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Fig. 4. Eosinophil adhesion via ß2 integrins is not required for PAF-stimulated alkalinization. Purified eosinophils were loaded with BCECF/AM and pretreated with or without anti-CD18 antibody (10 µg/ml) before being stimulated with control media, PAF (3 µM) or ionomycin (2 µM) at 37°C. Fluorescence was measured over 90 minutes and was converted to pH_i using a standard curve as described in Materials and Methods. Data are shown as mean±s.e.m. (A) Eosinophils stimulated with media control (open circles; n=4) showed no significant change in pH_i compared to pH_i at time 0. PAF-stimulated eosinophils ( ${bullet}$ ; n=4) showed a rapid and sustained cytosolic alkalinization compared to pH_i at time 0 and this was unaffected by pre-treatment with anti-CD18 antibody ( ${blacksquare}$ ; n=4). Initial pH_i values for media control, PAF, and PAF + anti-CD18 data sets were all 7.0±0.1. (B) Eosinophils stimulated with media control ( ${circ}$ ; n=4) showed no significant change in pH_i compared with pH_i at time 0. Stimulation with ionomycin in the absence ( ${blacksquare}$ ; n=4) or presence ( ${bullet}$ ; n=4) of anti-CD18 antibody resulted in cytosolic alkalinization, although the magnitude of alkalinization was lower in the presence of anti-CD18 antibody. Initial pH_i values for media control, ionomycin, and ionomycin + anti-CD18 were 7.0±0.1, 6.9±0.0, and 7.0±0.1, respectively. ^*P<0.05; ^**P<0.01 (compared to pH_i at time 0 or, if labeling a bracket, comparison of two final pH_i values after different stimuli); n.s., not statistically different.

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