Effects of NZ-107 on Airway Inflammation and Cell Activation in Guinea-pigs

Abstract— The effects of NZ-107 on some airway inflammation models and the generation of superoxide anion (O₂^?) were studied in guinea-pigs. Airway inflammation was caused by intra-tracheal injection of murine recombinant interleukin-5 (mrIL-5, 15 μg/animal), inhalation of platelet-activating factor (PAF, 0·003%) and intra-tracheal injection of leukotriene B₄ (LTB₄, 10 μg/animal). NZ-107 (4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone) at a dose of 50 mg kg^?1, intraperitoneally reduced mrIL-5- and PAF-induced eosinophilia. This compound at a dose of 25 and 50 mg kg^?1 also suppressed LTB₄-induced eosinophilia and neutrophilia in bronchoalveolar lavage fluid (BALF). On the other hand, prednisolone at a dose of 20 mg kg^?1, i.p., prevented the increased number of macrophages, eosinophils and neutrophils induced by mrIL-5, the increased number of eosinophils induced by PAF and the increased number of eosinophils and neutrophils induced by LTB₄ in BALF. Furthermore, both drugs reduced mrIL-5- or PAF-induced increase in the number of airway epithelial cells in BALF. The generation of O₂^? was measured by the method of cytochrome C reduction. NZ-107 (10–100 μg mL^?1) attenuated PAF- and FMLP-induced O₂^? production from macrophages and reduced PAF-induced O₂^? generation by eosinophils but had no effect on that from neutrophils. These results indicate that NZ-107 prevents the increased number of pulmonary eosinophils and airway epithelial cells and the activation of macrophages and eosinophils, suggesting that NZ-107 may be useful as a remedy for airway inflammatory diseases such as bronchial asthma.     

Effect of murine recombinant interleukin-5 on the cell population in guinea-pig airways.

1. An intratracheal injection of murine recombinant interleukin 5 (mrIL-5, 2-15 microgram/0.25 ml/animal) induced a dose-dependent increase in the number of macrophages, eosinophils, neutrophils and epithelial cells in the bronchoalveolar lavage fluid (BALF) of guinea-pigs 24 h after administration. Bovine serum albumin (15 micrograms/0.25 ml/animal), used as a reference material, did not cause any change of this type. 2. The intratracheal administration of mrIL-5 at a dose of 15 microgram showed a tendency to increase the number of these pulmonary inflammatory cells and epithelial cells in the BALF at 12 h with a significant increase observed at 24 h. 3. Prednisolone (20 mg kg-1, i.p.) inhibited the mrIL-5-induced increase in macrophages, eosinophils, neutrophils and epithelial cells. Ketotifen (2 mg kg-1, i.p.) reduced the mrIL-5-induced increase in the eosinophil, neutrophil and epithelial cell populations. The simultaneous injection of 2% disodium cromoglycate (DSCG) into the trachea prevented the mrIL-5-induced increase in the number of airway epithelial cells, without affecting changes in the other inflammatory leukocytes. 4. These results suggest that mrIL-5 is a potent inducer of lung inflammation, in terms of increased inflammatory leukocytes and epithelial cells in guinea-pig BALF. Prednisolone, DSCG and ketotifen are effective against mrIL-5-induced pulmonary inflammation, especially the desquamation of bronchial epithelial cells.     

Interference of cetirizine with the late eosinophil accumulation induced by either PAF or compound 48/80.

