Regulation of leukotriene and platelet-activating factor synthesis in human alveolar macrophages

It has been suggested that phospholipase A2 (PLA2) contributes to the regulation of leukotriene (LT) and platelet-activating factor (PAF) synthesis by controlling the release of their precursors, arachidonic acid (AA) and lysophosphatidylcholine (lysoPC), from membrane phospholipids. In rat alveolar macrophages (AMs), PLA2 appears to have a major role in LT synthesis but a more limited role in PAF synthesis. The present study was designed to define the role of PLA2 in LT and PAF synthesis in human AMs and determine whether differences exist between AMs obtained from normal subjects and those from patients with asthma. In the normal subjects, the calcium ionophore A23187 (Cal) increased AM PAF synthesis (percent incorporation of tritiated acetate) by 135% (p < 0.01) and LTB4 synthesis 88-fold (p < 0.001). Phorbol myristate acetate (PMA) had little effect alone, but it had a synergistic effect with Cal, increasing PAF synthesis by 466% and LTB4 synthesis to 229-fold above the control values (p < 0.001 for both). Ro 25-4331, a combined cytosolic (c) and secretory (s) PLA2 inhibitor, had little effect on the Cal-stimulated PAF synthesis, but it completely blocked the effect of PMA. It also blocked the Cal- and Cal+PMA-stimulated LTB4 synthesis. AACOCF3, a cPLA2 inhibitor, had no effect on either Cal or Cal+PMA-stimulated PAF synthesis. It reduced LTB4 synthesis, but it did so less effectively than Ro 25-4331. CoA-independent transacylase (CoAI-TA) activity in the AMs increased after stimulation and exposure to Ro 25-4331. SK&F 45905, a CoAI-TA inhibitor, reduced stimulated PAF synthesis by 30% to 40%. Patients with asthma had similar results except that cPLA2 had a greater role in stimulated LTB4 synthesis. These data indicate that PLA2 plays a direct role in human AM LT synthesis; both the cytosolic and secretory forms contribute to LT synthesis; PLA2 appears to have a more limited role in PAF synthesis, although it mediates the synergistic effect of PMA, probably via sPLA2; and CoAI-TA contributes to PAF synthesis during PLA2 inhibition. With the exception of the greater role for cPLA2 in stimulated LTB4 synthesis in the patients with asthma, the contributions of PLA2 and CoAI-TA to AM LT and PAF synthesis appear to be similar in normal subjects and patients with asthma.     

The effect of platelet-activating factor and its synthetic analogs on Ia-antigen expression by mouse peritoneal macrophages

Visual evoked potentials (VEPs) of the pattern shift reversal type were determined in a representative group of 57 prisoners of war (POWs) released in 1992 from detention camps in former Yugoslavia. The parameters were correlated with the conditions in four camps (1-4). All subjects were male, with a mean age of 34.75 years (SD +/- 8.92), average length of imprisonment 192.7 days (SD +/- 77.6), mean loss of body mass during imprisonment 19.32% (SD +/- 9.54), and the average number of reported blows to the head and neck was 25.7 (SD +/- 20.3). VEPs were determined on average 290.5 days after the last craniocerebral trauma caused by blows to the head and neck (SD +/- 152.0) i.e., on average 218.5 days after release from the camp (SD +/- 164.3). Although all the 57 POWs reported being maltreated to a certain extent, 14 reported being subjected to particularly brutal forms of torture, 5 had been held in solitary confinement and 25 had lost consciousness at least once. Solitary confinement and loss of consciousness had the most significant effect on VEPs, and the altered VEP parameters correlated significantly with the craniocerebral trauma experienced, loss of body mass and the length of time since the last craniocerebral trauma until examination, and from release until examination. However, the length of imprisonment and treatment in the camps did not have a significant effect on VEP parameters. The study confirmed that under such conditions the age of the subject is a risk factor. The results of this study also confirmed that prisoners in one camp had been subjected to the worst maltreatment.     

Heterogeneous platelet-activating factor (PAF) receptors and calcium increase in platelets and macrophages

We used the increase in cytosolic Ca2+ levels, [Ca2+]i, as a way to characterize PAF (platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) receptors in human platelets and rat and human macrophages. [Ca2+] was measured by means of the fluorescent probe fura-2/acetoxymethylester. PAF recognized heterogeneous receptors in human macrophages only (curve slope <1). The PAF antagonist SCH 37370 (1-acetyl-4(8-chloro-5,6-dihydro-11H-benzo[5.6]cyclohepta[1,2-b]pyridine -11-ylidine)piperidine) abolished [Ca2+]i elevation in human platelets, while in rat and human macrophages the maximal inhibition was 76% and 85%, respectively. On the contrary, the antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6Hthieno[3,2-f] [1,2,4]triazolo-[4,3-a] [1,4]-diazepin-2-yl]-1-(4-morpholiny)-1-propanon, apafant) totally inhibited the effect of PAF in both platelets and macrophages. The WEB 2086 concentration-response curves had a slope <1 in the three cell types, indicating interaction with heterogeneous receptors. Accordingly, 3H-WEB 2086 bound to two different classes of sites. Both phases of [Ca2+]i elevation (influx or release) were equally affected by the antagonists. These data support the notions that: 1) PAF receptors are heterogeneous; 2) the two antagonists have a different selectivity toward the receptor subtypes: WEB 2086 recognizes two different receptors both in platelets and in macrophages, while SCH 37370 does not discriminate between receptor subtypes in platelets, and only interacts with one subtype in macrophages; and 3) both SCH 37370 and WEB 2086 display different potencies in rat and human macrophages.     

Generation of reactive oxygen species and activity of platelet-activating factor acetylhydrolase in human monocyte-derived macrophages

Monocytes were prepared from healthy human volunteers and were allowed to differentiate into macrophages by adhesion to plastic surface and cultured over 7 days in presence of either 10% fetal calf serum (FCS), human control serum or serum from hyperlipaemic patients. Hyperlipaemic serum stimulated the differentiation (measured as an increase in cellular protein and DNA content) to a higher extent when compared to control serum and FCS. With all sera a marked increase of the cellular activity of the enzyme platelet-activating factor acetylhydrolase (PAF-AH) and a tremendous decrease in the capacity of cells to generate reactive oxygen species (ROS) was observed. After seven days of culture the increase in PAH-AH activity was about 19-fold with hyperlipaemic serum, 11-fold with control serum and 6-fold with FCS. During the same period of time ROS generation measured as zymosan-induced chemiluminescence decreased by about 98% and no significant differences between the three types of serum were found. The results indicate that the activity of PAF-AH and the capacity of ROS generation which are both assumed to play an important role in the oxidation of low-density lipoproteins (LDL) and thus in the development of atherosclerosis, change in opposite direction during the differentiation of blood monocytes into macrophages, and that hyperlipaemic serum stimulates PAF-AH activity but not ROS generation.     

Up-regulation of platelet-activating factor receptors in lung and alveolar macrophages in the bleomycin-hamster model of pulmonary fibrosis

Long-term exposure to agonist down-regulates receptor expression for many G protein-coupled receptors. This decrease in receptor density could occur through either an increase in receptor degradation or a decrease in receptor synthesis. We studied the mechanism of down-regulation of the alpha-2A and alpha-2B adrenergic receptor subtypes transfected into the Chinese hamster ovary cell line as well as the alpha-2A receptor endogenous to the HT29 cell line. The rate constants for receptor appearance and disappearance were calculated from the recovery of receptor expression after irreversible inactivation of the existing receptor population with an alkylating agent. In the presence of the agonist norepinephrine, the receptor subtypes in all three cell lines down-regulated to about 50% with a half-time of 2.5 hr. When recovering in the presence of norepinephrine after irreversible inactivation, the rate of receptor degradation increased approximately 2-fold for all three cell lines with little change in the rate of synthesis. During this recovery, the transfected alpha-2A receptor exhibited a half-life of 3.0 hr, which agrees with the 2.7-hr half-time of down-regulation in the presence of norepinephrine. In contrast, the transfected alpha-2B receptor and the endogenous alpha-2A receptor had a half-life of 1.2 hr and 8.9 hr, respectively. For only the endogenous alpha-2A receptor, pertussis toxin increased the half-time of down-regulation to 9.8 hr, similar to the 8.9-hr receptor half-life in the presence of norepinephrine during recovery after irreversible inactivation. Our results indicate that the mechanism of down-regulation of the alpha-2A and -2B adrenergic receptor subtypes is an increase in the rate of receptor degradation.     

