Exogenous Platelet-activating Factor Research Articles

Effect of platelet-activating factor-receptor antagonism on endotoxin-induced lung dysfunction in sheep.

To further define the role of platelet-activating factor (PAF) in endotoxin-induced lung dysfunction, we examined the effect of ABT-299, a specific and potent PAF-receptor antagonist, on the response to endotoxemia in six chronically instrumented awake sheep. We administered Escherichia coli endotoxin (0.5 microg/kg) intravenously with or without pretreatment with ABT-299 while monitoring mean pulmonary arterial pressure (Ppa), mean systemic arterial pressure (Psa), dynamic compliance of the lungs (Cdyn), and functional residual capacity (FRC). Endotoxin administration caused pulmonary hypertension, reduced Cdyn, leukopenia, and hypoxemia while having no significant effect on Psa or FRC. Administration of ABT-299 did not affect any of the measured variables at baseline. Pretreatment with ABT-299 attenuated the peak Ppa seen after endotoxin administration but had minimal effects on endotoxin-induced changes in Cdyn, white blood cell count, or alveolar-to-arterial oxygen difference. ABT-299 was shown to completely block the pulmonary hypertension and reduction in Cdyn seen after intravenous administration of exogenous PAF. We conclude that PAF does not play an essential role in the sheep's response to endotoxin.

PAF levels in saliva are regulated by inflammatory cells

Platelet activating factor (PAF), a powerful inflammatory phospholipid mediator, has been detected in normal human saliva and found to be increased in periodontitis. The cellular source of PAF in saliva is controversial although several data suggest an origin related to the presence of inflammatory cells. PAF levels in biological fluids are regulated by PAF‐producing cells and by the PAFdegrading acetylhydrolase. Although in normal human saliva acetylhydrolase activity is very low, no information is available on the levels of this enzyme in inflammatory conditions of the mouth. The aim of our study was to assess the contribution of inflammatory cells to the levels of PAF in saliva in normal subjects and in patients with periodontitis. PAF was measured by radioimmunoassay (RIA) in mixed uncentrifuged saliva and in cell‐free saliva from healthy subjects, before and after tooth brushing, and in patients with periodontitis. In healthy subjects PAF levels were significantly higher in whole saliva than in centifuged saliva (1.51 ±0.22 vs. 0.92±0.04 ng/ml, p<0.0039). A significant increase in the amount of PAF was detected in whole saliva, but not in centifuged saliva, 2 h after tooth brushing. In patients with periodontitis PAF levels were not different from those of healthy individuals when using centrifuged saliva but were significantly higher when using whole, uncentrifuged saliva. Exogenous radiolabelled PAF was degraded much more rapidly by the saliva of periodontitis patients than by that of normal subjects. In conclusion, our study shows that inflammatory cells regulate the levels of PAF in saliva contributing to its production and degradation. The differential degradation of PAF in normal and inflammatory saliva highlights the absolute need of a series of methodological precautions when performing studies on salivary PAF.

Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.

Apoptosis in vivo is followed almost inevitably by rapid uptake into adjacent phagocytic cells, a critical process in tissue remodeling, regulation of the immune response, or resolution of inflammation. Phagocytosis of apoptotic cells by macrophages has been suggested to be a quiet process that does not lead to production of inflammatory mediators. Here we show that phagocytosis of apoptotic neutrophils (in contrast to immunoglobulin G-opsonized apoptotic cells) actively inhibited the production of interleukin (IL)-1beta, IL-8, IL-10, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-alpha, as well as leukotriene C4 and thromboxane B2, by human monocyte-derived macrophages. In contrast, production of transforming growth factor (TGF)-beta1, prostaglandin E2, and platelet-activating factor (PAF) was increased. The latter appeared to be involved in the inhibition of proinflammatory cytokine production because addition of exogenous TGF-beta1, prostaglandin E2, or PAF resulted in inhibition of lipopolysaccharide-stimulated cytokine production. Furthermore, anti-TGF-beta antibody, indomethacin, or PAF receptor antagonists restored cytokine production in lipopolysaccharide-stimulated macrophages that had phagocytosed apoptotic cells. These results suggest that binding and/or phagocytosis of apoptotic cells induces active antiinflammatory or suppressive properties in human macrophages. Therefore, it is likely that resolution of inflammation depends not only on the removal of apoptotic cells but on active suppression of inflammatory mediator production. Disorders in either could result in chronic inflammatory diseases.

