title: 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 Ca(2+) concentration ([Ca(2+)](i)) when IgG-primed cells were activated with anti-canine IgG. Release of Ca(2+) from intracellular stores was analyzed with thapsigargin in the absence of extracellular Ca(2+). The Ca(2+) entry via store-operated Ca(2+) channel from the external environment was characterized using Ba(2+), Ni(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(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(2+)](i) elevation. When extracellular Ca(2+) was excluded by EGTA, a mild and transient increase in [Ca(2+)](i) was observed, indicating the release of Ca(2+) from anti-IgG-sensitive intracellular Ca(2+) stores. The constant Ba(2+) entry from external environment proved the Ca(2+) influx occurred mainly via a store-operated Ca(2+) channel which was inhibited by Ni(2+) and EGTA. Canine serum-sensitized cells showed a rapid and sustained increase in [Ca(2+)](i) upon both anti-IgG and anti-IgE stimulation. The [Ca(2+)](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(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(2+)](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(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.
authors.affiliation: Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Sato
authors.forename: Reiko
authors.initials: R
authors.lastname: Teshima
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Nakamura
authors.forename: Nobuo
authors.initials: N
authors.lastname: Sasaki
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Morita
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sawada
authors.forename: Seiichi
authors.initials: S
authors.lastname: Kitani
mesh_terms: D000166:Acridines / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000276:immunology; D004285:Dogs; D006636:Histamine Release / Q000187:drug effects; D007073:Immunoglobulin E / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism*; D034801:Mastocytoma / Q000276:immunology* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D019311:Staurosporine / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000166:Acridines; D007074:Immunoglobulin G; D009363:Neoplasm Proteins; C109226:ER 27319; D007073:Immunoglobulin E; D014443:Tyrosine; D019284:Thapsigargin; D019311:Staurosporine; D002118:Calcium