1. The effect of topical or systemic treatment with the histamine H1-receptor antagonist, cetirizine, on the rat pleural eosinophil accumulation induced by PAF or compound 48/80 was investigated. The number of pleural resident eosinophils increased 6 h after the intrathoracic (i.t.) injection of PAF (1 microgram/cavity), peaked within 24 h and persisted significantly augmented for at least 96 h. Compound 48/80 (25 micrograms/cavity) also produced a long lasting pleural eosinophilia but this was first noted only 24 h after stimulation. 2. Intraperitoneal pretreatment with cetirizine inhibited eosinophilia induced by either PAF (ED50 = 19 mg kg-1) or compound 48/80 (ED50 = 14 mg kg-1) whereas meclizine, another histamine H1-receptor antagonist, was inactive. 3. Administered locally, cetirizine (5 and 15 micrograms/cavity) also dose-dependently inhibited both PAF- and compound 48/80-induced eosinophilia, providing evidence that its inhibitory effect is not due to any action upon circulating eosinophils. Consistent with this result, incubation of isolated peritoneal eosinophils with cetirizine failed to modify in vitro eosinophil migration caused by PAF. 4. Since the late eosinophilia induced by PAF may depend on the synthesis of a transferable protein mediator, cetirizine was administered to donor or recipient rats in order to determine whether it interferes with the generation or with the expression of this protein. We showed that only the treatment of recipient rats abolished the transfer of the eosinophilotactic activity, indicating that cetirizine does not modify the generation but inhibits the expression of this activity. 5. Our findings indicate that cetirizine is able to inhibit eosinophil accumulation by acting locally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of prostaglandins in the down-regulation of allergic plasma leakage observed in rats undergoing pleural eosinophilia.

1. Recent evidence has implicated eosinophils in the inhibition of allergen-induced rat pleurisy, but the mechanism of this negative modulation is not completely understood. This study was undertaken in order to define the potential role of prostaglandins in this phenomenon. 2. Wistar rats were actively sensitized by subcutaneous injection of a mixture of ovalbumin and AI(OH)3 and challenged with an intrapleural (i.pl.) injection of ovalbumin (12 micrograms/cavity) 14 days later. 3. Analysis of the pleural fluid effluent revealed a massive mast cell degranulation and plasma protein extravasation 4 h post-challenge. We confirmed that concurrently with selective pleural fluid eosinophilia caused by platelet-activating factor (PAF), the pleural cavity became hyporesponsive to allergen-induced protein exudation and to the parallel reduction in the number of intact mast cells. 4. These hyporesponsive animals presented a significant augmentation in the pleural effluent level of prostaglandin E2 (PGE2), which increased with increasing numbers of eosinophils in the pleural cavity. Furthermore, pretreatment with either indomethacin or aspirin failed to modify allergen-induced exudation but reversed the exudatory hyporesponsiveness associated with eosinophil recruitment. 5. Allergic exudation was clearly down-regulated by the following pretreatments: (i) PGE2 (10 micrograms/cavity, i.pl.) plus rolipram (40 micrograms/cavity, i.pl.), (ii) misoprostol (200 micrograms kg-1, p.o.) or (iii) dibutyryl cyclic AMP (80 micrograms/cavity, i.pl.). 6. We conclude that prostaglandins may be implicated in the eosinophil-mediated inhibition of allergic pleurisy, probably acting via cyclic AMP signalling pathway activation.     

Eosinophil-eicosanoid interactions: inhibition of eosinophil chemotaxis in vivo by a LTD4-receptor antagonist

Chemotaxis of guinea-pig eosinophils in vivo has been studied in the guinea-pig conjunctiva. Guinea-pig eosinophils were labelled with 111In oxine and injected i.v. into recipient animals. Circulating radioactivity remained constant over a period of 4 h but dropped transiently in response to a bolus injection (1 nmol) of platelet activating factor (PAF), suggesting that the 111In-labelled eosinophils had retained their responsiveness to PAF. Leukotriene D4 (LTD4, 1 nmol/eye) and PAF (10 nmol/eye), but not histamine (5 nmol/eye), induced a significant 2.5-fold increase in conjunctival radioactivity, a measure of eosinophil chemotaxis in vivo, after 17 h. Antigen challenge (250 micrograms/eye) in ovalbumin-sensitized animals produced larger responses (8-fold increases) at the same time point. A specific LTD4 receptor antagonist MK-571 (10 micrograms/eye) completely inhibited in vivo chemotactic responses to LTD4, and partially inhibited (54%) the responses to ovalbumin (observations subsequently confirmed by histological studies). As eosinophils may play an important role in allergic diseases, the results with MK-571 indicate that selective and potent LTD4 receptor antagonists may provide a novel therapy for allergic conjunctivitis, rhinitis and asthma.     

