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First published online 26 October 2004
doi: 10.1242/jcs.01498

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Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils

¹ Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
² Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
³ Departments of Physiology and Animal Science, University of Minnesota, 1988 Fitch Avenue, St. Paul, MN 55108, USA

View larger version (15K): [in a new window]   Fig. 1. Cytosolic alkalinization occurs in human eosinophils stimulated with platelet-activating factor (PAF), but not with phorbol myristate acetate (PMA) or IL-5. Purified human eosinophils were loaded with BCECF/AM and stimulated with media control, PAF (3 µM), PMA (2 nM) or IL-5 (0.2 µM) at 37°C. Fluorescence was measured over 90 minutes and was converted to pHi using a standard curve as described in Materials and Methods. Data are shown as mean±s.e.m. (A) Eosinophils stimulated with media control (; n=17) showed no significant change in pHi compared to pHi at time 0. PAF-stimulated eosinophils (; n=17) showed a rapid and sustained cytosolic alkalinization compared to pHi at time 0 that reached a steady-state level approximately 1 hour after stimulation. Initial pHi values for media control and PAF data sets were both 7.1±0.0. (B) Eosinophils stimulated with media control (; n=11), PMA (; n=9), or IL-5 (; n=9) showed no significant changes in pHi compared with pHi values at time 0. Initial pHi values for media control, PMA and IL-5 data sets were 7.1±0.1, 7.1±0.1 and 7.2±0.1, respectively. *P<0.01 (compared to pHi at time 0).

View larger version (22K): [in a new window]   Fig. 2. Intracellular Ca2+ mobilization is associated with eosinophil cytosolic alkalinization. Purified human eosinophils were loaded with BCECF/AM and stimulated with thapsigargin (10 µM), ionomycin (2 µM) and EGTA (5 mM) at 37°C. Fluorescence was measured over 90 minutes and was converted to pHi using a standard curve as described in Materials and Methods. Data are shown as mean±s.e.m. Thapsigargin (; n=3) and ionomycin (; n=4) stimulation induced significant cytosolic alkalinization. Stimulation with ionomycin after EGTA pre-treatment (; n=4) resulted in complete inhibition of alkalinization and induced significant acidification relative to pHi at time 0. Initial pHi values for thapsigargin, ionomycin and ionomycin + EGTA data sets were all 7.1±0.1. *P<0.05; **P<0.01 (compared to pHi at time 0 or, when labeling the bracket, comparing two final pHi values from different stimuli); n.s., not statistically significant.

View larger version (19K): [in a new window]   Fig. 3. PAF, but not IL-5, stimulation of human eosinophils increases [Ca2+]i. Purified eosinophils were loaded with Fura-PE3/AM, plated onto glass coverslips at a concentration of 0.25x106/ml and allowed to attach to the glass before imaging at room temperature. Data are shown as mean±s.e.m. (A) Addition of PAF (3 µM) produced a rapid rise in [Ca2+]i that exhibited diminishing oscillations with time in the continuous presence of PAF (n=6, using cells from two isolations). (B) Eosinophils treated with IL-5 (0.2 µM) failed to show any increase in [Ca2+]i. Subsequent stimulation with 3 µM PAF produced an increase in [Ca2+]i indicating that the cells were capable of responding to calcium-mobilizing agonists (n=7, using cells from two isolations).

View larger version (19K): [in a new window]   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 pHi 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 pHi compared to pHi at time 0. PAF-stimulated eosinophils (; n=4) showed a rapid and sustained cytosolic alkalinization compared to pHi at time 0 and this was unaffected by pre-treatment with anti-CD18 antibody (; n=4). Initial pHi values for media control, PAF, and PAF + anti-CD18 data sets were all 7.0±0.1. (B) Eosinophils stimulated with media control (; n=4) showed no significant change in pHi compared with pHi at time 0. Stimulation with ionomycin in the absence (; n=4) or presence (; 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 pHi 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 pHi at time 0 or, if labeling a bracket, comparison of two final pHi values after different stimuli); n.s., not statistically different.

