Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Tumor necrosis factor-alpha regulates expression of receptors for formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and platelet-activating factor. Dissociation from priming in human polymorphonuclear neutrophils.

TNF-alpha enhances polymorphonuclear responses to many stimuli, including chemotactic peptide FMLP. It also promotes expression of FMLP receptors and thus may prime polymorphonuclear neutrophils to this and other agonists by up-regulating signal recognition molecules. However, we find that the cytokine's actions on FMLP receptors lagged priming of FMLP-induced degranulation. Moreover, TNF-alpha enhanced degranulation responses to leukotriene B4 and platelet-activating factor but paradoxically down-regulated leukotriene B4 receptors and only transiently up-regulated platelet-activating factor receptors. Hence, TNF-alpha has pleiotropic effects on receptor expression; these effects diverge from priming; and a large part of the primed state must reflect enhancement of post-receptor events.     

Regulation of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase (lyso-PAF-acetyltransferase) in exocrine glands. Evidence for an activation via phosphorylation by calcium/calmodulin-dependent protein kinase

Stimulation of secretion in guinea pig exocrine cells is associated with an enhanced synthesis in these cells of 1-O-alkyl-2-sn-acetyl-glycero-3-phosphocholines (PAF) from 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) (S?ling, H-D., and Fest, W. (1986) J. Biol. Chem. 261, 13916-13922). This results from a stimulation of the activity of lyso-1-alkylglycerophosphocholine acetyltransferase (EC 2.3.1.67). Here we have analyzed the effects of various agonists on the activity of this enzyme in guinea pig parotid gland microsomes. Carbamoylcholine leads within less than 30 s to a 2- to 4-fold activation of lyso-PAF-acetyltransferase, which persists after solubilization of the microsomal enzyme with octyl glucoside. The calcium ionophore A23187 has a similar though smaller effect. Neither isoproterenol (2 X 10(-5) M), which stimulates exocytosis more than carbachol, nor phorbol ester significantly affected lyso-PAF-acetyltransferase activity. Incubation of microsomes from unstimulated parotid gland acini with cAMP-dependent and calcium/calmodulin-dependent protein kinase resulted in a 4-fold and 2.9-fold activation of lyso-PAF-acetyltransferase activity, respectively. Protein kinase C had no significant effect. Activation with calcium/calmodulin-dependent protein kinase was inhibited by 40 microM trifluoperazine. When microsomes from carbachol-stimulated glands were used, in vitro activation of the enzyme by calcium/calmodulin-dependent protein kinase was almost abolished. Protein phosphatase 2A in vitro strongly reduced lyso-PAF-acetyltransferase activity in microsomes from both stimulated and unstimulated glands, whereas alkaline phosphatase and protein phosphatase 1 had only small effects. Following treatment with protein phosphatase 2A, enzyme activity in microsomes from stimulated glands could be enhanced more than 8-fold by subsequent incubation with calcium/calmodulin-dependent protein kinase. Although unsuccessful attempts have made it impossible so far to demonstrate directly the incorporation of phosphate into lyso-PAF-acetyltransferase, the results reported here strongly suggest that the enzyme in exocrine cells is regulated by phosphorylation-dephosphorylation and that a calcium/calmodulin-dependent protein kinase is responsible for the activation of the enzyme and type-2 protein phosphatases for its inactivation.     

Leukocyte inhibitory factor activates human neutrophils and macrophages to release leukotriene B4 and thromboxanes