Long-lasting inhibitory activity of the hetrazepinic BN 50730 on exudation and cellular alterations evoked by PAF and LPS.

1. Inhibitory effects of the hetrazepinic derivative BN 50730 on the rat pleural inflammatory response, triggered by PAF or lipopolysaccharides (LPS), were examined. The type of pharmacological blockade exerted by this antagonist in in vitro assays of eosinophil chemotaxis and platelet aggregation were also investigated. 2. Intrathoracic injection of PAF (1 microgram per cavity) caused a 4 fold increase in the extravasated protein within 15 min and led to a marked eosinophil accumulation 24 h post-challenge. BN 50730 (0.5-10 micrograms per cavity) inhibited exudation by PAF dose-dependently without modifying the response induced by histamine, bradykinin or 5-hydroxytryptamine (5-HT). 3. The kinetics of the inhibitory effect on exudation revealed that the actions of WEB 2086 and BN 52021 (10 micrograms per cavity) were over within 2 and 4 h respectively, whereas BN 50730 (10 micrograms per cavity) retained 80% of its inhibitory activity for 4 days. 4. Oral treatment with BN 50730 (10-20 mg kg-1, 1 h beforehand) suppressed the leucocyte accumulation and late eosinophilia observed 6 and 24 h after PAF respectively, but did not modify the eosinophilia induced by leukotriene B4 (LTB4) or bradykinin. BN 50730 also failed to reduce the eosinophil accumulation induced by LPS but drastically inhibited the neutrophil influx. 5. The pre-incubation of rat peritoneal eosinophils for 10 min with BN 50730 (30 nM-1 microM) dose-dependently inhibited the chemotaxis induced by PAF (0.1 microM) in vitro. The IC50 values for BN 52021, WEB 2086 and BN 50730 in this system were 5, 5 and 0.05 microM respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

银杏苦内酯对过敏性豚鼠气道内低密度嗜酸性粒细胞增多的调制作用

用卵蛋白吸入诱导豚鼠哮喘动物模型（哮喘组），然后，进行支气管肺泡灌洗，分析支气管肺泡灌洗细胞学改变。哮喘组支气管肺泡灌洗液中的细胞总数、嗜酸性粒细胞绝对计数及低密度嗜酸性粒细胞比例均显著高于正常对照组（Ｐ＜０．０５、Ｐ＜０．０１）。哮喘动物模型在吸入抗原激发之前，用血小板活化因子特异性拮抗剂银杏苦内酯（ＢＮ５２０２１）预处理后，支气管肺泡灌洗液中细胞总数、分类和嗜酸性粒细胞数量均与哮喘组差异无显著性。但低密度嗜酸性粒细胞比例却显著降低（Ｐ＜０．０５）。用地塞米松预处理动物模型（激素组）后，支气管肺泡灌洗液中的细胞总数、嗜酸性粒细胞数、细胞分类及低密度嗜酸性粒细胞比例均降至正常对照组水平。结果表明：银杏苦内酯能显著减少哮喘动物气道内“活化”状态的低密度嗜酸性粒细胞，可能是一种有用的哮喘抗炎药物。     

Effect of antiallergic drugs on interleukin 5-induced eosinophil infiltration of rat airways

Interleukin (IL)-5 is thought to play important roles in asthma and to be a potential therapeutic target. An intratracheal injection of murine recombinant IL-5 (3-30 microg/animal) induced a dose-dependent increase in the number of eosinophils in the bronchoalveolar lavage fluid of Brown Norway (BN) rats 24 h after administration. Bovine serum albumin (30pg/animal), used as reference material, did not cause any change. The reaction was not observed in F344 rats. The increase in the number of eosinophils did not accompany bronchial hyperreactivity in BN or F344 rats. Prednisolone (3-10 mg/kg, i.p.) and emedastine (30 mg/kg, p.o.) reduced the increased number of eosinophils induced by the IL-5 challenge. These results suggest that IL-5 is a potent inducer of eosinophils in the airway of BN rats. Prednisolone and emedastine are effective against IL-5-induced eosinophilia.     

