Figure 3.

Uptake of SOCS3-containing MPs by AECs inhibits target cell STAT3 activation. (A) AECs were pretreated with CM from AMs cultured overnight for the time intervals indicated, after which they were challenged with IL-6 for 1 h and lysates were subjected to WB for STAT3 activation; results are expressed as the percentage of the stimulated increase in cytokine-treated cells not receiving AM CM, indicated by the dashed line. (B) AECs were treated with or without AM CM for 2 h at 37°C or at 4°C, after which AEC lysate proteins were subjected to SOCS3 quantitation by ELISA. Data are expressed as ng/µg of total protein. (C) AM-derived MPs were labeled with FITC–annexin V and added to AECs at a ratio of 10:1 for 1 h at 37°C or at 4°C. Increases in fluorescence in AEC cultures were determined by flow cytometry and are depicted as histograms from a representative experiment (top) and mean fluorescence intensity (MFI; fold change versus background fluorescence of AECs alone) from three experiments (bottom). MFI of AECs receiving FITC–annexin V without MPs at 37°C was similar to background (not depicted). (D) MPs isolated from AM CM were incubated with AECs at a ratio of 10:1 for 2 h before stimulation with IL-6, and lysates were analyzed for STAT3 activation and expressed as the percentage of that determined in cytokine-treated AECs not pretreated with MPs. (E) AECs were pretreated with or without CM or with MP-depleted CM for 2 h at 37°C before stimulation with IL-6, after which lysates were analyzed for STAT3 activation and expressed as the percentage of that determined in cytokine-treated AECs not pretreated with CM. (A–E) Data represent the mean ± SE from at least three independent experiments (B–E) or are representative of two independent experiments (A). *, P < 0.05 versus AECs not pretreated with CM at 37°C (B), AECs incubated with FITC–annexin V–labeled MPs at 37°C (C), cytokine-stimulated AECs not pretreated with MPs (D), or AECs pretreated with CM (E).