Tumor necrosis factor-α develops late anaphylactic reaction through cytosolic phospholipase A₂ activation

Background: We have recently reported that tumor necrosis factor (TNF)-α plays an important role in the development of a late anaphylactic reaction, but the downstream pathway beyond TNF-α remains unclear. Objective: It was the aim of this study to examine whether TNF-α induces late-phase anaphylaxis via the activation of cytosolic phospholipase A ₂ (cPLA₂). Methods: Using a murine model of active systemic anaphylaxis to penicillin V, the induction of the late phase of anaphylaxis was quantified by measuring the increase in hematocrit value as well as the plasma level of platelet-activating factor in TNF-α knockout mice. Phosphorylation of mitogen-activated protein kinases (MAPKs) and cPLA ₂ was measured by immunoprecipitation. cPLA₂ activity was assessed by using 1-stearoyl-2-[1-¹⁴C] arachidonyl-sn-glycero-3- phosphocholine as the substrate. Results: Phosphorylation and enzymatic activity of cPLA₂, and phosphorylation of the 3 known MAPKs, i.e. p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase, were markedly increased in a TNF-α-dependent way in the lungs of mice undergoing anaphylaxis. A specific cPLA₂ inhibitor significantly attenuated the late anaphylactic symptoms. Either p38 or an ERK inhibitor significantly attenuated not only cPLA₂ phosphorylation and activity, but also the late-phase anaphylaxis. Conclusion: TNF-α-induces cPLA ₂ activation through the pathway involving p38 MAPK and ERK activation and appears to be the key mechanism leading to the development of late-phase anaphylaxis.