Predominance of endothelinA (ETA) receptors in ovine airway smooth muscle and their mediation of ET-1-induced contraction.

1. Platelet activating factor (PAF), leukotriene B4 (LTB4) and interleukin-5 (IL-5) are potent chemoattractants for guinea-pig eosinophils, which may be involved in eosinophil recruitment and up-regulation in allergic diseases. Eosinophils from the bronchoalveolar lavage fluid (BALF) of ovalbumin-sensitized guinea-pigs were collected 24 h after antigen provocation and migration induced by PAF, LTB4 and rhIL-5 was studied. 2. Total BALF content and distribution of eosinophils were greater in immunized, ovalbumin-challenged guinea-pigs (5.0 +/- 0.8 x 10(6)/guinea-pig; 12 +/- 1%) than in immunized, saline-challenged animals (3.0 +/- 0.7 x 10(6)/guinea-pig; 7 +/- 1%). 3. The chemoattraction of eosinophils isolated on a metrizamide gradient was studied in micro-Boyden chambers, results being expressed as the number of migrating cells (mean +/- s.e. mean). PAF and LTB4-induced migration of eosinophils from immunized and OA-challenged guinea-pigs were significantly enhanced, as compared to immunized and saline-challenged animals (170 +/- 36 vs 35 +/- 9 migrating eosinophils for 10 nM PAF; 271 +/- 60 vs 110 +/- 19 for 1 nM LTB4). 4. The IL-5 antibody TRFK-5, in vivo, reduced eosinophil recruitment in BALF of antigen-challenged immunized animals as well as the enhanced responsiveness of eosinophils from the challenged animals, suggesting a role for IL-5 in the priming of eosinophils in vivo. 5. In contrast to TRFK-5, nedocromil sodium reduced to a similar extent eosinophil, macrophage and lymphocyte recruitment into the BALF of antigen-challenged, but failed to down-regulate the enhanced responsiveness of eosinophils from the challenged animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eosinophil accumulation in pulmonary airways of guinea-pigs induced by exposure to an aerosol of platelet-activating factor: effect of anti-asthma drugs.

1. Exposure of guinea-pigs to aerosols of platelet activating factor (PAF) (0.01 to 100 micrograms ml-1) induced a dose-dependent increased incidence of eosinophils in bronchoalveolar lavage fluid (BAL) at 48 h. Total leucocyte numbers and the percentages of lymphocytes and neutrophils were unchanged in BAL fluid. 2. Increased numbers of eosinophils were detected in BAL 1 h after exposure to PAF but eosinophilia was not maximal until 48 h. One week after exposure to PAF, the percentage of eosinophils in BAL was within the normal range. 3. Depletion of circulating platelets or neutrophils by intravenous injection of specific antisera did not modify accumulation of eosinophils in the airway lumen following inhalation of PAF (10 micrograms ml-1). 4. PAF-induced pulmonary airway eosinophil accumulation was inhibited by treatment with SDZ 64-412, a selective PAF-antagonist, whether the compound was administered before, or 30 min after, inhalation of PAF. 5. Pulmonary airway eosinophil accumulation due to inhaled PAF (10 micrograms ml-1) was inhibited by prior treatment with aminophylline, cromoglycate, ketotifen, dexamethasone and AH 21-132. 6. Pulmonary airway eosinophil accumulation due to inhaled PAF (10 micrograms ml-1) was not inhibited by prior treatment with indomethacin, salbutamol or mepyramine.     

