IgG-Mediated Signal Transduction in Canine Mastocytoma-Derived Cells

Abstract

Background: We have reported canine cutaneous mastocytoma-derived cells named CM-MC sensitized with monomeric IgG released histamine upon anti-IgG stimulation. However, IgG or IgE-mediated signal transduction in the cells remains to be examined. Methods: Monomeric IgG-binding to cells was measured by flow cytometry using FITC-anti-IgG. IgG-mediated protein tyrosine phosphorylation was studied by Western blotting using anti-phosphotyrosine antibody. We monitored the intracellular Ca²⁺ concentration ([Ca²⁺]_i) when IgG-primed cells were activated with anti-canine IgG. Release of Ca²⁺ from intracellular stores was analyzed with thapsigargin in the absence of extracellular Ca²⁺. The Ca²⁺ entry via store-operated Ca²⁺ channel from the external environment was characterized using Ba²⁺, Ni²⁺ and EGTA. Cells sensitized with canine serum abundant in IgG and IgE or heat-inactivated serum were activated by anti-canine IgG or anti-canine IgE. The effect of extracellular Ca²⁺ and reaction time on IgG-mediated histamine release was examined. Staurosporine and ER-27319 were used to clarify the IgG-mediated protein tyrosine phosphorylation. Results: Abundant IgG-binding sites on the cell were detected by FACS analysis. Anti-IgG induced rapid protein tyrosine phosphorylation and [Ca²⁺]_i elevation. When extracellular Ca²⁺ was excluded by EGTA, a mild and transient increase in [Ca²⁺]_i was observed, indicating the release of Ca²⁺ from anti-IgG-sensitive intracellular Ca²⁺ stores. The constant Ba²⁺ entry from external environment proved the Ca²⁺ influx occurred mainly via a store-operated Ca²⁺ channel which was inhibited by Ni²⁺ and EGTA. Canine serum-sensitized cells showed a rapid and sustained increase in [Ca²⁺]_i upon both anti-IgG and anti-IgE stimulation. The [Ca²⁺]_i elevation induced by anti-IgE was decreased in the cells sensitized with heat-inactivated serum. Histamine release from CM-MCs was absolutely dependent on extracellular Ca²⁺, and reached equilibrium within 5 min. Staurosporine inhibited the tyrosine phosphorylation of 38-, 65-, 70-, 80-kD proteins. ER-27319 inhibited the tyrosine phosphorylation of 38- and 70-kD proteins. Staurosporine also inhibited IgG-mediated [Ca²⁺]_i elevation and histamine release in a dose-dependent manner. Conclusions: Canine cutaneous mastocytoma-derived (CM-MC) cells were activated by both IgG- and IgE-mediated mechanisms. IgG-mediated protein tyrosine phosphorylation and Ca²⁺ influx were similar to those mediated by IgE. CM-MC cells are useful for the study of allergic inflammation caused by IgG-dependent mechanisms.