SEQUENTIAL MORPHOLOGIC EVENTS DURING APOPTOSIS OF HUMAN NEUTROPHILS -MODULATION BY LIPOXYGENASE-DERIVED EICOSANOIDS

Dual-laser flow cytometry, based on the properties of the DNA-binding dyes Hoechst 33342 and propidium iodide, was used, with light and elec tron microscopy and DNA fragmentation studies, to define the influence of lipoxygenase-derived eicosanoids on apoptosis of human polymorphon uclear neutrophils (PMN) in vitro. Apoptosis was characterized by prog ression through an early apoptotic phase characterized by condensation of chromatin and coalescence of nuclear lobes, to a late apoptotic ph ase characterized by nuclear degradation and evanescence, and secondar y necrosis. Prolonged exposure of PMN to leukotriene B-4 (LTB(4)) affo rded dose-dependent inhibition of constitutive PMN apoptosis (percenta ge of normal and apoptotic PMN, respectively, after aging for 18 h: ve hicle, 30.5+/-2.7% and 61.8+/-3.2%; LTB(4) 10(-7) M 57.6+/-1.2% and 37 .6+/-1.0%) and apoptosis triggered by the classic peptide chemoattract ant FMLP. In contrast, apoptosis was not affected by the LTB(4) precur sor 5(S)-hydroxyeicosatetraenoic acid (HETE), the omega-oxidation LTB( 4) metabolites 20-hydroxy-LTB(4) and 20-carboxy-LTB(4), the cysteinyl leukotriene LTC(4), the 15-lipoxygenase product 15(S)-HETE, or the lip oxygenase interaction product lipoxin A(4). The anti-apoptotic effect of LTB(4) was mimicked by 20,20,20-trifluoro-LTB(4), LTB(4)-dimethylam ide, and 14,15-dehydro-LTB(4), and was blunted by pertussis toxin and genistein, inhibitors of G alpha i GTP-binding proteins and tyrosine k inases, respectively, but not by staurosporine, 15(S)-HETE, or lipoxin A(4). This unique pharmacologic profile suggested that LTB(4) attenua ted apoptosis through activation of cell surface receptors and signali ng events distinct from those involved with PMN trafficking, degranula tion, and respiratory bursts.