Effect of calcium,magnesium, and cytochalasin B on arachidonic acid-induced neutrophil aggregation

Suspended in media containing calcium and magnesium, human neutrophils aggregate briefly when exposed to arachidonic acid. The magnitude and duration of this response was enhanced by the presence of increased magnesium concentration, increased magnesium plus calcium concentrations, and cytochalasin B. The response was inhibited by increased calcium concentrations. When calcium, magnesium, or both bivalent cations were omitted from the cell suspension, the response did not occur. Since various chemotactic factors also briefly aggregate neutrophils and since these chemotactic factor-induced responses are similarly influenced by the bivalent cations and cytochalasin B, a close association between the bioactivities of arachidonic acid and chemotactic factors is evident. The aggregation response to both types of stimuli may share common intracellular mechanisms.Publication #1854 of the Research Institute of Scripps Clinic, La Jolla, California 92037.Supported by research grants: AI07007 (NIH); Office of Naval Research; Council for Tobacco Research.     

Substances which aggregate neutrophils. Mechanism of action.

Several agents which influence calcium fluxes in neutrophils were tested for their influence on human neutrophil aggregation. Formyl-methionyl-leucyl-phenylalanine, a synthetic chemotactic tripeptide, aggregated the cells. Cytochalasin B and high levels of extracellular calcium or phosphate enhanced this effect; 10(-6) M to 10(-5) M lanthanum inhibited it. In addition, the calcium ionophore A23187 aggregated the cells. Aggregation induced by the chemotactic factor and A23187 required extracellular calcium. These results correlate with the known or postulated ability of chemotactic factors, A23187, calcium, phosphate, lanthanum, and cytochalasin B to enhance or inhibit the influx and intracellular accumulation of the calcium ion. Transmembrane fluxes or intracellular levels of calcium may modulate PMN aggregation. Aggregation induced by the chemotactic tripeptide and A23187 also required extracellular magnesium. Since calcium and magnesium cannot substitute for each other in the aggregation response to the chemotactic factor or A23187, each bivalent cation must play a separate role in PMN aggregation. The role of magnesium is unknown. Since magnesium, unlike calcium, is known to be necessary for PMN adherence to glass, it may play a permissive role in PMN aggregation. Thus, magnesium may foster the formation of cell-cell adhesions. In addition to inhibiting chemotactic factor-induced aggregation at concentrations of 10(-6) M to 10(-5) M, lanthanum, at concentrations of 10(-4) M to 10(-3) M, aggregated the cells. Lanthanum-induced aggregation did not require extracellular calcium or magnesium. This aggregation may result from the formation of intercellular adhesions by the lanthanum ion directly.     

Animal model of human disease: trypanosomiasis, sleeping sickness

In the presence of Ca2+ and Mg2+, the chemotactic fragment of C5, the synthetic chemotactic oligopeptide formyl-methionyl-leucyl-phenyl-alanine, and the ionophore A23187 aggregated human neutrophils. Aggregation induced by the two chemotactic factors was transient and reversed within 2 to 4 minutes after exposure; aggregation induced by A23187 was sustained and continued to increase over 15 minutes. In the absence of the bivalent cations, none of these three agents aggregated the cells. If bivalent cations were added after cell contact with a chemotactic factor, aggregation was detected after, but not before, addition of the cations. Under these conditions, the magnitude of the aggregation response was sharply reduced: cells preincubated with a chemotactic factor for longer than 2 to 4 minutes aggregated minimally after addition of bivalent cations. Moreover, cells preincubated with a chemotactic factor for 4 minutes, exposed to bivalent cations, and then rechallenged with the same chemotactic factor also showed a minimal aggregation response, ie, the cells were "desensitized" to the original stimulus. However, cells desensitized to one of the chemotactic factors still aggregated prominently when exposed to the other chemotactic factor or to A23187. Cells could not be desensitized to the ionophore A23187. Desensitization of the neutrophil aggregation response closely resembles desensitization of mast cell and leukocyte degranulation. Degranulation and aggregation appear to be closely related cellular responses to immunologic stimuli. Both responses may reflect alterations in surface membrane permeability to bivalent cations and/or changes in surface membrane adhesiveness to other biologic membranes.     

A possible role of arachidonic acid in human neutrophil aggregation and degranulation.

