Phospholipase D activity facilitates Ca2+-induced aggregation and fusion of complex liposomes.

Phospholipase D (PLD) activation in stimulated neutrophils results in the conversion of membrane phosphatidylcholine (PC) to phosphatidic acid (PA). This change in membrane phospholipid composition has two potentially positive effects on degranulation. It 1) replaces a nonfusogenic phospholipid with a fusogenic one and 2) increases the potential for interactions between membranes and the annexins.

Unidirectional heterologous receptor desensitization between both the fMLP and C5a receptor and the IL-8 receptor.

During inflammation neutrophils receive multiple signals that are integrated, allowing a single modified response. One mechanism for this discrimination is receptor desensitization, a process whereby ligand-receptor binding is disassociated from cell activation. We examined the effect of heterologous receptor desensitization on neutrophil chemotaxis, calcium mobilization, and arachidonic acid production, using interleukin-8 (IL-8), C5a, and N-formyl-methionyl-leucyl-phenylalanine (fMLP).

PLA2 promotes fusion between PMN-specific granules and complex liposomes.

Neutrophil stimulation results in the activation of a variety of phospholipases, including phospholipase A2 (PLA2), which releases arachidonic acid from the 2 position of membrane phospholipids, leaving a lysophospholipid. Because arachidonic acid is known to be a potent fusogen in vitro, we examined the effect of metabolism by PLA2 on the fusion of complex liposomes (liposomes prepared with a phospholipid composition similar to that found in neutrophil plasma membrane). We observed that PLA2 augmented the fusion of complex liposomes with each other as well as with specific granules isolated from human neutrophils, lowering the Ca2+ requirement for fusion by three orders of magnitude.

Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils.

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles.