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The attainment of political stability in South Africa depends a lot on the ability of Pres. Nelson Mandela to revitalize the country's economy and to bring the benefits of economic progress to the poor majority. Economic revitalization in turn depends on the influx of foreign investments into South Africa. This represents a major challenge for Mandela, considering that Latin America, China, the Asian economic tigers, Eastern Europe and the former USSR are also vying for foreign investments. Foreign investment decisions would depend on comparative educational, skills, and wage levels; labor relations; and tax policies.

African National Congress leader Nelson Mandela has called for an end to economic sanctions against South Africa. The country's economy has deteriorated with a 46% unemployment rate, low direct foreign investment and serious balance of payment problems. The South African government must revise its economic policies that result in too much subsidies and over-regulation and protectionism. The US can help the South African economy by encouraging investments to the country and dismantling remaining sanctions.

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a > or =20 microm concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [(3)H]phorbol dibutyrate (PDB), increased binding of [(3)H]PDB within 15 s of exposure to > or =10-30 nm AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca(2+) chelators. The major metabolites of AA made by PMN, leukotriene B(4) and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha, beta(II), and delta to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca(2+)-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta(I) or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at > or =0.5 microm, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nm. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca(2+) or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.

Acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase, along with phospholipase A2, is a key regulator of platelet-activating factor biosynthesis via the remodeling pathway. We have now obtained evidence in human neutrophils indicating that this enzyme is regulated by a specific member of the mitogen-activated protein kinases, namely the p38 kinase. We earlier demonstrated that tumor necrosis factor-alpha (TNF-alpha) as well as N-formyl-methionyl-leucyl-phenylalanine treatment leads to increased phosphorylation and activation of p38 kinase in human neutrophils. Strikingly, in the present study these stimuli increased the catalytic activity of acetyltransferase up to 3-fold, whereas 4-phorbol 12-myristate 13-acetate, which activates the extracellular-regulated kinases (ERKs) but not p38 kinase, had no effect. Furthermore, a selective inhibitor of p38 kinase, SB 203580, was able to abolish the TNF-alpha- and N-formyl-methionyl-leucyl-phenylalanine-induced activation of acetyltransferase. The same effect was not observed in the presence of an inhibitor that blocked ERK activation (PD 98059). Complementing the findings in intact cells, we have shown that recombinant, activated p38 kinase added to microsomes in the presence of Mg2+ and ATP increased acetyltransferase activity to the same degree as in microsomes obtained from TNF-alpha-stimulated cells. No activation of acetyltransferase occurred upon treatment of microsomes with either recombinant, activated ERK-1 or ERK-2. Finally, the increases in acetyltransferase activity induced by TNF-alpha could be ablated by treating the microsomes with alkaline phosphatase. Thus acetyltransferase appears to be a downstream target for p38 kinase but not ERKs. These data from whole cells as well as cell-free systems fit a model wherein stimulus-induced acetyltransferase activation is mediated by a phosphorylation event catalyzed directly by p38 kinase.

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 stimulate human polymorphonuclear neutrophils (PMN) to translocate protein kinase C from the cytosol to plasmalemma as judged by their abilities to increase PMN binding of and receptor numbers for [3H]phorbol dibutyrate [( 3H]PDB) (O'Flaherty, J.T., Jacobson, D.P., Redman, J.F., and Rossi, A.G. (1990) J. Biol. Chem. 265, 9146-9152). Platelet-activating factor (PAF) had these same effects. Moreover, two potent PAF analogs (but not an inactive analog) increased [3H]PDB binding; a PAF antagonist blocked responses to PAF without altering those to fMLP; and PMN treated with PAF became desensitized to PAF while retaining sensitivity to fMLP. Indeed, PMN incubated with 1-100 nM PAF for 5-40 min had markedly enhanced [3H]PDB binding responses to fMLP. PAF thus acted through its receptors to stimulate and prime protein kinase C translocation. Its effects, however, did not necessarily proceed by a standard mechanism: Ca2(+)-depleted PMN failed to raise Fura-2-monitored cytosolic Ca2+ concentrations [( Ca2+]i), yet increased [3H]PDB binding and receptor numbers almost normally after PAF challenge. PAF also primed Ca2(+)-depleted PMN to fMLP. Nevertheless, [3H]PDB binding responses to PAF were blocked in PMN loaded with Ca2+ chelators, viz. Quin 2, Fura-2, or 5,5'-dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Exogenous Ca2+ reversed Quin 2 inhibition, and a weak chelator 4,4'-difluoro-BAPTA, lacked inhibitory actions. The chelators similarly influenced fMLP and leukotriene B4. Thus, PMN can by-pass [Ca2+]i to translocate protein kinase C. They may achieve this using a regulatable pool of Ca2+ that evades conventional [Ca2+]i monitors or a signal that needs cell Ca2+ to form and/or act. This signal may mediate function in Ca2(+)-depleted cells, the actions of [Ca2+]i-independent stimuli, cell priming, and protein kinase C movements that otherwise seem [Ca2+]i-induced.

