Fig. 6.
Fig. 6. Important role for Lyn tyrosine kinase in Fas receptor-mediated signaling in human eosinophils. (A) Freshly isolated eosinophils were stimulated with anti-Fas MoAb for the indicated times. The cell lysates were immunoprecipitated with an anti-Lyn MoAb and then analyzed by immunoblotting with a MoAb to ptyr, RC20. Increases in tyrosine phosphorylation of Lyn were observed within 3 minutes. The same Lyn immunoprecipitates were used for Western blotting with anti-Lyn MoAb to check for immunoprecipitation efficiency (bottom). The positions of molecular size standards (left). Data are representative of three independent experiments. (B) (Right panel) Eosinophils were cultured in the presence of 10 μmol/L Lyn antisense or sense oligodeoxynucleotides for 6 hours. Decreased Lyn expression in eosinophils cultured with Lyn antisense was observed. Lyn sense oligodeoxynucleotides had no or only little effects on Lyn protein expression. (Left panel) Cells were then stimulated with anti-Fas MoAb for 18 hours. Statistically significant inhibition of Fas receptor-mediated cell death was observed in Lyn antisense but not sense treated eosinophils. Data are from six independent experiments. *P < .05. (C) (Left panel) The ICE inhibitor Ac-YVAD-cmk blocked in a dose-dependent manner Fas receptor-mediated but not spontaneous eosinophils death. Purified eosinophils were cultured for 24 hours. Cell viability was assessed by uptake of 1 μmol/L ethidium bromide and flow cytometric analysis. Values are means ± SEM of three independent experiments. (Right panel) Ac-YVAD-cmk did not prevent activation of Lyn following Fas receptor cross-linking. Freshly isolated Ac-YVAD-cmk (0.1 μmol/L) -pretreated and untreated eosinophils were stimulated with anti-Fas MoAb for the indicated times. The cell lysates were immunoprecipitated with an anti-Lyn MoAb and then analyzed by immunoblotting with a MoAb to ptyr, RC20. Increases in tyrosine phosphorylation of Lyn were observed in both untreated and Ac-YVAD-cmk–treated eosinophils. The same Lyn immunoprecipitates were used for Western blotting with anti-Lyn MoAb to check for immunoprecipitation efficiency (bottom).

Important role for Lyn tyrosine kinase in Fas receptor-mediated signaling in human eosinophils. (A) Freshly isolated eosinophils were stimulated with anti-Fas MoAb for the indicated times. The cell lysates were immunoprecipitated with an anti-Lyn MoAb and then analyzed by immunoblotting with a MoAb to ptyr, RC20. Increases in tyrosine phosphorylation of Lyn were observed within 3 minutes. The same Lyn immunoprecipitates were used for Western blotting with anti-Lyn MoAb to check for immunoprecipitation efficiency (bottom). The positions of molecular size standards (left). Data are representative of three independent experiments. (B) (Right panel) Eosinophils were cultured in the presence of 10 μmol/L Lyn antisense or sense oligodeoxynucleotides for 6 hours. Decreased Lyn expression in eosinophils cultured with Lyn antisense was observed. Lyn sense oligodeoxynucleotides had no or only little effects on Lyn protein expression. (Left panel) Cells were then stimulated with anti-Fas MoAb for 18 hours. Statistically significant inhibition of Fas receptor-mediated cell death was observed in Lyn antisense but not sense treated eosinophils. Data are from six independent experiments. *P < .05. (C) (Left panel) The ICE inhibitor Ac-YVAD-cmk blocked in a dose-dependent manner Fas receptor-mediated but not spontaneous eosinophils death. Purified eosinophils were cultured for 24 hours. Cell viability was assessed by uptake of 1 μmol/L ethidium bromide and flow cytometric analysis. Values are means ± SEM of three independent experiments. (Right panel) Ac-YVAD-cmk did not prevent activation of Lyn following Fas receptor cross-linking. Freshly isolated Ac-YVAD-cmk (0.1 μmol/L) -pretreated and untreated eosinophils were stimulated with anti-Fas MoAb for the indicated times. The cell lysates were immunoprecipitated with an anti-Lyn MoAb and then analyzed by immunoblotting with a MoAb to ptyr, RC20. Increases in tyrosine phosphorylation of Lyn were observed in both untreated and Ac-YVAD-cmk–treated eosinophils. The same Lyn immunoprecipitates were used for Western blotting with anti-Lyn MoAb to check for immunoprecipitation efficiency (bottom).

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