Regulation of human eosinophil degranulation and activation by endogenous phospholipase A2

Abstract

The unique granular proteins of eosinophils may have a pathogenetic role in asthma and in the defense against parasitic infestations. However, the mechanisms regulating eosinophil degranulation are largely unknown. We examined the hypothesis that release of these proteins is regulated by endogenous activation of phospholipase A2. Human eosinophils (HE) were isolated from the peripheral blood of 42 subjects either by Percoll density separation or by negative-selection immunomagnetic fractionation. Eosinophil activation was initiated in vitro with 10-6 M FMLP and 5 μg/ml cytochalasin B and was assessed by measurement of eosinophil peroxidase (EPO), leukotriene C4 (LTC4) and superoxide radical (•O2-) secretion. Treatment of HE with 100 μM mepacrine before activation blocked EPO release (2.0±0.2 vs 10.2±2.1% cell content for activated HE, P < 0.004, n= 9), •O2- generation (2.6±0.9 vs 44.2±10.8 nmol/ml per 106 HE, P < 0.002, n = 5), and LTC4 secretion (68.2±32.2 vs 1,125.2±526.8 pg/ml per 106 HE, P < 0.04, n = 8). Pretreatment of HE with 100 μM 4-bromophenacyl bromide before activation similarly blocked EPO release, •O2- generation and LTC4 secretion. Addition of AA to HE after treatment with 100 μM mepacrine and before subsequent activation reversed the inhibition of both EPO (10.4±2.2% with 1 μM AA vs 2.0±0.2% for mepacrine, n = 5, P < 0.02) and LTC4 secretion (695.1±412.9 with 10 μM AA vs 68.2±32.2 pg/ml per 106 HE for mepacrine, n = 8, P < 0.04), but did not reverse inhibition of •O2- generation by mepacrine. We demonstrate that secretion of preformed cytotoxic proteins and •O2- by eosinophils is regulated endogenously by phospholipase A2.