Recent evidence has proved that cytokines can stimulate the production of 5-lipoxygenase products. Leukotriene B4 (LTB4) is a major mediator of leukocyte activation in acute inflammatory reactions, which produce chemotaxis, lysosomal enzyme release, and cell aggregation. Leukocyte inhibitory factor (LIF) also causes biological responses related to inflammation, i.e., LIF directly induces specific granule secretion by polymorphonuclears (PMNs) and potentiates many formyl-methionyl-leucyl-phenylalanine (FMLPs) mediated responses. Since arachidonic acid products are important mediators of inflammation, we have studied the effects of LIF on the arachidonic acid cascade products LTB4 and thromboxane A2 (TxA2). Resuspended at a final concentration of greater than 95% polymorphonuclear PMNs were isolated and tested with some cytokines on the release of LTB4 and TxA2. Peripheral blood mononuclear cells were isolated and seeded in Petri dishes and incubated for 60 min. Adherent macrophages were used for the cytokine stimulation study. Both types of leukocytes were treated with LIF, interleukin 6 (IL 6), and granulocyte-monocyte colony stimulating factor (GM-CSF) at different concentrations, and test agents A23187 and FMLP. Radioimmunoassay for LTB4 and TxB2 was determined by the resulting supernatants. Treatment of PMNs and macrophages with LIF at different concentrations proved to generate significant increases in LTB4 and TxA2 production. This was compared with IL 6 and GM-CSF, which had no effects. In these experiments, TxA2 generations could not be attributed to platelet contamination of PMN suspensions. The quantity of platelet contamination was not sufficient to influence how much TxB2 was produced. The similarities of LIF to other arachidonate stimulating cytokines suggest a similar mode of action in producing hematologic changes typical of tissue injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C activity appears to be required for the synthesis of platelet-activating factor and leukotriene B4 by human neutrophils

Human neutrophils synthesize platelet-activating factor (PAF) and leukotriene B4 (LTB4) when stimulated with the Ca2+ ionophore A23187. These processes are enhanced to a variable extent by phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C. The long chain amines sphingosine, stearylamine (Hannun, Y.A., Loomis, C.R., Merrill, A.H., Jr., and Bell, R.M. (1986) J. Biol. Chem. 261, 12604-12609), and palmitoylcarnitine competitively inhibit activation of purified protein kinase C in vitro and inhibit protein kinase C-mediated activation of the respiratory burst in human neutrophils (Wilson, E., Olcott, M.C., Bell, R.M., Merrill, A.H., Jr., and Lambeth, J.D. (1986) J. Biol. Chem. 261, 12616-12623). These amines were found to inhibit A23187-induced PAF and LTB4 synthesis. Inhibition of PAF and LTB4 synthesis occurred in parallel; half-maximal inhibition by sphingosine occurred at 7 microM, with complete inhibition at 15 microM. PMA by itself did not induce the synthesis of PAF or LTB4, although it did enhance PAF and LTB4 synthesis at suboptimal concentrations of A23187. PMA reversed long chain amine inhibition of PAF and LTB4 accumulation. Reversal of the inhibition of PAF and LTB4 accumulation occurred in parallel, was concentration-dependent, and was complete by approximately 3 x 10(-8) M PMA. The inactive 4 alpha-phorbol didecanoate ester did not reverse inhibition at these concentrations. Sphingosine completely prevented the A23187-induced release of [3H]arachidonate and its various metabolites from [3H]arachidonate-labeled cells. PMA, but not 4 alpha-phorbol didecanoate, restored arachidonate release and its metabolism. Therefore, while activation of protein kinase C is not sufficient to induce PAF and LTB4 synthesis, its action appears to be required to couple a rise in intracellular Ca2+ to their synthesis. This coupling occurs at the level of the initial reaction in the production of lipid mediators, a phospholipase A2-like activity that mobilizes the two substrates 1-O-alkyl-sn-glycero-3-phosphocholine and arachidonic acid from complex lipids.     

Leukotriene B4 stimulates the release of arachidonate in human neutrophils via the action of cytosolic phospholipase A2

