Primed neutrophils require phosphatidic acid for maximal receptor-activated elastase release.
BACKGROUND
Priming of neutrophils (PMNs) for protease release is believed to be central to the pathogenesis of PMN-mediated tissue injury observed in ARDS/MOF. Defining the intracellular signaling pathways involved with this excessive protease release may aid in establishing future therapies for ARDS. Phospholipase D (PLD) production of phosphatidic acid (PA) is thought to be pivotal in reactive oxygen species generation but its role in degranulation (i. e., protease release) remains unclear. Our hypothesis was that primed neutrophils require PLD production of PA for maximal activated release of elastase.


METHODS
Isolated human PMNs were incubated with a well described antagonist of PA production, ethanol (ETOH, 100-1000 mg/dL), and then primed (PAF, 200 nM) followed by activation (fMLP, 1 microM). To mimic fMLP receptor-dependent activation, PMNs were primed and then directly activated with exogenous dioctanoyl l-alpha-phosphatidic acid (PA8, 0.5-200 microM). To confirm the importance of PA in elastase release, PA8 was given to primed-activated PMNs after ethanol pretreatment in an attempt to recover the maximal response. Elastase release was measured by the cleavage of AAPV-pNA.


RESULTS
PA blockade with ETOH attenuated PAF-primed/fMLP-activated PMN elastase release in a dose-dependent manner. Exogenous PA8 reproduced maximally primed-activated PMN elastase release, and furthermore, PA8 was able to restore maximal elastase release following ethanol attenuation.


CONCLUSIONS
Elastase release from PAF-primed/ fMLP-activated neutrophils is dependent on PA production. Thus, PA production appears to be involved in both oxidant-dependent and independent mechanisms of neutrophil cytotoxicity and may be a potential therapeutic target in the treatment of hyperinflammatory diseases such as ARDS/MOF.
Denver Health Medical Center, University of Colorado Health Sciences Center, Denver, Colorado, 80204, USA.
D Y
DY
Tamura
E E
EE
Moore
D A
DA
Partrick
J L
JL
Johnson
G
G
Zallen
C C
CC
Silliman
D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism / Q000494:pharmacology*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology*; D018000:Receptors, Peptide / Q000502:physiology*
D010712:Phosphatidic Acids; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D019272:Leukocyte Elastase