Antibody to very late activation antigen 4 prevents antigen-induced bronchial hyperreactivity and cellular infiltration in the guinea pig airways.

This report examines the effect of an anti-VLA-4 monoclonal antibody (mAb) HP1/2 on antigen-induced bronchial hyperreactivity to methacholine, and on eosinophil and T lymphocyte infiltration in the airways of guinea pigs sensitized and challenged by aerosolized ovalbumin and used 24 h thereafter. The intravenous administration of 2.5 mg/kg of HP1/2, but not of its isotype-matched mAb 1E6, 1 h before and 4 h after antigen inhalation, markedly inhibited the increased bronchopulmonary responses to intravenous methacholine, as well as airway eosinophilia in bronchoalveolar lavage (BAL) fluid and in bronchial tissue. HP1/2 also suppressed the antigen-induced infiltration of the bronchial wall by CD4+ and CD8+ T lymphocytes, identified by immunohistochemical technique using specific mAbs that recognize antigenic epitopes of guinea pig T cells. Treatment with HP1/2 also resulted in a significant increase in the number of blood eosinophils, suggesting that inhibition by anti-VLA-4 mAb of eosinophil recruitment to the alveolar compartment may partially account for their accumulation in the circulation. These findings indicate that eosinophil and lymphocyte adhesion and subsequent infiltration into the guinea pig airways that follow antigen challenge are mediated by VLA-4. Furthermore, concomitant inhibition of antigen-induced bronchial hyperreactivity and of cellular infiltration by anti-VLA-4 mAb suggests a relationship between airway inflammation and modifications in the bronchopulmonary function.

Requirement for gammadelta T cells in allergic airway inflammation.

The factors that contribute to allergic asthma are unclear but the resulting condition is considered a consequence of a type-2 T helper (TH2) cell response. In a model of pulmonary allergic inflammation, mice that lacked gammadelta T cells had decreases in specific immunoglobulin E (IgE) and IgG1 and pulmonary interleukin-5 (IL-5) release as well as in eosinophil and T cell infiltration compared with wild-type mice. These responses were restored by administration of IL-4 to gammadelta T cell-deficient mice during the primary immunization. Thus, gammadelta T cells are essential for inducing IL-4-dependent IgE and IgG1 responses and for TH2-mediated airway inflammation to peptidic antigens.

Lipopolysaccharide from Escherichia coli reduces antigen-induced bronchoconstriction in actively sensitized guinea pigs.

Bronchoconstriction (BC) is the main feature of anaphylaxis in the guinea pig. Since LPS induces lung inflammation and antigen-induced BC depends on the endogenous formation of histamine and arachidonate metabolites, we studied whether LPS might modulate antigen-induced BC. Guinea pigs were sensitized subcutaneously with 10 micrograms ovalbumin (OA) on days 0 and 14. LPS (100 micrograms/kg) was injected intravenously on day 21, and daily injections of LPS were continued before the antigenic challenge on day 22, 23, 24, or 25. Intratracheal injection of 100 micrograms OA induced an abrupt and reversible BC. Single or repetitive injections of LPS reduced BC. LPS is likely to reduce the OA-induced BC by affecting the histamine-dependent component of BC, since (a) LPS induced a partial degranulation of lung mast cells; (b) BC is reduced by mepyramine, an histamine receptor antagonist; (c) LPS did not affect BC in mepyramine-treated guinea pigs; (d) LPS reduced histamine release by OA-stimulated guinea pig lungs in vitro. Moreover, the in vitro OA-induced production of arachidonate metabolites was also reduced by LPS. The decreased formation of TXB2 was not only secondary to a reduced release of histamine, since LPS inhibited TXB2 formation in the presence of mepyramine. Finally, the FMLP-induced BC and mediator release were inhibited by LPS, whereas the platelet activating factor-induced pulmonary responses were not. Thus, the protective effect of LPS is not antigen-specific and does not result from a general desensitization. These studies indicate that a single dose of LPS reduces the antigen-induced BC by reducing histamine release from lung mast cells, although a decreased formation of eicosanoids may contribute to the protective effect of LPS.

In vivo neutralization of eosinophil-derived major basic protein inhibits antigen-induced bronchial hyperreactivity in sensitized guinea pigs.