Leukotriene B₄ (LTB₄) is a potent lipid mediator of inflammation and is involved in the receptor-mediated activation of a number of leukocyte responses including degranulation, superoxide formation, and chemotaxis. In the present research, stimulation of unprimed polymorphonuclear leukocytes (neutrophils) with LTB₄ results in the transient release of arachidonate as measured by mass. This release of arachidonate was maximal at an LTB₄ concentration of 50–75 nM and peaked at 45 s after stimulation with LTB₄. The transient nature of this release can be attributed, in part, to a fast (<60 s) metabolism of the added LTB₄. Moreover, the inhibition of the reacylation of the released arachidonate with thimerosal results in greater than 4-times as much arachidonate released. Thus, a rapid reacylation of the released arachidonate also contributes to the transient nature of its measured release. Multiple additions of LTB₄, which would be expected to more closely resemble the situation in vivo where the cell may come into contact with an environment where LTB₄ is in near constant supply, yielded a more sustained release of arachidonate. No release of [³H]arachidonate was observed when using [³H]arachidonate-labeled cells. This indicates that the release of arachidonate as measured by mass is most probably the result of hydrolysis of arachidonate-containing phosphatidylethanolamine within the cell since the radiolabeled arachidonate is almost exclusively incorporated into phosphatidylcholine and phosphatidylinositol pools under the non-equilibrium radiolabeling conditions used. Consistent with the role of cytosolic phospholipase A₂ (cPLA₂) in the release of arachidonate, potent inhibition of the LTB₄-stimulated release was observed with methylarachidonylfluorophosphonate, an inhibitor of cPLA₂ (IC₅₀ of 1 μM). The bromoenol lactone of the calcium-independent phosphospholipase A₂. failed to affect LTB₄-stimulated release of arachidonate in these cells.     

Metabolism of platelet-activating factor in human platelets. Transacylase-mediated synthesis of 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine

The present study demonstrates that inactivation of exogenous 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC; platelet-activating factor) by human platelets is mediated by the sequential action of two enzymes, 1) a Ca2+-independent acetylhydrolase recovered in the cytosolic fraction of platelets that deacylates alkylacetyl-GPC forming alkyllyso-GPC and 2) a CoA-independent, N-ethylmaleimide-sensitive transacylase associated with platelet membranes that incorporates a long-chain fatty acid into alkyllyso-GPC to produce alkylacyl-GPC. Separation of platelet phospholipids and subsequent resolution into individual molecular species by high-performance liquid chromatography revealed that the newly formed alkylacyl-GPC was exclusively alkylarachidonoyl-GPC and that the arachidonoyl group for acylation of alkyllyso-GPC was provided by phosphatidylcholine. We conclude that the previously described platelet arachidonoyl transacylase (Kramer, R.M., and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811) may play an important role in the metabolism of platelet-activating factor.     

Phospholipase D activation by platelet-activating factor, leukotriene B4, and formyl-methionyl-leucyl-phenylalanine in rabbit neutrophils. Phospholipase D activation is involved in enzyme release.

Lipid chemoattractants, such as platelet-activating factor and leukotriene B4, as well as the peptide chemoattractant FMLP, were found to stimulate [3H]phosphatidic acid ([3H]PA) formation in 1-O-[3H]octadecyl-lyso platelet-activating factor-labeled rabbit neutrophils. The stimulation of [3H]PA formation appears to result from the activation of phospholipase D (PLD), because in the presence of ethanol, chemoattractant stimulation produced [3H]phosphatidylethanol, the characteristic compound produced by PLD at the expense of [3H]PA formation. The PLD activation by all chemoattractants tested was primed by cytochalasin B and revealed a similar time dependence. However, lipid chemoattractants were less potent as compared with FMLP, and the maximal stimulation by the former was lower than that by the latter. From these results, it is concluded that the mechanism of PLD activation by lipid chemoattractants is similar to, but different from, that by FMLP. Cytochalasin B stimulated degranulation and [3H]PA formation in agonist-stimulated neutrophils, and their stimulations were well correlated. Ethanol inhibited both agonist-stimulated [3H]PA formation and degranulation in a concentration-dependent manner, but the inhibition in degranulation was much less than that in [3H]PA formation. These results suggest that PLD activation is involved in degranulation, but another signaling pathway may also be required for full stimulation of degranulation. When the radiolabeled neutrophils were stimulated by chemoattractants for 5 min, 1,2-[3H]diglyceride was found to accumulate. The accumulation was inhibited by either ethanol or the phosphatidate phosphohydrolase inhibitor propranolol, which indicates that PA produced by PLD can be converted to 1,2-diglyceride by phosphatidate phosphohydrolase. Under these conditions, propranolol did not inhibit degranulation stimulated by chemoattractants. These results indicate that PA produced by PLD is more important than its metabolite diglyceride for the degranulation of rabbit neutrophils.     

