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 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.


METHODS
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.
Palmantier
R
R
Centre de Recherche en Inflammation, Immunologie et Rhumatologie, Centre Hospitalier, Université Laval, Québec, Canada.
Surette
M E
ME
Sanchez
A
A
Braquet
P
P
Borgeat
P
P
D001094:Arachidonate 5-Lipoxygenase / Q000097:blood*; D000001:Calcimycin / Q000494:pharmacology; D015936:Complement C5a / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D016209:Interleukin-8 / Q000494:pharmacology; D007700:Kinetics; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015054:Zymosan / Q000494:pharmacology
D016209:Interleukin-8; D010972:Platelet Activating Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D000001:Calcimycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D015054:Zymosan; D001094:Arachidonate 5-Lipoxygenase