This study examines the effect of purified rabbit antiguinea pig eosinophil-derived major basic protein (MBP) Ig on antigen-induced bronchial hyperreactivity to inhaled acetylcholine in aerosol-sensitized guinea pigs. Ovalbumin inhalation by sensitized guinea pigs induced a rise in the numbers of eosinophils and in the levels of MBP in the bronchoalveolar lavage fluid, which peaked at 24 h and resolved at 72 h. Antigen-challenged animals exhibited bronchial hyperreactivity to inhale acetylcholine at 72 h, but not at 6 or 24 h. The intranasal administration of 200 microliter of purified rabbit anti-guinea pig MBP Ig, at 2.5 mg/ml, but not of the control preimmune rabbit Ig, 1 h before and 5 h after ovalbumin inhalation suppressed bronchial hyperreactivity to acetylcholine at 72 h without affecting the number of eosinophils accumulating in the bronchoalveolar lavage fluid. These findings indicate that antigen challenge in sensitized guinea pigs is followed by early eosinophil infiltration and activation within the airways and by late bronchial hyperreactivity. Neutralization of endogenously secreted MBP by a specific antiserum prevented antigen-induced bronchial hyperreactivity, suggesting that eosinophil degranulation plays an important role in the alterations of bronchopulmonary function in the guinea pig.

Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice.

This report examines the effect of recombinant murine (rm) IL-10 on antigen-induced cellular recruitment into the airways of sensitized Balb/c mice. The intranasal instillation of 10 micrograms ovalbumin induced an early (6-24 h) increase in the number of neutrophils, and a late rise (24-96 h) in that of eosinophils in the bronchoalveolar lavage (BAL) fluid and bronchial tissue. A single intranasal instillation of 0.01-0.1 microgram of rmIL-10, administered concurrently with ovalbumin, but not 1 or 3 h thereafter, dose-dependently inhibited both airway neutrophilia and eosinophilia. This phenomenon was suppressed by treating the sensitized mice with 1 mg/mouse of a neutralizing anti-IL-10 mAb, which increased significantly ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These results suggest that antigen stimulation may trigger the in vivo generation of IL-10, which, in turn, participates in the leukocyte infiltration into the airways. rmIL-10 also reduced TNF-alpha release in the BAL fluid observed 1 and 3 h after antigen challenge. Furthermore, the intranasal instillation of an anti-TNF-alpha antiserum to sensitized mice markedly reduced ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These findings indicate that leukocyte infiltration into the airways of antigen-challenged mice is regulated by IL-10. Furthermore, inhibition of TNF-alpha production by rmIL-10 suggests that allergic airway inflammation and TNF-alpha formation are parallel events in this model.

Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction.

Lyso-phospholipids exert a major injurious effect on lung cell membranes during Acute Respiratory Distress Syndrome (ARDS), but the mechanisms leading to their in vivo generation are still unknown. Intratracheal administration of LPS to guinea pigs induced the secretion of type II secretory phospholipase A2 (sPLA2-II) accompanied by a marked increase in fatty acid and lyso-phosphatidylcholine (lyso-PC) levels in the bronchoalveolar lavage fluid (BALF). Administration of LY311727, a specific sPLA2-II inhibitor, reduced by 60% the mass of free fatty acid and lyso-PC content in BALF. Gas chromatography/mass spectrometry analysis revealed that palmitic acid and palmitoyl-2-lyso-PC were the predominant lipid derivatives released in BALF. A similar pattern was observed after the intratracheal administration of recombinant guinea pig (r-GP) sPLA2-II and was accompanied by a 50-60% loss of surfactant phospholipid content, suggesting that surfactant is a major lung target of sPLA2-II. In confirmation, r-GP sPLA2-II was able to hydrolyze surfactant phospholipids in vitro. This hydrolysis was inhibited by surfactant protein A (SP-A) through a direct and selective protein-protein interaction between SP-A and sPLA2-II. Hence, our study reports an in vivo direct causal relationship between sPLA2-II and early surfactant degradation and a new process of regulation for sPLA2-II activity. Anti-sPLA2-II strategy may represent a novel therapeutic approach in lung injury, such as ARDS.

Clostridiopeptidase B inhibition by plasma marcroglobulins and microbial antiproteases.

