Osmotic stimulation of the Na+/H+ exchanger NHE1: relationship to the activation of three MAPK pathways.

The Na+/H+ exchanger (NHE) becomes activated by hyperosmolar stress, thereby contributing to cell volume regulation. The signaling pathway(s) responsible for the shrinkage-induced activation of NHE, however, remain unknown. A family of mitogen-activated protein kinases (MAPK), encompassing p42/p44 Erk, p38 MAPK and SAPK, has been implicated in a variety of cellular responses to changes in osmolarity. We therefore investigated whether these kinases similarly signal the hyperosmotic activation of NHE. The time course and osmolyte concentration dependence of hypertonic activation of NHE and of the three sub-families of MAPK were compared in U937 cells. The temporal course and dependence on osmolarity of Erk and p38 MAPK activation were found to be similar to that of NHE stimulation. However, while pretreatment of U937 cells with the kinase inhibitors PD98059 and SB203580 abrogated the osmotic activation of Erk and p38 MAPK, respectively, it did not prevent the associated stimulation of NHE. Thus, Erk1/2 and/or p38 MAPK are unlikely to mediate the osmotic regulation of NHE. The kinetics of NHE activation by hyperosmolarity appeared to precede SAPK activation. In addition, hyperosmotic activation of NHE persisted in mouse embryonic fibroblasts lacking SEK1/MKK4, an upstream activator of SAPK. Moreover, shrinkage-induced activation of NHE still occurred in COS-7 cells that were transiently transfected with a dominant-negative form of SEK1/MKK4 (SEK1/MKK4-A/L) that is expected to inhibit other isoforms of SEK as well. Together, these results demonstrate that the stimulation of NHE and the activation of Erk, p38 MAPK and SAPK are parallel but independent events.

Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils.

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2), calcium-independent PLA2, and cytosolic PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA2 activity and AA, 70% of sPLA2 activity remained associated with the cell. Treatment with the cell-impermeable sPLA2 inhibitors DTT or LY311-727, or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity, but had minimal effect on neutrophil AA mass release. In contrast, coincubation of streptolysin-O toxin-permeabilized neutrophils with DTT, LY311-727, or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled, fMLP-stimulated cells. Exposure to fMLP resulted in a decrease in the electrophoretic mobility of cPLA2, a finding consistent with cPLA2 phosphorylation, and stimulated the translocation of cPLA2 from cytosolic to microsomal and nuclear compartments. The role of cPLA2 was further evaluated with the cPLA2 inhibitor methyl arachidonyl fluorophosphonate, which attenuated cPLA2 activity in vitro and decreased fMLP-stimulated AA mass release by intact neutrophils, but had no effect on neutrophil sPLA2 activity. Inhibition of calcium-independent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release. These results indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human neutrophils.

Activation of leukotriene synthesis in human neutrophils by exogenous arachidonic acid: inhibition by adenosine A(2a) receptor agonists and crucial role of autocrine activation by leukotriene B(4).

We report here that the apparent inability of isolated human polymorphonuclear leukocytes (PMNs) to efficiently transform arachidonic acid (AA) is the consequence of A(2a) receptor engagement by endogenous adenosine accumulating in incubation media. Indeed, when adenosine is eliminated from PMN suspensions by the addition of adenosine deaminase, or when cells are incubated with adenosine A(2a) receptor antagonists, important quantities (40-80 pmol/10(6) cells) of 5-lipoxygenase products are synthesized by PMN incubated with 1 to 5 microM exogenous AA. The selective A(2a) receptor agonist CGS21680 was a very potent inhibitor of the AA-induced leukotriene (LT) synthesis, showing an IC(50) of approximately 1 nM. The mechanism of AA-induced stimulation of LT synthesis observed in the absence of extracellular adenosine was investigated. In adenosine deaminase-treated PMN, exogenous AA induced Ca(2+) mobilization and the translocation of 5-lipoxygenase to nuclear structures. A time lag of 20 to 60 s (variable between PMN preparations) was observed consistently between the addition of AA and the elevation of intracellular Ca(2+) concentration (and LT synthesis), indicating that AA itself did not trigger the Ca(2+) mobilization in PMN. This AA-induced Ca(2+) mobilization, as well as the corresponding 5-lipoxygenase translocation and stimulation of LT synthesis, was blocked efficiently by the LT synthesis inhibitor MK0591, the LTB(4) receptor antagonists CP105696 and LY223982, and the LTA(4) hydrolase inhibitor SC57461A. These data demonstrate that AA is a highly potent and effective activator of LT synthesis and acts through a mechanism that requires an autocrine stimulatory loop by LTB(4).

