Jean Lefort

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.

BACKGROUND AND PRESENT STATUS OF RESEARCH ON PLATELET‐ACTIVATING FACTOR (PAF‐ACETHER)

A leukocyte-dependent mechanism capable of releasing histamine from platelets was first reported in 1966, and a soluble principal intermediate between rabbit leukocytes and platelets was detected in 1971.’e2 It was at that time described as “lytic” and was not characterized. One of us described the methodology for obtaining this substance routinely, started its characterization, named it platelet-activating factor (PAF), and showed that it was released from rabbit basophils through an IgE-dependent proces~ .~ Its presence was subsequently demonstrated in human leukocytes, and aggregation and the release reaction were shown for human platelet^.^ Finally, most of its known physicochemical characteristics, including its phospholipid nature, were described (see refs. in 5) . At this stage, we knew that PAF was a glycerophospholipid with a choline polar head group, an ester-linked acyl chain on the carbon 2, and no ester link at the carbon 1 position.6 A new class of phospholipid mediator was therefore proposed. Finally, the structure of the mediator was elucidated (FIG. 1 ) as being 1 -O-alkyl-2-acetyl-glyceryl-3-phosphorylcholine, and its total synthesis was achieved.’-g PAF is therefore now termed PAFacether, since it is an ether-lipid and has an acetate residue. We think it is preferable to keep to the established name, and not to replace it by the initials of the now recognized chemical name, in order to preserve a link with previous accomplishments and reduce literature confusion. For similar reasons prostaglandins or heparin, for instance, were not renamed when structures became available. Given the numerous substances present in biological fluids and cell supernatants that can activate platelets, it was necessary to define PAF-acether strictly. Even before knowing its structure, we used the following criteria to distinguish it from arachidonic acid, thrombin, ADP or prostaglandins:

Cutting Edge: Invariant Vα14 NKT Cells Are Required for Allergen-Induced Airway Inflammation and Hyperreactivity in an Experimental Asthma Model

Airway hyperreactivity (AHR), eosinophilic inflammation with a Th2-type cytokine profile, and specific Th2-mediated IgE production characterize allergic asthma. In this paper, we show that OVA-immunized Jα18−/− mice, which are exclusively deficient in the invariant Vα14+ (iVα14), CD1d-restricted NKT cells, exhibit impaired AHR and airway eosinophilia, decreased IL-4 and IL-5 production in bronchoalveolar lavage fluid, and reduced OVA-specific IgE compared with wild-type (WT) littermates. Adoptive transfer of WT iVα14 NKT cells fully reconstitutes the capacity of Jα18−/− mice to develop allergic asthma. Also, specific tetramer staining shows that OVA-immunized WT mice have activated (CD69+) iVα14 NKT cells. Importantly, anti-CD1d mAb treatment blocked the ability of iVα14 T cells to amplify eosinophil recruitment to airways, and both Th2 cytokine and IgE production following OVA challenge. In conclusion, these findings clearly demonstrate that iVα14 NKT cells are required to participate in allergen-induced Th2 airway inflammation through a CD1d-dependent mechanism.

Bacterial Lipopolysaccharide Signaling Through Toll-Like Receptor 4 Suppresses Asthma-Like Responses Via Nitric Oxide Synthase 2 Activity

Asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. Microbial infections could prevent allergic responses by inducing the secretion of the type 1 cytokines, IL-12 and IFN-γ. In this study, we examined whether administration of bacterial LPS, a prototypic bacterial product that activates innate immune cells via the Toll-like receptor 4 (TLR4) could suppress early and late allergic responses in a murine model of asthma. We report that LPS administration suppresses the IgE-mediated and mast cell-dependent passive cutaneous anaphylaxis, pulmonary inflammation, airway eosinophilia, mucus production, and airway hyperactivity. The suppression of asthma-like responses was not due to Th1 shift as it persisted in IL-12−/− or IFN-γ−/− mice. However, the suppressive effect of LPS was not observed in TLR4- or NO synthase 2-deficient mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses in vivo via the TLR4-dependent pathway that triggers NO synthase 2 activity.

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.

Effect of cyclo‐oxygenase inhibitors and modulators of cyclic AMP formation on lipopolysaccharide‐induced neutrophil infiltration in mouse lung

1 The adult respiratory distress syndrome (ARDS) is an acute lung inflammation developed after direct or indirect contact with pathogenic agents. In the present study, a mouse model was developed to mimic this condition using aerosolized bacterial lipopolysaccharide (LPS) and to investigate the mechanisms involved in the lung inflammatory response. 2 Inhalation of LPS led to a time and dose‐dependent increase in tumour necrosis factor‐α (TNF‐α) production and neutrophil recruitment into the bronchoalveolar lavage fluid (BALF) of Balb/c mice. Under the same conditions, neutrophil infiltration was also found in the BALF of the LPS‐sensitive mouse strain C3H/HeN, but was absent in the LPS‐resistant strain C3H/HeJ. Intranasal administration of murine recombinant TNF‐α also triggered neutrophil recruitment. 3 One hour after inhalation of LPS, half of the maximal level of TNF‐α was measured in the BALF, but only a few neutrophils were detected at this time. The peak TNF‐α concentration was reached at 3 h, when the neutrophil amount started to increase. At 24 h, maximal neutrophil number was found in the BALF and TNF‐α was no longer present. 4 Pretreatment of mice under different experimental conditions demonstrated that: (a) cycloheximide almost completely blocks both neutrophil recruitment and TNF‐α production; (b) anti TNF‐α antibodies block neutrophil recruitment; (c) indomethacin or aspirin enhance by two fold neutrophil recruitment; (d) indomethacin significantly increases TNF‐α production 1 h after inhalation of LPS; (e) dibutyryl cyclic AMP and prostaglandin E2 (PGE2) block both neutrophil recruitment and TNF‐α production. 5 It is concluded that aerosolized LPS in mice triggers an acute lung inflammation which can be used as a potential model of inhalational ARDS and that, strategies leading to the elevation of cyclic AMP levels in vivo can be effective in modulating LPS‐induced TNF‐α synthesis and neutrophil recruitment.

α‐Galactosylceramide‐induced iNKT cells suppress experimental allergic asthma in sensitized mice: Role of IFN‐γ

Allergic asthma is a multifaceted syndrome consisting of eosinophil‐rich airway inflammation, bronchospasm, and airway hyper‐responsiveness (AHR). Using a mouse model of allergic asthma, we previously reported that invariant NKT (iNKT) cells increase the severity of this disease. Herein, we demonstrate that a single i.v. injection of α‐galactosylceramide (α‐GalCer), 1 h before the first airway allergen challenge of OVA‐sensitized mice, abrogates elicitation of AHR, airway eosinophilia, IL‐4 and IL‐5 production in bronchoalveolar lavage fluid, and specific anti‐OVA IgE antibodies. Further, α‐GalCer administered intranasally also strongly inhibited the major symptoms of asthma in sensitized and challenged mice. α‐GalCer treatment induces iNKT cell accumulation in the lungs, and shifts their cytokine profile from pro‐asthmatic IL‐4 to a protective IFN‐γ production. The role of IFN‐γ from iNKT cells in protection was shown by adoptive transfer of sorted iNKT cells from OVA‐sensitized and α‐GalCer‐treated mice which protected immunized recipients from manifesting asthma by an IFN‐γ‐dependent pathway. Our findings demonstrate for the first time that α‐GalCer administered locally inhibits asthma symptoms, even in predisposed asthmatic mice, through an iNKT cell‐ and IFN‐γ‐dependent pathway.

Effect of antigen provocation of IL-5 transgenic mice on eosinophil mobilization and bronchial hyperresponsiveness

We investigated whether allergen-induced eosinophil recruitment into mouse airways modifies the in vivo bronchopulmonary responses to standard agonists, and adaptation of a technique described for larger animals. Swiss, CBA, and IL-5 transgenic mice were immunized with ovalbumin and challenged intranasally after 14 days. Immunization alone was followed by increased eosinophil counts in bone marrow and blood, whereas antigenic challenge induced eosinophil infiltration in lungs and bronchoalveolar lavage fluid, which was suppressed by dexamethasone. Despite the high eosinophil counts, no bronchopulmonary hyperreactivity to methacholine or serotonin was detected 3 to 96 hours after antigenic provocation. Our results demonstrate that immunization augments the production of eosinophils by mice, which is further increased by antigenic challenge, but that eosinophil overproduction and lung infiltration, per se, are not sufficient to induce bronchopulmonary hyperreactivity, even in constitutively hypereosinophilic IL-5 transgenic mice.