View larger version (21K): [in a new window]   Fig. 5. PAF-stimulated cytosolic alkalinization is not blocked by inhibiting known proton-efflux mechanisms. Purified human eosinophils were loaded with BCECF/AM and stimulated with media control or PAF (3 µM) in the presence or absence of EIPA (100 µM), Zn2+ (250 µM), bafilomycin (2.5 µM), or folimycin (2.5 µM) at 37°C. Fluorescence was measured over 90 minutes and was converted to pHi using a standard curve as described in Materials and Methods. Data are shown as mean±s.e.m. (A) Eosinophils stimulated with media alone (n=5) showed no significant changes in pHi compared with pHi at time 0. Eosinophils stimulated with PAF alone (n=3) or in the presence of EIPA (n=3), Na+-free media (n=3) or Zn2+ (n=3) as indicated, all show significant alkalinization compared to pHi at time 0. Initial pHi values for EIPA, Na+-free media, and Zn2+ data sets were 6.8±0.0, 6.9±0.1 and 6.7±0.0, respectively. None of the inhibitors resulted in a final pHi that was significantly different from that induced by PAF alone. (B) Eosinophils stimulated with media control (n=6) showed no significant changes in pHi compared with pHi values at time 0. Eosinophils stimulated with PAF alone (n=7) or in the presence of bafilomycin (n=7) or folimycin (n=5) showed similar levels of alkalinization. Initial pHi values for media control, PAF, PAF + bafilomycin and PAF + folimycin data sets were all 7.0±0.1. There were no significant differences in final pHi in when cells were stimulated with PAF, PAF + bafilomycin or PAF + folimycin. *P<0.01 (compared to pHi at time 0); n.s., not statistically different.

View larger version (99K): [in a new window]   Fig. 6. Eosinophil granules are apparent intracellular sites of acidification. Purified eosinophils were loaded with LysoSensor Yellow/Blue DND-160 and stimulated with media control, PAF (3 µM) or IL-5 (0.2 µM) at room temperature (n=10-15 images from four individual isolations for each condition). Confocal microscopy was used to document fluorescence changes under each condition and overlays of the blue and yellow fluorescence of each image are shown. Original images were taken at a magnification of x630. (A) Low power confocal images of eosinophils stimulated with PAF for varying amounts of time (0-15 minutes). (B) Representative images of eosinophils stimulated with IL-5 or PAF for 40 minutes at room temperature. Note the granular appearance of the dye, consistent with accumulation of the dye within the characteristic secondary granules of the eosinophil.

View larger version (94K): [in a new window]   Fig. 7. Eosinophils express V-ATPase subunit E in whole cell lysates. Eosinophil whole cell lysates were prepared and examined for V-ATPase subunit E by western blotting as described in Materials and Methods. Commercially available mouse brain extract was used as a positive control (+). Shown are representative control (nIgG) and test (V-ATPase E) blots from three independent experiments using eosinophils. Each Eo Lysates lane represents an eosinophil extract from a separate donor. A band specific to the V-ATPase E blot is seen around 30 kDa in the positive control and in each eosinophil extract lane (arrow). The non-specific band around 48 kDa in the eosinophil extracts is probably eosinophil peroxidase (EPO).

View larger version (39K): [in a new window]   Fig. 8. The V-ATPase subunit E is localized to granular compartments in eosinophils. Eosinophils were isolated and stained for the V-ATPase subunit E and were analyzed by confocal microscopy as described in Materials and Methods. Shown are representative images from four independent experiments. Original images were taken at a magnification of x630. (A) Eosinophils stained with an isotype control antibody followed by FITC-conjugated secondary antibody. (B) Eosinophils stained with anti-V-ATPase subunit E followed by FITC-conjugated secondary antibody show a granular staining.