Adenosine. An endogenous inhibitor of arachidonic acid release and leukotriene biosynthesis in human neutrophils.

Insufficient arachidonic acid availability limits the biosynthesis of leukotriene B2 (LTB4) in polymorphonuclear leukocytes (PMN) stimulated with soluble agonists. We report that endogenous adenosine (Ado) present in human PMN suspensions suppresses LTB4 biosynthesis induced by platelet-activating factor (PAF). The blockade of the effects of Ado with an antagonist, theophylline, during the incubation of PMN resulted in significant enhancement of arachidonic acid release and LTB4 biosynthesis upon PAF stimulation. The enhancement of LTB4 biosynthesis in theophylline-treated PMN was reversed upon addition of exogenous Ado and analogues of Ado; 5'(N-ethyl)caboxamidoadenosine (IC50 = 6 nM) was more potent than Ado (IC50 = 60 nM) which was more potent than N6-cyclopentyladenosine (IC50 = 330 nM) in inhibiting LTB4 biosynthesis, a pharmacological profile which is consistent with the involvement of the Ado A2 receptor type. The mechanism of inhibition of arachidonic acid release by Ado was investigated. Immunoblot analysis of cytosolic phospholipase A2 (cPLA2) in PMN fractions demonstrated that theophylline failed to further increase the translocation of the enzyme to particulate fractions (12,000 x g and 180,000 x g pellets) upon PAF stimulation. Moreover, the stimulation of intact PMN with PAF caused a decreased electrophoretic mobility of the cPLA2 and the presence of theophylline did not alter this mobility shift. Together, these results demonstrate that elevated endogenous Ado, acting through A2 receptors, suppresses arachidonic acid release and LTB4 biosynthesis induced by PAF. These data provide an explanation for the relative inability of soluble agonists to trigger leukotriene biosynthesis in human PMN suspensions and support the concept that Ado, by suppressing PMN functions, acts as a physiological anti-inflammatory agent.

Loading pyranine via purinergic receptors or hypotonic stress for measurement of cytosolic pH by imaging.

Although used extensively for the measurement of intracellular pH, derivatives of fluorescein such as 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have suboptimal sensitivity and can generate toxic photoproducts. These limitations can be overcome using the pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine), which has improved spectroscopic properties. However, the use of pyranine has been limited by the difficulties encountered in delivering this highly hydrophilic dye to the cell interior. We describe a strategy for intracellular delivery of pyranine based on the reversible activation of purinergic P2x7 receptors, which allow permeation of the dye into otherwise intact cells. When loaded into J774 or RAW cells by this method, pyranine is not only more sensitive than BCECF (the dynamic range is approximately 7-fold greater), but is retained better and is less toxic. Pyranine was distributed throughout the cytosol but was not detectable in endomembrane compartments. Repeated illumination resulted in blebbing and loss of functional responsiveness of cells loaded with BCECF, whereas comparably irradiated cells loaded with pyranine remained healthy and responsive. Pyranine can also be loaded into cells not expressing P2x7 receptors by brief exposure to a hypotonic solution. The properties of cells labeled by this method are similar to those loaded via purinergic receptors and compare favorably with those of BCECF-loaded cells. Pyranine thus provides a useful alternative to fluorescein derivatives for the measurement of intracellular pH, particularly when using the high excitation intensities required for microscopic digital imaging.

Suppression of leukotriene B4 biosynthesis by endogenous adenosine in ligand-activated human neutrophils.

Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs). The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs. Measurements of Ado in PMN resuspended in Hanks' buffered salt solution (HBSS) or plasma showed a cell concentration- and time-dependent accumulation of the nucleoside. The removal of endogenous Ado with either Ado deaminase or the blockade of its action by the Ado A2a receptor antagonist, 8-(3-chlorostyryl) caffeine, markedly increased LTB4 biosynthesis upon ligand stimulation in HBSS. Similarly, LTB4 synthesis by ligand-stimulated PMNs in plasma (containing recombinant LTA4 hydrolase to allow the conversion of protein-bound LTA4) was strongly enhanced by addition of Ado deaminase. Addition of red blood cells to suspensions of PMNs in plasma mimicked the effect of adding Ado deaminase and LTA4 hydrolase in enhancing LTB4 biosynthesis upon ligand stimulation. This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase. These results demonstrate that endogenous Ado efficiently downregulates ligand-stimulated LTB4 biosynthesis in PMN suspensions, pointing out a potentially important regulatory function of Ado in inflammatory exudates. These results also unveil a dual role for red blood cells in upregulating LTB4 biosynthesis, namely, the removal of endogenous Ado and the conversion of LTA4 released by activated PMNs.

Induction of tyrosine phosphorylation and Na+/H+ exchanger activation during shrinkage of human neutrophils.

The ubiquitous isoform of the Na+/H+ exchanger (NHE1) is essential for the regulation of cellular volume. The underlying molecular mechanism, which is poorly understood, was studied in human polymorphonuclear leukocytes (PMN). Suspension of PMN in hypertonic media induced rapid cellular shrinkage and activation of NHE1, which is measurable as a cytosolic alkalinization. Concomitantly, hypertonic stress also induced extensive tyrosine phosphorylation of several proteins. Pretreatment of PMN with genistein, a tyrosine kinase inhibitor, prevented not only the tyrosine phosphorylation in response to a hypertonic shock but also the activation of NHE1. The signal elicited by hyperosmolarity that induces activation of tyrosine kinases and NHE1 was investigated. Methods were devised to change medium osmolarity without altering cell volume and vice versa. Increasing medium and intracellular osmolarity in normovolemic cells failed to activate tyrosine kinases or NHE1. However, shrinkage of cells under iso-osmotic conditions stimulated both tyrosine phosphorylation and NHE1 activity. These findings imply that cells detect alterations in cell size but not changes in osmolarity or ionic strength. The identity of the proteins that were tyrosine-phosphorylated in response to cell shrinkage was also investigated. Unexpectedly, the mitogen-activated protein kinases SAPK, p38, erk1, and erk2 were not detectably phosphorylated or activated. In contrast, the tyrosine kinases p59(fgr) and p56/59(hck) were phosphorylated and activated upon hypertonic challenge. We propose that cells respond to alterations in cell size, but not to changes in osmolarity, with increased tyrosine phosphorylation, which in turn leads to the activation of NHE1. The resulting changes in ion content and cytosolic pH contribute to the restoration of cell volume in shrunken cells.

Chemotactic peptide N-formyl-met-leu-phe activation of p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase-2 in human neutrophils.

Activation of polymorphonuclear leukocytes (PMN) by chemotactic peptides initiates a series of functional responses that serve to eliminate pathogens. The intermediate steps that link engagement of the chemoattractant receptor to the microbicidal responses involve protein kinases that have yet to be identified. In this study we detected in human PMN the presence of p38 mitogen-activated protein kinase (MAPK), which became rapidly tyrosine phosphorylated and activated in response to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Pretreatment of PMN with wortmannin, a phosphatidylinositol 3-kinase inhibitor, or bis-indolylmaleimide, a protein kinase C antagonist, resulted in partial inhibition of p38 phosphorylation upon fMLP stimulation. Similarly, phosphorylation of p38 was only partially inhibited when the fMLP-induced cytosolic calcium transient was prevented. Stimulation of PMN by the chemoattractant also resulted in the rapid phosphorylation and activation of MAPK-activated protein kinase-2 (MAPKAPK-2), which was completely inhibited by the specific p38 inhibitor, SB203580. The physical interaction of p38 with MAPKAPK-2 was studied by coimmunoprecipitation. These two kinases were found to be associated in unstimulated PMN but dissociated upon activation of the cells by fMLP. Together these findings demonstrate the activation of p38 by chemotactic peptides in human PMN by a process involving phosphatidylinositol 3-kinase, protein kinase C, and calcium. p38, in turn, is an upstream activator of MAPKAPK-2.

