B.B. Vargaftig

Platelet-activating factor induces a platelet-dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives.

Abstract Platelet-activating factor (PAF-acether) is a phospholipid derivative released from stimulated basophils, macrophages and platelets. It induced the release of ATP from, and aggregated guinea-pig platelets. These effects were inhibited by neither aspirin nor eicosatetraynoic acid, ruling out the participation of platelet cyclooxygenase and lipoxygenase. Accordingly, no thromboxane A2 activity was found in incubates of guinea-pig platelet-rich plasma with PAF-acether. Prostacyclin suppressed in vitro platelet activation by PAF-acether, which therefore appears to be controled by platelet cyclic AMP levels. PAF-acether injected i.v. to guinea pigs induced hypotension and an increase of bronchial resistance to inflation accompanied by thrombocytopenia. The latter, as well as bronchoconstriction were inhibited by prostacyclin, whereas doses of aspirin which suppress the in vivo platelet and bronchial effects of the TxA2 precursor arachidonic acid were inactive against PAF-acether. Bronchoconstriction due to PAF-acether was platelet-dependent as it was suppressed by immune platelet depletion. The hypotensive effect of PAF-acether was not inhibited after platelet depletion. The pharmacological properties of PAF-acether are compatible with its proposed role in the bronchoconstriction of asthma which is probably caused by a still unidentified factor.

Platelet-activating factor (PAF-acether) secretion from platelets: effect of aggregating agents.

Platelet‐activating factor is a 1.0‐alkyl‐2 acetyl analogue of phosphatidylcholine (PAF‐acether) which triggers platelet aggregation independently from ADP release and thromboxane A2 (T×A2) formation. PAF‐acether was described initially as being secreted by rabbit basophils during an immunological challenge. We have now found that it is also formed by washed rabbit platelets stimulated by the calcium ionophore A23187, thrombin and collagen. These three agents are known to trigger platelet aggregation independently from the release of ADP and from the formation of T×A2. By contrast, ADP and arachidonic acid, the precursor of T×A2, which do not share these properties, and PAF‐acether itself, were unable to induce PAF‐acether formation. Our results suggest that PAF‐acether may be the mediator responsible for AIIP and T×A2 independent‐aggregation.

The platelet‐independent release of thromboxane A2 by Paf‐acether from guinea‐pig lungs involves mechanisms distinct from those for leukotriene

1 Intra‐arterial injections of platelet‐activating factor (Paf‐acether, 10–300 ng) to the perfused guinea‐pig lung induced a dose‐related bronchoconstriction, followed by contraction of the rat aorta superfused with the lung effluent, indicating the release of thromboxane A2 (TXA2) activity. These effects were matched with injections of bradykinin (Bk) at 100–1000 ng, leukotriene C4 (LTC4) at 10–300 ng or arachidonic acid (AA) at 30–300 μg. 2 Repeated doses of Paf‐acether led to a specific desensitization of the release of TXA2, under conditions where Bk, LTC4 and arachidonic acid retained their ability to release TXA2. 3 Bronchoconstriction and the release of TXA2 induced by Paf‐acether were suppressed when the lungs were perfused with acetylsalicylic acid, but not with salicylic acid. 4 The phospholipase A2 inhibitor, p‐bromophenacyl bromide suppressed the release of TXA2 by Bk, but did not interfere with its formation from AA, nor with its release with Paf‐acether and LTC4. The lipoxygenase inhibitor, nordihydroguaiaretic acid, inhibited to a similar extent the release of TXA2 by Bk, LTC4 and Paf‐acether but also reduced directly the formation of TXA2 from arachidonic acid, invalidating its use as a specific antilipoxygenase agent. 5 The leukotriene C4/D4 antagonist, FPL 55712, suppressed the TXA2 releasing effects of LTC4, and was completely inactive against Paf‐acether, Bk or arachidonic acid. 6 The aerosol of Paf‐acether was tested in the anaesthetized guinea‐pig and resulted in bronchoconstriction, unaccompanied by thrombocytopenia. Unlike bronchoconstriction induced by intravenous Paf‐acether, which is refractory to cyclo‐oxygenase inhibitors, the effects of the aerosol were suppressed by aspirin. Platelet depletion, which blocks the intravenous effects of Paf‐acether, failed to interfere with those of the aerosol. 7 Paf‐acether induced a marked contraction of the superfused guinea‐pig isolated parenchyma lung strip, which was followed by total and irreversible desensitization to itself. The contractile effect was not inhibited by aspirin or indomethacin, atropine, mepyramine, methysergide, phenoxybenzamine or propranolol, indicating that cyclo‐oxygenase products, cholinergic stimuli, histamine, 5‐hydroxytryptamine and catecholamine mechanisms are not involved. 8 Our results indicate that Paf‐acether interacts with pulmonary sites distinct from those for Bk, LTC4 or AA, since no cross‐desensitization between Paf‐acether and the other agonists was noted, p‐bromophenacyl bromide inhibited Bk only and FPL 55712 inhibited only LTC4. The phospholipase A2 involved with the release of the arachidonate needed for the formation of TXA2 by Paf‐acether or LTC4‐stimulated lungs may differ from the enzyme accounting for its formation by Bk. The cellular sites with which Paf‐acether interacts may also be distinct and less readily accessible to p‐bromophenacyl bromide.

