Platelet-activating Factor Challenge Research Articles

A histamine-independent itch pathway is required for allergic ocular itch

Non-histaminergic TRPA1 neural pathway is required for the development of allergic ocular itch. Pharmacological inhibition of TRPA1 channel can lead to novel therapeutic strategies to treat ocular itch in severe allergic conjunctivitis.

Evaluation of nasal symptoms induced by platelet activating factor, after nasal challenge in both healthy and allergic rhinitis subjects pretreated with rupatadine, levocetirizine or placebo in a cross-over study design

BackgroundPlatelet-activating factor (PAF) is produced by most inflammatory cells and it is involved in inflammatory and allergic reactions. We aimed to assess the anti-PAF effects of rupatadine and levocetirizine in the upper airways.FindingsHealthy volunteers (HV, N = 10) and seasonal allergic rhinitis (SAR, N = 10) asymptomatic patients were treated out of the pollen season with either rupatadine 20 mg, levocetirizine 10 mg, or placebo once a day during 5 days prior to the PAF nasal challenge. Total 4-nasal symptom score (T4SS) and nasal patency (Vol2-5, by acoustic rhinometry) were assessed from 0 to 240 minutes after a repeated PAF challenge. In SAR patients but not in HV, both rupatadine and levocetirizine showed a trend to decrease PAF-induced T4SS from 60 to 120 minutes. Rupatadine but not levocetirizine caused a significant reduction (p < 0.05) of T4SS area under the curve compared to placebo. Rupatadine and levocetirizine caused no significant changes on nasal patency compared to placebo.ConclusionsThese results suggest that both rupatadine and levocetirizine showed a tendency decrease toward nasal symptoms, but only rupatadine significally reduces the overall nasal symptoms (AUC) induced by PAF in SAR patients.

Induction effect of platelet-activating factor on Src-suppressed C kinase substrate gene expression in rat pulmonary microvascular endothelial cells

To investigate the effect of platelet-activating factor (PAF) on the production of Src-suppressed C kinase substrate (SSeCKS) mRNA in rat pulmonary microvascular endothelial cell (RPMVEC) and its signal transduction pathways. Cellular in situ hybridization was performed to reveal changes in SSeCKS mRNA expression in the cultured RPMVECs after giving PAF stimulation. Normal RPMVECs expressed SSeCKS mRNA at a low level, which appeared throughout the cytoplasm with specific hybridization signals. 1.5 hours of 10(-10), 10(-9), 10(-8), 10(-7) mol/L PAF incubation induced a progressive increase in SSeCKS mRNA expression. When compared to the normal control group each difference had statistical significance (0.054 9 + or - 0.000 7, 0.059 9 + or - 0.001 8, 0.069 0 + or - 0.001 8, 0.075 4 + or - 0.001 9 vs. 0.036 8 + or - 0.003 7, respectively, all P<0.05). Within 0.5, 1.5, 3, 6, 12, 24 hours of 10(-7) mol/L PAF challenge, the level of SSeCKS mRNA expression raised at 0.5 hour markedly (0.071 0 + or - 0.001 5), peaked at 1.5 hours (0.075 6 + or - 0.001 7), then began to decline gradually, and still persisted at a higher level than the normal control group until 24 hours (0.043 9 + or - 0.001 0 vs. 0.038 2 + or - 0.004 1, all P<0.05). Pre-incubation of 10 micromol/L pyrrolidine dithiocarbamate (PDTC) that inhibits activity of nuclear factor-KappaB (NF-KappaB) in RPMVECs caused a conspicuous attenuation of PAF-induced SSeCKS mRNA expression (0.049 7 + or - 0.003 2 vs. 0.071 9 + or - 0.001 9, P<0.05), whereas no change of PAF-induced effect was found by pretreatment of protein kinase C (PKC) inhibitor bis-indolylmaleimide (BIM, 0.069 7 + or - 0.002 1 vs. 0.071 9 + or - 0.001 9, P>0.05). PAF can up regulate the expression of SSeCKS mRNA in a dose- and time-dependent manner in RPMVECs. It is NF-KappaB rather than PKC signal pathway that is involved in modulation of the intracellular signaling process.