Stimulation of platelet-activating factor synthesis by neurotransmitters in salivary glands

Platelet-activating factor (PAF), a phospholipid mediator exhibiting potent biological activities, has been shown to stimulate amylase release from the pancreas and salivary glands. The capacity of salivary glands for PAF biosynthesis in response to stimulation has also been demonstrated. To elucidate the role of PAF in salivary glands, we studied the regulation of platelet-activating factor synthesis by the autonomic nervous system in canine salivary glands. Acetylcholine and ionomycin stimulated PAF production in dispersed cells from parotid, submandibular, and sublingual glands of dogs. Norepinephrine and phenylephrine, but not isoproterenol, also stimulated PAF production in submandibular gland cells. Norepinephrine-induced PAF production was blocked by phentolamine but not by propranolol. Acetylcholine and norepinephrine increased both the PAF production and liberation of [14C]arachidonic acid from cells pre-labeled with [14C]arachidonic acid in the presence of Ca2+ in the medium. These stimulants increased [14C]arachidonic acid liberation without the accompanying production of PAF in Ca(2+)-deprived medium. No activators or inhibitors of protein kinase C produced or affected acetylcholine-induced PAF production. Lyso-PAF:acetyl-CoA acetyltransferase was activated in the cells treated with acetylcholine, norepinephrine, isoproterenol, and 8Br-cyclic AMP. Deprivation of Ca2+ in the medium markedly reduced acetylcholine-induced activation of the transferase, but little affected norepinephrine-, isoproterenol-, and 8Br-cyclic AMP-induced activation. Dithiothreitol-insensitive cholinephosphotransferase activity was also increased by acetylcholine, norepinephrine, isoproterenol, and 8Br-cyclic AMP, and the deprivation of Ca2+ in the medium further increased the activation of the enzyme activity by these agents. These results suggest that PAF synthesis in canine salivary glands is under the control of muscarinic cholinergic and alpha-adrenergic systems via Ca(2+)-dependent remodeling pathways, and that the independent activation of either phospholipase A2 or acetyltransferase is insufficient for PAF production in submandibular gland cells, i.e., the concurrent activation of these enzymes is required.     

Modulatory effect of interleukin-10 on the production of platelet-activating factor and superoxide anions by human leucocytes

We observed that human monocytes (MO) and polymorphonuclear neutrophils (PMN) stimulated by lipopolysaccharide (LPS) produce platelet-activating factor (PAF) in a pattern characterized by an early and a delayed peak of synthesis. The early peak of PAF synthesis was due to a direct stimulation of these cells through mCD14 receptor as it was inhibited by anti-CD14 monoclonal antibody. The delayed and sustained peak of PAF synthesis was dependent on protein synthesis and cytokine production as shown by the inhibitory effect of cycloheximide on both MO and PMN, and of anti-tumour necrosis factor-alpha (anti-TNF-alpha) and of anti-interleukin-8 (anti-IL-8) neutralizing antibodies on MO and PMN respectively. IL-10 completely prevented this second, cytokine-dependent peak of PAF synthesis. In contrast, IL-10 markedly enhanced the first peak of PAF synthesis both in MO and PMN. Moreover, IL-10 was shown to modulate the production of superoxide anions (O2-) on both MO and PMN. As suggested by previous studies, IL-10 inhibited the delayed production of O2-. In the present study, we observed that IL-10 directly stimulated an early production of O2-. In addition, IL-10 enhanced the synthesis of O2- by MO and PMN challenged with LPS. The IL-10-induced O2- production was dependent, at least in part, from its effect on PAF synthesis, as it was inhibited by the PAF receptor antagonist WEB 2170. These results suggest that IL-10 may upregulate the early synthesis of PAF and O2- triggered by direct LPS stimulation, whereas it may downregulate the delayed production of these mediators.     