Clostridiopeptidase B (EC 3.4.22.8) was not inhibited by stoichiometric amounts of lima bean trypsin inhibitor, ovomucoid trypsin inhibitor, Kuntiz bovine trypsin inhibotor, Kunitz soybean trypsin inhibitor or ovoinhibitor. Activity was diminished at relatively high concentrations of the three latter inhibitors. Human plasma alpha 2-macroglobulin inhibited both the amidase and protease activity of the enzyme. Rat and dog plasmas contained high molecular weight inhibitors, presumably macroglobulins as well. Inhibition by this component was greater in rat plasma than in dog plasma, which may be related to the observation that clostridiopeptidase B-induced generation of kinin activity is indirect in the former plasma, but direct in the later. Leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) and antipain ([S)-1-carboxy-2-phenylethyl] carbamoyl-L-arginyl-L-valyl-L-argininal) inhibited clostridiopeptidase B (Ki of 2 . 10(-8) and 3 . 10(-8) M, respectively). They were potent inhibitors of clostridiopeptidase B-induced kinin release in dog plasma.

Reversible inhibition by protamine of human synovial and rabbit platelet secretory phospholipase A2.

We investigated the effects of protamine on the release and the activity of 14 kDa type II phospholipase A2 (sPLA2). Protamine blocks both release and activity of sPLA2 from thrombin-stimulated platelets in a concentration-dependent manner. Heparin, an anionic sulfate polysaccharide which has a high affinity for this enzyme, has no inhibitory effect on sPLA2 by itself but it is able to reverse the inhibitory effect of protamine. The liberation by thrombin of platelet factor 4, an alpha-granule constituent, unlike to that of ATP stored in dense bodies, was suppressed by protamine. Platelet aggregation, determined in parallel, was not affected by protamine. Also, protamine did not inhibit platelet arachidonic acid liberation, which is mainly produced by cytosolic PLA2. The non-proteinaceous polycationic hexadimethrine and acidic protein casein failed to inhibit platelet sPLA2 activity. By contrast, the basic polypeptides poly(L-arginine) and poly(L-lysine) potently inhibited sPLA2 activity, indicating the important role of basic amino acids in the inhibitory effect evoked by protamine. Activities of the human recombinant sPLA2 and the unpurified synovial enzyme of patients with rheumatoid arthritis were also inhibited by the same range of protamine, poly(L-arginine) and poly(L-lysine) concentrations. Our results demonstrate that protamine, unlike heparin, blocks platelet sPLA2 release and exerts a reversible inhibitory effect on its activity, probably through the interaction of basic amino acids with the enzyme.

Changes induced by PAF-acether in diacyl and ether phospholipids from guinea-pig alveolar macrophages.

The release and the mobilization of arachidonic acid from guinea-pig alveolar macrophages labeled with [1-14C]arachidonic acid for short (1 h) and long (18 h) periods and stimulated with PAF-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was studied. After short labeling periods arachidonic acid was primarily incorporated into alkylacyl- and diacylglycerophosphocholine (alkylacylGPC, diacylGPC) and glycerophosphoinositol (GPI), whereas after long labeling periods arachidonic acid was mainly incorporated into alkenylacylglycerophosphoethanolamine (alkenylacylGPE). In macrophages labeled for 1 h, PAF-acether (1 microM) induced a significant decrease in the amount of arachidonic acid esterified into diacyl- and alkylacylGPC and GPI, as well as a significant increase of arachidonate transferred into alkenylacylGPE. No significant decrease in arachidonate esterified in GPC fractions and in GPI was induced by PAF-acether in macrophages labeled for 18 h, whereas the increased transfer of the fatty acid into alkenylacylGPE was still measurable. This study shows that PAF-acether induces the release and the mobilization of newly incorporated arachidonic acid in alveolar macrophages. When cells are labeled for long periods and the majority of arachidonic acid is retained in ether-linked phospholipids, no PAF-acether-induced release of arachidonate was obtained, whereas its transfer was maintained.

1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine is the precursor of platelet-activating factor in stimulated rabbit platelets. Evidence for an alkylacetyl-glycerophosphorylcholine cycle.