Platelet activating factor‐induced neuronal apoptosis is initiated independently of its G‐protein coupled PAF receptor and is inhibited by the benzoate orsellinic acid

The bioactive lipid mediator platelet activating factor (PAF) is recognized as a key effecter of neuronal apoptosis, yet it is not clear whether its G-protein coupled receptor (PAFR) initiates or prevents PAF neurotoxicity. Using PAFR-/- and congenic wild-type mice, we show that PAF triggers caspase-3/7 activity and neuronal death in PAFR-/- but not PAFR+/+ cerebellar granule neurons. Restoring receptor expression by recombinant adenoviral infection protected cells from PAF challenge. Neuronal death was not mediated by nitric oxide or N-methyl-d-aspartate receptor signaling given that N-nitro-l-arginine methyl ester and MK-801 did not inhibit PAF-induced neuronal loss in PAFR-/- neurons. To intervene in PAFR-independent neurotoxicity, the anti-apoptotic actions of three structurally distinct PAF antagonists were compared to a panel of plant and fungal benzoic acid derivatives. We found that the PAF antagonist BN 52021 but not FR 49175 or CV 3988 inhibited PAFR-independent neurotoxicity. Orsellinic acid, a fungal-derived benzoic acid, blocked PAF-mediated neuronal apoptosis without affecting PAFR-mediated neuroprotection. These findings demonstrate that PAF can transduce apoptotic death in primary neurons independently of its G-protein coupled receptor, that PAFR activation is neuroprotective, and that orsellinic acid effectively attenuates PAFR-independent neuronal apoptosis.

Tetrahydrobiopterin prevents platelet-activating factor–induced intestinal hypoperfusion and necrosis: Role of neuronal nitric oxide synthase

We reported previously that neuronal nitric oxide synthase (nNOS) is the predominant NOS in rat small intestine and is down-regulated by platelet-activating factor (PAF). The severity of the bowel injury induced by PAF is inversely related to its suppressing effect on nNOS. Here, we investigated whether intestinal perfusion is regulated by nNOS and whether tetrahydrobiopterin, a co-factor and stabilizer of nNOS, reverses PAF-induced intestinal hypoperfusion and injury. Animal laboratory. We first examined nNOS regulation of splanchnic blood flow by measuring the perfusion of the heart, lung, ileum, and kidney in rats after a nNOS inhibitor. We then examined the protective effect of tetrahydrobiopterin on PAF-induced bowel injury, mesenteric hypoperfusion, and systemic inflammation. Adult male Sprague-Dawley rats. In part 1 of the experiment, rats were given 7-nitroindazole (a specific nNOS inhibitor, 50 mg.kg.day). In part 2 of the experiment, rats were treated with tetrahydrobiopterin (20 mg/kg) 5 mins before and 30 mins after PAF challenge (2.2 microg/kg, intravenously) Perfusion of the heart, lung, ileum, and kidney was measured at 1 and 4 days after 7-nitroindazole, using fluorescent microspheres. Intestinal injury and inflammation (myeloperoxidase content), blood perfusion, calcium dependent-NOS activity, and systemic inflammation (hypotension and hematocrit increase) were assessed 1 hr after PAF with and without tetrahydrobiopterin treatment. In part 1 of the experiment, 7-nitroindazole induced a long-lasting reduction of blood perfusion and inducible NOS expression selectively in the ileum but not in nonsplanchnic organs such as heart, lungs, and kidneys. In part 2, tetrahydrobiopterin protected against PAF-induced intestinal necrosis, hypoperfusion, neutrophil influx, and NOS suppression. It also reversed hypotension and hemoconcentration. Sepiapterin (2 mg/kg, stable tetrahydrobiopterin precursor) also attenuated PAF-induced intestinal injury. We conclude that nNOS selectively regulates intestinal perfusion. Tetrahydrobiopterin prevents PAF-induced intestinal injury, probably by stabilizing nNOS and maintaining intestinal perfusion.

