Priming for the synthesis of 5-lipoxygenase products in human blood ex vivo by human granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha

Abstract

Background: Previous studies reported the priming effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF alpha) on leukotriene synthesis by isolated polymorphonuclear leukocytes; however, little is known as of yet of these biologic effects of the two cytokines in a physiologic environment.

Experimental design: In this study, we investigate the effects of GM-CSF and TNF alpha on the synthesis of 5-lipoxygenase (5-LO) products in heparinized blood stimulated ex vivo, using reverse phase high performance liquid chromatography analysis of deproteinized plasma samples.

Results: Stimulation of blood with f-Met-Leu-Phe resulted in the accumulation of up to 30 pmol of 5-LO products/ml of plasma. Preincubation of blood with 100 pM GM-CSF or 1.2 nM (200 units/ml) TNF alpha for 30 minutes at 37 degrees C before stimulation with f-Met-Leu-Phe resulted in a marked enhancement (> 5-fold) of the synthesis of leukotriene B4 and 5(S)-hydroxyeicosatetraenoic acid, which were formed in equivalent amounts. GM-CSF and TNF alpha priming activities were detectable at concentrations as low as 3 pM and 6 pM (1 unit/ml), respectively. The preincubation times required for optimal priming by GM-CSF and TNF alpha were different (40 and 10 minutes, respectively), and the effects of the two cytokines on leukotriene B4 and 5(S)-hydroxyeicosatetraenoic acid synthesis were additive, suggesting different priming mechanisms. The synthesis of 5-LO products in primed blood was also induced by platelet-activating factor, the complement fragment C5a, the particulate stimulus zymosan, and the ionophore A23187, but not by interleukin-8. Polymorphonuclear leukocytes and mononuclear cells accounted for 80% and 20% of the synthesis of 5-LO products, respectively.

Conclusions: These data demonstrate that GM-CSF and TNF alpha exert very potent priming effects on the biosynthesis of 5-LO products in whole blood stimulated by various stimuli and strongly support that these cytokines could be important modulators of lipid mediator synthesis in physiologic and pathophysiologic conditions.