ROLE OF PLATELETS IN ASPIRIN‐SENSITIVE BRONCHOCONSTRICTION IN THE GUINEA‐PIG; INTERACTIONS WITH SALICYLIC ACID

1 The bronchoconstriction caused in the guinea‐pig by arachidonic acid (AA), bradykinin, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) was correlated with effects on platelets. ATP and ADP produced a brief thrombocytopenia and AA a more prolonged one. Bradykinin had no effect on platelets. 2 Aspirin inhibited bronchoconstriction and thrombocytopenia produced by AA and part of the bronchoconstriction produced by ATP, but had no effect against ADP. Thrombocytopenia produced by ADP and ATP was not affected by aspirin or indomethacin. 3 Platelet depletion by antiserum prevented bronchoconstriction in response to ADP and to ATP, but not in response to bradykinin or to AA, showing that platelets are not involved in aspirin‐sensitive bronchoconstriction. Infusions of ADP reduced bronchoconstriction and thrombocytopenia in response to ADP itself and to ATP, but not to AA. Bronchoconstriction by ADP or ATP involves an action on platelets. Only that due to ATP is partially dependent on the activity of prostaglandin synthetase. 4 ATP induced aggregation in vitro in guinea‐pig platelet‐rich plasma (PRP). Rabbit PRP responded only when ATP was first incubated with guinea‐pig plasma. The aggregating compound formed was probably ADP, since it was destroyed by apyrase. Its formation was not inhibited by aspirin or indomethacin, indicating that aspirin inhibits ATP‐induced bronchoconstriction by a different mechanism. 5 The aggregating effect of ATP on guinea‐pig platelets was inhibited by concentrations of apyrase that block ADP‐induced aggregation, and potentiated by lower concentrations of apyrase. 6 Adenosine 5′‐tetraphosphate did not aggregate platelets in vivo or in vitro. In vitro aggregation occurred when apyrase was added, suggesting transformation into ADP. Adenosine 5′‐tetraphosphate and apyrase inhibited aggregation due to ADP, but failed to affect that due to AA. This suggests that aggregation involving products of prostaglandin synthesis does not require ADP. 7 Salicylic acid did not interfere with bronchoconstriction or aggregation due to AA, but prevented inhibition by aspirin when the weight ratio, salicylic acid:aspirin was 4:1. Salicylic acid may be useful in studies of potential inhibitors of thromboxane A2 synthesis and of thromboxane A2‐depen‐dent processes in vivo and in vitro.

Interference by the novel PAF-acether antagonist WEB 2086 with the bronchopulmonary responses to PAF-acether and to active and passive anaphylactic shock in guinea-pigs

The interaction between the triazolothienodiazepine WEB 2086 and the in vitro and in vivo bronchopulmonary effects of PAF-acether and active/passive anaphylaxis in the guinea-pig was studied. WEB 2086 (1-100 nM) inhibited PAF-acether (10-100 ng)-induced bronchoconstriction and TXB2 release from isolated and perfused guinea-pig lungs without affecting the response to 100 micrograms arachidonic acid. In addition, 1-10 microM WEB 2086 significantly reduced antigen-induced TXB2 and histamine release from lungs from actively and passively sensitized guinea-pigs. In the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), mepyramine, methysergide, indomethacin and atropine, WEB 2086 (20-50 microM) inhibited by 30-40% the residual contraction of lung parenchyma strips from guinea-pigs actively sensitized by 0.1-10 micrograms antigen. In vivo, WEB 2086 (0.1-1 mg/kg) reversed or abolished the bronchoconstriction, hypotension, thrombocytopenia and leukopenia evoked by perfusion of PAF-acether (3 or 44 ng/kg per min). At 3 mg/kg, WEB 2086 also markedly decreased the bronchoconstriction and leukopenia induced by 100 micrograms/kg antigen in mepyramine (5 micrograms/kg)-treated passively sensitized guinea-pigs. In contrast, WEB 2086 was ineffective against active anaphylaxis in vivo. These results demonstrate that WEB 2086 antagonizes the bronchopulmonary effects due to PAF-acether and to anaphylactic shock in the guinea-pig.