The metabolism of [3H]PAF-acether ([1',2'-3H]alkyl-2-acetyl-sn-glycero-3-phosphorylcholine ([3H]alkylacetyl-GPC)) by rabbit platelets was investigated using thin-layer chromatography and high-performance liquid chromatography followed by radioactivity detection. After 2 h of incubation at 37 degrees C, 90 +/- 5.3% of [3H]PAF-acether taken up by the platelets were converted into a product identified as sn-2 long-chain acyl analogue ([3H]alkylacyl-GPC) which was incorporated in the membranes. This conversion was independent from extracellular calcium and was completely inhibited by platelet pre-exposure to 2 mM phenylmethylsulfonyl fluoride, a serine hydrolase inhibitor, which failed to inhibit the uptake of [3H]PAF-acether by the cells. The 2-deacetylated derivative, lyso-[3H]PAF-acether was found to be an intermediate of the conversion of [3H]PAF-acether into [3H]alkylacyl-GPC in platelet homogenates. Platelet stimulation with 2.5 U/ml of thrombin induced a reduction (16.5 +/- 2.2%) of its content of [3H]alkylacyl-GPC, accompanied by the release of [3H]PAF-acether and lyso-[3H]PAF-acether to the medium. These effects were suppressed by the phospholipase A2 inhibitor, p-bromophenacyl bromide. Our results demonstrate that intact platelets convert exogenous PAF-acether into alkylacyl-GPC, which can serve as the precursor of PAF-acether released during stimulation. The existence of a metabolic cycle for the uptake, the release and the inactivation of PAF-acether by platelets is suggested.

Platelet secretory phospholipase A2 fails to induce rabbit platelet activation and to release arachidonic acid in contrast with venom phospholipases A2.

The ability of platelet secretory phospholipase A2 (sPLA2) to induce platelet activation was investigated. sPLA2 (group II) contained in an activated platelet supernatant, as well as high concentrations of purified recombinant platelet sPLA2, failed to induce platelet activation. Furthermore, sPLA2 did not modify platelet activation induced by various agonists. The possible relationship between the failure of this enzyme to induce platelet activation and its origin (mammalian) or its structural group (group II) was then investigated, using pancreatic PLA2s (group I) and venom PLA2s from groups I, II and III. All venom PLA2s induced platelet activation that was accompanied by the liberation of arachidonic acid and was abolished by aspirin. In contrast, as observed for platelet sPLA2, enzymes from hog or bovine pancreas were unable to induce platelet activation even when used at high concentrations. Interestingly, PLA2 able to induce platelet activation efficiently hydrolyse phosphatidylcholine, while those inactive on platelets did not. Taken together, these results suggest that the catalytic activity of added PLA2 is necessary but not sufficient to induce platelet activation. Moreover, the ability of PLA2 to induce platelet activation is not related to its structural group (I, II, III) but rather to its origin (venom vs. mammalian) and capacity to hydrolyse phosphatidylcholine, the major phospholipid of the outer leaflet of the plasma membrane.

Formation of LTB4 by fMLP-stimulated alveolar macrophages accounts for eosinophil migration in vitro.

Guinea pig alveolar macrophages obtained by bronchoalveolar lavage were isolated by adherence for 2 h and stimulated with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) for different time intervals. The supernatants then were tested for their chemotactic effect on guinea pig peritoneal normodense eosinophils and for release of thromboxane B2, leukotriene B4 (LTB4), and platelet activating factor (PAF). The supernatant from fMLP-stimulated alveolar macrophages induced a significant eosinophil attraction (96.0 +/- 11.9, number of migrating eosinophils [mean +/- SEM], n = 17) as compared to unstimulated macrophages (4.8 +/- 1.4, n = 15). This effect was not accounted for by fMLP carry-over to the macrophages because, in contrast to human eosinophils, fMLP has no chemotactic effect on guinea pig eosinophils. Pretreatment of eosinophils with BN 52021 (100 microM), a specific PAF antagonist, and with indomethacin (10 microM), a cyclooxygenase inhibitor, failed to inhibit migration of eosinophils induced by supernatants from either stimulated or unstimulated alveolar macrophages. In contrast, inhibition of the 5-lipoxygenase enzyme with N-(3-phenoxycinamyl)-acetohydroxamic acid (1 microM) suppressed eosinophil migration by alveolar macrophage supernatants (94.1 +/- 2.6% of inhibition, n = 6). Desensitization of eosinophils by and to LTB4 (10 nM) inhibited migration induced by supernatants from stimulated alveolar macrophages (87.5 +/- 5.4% of desensitization toward LTB4 and 83.1 +/- 5.4% of desensitization toward supernatants, n = 5). Under the present experimental conditions, LTB4 is the only agent implicated in eosinophil migration induced by supernatants from fMLP-stimulated alveolar macrophages.

Selective Mycobacterium avium-induced production of nitric oxide by human monocyte-derived macrophages.

