Bcl-xL, but not Bcl-xS, protein is significantly expressed by purified blood eosinophils. (A) Immunocytochemistry. Bcl-x (the antibody used reacts with both Bcl-xL and Bcl-xS proteins) proteins were expressed in freshly isolated peripheral blood eosinophils (0), and levels rapidly decreased under conditions of cytokine withdrawal in vitro, but were maintained in the presence GM-CSF. (B) Flow cytometry. The Bcl-x protein expression observed in freshly isolated peripheral blood eosinophils (0) was undetectable in the absence of eosinophil hematopoietins and appeared to be increased by cytokine exposure in cultured eosinophils after 20 hours. (C) Immunoprecipitation and immunoblot. Bcl-xL (arrow), but not Bcl-xS(arrowhead), protein expression was observed in freshly isolated peripheral blood eosinophils (0). Bcl-xL protein expression was increased after GM-CSF stimulation but decreased after cytokine withdrawal in vitro. (Right) The positions of molecular size standards. (D) Semiquantitative RT-PCR. Bcl-xL protein expression correlates with the expression of Bcl-xL mRNA. Cytokine withdrawal in vitro decreased Bcl-xL mRNA in eosinophils, whereas stimulation with GM-CSF or IL-5 (not presented) increased Bcl-xL mRNA expression. (Top) Numbers of PCR cycles. (E and F) Immunocytochemistry and flow cytometry. No detectable Bcl-xS (the antibody used reacts specifically with Bcl-xS) protein expression was observed in freshly isolated peripheral blood eosinophils (0). (A, B, E, and F) are representative of eight independent experiments, (C and D) of three independent experiments.
