Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked.
Neutrophils phagocytize high-valency immune complexes (HIC) by an Fc receptor-mediated mechanism. Engaging Fc receptors in this manner induces PMN to generate superoxide and release the contents of both their specific and azurophilic granules. Signaling events that precede and accompany PMN secretion include activation of phospholipase D (PLD), as well as changes in cytoplasmic [Ca2+] (delta[Ca2+]in) and pH (delta pHin). Although the role of PLD and delta[Ca2+]in in mediating Fc receptor-mediated PMN secretion has been studied, whether pHin plays a regulatory role has not yet been defined. HIC-stimulated PMN undergo an intracellular acidification followed by a prolonged Na+/H+ antiport-mediated alkalinization. To investigate the role of the pH transient in controlling degranulation, the Na+/H+ antiport was inhibited either with 100 microM dimethylamiloride (DMA) or by substituting N-methyl-glucamine for extracellular sodium. Blocking the antiport with DMA led to hyperacidified PMN, which exhibited an increase in degranulation, but did not affect generation of superoxide. DMA did not alter the ability of neutrophils to phagocytose and oxidize dichlorodihydrofluoresceinated HIC, suggesting the increase in degranulation was not the result of failed phagocytosis. Investigation into whether the observed increase in degranulation when the antiport was blocked was mediated by PLD or delta[Ca2+]in revealed that blocking the antiport increased HIC-induced PLD activity but had no effect on HIC-induced delta[Ca2+]in. Blocking the Na+/H+ antiport by ion substitution caused similar effects on PMN signaling and secretion as was seen with DMA. These results indicate that Na+/H+ antiport activity is not necessary for degranulation or superoxide release in HIC-stimulated PMN and that hyperacidification of the cytoplasm can modulate degranulation. Therefore, pHin, via its effect on PLD, may be a control point of degranulation and may represent one way that neutrophils achieve differential control of their antibacterial products.
Department of Biochemistry, Boston University School of Medicine, MA 02118, USA.
A T
AT
Gewirtz
K F
KF
Seetoo
E R
ER
Simons
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000936:Antigen-Antibody Complex / Q000494:pharmacology; D015550:Cell Degranulation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology*; D010739:Phospholipase D / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*; D013481:Superoxides / Q000378:metabolism
D000936:Antigen-Antibody Complex; D017923:Sodium-Hydrogen Exchangers; D013481:Superoxides; C039465:5-dimethylamiloride; D000584:Amiloride; D010739:Phospholipase D