Infection with a virulent strain of Mycobacterium avium, but not with virulent Mycobacterium tuberculosis or avirulent Mycobacterium smegmatis, induced the formation of nitric oxide by human monocyte-derived macrophages. This process was not affected by lipopolysaccharide or cytokines such as interferon-gamma or tumor necrosis factor alpha. M. avium-induced nitric oxide production was significantly decreased by NG-monomethyl-L-arginine, a potent inhibitor of nitric oxide synthase activity, without any significant enhancement of intramacrophagic mycobacterial growth. Infection with all the three mycobacterial species induced a significant activation of phospholipase A2 activity of macrophages as evidenced by the increased release of thromboxane A2. Finally, nitric oxide production by human monocyte-derived macrophages required infection with live M. avium, as neither gamma-irradiated M. avium nor the subcellular fractions of this microorganism (cell wall, cytosol) were able to trigger nitric oxide synthesis.

Role of PAF in the allergic pleurisy caused by ovalbumin in actively sensitized rats.

Selective platelet-activating factor (PAF) antagonists and autodesensitization to this lipid were used to investigate the role of PAF in antigen-induced pleurisy in the rat. Pleural inflammation was triggered by the intrathoracic (i.t.) injection of ovalbumin (12 micrograms/cavity) into animals actively sensitized 14 days before. Successive daily i.t. injections of PAF (1 microgram/cavity) led to selective autodesensitization, which was apparent after the third injection and maximal after the fifth. The PAF antagonists BN 52021 and WEB 2086 inhibited the late pleural eosinophil accumulation caused by antigen but, as also noted with WEB 2170, failed to modify the early antigen-induced plasma exudation and leukocyte infiltration. In contrast to the antagonists, desensitization to PAF was clearly effective against these early alterations. To further investigate this discrepancy, the antigenic challenge was performed 24 h after a single prestimulation with PAF, when sensitivity to the lipid was still intact. Under this condition, plasma exudation and cellular influx triggered by the antigen were also abrogated, indicating that this protective effect was accounted for by a mechanism other than refractoriness to PAF. Because 24 h after PAF injection only eosinophil counts remained elevated, an alternative eosinophilotactic substance was used to further study the mechanism of PAF versus antigen-induced pleural inflammation. Prior treatment with the peptide Ala-Gly-Ser-Glu (ECF-A, 20 micrograms/cavity) also inhibited the allergic pleurisy, whereas the noneosinophilotactic substances histamine (200 micrograms/cavity) and serotonin (100 micrograms/cavity) were inactive. Furthermore, drugs that share the ability to impair PAF-induced eosinophilia, including azelastine and cetirizine, prevented the inhibitory effect of PAF on the antigen-induced pleurisy. These findings suggest that PAF may account for the late eosinophilia, but not for the acute phase of the rat allergic pleurisy, which is clearly attenuated by PAF or ECF-A pretreatment.

Gamma-thrombin-induced phospholipase A2 activation in rabbit platelets: comparison with alpha-thrombin.

gamma-Thrombin stimulated release of [3H]arachidonic acid ([3H]AA) accompanied by a significant production of PAF and lyso-PAF by rabbit platelets. These responses, which reflect PLA2 activation, were observed after a prolonged lag and to a lower extent when compared to those induced by alpha-thrombin which evoked a much higher elevation in intracellular calcium. This elevation together with [3H]AA release were markedly reduced by EDTA. However, addition of ionophore A23187 enhanced the release of [3H]AA by gamma-thrombin to the levels similar to those of alpha-thrombin. We conclude that gamma-thrombin is able to activate PLA2 and suggest that calcium influx may be a limiting factor for this activation.

The effects of allergen and anti-allergic drugs on murine hemopoietic cells: moving targets, unusual mechanisms, and changing paradigms.

We used a variety of techniques to evaluate the effects of airway allergen exposure in mice on the responses of hemopoietic cells to cytokines and drugs in vitro and in vivo. Initial studies have shown that allergen exposure of sensitized mice leads to release of circulating mediators, that induce rapid upregulation of bone-marrow responses to IL-5 and GM-CSF. This may be related to glucocorticoids, because exogenous dexamethasone has similar effects on cultured murine bone-marrow, and because stress-induced glucocorticoids, in naïve or sensitized mice, have effects indistinguishable from those of allergen challenge in sensitized animals. Upregulation of eosinophil production is associated with an increased expression of alpha4 integrins, which may contribute to retention of these cells in the bone-marrow. Glucocorticoids regulate the adhesiveness, maturation and survival of eosinophils in murine bone-marrow culture, partly by counteracting the actions of Prostaglandin E2 and possibly other prostanoids. Allergen exposure of sensitized mice leads to accumulation of hemopoietic progenitors in the lungs, which differ from those in bone-marrow in growth properties and sensitivity to glucocorticoids. Lung transplantation has been used to demonstrate that the lung acts as a source of endocrine factors that promote hemopoietic cell accumulation, independently of damage caused by local allergic inflammation.