Chemotactic factors stimulate neutrophils to aggregate and, in the presence of cytochalasin B, to degranulate. Recently, the authors found that arachidonic acid also stimulates human neutrophils to aggregate but does not stimulate cytochalasin-B-treated or untreated cells to degranulate. In this report the authors examined the effect of three blockers of arachidonic acid metabolism on these cellular responses. It was found that the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid and indomethacin, but not aspirin, inhibited no only the arachidonic-acid-induced aggregation response but also the degranulation responses evoked by C5a or a synthetic oligopeptide chemotactic factor. These results suggest that arachidonic acid may be a precursor of bioactive metabolites that stimulate the aggregation and foster the degranulation responses of neutrophils. Thus, these metabolites may be mediators of neutrophil function. Agents that block their formation may thereby inhibit aggregation and degranulation.     

Influence of calcium, magnesium, lanthanum and A23187 on the aggregation of polymorphonuclear neutrophils.

Chemotactic factors transiently aggregate human polymorphonuclear neutrophils. We have found that the aggregation induced by the chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine, depends on the extracellular calcium and magnesium concentrations and is inhibited by 1 and 10 M lanthanum chloride. The ionophore A23187 and 1 and 0.1 mM lanthanum chloride aggregate the cells. A23187-induced aggregation is dependent on extracellular calcium and magnesium whereas lanthanum-induced aggregation is not.     

Influence of Calcium, Magnesium, Lanthanum and A23187 on the Aggregation of Polymcrphonuclear Neutrophils

Chemotactic factors transiently aggregate human polymorphonuclear neutrophils. We have found that the aggregation induced by the chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine, depends on the extracellular calcium and magnesium concentrations and is inhibited by 1 and 10 M lanthanum chloride. The ionophore A23187 and 1 and 0.1 mM lanthanum chloride aggregate the cells. A23187-induced aggregation is dependent on extracellular calcium and magnesium whereas lanthanum-induced aggregation is not.     

Neutrophil-aggregating activity of monohydroxyeicosatetraenoic acids.

The following oxidative derivatives of arachidonic acid were prepared and assayed for their ability to aggregate cytochalasin-B-pretreated human neutrophils: 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids. The compounds were prepared by oxidation of arachidonic acid and purified by direct and reverse phase high performance liquid chromatography. Each lipid was racemic at the hydroxy residue and had a cistrans conjugated double bond adjacent to the hydroxy residue. Except for racemization, therefore, they were identical to hydroxyeicosatetraenoic acids generated by neutrophils exposed to diverse aggregating stimuli. In addition, 15-L-hydroxyeicosatetraenoic acid was prepared from soybean lipoxygenase. Of these 7 fatty acid preparations, only 5- and 12-hydroxyeicosatetraenoic acid aggregated the cells. Thus, the bioactions of these lipids are crucially dependent upon the position of the hydroxy residue. The 5- and 12-hydroxy derivatives were potent aggregating agents, inducing half-maximal responses at 200 and 40 nM, respectively. Their bioactions required extracellular calcium and magnesium. And the response to both fatty acids was effectively blocked by three inhibitors of cellular arachidonic acid metabolism: nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, and indomethacin. The 5- and 12- hydroxyeicosatetraenoic acids, therefore, may induce neutrophils to metabolize their endogenous arachidonate. Alternatively, the two hydroxy acids themselves may be further metabolized through pathways inhibited by arachidonate antimetabolites into a final mediator(s) of aggregate formation.     

Phorbol ester-induced adhesion (binding) among human mononuclear leukocytes requires extracellular Mg++ and is sensitive to protein kinase C, lipoxygenase, and ATPase inhibitors

Under gentle shaking, the phorbol 12,13-dibutyrate (P(Bu)2)-induced adhesion among human blood mononuclear leukocytes started within a few minutes, increased with time and was almost complete after 12 h. During this moment and thereafter more than 60% of the cells were in aggregates. Induction of the cell aggregation by 20 min treatment with P(Bu)2 did not occur in Ca++/Mg++-free medium but was almost complete with Mg++ alone and reached its maximal manifestation with both divalent cations present. The intracellular Ca++ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate had a minimal inhibitory effect, and simultaneous treatment of the cells with Ca++ ionophore A23187 and P(Bu)2 did not increase the intercellular binding, whereas A23187 alone induced some cell aggregation. Retinal, a protein kinase C inhibitor, inhibited the intercellular adhesion by more than 50%, and treatment of intact cells with 1-oleoyl-2-acetyl-glycerol, an activator of the enzyme, induced some cell aggregation that was slightly increased when A23187 was added simultaneously. Nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid and 5,8,11,14-eicosatetraynoic acid, which inhibit lipoxygenation of arachidonic acid, reduced the cell aggregation in contrast to indomethacin and acetylsalicylic acid that had no effect. Cellular ATPases, inhibited by quercetin, but not the ouabain-sensitive Na+, K+-ATPase, appeared to participate, whereas the amiloride-sensitive plasma membrane Na+/H+ exchanger and the intracellular levels of cGMP did not seem to influence the system.     