Effect of platelet-activating factor on the growth of human erythroid and myeloid CD34+ progenitors.

We have assessed the effect of platelet-activating factor (PAF), a biologically active phospholipid present in the human marrow, on the growth of human marrow and blood CD34+ progenitors. While the metabolization rate of PAF by CD34+ cells is low (weak acetylhydrolase and acylation processes) it is readily catabolized by the acetylhydrolase activity present in the growth medium (10% fetal calf serum + 10% 5637-conditioned medium). Treatment of marrow CD34+ cells with the non-metabolizable PAF agonist C-PAF (1 nM to 100 nM) immediately before semi-solid culture significantly (P < 0.01) decreased the number of BFU-E but not of CFU-GM colonies. Treatment of marrow or blood CD34+ cells with C-PAF (10-100 nM) for 3 days in liquid medium before semi-solid culture significantly (P < 0.01) decreased the number of BFU-E and CFU-GM colonies. Treatment of blood CD34+ cells with the two PAF receptor antagonists CV 3988 and BN 52021 (1 microM) had no significant effect on the number of BFU-E and CFU-GM colonies suggesting no role of endogenous PAF in these processes. These results show that exogenous PAF downregulates human erythropoiesis and myelopoiesis, a result that might be of importance during inflammatory states.

Relevance of platelet-activating factor in inflammation and sepsis: Mechanisms and kinetics of removal in extracorporeal treatments

Sepsis can be considered a systemic inflammatory response syndrome (SIRS) caused by infection. When an excessive and/or persistent activation of humoral and cellular mechanisms of host defense is present, an exaggerated and generalized activation of inflammatory mechanisms can lead to a multiple organ dysfunction syndrome. Mediators thought to be involved in this syndrome include the major plasma cascade systems (complement, coagulation, and fibrinolytic systems) and soluble cell-derived mediators (cytokines, reactive oxygen species, platelet-activating factor (PAF), arachidonic acid metabolites, and nitric oxide and related compounds). Several findings indicate that among these mediators, PAF may exert an important role in the pathophysiology of septic shock. Evidence is accumulating that in human sepsis this scenario is far more complicated and that removal of inflammatory mediator excess from plasma, rather than blockade of their potentially beneficial local production, might provide a rationale for the use of continuous renal replacement therapy (CRRT). There is an emerging view that CRRT should be considered in the light of broader concept (ie, the use of blood purification for the treatment of sepsis). Recent studies, performed in an experimental model of continuous arteriovenous hemofiltration with exogenous PAF, demonstrated that polysulfone membranes can adsorb substantial amounts of biologically active PAF. These studies also showed that the removal of this mediator occurs by a two-step process involving early adsorption followed by ultrafiltration. Although the removal of cytokines, such as tumor necrosis factor-α (TNF-α), remains controversial, mainly because of differences in membrane used, operational conditions, and inter- and intra-assay variability, the crucial point is that no evidence has yet been given to show real benefit from CRRT in significantly reducing the plasma concentration of cytokines. The net advantage of CRRT, however, may not only be the removal of cytokines per se, but also the simultaneous elimination of cytokine-inducing substances. Experimental and human studies will be discussed as to whether extracorporeal treatments may remove an excess of circulating cytokines, either by increasing the turnover rate (the so-called high-volume hemofiltration), or by using sorbent systems to regenerate plasma filtrate.

A protective role of platelet-activating factor in murine candidiasis.

Platelet-activating factor (PAF) is a potent phospholipid-derived modulator of immunological and inflammatory processes. In this study, the role of exogenous and endogenous PAF in resistance to infection with Candida albicans was investigated. Administration of PAF following a lethal challenge of C. albicans significantly protected mice from death and reduced the number of organisms in the kidneys. Neutralization of endogenous PAF with the PAF antagonist BN50739 shortened the mean survival time and increased the number of C. albicans cells per kidney. Shortly after infection of mice (30 min), significant levels of PAF were detected in the serum. PAF-induced protection appears to be mediated through the actions of tumor necrosis factor alpha (TNF-alpha), since pretreatment with anti-TNF-alpha before each injection of PAF abrogated the majority of PAF-induced enhanced resistance. Administration of PAF in vivo elevated serum TNF-alpha levels and TNF-alpha mRNA expression in the kidney. Production of TNF-alpha was markedly diminished by pretreatment with the PAF antagonist BN50739 prior to infection with C. albicans. We conclude that PAF, which is produced during infection with C. albicans, plays an important role in determining the level of resistance to this infectious microorganism. This effect of PAF appears to be mediated, at least in part, through the induction of TNF-alpha.

