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Role of diradylglycerol formation in H2O2 and lactoferrin release in adherent human polymorphonuclear leukocytes
T Nakamura1, S J Suchard, A Abe
1Department of Pediatrics, University of Michigan, Ann Arbor.
Abstract:
Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the release of H2O2 in response to fMLP. Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin-containing specific granules and activation of phospholipase D (PLD). We also found that chelation of intracellular calcium blocked both lactoferrin and H2O2 release in stimulated PMNs in spite of the fact that adhesion and spreading remained normal. Since diradylglycerol (DRG) formation has been implicated in PMN secretion and oxidant release, we determined the effect of intracellular calcium chelation on PLD activation and DRG formation to ascertain whether DRG formation was coupled to lactoferrin and H2O2 release. We observed that chelation of intracellular calcium with bis-(O-aminophenoxy)-ethanol-N,N;N'-tetraacetic acid (BAPTA) prevented PLD activation as monitored by inhibition of phosphatidylethanol formation. Formation of DRG derived from phosphatidic acid (PA) was also inhibited in the presence of BAPTA. Following the addition of the calcium ionophore ionomycin to the BAPTA-treated PMNs, lactoferrin and H2O2 release was coincident with the onset of DRG formation. Also the addition of sn-1,2-didecanoylglycerol to the BAPTA-treated PMNs stimulated them to release H2O2. Our studies support the hypothesis that DRG derived from PLD activation is required for degranulation of specific granules and associated H2O2 release from adherent PMNs.
Insights
Intracellular calcium is crucial for polymorphonuclear leukocytes (PMNs) to release hydrogen peroxide (H2O2) and lactoferrin. Diradylglycerol (DRG) formation, triggered by phospholipase D (PLD) activation, is essential for this degranulation process in adherent PMNs.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMNs) exhibit delayed hydrogen peroxide (H2O2) release when adhering to fibrinogen.
- H2O2 release in adherent PMNs is linked to specific granule exocytosis and phospholipase D (PLD) activation.
- Intracellular calcium chelation inhibits lactoferrin and H2O2 release without affecting PMN adhesion or spreading.
Purpose of the Study:
- To investigate the role of intracellular calcium in PLD activation and diradylglycerol (DRG) formation.
- To determine if DRG formation is coupled to lactoferrin and H2O2 release in stimulated PMNs.
Main Methods:
- Chelation of intracellular calcium using BAPTA.
- Monitoring PLD activation via phosphatidylethanol formation.
- Assessing DRG formation from phosphatidic acid (PA).
- Stimulating PMNs with ionomycin and sn-1,2-didecanoylglycerol.
Main Results:
- BAPTA prevented PLD activation and subsequent DRG formation.
- Restoring calcium with ionomycin initiated DRG formation, coinciding with lactoferrin and H2O2 release.
- Exogenous sn-1,2-didecanoylglycerol stimulated H2O2 release in BAPTA-treated PMNs.
Conclusions:
- Diradylglycerol (DRG) formation, derived from PLD activation, is a necessary step for specific granule degranulation and H2O2 release in adherent PMNs.
- Intracellular calcium signaling is essential for PLD activation and subsequent DRG-mediated degranulation in PMNs.