1-O-Alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (i.e., platelet-activating factor) was prepared and confirmed to possess potent platelet aggregating activity. It was also potent in aggregating and degranulating rabbit and human neutrophils. When injected into rabbits, the lipid induced profound neutropenia, thrombocytopenia, and anaphylactic symptoms. The lyso derivative of this lipid, 1-O-alkyl-sn-glyceryl-3-phosphorylcholine, was inactive or several orders of magnitude weaker in inducing these responses. The acetylated lipid appears to be a potent stimulator of both platelets and neutrophils. Its anaphylactic-like toxicity may be related, at least in part, to its ability to aggregate or otherwise stimulate these cells.

Chemotactic factors and arachidonic acid aggregate neutrophils; indomethacin and 5,8,11,14-eicosatetraynoic acid, two blockers of cellular arachidonate metabolism, inhibit these responses. Additionally, A23187, an ionophore which specifically transports bivalent cations into cells, also aggregates the neutrophils, and this response, as well as the response to chemotactic factors, requires the presence of extracellular calcium and magnesium. In this report these relationships were further studied. It was found that human neutrophil aggregation stimulated by arachidonic acid also required calcium and magnesium. Furthermore, cells preincubated with arachidonate for 4 min before exposure to the bivalent cations did not aggregate before or after this exposure and, following this exposure, were refractory to subsequent stimulation by more arachidonate, a chemotactic tripeptide (formylmethionylleucylphenylalanine), or A23187, i.e., these cells had become nonselectively desensitized to the aggregating agents. Finally, indomethacin and 5,8,11,14-eicoasatetraynoic acid inhibited the aggregation response stimulated by A23187 and their potency in doing so paralleled their potency in inhibiting the responses induced by arachidonate or the chemotactic tripeptide. Thus, the neutrophil aggregation responses induced by arachidonate, chemotactic factor, and A23187 were similarly influenced by preincubating the cells with arachidonate, had similar requirements for calcium and magnesium, and were similarly inhibited by blockers of arachidonate metabolism. It appears that bivalent cations and arachidonic acid play essential and, perhaps, interacting roles in the aggregation response to diverse stimuli.

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.

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.

The ability of several chemotactic factors to alter polymorphonuclear neutrophil (PMN) adhesiveness to nylon fibers was studied. Partly purified bacterial chemotactic factor, the isolated chemotactic fragment of human C5, and the chemotactic synthetic tripeptide, formyl-methionyl-leucylphenylalanine, transiently enhanced the nylon fiber adhesiveness of rabbit peritoneal PMNs. The capacity of these chemotactic factors to augment PMN adherence closely paralleled their ability to aggregate PMNs in suspension and to induce neutropenia when infused into rabbits. However, at least a portion of the adherence-augmenting capacity of these agents was independent of their ability to induce PMN aggregation. Thus, chemotactic factors appear to transiently enhance PMN adhesiveness to a variety of surfaces. This hyperadhesiveness may underlie the augmented nylon fiber adherence, aggregation, and neutropenia induced by these factors.