Involvement of platelet-activating factor (PAF) in glutamate neurotoxicity in rat neuronal cultures

To clarify the role of platelet-activating factor (PAF) in glutamate neurotoxicity, in vitro experiments using primary neuronal cultures were performed. The anti-PAF immunoglobulin-G (aPAF-IgG) and the three PAF receptor antagonists (BN52021, CV6209, and E5880) were tested for their neuroprotective activity in primary neuronal cultures isolated from embryonic rat cerebral cortex. The cultured neurons were exposed to glutamate (1 mM) for 60 min. Twenty-four hours after this exposure, aPAF-IgG demonstrated evidence of protective effects against neuronal damage in a dose-dependent manner. Protective effects also were observed in cultures treated with the three PAF antagonists ( P<0.05 at 1 μg/ml aPAF-IgG, P<0.01 at 100 μM BN52021, P<0.05 at 10 nM CV6209 and P<0.01 at 10 nM E5880). The Fura-2 assay was used to estimate whether low dosages of exogenous PAF affect cultured neurons. The cultured neurons were loaded with Fura-2/AM. After preincubation for 120 min, the Fura-2-loaded neurons were exposed to various concentrations of PAF for 60 min. By measuring the fluorescent intensity of the medium as representing the amount of Fura-2 released from damaged neurons, we detected an increased release of Fura-2, even at low doses of PAF ( P<0.01 at 10 nM PAF). We further studied PAF production by neurons in response to glutamate. The level of PAF measured in the medium exposed to glutamate was significantly higher than the level in the medium unexposed to glutamate ( P<0.05). Our results suggest an important role of PAF in glutamate neurotoxicity. © 1997 Elsevier Science B.V. All rights reserved.

Deficiency of plasma platelet-activating factor acetylhydrolase: roles of blood cells.

Platelet-activating factor (PAF), a potent mediator of inflammation and circulatory shock, is inactivated by the enzyme PAF acetylhydrolase. Plasma PAF acetylhydrolase deficiency occurs even in healthy subjects. We hypothesized that erythrocyte PAF acetylhydrolase could play a supplementary role in this plasma acetylhydrolase deficiency. We examined 1,030 subjects who participated in mass checkups, and assayed plasma and erythrocyte PAF acetylhydrolase. We also investigated the degradation of exogenous PAF by erythrocytes or other blood cells obtained from subjects who exhibited the plasma enzyme deficiency. The incidence of the plasma enzyme deficiency in this general Japanese population was 4.7% (48/1,030). None of the subjects with the deficiency had a history of allergy, circulatory shock, or chronic inflammatory diseases. The mean values for erythrocyte cytosolic PAF acetylhydrolase activity in the normal and deficient subjects were 0.51 +/- 0.15 (SD) and 0.71 +/- 0.28 nkat (nmol/s)/g protein, respectively, and the difference was significant (P < 0.001, Mann-Whitney U-test). The half-life of 10 nmol/l [3H]PAF in plasma from normal subjects was about 5 min, and the half-life in whole blood or erythrocyte suspension in autologous plasma was almost the same as that in plasma. In plasma from deficient subjects, unchanged PAF virtually remained and the degradation in whole blood or erythrocyte suspension was a little faster than in plasma. We conclude that erythrocytes contribute only little to PAF metabolism in normal blood but they account for almost all of the slow PAF degradation in blood from subjects deficient in plasma PAF acetylhydrolase.

Deficiency of plasma platelet‐activating factor acetylhydrolase: Roles of blood cells

