Eric Krump

Adenosine A2 receptor-induced inhibition of leukotriene B4 synthesis in whole blood ex vivo.

1 Engagement of adenosine A2 receptors suppresses several leukocyte functions. In the present study, we examined the effect of adenosine on the inhibition of leukotriene B4 (LTB4) synthesis in heparinized human whole blood, pretreated with lipopolysaccharide (LPS) and tumour necrosis factor α (TNF‐α) and stimulated with the chemotactic peptide, N‐formyl‐Met‐Leu‐Phe (FMLP). 2 The FMLP‐induced synthesis of LTB4 in whole blood pretreated with LPS and TNF‐α was dose‐dependently inhibited by adenosine analogues in the following order of potency; 5′(N‐ethyl)carbox‐ amidoadenosine (NECA) ≅ CGS 21680 > 2‐Cl‐adenosine > N6‐cyclopentyladenosine (CPA), indicating the involvement of the adenosine A2 receptor subtype. The IC50 values for NECA, CGS 21680, 2‐Cl‐ adenosine, and CPA were 6nM, 9nM, 180nM, and 990 nM, respectively. 3 Dipyridamole, an agent that blocks the cellular uptake of adenosine by red cells and causes its accumulation in plasma, also inhibited the synthesis of LTB4 in LPS and TNF‐α‐treated whole blood stimulated by FMLP; moreover, this inhibition was reversed upon addition of adenosine deaminase. 4 A highly selective antagonist of the adenosine A2 receptor, 8‐(3‐chlorostyryl)caffeine (CSC), reversed the inhibition of LTB4 synthesis by 2‐Cl‐adenosine and dipyridamole in LPS and TNF‐α‐treated whole blood, stimulated by FMLP. 5 LTB4 synthesis in whole blood originates predominantly from neutrophils and to a lesser extent from monocytes. 2‐Cl‐adenosine also inhibited the synthesis of LTB4 induced by FMLP in these isolated LPS and TNF‐α‐treated cells; however, 2‐Cl‐adenosine was a more potent inhibitor of LTB4 synthesis in neutrophils than monocytes. 6 The present data demonstrate that adenosine, acting through A2 receptors, exerts a potent inhibitory effect on the synthesis of LTB4 and thus contribute to the understanding of its anti‐inflammatory properties.

Kinetics of 5-lipoxygenase activation, arachidonic acid release, and leukotriene synthesis in human neutrophils: Effects of granulocyte-macrophage colony-stimulating factor

The kinetics of 5-lipoxygenase (5-LO) activation in human neutrophils was compared with that of arachidonic acid (AA) release and leukotriene (LT) B4 synthesis, and the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on these processes was examined. The soluble agonists N-formyl-Met-Leu-Phe and platelet-activating factor stimulated 5-LO activity, which peaked within 10 s and then rapidly declined. At all time points investigated, 5-LO activity was greater in GM-CSF-treated neutrophils. The release of AA was detectable only in GM-CSF-treated neutrophils and peaked 1 min after the agonist stimulation. Accordingly, synthesis of LTB4 was detected only in GM-CSF-treated neutrophils. By comparison, 100 nM of ionomycin induced a greater and sustained activation of 5-LO, resulting in a greater synthesis of LTB4. These results show that 5-LO activation is immediate and transient in response to soluble agonists and that temporal dissociation with the release of AA limits LTB4 synthesis.

Enhancement of human neutrophil leukotriene synthesis by human granulocyte-macrophage colony-stimulating factor

Colony-stimulating factors (CSFs), a group of cytokines derived from activated T lymphocytes, macrophages, fibroblasts and endothelial cells, are involved in the proliferation and maturation of progenitor cells of the haematopoietic system [1]. CSFs also have both in vivo and in vitro actions on mature immune cells such as neutrophils, eosinophils and macrophages and may therefore modulate inflammatory responses. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22000 Da glycoprotein whose known biological actions have recently been extended to include amplification of several important leukocyte responses to inflammatory mediators such as N-formyl-Methionyl-Leucyl-Phenylalanine (fMet-LeuPhe), PAF-acether, leukotriene B 4 as well as phagocytic stimuli [2, 3]. For example, pretreatment of neutrophils with GM-CSF results in increased superoxide production in response to PAF-acether, fMet-Leu-Phe, C5a and leukotriene B4 [2] and enhances the ingestion of Staphylococcus Aureus [31. Products of the 5-1ipoxygenase system play an important role in inflammation [4]. In light of the possible association between GM-CSF and leukotrienes on myelopoiesis [5] and neutrophil activation, we sought to characterize the effect of GMCSF on neutrophil leukotriene synthesis. In addition, to better understand the mechanism of the effect of GM-CSF on leukotriene synthesis we compared the effect of pretreatment with GMCSF on leukotriene synthesis in response to A23187 and a receptor-dependent stimulus the chemotactic peptide fMet-Leu-Phe. The recent findings of enhanced intracellular calcium mobilization by chemotactic factors in GM-CSF pretreated neutrophils [6] provided an additional basis for the present studies since both release of endogenous arachidonic acid and activation of the 5-1ipoxygenase are calcium-dependent processes.