Previous studies have concluded that cytosolic Ca2+ [(Ca2+]i) transients are essential for neutrophils (PMN) to degranulate and make superoxide anion when challenged with the receptor agonists N-formyl-methionyl-leucyl-phenylalanine, platelet-activating factor and leukotriene B4. This view is based on the profound unresponsiveness of PMN that have their [Ca2+]i fixed at resting levels by removing storage Ca2+ and loading the cells with greater than or equal to 20 microM of a Ca2+ chelator, quin2 AM. We too observed this unresponsive state in PMN loaded with 10-32 microM-quin2 AM, fura-2 AM or 1,2-bis-(2-aminophenoxy) ethane-NNN'N'-tetra-acetic acid (BAPTA). When loaded with less than or equal to 1 microM fura-2 AM, however, Ca(2+)-depleted PMN failed to alter [Ca2+]i appreciably, yet still had substantial degranulation and superoxide-anion-generating responses to the receptor agonists. Function thus did not require [Ca2+]i transients. Moreover, Ca(2+)-depleted PMN had 20-35% decreases in receptor numbers for each of the three agonists, and chelator loading of these cells decreased receptor availability by 30-50%. All receptor losses were reversed by incubating PMN with Ca2+ at 37 degrees C, but not at 4 degrees C, and agonist binding at 4 degrees C was not influenced by the presence or absence of extracellular Ca2+. Ca2+ thus caused PMN to up-regulate their agonist receptors at 37 degrees C, and the effect persisted at 4 degrees C regardless of ambient Ca2+. We conclude that Ca2+ acts in at least three ways to regulate responses to receptor agonists. First, some pool of (probably cellular) Ca2+ maintains receptor expression. Second, [Ca2+]i transients potentiate, but are not required for, function. The [Ca2+]i pool may or may not be the same as that influencing receptors. Finally, another pool(s) of Ca2+ signals or permits responses. This last pool, rather than [Ca2+]i transients, appears essential for the bioactions of standard Ca(2+)-mobilizing stimuli.

5-Hydroxy- and 5-oxo-eicosatetraenoate (5-HETE and 5-oxoETE) activate polymorphonuclear neutrophils (PMNs) through a common, receptor-like recognition system. To define this system, we examined the interaction of these eicosanoids with human PMNs. PMNs esterified 5-[3H]HETE to glycerolipids at 37 and 4 degreesC. At 37 but not 4 degreesC, the cells also hydroxylated the label to 5, 20-[3H]diHETE. The acyl:CoA synthetase blocker, triacsin C, inhibited esterification but also led to an increase in the hydroxylation of the label. PMNs processed 5-[3H]oxoETE through the same pathways but only or principally after reducing it to 5-[3H]HETE (37 or 4 degreesC). In the presence of these varying metabolic reactions, PMNs (37 or 4 degreesC; +/- triacsin C) could not be shown to receptor bind either radiolabel. Plasma membranes isolated from PMNs esterified but unlike whole cells did not reduce or hydroxylate 5-[3H]oxoETE. Triacsin C blocked esterification, thereby rendering the membranes unable to metabolize this radiolabel. Indeed, triacsin C-treated membranes bound (Kd = 3.8 nM) 5-[3H]oxoETE specifically and reversibly to 86 pmol of sites per 25 micrograms of membrane protein. 5-OxoETE, 5-HETE, and 5,15-diHETE displaced this binding at concentrations correlating with their potency in eliciting PMN Ca2+ transients. GTP and GTPgammaS, but not ATP or ATPgammaS, also reduced 5-[3H]oxoETE binding, whereas 15-HETE, leukotriene B4, platelet-activating factor, IL-8, C5a, and N-formyl-Met-Leu-Phe lacked this effect. We conclude that PMNs and their plasma membranes use an acyl:CoA synthetase-dependent route to esterify 5-HETE and 5-oxoETE into lipids. Blockade of the synthetase uncovers cryptic plasmalemma sites that bind 5-oxoETE with exquisite specificity. These sites apparently mediate responses to the 5-oxo class of eicosanoids and are likely members of the serpentine superfamily of G protein-linked receptors.

