uncoated plates compared with medium alone at 3.5 hrs (202±6 vs 0 pg/ml, laminin; 178±11 vs 0 pg/ml, uncoated, respectively, p < 0.001). In addition, the production of IL-6 on either laminin- or uncoated plates was undetectable in the presence of either CC or BIM. Conclusions: These findings demonstrate that PMA induces mast cell adherence to laminin and stimulates the production of IL-6 by a PKC-dependent pathway.