Neutrophil aggregation and degranulation. Effect of arachidonic acid.

In response to aggregating and degranulating stimuli, platelets metabolize endogenous arachidonic acid to bioactive derivatives. These derivatives can stimulate platelets to degranulate and aggregate and, therefore, may be mediators of the platelet response. Because exogenous arachidonic acid also stimulates platelets to degranulate, aggregate, and form these mediators, we examined the effect of adding arachidonic acid to purified human neutrophil suspensions. Micromolar concentrations of arachidonic acid stimulated neutrophils to aggregate but not to degranulate. Cytochalasin B, a potentiator of neutrophil responses to chemotactic factors, also potentiated the arachidonic-acid-induced aggregation response; 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism, blocked this response. Aggregation of neutrophils, was not stimulated by several fatty acids with structural similarity to arachidonic acid. These results suggest that metabolic derivatives of arachidonic acid may be active in stimulating certain neutrophil responses. The role of these derivatives in mediating neutrophil responses to various stimuli needs to be examined.     

Horse eosinophil degranulation induced by the ionophore A23187. Ultrastructure and role of phospholipase A2

Horse eosinophils stimulated with the calcium ionophore A23187 were examined by transmission and scanning electron microscopy. Secretion was characterized by granule movement to the cell periphery and fusion of adjacent granules. The granules became swollen and less electron-dense as their contents were released into large intracellular vacuoles, which opened to the outside of the cell through surface pores. A23187-induced eosinophil peroxidase (EPO) release, as measured by guaiacol oxidation, was blocked by eicosa-5,8,11,14-tetraynoic acid (ETYA) (which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism) but not by indomethacin (which inhibits only the cyclooxygenase pathway). Highly purified porcine phospholipase A2 induced noncytotoxic eosinophil degranulation (as measured by the release of EPO without the concomitant release of the cytoplasmic marker lactate dehydrogenase), which was blocked by pretreatment of the enzyme with the phospholipase A2 inhibitor 4-bromophenacyl bromide. These results suggest that calcium-dependent activation of phospholipase A2 and generation of lipoxygenase products of arachidonic acid metabolism are important in the initiation of eosinophil degranulation.     

Mechanism of leukotriene generation in polymorphonuclear leukocytes by staphylococcal alpha-toxin

The effects of staphylococcal alpha-toxin on arachidonic acid metabolism in rabbit polymorphonuclear leukocytes (PMNs) were investigated and compared with those of the ionophore A23187 and the chemotactic tripeptide formylmethionyl-leucyl-phenylalanine (fMLP). Sublytic amounts of alpha-toxin stimulated the release of leukotriene B4 (LTB4) in PMNs in a dose-dependent manner. The toxin was several times more potent than fMLP but was not as effective as the ionophore. Preincubation of the toxin with neutralizing antibodies abolished the effect. Extracellular calcium was strictly required for eliciting LTB4 generation. Verapamil, a calcium channel blocker, inhibited fMLP-mediated LTB4 generation but had no effect on alpha-toxin- or A23187-exposed PMNs. Agents such as trifluoperazine and N-6(aminohexyl)-5-chloro-1-naphthalene sulfonamid that interfered with calmodulin activity, however, inhibited LTB4 generation in all cases. One minute after the addition of alpha-toxin, PMNs exhibited a severalfold enhancement in passive permeability to 45Ca2+. In addition, these cells became permeable to sucrose but not to inulin or dextran. The influx pattern was consistent with the previous observation that alpha-toxin creates discrete transmembrane channels in erythrocytes with an effective internal diameter of 2 to 3 nm. The results suggest that alpha-toxin triggers the arachidonic acid pathway in PMNs by facilitating calcium influx into the cells, possibly via transmembrane toxin pores that serve as calcium gates. Generation of arachidonic acid metabolites in PMNs by sublytic amounts of alpha-toxin may represent an important cellular reaction that generally occurs during infections with Staphylococcus aureus.     