In the presence of extracellular calcium and magnesium, a series of chemotactic oligopeptides and C5a caused aggregation of human polymorphonuclear neutrophils (PMNs). This cellular response developed rapidly and began to reverse 2 min after exposure to the chemotactin. In the absence of the bivalent cations, none of the chemotactins stimulated the aggregation response. If cells were first exposed to a chemotactin and then treated with calcium and magnesium, aggregation was detected only after addition of the cations, and the magnitude of the response fell sharply as the interval between the addition of chemotactin and addition of cations was lengthened: when this interval exceeded 2 min, aggregation was barely detectable. This loss of reactivity persisted even when cells were re-exposed to fresh chemotactic factor and washed between the first and second exposures. In all instances, however, loss of cellular reactivity was highly selective: cells preincubated with any chemotactic oligopeptide were hyporesponsive to subsequent stimulation with an oligopeptide but remained fully responsive to C5a; cells preincubated with C5A were hyporesponsive to C5a but retained their responsitivity to the oligopeptides. Because this selectivity parallels the known specificities of these chemotactic factors for their receptors in or on the neutrophil, desensitization may reflect functional loss of receptors after stimulation. Alternatively, this selectivity may indicate that morphologically identical neutrophils contain subpopulations of cells with varying reactivities to receptor-bound chemotactic factors. In either event, desensitization may be useful in functionally defining chemotactic factors and their respective receptors. The rapidity of development of desensitization suggests that it may operate to limit or moderate various in vitro and in vivo neutrophil responses to chemotactic factors.

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.

Protein kinase C (PKC) (Ca2+/phospholipid-dependent enzyme) activators stimulated human neutrophils to reduce the availability of high affinity receptors for platelet-activating factor. These effects were concentration dependent, irreversible, temperature sensitive, and antagonized by a PKC blocker. The activators also inhibited 1-O-alkyl-65% hexadecyl, 25% octadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF)-induced Ca2+transients; this inhibition correlated precisely with receptor depletion. Contrastingly, PKC activators could enhance as well as inhibit PAF-induced degranulation. Inhibition of degranulation occurred only at concentrations of the activators which depressed high affinity PAF binding by > 75%. Cells treated with lesser activator concentrations responded to PAF with reduced but still substantial rises in cytosolic Ca2+, markedly increased degranulation, and markedly increased PKC mobilization. The last two responses, however, failed to occur in cells that were (a) calcium depleted, (b) treated with high activator concentrations (which inhibited virtually all PAF binding and PAF-induced Ca2+transients), or (c) treated with PAF 5 min before a PKC activator (PAF-induced rises in cytosolic Ca2+reversed in < 5 min). Thus, activated PKC down-regulates high affinity PAF receptors. This tends to reduce neutrophil responses to PAF. On the other hand, PAF, perhaps by raising cytosolic Ca2+, acts synergistically with PKC activators in mobilizing PKC. This may tend to enhance function but seems capable of influencing only those responses that are elicited by PKC activators (e.g. degranulation but not Ca2+transients). The complex and bidirectional effects of PKC activators on other receptor-mediated, calcium mobilizing agonists in various cell types may reflect these opposing mechanisms.

In this study, the molecular species distribution of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) synthesized by rabbit peritoneal and human peripheral neutrophils was examined. Radiolabeled platelet-activating factor synthesized from [3H]acetate by neutrophils stimulated with ionophore A23187, opsonized zymosan, or N-formyl-methionyl-leucyl-phenylalanine was separated into the individual molecular species on the basis of length and degree of unsaturation of the 1-O-alkyl chain using reverse-phase high-performance liquid chromatography. The predominant alkyl chains in the labeled platelet-activating factor synthesized by ionophore- or zymosan-stimulated rabbit cells were 15:0 (4%), 16:0 (43%), 18:0 (11%), and 18:1 (26%). This is in contrast to the alkyl chain distribution of the widely accepted precursor of platelet-activating factor, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, present in the cell. The major alkyl chains contained in the labeled platelet-activating factor synthesized by ionophore-, zymosan-, or N-formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils were 16:0 (40%), 17:0 (8 and 5%; two isomers), 18:0 (16%), and 18:1 (18%). As found with the rabbit cells, this distribution differs from the alkyl chain content of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine in human neutrophils, which contains less than 1% of each 17:0 isomer. We demonstrate here that the platelet-activating factor synthesized by rabbit peritoneal and human peripheral polymorphonuclear neutrophils is heterogeneous and that some selectivity exists in the choice of the 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine precursor for platelet-activating factor synthesis in these cells. It also appears that the molecular species distribution of platelet-activating factor is independent of the stimulus used to elicit its synthesis.