Characterization and pharmacological modulation of antigen-induced peritonitis in actively sensitized mice.

1. The intraperitoneal (i.p.) injection of 1 or 10 micrograms ovalbumin to sensitized Balb/c mice led to an acute histamine release, firstly evidenced 1 min after the challenge and returning to basal levels 30 min thereafter. This phenomenon was unaccompanied by protein extravasation. A dose-dependent increase in the amounts of immunoreactive leukotriene (LT) C4 and LTB4 was observed in the peritoneal washing from sensitized mice 6 h after 1 or 10 micrograms ovalbumin administration. In separate experiments, the i.p. administration of 1 mg activated zymosan to non-immunized mice was followed by a marked protein extravasation, and by immunoreactive LTC4 and LTB4, but not histamine, release in mouse peritoneum 1 h after its injection. 2. Mediator release in the mice peritoneal cavity was concomitant with a transient neutrophil infiltration, which peaked at 6 h and returned to basal levels therefore. An intense eosinophil accumulation starting at 24 h, peaking at 48 h and returning to basal values at 164 h, was also observed. 3. Ovalbumin (1 microgram)-induced eosinophilia, observed at 24 h, was reduced by the pretreatment of the animals with dexamethasone (1 mg kg-1, s.c.) or with the 5-lipoxygenase inhibitor, BWA4C (20 mg kg-1, s.c.), whereas indomethacin (2 mg kg-1, s.c.) and the platelet-activating factor (PAF)-antagonist SR 27417 (10 mg kg-1, s.c.) were ineffective. These results indicate that metabolites of arachidonic acid of lipoxygenase pathway, but not cyclo-oxygenase derivatives or PAF, mediate antigen-induced eosinophil accumulation in the mouse peritoneum.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-5 accounts for the mouse pleural eosinophil accumulation triggered by antigen but not by LPS

The involvement of interleukin-5 (IL-5) in the pleural eosinophilia induced by LPS or allergen was investigated. The number of pleural eosinophils in actively sensitized mice increased 24 h after the intrathoracic (i.t) injection of ovalbumin (12 mg/cavity), peaked within 72 h, and persisted significantly increased for at least 120 h. Despite being less intense, the i.t. injection of LPS (250 ng/cavity) also increased the number of pleural eosinophils at 24 h, returning to basal levels within 72 h. Intraperitoneal pretreatment with monoclonal antibody to IL-5 (TRFK-4 and TRFK-5, 500 mg/kg) suppressed the eosinophil accumulation induced by IL-5 (200 units/cavity) or ovalbumin, but had no effect on the LPS-induced eosinophilia. Transfer of the cell-free pleural washing from LPS-treated donor mice to naive recipient animals led to a selective increase in the eosinophil counts. The co-incubation of the pleural washing from LPS-treated animals with monoclonal antibody to IL-5 failed to modify the pheonomenon. The results indicate that IL-5 plays an important role in the antigen-induced accumulation of eosinophils in vivo, but not in the eosinophilia triggered by LPS.     

Effect of a novel anti-allergic agent, HSR-609, on antigen-induced airway hyperresponsiveness in mice