Ca2+-channel blockers modulate the oxidative burst induced with arachidonic acid in macrophage-like P 388D1 cells. Interaction with peripheral benzodiazepines

Ca2+-channel blockers are shown to modulate, like peripheral benzodiazepines, the oxidative burst induced by arachidonic acid in the macrophage-like P 388D1 cell line. Nifedipine (1 and 10 nM) enhanced the cellular response to arachidonate; this stimulation by nifedipine was reversed by PK 1195, a specific antagonist for the peripheral benzodiazepine binding site. Verapamil (5 microM), on the other hand, antagonized the stimulation of the oxidative burst by benzodiazepines. The data suggest some possible interaction of Ca2+-channel blockers at the benzodiazepine binding site or at a secondary step in the activation mechanism. None of the molecules modulated the calcium influx triggered by arachidonate. Instead, the oxidative response of P 388D1 cells to arachidonate was inhibited by K+-channel blockers, quinidine and tetraethylammonium bromide.     

Role of phospholipase A2 and pertussis toxin-sensitive G proteins in histamine secretion induced by substance P

Incubation of mast cells with substance P or ionophore A23187 resulted in histamine release and arachidonic acid liberation from purified rat peritoneal mast cells. The treatment of mast cells with 100 ng/ml of pertussis toxin for 2h inhibited the effect of substance P on both histamine release and arachidonic acid liberation, but the response to the ionophore A23187 was not affected. Para-bromophenacyl bromide, a selective inhibitor of phospholipase A₂, inhibited similarly histamine and arachidonate release induced by substance P but not that evoked by ionophore A23187. These results suggest that phospholipase A₂ plays a key role in the histamine secretion induced by substance P.

     

Eicosanoid synthesis and release from primary cultures of rat central nervous system astrocytes and meningeal cells

Primary cultures of astrocytes and meningeal cells derived from neonatal rat brain synthesize and release thromboxane A2 and prostacyclin, respectively. Exogenously supplied arachidonic acid and the calcium ionophore, A23187, promote the release of eicosanoids; these effects are blocked by indomethacin and the calcium chelator, ethyleneglycoltetraacetic acid. The finding that astrocytes synthesize and release thromboxane A2 is discussed in the light of our recent findings of receptor-linked membrane phospholipid turnover in these cells.     

Modulation of the eosinophil chemotactic factor (ECF) release from various cells and their subcellular components by phospholipids

An eosinophil chemotactic factor (ECF) can be released from human polymorphonuclear neutrophils (PMN), rat mononuclear and rat mast cells by the calcium ionophore (A23187), during phagocytosis, by arachidonic acid and phospholipase A2. It has been suggested that stimuli such as the ionophore and the phagocytic event lead to phospholipid turnover with the generation of arachidonic acid which is subsequently transformed by a lipoxygenase-like enzyme into ECF. Addition of phospholipids such as phosphatidylethanolamine and phosphatidylinositol during ionophore stimulation of various cells increased the ECF release significantly. ECF activity is also enhanced in the presence of indomethacin at concentrations which inhibit prostaglandin synthesis. With bromphenylacylbromide and eicosatetraynoic acid, ECF generation as well as the chemotaxis of eosinophils is inhibited suggesting that the phospholipase A2-arachidonic acid pathway represents a common link for ECF release as well as for the chemotaxis of eosinophils. From the cytosol of human PMN an ECF-containing enzyme was obtained. Incubation of phospholipase A2 and phospholipids with the ECF-converting enzyme led to potent ECF indicating that addition of phospholipids provides the soluble ECF-generating system with an additional source of arachidonic acid. The data represent a molecular approach to analyze the mechanisms of ECF release from soluble components after immunological triggering of the cells.     

