Introduction Eosinophils account for approximately 2-3% of peripheral white blood cells and are involved in clearing helminth infections (reviewed by Giembycz and Lindsay, 1999). However, in populations where helminth infections are non- endemic, eosinophils are typically associated with the lung pathologies observed in asthma and other eosinophil- associated diseases. Activated eosinophils in all situations can severely damage pathogen and host cells by several effector mechanisms, including superoxide production and the release of toxic granule proteins (reviewed in Giembycz and Lindsay, 1999). Activation of human eosinophils in vitro with platelet- activating factor (PAF) or IL-5 increases superoxide production by stimulating NADPH oxidase activity (Bankers-Fulbright et al., 2001; Bankers-Fulbright et al., 2003). Plasma membrane NADPH oxidase activation produces a rapid transfer of electrons from intracellular NADPH to extracellular oxygen, forming the superoxide anion. Concurrently, protons are generated in the cytosol as NADPH is converted to NADP + and H + . Estimates of the rate of free proton generation indicate that catastrophic acidification would occur within the cell unless an efficient proton extruding mechanism was activated in parallel with electron transport (Rotstein et al., 1987). Several studies have shown that a voltage-dependent, Zn 2+ -sensitive plasma membrane proton channel is activated in parallel with the increase in NADPH oxidase activity (Cherny et al., 2001; Cherny et al., 2003; DeCoursey et al., 2001; DeCoursey et al., 2003; Gordienko et al., 1996; Petheo et al., 2003). This channel is regulated by mediators that increase PKCδ activity and, following PKC activation, the cytosolic pH (pH i ) does not significantly change in response to increases in superoxide production (Bankers-Fulbright et al., 2001). Thus, proton channels play a major role in the control of pH i in activated human eosinophils. Other mechanisms responsible for pH i regulation in resting and activated eosinophils have not been investigated in detail, but it is likely that they share some similarity with neutrophils (Coakley et al., 2002; DeCoursey and Cherny, 1994; Demaurex et al., 1996; Fukushima et al., 1996; Grinstein et al., 1986; Grinstein et al., 1991). Early investigations of pH i regulation in neutrophils during the oxidative burst revealed that Na + -H + exchange (NHE-1) activity as well as the activation of proton channels was essential to prevent intracellular acidification (Coakley et al., 2002; DeCoursey and Cherny, 1994; Demaurex et al, 1996; Fukushima et al., 1996). In addition, a 5749 The effects of platelet-activating factor (PAF) and IL-5 on intracellular pH were investigated in human eosinophils. Purified peripheral blood eosinophils were loaded with the ratiometric fluorescent pH indicator BCECF-AM ester. Stimulation of eosinophils with PAF produced time- dependent alkalinization of the cytoplasm from an initial pH of 7.1±0.04 to 7.5±0.05. A similar alkalinization response was produced by the calcium ionophore, ionomycin and by the calcium ATPase inhibitor, thapsigargin. These compounds as well as PAF produce significant increases in cytoplasmic calcium ([Ca 2+ ] i ). In contrast, IL-5 and the protein kinase C (PKC) activator phorbol myristate acetate (PMA) did not produce cytoplasmic alkalinization and had no effect on [Ca 2+ ] i in eosinophils. PAF-stimulated alkalinization was not inhibited under conditions that blocked plasma membrane Na + -H + exchange, proton channel or plasma membrane H + - ATPase activities. Measurements of intragranule pH with a cell permeant pH indicator (LysoSensor Yellow/Blue DND-160), which partitions into intracellular acidic compartments, revealed that PAF-stimulated cytosolic alkalinization correlated with intragranule acidification. These results suggest that the increase in [Ca 2+ ] i after PAF stimulation activates a H + -ATPase present in the granule membranes, leading to enhanced granule acidification and cytoplasmic alkalinization. We propose that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins. Key words: Eosinophils, pH regulation, IL-5, PAF, H + -ATPase Summary Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils Jennifer L. Bankers-Fulbright 1 , Gail M. Kephart 1 , Kathleen R. Bartemes 1 , Hirohito Kita 1,2 and Scott M. O’Grady 3, * 1 Department of Medicine and 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 *Author for correspondence (e-mail: ograd001@umn.edu) Accepted 17 August 2004 Journal of Cell Science 117, 5749-5757 Published by The Company of Biologists 2004 doi:10.1242/jcs.01498 Research Article