lactoferrin and diarachidonyl-diglyceride

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.

Topics: Amino Acid Sequence; Calcium; Cell Adhesion; Cells, Cultured; Chelating Agents; Diglycerides; Egtazic Acid; Enzyme Activation; Exocytosis; Fibrinogen; Humans; Hydrogen Peroxide; Indicators and Reagents; Intracellular Fluid; Lactoferrin; Molecular Sequence Data; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipase D

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.

Topics: Cell Adhesion; Diglycerides; Ethanol; Fibrinogen; Humans; Hydrogen Peroxide; In Vitro Techniques; Kinetics; Lactoferrin; Myristic Acid; Myristic Acids; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipase D; Propranolol