Platelet-activating factor (PAF), a potent mediator of inflammation and circulatory shock, is inactivated by the enzyme PAF acetylhydrolase. Plasma PAF acetylhydrolase deficiency occurs even in healthy subjects. We hypothesized that erythrocyte PAF acetylhydrolase could play a supplementary role in this plasma acetylhydrolase deficiency. We examined 1,030 subjects who participated in mass checkups, and assayed plasma and erythrocyte PAF acetylhydrolase. We also investigated the degradation of exogenous PAF by erythrocytes or other blood cells obtained from subjects who exhibited the plasma enzyme deficiency. The incidence of the plasma enzyme deficiency in this general Japanese population was 4.7% (48/1,030). None of the subjects with the deficiency had a history of allergy, circulatory shock, or chronic inflammatory diseases. The mean values for erythrocyte cytosolic PAF acetylhydrolase activity in the normal and deficient subjects were 0.51 ± 0.15 (SD) and 0.71 ± 0.28 nkat (nmol/s)/g protein, respectively, and the difference was significant (P < 0.001, Mann-Whitney U-test). The half-life of 10 nmol/l [3H]PAF in plasma from normal subjects was about 5 min, and the half-life in whole blood or erythrocyte suspension in autologous plasma was almost the same as that in plasma. In plasma from deficient subjects, unchanged PAF virtually remained and the degradation in whole blood or erythrocyte suspension was a little faster than in plasma. We conclude that erythrocytes contribute only little to PAF metabolism in normal blood but they account for almost all of the slow PAF degradation in blood from subjects deficient in plasma PAF acetylhydrolase. © 1996 Wiley-Liss, Inc.

Platelet-activating factor augments lipopolysaccharide-induced nitric oxide formation by rat Kupffer cells.

Acute endotoxic shock is accompanied by an increase in the production of nitric oxide (NO) by several different hepatic cell types. Platelet-activating factor (PAF) is a potent proinflammatory mediator with many pathophysiological actions and, in fact, elevated plasma and tissue levels of PAF are observed in animal models of endotoxic shock. The current study demonstrates that PAF induced nitrite formation, the end product of nitric oxide synthesis, by Kupffer cells in a dose- and time-dependent manner. Moreover, PAF was seen to initiate NO synthase gene expression and protein synthesis. PAF augmented lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase messenger RNA (mRNA), protein, nitrite and cyclic guanosine monophosphate (cGMP) levels in Kupffer cells. Treatment of Kupffer cells with actinomycin D or cycloheximide inhibited PAF- and LPS-stimulated nitrite and nitric oxide synthase protein formation confirming that de novo synthesis of the enzyme occurred. In Kupffer cells, the presence of an arginine analog, NG-methyl-L-arginine, attenuated nitrite formation induced by PAF and LPS alone or in combination. L-arginine is the principal substrate for nitric oxide synthase. PAF and LPS individually and in combination induced a time-dependent uptake of L-[3H]-arginine into the Kupffer cell, and this response was sensitive to cycloheximide. The current study indicates that exogenous PAF contributes to the induction of nitric oxide synthase by LPS in cultured rat Kupffer cells.

Platelet-activating factor augments lipopolysaccharide-induced nitric oxide formation by rat Kupffer cells

Acute endotoxic shock is accompanied by an increase in the production of nitric oxide (NO) by several different hepatic cell types. Platelet-activating factor (PAF) is a potent proinflammatory mediator with many pathophysiological actions and, in fact, elevated plasma and tissue levels of PAF are observed in animal models of endotoxic shock. The current study demonstrates that PAF induced nitrite formation, the end product of nitric oxide synthesis, by Kupffer cells in a dose- and time- dependent manner. Moreover, PAF was seen to initiate NO synthase gene expression and protein synthesis. PAF augmented lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase messenger RNA (mRNA), protein, nitrite and cyclic guanosine monophosphate (cGMP) levels in Kupffer cells. Treatment of Kupffer cells with actinomycin D or cycloheximide inhibited PAF- and LPS-stimulated nitrite and nitric oxide synthase protein formation confirming that de novo synthesis of the enzyme occurred. In Kupffer cells, the presence of an arginine analog, NG-methyl-L-arginine, attenuated nitrite formation induced by PAF and LPS alone or in combination. L-arginine is the principal substrate for nitric oxide synthase. PAF and LPS individually and in combination induced a time-dependent uptake of L-[3H]-arginine into the Kupffer cell, and this response was sensitive to cycloheximide. The current study indicates that exogenous PAF contributes to the induction of nitric oxide synthase by LPS in cultured rat Kupffer cells. (Hepatology 1996 Jun;23(6):1622-30)

Attenuation of endotoxin-induced pathophysiology by a new potent PAF receptor antagonist.