Platelet-activating factor production in stimulated macrophages is down-regulated by concurrently produced prostaglandin E2

When rat peritoneal macrophages were incubated in medium containing 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, production of cell-associated platelet-activating factor (PAF) and extracellular prostaglandin E2 (PGE2) increased. In the presence of the cyclooxygenase inhibitor indomethacin, TPA-induced PAF production was further enhanced dose-dependently in accordance with decrease of PGE2 levels. In addition, indomethacin further enhanced PAF production that was stimulated by the protein kinase C activators, aplysiatoxin and teleocidin. Other cyclooxygenase inhibitors such as naproxen and ibuprofen also enhanced TPA-stimulated PAF production in accordance with inhibition of PGE2 production. Cyclooxygenase inhibitor-induced enhancement of PAF production was markedly prevented by exogenous PGE2. Exogenous arachidonic acid also inhibited TPA-induced PAF production in parallel with increase in PGE2 levels. Inhibition of PAF production by exogenous arachidonic acid was abolished by indomethacin. Furthermore, PAF production stimulated by the endomembrane Ca+2-ATPase inhibitors thapsigargin or thapsigargicin, or by the Ca+2 ionophore A23187, was also enhanced by indomethacin in compensation for the decrease in PGE2 production. In addition, the adenylate cyclase activator forskolin, or the cyclic adenosine monophosphate (cAMP) analogues 8-bromo cAMP and dibutyryl cAMP inhibited thapsigargin-induced PAF production. TPA-induced accumulation of intracellular cAMP was inhibited by indomethacin, and indomethacin-induced decrease of cAMP level was reversed by exogenous PGE2. These results suggested that concurrently produced PGE2 in stimulated macrophages down-regulates PAF production via adenylate cyclase and cAMP pathway.     

Human macrophages induced in vitro by macrophage colony-stimulating factor are deficient in IL-12 production

IL-12 is important for Th1 differentiation. Myeloid-derived antigen-presenting cells (APC) such as monocytes, macrophages (Mphi) and dendritic cells (DC) are believed to be major sources of IL-12 in vivo. We have compared IL-12 production of fresh monocytes with Mphi differentiated in vitro using macrophage colony-stimulating factor (M-CSF) or human plasma, and in vitro generated dendritic cells, since these differentiated cell types represent APC at sites of antigen challenge. Macrophages stimulated with lipopolysaccharide (LPS) or heat-killed Listeria monocytogenes in the presence or absence of IFN-gamma produced minimal IL-12 p70 by comparison with DC or monocytes, despite comparable production of TNF-alpha. M-CSF-induced Mphi produced low levels of IL-10 constitutively and high levels after stimulation with LPS, but neutralization of IL-10 did not augment Mphi IL-12 production. Exposure of Mphi to TNF-alpha, granulocyte-macrophage CSF or IFN-gamma did not substantially up-regulate IL-12. Therefore M-CSF induces a differentiated Mphi phenotype in which IL-12 production is down-regulated, perhaps irreversibly. This may be the default pathway for monocyte-Mphi development in the absence of inflammation.     

Characterization of B lymphocytes rescued from apoptosis by platelet-activating factor

B lymphocyte development is characterized by deletion via apoptosis of immature cells that are insufficiently stimulated. We have previously demonstrated that crosslinking of the B cell receptor (BCR) using anti-IgM antibodies (alphaIgM) (2 microg/ml) in Ramos B lymphoblastoid cells causes deletion of 30-40% of cells by apoptosis in 24 h. Addition of the potent lipid mediator platelet-activating factor (10(-7) M) to alphaIgM stimulated Ramos cells significantly decreases the number of apoptotic cells as measured by annexin V labeling. We have characterized the phenotype of Ramos cells that have not become apoptotic following BCR stimulation. In these cells, there is a significant decrease in the surface expression of the VLA-4 adhesion molecule (31% of control expression) and surface IgM expression (sIgM) (53% of control expression). Significantly fewer cells co-incubated with platelet-activating factor (PAF) underwent apoptosis, and the remaining cells maintained control levels of VLA-4 (104% of control expression) and sIgM expression (104% of control). All of these protective effects were inhibited by the specific PAF receptor antagonist, WEB 2170. The action of PAF on alphaIgM induced apoptosis was not inhibited by either cycloheximide or cytochalasin B, suggesting that de novo protein synthesis and F-actin polymerization were not implicated in the rescue of Ramos cells by PAF. In contrast, the ability of PAF to maintain sIgM and VLA-4 expression at control levels was inhibited by cycloheximide (7. 5 microg/ml). Cytochalasin B (5 microg/ml) had no effect on sIgM expression but blocked the decrease in VLA-4 expression mediated by alphaIgM. These data indicate that PAF's effect on rescuing and maintaining alphaIgM stimulated Ramos B cells is mediated via at least two pathways. Abrogation of apoptosis does not require de novo protein synthesis, while maintenance of sIgM and VLA-4 expression requires protein synthesis.     