Activation of the human neutrophil 5-lipoxygenase by exogenous arachidonic acid: involvement of pertussis toxin-sensitive guanine nucleotide-binding proteins.

1 The mechanism by which incubation of human peripheral blood neutrophils with exogenous arachidonic acid leads to 5‐lipoxygenase product synthesis was investigated. 2 Incubation of neutrophils with arachidonic acid caused a concentration‐ and time‐dependent synthesis of leukotriene B4, its Ω‐oxidation products, and 5‐hydroxyeicosatetraenoic acid. 3 The threshold concentration of arachidonic acid required for this effect was equal to, or greater than 3.3 μm and the synthesis increased with up to 33 μm arachidonic acid, the highest concentration used. Synthesis induced by arachidonic acid increased with time for up to 15 min and the major products detected were the Ω‐oxidation products of leukotriene B4. 4 Pre‐incubation of neutrophils with pertussis toxin inhibited the synthesis of 5‐lipoxygenase products induced by arachidonic acid by 75% or more, but had no effect on either arachidonic acid‐induced synthesis of the 15‐lipoxygenase product, 15‐hydroxyeicosatetraenoic acid, or activation of the 5‐lipoxygenase induced by the calcium ionophore A23187. 5 Pre‐incubation of neutrophils with granulocyte‐macrophage colony‐stimulating factor lead to enhanced leukotriene synthesis in response to arachidonic acid. 6 These results imply that exogenous arachidonic acid is not only used as a substrate, but also activates the 5‐lipoxygenase. Possible mechanisms of action are discussed.

C2-ceramide primes specifically for the superoxide anion production induced by N-formylmethionylleucyl phenylalanine (fMLP) in human neutrophils

Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C2-ceramide modulates some of the effects of N-formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O2-) production. The preincubation of PMN for 15 min with C2-ceramide increased by up to almost 3-fold the amounts of O2- generated in response to 0.1 and 1 microM fMLP. Priming was detected at C2-ceramide concentrations of 2 microM to 4 microM per million PMN. Though less potent than C2-ceramide, C6-ceramide (N-hexanoylsphingosine) could prime for O2- generated in response to 0.1 microM fMLP, with maximal effects obtained at 10-20 microM. In contrast, micromolar concentrations of sphingosine, dihydroceramide, and ceramide-phosphate, failed to exert any potentiating effect on fMLP-induced O2- generation. As expected, TNF-alpha (1000 U/ml), also primed for fMLP-induced O2- production; however, the combination of TNF-alpha and C2-ceramide showed no additive effect. Moreover, S. aureus sphingomyelinase (0.1 U/ml), was unable to reproduce the priming effects of C2-ceramide and TNF-alpha. C2-ceramide at 2 microM did not enhance the production of O2- induced by 100 nM recombinant human interleukin-8 (IL-8), leukotriene B4 (LTB4), platelet-activating factor (PAF) or 20 mM sodium fluoride (NaF). Furthermore, C2-ceramide (2 microM) did not enhance the mobilization of calcium, the release of arachidonic acid or the accumulation of phosphatidylethanol, induced by 100 nM fMLP. This suggests that probably neither phospholipases C, A2 or D (PLC, PLA2, PLD) were involved in the priming effect by C2-ceramide. However, C2-ceramide inhibited in a dose-related manner the production of O2- induced by phorbol 12-myristate 13-acetate (PMA) and mezerein. Furthermore, PMA-stimulated PLD activity was also significantly reduced by a preincubation of PMN with C2-ceramide. The priming of O2- production by C2-ceramide could involve yet unidentified mechanisms specific for fMLP, or it might imply that cytokines such as TNF-alpha have different mechanisms than C2-ceramide.