The role of platelet-activating factor (PAF) as a mediator of endotoxin-induced pathophysiology has been studied in several animal models with conflicting results. We evaluated the effect of a new, potent, and specific PAF receptor antagonist, ABT-299 (Abbott Laboratories) against endotoxin (lipopolysaccharide; LPS)-induced cardiopulmonary dysfunction in a porcine model. In initial experiments, the potency of ABT-299 was confirmed in vitro by its ability to inhibit PAF-induced porcine platelet aggregation at an IC50 of .047 +/- .01 microM, and in vivo by the ability of low doses (.12 mg/kg + .03 mg/kg/h) to block the cardiopulmonary pathologic response to exogenous PAF infusion. To evaluate the effect of ABT-299 administration during endotoxemia, pigs were randomly assigned to one of three groups: controls (n = 7), LPS (n = 9), or ABT-299 + LPS (n =7). ABT-299 was given at 1.0 mg/kg from -0.5 to 0 h plus .3 mg/kg/h from 0 to 6 h. LPS was given at .5 micrograms/kg/hr from 0 to 6 h. ABT-299 reduced the early LPS-induced fall in cardiac index and stroke volume, pulmonary hypertension and vasoconstriction, bronchoconstriction, and hypoxemia. Administration of LPS resulted in 44% mortality (before 6 h), which was blocked by ABT-299. Results with this antagonist indicate that PAF contributes to endotoxin-induced cardiopulmonary dysfunction in the pig, and is associated with mortality in this model.

Interaction of Platelet-Activating Factor with Rat Hepatocytes: Uptake, Translocation, Metabolism, and Effects on PAF-Acetylhydrolase Secretion and Protein Tyrosine Phosphorylation

In the present study the interaction of the phospholipid mediator platelet-activating factor (PAF) with rat hepatocytes in primary culture was examined. Following exposure to hepatocytes, exogenous [3H]alkyl-PAF was metabolized rapidly to [3H]lyso-PAF, the content of which was raised in the outer leaflet of the plasma membrane within the initial 5 min of incubation. Thereafter [3H]lyso-PAF was translocated into cells with concomitant reacylation to [3H]alkyl-acyl-glycerophosphocholine. A portion of untransformed [3H]PAF accumulated in the outer leaflet, and only a small amount of the [3H]PAF was translocated into the inner leaflet of the plasma membrane. Detectable levels of [3H]lyso-PAF were found in the medium of hepatocyte cultures at all times of incubation. These findings suggest that at least a portion of the cellular PAF-acetylhydrolase (PAF-AH) activity is located in the outer leaflet of the plasma membrane and can be secreted into the medium. Indeed, rat hepatocytes in culture released PAF-AH into the medium in a time-dependent fashion. Incubation of hepatocytes with exogenous PAF increased secretion of PAF-AH, whereas lyso-PAF and the nonhydrolyzable analog methylcarbamyl-PAF significantly reduced secretion. The structurally related PAF receptor antagonist CV 3988 markedly inhibited the activity of PAF-AH and also diminished its release by hepatocytes. In contrast, BN 50739 and WEB 2170, thienotriazolodiazepine PAF receptor antagonists, did not affect the PAF-AH activity, but increased its secretion by the cells. A full-length 3.8-kb mRNA encoding the cell surface PAF receptor was absent in hepatocytes as indicated by Northern blot analysis using the rat PAF receptor cDNA, whereas PAF receptor mRNA was readily detected in Kupffer cells. Upon incubation with hepatocytes, PAF induced tyrosine phosphorylation of proteins with molecular masses of 120–130 and 160–180 kDa and dephosphorylation of 80- to 90-kDa proteins; these responses were not inhibited by WEB 2170 and BN 50739. The protein tyrosine kinase inhibitor genistein abolished the release of free arachidonic acid, suggesting a crucial role for tyrosine phosphorylation in PAF-induced phospholipase A2activation in rat hepatocytes. Taken together, our data indicate that the interaction of PAF with rat hepatocytes is dependent upon its metabolism, involves protein tyrosine phosphorylation/dephosphorylation and arachidonic acid release, and does not involve the heteromeric G-protein-coupled PAF receptor which has been characterized in Kupffer cells. This metabolically regulated mechanism for PAF action on hepatocytes may be of potential biological importance in the liver under normal and pathological conditions.