Colocalization of cytosolic phospholipase A2, 5-lipoxygenase, and 5-lipoxygenase-activating protein at the nuclear membrane of A23187-stimulated human neutrophils.

The distribution of cytosolic phospholipase A2 (cPLA2), arachidonate 5-lipoxygenase, and 5-lipoxygenase-activating protein (5-LAP) was investigated in subcellular fractions of human neutrophils disrupted by three techniques. As determined by immunoblot analysis, the bulk of cPLA2 and 5-lipoxygenase was detected in cytosolic fractions of unstimulated neutrophils disrupted by sonication or cavitation. After cell stimulation with the calcium ionophore A23187, both proteins accumulated primarily in nuclei-containing fractions; this accumulation was accompanied by a loss of these enzymes from cytosolic fractions. Further resolution of nuclear fractions revealed that 5-lipoxygenase and cPLA2 were localized in a fraction that contained nuclear membranes. In comparison, 5-LAP was localized to the nuclear-membrane fraction of resting and activated neutrophils, as determined by immunoblotting and photoaffinity labeling. In agreement with the immunoblot data, A23187 stimulation markedly enhanced 5-lipoxygenase enzymatic activity in the nuclear-membrane fraction, which was accompanied by decreased cytosolic 5-lipoxygenase activity. Similarly, neutrophil activation caused increased phosphorylation of cPLA2, a process that is known to result in enhanced catalytic activity. Our data demonstrate that in activated human neutrophils, the key proteins involved in leukotriene synthesis colocalize at the nuclear membrane, in a catalytically active state.

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 alpha (TNF-alpha) 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-alpha was dose-dependently inhibited by adenosine analogues in the following order of potency; 5'(N-ethyl)carboxamidoadenosine (NECA) approximately equal to 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 6 nM, 9 nM, 180 nM, 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-alpha-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-alpha-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-alpha-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.

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.

Leukotriene synthesis in calcium-depleted human neutrophils: arachidonic acid release correlates with calcium influx.

The relationship between intracellular calcium concentration ([Ca2+]i), the release of arachidonic acid and the synthesis of leukotriene B4 (LTB4) was investigated using Ca(2+)-depleted human polymorphonuclear leucocytes (PMNs) in which [Ca2+]i can be manipulated by varying the concentration of exogenous Ca2+ added with agonists. In this model, Ca2+, platelet-activating factor (PAF) and N-formyl-Met-Leu-Phe (FMLP), added alone, were unable to induce arachidonic acid release or LTB4 synthesis, as assessed by measurements of the products by MS and HPLC, respectively. However, the simultaneous addition of Ca2+ and either PAF or FMLP to these Ca(2+)-depleted PMNs resulted in an influx of Ca2+ proportional to the extracellular concentration of Ca2+ and caused a substantial release of arachidonic acid and synthesis of LTB4. The [Ca2+]i values for threshold and maximal arachidonic acid release were found to be 150 nM and 350 nM respectively, suggesting the involvement of cytosolic phospholipase A2 (cPLA2). Under stimulatory conditions resulting in similar [Ca2+]i, Ca(2+)-depleted PMNs released significant amounts of arachidonic acid but normal (Ca(2+)-repleted) PMNs did not, indicating that Ca2+ depletion of PMNs altered the normal regulation of arachidonic acid release and facilitated the release of the fatty acid upon stimulation with agonists. cPLA2 and mitogen-activated protein kinase (MAP kinase) phosphorylation, as assessed by changes of electrophoretic mobility, occurred in both Ca(2+)-depleted and Ca(2+)-depleted PMNs upon addition of agonist. These data demonstrate that in Ca(2+)-depleted PMNs stimulated with agonists, arachidonic acid release and LTB4 synthesis correlated with extracellular Ca2+ influx.