Lack of systemic oxidative stress during PAF challenge in mild asthma

To further establish the role of oxidative stress in the pathogenesis of acute bronchial asthma, we investigated the effects of platelet-activating factor (PAF) challenge on systemic oxidant-antioxidant balance in 12 asthmatic patients (age, 25+/-3[SEM] yr; FEV1, 95+/-10% predicted), using a double blinded, controlled with Lyso-PAF (L-PAF), cross-over design. Respiratory system resistance (Rrs), arterial blood gases, peripheral blood neutrophils and oxidant-antioxidant balance, including thiobarbituric acid (TBA)-malondialdehyde (MDA) adducts, protein sulphydryls and Trolox equivalent antioxidant capacity (TEAC), were assessed at baseline and 5, 15 and 45 min after PAF and L-PAF (18 microg each) bronchoprovocation. Urinary leukotriene E4 (uLTE4) elimination was measured 120 min after challenge. Compared with baseline, as expected, PAF increased significantly Rrs and AaPO2 and decreased PaO2 and peripheral blood neutrophils along with a rebound neutrophilia and increased uLTE4. By contrast, markers of systemic oxidative stress remained unaltered throughout the study. Unlike PAF, L-PAF-induced changes were negligible. We conclude that there is no systemic oxidant-antioxidant imbalance during acute bronchoconstriction induced by PAF in these patients with mild asthma.

Ketoprofen and Phenylbutazone Attenuation of PAF-induced Lung Inflammation in Calves

The purposes of this study were: (1) to investigate which arachidonic acid metabolites contributed to platelet-activating factor (PAF) induced pulmonary dysfunction; and (2) to compare the effect of two non-steroidal anti-inflammatory drugs, phenylbutazone and ketoprofen in a model of PAF-induced reversible lung inflammation in six calves.In placebo and phenylbutazone groups, PAF infusion induced significant dysfunctions in the pattern of breathing, mechanics of breathing and gas exchange. These dysfunctions were prevented by ketoprofen pretreatment, except for the mechanics of breathing which was moderately but significantly altered by the PAF challenge. In all calves, leukotriene (LT) B4plasma concentrations did not significantly increase above baseline values at any time. Prostaglandin (PG) E2plasma concentrations showed a minor significant increase in phenylbutazone pretreated calves (55.8∓25.8pg/mL from 36.7∓16.13pg/mL). Thromboxane (TX) B2plasma concentration was significantly increased during PAF challenge in placebo- and phenylbutazone-pretreated groups, but not in ketoprofen-pretreated calves (1580.0∓1370 from 42.7∓10.7pg/mL; 2340∓477 from 63∓32pg/mL; and 36.5∓4.12 from 39.3∓12.0pg/mL, respectively).These data suggest that TXA2is an important cyclooxygenase metabolite of arachidonic acid produced in response to PAF and that ketoprofen (intramuscular injection, 3mg/kg) is more effective than phenylbutazone (intramuscular injection, 10mg/kg) in preventing respiratory dysfunctions induced by the PAF challenge 30min after drug administration. Ketoprofen did not suppress totally the PAF-induced changes in mechanics of breathing, which suggests that PAF or a secondary release of mediators could have a direct action on airway smooth muscle.

Protective effects of WEB 2086 (PAF antagonist) and ketoprofen (NSAID) on PAF-induced changes in the morphological ultrastructure of blood platelets in calves.

The ultrastructure of bovine platelets was examined by transmission electron microscopy without any pretreatment (control), and after WEB 2086 (a triazolodiazepine) or ketoprofen (NSAID) pretreatment, followed by PAF infusion. The blood platelet count was also investigated. The group of calves that received WEB 2086 pretreatment before platelet-activating factor (PAF) infusion did not show a decreased number of platelets. However, in the other group, with ketoprofen pretreatment before PAF infusion, there was a rapid decrease from 1 to 3 min, while from 5 min the number of platelets recovered to the normal value. Electron microscopy revealed that pretreatment with WEB 2086 followed by PAF infusion did not alter the morphological ultrastructure of bovine platelets, except that the microtubules were briefly modified from 1 until 3 min after PAF challenge. After ketoprofen pretreatment, bovine platelets kept their regular shape, the number of dense bodies was not significantly altered, the number of mitochondria was maintained from 5 min after PAF infusion, giant platelets were not observed and the Golgi apparatus was rarely visible. Thus pretreatment with WEB 2086 and ketoprofen before PAF infusion had a protective activity on the ultrastructure of bovine platelets and, in cattle, pretreatment with WEB 2086 and ketoprofen before PAF challenge prevented the thrombocytopenia induced by PAF.