Human polymorphonuclear leukocytes (PMN) incubated with 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-[3H]alkyl-2-acetyl-GPC; platelet activating factor) inactivated the compound by removing the acetyl group and replacing it with a long chain acyl residue. The nature of the acyl group added at the 2-position of the 1-O-[3H]alkyl-2-acyl-GPC formed was examined by argentation chromatography and by reverse phase high performance liquid chromatography. A striking selectivity for arachidonate was observed in the acylation reaction. The major labeled component of the starting material was the 1-O-hexadecyl-linked species; high performance liquid chromatography analysis revealed that 75 to 80% of this component was acylated by arachidonate. Similarly, based on argentation thin layer chromatography, approximately 80% of the total starting material was acylated by tetraenoic acyl residues. The incorporation of 1-O-[3H]alkyl-2-lyso-GPC into 1-O-alkyl-2-acyl-GPC by the PMN was compared; no difference in the acylation pattern was observed with the 2-acetyl and 2-lyso precursors. Thus, activation of the PMN does not appear to be required to elicit the selectivity for arachidonate. When labeled 1-palmitoyl-2-lyso-GPC was compared in the system under the same conditions, it was also preferentially acylated by arachidonate; thus, it is not clear at this time whether or not the selectivity for arachidonate is physiologically limited to platelet activating factor. Our findings suggest a close relationship exists between the metabolism of platelet activating factor and arachidonate in human PMN.

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (AAGPC) triggered the release of [3H]arachidonate but not [14C]stearate from cellular phospholipids in cytochalasin B-treated rabbit polymorphonuclear leukocytes. Concentrations of AAGPC up to 20 nM caused a dose-dependent release and subsequent metabolism of the released [3H]arachidonic acid. Most of the release of the [3H]arachidonate had taken place within the first 2 min of stimulation. Phosphatidylinositol and phosphatidylcholine served as the sources of [3H]arachidonate with about 50% of the label coming from each pool. Challenge of cytochalasin B-treated polymorphonuclear leukocytes with AAPGC led to the production of [3H]hydroxyeicosatetraenoic acids and [3H]dihydroxyeicosatetraenoic acids. No significant production of [3H]prostaglandins or [3H]thromboxanes was detected. AAGPC also caused a dose-dependent degranulation of cytochalasin B-treated rabbit polymorphonuclear leukocytes as shown by the release of beta-glucuronidase and lysozyme. Both the AAGPC-stimulated production of arachidonate metabolites and the degranulation response were blocked by eicosatetraynoic acid and non-dihydroguaiaretic acid at similar inhibitor concentrations. These findings suggest the bioactions of AAGPC on polymorphonuclear leukocytes may be mediated by the release of arachidonic acid and the production of mono- and dihydroxyeicosatetraenoic acids.