1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine. A common source of platelet-activating factor and arachidonate in human polymorphonuclear leukocytes

1-O-[3H]Alkyl-2-lyso-sn-glycero-3-phosphocholine (1-O-[3H]alkyl-2-lyso-GPC) incubated with human polymorphonuclear leukocytes (PMN) for 30 min is metabolized to 1-O-alkyl-2-acyl-GPC containing greater than 80% arachidonate at the 2 position (Chilton, F. H., O'Flaherty, J. T., Ellis, J. M., Swendsen, C. L., and Wykle, R. L. (1983) J. Biol. Chem. 258, 7268-7271). PMN containing 1-O-[3H]alkyl-2-arachidonoyl-GPC incorporated into their cellular phospholipids in this manner were stimulated with Ca2+ ionophore (A23187). Within 5 min after stimulation, 14%, 7%, and 7% of the total 1-O-[3H]alkyl-2-arachidonoyl-GPC in the cells had been converted to 1-O-[3H]alkyl-2-acetyl-GPC (platelet-activating factor), 1-O-[3H]alkyl-2-lyso-GPC, and 3H-labeled neutral lipid, respectively. Stimulation by opsonized zymosan yielded similar results. In related studies, cells were labeled with 1-O-hexadecyl-2-arachidonoyl-GPC containing a [methyl-14C] choline moiety. The nature of the long-chain acyl residues in the sn-2 position of the labeled 1-O-hexadecyl-2-acyl-GPC remaining after stimulation with A23187 was examined. Analysis by high-performance liquid chromatography using synthetic 1-O-hexadecyl-2-acyl-GPC standards indicated there is a time-dependent loss of arachidonate from the 2 position of the labeled 1-O-hexadecyl-2-arachidonoyl-GPC followed by reacylation by other fatty acids (primarily linoleic and oleic). This shift in the acylation pattern exhibited after Ca2+ ionophore stimulation was further examined in PMN preincubated with A23187 and subsequently incubated with labeled 1-O-alkyl-2-lyso-GPC; the stimulated cells produced 1-O-[3H]alkyl-2-acetyl-GPC (greater than 15% of total label) and 1-O-[3H]alkyl-2-acyl-GPC containing linoleic acid and oleic acid, rather than arachidonic acid in the sn-2 position. The findings demonstrate that upon stimulation of PMN, 1-O-alkyl-2-arachidonoyl-GPC can yield arachidonate and 1-O-alkyl-2-lyso-GPC; the 1-O-alkyl-2-lyso-GPC formed may be acetylated producing platelet-activating factor or reacylated with fatty acyl residues other than arachidonate.     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human fetal membranes and decidua vera

We have previously reported that platelet-activating factor (PAF) is present in human amniotic fluid obtained from women in labor. We have also demonstrated that PAF, lyso-PAF, and alkyl acyl-sn-glycero-3-phosphocholine (AA-GPC) are present in human amnion tissue. In the reported study, we have investigated the enzymes involved in PAF metabolism in amnion tissue and their regulation. A phospholipase A2 activity has been demonstrated in amnion tissue which cleaves alkyl acyl (long-chain) sn-glycero-3-phosphocholine. The enzyme activity is not altered by Ca2+ and is distinctly different from the phospholipase A2 that we have previously characterized in this tissue. Amnion tissue contains acetyltransferase activity which requires Ca2+ and is associated with the microsomal fraction. Acetylhydrolase is also present in the cytosolic fraction of amnion tissue. Acetylhydrolase activity has also been demonstrated in amniotic fluid. The affinities of acetyltransferase (for lyso-PAF) and acetylhydrolase (for PAF) were unaffected by Ca2+. In the presence of Ca2+, however, the specific activity of acetyltransferase was increased four- to fivefold while that of acetylhydrolase was unaffected. Acetyltransferase and acetylhydrolase activities in fetal membranes and decidua were similar and were unchanged with gestational age. The possible role of PAF in the initiation of human parturition is discussed.     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human promyelocytic leukemic HL60 cells. Stimulated expression of phospholipase A2 and acetyltransferase requires differentiation