Pharmacology of ABT-491, a highly potent platelet-activating factor receptor antagonist

ABT-491 (4-ethynyl- N, N-dimethyl-3-[3-fluoro-4-[(2-methyl-1 H-imidazo-[4,5- c]pyridin-1-yl)methyl]benzoyl]-1 H-indole-1-carboxamide hydrochloride) is a novel PAF (platelet-activating factor) receptor antagonist with a K i for inhibiting PAF binding to human platelets of 0.6 nM. Binding kinetics of ABT-491 to the PAF receptor is consistent with a relatively slow off-rate of the antagonist when compared to PAF. Inhibition of PAF binding is selective and is correlated with functional antagonism of PAF-mediated cellular responses (Ca 2+ mobilization, priming, and degranulation). Administration of ABT-491 in vivo leads to potent inhibition of PAF-induced inflammatory responses (increased vascular permeability, hypotension, and edema) and PAF-induced lethality. Oral potency (ED 50) was between 0.03 and 0.4 mg/kg in rat, mouse, and guinea-pig. When administered intravenously in these species, ABT-491 exhibited ED 50 values between 0.005 and 0.016 mg/kg. An oral dose of 0.5 mg/kg in rat provided >50% protection for 8 h against cutaneous PAF challenge. ABT-491 administered orally was also effective in inhibiting lipopolysaccharide-induced hypotension (ED 50=0.04 mg/kg), gastrointestinal damage (0.05 mg/kg, 79% inhibition), and lethality (1 mg/kg, 85% vs. 57% survival). The potency of this novel antagonist suggests that ABT-491 will be useful in the treatment of PAF-mediated diseases. © 1997 Elsevier Science B.V.

Effects of aminoguanidine on systemic inflammatory response syndrome induced by platelet activating factor and by lipopolysaccharide in rats

We investigated the effects of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide (NO) synthase, on the systemic inflammatory response syndrome induced by platelet activating factor (PAF) and by lipopolysaccharide in rats, with emphasis on NO production in vivo. Aminoguanidine treatment improved survival rates after lipopolysaccharide challenge, whereas it aggravated the lethality caused by PAF. Lipopolysaccharide induced a marked increase in the concentrations of nitrate and nitrite in plasma compared with vehicle administration, and the increase was prevented by aminoguanidine. In contrast, PAF challenge with or without aminoguanidine did not affect the concentrations of nitrate and nitrite in plasma compared with vehicle administration. These results suggest that NO derived from inducible NO synthase is not a major participant in the systemic inflammatory response syndrome induced by PAF. Aminoguanidine is not likely to provide beneficial effects in conditions where PAF is produced and the concentrations of nitrate and nitrite in plasma are not significantly increased. © 1997 Elsevier Science B.V.

Role of superoxide and nitric oxide in platelet-activating factor-induced acute lung injury, hypotension, and mortality in rats.

