Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony-stimulating factor and stimulated with the complement component C5A and FMLP as second signals.

Abstract

Neutrophils (PMN) preincubated with recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) for 2 h and then stimulated with the chemotactic factors, C5a or FMLP, produce substantial amounts of the lipoxygenase products 5-Hete, LTB4, and omega-oxidised LTB4 metabolites (4.36 +/- 0.95 (SEM) pM (n = 21) LTB4 and LTB4 metabolites/10(6) PMN). No lipoxygenase metabolites are detected by HPLC and RIA if purified PMN are stimulated by either GM- CSF or chemotactic factors in the absence of exogenous arachidonate. The priming effect of GM-CSF upon chemotactic factor induced generation of lipid mediators is a relatively slow process, clearly evident after 1 h and optimal after 2 h. Leukotriene generation is measurable with 0.8 U GM-CSF/10(6) PMN and is maximal with 80 U (10(-11)-10(-9) M). Upon activation of primed PMN with chemotactic factors, leukotriene synthesis is induced very rapidly. Already 2.5 min after activation the major lipoxygenase metabolites present are 20-OH LTB4 and 20-COOH LTB4. Our study shows that the synthesis of lipoxygenase metabolites from endogeneous AA can be initiated in PMN through receptor mediated processes by the appropriately timed combination of biological soluble inflammatory mediator peptides. Furthermore, these results indicate that GM-CSF not only enhances effector cell functions but can qualitatively change the mediator profile formed after activation with a second triggering signal. Such a mechanism might be important in amplifying inflammatory responses. Alternatively, lipid mediators formed might also have an intracellular or autocoid role and be responsible for the enhancement of other PMN functions like oxygen radical release.

The monocyte-derived neutrophil activating peptide (NAP/interleukin 8) stimulates human neutrophil arachidonate-5-lipoxygenase, but not the release of cellular arachidonate.

“…Apparently, excessive amounts of exogenous AA (30-250 1.M) are necessary in order to obtain significant production of LTB4 or its metabolites following PMNL stimulation with chemotactic factors (25,26,40). Moreover, it has been demonstrated that AA itself is able to activate PMNL-5-LO at high concentrations when given in ethanolic solution (41), possibly due to membrane perturbation, increased trans-membrane penetration of AA (42), or induction of additional cellular AA release by the vehicle (43).…”

Thrombin‐activated platelets promote leukotriene B4 synthesis in polymorphonuclear leucocytes stimulated by physiological agonists

“…GM-CSF-treated or 'primed' PMNL show increased functional responses to a second stimulation, such as enhanced superoxide anion formation and enzyme release in response to FMLP (Weisbart et al, 1987;Kaufman et al, 1989), increased phagocytosis (Lopez et al, 1986) and also increased leukotriene synthesis in response to soluble agonists (FMLP, C5a, PAF) and the ionophore A23187 (Silberstein et al, 1986;Dahinden et al, 1988;Dipersio et al, 1988). In the present study, GM-CSF-priming of PMNL led to the expected increase of leukotriene synthesis in response to FMLP (Figures 2a and 3).…”

The Role of Formylpeptide Receptors, C5a Receptors, and Cytosolic-Free Calcium in Neutrophil Priming

“…The use of C5a and FMLP allowed us to detect macrophage colony-stimulating factor [29][30][31][32], tumor necrosis factor-a [33], and endotoxin [10,11,28], have been shown to enhance the response triggered by a second agonist. Priming with EAS occurs rapidly (figure 3), whereas prolonged incubation is required for the action of lipopolysaccharide or cytokines [7,10,11,28].…”