The effects of a newly synthesized anti-allergic agent, HSR-609, on allergic airway hyperresponsiveness and airway inflammation have been studied in sensitized mice. The effects were compared with those of two histamine H(1) receptor antagonists, cetirizine and terfenadine, and prednisolone. Three inhalations of antigen caused an increase in leukocytes (including eosinophils) with increases in IL-5 in BALF and airway hyperresponsiveness to acetylcholine in BALB/c mice. All drugs were orally administered once a day for 10 days from the day before the first inhalation of antigen. HSR-609 (10 mg/kg) and prednisolone (5 mg/kg) significantly inhibited the antigen-induced airway hyperresponsiveness, whereas cetirizine (10 mg/kg) or terfenadine (100 mg/kg) did not affect this airway response. At the same time HSR-609 inhibited the antigen-induced eosinophilia and IL-5 production in BALF. Prednisolone also showed an inhibitory effect on the airway eosinophilia and IL-5 production but not cetirizine and terfenadine in the same experiments. In addition, HSR-609 (p.o.) dose-dependently suppressed the accumulation of eosinophils elicited by antigen-stimulated D10G4.1 cells, a murine Th2 clone, in peritoneal cavity lavage fluid in AKR/J mice. These results suggest that HSR-609 inhibits allergic airway hyperresponsiveness to acetylcholine probably because of the inhibition of Th2-dependent eosinophilia caused by IL-5. In addition, effects of HSR-609 were different from those of cetirizine and terfenadine concerning the inhibition of antigen-induced airway hyperresponsiveness in mice.     

Nedocromil sodium prevents in vivo generation of the eosinophilotactic substance induced by PAF but fails to antagonize its effects.

1 The intrathoracic injection of platelet activating factor (PAF) into rats induced a decrease in the pleural leucocyte numbers within 15 min, accompanied by a marked exudation, maximal 1 h later. After 6 h, concomitantly with the reduction of exudation, a marked increase in the number of mononuclear cells, neutrophils and eosinophils was observed. Within 24 h, the pleural eosinophil accumulation peaked and persisted up to 96 h. 2 Topical treatment with nedocromil sodium affected pleural exudation by PAF under conditions where systemic meclizine was ineffective. Nedocromil sodium blocked, dose-dependently, the increase in the pleural content of mononuclear cells, neutrophils and eosinophils, observed 6 h after PAF administration, as well as the eosinophilia 24 h later. Moreover, the co-incubation of peritoneal eosinophils with nedocromil sodium did not interfere with the migration triggered by PAF. 3 The transfer of the 6 h-PAF pleural washings from donor to recipient rats caused a selective pleural eosinophilia, which was clearly inhibited when nedocromil sodium was administered to donor, but not to recipient animals, showing that this drug interferes with the generation rather than with the expression of the eosinophilotactic activity(ies). 4 These findings indicate that the nedocromil sodium interferes with PAF-induced exudation and leucocyte accumulation, by a mechanism other than its ability to reduce the local effects of histamine and which may relate to suppression of the eosinophilotactic principle generation.     

Systemic and local dexamethasone treatments prevent allergic eosinophilia in rats via distinct mechanisms.

We have studied the effect of local and systemic treatment with dexamethasone for prevention of the pleural eosinophilia triggered by allergen in actively sensitised Wistar rats. Parallel changes in blood and marrow eosinophil numbers were assessed for comparison. The intrapleural (i.pl.) injection of ovalbumin into ovalbumin-sensitised animals led to a long-lasting pleural fluid eosinophilia which peaked from 24 to 72 h post-challenge. At these time points, there was a significant 2- to 3-fold increase in the blood eosinophil numbers, whereas the bone marrow number of mature eosinophils remained unaltered. Systemic treatment with dexamethasone (0.05-0.5 mg/kg, i.p.) abolished the pleural and blood eosinophilia observed 24 and 48 h post-challenge, also causing a significant reduction in marrow eosinophil numbers. Despite being unable to alter blood and bone marrow eosinophil numbers, the local i.pl. administration of dexamethasone (2.5-10 microg/cavity) inhibited dose dependently the allergen-induced pleural eosinophil influx at 24 h but not at 48 h post-challenge. This treatment also shortened the time course of eosinophil accumulation in the pleural space from the 48 h time point on. We conclude that the effect of systemic but not of local treatment with dexamethasone on allergen-induced eosinophil recruitment is well correlated with the inhibition of eosinophil production in bone marrow. In contrast, low amounts of dexamethasone injected into the pleural space seem to affect locally eosinophil recruitment and survival.     

