Secretory and cytosolic phospholipases A2 are activated during TNF priming of human neutrophils.
Cytokines alter neutrophil (PMN) function during inflammation, and Tumor Necrosis Factor (TNF) in vitro primes PMN such that receptor-mediated stimulation causes markedly enhanced release of arachidonic acid. We hypothesized that two Ca(2+)-dependent PLA2's in PMN might be activated during priming of the cell, thus affecting arachidonate release. A low molecular weight, secretory PLA2 was identified by enzymatic activity in the cell free supernates of primed or stimulated PMN, and in PMN disrupted by nitrogen cavitation. The enzymatic activity was calcium-dependent, acid stable, destroyed by dithiothreitol, and blocked by anti-sPLA2 antibodies. TNF caused secretion of sPLA2 and also caused an increase in cell-associated sPLA2 enzymatic activity. Activation and release were maximal with fMLP stimulation of TNF-primed PMN. Neutrophils also contained a cytosolic PLA2 (cPLA2) characterized by enzymatic activity which was calcium dependent, enhanced by dithiothreitol, and blocked by anti-cPLA2 antibody. TNF caused a doubling of cPLA2 enzymatic activity which was associated with phosphorylation of the enzyme as judged by a migration shift on Western blots. Thus, TNF priming of human PMN caused marked increase in fMLP stimulated AA release in parallel to enhanced activity of two different PLA2's.
Seeds
M C
MC
Department of Internal Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157-1054, USA. mseeds@bgsm.edu
Jones
D F
DF
Chilton
F H
FH
Bass
D A
DA
D016718:Arachidonic Acid / Q000097:blood; D002469:Cell Separation; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D010741:Phospholipases A / Q000097:blood* / Q000378:metabolism; D054467:Phospholipases A2; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010741:Phospholipases A; D054467:Phospholipases A2