Differential inhibition by two hetrazepine PAF antagonists of acute inflammation in the mouse

1 The injection of 100 or 300 μg of carrageenin into the mouse paw or pleural cavity produced a delayed inflammatory reaction at 48 h while platelet activating factor (PAF)‐induced paw oedema and pleurisy were maximal 30 min after its injection. 2 The PAF antagonist, WEB 2086, failed to inhibit mouse paw oedema and pleurisy induced by PAF, but reduced the first phase of oedema (1–4 h) induced by carrageenin without interfering with the second one (48–72 h). In contrast, another structurally‐related PAF antagonist, WEB 2170, inhibited dose‐dependently both oedema and pleurisy induced by PAF and by carrageenin (48 h). 3 Repeated injections of PAF into the mouse paw or pleural cavity led to significant auto‐desensitization. The animals desensitized to PAF and injected with carrageenin also displayed a significantly reduced oedema. 4 Our results suggest that PAF may be involved in the inflammatory response to carrageenin in mice. Furthermore, because the different receptor antagonists displayed distinct effects against PAF itself, different sites for in vivo interaction of PAF are available and are species‐ and drug‐dependent.

Desensitization to PAF-induced bronchoconstriction and to activation of alveolar macrophages by repeated inhalations of PAF in the guinea pig.

Guinea-pig alveolar macrophages are activated in the presence of PAF-acether (PAF), as shown by O2.- production, suggesting that these cells, abundant in the lungs, are involved in PAF-induced bronchoconstriction. Alveolar macrophages collected after in vivo desensitization to the bronchoconstrictor effect of PAF became refractory to it in vitro, whereas the O2.- production in response to f-met-leu-phe persisted, although it was diminished suggesting a partial cross-desensitization. A similar desensitization to PAF was also observed in alveolar macrophages in vitro, demonstrating a stimulus-specific process. This study suggests that alveolar macrophages may be involved in bronchoconstriction induced by aerosol of PAF.

Drug modulation of antigen-induced paw oedema in guinea-pigs: effects of lipopolysaccharide, tumour necrosis factor and leucocyte depletion.

1 In guinea‐pigs previously sensitized with ovalbumin, the intra‐plantar administration of the antigen induced dose‐dependent and sustained oedema. An intense infiltrate of neutrophils and eosinophils was observed at the peak of the oedema (4 h). 2 Oedema induced by ovalbumin at the doses of 50 or 200 μg/paw was not inhibited by antihistamines (meclizine and cetirizine), a PAF antagonist (BN 50730), a cyclo‐oxygenase inhibitor (indomethacin), a lipoxygenase inhibitor (MK‐886), a dual type lipo‐ and cyclo‐oxygenase inhibitor (NDGA), a bradykinin antagonist (Hoe 140) or the combination of cetirizine, MK‐886, indomethacin and BN 50730. These drugs did inhibit paw oedema induced by their specific agonists or by carrageenin. These results suggest that histamine, PAF, prostaglandins, leukotrienes or bradykinin are not important in the development of immune paw oedema in guinea‐pigs. 4 Dexamethasone (10 mg kg−1) inhibited oedema induced by ovalbumin (50 or 200 μg/paw, P < 0.05). This effect apparently does not result from inhibition of arachidonate metabolism, since indomethacin, MK‐886 and NDGA were without effect. 5 Oedema induced by ovalbumin (50 or 200 μg/paw) was also inhibited by azelastine. This effect was not due to the anti‐histaminic property of azelastine since two other potent‐antihistamines, meclizine and cetirizine, were ineffective. 6 Intravenous injection of lipopolysaccharide (LPS) dose‐dependently inhibited the oedema induced by ovalbumin (200 μg/paw). This effect could not be attributed to hypotension or leucopenia since the maximal dose applied (81 μg kg−1) did not induce significant changes in the blood pressure or in the white blood cell levels of the animals. It is suggested that the effect of LPS is mediated by the endogenous release of cytokines, including tumour necrosis factor (TNFα). Murine TNFα dose‐dependently (9–81 μg kg−1) inhibited the paw oedema induced by ovalbumin. 7 The anti‐oedematogenic effects of LPS and/or TNFα are possibly associated with their capacity to inhibit leucocyte emigration. Accordingly, guinea‐pigs rendered leucopenic with vinblastine exhibited less intense oedema after ovalbumin. Vinblastine did not affect oedema induced by PAF or bradykinin, indicating that vascular responsiveness was not involved.