Degranulation of rabbit platelets with PAF-acether: a new procedure for unravelling the mode of action of platelet-activating substances.

Aggregation and secretion of ATP induced by thrombin, collagen, the snake venom component convulxin and platelet-activating factor (PAF-acether) were studied after the exposure of rabbit platelets to 1 microM of PAF-acether. This concentration, which is around 6 orders of magnitude above the concentration needed to induce full aggregation, was required to remove most of the releasable ATP from the platelets. The depleted platelets aggregated to PAF-acether, to thrombin and to convulxin under conditions where only very low amounts of ATP were secreted, confirming that these agents do not require the release of dense body components to trigger aggregation. Furthermore, when exposure to PAF-acether was associated to inactivation of platelet cyclooxygenase with aspirin, aggregation to thrombin persisted, validating the claim that thrombin induces aggregation by a third pathway unrelated to ADP and to thromboxane A2. Aggregation by collagen was markedly reduced by exposure of the platelets to PAF-acether or to aspirin; when both procedures were associated, aggregation was suppressed. Failure to desensitize the rabbit platelets to PAF-acether upon exposure to high amounts of it indicates the absence of irreversible membrane changes due to PAF-acether, and allows its use as a depleting procedure for the dense body materials, which does not affect platelet membrane components as is the case for thrombin.

PAF-acether may not mediate the third pathway of platelet aggregation since self-desensitization reduces the effects of low thrombin but enhances those of convulxin.

PAF-acether (platelet-activating factor) was hypothesized as the mediator of the ADP and thromboxane-independent activation of platelets induced by thrombin (Thr) and by the snake venom glycoprotein convulxin (Cx). Aspirinized rabbit platelets self-desensitized to PAF-acether were less responsive to low amounts of Thr, as expected if PAF-acether would be formed, but were hyper-reactive to Cx, in contradiction with its hypothesized mediating role. Aggregation by higher concentrations of Thr overcame inhibition. Experiments with ADP-depleted platelets showed that secretion is neither involved with desensitization to PAF-acether nor with hyper-reactivity to Cx. Those effects required the presence of PAF-acether in the platelet suspension and persisted when transformation of PAF-acether into its recognized metabolite alkyl-acyl-glycerophosphorylcholine was inhibited. The ADP and thromboxane-independent activation of rabbit platelets by low and medium concentrations of Thr may be accounted for by platelet formation of PAF-acether, but overall the contrasting effects of platelet desensitization to PAF-acether on responsiveness to Thr and to Cx suggest that the third pathway of aggregation requires other explanations.

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. Salicyclic acid may be useful in studies of potential inhibitors of thromboxane A2 synthesis and of thromboxane A2-dependent processes in vivo and in vitro.

Inhibition by non-steroid anti-inflammatory agents of rabbit aorta contracting activity generated in blood by slow reacting substance C.

1 A crude and a partially purified preparation of slow reacting substance C (SRS-C) as well as arachidonic acid decreased resistance to perfusion of the dog hind paw. This effect was suppressed by treatment with non-steroid anti-inflammatory drugs.2 Injections of SRS-C or of arachidonic acid induced marked and reproducible contractions of strips of rabbit aorta and a rat stomach which were bathed in blood from an anaesthetized dog. The effect on the rabbit aorta is attributed to formation of a rabbit aorta contracting substance (RCS). The contractions were suppressed when the dog was treated with a non-steroid anti-inflammatory drug.3 Incubation of blood or of platelet-rich plasma with SRS-C or arachidonic acid resulted in the formation of similar materials. This formation was suppressed by anti-inflammatory drugs.4 SRS-C, linoleic, linolenic, and arachidonic acids are suitable substrates for soybean lipoxidase for the generation of RCS.5 It is suggested that RCS and prostaglandin are formed within platelets, when SRS-C or arachidonic acid are injected into animals or added in vitro. Non-steroid anti-inflammatory drugs suppress these effects, possibly by inhibiting prostaglandin synthetase.