Platelet-activating factor (PAF) desensitizes as well as stimulates its various target cells, We find that human polymorphonuclear neutrophils (PMN) exposed to PAF became maximally unresponsive to a second PAF challenge within 15-90 s in assays of Ca2+ mobilization and degranulation. The cells regained full PAF-sensitivity over the ensuing 20-40 min. These effects correlated with changes in PAF receptor availability. PMN treated with PAF, washed in regular buffer and assayed for PAF binding exhibited falls (maximal in 15 s), followed by rises (reaching control levels by 60 min), in the number of high-affinity PAF receptors. However, tracking studies showed that [3H]PAF accumulated on the cell surface for approximately 2 min before being internalized. Regular-buffer washes did not remove this superficial PAF, whereas a washing regimen using excess albumin to adsorb PAF removed 99% of the surface compound. PMN washed by the latter regimen after PAF exposure lost PAF receptors relatively slowly (maximal at approximately 5 min), but the ultimate extent of this loss and the rate at which receptor expression normalized were similar to those of cells washed in regular buffer. Neither cycloheximide nor actinomycin D influenced the course of the receptor changes, but two protein kinase C (PKC) blockers, staurosporine and 1-(5-isoquinolinesulphonyl)piperazine, inhibited the receptor-receptor-depleting actions of PAF. Indeed, a phorbol diester activator of PKC also caused PMN to decrease high-affinity PAF receptor numbers, and the two PKC blockers antagonized this action at concentrations that inhibited PAF-induced PAF receptor losses. We conclude that: (a) PAF induces PMN to down-regulate and then to re-express PAF receptors independently of protein synthesis; (b) these changes are likely to underlie the later stages and reversal of desensitization; (c) the onset (t < or = 2 min) of desensitization, however, precedes receptor down-regulation and must be due to receptor uncoupling from transductional elements; and (d) down-regulation of receptors for PAF appears to be mediated by PKC and/or elements inhibited by PKC blockers.

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B4 (LTB4). All three eicosanoids stimulate Ca2+ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB4, 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB4, GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA2, as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [14C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca2+ depletion, inhibited MAPK responses to 5-oxoETE and LTB4, indicating that responses to both agents are coupled through G proteins but not dependent upon Ca2+ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3- and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB4 antagonist, blocked the responses to LTB4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB4. The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation.

Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a 2-fold increase in the activity of a cytosolic phospholipase A2 (PLA2) when measured using sonicated vesicles of 1-stearoyl-2-[14C]arachidonoylphosphatidylcholine as substrate. A similar increase in cytosolic PLA2 activity was induced by stimulation of neutrophils with leukotriene B4 (LTB4), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE). LTB4 was the most potent of the agonists, showing maximal effect at 1 nM. Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevented the AA-induced increase in PLA2 activity but had no effect on the response induced by LTB4. Furthermore, pretreatment of neutrophils with a LTB4-receptor antagonist, LY 255283, blocked the AA- and LTB4-induced activation of PLA2 but did not influence the action of 5-HETE. Treatment of neutrophils with pancreatic PLA2 also induced an increase in the activity of the cytosolic PLA2; this response was inhibited by both eicosatetraynoic acid or LY 255283. The increases in PLA2 activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA2, as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA2 likely underlies its increase in catalytic activity. Although stimulation of neutrophils with individual lipoxygenase metabolites did not induce significant mobilization of endogenous AA, they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA as determined by mass spectrometry analysis. Our findings support a positive-feedback model in which stimulus-induced release of AA or exocytosis of secretory PLA2 modulate the activity of the cytosolic 85-kDa PLA2 by initiating the formation of LTB4. The nascent LTB4 is then released to act on the LTB4 receptor and thereby promote further activation of the 85-kDa PLA2. Since 5-HETE and LTB4 are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA2 plays a role in platelet-activating factor synthesis.

The influences of extracellular Ca2+ and Mg2+ on chemotactic factor-induced rabbit polymorphonuclear neutrophil (PMN) aggregation was studied using a recently described Coulter counter assay technique. Compared with those of other PMN functional assays (adhesion, chemotaxis, degranulation, and phagocytosis), the cation requirements for cell aggregation appear unique. Chemotactic factor-induced aggregation did not occur in the absence of either cation. When the concentration of both cations was increased equivalently, aggregation increased. The effect plateaued at 2.8 mM of Ca2+ and Mg2+. When the concentration of one cation alone was increased, aggregation peaked. Further increases inhibited maximal aggregation. By systematic variation of the cation concentrations, optimal aggregation was found at Ca2+ and Mg2+ concentrations of 2.8 mM. At these concentrations, significant aggregation was induced with chemotactic doses of bacterial factor, the chemotactic fragment of human C5, and the synthetic chemotactic tripeptide, formyl-met-leu-phe. Thus, under these conditions, chemotactic factor-induced-aggregation of neutrophils may be a useful indicator of interactions of chemotactic factors with neutrophils.

