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

BACKGROUND: Previous studies reported the priming effects of granulocy te-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis fa ctor-alpha (TNF alpha) on leukotriene synthesis by isolated polymorpho nuclear leukocytes; however, little is known as of yet of these biolog ic effects of the two cytokines in a physiologic environment. EXPERIME NTAL DESIGN: In this study, we investigate the effects of GM-CSF and T NF alpha on the synthesis of 5-lipoxygenase (5-LO) products in heparin ized blood stimulated ex vivo, using reverse phase high performance li quid 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 ma rked enhancement (>5-fold) of the synthesis of leukotriene B-4 and 5(S )-hydroxyeicosatetraenoic acid, which were formed in equivalent amount s. GM-CSF and TNF alpha priming activities were detectable at concentr ations as low as 3 pM and 6 pM (1 unit/ml), respectively. The preincub ation times required for optimal priming by GM-CSF and TNF alpha were different (40 and 10 minutes, respectively), and the effects of the tw o cytokines on leukotriene B-4 and 5(S)-hydroxyeicosatetraenoic acid s ynthesis were additive, suggesting different priming mechanisms. The s ynthesis of 5-LO products in primed blood was also induced by platelet -activating factor, the complement fragment C5a, the particulate stimu lus zymosan, and the ionophore A23187, but not by interleukin-8. Polym orphonuclear 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 e ffects 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 pa thophysiologic conditions.