Human promyelocytic leukemia (HL60) cells can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide. The addition of N-formylMet-Leu-Phe or the Ca2+ ionophore A23187 to these differentiated cells generated 15-30 pmol of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC)/10(6) cells as quantified by platelet aggregation assays. Under identical conditions, uninduced cells produced little alkylacetyl-GPC. Upon the addition of ionophore A23187, differentiated cells, and not uninduced ones, released [14C]arachidonate from prelabeled phospholipids including ether-linked phosphatidylcholines, formed both 3H-labeled 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC) and [3H]alkylacetyl-GPC from endogenous 3H-labeled 1-O-alkyl-2-(long chain) acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), and incorporated exogenously added [3H]acetate or [3H]alkyllyso-GPC into alkylacetyl-GPC. These results are suggestive that both phospholipase A2 and acetyltransferase activities are involved in alkylacetyl-GPC biosynthesis by HL60 cells and that these activities appear during differentiation. However, when measured in cell extracts, the activities of phospholipase A2 and acetyltransferase of uninduced cells were virtually indistinguishable from those of differentiated cells. Uninduced cells exhibited enhanced incorporation of [3H]alkyllyso-GPC or [3H]alkylacetyl-GPC into alkylacyl-GPC and of [14C]arachidonate and [14C]oleate into various phospholipids including phosphatidylcholine. However, such enhanced expression of acylation reactions could not account for the lack of accumulation of arachidonate or of alkylacetyl-GPC by uninduced cells. Furthermore, analyses of phospholipid classes by phosphorus determination showed no significant alterations in phospholipid composition of HL60 cells during differentiation. Together these data are suggestive that mechanisms regulating the activation of phospholipase A2 and acetyltransferase activities are defective in uninduced cells and that an increased concentration of cytosolic free Ca2+ alone is not a sufficient requirement for these mechanisms.     

Tumor necrosis factor stimulates ornithine decarboxylase activity in human fibroblasts and tumor target cells

The activity of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), has been shown to be rapidly modulated by a variety of growth regulatory molecules. In this report the effect of the growth modulatory peptide, tumor necrosis factor, on ODC activity was examined on two cell lines which express equivalent TNF binding properties, but differ in their growth response when exposed to this factor. TNF treatment of WI-38 fibroblasts stimulated both their growth and induced ODC activity 5-10-fold when measured 6-24 h after TNF incubation. TNF induced cytotoxicity in ME-180 cervical carcinoma cells and, interestingly, stimulated both ODC activity (3-6-fold) and putrescine accumulation when measured prior to the onset of cytotoxicity. Induction of ODC was TNF concentration-dependent and paralleled the concentration-dependency for cytotoxicity. Based upon studies with cycloheximide, de novo protein biosynthesis was required for TNF-mediated ODC induction in ME-180 cells. The effects of other growth inhibitory peptides and growth factors were analyzed for their combined effect on ODC activity in TNF-treated or untreated ME-180 cells. Interferon gamma treatment had no significant effect on basal ODC activity but inhibited TNF-mediated ODC induction by approximately 50%. EGF treatment resulted in a potent stimulation of ODC activity which was not affected by TNF pre-treatment or coadministration on ME-180 cells. These results suggest that TNF has properties which are similar to those of a growth factor and distinct from those of other growth inhibitory peptides. The early growth factor-like actions of TNF occur on both normal fibroblasts and some tumor cells and evidence suggests that these effects are antagonistic to the antiproliferative effects of TNF.     