Activity of a novel thienodiazepine derivative as a platelet-activating factor antagonist in guinea pig lungs. Effects on platelet-activating factor and allergen induced eosinophil accumulation

Platelet-activating factor (PAF) inhalation in guinea pigs caused a significant increase in the number of eosinophils recovered from bronchoalveolar lavage fluid (BALF). Oral administration of (S)-(+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8,11- dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3':4,5]thieno[3,2-f] [1,2,4]triazolo[4,3-a][1,4]diazepine (E-6123), a novel PAF antagonist, at the dose of 100 micrograms/kg completely inhibited the PAF-induced eosinophil accumulation. Antigen inhalation in passively sensitized guinea pigs caused a significant increase in lung contents of PAF at 5 min, and accumulation of eosinophils in the bronchi 1 and 2 days thereafter, E-6123 inhibited the antigen-induced eosinophil accumulation and the maximum inhibition was approximately 65%. On the other hand, methylprednisolone completely inhibited the antigen-induced eosinophil accumulation. The results suggest that PAF is a potent attractant of eosinophils and is involved in antigen-induced eosinophil infiltration into bronchi. The results also suggest that E-6123 may be of therapeutic value in the treatment of asthma exhibiting eosinophil recruitment in airways.     

Effect of human recombinant interleukin-5 on in vitro responsiveness to PAF of lung from actively sensitized guinea-pigs.

1. The intra-tracheal (i.t.) administration of human recombinant interleukin-5 (rhIL-5; 100 or 300 ng) to isolated perfused lungs from guinea-pigs actively sensitized to ovalbumin induced an increased bronchoconstriction and release of thromboxane A2 (TXA2) and histamine into the lung effluent following the subsequent (10 min) intra-arterial injection of platelet-activating factor (PAF). Lung responses to 5-hydroxytryptamine were unaffected by rhIL-5. 2. Hyperresponsiveness to PAF was observed when the lungs were obtained from guinea-pigs used 2 or 7 days after a booster injection of the antigen and, to a lower extent, when they were from animals sensitized by a single antigen administration. By contrast, rhIL-5 did not modify the responses to PAF of lungs from passively sensitized or from adjuvant-treated guinea-pigs, suggesting that immunological stimulation is required to allow the expression of synergism between rhIL-5 and PAF. 3. Guinea-pigs killed 2 and 7 days after the booster injection of the antigen exhibited a marked increase in the number of eosinophils in the bronchoalveolar lavage fluid (BAL), as compared to non-sensitized animals. 4. Our results demonstrate that rhIL-5 and PAF act synergistically to induce enhanced bronchoconstriction and mediator release exclusively when lungs are obtained from guinea-pigs sensitized once to ovalbumin and then boosted. Since recruitment of eosinophils into the airways and the development of hyperresponsiveness to PAF are concomitant, it is suggested that eosinophils are the target cells for interaction between rhIL-5 and PAF.     

Granulocyte chemotaxis in the canine trachea: inhibition by lipid mediator antagonists and systemic inhibitors.

