Fig. 4.

IL-6Rα is synthesized from constitutive mRNA by human PMNs in response to inflammatory signaling to mTOR. PMNs were incubated with control buffer, PAF, or other inflammatory agonists in suspension, and IL-6Rα protein was detected by Western analysis with the same antibody used for immunocytochemical studies (Fig. 3). (A) PMNs were incubated with control buffer (lane C) or increasing concentrations of PAF for 1 h. (B) PMNs were incubated with control buffer, 100 nM PAF, PAF that had been preincubated with recombinant human PAF acetylhydrolase (rPAF-AH), or the indicated agonists. (C) HUVEC monolayers were incubated with PMNs activated with 100 nM PAF alone or after pretreatment with rapamycin by using separated chambers and were then blotted for IL-6Rα. Control HUVEC were incubated in the absence of PAF-stimulated PMNs. Lane 1, control HUVEC; lane 2, HUVEC incubated with PMNs pretreated with rapamycin before stimulation with PAF; lane 3, HUVEC incubated with PAF-stimulated PMNs. (D) PMNs were pretreated with control buffer or actinomycin D followed by activation with 100 nM PAF for the times shown. Blots from PMNs activated with PAF alone or after pretreatment with actinomycin D are shown above and below one another at each time point. Control PMNs were lysed in the absence of actinomycin D or PAF and blotted (leftmost lane in Upper). In some experiments IL-6Rα stains as a doublet or triplet on immunoblotting, as shown. (E) PMNs were pretreated with control buffer or rapamycin and then activated with 100 nM PAF for the times indicated. In a control experiment, neither actinomycin D nor rapamycin alone in the absence of PAF induced an IL-6Rα band (data not shown).