Microvascular mechanisms of histamine‐induced potentiation of leukocyte adhesion evoked by chemoattractants

1. Intravital microscopy of the rat mesentery was used to examine interactions between histamine and the chemoattractant leukotriene B4 (LTB4) with regard to leukocyte adhesion in postcapillary venules. 2. Topical administration of histamine caused a four fold potentiation of LTB4-induced leukocyte adhesion. 3. Histamine significantly increased the rolling leukocyte flux by 25%, and this effect of histamine on rolling was strictly blood flow-dependent, i.e. we found significant positive correlations between both blood flow and total leukocyte flux and between total and rolling leukocyte flux, while no changes in leukocyte rolling fraction or rolling velocity were observed. Furthermore, histamine caused a clear-cut increase in venular plasma protein leakage. 4. The platelet-activating factor (PAF) receptor antagonist WEB 2086, which effectively inhibited adhesion of leukocytes evoked by exogenous PAF, did not reduce the potentiating effect of histamine on LTB4-induced leukocyte adhesion. 5. The vasodilator acetylcholine (ACh) caused a moderate enhancement of LTB4 induced leukocyte adhesion in proportion to its blood flow-dependent 40% increase in rolling leukocyte flux. In contrast to histamine, ACh did not provoke vascular leakage of plasma proteins. 6. Taken together, our findings suggest that histamine plays an important pro-inflammatory role in tissues where leukocyte rolling is already present, by potentiating chemoattractant-induced firm leukocyte adhesion through a combination of microcirculatory changes such as increased rolling leukocyte flux and vascular permeability.

Differential metabolism of exogenous platelet-activating factor by glandular epithelial and stromal cells of rabbit endometrium.

Significant changes in platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) concentration have been observed in rabbit endometrium during the preimplantation period, but, under in vitro conditions, constitutive PAF biosynthesis by isolated endometrial tissues was not easily demonstrable. Relative changes in enzymes involved in the synthesis and metabolism of PAF in the tissues may account for this disparity. In addition, during this period of preimplantation, marked changes in PAF receptor concentration have been noted. The present study examines the factors that may modulate the metabolism of exogenous [3H]PAF in the endometrium of rabbits on day 6 of pregnancy. Since preferential [3H]PAF binding in situ by the glandular epithelial, but not by the stromal, cells was demonstrated, their cell-specific metabolism of exogenous [3H]PAF was also examined. After entry into the endometrial cell, [3H]PAF was rapidly metabolized by the sequential action of cytosolic Ca(2+)-independent acetylhydrolase to [3H]lyso-PAF and this was in turn acylated by membrane-associated transacylase to [3H]alkylacyl-glycerylphosphorylcholine. PAF resynthesis was not observed and, in stromal cells, there was a significant build-up of [3H]lyso-PAF, suggesting that lyso-PAF:acetyl-CoA acetyl-transferase may be a limiting factor. In the glandular epithelial cells, however, there was a significant accumulation of a neutral lipid without a significant build-up of [3H]lyso-PAF or [3H]PAF. The neutral lipid co-migrated with the product of phospholipase C-catalysed metabolism of PAF and authentic 1-O-hexadecyl-2-acetyl-glycerol. In addition, the elution times of phospholipase C digestion of C18 PAF and the neutral lipid produced by cellular metabolism of [3H]PAF, determined by gas chromatography/flame ionization detection, were similar. It seems that it is the synthesis of the neutral lipid from reacetylated [3H]lyso-PAF that prevented [3H]PAF accumulation under in vitro conditions. This is the first documentation of the synthesis of this lipid in the mammalian uterus. The lipid may serve as the precursor for de novo PAF synthesis in the glandular epithelial cells during endometrial proliferation.

Platelet-activating factor increases vascular resistance in rat hindquarters by thromboxane A2

The effects of platelet-activating factor (PAF) on vascular resistance and capillary permeability were studied in the isolated rat hindquarter. Six groups were studied (n = 30): control; PAF alone (1.4 microM); and PAF (1.4 microM) pretreated with ibuprofen (30 mg/kg), thromboxane A2 (TxA2)-receptor antagonist (BM-13505, 2 mg/kg), PAF-receptor antagonist (WEB-2086, 5 mg/kg), or dexamethasone (5 mg/kg). The vascular resistance was calculated, and the reflection coefficient (sigma) was determined as an index of capillary permeability. Exogenous PAF caused a threefold increase in vascular resistance peaking at 5 min and a 2.5-fold increase in capillary permeability. The increased vascular resistance caused by PAF alone was significantly attenuated by ibuprofen, BM-13505, and dexamethasone. The PAF-induced permeability was neither attenuated by ibuprofen nor BM-13505. However, both the increased vascular resistance and permeability were blocked and attenuated by WEB-2086 and dexamethasone, respectively. We conclude that TxA2 mediates the PAF-induced increased vascular resistance; however, the increased vascular permeability is independent of the formation of TxA2 in the isolated hindquarter.