Blockade by fenspiride of endotoxin-induced neutrophil migration in the rat

Fenspiride, an antiinflammatory drug with low anti-cyclooxygenase activity, administered orally at 60-200 mg/kg inhibited neutrophil migration into peritoneal and air pouches cavities as well as exudation into peritoneal cavities induced by endotoxin but not induced by carrageenin. Up to 100 microM, fenspiride failed to inhibit the in vitro release of a neutrophil chemotactic activity by endotoxin-stimulated macrophages and the in vivo migration into the peritoneal cavities induced by the supernatant of those macrophages. The release of tumour necrosis factor by stimulated macrophages was inhibited by fenspiride in a dose-dependent manner. These results suggest that the antiinflammatory effects of fenspiride are associated with the inhibition of the tumour necrosis factor release by resident macrophages.

Histamine and leukotriene-independent guinea-pig anaphylactic shock unaccounted for by Paf-acether.

1 Ovalbumin induced dose‐dependent contractions of lung parenchyma strips from sensitized guinea‐pigs, whereas Paf‐acether (1‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphoryl choline), a potential mediator of immediate hypersensitivity, induced a single contraction, followed by specific desensitization to a second exposure. 2 Lung strips desensitized to Paf‐acether contracted to ovalbumin as did non‐desensitized controls, even in the presence of inhibitors of other mediators of anaphylaxis. 3 Contractions by Paf‐acether and by ovalbumin were reduced by nordihydroguaiaretic acid (NDGA) and by the phospholipase A2 inhibitor p‐bromophenacyl bromide (0.1–0.3 mm). Three other anti‐lipoxygenase agents (diethylcarbamazine, 5mm; eicosatetraynoic acid and BW755c, 0.1 mm), reduced the contractions by ovalbumin but also those due to acetylcholine, indicating non‐specific effects. Neither the anti‐allergic compound sodium cromoglycate (3 mm) nor the anti‐leukotriene agent, FPL 55712 (0.01 mm), inhibited the contractions by ovalbumin or by Paf‐acether. 4 A sensitized strip stimulated with ovalbumin released substances which contracted a non‐sensitized strip mounted in the same organ bath. The contractions of the non‐sensitized strip were abolished by FPL 55712 (0.01 mm), by NDGA and BW755c, (0.1 mm), whereas those of the sensitized one were unaffected. Leukotrienes are formed by the lung strips during shock but alone, they do not explain the contractile activity. 5 The intravenous administration of ovalbumin (1 mg kg−1) led to bronchoconstriction and thrombocytopenia, which were not modified by the anti‐leukotriene substance FPL 55712 nor by aspirin. Bronchoconstriction was suppressed if FPL 55712 was used in combination with aspirin (20 mg kg−1), mepyramine and methysergide (200 μg kg−1 of either). Pretreatment of the guinea‐pigs with propranolol reduced this inhibition to approximately 60%. In no instance was thrombocytopenia prevented. 6 In vitro contractions of the actively sensitized lung strip are not fully accounted for by histamine, FPL 55712‐inhibitable leukotrienes or Paf‐acether, whereas in systemic anaphylaxis histamine and leukotrienes (inhibited respectively by mepyramine and by FPL 55712) have a significant role.

Involvement of leukotrienes and PAF-acether in the increased microvascular permeability of the rabbit retina

The effects of intravenous injection of PAF-acether and of leukotrienes LTB4 and LTD4 on rabbit retina were determinedin vivo in the unanaesthetized animal. PAF-acether induced an intense ischemia accompanied by a marked plasma leakage. The extravasation remained both in the platelet-depleted rabbit where a sludge phenomenon appeared and in the reserpine-treated animal. Prostacyclin injected after PAF-acether restored only partially the normal retinal appearance.The effect of LTC4 and LTD4 was markedly less intense than PAF-acether which is not too surprising in this species, given the previous results.

Interference of the PAF-acether antagonist BN 52021 with passive anaphylaxis in the guinea-pig

PAF-acether may be involved in anaphylaxis and asthma. We tested the new PAF-acether antagonist BN 52021 against the effects of antigen in passively sensitized guinea-pigs. Bronchoconstriction by ovalbumin administered i.v. (1 mg/kg) or by aerosol (1 or 10 mg/ml for a period of 1 min) was significantly reduced by BN 52021 (1-10 mg/kg), which did not inhibit drop of leukocyte counts after the i.v. challenge. In both cases, when the guinea-pigs were pretreated by propranolol, high amounts of BN 52021 became ineffective against shock. The reduction of the anaphylactic bronchoconstriction, induced by the combination of mepyramine, aspirin and FPL 55712 was not improved by BN 52021. Tested on isolated lung strips from passively sensitized guinea-pig, BN 52021, at a concentration which inhibits PAF-induced contraction (0.1 mM), did not inhibit the anaphylactic contraction triggered by the administration of ovalbumin (10 micrograms/ml) nor the accompanying release of histamine and thromboxane. In contrast, BN 52021 (30 microM) significantly reduced the anaphylactic release of histamine and of thromboxane from perfused lungs of passively sensitized guinea-pigs. The results with the isolated lung strips and the propranolol-treated guinea-pigs in vivo suggest a dissociation between the anti-anaphylactic and the anti-PAF-acether properties of BN 52021.