Metabolism of platelet-activating factor in rat epididymal spermatozoa

A role for platelet-activating factor (PAF) in sperm function has been proposed. In the present investigation the metabolism of PAF was examined in sperm and epididymal tissue. The major regulatory enzymes for the synthesis of PAF via the de novo pathway have been established in spermatozoa; these include acetyltransferase and cholinephosphotransferase specific for PAF biosynthesis. De novo acetyltransferase activity for PAF biosynthesis in spermatozoa was significantly higher than that of the acetyltransferase of the remodeling pathway. A metabolic pathway was described for the catabolism of PAF in sperm, involving PAF-acetylhydrolase (-AH), lysophospholipase D, and a phosphohydrolase. An isozyme of PAF-AH similar to that reported in sera was also demonstrated in epididymal fluid and tissue. This isozyme was distinctly different from that found in the spermatozoa. The partial inactivation of PAF-AH by the vaginal pH, and/or its detachment from sperm during migration to the site of fertilization, may allow increased motility and migration to the site of fertilization. It is suggested that a decapacitation factor previously described may be related to PAF-AH.     

Vitamin C blocks inflammatory platelet-activating factor mimetics created by cigarette smoking

Cigarette smoking within minutes induces leukocyte adhesion to the vascular wall and formation of intravascular leukocyte-platelet aggregates. We find this is inhibited by platelet-activating factor (PAF) receptor antagonists, and correlates with the accumulation of PAF-like mediators in the blood of cigarette smoke-exposed hamsters. These mediators were PAF-like lipids, formed by nonenzymatic oxidative modification of existing phospholipids, that were distinct from biosynthetic PAF. These PAF-like lipids induced isolated human monocytes and platelets to aggregate, which greatly increased their secretion of IL-8 and macrophage inflammatory protein-1alpha. Both events were blocked by a PAF receptor antagonist. Similarly, blocking the PAF receptor in vivo blocked smoke-induced leukocyte aggregation and pavementing along the vascular wall. Dietary supplementation with the antioxidant vitamin C prevented the accumulation of PAF-like lipids, and it prevented cigarette smoke-induced leukocyte adhesion to the vascular wall and formation of leukocyte-platelet aggregates. This is the first in vivo demonstration of inflammatory phospholipid oxidation products and it suggests a molecular mechanism coupling cigarette smoke with rapid inflammatory changes. Inhibition of PAF-like lipid formation and their intravascular sequela by vitamin C suggests a simple dietary means to reduce smoking-related cardiovascular disease.     

Ginkgolides antagonizing some effects of platelet-activating factor in vitro

The mixture of platelet-activating factor (PAF) and platelets produced significant contraction of guinea pigs' bronchus, while the contraction induced by PAF alone was mild relatively, the IC50 were 6.14 x 10(-7) mol/L and 6.32 x 10(-4) mol/L respectively. There was significant difference between these two groups (P < 0.05). When the platelets were pre-incubated with ginkgolides for 10 minutes in Tris-Tyrode's buffered saline, effects of the PAF and platelets mixture were significantly inhibited (P < 0.01). Exposure of guinea pigs' bronchus to PAF in vitro resulted in a loss of beta-adrenergic receptors and responses to isoproterenol, and this effect of PAF was prevented by prior incubation of the guinea pigs' bronchus with ginkgolides (P < 0.05). The results showed ginkgolides were a potent PAF antagonist.     