Release of platelet activating factor (PAF) and eicosanoids in UVC-irradiated corneal stromal cells.

Ultraviolet (UV) irradiation provokes acute inflammation of the eye, and can be used to model processes that occur in response to damage to the anterior segment. This study characterized ultraviolet-C (UVC, 254 nm) irradiation-induced PAF synthesis, and arachidonic acid (20:4) and eicosanoid release in rabbit corneal stromal cells maintained in vitro. PAF was measured by radioimmunoassay (RIA) after exposing cultured corneal stromal cells to UVC irradiation (20 min, 2, 5, 10 mW/cm2). 14C-20:4-labeled stromal cells were also stimulated with UVC and radiolabeled phospholipids, neutral lipids and eicosanoids were measured. Synthesis of cell-associated and secreted PAF from corneal stromal cells was increased by UV irradiation. UV irradiation (254 nm, 5mW/cm2) enhanced 20:4 release from triacylglycerols, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine, and increased levels of 20:4-diacylglycerol and unesterified 20:4. The released 20:4 entered both the cyclooxygenase and lipoxygenase pathways after UVC irradiation. The PAF antagonist, BN52021 (10 microM) reduced UVC irradiation-induced stimulation of prostaglandin production, but failed to inhibit UVC-induced 20:4 release and synthesis of lipoxygenase products. Furthermore, exogenous PAF (1 microM) stimulated prostaglandin production, but did not increase the synthesis of lipoxygenase products from radiolabeled 20:4. The effects of PAF on prostaglandin synthesis were inhibited by BN52021. These findings indicate that responses to injury in cultured corneal stromal cells include PAF synthesis, release of 20:4 from glycerolipids, accumulation of diacylglycerol and synthesis of eicosanoids. The data further suggest that during UVC irradiation in vitro, PAF is not a primary or initial mediator of 20:4 release and synthesis of lipoxygenase products, but may mediate UVC-induced prostaglandin synthesis.

Production and metabolism of platelet-activating factor in the normal and ischemic fetal rat brain.

Production and metabolism of platelet-activating factor (PAF) in the fetal rat brain under normal and under ischemic stress conditions were examined. Endogenous PAF levels, determined by a bioassay using PAF-stimulated platelet release of [3H]serotonin, averaged 2.32 +/- 2.14 pg/mg in control brains and was reduced to 1.10 +/- 1.06 pg/mg after 20 min of maternal-fetal blood flow occlusion. [3H]PAF administered intracranially into the fetuses in utero was removed in a biphasic, time-dependent manner: a rapid component with an estimated elimination rate constant of 0.067 min-1 and t1/2 of 10 min and a slower component with an elimination rate of 0.017 min-1 and t1/2 of 41 min. In fetal brains subjected to ischemia a delayed elimination of [3H]PAF was noticed in the slow component (t1/2 = 59 min), indicating a possible difference between the clearance of exogenous and endogenous PAF. The disappearance of [3H]PAF was accompanied by an increase in the radioactivity associated with lyso-PAF that reached a plateau after 2.5 min, possibly indicating the degradation of the fast component. A steady increase in the alkyl-acyl-glycerophosphorylcholine radioactivity commenced after 5 min and continued up to 30 min. The endogenous production of PAF and the rapid degradation due to maternal-fetal blood flow occlusion indicate an additional target for therapeutic intervention in the pathology of intrauterine ischemia. Addition of the calcium ionophore A23187 stimulated in vitro formation of PAF and lyso-PAF from [3H]-choline-labeled fetal brain phospholipids, suggesting that intracellular calcium may play a major stimulatory role in PAF production.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of the PAF receptor antagonist UK‐74,505 on neutrophil and eosinophil accumulation in guinea‐pig skin