1-O-[3H]Alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-[3H]alkyl-2-acetyl-GPC) incubated with rabbit polymorphonuclear leukocytes was metabolized to 1-O-alkyl-2-acyl-GPC containing long chain groups at the 2 position. Within 5 min, 60% of the total label added to the cell suspension was found in the 1-O-[3H]alkyl-2-acyl-GPC. At earlier time points, 1-O-[3H]alkyl-2-lyso-GPC was present. No metabolites were detected that would indicate the ether bond had been cleaved or the polar head group had been altered by the cells. At subaggregating and subdegranulating concentrations of labeled 1-O-[3H]alkyl-2-acyl-GPC, the same pattern of product formation was observed. Furthermore, when 1-O-[3H]alkyl-2-lyso-GPC was added to the cells, it was taken up and acylated in the same manner as that from 1-O-alkyl-2-acetyl-GPC. The nature of the long chain acyl residues incorporated into the 2 position was then examined by argentation chromatography and high performance liquid chromatography. Argentation chromatography of 1-O-[3H]alkyl-2-acyl-3-acetylglycerols obtained from 1-O-[3H]alkyl-2-acyl-GPC after acetolysis indicated that mono-, di-, and tetraenoic fatty acids were the predominant molecular species being incorporated into the 2 position of the molecule (18, 55, and 18%, respectively). Furthermore, high performance liquid chromatographic analysis using synthetic 1-O-alkyl-2-acyl-GPC standards indicated that three fatty acids, linoleic, arachidonic, and oleic (50, 15, and 12%, respectively), were the major chains being incorporated into the 1-O-hexadecyl-linked species. Analysis of 1-O-[3H]alkyl-2-acyl-GPC derived from exogenously added 1-O-[3H]alkyl-2-lyso-GPC revealed the same distribution of acyl groups linked to the 2 position. The findings are consistent with a pathway in which two enzymatic activities are responsible for the metabolism of exogenous platelet-activating factor in the rabbit neutrophils: one that hydrolyzes the acetyl residue (acetylhydrolase) and another that transfers a fatty acyl chain to the 1-O-alkyl-2-lyso-GPC formed (possibly acyl-CoA:1-O-alkyl-2-lyso-GPC acyltransferase). These events appear to play an important role in inactivating this potentially lethal phospholipid.

The synthesis of platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was studied in rabbit peritoneal polymorphonuclear neutrophils. Upon stimulation with ionophore A23187 and Ca2+, these cells are able to incorporate [3H]acetate or 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine into platelet activating factor. Under the same incubation conditions, however, the cells do not synthesize platelet activating factor from [14C]hexadecanol, which is an immediate precursor of O-alkyl chains in the de novo pathway. In the absence of ionophore, [14C] hexadecanol is incorporated into 1-O-alkyl-2-acyl-sn-glycerol-3-phosphate and subsequently into the 1-O-alkyl-linked choline and ethanolamine phosphoglyceride pools. However, in the presence of ionophore, [14C] hexadecanol incorporation is limited to phosphatidic acid, perhaps due to the inhibition of choline phosphotransferase. These findings provide strong evidence that platelet activating factor is synthesized by a deacylation-reacylation mechanism. Upon stimulation, these cells can utilize both plausible substrates of this pathway to make the final product, while under the same conditions it appears that a key step of the de novo pathway is inhibited.

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The ability of several stimuli, all of which have been reported to stimulate eosinophil motility, to augment uptake of extracellular deoxyglucose (DOG), a glucose analogue, was studied. DOG uptake was stimulated in a concentration-dependent manner by the more potent chemotaxins-zymosan-activated serum, partially purified C5a, N-formylmethionyl- Ieucyl-phenylalanine, and 5- and ll-hydroxyeicosatetraenoic acids. Less potent stimuli - the eosinophil chemotactic factor of anaphylaxis, histamine, and prostaglandin E2 - did not stimulate DOG uptake. However, the correlation between the abilities of the agents to stimulate motility and DOG uptake was not completely uniform. Prostaglandin F2α was a potent stimulant of DOG uptake yet is known to enhance only weakly chemokinesis and to be not chemotactic for eosinophils. The combined data from this and other studies indicate that these stimuli elicit specific and different sets of functional responses from eosinophils.