Effects of the lonophore A23187 on blood platelets I. Influence on aggregation and secretion

A23187 is an antibiotic ionophore which transports divalent cations across cell membranes into the cytoplasm and releases cations from intracellular storage sites. The present investigation has studied the influence of A23187 on blood platelet aggregation and secretion. A23187 added to stirred C-PRP produced concentration-dependent aggregation and release of ¹⁴C-serotonin. Calcium ions potentiated the response of platelets in washed suspensions to A23187, but they were not required for ionophore-induced aggregation or release. N-ethylmaleimide and agents which increase the level of cyclic 3′,5′-adenosine monophosphate (cAMP) in platelets were effective inhibitors of A23187-induced aggregation and secretion.Platelets incubated with ⁴⁵Ca⁺⁺ before addition of A23187 increased their content of isotope by 50% within 45 seconds after exposure to the ionophore. The results indicate that an increase in the cytoplasmic concentration of calcium ions from intracellular sources may be the critical event in triggering platelet contraction and the release reaction. Increased concentrations of cAMP may inhibit the platelet response to A23187 and other aggregating agents by stimulating a calcium extrusion pump.     

Structural requirements for the activity of an immunologically generated lipid chemotactic factor

The predominant lipid chemotactic factor (LCFR) generated by IgGa-dependent immunological challenge of the rat peritoneal cavity was resolved from other lipid mediators released simultaneously into the peritoneal cavity and from previously described lipid chemotactic factors by sequential chromatography on Amberlite XAD-8, DE-52, silicic acid, and silica gel H plates, and by Zorbax C-18 reverse phase high pressure liquid chromatography. The incubation of purified rat peritoneal mononuclear leucocytes, but not mast cells, with calcium ionophore A23187 in vitro resulted in the generation of LCFR activity which was chromatographically identical to the LCFR released in vivo. That indomethacin inhibited the appearance of LCFR both in vivo and in vitro indicated a dependence on the cyclo-oxygenation of arachidonic acid. Because of the limited quantities of highly purified LCFR, the critical determinants of human neutrophil chemotactic activity were studied by examining the functional effects of chemical modification of specific substituents. The chemotactic activity of LCFR was diminished significantly by acetylation with acetic anhydride in pyridine or methylation with ethereal diazomethane, but was not influenced by 0.1 M periodate or acid or base hydrolysis, suggesting that LCFR is a complex fatty acid containing a hydroxyl or amino group. The addition of acetylated LCFR to the stimulus compartment at a 0.3-0.5 molar ratio with native LCFR diminished the neutrophil chemotactic response by 50%, without affecting a comparable response to f-Met-Leu-Ala-Phe. Thus LCFR is a unique lipid chemotactic factor which is distinct from other lipid mediators and accounts for the bulk of the PMN leucocyte chemotactic activity released by immunological challenge of the rat peritoneal cavity.     

Defective platelet aggregation to the calcium ionophore A23187 in a patient with a lifelong bleeding disorder

A patient with a lifelong bleeding disorder is presented with a prolonged bleeding time and abnormal aggregation and secretion responses to arachidonic acid, thromboxane A2, PAF-acether and the divalent calcium ionophore A23187. Platelet alpha and dense granule contents and morphology appear normal. The proposed defect is due to an abnormality of a platelet intracellular calcium dependent process.     

Regulation of canine platelet function II. Catecholamines

The action of epinephrine (E) on canine platelet aggregation is described. Although E did not induce a change in platelet shape or aggregation, potentiation of aggregation induced by the following agents was observed at physiological E concentrations (that is, less than 10 nM/1): arachidonic acid; the dense granule agonists, ADP and serotonin (5-HT); and collagen. Epinephrine-induced potentiation was in part independent of formation of arachidonic acid metabolites, and E potentiated the aggregating action of the bivalent cationophore A23187. Potentiation was inhibited by alpha-adrenergic receptor antagonists phenoxybenzamine, phentolamine, and ergotamine, and mimicked by alpha-adrenergic receptor agonists norepinephrine, clonidine, and in some cases, phenylephrine. The beta-adrenergic receptor agonists isoproterenol and dobutamine inhibited ADP-induced aggregation, and this action was presented by pretreating the platelets with propranolol and dichloroisoproterenol. An augmentation of the aggregation response of platelets to arachidonic acid was observed in blood samples withdrawn when circulating catecholamines were elevated. The physiological implication of epinephrine acting as a gain controller that alters the relationship between actuating signal and the platelet response to an agonist is discussed.     

Arachidonic acid aggregates neutrophils

Arachidonic acid, but not several structurally similar fatty acids, stimulated neutrophils in suspension to aggregate; this effect was blocked by 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism. Analagous to platelets, arachidonate may be a precursor of active metabolites which mediate neutrophil responses.This work was supported in part by NIH grants AI-09651, HL-05474, HL-22437 and HL-07202.