1. The effect of the dihydropyridine, platelet activating factor (PAF) receptor antagonist, UK-74,505, on leucocyte accumulation and oedema formation in guinea-pig skin was investigated. The inflammatory reactions studied were elicited by exogenous mediators, a passive cutaneous anaphylactic (PCA) reaction and zymosan particles. 2. Leucocyte accumulation and oedema formation were measured as the local accumulation of i.v. administered 111In-labelled neutrophils or eosinophils together with 125I-labelled albumin. UK-74,505 was either administered i.v. or used to pretreat the radiolabelled leucocytes in vitro prior to their last wash and injection into recipient animals. 3. In vitro, UK-74,505 inhibited PAF-induced elevations in cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2-loaded guinea-pig neutrophils and eosinophils with IC50 values of 10(-9) M and 7 x 10(-9) M respectively. Neutrophils and eosinophils pretreated with 10(-7) M and 10(-6) M UK-74,505 respectively, and maintained at 37 degrees C, were unresponsive to PAF for the 4 h period investigated. 4. In vivo, using 2 h test periods, i.v. UK-74,505 (0.5 and 2.5 mg kg-1) inhibited the accumulation of 111In-neutrophils, 111In-eosinophils and oedema formation induced by intradermal PAF, but had no effect on responses elicited by leukotriene B4 (LTB4) and zymosan-activated plasma (ZAP, used as a source of C5a des Arg). UK-74,505 (2.5 mg kg-1) was also without an effect on response induced by a PCA reaction but significantly suppressed the 111In-eosinophil accumulation following the intradermal administration of zymosan particles. The 111In-neutrophil accumulation induced by zymosan particles was not, however, affected by UK-74,505. 5. In a second series of in vivo experiments, "'In-leucocytes were pretreated in vitro with UK-74,505 prior to their last wash and injection into recipient animals. Radiolabelled neutrophils, and eosinophils were pretreated with 10-7 M and 10-6 M UK-74,505 respectively, concentrations previously shown to block the leucocyte responses to PAF in vitro for up to 4 h. The in vitro pretreatment of the cells with the PAF antagonist, whilst not affecting the responses to intradermally-injected PAF, suppressed the"'In-eosinophil accumulation response induced by zymosan particles.6. The results of this study indicate that PAF is not involved in neutrophil accumulation, eosinophil accumulation and oedema formation induced by LTB4, ZAP and a PCA reaction. Endogenous PAF does, however, appear to have a role in zymosan-induced eosinophil accumulation but not neutrophil accumulation, suggesting the existence of different inflammatory pathways in the induction of neutrophil and eosinophil accumulation in vivo. Furthermore, while leucocyte accumulation induced by exogenous PAF does not appear to involve leucocyte PAF receptors, the mechanism by which endogenous PAF mediates the zymosan-induced eosinophil accumulation appears dependent on the expression of PAF receptors on eosinophils.

Excretion of platelet activating factor–acetylhydrolase and phospholipase A2 into nasal fluids after allergenic challenge: Possible role in the regulation of platelet activating factor release

Platelet activating factor (PAF), a proinflammatory mediator synthesized through a phospholipase A2 (PLA2)-dependent reaction, is hydrolyzed into its inactive metabolite, lyso-PAF, by a specific acetylhydrolase. Previous studies have shown that allergen challenge of patients with allergic rhinitis leads to an increase of the concentrations of lyso-PAF in nasal lavage fluid (NLF), whereas PAF is detected only marginally. PAF-hydrolyzing enzymes are expected to be released on allergenic challenge, to account for the reduced concentrations of PAF in NLF. Here, we show that allergen challenge of patients with allergic rhinitis induces an increase of acetylhydrolase-like activity in NLF, which peaks within 10 minutes and returns to basal values 1 hour later. Acetylhydrolase hydrolyzed exogenous PAF with a complete loss of its ability to induce platelet aggregation. Allergen challenge also led to a parallel release of a PLA2 in nasal fluids. This enzyme preferentially hydrolyzes negatively charged phospholipids (phosphatidic acid monomethyl ester and phosphatidylgylcerol) versus phosphatidylcholine. More interestingly, the hydrolysis of phosphatidic acid monomethyl ester and phosphatidylglycerol by NLF was completely abolished by the addition of ethylenediaminetetraacetic acid which had no effect on the hydrolysis of PAF, indicating that the PLA2 secreted in nasal fluids is not involved in the degradation of PAF. Finally, our results show that allergen-induced increase in the concentrations of lyso-PAF and prostaglandin D2 occurred with a kinetic similar to that of tosyl-L-arginine-methyl-ester esterase, suggesting that mast cells are implicated in this process. Although no direct relationship was demonstrated between the absence of PAF and the increase of acetylhydrolase levels in NLF, we suggest a potential role for this enzyme in the inactivation of PAF if the latter is released in the nasal lumen. (J ALLERGY CLIN IMMUNOL 1994;94:109-19.)