Inhibition of Na+,K(+)-ATPase by platelet-activating factor in dog submandibular glands

Physiological stimulation of dog submandibular gland has been shown to generate platelet-activating factor (PAF). However, PAF is not released from cells in the tissue. To assess its intracellular activity, the effect of PAF on Na+,K(+)-ATPase was examined in dog submandibular gland cells. PAF inhibited Na+,K(+)-ATPase in membrane preparations, and the inhibitory effect was dependent on the protein concentration in the enzyme preparation. The inhibitory effect of a low concentration of PAF was antagonized by a PAF-receptor antagonist, BN 50,739, but at high concentrations, PAF was not antagonized. Kinetic analysis of PAF inhibition of Na+,K(+)-ATPase suggests that the inhibition of Na+,K(+)-ATPase by PAF is not due to competition by PAF at K(+)- or Na(+)-binding sites on the enzyme, but by complex inhibitory mechanisms. These results suggest that PAF may interact with specific and nonspecific site of action resulting in the inhibition of Na+,K(+)-ATPase. Ouabain increased mucin release from dog submandibular gland cells. Because Na+,K(+)-ATPase and ion exchange pathways are important in the secretory responses of acinar cells, PAF may regulate intracellularly the secretory function of acinar cells by modulating Na+,K(+)-ATPase and ionic homeostasis.     

Regulation of TNF-alpha production in activated mouse macrophages by progesterone

The purpose of this study was to investigate the relationships between macrophage production of TNF-alpha and female hormones. Northern blot hybridization experiments showed that the female sex steroid hormone, progesterone, decreases steady state levels of TNF-alpha mRNA in LPS-activated mouse macrophages (RAW 264.7 and ANA-1 cells) in vitro. The production of intracellular and secreted TNF-alpha protein, as determined by ELISA, was decreased in both progesterone- and dexamethasone-treated, LPS-stimulated macrophages. Estrogen had no effect on expression of the TNF-alpha gene in mouse macrophages and did not alter progesterone-mediated suppression. Additional experiments conducted to investigate the mechanism of action of progesterone showed that this hormone, like dexamethasone, elevates steady state mRNA levels of IkappaB alpha and increases the levels of IkappaB alpha protein that are translocated from the cytoplasm to the nucleus. Thus, progesterone is a potent inhibitor of steady state levels TNF-alpha mRNA and TNF-alpha protein production in activated macrophages and may achieve this result through effects on an inhibitor of NF-kappaB.     

Stimulation of nitric oxide production in macrophages by Babesia bovis

Gamma interferon (IFN-gamma)-activated macrophages are believed to play a key role in resistance to Babesia bovis through parasite suppression by macrophage secretory products. However, relatively little is known about interactions between this intraerythrocytic parasite and the macrophages of its bovine host. In this study, we examined the in vitro effect of intact and fractionated B. bovis merozoites on bovine macrophage nitric oxide (NO) production. In the presence of IFN-gamma, B. bovis merozoites stimulated NO production, as indicated by the presence of increased L-arginine-dependent nitrite (NO2-) levels in culture supernatants of macrophages isolated from several cattle. The merozoite crude membrane (CM) fraction stimulated greater production of NO, in a dose-dependent manner, than did the merozoite homogenate or the soluble, cytosolic high-speed supernatant fraction. Stimulation of NO production by CM was enhanced by as little as 1 U of IFN-gamma per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by either B. bovis-parasitized erythrocytes and IFN-gamma or CM was also observed. B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-gamma, were also found to induce L-arginine-dependent NO2- production. Supernatants that induced the highest levels of NO also contained biologically active TNF. These results show that B. bovis merozoites and antigen-stimulated B. bovis-immune T cells can induce the production of NO, a molecule implicated in both protection and pathologic changes associated with hemoprotozoan parasite infections.     

The vascular distribution of the platelet-activating factor receptor

Although the platelet-activating factor (PAF) is the most active inflammatory mediator known to date, little is known about its effects on the vascular endothelium and about the cellular and subcellular distribution of its receptor, already identified as a membrane protein of approximately 39 kDa. To better understand its functions we decided: i) to study PAF effects on a model microvascular bed (the rat cremaster), ii) to raise monoclonal antibodies against synthetic peptides reproducing short segments (14 and 16 amino acids) at the N and C terminal parts of PAF-receptor (PAF-R), iii) to determine the distribution of PAF-R on a number of microvascular beds. Topical application of the PAF on the cremaster led promptly to: i) opening of the venular and capillary endothelial junctions; ii) fenestration of the endothelium and iii) swelling, clustering and fusion of endothelial plasmalemmal vesicles. With the anti-N terminal antibody, we localized PAF-R by immunofluorescence on semithin frozen sections of lung, heart, diaphragm, kidney, and brain specimens. With the exception of brain, the signal was restricted primarily to the vascular endothelium. Using immunogold procedures, we localized the PAF-R in small clusters on endothelial surfaces and found it associated preferentially with the plasmalemma proper, rather than to any differentiated microdomain. A morphometric analysis revealed a greater signal density at the level of the venular endothelium than at the level of the endothelium of any other segment of the microvasculature. With the same antibody, we immunoprecipitated PAF-R from whole homogenates of the same tissues. The results obtained were in general agreement with the immunofluorescence tests.     

