NA+ H+ EXCHANGE ACTIVITY DURING PHAGOCYTOSIS IN HUMAN NEUTROPHILS - ROLE OF FC-GAMMA RECEPTORS AND TYROSINE KINASES/

In neutrophils, binding and phagocytosis facilitate subsequent intrace llular killing of microorgan isms. Activity of Na+/H+ exchangers (NHEs ) participates in these events, especially in regulation of intracellu lar pH (pH(i)) by compensating for the H+ load generated by the respir atory burst, Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutr ophils, The purpose of this study was to identify which NHE(s) are exp ressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis, Exposure of cells to the phagocytic sti mulus opsonized zymosan (OpZ) induced a transient cytosolic acidificat ion followed by a prolonged alkalinization, The latter was inhibited i n Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange, Reverse transcriptase PCR and cDNA sequ encing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed, Immunoblotting of purified p lasma membranes with isoform-specific antibodies confirmed the presenc e of NHE-1 protein in neutrophils, Since phagocytosis involves Fc gamm a (Fc gamma R) and complement receptors such as CR3 (a beta(2) integri n) 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 antibo dies (mAb) directed against Fc gamma Rs (Fc gamma RII > Fc gamma RIII) but not beta(2) integrins induced an amiloride-sensitive cytosolic al kalinization, However, anti-beta(2) integrin mAb diminished OpZ-induce d alkalinization suggesting that NHE-1 activation involved cooperation between integrins and Fc gamma Rs, The tyrosine kinase inhibitors gen istein and herbimycin blocked cytosolic alkalinization after OpZ or Fc gamma R cross-linking suggesting that tyrosine phosphorylation was in volved 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 Fc gamma R cross-linking, In summary, Fc gamma Rs and beta(2) integ rins cooperate in activation of NHE-1 in neutrophils during phagocytos is by a signaling pathway involving tyrosine phosphorylation.