Inflammation of the airways contributes to the multicomponent disease known as asthma. The primary cells that infiltrate the airways in response to antigen exposure are PMNs and eosinophils, cells that can release cellular components, and damage the airways. We adapted a double-balloon endotracheal tube to study the cellular response to three de novo synthesized lipid mediators (LTB4, PAF-acether and 15 HETE) found in respiratory fluids following antigen exposure. In random repeat challenges in groups of 7 dogs using mongrel dogs at 240 min following exposure to 10(-6) M agonists, the PMN content of the perfused fluid was 870 +/- 240, 1632 +/- 883, 515 +/- 395, and 1575 +/- 214 cells/ml/5 high power fields for vehicle, LTB4, PAF, and 15 HETE respectively. Eosinophils that infiltrated the lumen at 240 min were 162 +/- 23, 608 +/- 287, 502 +/- 23, 115 +/- 14 cells/ml/5 HPF for vehicle, LTB4, PAF, and 15 HETE respectively. Thus LTB4 and PAF-acether significantly (p less than 0.05) increased eosinophils, and LTB4 and 15 HETE increased PMNs (p less than 0.05). After determining the agonist response for the 3 agonists we included 2 specific antagonists in the perfusate. The LTB4 antagonist U-75,302 10(-5) M, and the PAF antagonist L 652,731 10(-5) M in chambers containing LTB4 and PAF-acether respectively blocked significantly the influx of PMNs and eosinophils compared to vehicle (p less than 0.01). Methylprednisolone 5 mg/kg i.m.--18 hrs blocked eosinophilia to PAF and LTB4. Oral U-78,517F a Trolox amine lazaroid, active as an inhibitor of lipid peroxidation, 30 mg/kg--18 hrs significantly blocked eosinophilia to PAF-acether and LTB4 directed chemotaxis compared to vehicle (p less than 0.05) but not 15 HETE. Specificity was shown for each antagonist since the PAF and LTB4 antagonists did not block the opposite agonist. Use of this novel in vivo chemotaxis model allows the additional advantage of studying chemotaxis in living tissue.     

Pretreatment with rh-GMCSF, but not rh-IL3, enhances PAF-induced eosinophil accumulation in guinea-pig airways.

Intraperitoneal injections of recombinant human granulocyte-macrophage colony stimulating factor (rh-GMCSF, 50 micrograms/kg-1 daily) or interleukin-3 (rh-IL3, 50 micrograms kg-1 daily) for two days, induced an increase in the percentage of bone marrow and pulmonary airway eosinophils in the guinea-pig. In addition, rh-IL3-treated animals exhibited an increase (21%) in blood neutrophils. Exposure of guinea-pigs to an aerosol of platelet activating factor (PAF) gives rise to a selective pulmonary eosinophil accumulation, maximal at 48 h. The eosinophilic response to PAF was significantly enhanced in rh-GMCSF-treated guinea-pigs but was suppressed in rh-IL3-treated animals.     

Effects of ZCR-2060 on Allergic Airway Inflammation and Cell Activation in Guinea-pigs

Abstract— The effects of 2-(2-(4-(diphenylmethyl)-1-piperadinyl) ethoxy) benzoic acid malate (ZCR-2060) on allergic airway inflammation and inflammatory cell activation in guinea-pigs were studied. Allergic airway inflammation was induced by inhalation of antigen into actively-sensitized animals and the increase in inflammatory cells into bronchoalveolar lavage fluid (BALF) was measured. Aeroantigen-induced infiltration of inflammatory cells, especially eosinophils and neutrophils, in BALF gradually increased, and reached a peak at 6 or 9 h after the challenge. ZCR-2060 (1 mg kg^?1 p.o.) clearly inhibited the increase of eosinophil numbers in BALF. Moreover, the effect of ZCR-2060 on inflammatory cell activation in terms of chemotaxis and superoxide generation in-vitro was studied. ZCR-2060 (10^?6-10^?4 m ) inhibited the platelet-activating factor (PAF)-induced chemotaxis of eosinophils and neutrophils, but did not inhibit the leukotriene B₄-induced chemotaxis of eosinophils and the formyl-Met-Leu-Phe-induced chemotaxis of neutrophils. PAF-induced superoxide anion generation by eosinophils, neutrophils and alveolar macrophages was inhibited by ZCR-2060 (10^?6-10^?4 m ). However, ZCR-2060 did not affect phorbol myristate acetate-induced superoxide anion generation by eosinophils, neutrophils and alveolar macrophages. These results indicate that ZCR-2060 inhibits allergic airway inflammation, and PAF-induced inflammatory cell activation in guinea-pigs. ZCR-2060 may prove useful for the treatment of allergic airway inflammation or allergic disorders, especially inflammatory cell infiltration and activation.