The structure and function of platelet-activating factor acetylhydrolases

Platelet-activating factor acetylhydrolases (PAF-AHs, EC 3.1.1.47) constitute a unique and biologically important family of phospholipase A2s. They are related to neither the well-characterized secretory nor cytosolic PLA2s, and unlike them do not require Ca2+ for catalytic activity. The distinguishing property of PAF-AHs is their unique substrate specificity: they act on the phospholipid platelet-activating factor (PAF), and in some cases on proinflammatory polar phospholipids, from which they remove a short acyl moiety--acetyl in the case of PAF--located at the sn-2 position. Because PAF is found both in the plasma and in the cytosol of many tissues, PAF-acetylhydrolases are equally widely distributed in an animal organism. Recent crystallographic studies shed new light on the complex structure-function relationships in PAF-AHs.     

Characterization of platelet-activating factor synthesis in glomerular endothelial cell lines

Platelet-activating factor synthesis in two transformed lines of glomerular endothelial cells was characterized and contrasted with platelet-activating factor production in macrovascular-derived endothelial cells as well as with glomerular cells of mesenchymal origin. Platelet-activating factor synthesis was assessed in intact cells and in cell-free preparations. Glomerular endothelial cells constitutively synthesize bio-active alkyl-PAF, and this basal activity can be chronically augmented by various inflammatory and thrombotic agents. In contrast, thrombin-mediated platelet-activating factor formation in bovine pulmonary aortic endothelial cells as well as in glomerular mesangial cells is acute and transient. The potential role of anti-inflammatory prostanoids to function as negative feedback modulators of thrombin- or endothelin-mediated platelet-activating factor synthesis was also investigated, as the synthesis of platelet-activating factor is often associated with the formation of these prostanoids. Indomethacin augmented receptor-mediated platelet-activating factor synthesis while prostanoids of the E and I series reduced agonist-stimulated PAF synthesis. In summary, the unique capacity of glomerular endothelial cells to respond to inflammatory stimuli with sustained platelet-activating factor synthesis is a clear indication of this cell's pivotal role in augmenting the inflammatory response in the limited environment of the glomerulus.     

Potential angiogenic role of platelet-activating factor in human breast cancer

This study investigated the presence of platelet-activating factor (PAF) in the lipid extracts of 18 primary breast carcinomas and 20 control breast tissues. The amount of PAF detected in breast carcinomas was significantly higher than in controls. The mass spectrometric analysis of PAF-bioactive lipid extract from breast carcinomas showed the presence of several molecular species of PAF, including C16-alkylPAF, C18-lysophosphatidylcholine (LPC), C16-LPC, lyso-PAF, and C16-acylPAF. The amount of bioactive PAF extracted from breast specimens significantly correlated with tumor vascularization revealed by the number of CD34-and CD31-positive cells. As C16-alkylPAF was previously shown to induce angiogenesis in vivo, we evaluated whether the thin layer chromatography-purified lipid extracts of breast specimens elicited neoangiogenesis in a murine model of subcutaneous Matrigel injection. The lipid extracts from specimens of breast carcinoma containing high levels of PAF bioactivity, but not from breast carcinomas containing low levels of PAF bioactivity or from normal breast tissue, induced a significant angiogenic response. This angiogenic response was significantly inhibited by the PAF receptor antagonist WEB 2170. T47D and MCF7 breast cancer cell lines, but not an immortalized nontumor breast cell line (MCF10), released PAF in the culture medium. A significant in vivo neoangiogenic response, inhibited by WEB 2170, was elicited by T47D and MCF7 but not by MCF10 culture medium. These results indicate that an increased concentration of PAF is present in tumors with high microvessel density and that PAF may account for the neoangiogenic activity induced in mice by the lipid extracts obtained from breast cancer. A contribution of PAF in the neovascularization of human breast cancer is suggested.