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- Title
IgG-Mediated Signal Transduction in Canine Mastocytoma-Derived Cells.
- Authors
Sato, Yoshitaka; Teshima, Reiko; Nakamura, Ryosuke; Sasaki, Nobuo; Morita, Yutaka; Sawada, Jun-ichi; Kitani, Seiichi
- 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 Ca[sup 2+] concentration ([Ca[sup 2+] ][sub i] ) when IgG-primed cells were activated with anti-canine IgG. Release of Ca[sup 2+] from intracellular stores was analyzed with thapsigargin in the absence of extracellular Ca[sup 2+] . The Ca[sup 2+] entry via store-operated Ca[sup 2+] channel from the external environment was characterized using Ba[sup 2+] , Ni[sup 2+] 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 Ca[sup 2+] 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 [Ca[sup 2+] ][sub i] elevation. When extracellular Ca[sup 2+] was excluded by EGTA, a mild and transient increase in [Ca[sup 2+] ][sub i] was observed, indicating the release of Ca[sup 2+] from anti-IgG-sensitive intracellular Ca[sup 2+] stores. The constant Ba[sup 2+] entry from external environment proved the Ca[sup 2+] influx occurred mainly via a store-operated Ca[sup 2+] channel which was inhibited by Ni[sup 2+] and EGTA. Canine serum-sensitized cells showed a rapid and sustained increase in [Ca[sup 2+] ][sub i] upon both anti-IgG and anti-IgE stimulation. The [Ca[sup 2+] ][sub 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 Ca[sup 2+] , 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 [Ca[sup 2+] ][sub 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 Ca[sup 2+] influx were similar to those mediated by IgE. CM-MC cells are useful for the study of allergic inflammation caused by IgG-dependent mechanisms.Copyright © 2002 S. Karger AG, Basel
- Subjects
DOG diseases; MAST cell disease; IMMUNOGLOBULIN G; TYROSINE; PHOSPHORYLATION; ALLERGIES
- Publication
International Archives of Allergy & Immunology, 2002, Vol 129, Issue 4, p305
- ISSN
1018-2438
- Publication type
Article
- DOI
10.1159/000067587