Na+/H+ exchange activity during phagocytosis in human neutrophils: Role of Fcγ receptors and tyrosine kinases

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 (pH(i)) 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 Fcγ (FcγR) and complement receptors such as CR3 (a β2 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 FcγRs (FcγRII > FcγRIII) but not β2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-β2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and FcγRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcγR 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 Ca2+ nor buffering of changes in [Ca2+](i) inhibited alkalinization after OpZ or FcγR cross-linking. In summary, FcγRs and β2 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation.