GTP regulation of platelet-activating factor binding to human neutrophil membranes

Radiolabeled ligand binding studies showed that specific receptors for platelet-activating factor are present in human neutrophil membranes. GTP at 10(-7) to 10(-3) M decreased the specific binding of platelet-activating factor to neutrophil membranes in a dose-dependent manner. Inhibition of platelet-activating factor binding was also induced by other guanine nucleotides but not by adenine nucleotides. Our results suggest that platelet-activating factor receptor in human neutrophil membranes may be coupled to a guanine nucleotide binding protein.     

Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism.

Cytosolic phospholipase A2 initiates the biosynthesis of prostaglandins, leukotrienes, and platelet-activating factor (PAF), mediators of the pathophysiology of asthma and arthritis. Here, we report the X-ray crystal structure of human cPLA2 at 2.5 A. cPLA2 consists of an N-terminal calcium-dependent lipid-binding/C2 domain and a catalytic unit whose topology is distinct from that of other lipases. An unusual Ser-Asp dyad located in a deep cleft at the center of a predominantly hydrophobic funnel selectively cleaves arachidonyl phospholipids. The structure reveals a flexible lid that must move to allow substrate access to the active site, thus explaining the interfacial activation of this important lipase.     

Human spermatozoa produce C16-platelet-activating factor.

Recent data indicate that human spermatozoa produce platelet-activating factor as determined by the rabbit platelet [3H]serotonin release bioassay. In this report, we examined by fast atom bombardment/mass spectrometry the molecular species of platelet-activating factor generated by these germ cells. Extracted spermatozoal samples that contained platelet-activating factor bioactivity underwent straight-phase high-performance liquid chromatography, and fractions which coeluted with authentic C16- and C18-platelet-activating factor standards were subjected to fast atom bombardment/mass spectrometry. Our mass spectral data indicate that human spermatozoa synthesize C16-platelet-activating factor but not C18-platelet-activating factor.     

Occurrence of an endogenous inhibitor of platelet-activating factor in rat liver

Hepatocytes obtained from livers derived from fed rats perfused with a collagenase-containing mixture were found to contain significant levels of platelet-activating factor activity as isolated by Silica Gel G thin layer chromatography. However, when soybean trypsin inhibitor was included in the collagenase-containing perfusion medium for hepatocyte preparation, platelet-activating factor activity could not be detected on Silica Gel G chromatograms. Examination of the lipids extracted from freeze clamped perfused rat livers revealed low, but detectable, levels of platelet-activating factor. Further investigation of these observations indicated that a lipid-like inhibitor was present in freeze-clamped perfused livers as well as in hepatocytes isolated in the presence of soybean trypsin inhibitor. In each instance platelet-activating factor and this newly discovered inhibitor, which comigrated at the same RF value on Silica Gel G thin layer chromatography plates, could be separated by further chromatography on high performance thin layer plates. The present study shows that platelet-activating factor is present in unstimulated liver and that its detection is masked by an endogenous lipid-like inhibitor.     

Mechanism involved in the mobilization of neutrophil calcium by 5-hydroxyeicosatetraenoate

5-Hydroxyeicosatetraenoate (5-HETE), like leukotriene B4 and platelet-activating factor, stimulated human polymorphonuclear neutrophils to mobilize intracellular calcium. The three compounds acted through mechanisms that were inhibited by pertussis toxin, cholera toxin, and PMA. Each agonist, furthermore, desensitized (or down-regulated) the neutrophil's calcium mobilization response to a second challenge with the same agonist. However, 5-HETE and leukotriene B4 had little or no activity in cross-desensitizing neutrophil responses to each other or to platelet-activating factor. Furthermore, 5-HETE interfered minimally or not at all with the binding of radiolabeled leukotriene B4 and platelet-activating factor to their respective receptors on neutrophils. Thus, 5-HETE mobilizes neutrophil calcium by a mechanism different from those used by leukotriene B4 and platelet-activating factor. This mechanism appears to involve specific 5-HETE receptors that couple to pertussis toxin-inhibitable, GTP-binding proteins.     

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.     