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.

Granulocyte-macrophage colony-stimulating factor increases the synthesis of leukotriene B4 by human neutrophils in response to platelet-activating factor. Enhancement of both arachidonic acid availability and 5-lipoxygenase activation.

Granulocyte-macrophage CSF (GM-CSF) primes human neutrophils for increased functional responsiveness to a variety of inflammatory agonists. In the present report, we have investigated the effect of human GM-CSF on the ability of platelet-activating factor (PAF) to induce the synthesis of 5-lipoxygenase products in human neutrophils. Human neutrophils stimulated with PAF in the range of 10(-5) to 10(-7) M for 15 min released small quantities of leukotriene B4 and its omega-oxidation products, 20-OH- and 20-COOH-leukotriene B4 in amounts that were detectable by enzyme immunoassay. Preincubation of normal peripheral blood neutrophils with human rGM-CSF enhanced the synthesis of the 5-lipoxygenase products in a time- and dose-dependent manner. Treatment with GM-CSF enabled their detection in response to lower concentrations of PAF (greater than or equal to 10(-9) M). The PAF receptor antagonist BN52021 inhibited the synthesis of 5-lipoxygenase products by GM-CSF-treated neutrophils in response to PAF. In addition to its effect on PAF-induced leukotriene synthesis, GM-CSF also augmented intracellular calcium mobilization by PAF. This observation prompted us to examine the effect of GM-CSF on two calcium-dependent events that are essential for leukotriene synthesis, arachidonic acid liberation, and 5-lipoxygenase activation. GM-CSF by itself, did not directly activate either of these two processes, however, it consistently and markedly enhanced the ability of PAF to do so. These results indicate that preincubation of peripheral blood neutrophils with GM-CSF enhances the ability of PAF to stimulate leukotriene synthesis by increasing both arachidonic acid availability and 5-lipoxygenase activation in response to PAF. These observations provide additional evidence of an important role for GM-CSF in the modulation of inflammatory responses to endogenous agonists through enhancement of the production of potent cellular inflammatory mediators such as leukotrienes.

Enhancement of platelet-activating factor-induced leukotriene synthesis in neutrophils by granulocyte-macrophage colony-stimulating factor (GM-CSF): studies on the mechanism of action of GM-CSF.

Preincubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) increased the synthesis of leukotriene B4 and its omega-oxidation products by neutrophils in response to 10(-7) M platelet-activating factor (PAF) by more than 10-fold compared to untreated cells. Pretreatment with GM-CSF also enabled the detection of these products in response to lower concentrations of PAF (greater than or equal to 10(-9) M), under which conditions synthesis of 5-lipoxygenase products was not observed in non-GM-CSF-treated cells. While PAF induced the release of arachidonic acid from neutrophils, GM-CSF alone did not stimulate the release of detectable amounts of the fatty acid. However, the levels of free arachidonic acid in response to PAF were enhanced in neutrophils pretreated with GM-CSF. Similarly, GM-CSF alone did not directly activate the 5-lipoxygenase as determined by the 5-lipoxygenase-mediated transformation of exogenous 15-hydroperoxyeicosatetraenoic acid into 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE). However, stimulation of neutrophils with PAF resulted in the transformation of the 15-hydroperoxy acid into the 5,15-diHETE, and furthermore, preincubation of neutrophils with GM-CSF resulted in enhanced formation of 5,15-diHETE in response to PAF. Taken together, these data indicate that GM-CSF does not enhance leukotriene synthesis in neutrophils in response to PAF by either directly activating the 5-lipoxygenase or stimulating the liberation of endogenous arachidonic acid, but rather by augmenting the effect of PAF on these two responses.

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 omega-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 microM and the synthesis increased with up to 33 microM 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 omega-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.