Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases.

Abstract

Abstract. In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H ÷ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H ÷ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H + exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fc~/(Fc~R) and complement receptors such as CR3 (a 132 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against Fc~/Rs (Fc-,/RII > Fc-yRIII) but not 132 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-132 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and Fc~Rs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcvR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-1 activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca 2÷ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc~R cross-linking. In summary, Fc~/Rs and 132 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation. NEUTROPmLS react to invading microorganisms and mediators present within an inflammatory milieu with a variety of rapid and coordinated responses which include movement of cells out of the vascular space along a gradient of chemotactic molecules followed by phagocytosis and killing of the microorganisms. This bactericidal function is effected by complex processes involving secretion of proteolytic enzymes and reactive oxygen intermediates (produced by the NADPH oxidase) into the phagolysosome (for review see Sha'afi and Molski, 1988). During these processes, dynamic alterations occur in leu-

Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis

“…In cells exhibiting recovery, the nadir occurred after 3.4 Ϯ 2.0 min (SD, n ϭ 58). The large decrease in pH i in phagocytosing neutrophils was unexpected and greatly exceeds reported responses (1,(3)(4)(5)7). This apparent discrepancy is likely attributable mainly to the smearing of time courses of individual cells by averaging, inclusion of nonresponding cells, and use of soluble stimuli in previous studies.…”

“…For 3 decades, pH i during the respiratory burst has been characterized as a small (0.05-0.1 unit) transient drop in pH i that is followed by larger (0.16-0.60 unit) prolonged alkalinization caused by Na ϩ /H ϩ antiport activity, whether the stimulus is fMLF (fMetLeuPhe, a chemotactic peptide), PMA (a PKC-activating phorbol ester), phospholipase C, or OPZ (opsonized zymosan) (1)(2)(3)(4)(5)(6)(7). In most studies, soluble stimuli were applied to populations of neutrophils.…”

“…In most studies, soluble stimuli were applied to populations of neutrophils. When phagocytosis was studied, the responses of many cells were averaged (5,8). A powerful new imaging approach, confocal microscopy using the fluorescent pH indicator SNARF-1 enhanced by shifted excitation and emission ratioing (SEER) (9,10) enabled us to examine the responses of individual cells with high spatial and temporal resolution.…”

Rapid activation of sodium–proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades

“…Those similarities provide evidence to suggest that ERK may be a direct proximal component of an NHE regulatory pathway [5,6]. There is a growing awareness that tyrosine phosphorylation cycles are critical in regulating NHE activities in a number of cell types [6][7][8][9][10], as has also been shown for ERK [11]. Other studies have demonstrated that NHE and ERK activities are modulated by overlapping upstream enzymes, including phosphoinositide 3h-kinase (PI-3K), phospholipase Cγ, and PKC [12][13][14][15][16][17][18][19].…”

Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in 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).…”