Secretory and cytosolic phospholipases A 2 are activated during TNF priming of human neutrophils

Abstract

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 PLA 2's in PMN might be activated during priming of the cell, thus affecting arachidonate release. A low molecular weight, secretory PLA 2 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-sPLA 2 antibodies. TNF caused secretion of sPLA 2 and also caused an increase in cell-associated sPLA 2 enzymatic activity. Activation and release were maximal with fMLP stimulation of TNF-primed PMN. Neutrophils also contained a cytosolic PLA 2 (cPLA 2) characterized by enzymatic activity which was calcium dependent, enhanced by dithiothreitol, and blocked by anti-cPLA 2 antibody. TNF caused a doubling of cPLA 2 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 PLA 2's.