To investigate the role of superoxide and nitric oxide in platelet-activating factor-induced acute lung injury, hypotension, and mortality. Prospective, randomized, controlled, experimental study. University research laboratory. Anesthetized male Wistar rats (180 to 220 g) were studied. In the first set of experiments, animals were divided into three groups. Group 1 received platelet-activating factor (2 microg/kg i.v.). Group 2 received recombinant human superoxide dismutase (50,000 U/kg i.v.) 30 mins before platelet-activating factor injection. Group 3 received vehicle agents. In the second set of experiments, animals were divided into six groups that received N(G)-nitro-L-arginine (L-NNA), a selective inhibitor of nitric oxide synthesis, or L-arginine, the physiologic precursor of nitric oxide synthesis: a) vehicles (i.v.); b) vehicle plus L-arginine (100 mg/kg i.v.); c) vehicle plus L-NNA (10 mg/kg i.v.); d) vehicle plus platelet-activating factor (2 microg/kg i.v.); e) L-arginine plus platelet-activating factor; and f) L-NNA plus platelet-activating factor. The first intravenous administration was given 5 mins before the second intravenous injection for each group. In the first set of experiments, vascular labeling with Monastral blue B demonstrated diffuse microvascular injury in the alveolar capillary beds 2 hrs after platelet-activating factor challenge. Thiobarbituric acid-reactive substances in the lung significantly increased at 2 hrs after platelet-activating factor injection. Platelet-activating factor treatment also resulted in an increased concentration of total protein, albumin, and Evans blue dye in bronchoalveolar lavage fluid at 2 hrs after administration, suggesting platelet-activating factor induction of increased alveolar permeability. The platelet-activating factor-induced alveolar microvascular injury, lipid peroxidation, and increased alveolar permeability were inhibited by pretreatment with recombinant human superoxide dismutase. Although L-NNA alone did not affect alveolar permeability in the second set of experiments, L-NNA treatment before platelet-activating factor challenge significantly aggravated platelet-activating factor-induced increased alveolar permeability 2 hrs after platelet-activating factor challenge. Platelet-activating factor also produced a rapid decrease in blood pressure that was not ameliorated by treatment with L-NNA. However, L-NNA pretreatment was associated with a significant increase in platelet-activating factor-caused mortality within 6 hrs. All rats survived with L-arginine treatment before platelet-activating factor challenge. L-NNA treatment decreased nitrate/nitrite concentration, an index of total nitric oxide production, in plasma. These results indicate that superoxide, the derived active oxygen species, and lipid peroxidation are implicated in the pathogenesis of platelet-activating factor-induced acute lung injury. Nitric oxide does not play a major role in platelet-activating factor-induced hypotension. Nitric oxide appears to play a protective role in the acute lung injury and mortality induced by platelet-activating factor.

Nasal neutrophilia and release of myeloperoxidase induced by nasal challenge with platelet activating factor: Different degrees of responsiveness in atopic and nonatopic subjects

Nasal challenge with platelet activating factor (PAF) is able to induce local neutrophilia, with a different degree of responsiveness in atopic subjects and in nonatopic subjects. We investigated whether nasal accumulation of neutrophils induced by PAF is accompanied by the release of neutrophil-derived mediators. Nasal lavages were performed before and after challenge with PAF (500 nmol), lyso-PAF (500 nmol), and saline solution in 10 patients with allergic rhinitis and 10 normal subjects to evaluate changes in neutrophil counts and the release of myeloperoxidase (MPO) and immunoreactive leukotriene B4. PAF caused neutrophilia, which appeared after 30 minutes in atopic subjects and after 3 hours in nonatopic subjects. Furthermore, when compared with saline insufflation, PAF caused a significant release of MPO in the nasal lavage fluids collected 30 minutes, 3 hours, and 24 hours after challenge in atopic subjects and 3 hours after challenge in nonatopic subjects, with higher values in the former than in the latter. Neutrophil counts correlated with MPO levels in the nasal lavages collected after PAF challenge. A lower degree of neutrophilia was found 3 hours after stimulation with lyso-PAF in both groups of subjects, with a marginal release of MPO in atopic subjects only. No significant increase of immunoreactive leukotriene B4 levels in nasal lavages was found after challenge with either PAF or lyso-PAF. These results indicate that PAF-induced neutrophilia in the nose is accompanied by the release of MPO, which appears earlier and is more marked in atopic subjects than in nonatopic subjects.

Ovine endometrium synthesizes and releases platelet-activating factor, which can cause the release of prostaglandin F2 alpha by the uterus in situ.