Human granulocyte-macrophage colony-stimulating factor and other cytokines prime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis

Human recombinant granulocyte-macrophage CSF (GM-CSF) "primes" neutrophils for enhanced biologic responses to a number of secondary stimuli. Here, we examined the properties of neutrophil priming by GM-CSF and other growth factors such as human rTNF and granulocyte CSF. Although GM-CSF has a negligible direct effect on [3H]arachidonic acid release, it enhances or "primes" neutrophils for three- to fivefold increased release of [3H]arachidonic acid, induced by 1.0 microM A23187 and the chemotactants FMLP, platelet-activating factor, and leukotriene B4 (LTB4) (all 0.1 microM). The priming effects of GM-CSF were concentration- and time-dependent (maximum 100 pM, 1 h at 23 degrees C), and consistent with the determined dissociation constant of the human GM-CSF receptor. Indomethacin (10(-8) M), cycloheximide (100 micrograms/ml), and pertussis toxin (200 ng/ml, 2 h at 37 degrees C) had no effect on GM-CSF-, A23187, or platelet-activating factor-induced [3H]arachidonic acid release. The lipoxygenase inhibitor, nordihydroguaiaretic acid, however, totally abolished A23187-induced [3H]arachidonic acid release from both diluent- and GM-CSF-treated neutrophils. Consistent with this observation, we found that GM-CSF-pretreated neutrophils synthesize increased levels of LTB4 after stimulation with A23187 and chemotactic factors. GM-CSF enhances neutrophil arachidonic acid release and LTB4 synthesis, and thereby may amplify the inflammatory response to chemotactic factors and other physiologically relevant stimuli.     

Binding and internalization of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in washed rabbit platelets

The binding profile of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, platelet-activating factor) to washed rabbit platelets was investigated through the use of structural analogs of AGEPC, e.g. U66985, which specifically suppressed AGEPC biological activities on rabbit platelets. This interaction of AGEPC with platelets could be divided into three different components termed A, B, and C. Component A was considered as one of high affinity (Kd = 0.5 X 10(-9) M) and with a low capacity (about 400 sites/platelet). The binding of AGEPC to component A was reversible and was blocked by the inhibitory analogs of AGEPC. This was considered to be the AGEPC receptor site(s). Component B was irreversible in nature and was presumed to be associated with internalization of AGEPC. The latter process was sensitive to the structural inhibitors. Component C was not affected by the inhibitors and probably represented a nonspecific binding to the lipid layer of the membrane. The binding profile of 1-O-alkyl-2-(lyso)-sn-glycero-3-phosphocholine, a biologically inactive and noninhibitory analog of AGEPC, was observed to consist of a single component and was (also) unaffected by the inhibitors. Internalization of AGEPC into rabbit platelets was further examined by the bovine serum albumin extraction method, which was originally developed by Mohandas et al. (Mohandas, N., Wyatt, J., Mel, S. F., Rossi, M. E., and Shohet, S. B. (1982) J. Biol. Chem. 257, 6537-6543). AGEPC was instantly taken up by the cell and internalization into its membrane, where it remained and was not released into cytosol. The internalization of AGEPC was suppressed by pretreating the cells with AGEPC analogs. In platelets desensitized to AGEPC, no down-regulation of the receptor site(s) was observed. The internalization of AGEPC in the desensitized cells was clearly enhanced and this was obvious even in the presence of the AGEPC inhibitor(s). Even in the presence of the inhibitors, effective internalization of AGEPC was also evident in thrombin-treated cells. These results suggested that the internalization of AGEPC was irreversibly enhanced in the platelets which were activated by AGEPC itself as well as by thrombin.     

Tumor necrosis factor stimulates interleukin-1 and prostaglandin E2 production in resting macrophages

We have investigated the effect of tumor necrosis factor on the release of interleukin-1 and PGE2 from murine resident peritoneal macrophages. Tumor necrosis factor causes an increase in the production of interleukin-1 and PGE2 with a maximum induction for both noted at 5.9 X 10(-8) M. While indomethacin decreased tumor necrosis factor induced PGE2 production, this cyclooxygenase inhibitor augmented tumor necrosis factor induced interleukin-1 production. Our data suggests that tumor necrosis factor may be an important immunopotentiating agent in addition to its previously described cytolytic and metabolic activities.     