Inflammation and the mechanism of action of anti-inflammatory drugs

Inflammation is caused by release of chemicals from tissues and migrating cells. Most strongly implicated are the prostaglandins (PGs), leukotrienes (LTs), histamine, bradykinin, and, more recently, platelet-activating factor (PAF) and interleukin-1. Evidence for their involvement comes from studies with competitive antagonists for their receptors and inhibitors of their synthesis. H1 histamine antagonists are effective for hay fever and some skin allergies such as urticaria, which indicates the importance of histamine in these conditions. Symptoms of rheumatoid arthritis are alleviated by the aspirinlike anti-inflammatory drugs, which inhibit the cyclo-oxygenase enzyme and reduce synthesis of prostanoids. Corticosteroids prevent the formation of both PGs and LTs by causing the release of lipocortin, which by inhibition of phospholipase A2 reduces arachidonic acid release. They suppress the inflammation of rheumatoid arthritis and asthma. Currently, high doses of nonsedating H1 antihistamines and PAF antagonists are being tested for the treatment of allergic asthma.     

Effects of the platelet-activating factor antagonist BN 52021 on the hemodynamics of rats with experimental cirrhosis of the liver

Previous studies from this laboratory have shown that rats with experimental cirrhosis of the liver induced by the combined administration of oral phenobarbital and inhaled carbon tetrachloride show an hyperdynamic status with enhanced cardiac output (CO), and decreased mean arterial pressure (MAP) and peripheral vascular resistance (PVR). Cirrhotic rats also showed an increased vascular permeability. All these phenomena are similar to some of the known effects of the systemic infusion of low doses of synthetic platelet-activating factor into the systemic circulation of normal rats. The measurement of the levels of platelet-activating factor in samples of blood demonstrated significantly higher levels in cirrhotic (2.65 +/- 0.39; n = 10) than in control rats (1.50 +/- 0.57 ng/ml; n = 10; p less than 0.05). The hemodynamic changes induced by the intravenous injection of the platelet-activating factor receptor antagonist BN 52021 (5 mg/kg body weight) have been measured in 10 control and 10 cirrhotic male Wistar rats, using a radioactive microsphere technique. BN 52021 induced no significant hemodynamic changes in control animals. However, in cirrhotic animals it induced a significant decrease in CO with increase in PVR. MAP increased slightly but not significantly. From these data it can be deduced that platelet-activating factor plays a role in the hemodynamic derangement shown by cirrhotic rats and that these derangement can be reversed by BN 52021, a highly selective antagonist of the platelet-activating factor receptor.     

Effect of platelet-activating factor on arachidonic acid metabolism in renal epithelial cells

We studied the effects of platelet-activating factor (PAF-acether) on phospholipase activity in renal epithelial cells. When platelet-activating factor was added to renal cells prelabeled with [3H]arachidonic acid, it induced the rapid hydrolysis of phospholipids. Up to 26% of incorporated [3H]arachidonic acid was released into the medium from renal cells. After the addition of PAF-acether, the degradation of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were observed. The amount of [3H]arachidonic acid released were comparable to the losses of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. In renal cells biosynthetically labeled by incorporation of [3H]choline into cellular phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, the range of concentrations of PAF-acether-induced hydrolysis of labeled phosphatidylcholine were approximately equal to the amounts of lysophosphatidylcholine produced. We also observed a transient rise of diacylglycerol after the addition of platelet-activating factor to these cells. To test for action of phospholipase C, the accumulations of [3H]choline, [3H]inositol and [3H]ethanolamine were determined. The radioactivities in choline and ethanolamine showed little or no change. An increase in inositol was detectable within 1 min and it peaked at 3 min. These results indicate that platelet-activating factor stimulates phospholipase A2 and phosphatidylinositol-specific phospholipase C activity in renal epithelial cells. These phospholipase activities were Ca2+ dependent. Moreover, PAF-acether enhanced changes in cell-associated Ca2+. These results suggest that the increased Ca2+ permeability of cell membrane stimulates phospholipases A2 and C in renal epithelial cells. Prostaglandin biosynthesis was also enhanced in these cells by platelet-activating factor.     

Human eosinophil, but not neutrophil, adherence to IL-1-stimulated human umbilical vascular endothelial cells is alpha 4 beta 1 (very late antigen-4) dependent.

Eosinophils, through their ability to generate an array of potent mediators, are thought to be the major effector cells in a number of conditions, including parasitic infection, asthma, and other allergic diseases. The mechanism(s) by which eosinophils, as opposed to neutrophils, accumulate at inflammatory sites is unknown. One possible mechanism would be an eosinophil-specific pathway of adhesion to vascular endothelium. In this study we have demonstrated that human eosinophils, but not neutrophils, constitutively express alpha 4 beta 1 (CD49d/CD29). Expression was not increased on low density eosinophils or normal density cells stimulated with platelet-activating factor. Eosinophils, but not neutrophils, specifically adhered to COS cells transfected with vascular adhesion molecule-1 in a alpha 4 beta 1-dependent manner. Eosinophil, but not neutrophil, adhesion to IL-1 stimulated human umbilical vascular endothelial cells was significantly inhibited by alpha 4 beta 1 mAb at both 5 h (p less than 0.05) and 20 h (p less than 0.001). Inhibition of both resting and platelet-activating factor-(10(-7) M) stimulated eosinophil adhesion was observed. We conclude that the alpha 4 beta 1/vascular adhesion molecule-1 adhesion pathway may be involved in specific eosinophil, as opposed to neutrophil, migration into sites of eosinophilic inflammation.     

Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor.

Although platelet-activating factor (PAF) has been shown to exert pleiotropic effects on isolated cells or tissues, controversy still exists as to whether it plays significant pathophysiological roles in vivo. To answer this question, we established transgenic mice over-expressing a guinea-pig PAF receptor (PAFR). The transgenic mice showed a bronchial hyperreactivity to methacholine and an increased mortality when exposed to bacterial endotoxin. An aberrant melanogenesis and proliferative abnormalities in the skin were also observed in the transgenic mice, some of which spontaneously bore melanocytic tumors in the dermis after aging. Thus, PAFR transgenic mice proved to be a useful model for studying the basic pathophysiology of bronchial asthma and endotoxin-induced death, and screening of therapeutics for these disorders. Furthermore, our findings provide new insights regarding the role of PAF in the morphogenesis of dermal tissues as well as the mitogenic activity of PAF and PAFR in vivo.     

The priming of neutrophils by lipopolysaccharide for production of intracellular platelet-activating factor. Potential role in mediation of enhanced superoxide secretion

LPS priming of the neutrophil results in enhanced release of superoxide upon subsequent stimulation, but the mechanism of this effect remains obscure. The recent recognition that neutrophils synthesize and retain platelet-activating factor within the cell led us to hypothesize that enhanced synthesis of platelet-activating factor in the LPS-primed cell might account for the observed effects of lipopolysaccharide. Using human neutrophils isolated on plasma-Percoll gradients, we found that incubation with 100 ng/ml LPS for 60 min resulted in a small but significant increase in intracellular platelet-activating factor assessed after lipid extraction, TLC, and bioassay. The further stimulation of primed neutrophils with FMLP resulted in a marked increase in neutrophil platelet-activating factor compared with non-LPS-treated controls. The priming effect of LPS was time dependent (30 to 60 min), dose dependent, and inhibited at 0 degree C and did not require protein synthesis. Platelet-activating factor so generated was not released but rather retained within the neutrophil, and the molecular species of platelet-activating factor produced was predominantly 1-O-hexadecyl-2-acetyl-sn-3-phosphorylcholine. Platelet-activating factor production in LPS-treated neutrophils was also enhanced by PMA, suggesting that receptor-mediated events could not account exclusively for the enhancement. Considering the ability of nanomolar concentrations of exogenously added platelet-activating factor to prime the neutrophil for enhanced release of superoxide, the rapid intracellular accumulation of platelet-activating factor that accompanies stimulation of an LPS-primed cell by FMLP may modulate the secretory events that accompany such stimulation.     

Platelet-activating factor and leukotriene biosynthesis is inhibited in polymorphonuclear leukocytes depleted of arachidonic acid

Rat peripheral or elicited polymorphonuclear leukocytes 90% deficient in arachidonic acid incorporate, after stimulation with the calcium ionophore A23187, 86% less acetate into platelet-activating factor than control. The total amount of platelet-activating factor in the ionophore stimulated elicited polymorphonuclear leukocytes deficient in arachidonate, measured by gas chromatography-negative ion chemical ionization mass spectrometry, was 84% less than that of control. The mass spectrometry also revealed the presence of various molecular species of platelet-activating factor ranging from 1-O-tetradecyl to 1-O-nonadecyl forms in both the deficient and control cells. However, the 1-O-hexadecyl was the predominate molecular species representing 79 and 96% of the total platelet-activating factor in the respective deficient and control cells. Other molecular species were less than 1.5 and 8.5% of the total for control and deficient polymorphonuclear leukocytes, respectively. Leukotriene B4 formation was also inhibited by 90% in the deficient cells. Both platelet-activating factor and leukotriene B4 biosynthesis could be partially restored in arachidonic acid-deficient cells by prelabeling the cells with arachidonate. This represents the first dietary link with platelet-activating factor biosynthesis.     

Recombinant human platelet-activating factor-acetylhydrolase inhibits airway inflammation and hyperreactivity in mouse asthma model

Numerous in vitro and in vivo studies in both animal models and human asthmatics have implicated platelet-activating factor (PAF) as an important inflammatory mediator in asthma. In a murine asthma model, we examined the anti-inflammatory activities of recombinant human PAF-acetylhydrolase (rPAF-AH), which converts PAF to biologically inactive lyso-PAF. In this model, mice sensitized to OVA by i.p. and intranasal (i.n.) routes are challenged with the allergen by i.n. administration. The OVA challenge elicits an eosinophil infiltration into the lungs with widespread mucus occlusion of the airways and results in bronchial hyperreactivity. The administration of rPAF-AH had a marked effect on late-phase pulmonary inflammation, which included a significant reduction in airway eosinophil infiltration, mucus hypersecretion, and airway hyperreactivity in response to methacholine challenge. These studies demonstrate that elevating plasma levels of PAF-AH through the administration of rPAF-AH is effective in blocking the late-phase pulmonary inflammation that occurs in this murine allergen-challenge asthma model. These results suggest that rPAF-AH may have therapeutic effects in patients with allergic airway inflammation.     

A superfusion bioassay for platelet-activating factor

A superfusion bioassay for platelet-activating factor is described using various types of tissues. By washing the tissue with 0.1-0.5% bovine serum albumin for 2-3 min after each addition of platelet-activating factor, desensitization did not develop in most tissues studied. Because of the ability to apply a sample directly onto an assay tissue with negligible dilution, this bioassay can detect smaller amounts of platelet-activating factor than those previously reported in which an organ bath was utilized. The ascending colon of the rat and dog appeared to be the most sensitive of the tissues tested, with a limited of detectability in the range of 100-500 fg. Repeated additions of platelet-activating factor could be made for up to 4 h without desensitization. Release of platelet-activating factor from samples of rat stomach was measured using the superfusion bioassay and a platelet aggregation bioassay. There was a significant correlation (r = 0.96; p less than 0.01) between the values obtained using the two assay systems. Thus, the sensitivity, the reproducibility, and the inexpensive nature of this bioassay make it an attractive alternative to existing bioassays for platelet-activating factor.     

The disabled dendritic cell.

Dendritic cells are important antigen-presenting cells of the immune system that induce and modulate immune responses. They interact with T and B lymphocytes as well as with natural killer cells to promote activation and differentiation of these cells. Dendritic cells generated in vitro from monocytes by use of the cytokines GM-CSF and IL-4 are increasingly used clinically to enhance antitumor immunity in cancer patients. However, recent studies revealed that the functional repertoire of monocyte-derived dendritic cells may be incomplete. Important functions of monocyte-derived dendritic cells such as migration or the ability to induce natural killer cell activation or type 2 T helper cell differentiation appear to be impaired. We propose that all these deficiencies relate to a single biochemical deficiency of monocyte-derived dendritic cells. IL-4, which is used to generate monocyte-derived dendritic cells, suppresses phospholipase A2, the enzyme that liberates arachidonic acid from membrane phospholipids and contributes to the synthesis of platelet-activating factor. Monocyte-derived dendritic cells must therefore fail to generate platelet-activating factor as well as arachidonic acid derivatives such as prostaglandins, leukotrienes, and lipoxins, collectively referred to as eicosanoids. Since eicosanoids and platelet-activating factor are known to play an important role in processes such as leukocyte migration, natural killer cell activation, and type 2 T helper cell differentiation, the deficiency in eicosanoid and platelet-activating factor biosynthesis may be responsible for the observed handicaps of monocyte-derived dendritic cells.     