Explanted endometrial tissue from ovariectomized ewes that had received hormone replacement to mimic the luteal phase released platelet-activating factor (PAF) into medium in vitro. On Day 14, 310.1 (261-2185), and on Day 15, 424.4 (14.1-590.7) pg PAF (median 25th-75th percentile; p > 0.05) was released per 100 mg endometrial tissue after a 20-min incubation. A regulatory and final enzyme in the PAF biosynthetic pathway, lysoPAF:acetyltransferase, was also present with a specific activity of 0.533 +/- 0.124 pmol acetate/50 micrograms protein/30 min in Day 14 endometrial tissue and 0.810 +/- 0.468 in Day 15 tissue (mean +/- SEM; p > 0.05). PAF:acetylhydrolase, the metabolic enzyme regulating PAF's half-life, was present in uterine luminal washings, with a specific activity of 3.78 +/- 1.37 nmol acetate released/min/mg protein on Day 14 and 3.41 +/- 0.34 on Day 15 (mean +/- SEM; p > 0.05). Intrauterine infusion of 50-400 micrograms PAF caused a dose-dependent release of prostaglandin (PG) F2 alpha (measured as venous 14-dihydro-15-keto-prostaglandin F2 alpha, PGFM) within 10 min on Days 13-15. The enantiomeric form of PAF was significantly less effective in inducing a rise in venous PGFM. WEB 2086 is reported to be a competitive receptor antagonist for PAF, but a 10-fold excess of WEB 2086 failed to inhibit PAF-induced release of PGFM. It was observed that this dose of WEB alone induced PGFM release, suggesting that in this model this agent may not work as a true competitive antagonist. Repeat challenges at intervals of 0, 90, and 120 min with either 200 micrograms PAF or micrograms oxytocin resulted in a marked tachyphylaxis of response by the third challenge. This desensitization after repeated PAF challenge suggests a specificity of its actions. Infusion of 200 micrograms PAF on Day 14 or 15 at five 60-min intervals resulted in a tachyphylaxis such that by the fourth and fifth challenges, essentially no response was observed. The tachyphylaxis that was induced by four repeat challenges with PAF had no apparent effect on the capacity of the uterus to respond to a fifth challenge with oxytocin. Thus, PAF and oxytocin both caused homologous desensitization of their own capacity to mobilize uterine PGF2 alpha, but PAF did not cause heterologous desensitization of the response to oxytocin. This failure of heterologous desensitization suggests differences in the mechanism of action of the two ligands. Kinetic binding studies showed that PAF did not compete for the oxytocin receptor in vitro. This result demonstrated that the ovine endometrium produces PAF and responds to it by the release of PGF2 alpha in situ. The dynamics of the response elicited by PAF were similar to those of oxytocin, yet the two mediators apparently act separately. PAF may be a modulator of PGF2 alpha release by the ovine uterus during the luteal phase.

L-type calcium channel blockers modulate the microvascular hyperpermeability induced by platelet-activating factor in vivo.

Platelet-activating factor (PAF) is a potent phospholipid mediator of the microvascular dysfunction associated with ischemia-reperfusion injury. Because changes in cytosolic-free Ca2+ concentration are essential in PAF cellular signaling, we formulated the hypothesis that blockade of Ca2+ entry may inhibit the PAF-induced microvascular dysfunction. To investigate this hypothesis two L-type calcium channel blockers, verapamil and nifedipine, were applied to the hamster cheek pouch before the topical PAF challenge was undertaken. Permeability was assessed by measurement of the plasma clearance of fluorescein isothiocyanate dextran, 150,000 mol wt. The arteriolar diameter was measured simultaneously to evaluate the effects of L-type calcium channel blockers on PAF-induced vasoconstriction. Baseline clearance was 498.7 +/- 225.0 nl/60 min/gm (mean +/- SE). PAF at 10(-8) mol/L (n = 5) increased clearance to 3753.8 +/- 572.8 nl/60 min/gm (p < 0.01). Pretreatment with verapamil (2 mg/kg; n = 5) significantly reduced the increase in permeability caused by 10(-8) mol/L PAF (1909.1 +/- 620.2 nl/60 min/gm; p < 0.05). Nifedipine (5-10(-6) mol/L; n = 5) also significantly attenuated the impact of 10(-8) mol/L PAF (2037.2 +/- 427.5 nl/60 min/gm; p < 0.05). Neither verapamil nor nifedipine affected PAF-induced vasoconstriction. The significant inhibition of the increase in permeability by the L-type calcium channel blockers suggests that these compounds may be useful in the management of PAF-induced hyperpermeability.

Platelet‐activating factor: an inflammatory mediator in the acute phase of allergic conjunctivitis in a guinea‐pig model

