Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked.

Neutrophils phagocytize high-valency im- mune 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 gran- ules. Signaling events that precede and accompany PMN secretion include activation of phospholipase D (PLD), as well as changes in cytoplasmic (Ca 21 ) (D(Ca 21 )in) and pH (DpHin). Although the role of PLD and D(Ca 21 )in in mediating Fc receptor-mediated PMN secretion has been studied, whether pHin plays a regu- latory role has not yet been defined. HIC-stimulated PMN undergo an intracellular acidification followed by a prolonged Na 1 /H 1 antiport-mediated alkalinization. To investigate the role of the pH transient in controlling degranulation, the Na 1 /H 1 antiport was inhibited ei- ther with 100 mM dimethylamiloride (DMA) or by substituting N-methyl-glucamine for extracellular so- dium. Blocking the antiport with DMA led to hyper- acidified PMN, which exhibited an increase in degranu- lation, but did not affect generation of superoxide. DMA did not alter the ability of neutrophils to phagocy- tose 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 anti- port was blocked was mediated by PLD or D(Ca 21 )in revealed that blocking the antiport increased HIC- induced PLD activity but had no effect on HIC- induced D(Ca 21 )in. Blocking the Na 1 /H 1 antiport by ion substitution caused similar effects on PMN signal- ing and secretion as was seen with DMA. These results indicate that Na 1 /H 1 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 neutro- phils achieve differential control of their antibacterial products. J. Leukoc. Biol. 64: 98-103; 1998.