Calcium ionophore potentiates chemotactic peptide and platelet activating factor in stimulating thromboxane B2 and leukotriene B4 biosynthesis in human neutrophils

Formyl-Met-Leu-Phe (FMLP) and platelet activating factor (PAF) stimulated the synthesis of thromboxane B2 (TXB2) and leukotriene B4 (LTB4) to a small degree in human neutrophils. Calcium ionophore A-23187 enhanced synergistically both FMLP and PAF induced eicosanoid synthesis, whereas phorbol ester PMA attenuated PAF but not FMLP stimulated arachidonate metabolism. These results suggest that calcium mobilization may be a rate limiting step in FMLP and PAF induced synthesis of TXB2 and LTB4 and that protein kinase C activation may play a negative regulatory role in PAF stimulated eicosanoid synthesis.     

Spontaneous and protein-catalyzed transfer of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) between phospholipid bilayers

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor or alkylacetyl-GPC), a bioactive phospholipid that possesses hypotensive, platelet-aggregating and inflammatory properties, is known to be secreted by a variety of cell types. The biological activity of alkylacetyl-GPC is related to a precise chemical structure that implicates interaction with proteins. We have studied the spontaneous and protein-catalyzed transfer of alkylacetyl-GPC between phospholipid vesicles and have demonstrated the following: 1. There are at least two transferable pools of alkylacetyl-GPC in sonicated phospholipid vesicles. 2. These two pools differ in the rate at which they dissociate from the vesicles; one pool equilibrates between donor and acceptor vesicles instantaneously while the other pool is transferred much more slowly. 3. Dialysis of alkylacetyl-GPC between phospholipid vesicles through the aqueous phase is slow. 4. A protein fraction derived from rat lung cytosol catalyzes the transfer of the nonequilibrating pool of alkylacetyl-GPC between phospholipid vesicles; this transfer is superimposed on the spontaneous transfer and is unchanged in experiments using vesicles from which the rapidly equilibrating pool has been removed.     

Production of platelet-activating factor by stimulated human polymorphonuclear leukocytes. Correlation of synthesis with release, functional events, and leukotriene B4 metabolism

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation that is synthesized by several human cell types including polymorphonuclear leukocytes (PMN). We examined the synthesis and release of PAF by stimulated human PMN under several conditions, assayed by the incorporation of [3H]acetate into PAF and by bioassay. PAF synthesis was induced by calcium ionophore A23187 (IoA), opsonized zymosan (OpsZ), and N-formyl-methionyl-leucyl-phenylalanine (FMLP) with the relative order of potency IoA much greater than OpsZ greater than FMLP. A variety of other agonists, including phorbol myristate acetate, an activator of protein kinase C and of PMN functional responses, did not stimulate PAF synthesis. PAF synthesis by PMN in response to IoA, OpsZ, and FMLP was concentration- and time-dependent but release of the phospholipid was not: little PAF (1 to 10%) was released from PMN in suspension regardless of the total amount produced, the agonist, its concentration, the time of incubation, or the concentration of extracellular albumin. This was also the case with functionally altered neutrophils that had been "primed" with cytochalasin B or lipopolysaccharide or that had adhered to surfaces. PAF synthesis was tightly coupled with leukotriene B4 production by adherent PMN as well as by neutrophils in suspension, supporting the hypothesis that the two lipid autacoids may be derived from a common precursor. However, PAF synthesis could be dissociated from aggregation and surface adhesion, indicating that it is not absolutely required for these responses of activated PMN. The total amount of PAF that accumulated, but not the percentage that was released, was altered in adherent PMN compared to cells in suspension. These experiments demonstrate that PAF production and its subsequent processing by human neutrophils are highly regulated events. PAF synthesis is associated with PMN activation, but it is not a requisite for early adhesive responses of neutrophils. Because little of the PAF produced by stimulated PMN is released from the cells, it appears that PAF has an intracellular role in PMN function and/or that it may have novel intercellular effects that do not require release into the fluid phase.