The role of platelet-activating factor (PAF) as a mediator of increased conjunctival vascular permeability was investigated in a guinea-pig model of immediate hypersensitivity. Vascular permeability of the conjunctiva was determined by measuring the albumin content in lavage fluid (LF) after topical challenge with either PAF or ovalbumin. PAF produced a dose-dependent increase of the vascular permeability within minutes. Topical pretreatment with levocabastine, a potent histamine H1-antagonist demonstrated no effect towards the vascular permeability in response to PAF provocation. Pretreatment with eyedrops containing the specific PAF antagonist BN 52021 (1%) showed a significant inhibition of the vascular permeability (60.2%) and the clinical score (27.5%) after PAF challenge. In sensitized guinea-pigs, levocabastine showed a marked inhibition of both the vascular permeability (80.5%) and the clinical score (70%) after topical challenge with ovalbumin. BN 2021, although to a lesser extent, showed a similar effect towards the vascular permeability (26.8%) and the clinical score (28%) after antigen provocation. When BN 52021 and levocabastine were administered in combination, the vascular permeability was significantly decreased after antigen challenge in comparison with eyes pretreated with levocabastine alone. These results indicate that PAF plays a role in the acute phase of allergic conjunctivitis in the guinea-pig.

Acute Bronchial and Hematologic Effects Following Inhalation of a Single Dose of PAF

This study compared the acute bronchial and hematologic effects of inhaled platelet activating factor (PAF) (30 micrograms as a single dose) in 19 patients with mild asthma and 19 normal subjects. Each subject underwent a methacholine bronchial challenge 1 week before PAF challenge to determine the concentration of methacholine causing a 20 percent fall in FEV1 (PC20M). On the day of PAF challenge, specific conductance (SGaw), FEV1, FEF25-75, and platelet and leukocyte counts were measured before, and 5, 10, 15, and 20 min after PAF inhalation. Changes in pulmonary and hematologic parameters were expressed as percent of control (saline solution/ethanol solution). Unlike normal subjects, subjects with asthma had bronchial hyperresponsiveness to methacholine: geometric mean (range): 0.59 mg/ml (0.07 to 9.8) vs > 32 mg/ml. Acute bronchial obstruction over the first 20 min after PAF inhalation was more pronounced in asthmatics than in normal subjects whatever the functional index considered (p < 0.01). In asthmatics (n = 19), mean (SEM) maximal fall in SGaw, FEV1, and FEF25-75 reached 50 percent (6), 11 percent (4), and 19 percent (5), respectively, while in normal subjects (n = 19) the maximal decreases were 24 percent (6), 4 percent (1), and 6 percent (1), respectively. In asthmatics, no correlation was found between log PC20M and log fall in FEV1 after PAF (r = 0.04 p > 0.05). In asthmatics and normal subjects, inhaled PAF caused a transient fall in neutrophils and monocytes by 5 min followed by a full recovery at 15 min and 20 min. These hematologic changes were not significantly different between the two groups. While not correlated with their airway responsiveness to methacholine, asthmatics, compared with normal subjects, develop an exaggerated acute airway obstruction in response to PAF. In contrast, hematologic changes induced by PAF do not differ between asthmatics and normal subjects.

Nasal eosinophilia induced by PAF-acether is accompanied by the release of eosinophil cationic protein.

It has been demonstrated that platelet-activating factor (PAF)-acether can induce nasal neutrophilia and eosinophilia, with a different degree of responsiveness in atopic and in nonatopic subjects. The aim of this study was to evaluate whether PAF can also induce the release of secondary mediators in the human nose. Ten patients with allergic rhinitis and 10 normal subjects underwent nasal challenge with PAF (500 nmol), lyso-PAF (500 nmol) and saline solution. Nasal lavages were performed before and after challenge to evaluate changes in nasal cytology and release of histamine, immunoreactive leukotriene (iLT) C4 and eosinophil cationic protein (ECP). PAF caused neutrophilia and eosinophilia, which appeared earlier in atopic than in nonatopic subjects (30 min vs 1 h), and peaked 3 h after challenge in both groups. Lyso-PAF caused mild neutrophilia, which appeared 3 h after challenge in both groups; an increase in eosinophil counts was observed 3 h after challenge in atopic subjects, but not in nonatopic subjects. PAF insufflation caused a significant release of ECP in nasal lavage fluids 30 min and 3 h after challenge in atopic subjects, and 3 h after challenge in nonatopic subjects. ECP levels in the nasal lavages collected 30 min and 3 h after challenge with PAF were higher in atopic than in nonatopic subjects. Eosinophil counts correlated with ECP levels in the nasal lavages collected 30 min after PAF challenge in atopic subjects. Nasal challenge with lyso-PAF did not provoke any release of ECP. No significant increase of histamine and iLTC4 levels in nasal lavages was found after challenge with either PAF or lyso-PAF.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural characterization of platelet-activating factor-stimulated human eosinophils from patients with asthma.

