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Phospholipase D-mediated diradylglycerol formation coincides with H2O2 and lactoferrin release in adherent human
S J Suchard1, T Nakamura, A Abe
1Department of Pediatrics, University of Michigan, Ann Arbor 48109.
Abstract:
Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the onset of the respiratory burst in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP). Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin-containing specific granules. Since diradylglycerol (DRG) has been implicated in PMN secretion and oxidant release, we measured DRG formation during PMN adhesion to fibrinogen. PMNs were added to fibrinogen-coated plastic in the presence of fMLP, and H2O2 release, lactoferrin release, and DRG formation measured over a time course of 120 min. H2O2 and lactoferrin release were not apparent until 45-60 min, reaching maximal levels by 120 min. In contrast, DRG concentration increased by 15-30 min, from 275 +/- 27 pmol/mg of protein in resting cells to 600 +/- 173 pmol/mg protein in cells exposed to fMLP. DRG levels returned to base line by 30-45 min (383 +/- 32 pmol/mg of protein) before increasing again between 60 and 120 min (944 +/- 230 pmol/mg of protein and 1632 +/- 351 pmol/mg of protein, respectively). Propranolol, an inhibitor of phosphatidate phosphohydrolase, caused a dose-dependent inhibition of both H2O2 and lactoferrin release, with maximal inhibition at 50-100 microM. Propranolol also inhibited the second, but not the first phase of DRG formation. Similarly, ethanol treatment completely blocked H2O2 and lactoferrin release, and the second phase of DRG formation. In the presence of ethanol, phospholipase D (PLD)-mediated formation of [3H]phosphatidylethanol from 3H-O-alkyl-phosphatidylcholine corresponded to the second, but not the first, phase of DRG formation (23,169 +/- 2,017 cpm/mg protein, ethanol versus 2,696 +/- 261 cpm/mg protein, control). These data indicate that DRG, generated through the activation of PLD, plays an important role in degranulation and oxidant release in adherent PMNs.
Insights
Diradylglycerol (DRG) formation, stimulated by phospholipase D (PLD), is crucial for the delayed respiratory burst and lactoferrin release in adherent polymorphonuclear leukocytes (PMNs). This study reveals DRG
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMNs) exhibit a delayed respiratory burst upon adhesion to fibrinogen.
- This delay is linked to the exocytosis of lactoferrin from specific granules.
- Diradylglycerol (DRG) is implicated in PMN secretion and oxidant release.
Purpose of the Study:
- To investigate the role of DRG formation in the delayed respiratory burst and degranulation of adherent PMNs.
- To elucidate the signaling pathways involved in DRG generation and its subsequent effects on PMN function.
Main Methods:
- PMNs were adhered to fibrinogen-coated surfaces and stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP).
- Measurements included H2O2 release, lactoferrin release, and DRG formation over a 120-minute time course.
- Inhibitors such as propranolol and ethanol were used to probe the involvement of specific enzymes like phospholipase D (PLD).
Main Results:
- DRG levels increased early (15-30 min), preceding the delayed H2O2 and lactoferrin release (45-60 min).
- A second phase of DRG formation occurred later, correlating with maximal H2O2 and lactoferrin release.
- Inhibition of phosphatidate phosphohydrolase (with propranolol) or PLD (with ethanol) blocked the second DRG phase and subsequent oxidant/lactoferrin release.
Conclusions:
- DRG, generated via PLD activation, plays a critical role in the degranulation and oxidant release of adherent PMNs.
- The findings highlight a two-phase mechanism involving DRG in regulating PMN inflammatory responses.