Leukotriene B₄ activates the NADPH oxidase in eosinophils by a pertussis toxin-sensitive mechanism that is largely independent of arachidonic acid mobilization

Experiments were designed to investigate whether leukotriene (LTB₄) receptors can couple directly to phospholipase A₂ (PLA₂) in guinea pig eosinophils and the role of endogenous arachidonic acid (AA) in LTB₄- induced activation of the NADPH oxidase. LTB₄ (EC₅₀ ~ 16 nM) and AA (EC₅₀ ~ 6 μM) generated hydrogen peroxide (H₂O₂) in a concentration- dependent manner and at an equivalent maximum rate (5-6 nmol/min/10⁶ cells). LTB₄ stimulated PLA₂ over a similar concentration range that activated the NADPH oxidase, although kinetic studies revealed that the release of [³H]AA (t 1/4 ~ 2 s) preceded H₂O₂ generation (t 1/4 > 30 s). Pretreatment of eosinophils with pertussis toxin abolished the increase in inositol(1,4,5)trisphosphate mass, [Ca²⁺](c), [³H]AA release, and H₂O₂ generation evoked by LTB₄. Qualitatively identical results were obtained in eosinophils in which phospholipase C (PLC) was desensitized by 4β-phorbol 12,13-dibutyrate with the exception that [³H]AA release was largely unaffected. Additional studies performed with the protein kinase C inhibitor, Ro 31-8220, and under conditions in which Ca²⁺ mobilization was abolished, provided further evidence that LTB₄ released [³H]AA independently of signal molecules derived from the hydrolysis ofphosphatidylinositol(4,5)bisphosphate by PLC. Pretreatment of eosinophils with the PLA₂ inhibitor, mepacrine, abolished LTB₄-induced [³H]AA release at a concentration that inhibited H₂O₂ by only 36%. Collectively, the results of this study indicate that agonism of LTB₄ receptors on guinea pig eosinophils mobilizes AA by a mechanism that does not involve the activation of PLC. In addition, although LTB₄ effectively stimulated PLA₂, a central role for AA in the activation of the NADPH oxidase was excluded.