1. Purified human eosinophils from asthmatic patients were stimulated with platelet-activating factor in vitro and examined for morphological changes by transmission electron and light microscopy. Changes were also evaluated by morphometric analysis and were related to the platelet-activating factor-stimulated release of granular eosinophil cationic protein. 2. Stimulation of eosinophils with platelet-activating factor induced a dose-dependent shape change, including the elongation of cells, loss of microvilli and the formation of lamellipodia. This effect was maximal at 25 min and was reversible. 3. Stimulation with platelet-activating factor also induced granule movement to the cell periphery and fusion of adjacent granules. Granules became swollen and vesiculated, whereas both the matrix and core showed evidence of solubilization. 4. There was a time-dependent secretion of eosinophilic cationic protein from human eosinophils upon stimulation with platelet-activating factor which occurred without significant lactate dehydrogenase release. 5. Morphometric analysis of the transmission electron micrographs indicated a significant reduction in cytoplasmic area after 10 min of incubation with platelet-activating factor from 39.0 +/- 1.7 microns 2 for untreated eosinophils to 33.2 +/- 2.3 microns 2 (P < 0.02) for platelet-activating factor-treated cells, underscoring the observation that the cells change from spherical to ellipsoidal. No significant increase in the perimeter of the cells was found. 6. The number of granule-profiles in platelet-activating factor-stimulated eosinophils was slightly reduced when compared with control, and an increase in granule area was observed 10 min after platelet-activating factor challenge (0.215 +/- 0.011 microns 2 versus 0.246 +/- 0.016 microns 2).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a 5-lipoxygenase inhibitor and leukotriene antagonist (PF 5901) on PAF-induced airway responses in neonatally immunized rabbits.

1. Aerosol administration of platelet activating factor (PAF) (80 micrograms ml-1 for 60 min) to neonatally immunized rabbits caused bronchoconstriction which was far in excess of that produced by a comparable aerosol of bovine serum albumin (BSA), the carrier molecule for PAF. Bronchoconstriction of a similar magnitude was elicited by PAF in immunized, sham-immunized and normal rabbits. 2. Aerosol administration of PAF to immunized rabbits induced enhanced airway responsiveness to inhaled histamine in all animals tested, 24 h and 72 h after exposure. In not all cases had airways responsiveness returned to basal levels at 1 week following PAF challenge. In contrast, following exposure of immunized rabbits to BSA, no significant changes in airway responsiveness to histamine were evident at any of the measured time points. 3. A significant increase in the total number of inflammatory cells recovered in bronchoalveolar lavage (BAL) fluid was determined 24 h and 72 h following PAF exposure in immunized rabbits. This was associated with a significant increase in the number of neutrophils and eosinophils. Similar changes were observed following exposure of PAF to normal and sham-immunized rabbits. No change in the total number of inflammatory cells was obtained in BAL after BSA challenge to immunized rabbits; however, neutrophil numbers were significantly increased. 4. PF 5901, a specific inhibitor of the 5-lipoxygenase pathway of arachidonic acid metabolism and a leukotriene D4 antagonist, at a dose of 10 mg (direct intratracheal administration) significantly inhibited the airway resistance (RL) component of the bronchoconstriction induced by PAF in neonatally-immunized rabbits. Doses of 10 mg, 3 mg and 1 mg PF 5901 (direct intratracheal administration) were sufficient to inhibit significantly the PAF-induced increase in airways responsiveness to inhaled histamine in immunized rabbits. PF 5901 however, failed to alter the pulmonary cell infiltration induced by PAF,as assessed by BAL.5. We suggest from the results of the present study that PAF induces consistent and long-lasting increases in airways responsiveness to histamine in immunized rabbits, which is mediated, at least in part, by products of the 5-lipoxygenase metabolic pathway. Furthermore, the inability of PF 5901 to inhibit the influx of inflammatory cells into the airway lumen following PAF challenge may suggest that bronchial hyperresponsiveness and cellular infiltration are not strictly associated events.

Regulation of platelet-activating-factor receptors and the desensitization response in polymorphonuclear neutrophils.

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