The Ile198Thr and Ala379Val variants of plasmatic PAF-acetylhydrolase impair catalytical activities and are associated with atopy and asthma

The platelet-activating factor (PAF) represents a phospholipid with complex biological functions, including involvement in inflammatory processes. The degrading enzyme PAF acetylhydrolase (PAFAH) represents a candidate for asthma and other atopic diseases. Two loss-of-function mutations of PAFAH are associated with severe asthma in Japanese individuals. Our aim was to look for further PAFAH variants in white populations, their possible association with atopic and asthmatic phenotypes, and their functional importance. We picked up three common variants in the PAFAH gene: Arg92His (exon 4), Ile198Thr (exon 7), and Ala379Val (exon 11). The known loss-of-function mutations were not seen. The variant allele Thr198 was found to be highly associated with total IgE concentrations in an atopic population (P=.009) and with "atopic asthma" in an asthmatic population (P=.008). The variant allele Val379 was found to be highly associated with "specific sensitization" in the atopic population (P=.002) and with "asthma" in the asthmatic population (P=.003). By use of recombinant PAFAH enzymes, the variant Val379 showed increased (14 microM) and Thr198 markedly increased (42 microM) KM values compared to the wild type (7 microM); furthermore, Vmax of Val379 was highly increased (132%). Thr198 and Val379 influence plasmatic PAFAH toward lower substrate affinities and therefore are very likely to prolong the activities of PAF. At the same time, they are associated with an increased risk to develop asthma and atopy. Thus, two PAFAH variants seem to play a key role in atopic and asthmatic processes in Caucasian populations.     

Inhibitory cytokines in asthma

Although research in asthma has concentrated on complex proinflammatory mechanisms, it is likely that defective expression of cytokines that inhibit allergic inflammation, such as interleukin 10, interleukin 12 and interferon γ, might also be important, particularly in determining disease severity and persistence of inflammation in the airways. Therapy based on these cytokines might also be useful, with the advantage that it restores the balance of endogenous cytokines. We discuss the therapeutic potential of these and other inhibitory cytokines in the treatment of asthma, particularly in patients with severe disease that is poorly responsive to conventional therapy, or as a disease-modifying treatment when used early.     

Calcium-ionophore-induced formation of platelet-activating factor and leukotrienes by horse eosinophils: a comparative study

Horse eosinophils preincubated with 3H-labelled acetate and stimulated with the Ca2+ ionophores ionomycin or A23187 form a radioactive compound, which we have shown to be 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine (platelet-activating factor). We could detect no 1-O-acyl-2-[3H]acetyl-glycero-3-phosphocholine in the radioactive fraction. The formation of platelet-activating factor was strongly correlated to the generation of leukotriene C4, the main arachidonate metabolite in horse eosinophils, suggesting that platelet-activating factor and leukotriene C4 have a common precursor pool (1-O-alkyl-2-arachidonyl-glycero-3-phosphocholine) and a common regulation of synthesis. Even though both ionomycin and A23187 are described as Ca2+ ionophores, they have a series of significantly different effects on the eosinophils with respect to formation of platelet-activating factor and leukotriene C4. While A23187 induces an asymptotic maximum of mediator formation at concentrations higher than 20 microM, ionomycin expressed a narrow optimum at 2 microM. The effects of exogenous pH on the release of mediators also differ strongly between the two ionophores: for A23187 the effects are the same for both mediators but when ionomycin is used as stimulant, generation of platelet-activating factor and leukotriene C4 are affected differently.