REGULATION OF HUMAN EOSINOPHIL DEGRANULATION AND ACTIVATION BY ENDOGENOUS PHOSPHOLIPASE-A(2)

The unique granular proteins of eosinophils may have a pathogenetic ro le in asthma and in the defense against parasitic infestations. Howeve r, the mechanisms regulating eosinophil degranulation are largely unkn own. We examined the hypothesis that release of these proteins is regu lated 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 fra ctionation. Eosinophil activation was initiated in vitro with 10(-6) M FMLP and 5 mug/ml cytochalasin B and was assessed by measurement of e osinophil peroxidase (EPO), leukotriene C4 (LTC4) and superoxide radic al (.O2-) secretion. Treatment of HE with 100 muM mepacrine before act ivation 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 10(6) HE, P < 0.002, n = 5), and LTC4 secretion (68.2 +/-32.2 vs 1, 1 25.2+/-526.8 pg/ml per 10(6) HE, P < 0.04, n 8). Pretr eatment of HE with 100 muM 4-bromophenacyl bromide before activation s imilarly blocked EPO release, .O2- generation and LTC4 secretion. Addi tion of AA to HE after treatment with 100 muM mepacrine and before sub sequent activation reversed the inhibition of both EPO (10.4+/-2.2% wi th 1 muM AA vs 2.0+/-0.2% for mepacrine, n = 5, P < 0.02) and LTC4 sec retion (695.1+/-412.9 with 10 muM AA vs 68.2+/-32.2 pg/ml per 10(6) HE for mepacrine, n = 8, P < 0.04), but did not reverse inhibition of .O 2- generation by mepacrine. We demonstrate that secretion of preformed cytotoxic proteins and .O2- by eosinophils is regulated endogenously by phospholipase A2.