Marcia C.R. Lima

Pharmacological modulation of Paf‐induced rat pleurisy and its role in inflammation by zymosan

1 The intrapleural injection of Paf‐acether into rats caused, at 30 min, a marked exudation accompanied by a reduction in the pleural leucocyte count. At 6 h, the exudate volume had decreased and a significant increase in the total leucocyte count, particularly eosinophils was noted. 2 Two Paf‐acether antagonists, WEB 2086 and 48740 RP abrogated the pleural leucopenia observed 30 min after Paf‐acether administration, whereas the exudation was inhibited only by the former. Pleurisy was also reduced by about 60% with dexamethasone, by about 45% with BW 755C or LY 171883, a mixed cyclo‐oxygenase/lipoxygenase inhibitor and a peptido‐leukotriene antagonist respectively, and by about 30% with indomethacin, flurbiprofen or piroxicam. 3 Repeated daily intrapleural injections of Paf‐acether led to a state of progressive desensitization to Paf‐acether itself, whereas responsiveness to 5‐hydroxytryptamine was maintained. In addition, the Paf‐induced auto‐desensitization was largely inhibited by WEB 2086. 4 Pleurisy induced by zymosan, but not by carrageenin, was significantly reduced in Paf‐acether‐desensitized animals. These results were consistent with those obtained with WEB 2086 which supressed zymosan‐induced but not carrageenin‐induced pleurisy. 5 This study suggests that Paf‐acether‐induced pleurisy in the rat may be mediated by lipoxygenase arachidonic acid metabolites and that pleurisy induced by zymosan, but not by carrageenin, is largely dependent upon Paf‐acether.

EFFECT OF AZELASTINE ON PLATELET-ACTIVATING FACTOR AND ANTIGEN-INDUCED PLEURISY IN RATS

The interference of azelastine with pleurisy induced by antigen was investigated in actively sensitized rats. The antigenic challenge (ovalbumin, 12 micrograms/cavity) caused early plasma leakage, which peaked within 4 h, accompanied by intense neutrophil infiltration. Pleural exudate decayed 24 h after antigen provocation, when a long-lasting increase in the number of resident eosinophils was observed. Oral pretreatment with azelastine (1-10 mg/kg) dose dependently inhibited the vasopermeation (ED50 = 4.2 mg/kg) and reduced the pleural exudate (ED50 = 6.8 mg/kg) induced by the antigen. In contrast, azelastine (10 mg/kg) failed to modify the neutrophil influx observed at 4 h and the eosinophil accumulation detected at 24 h. Azelastine was also effective against rat pleurisy induced by either platelet-activating factor (PAF-acether), histamine or serotonin. It reduced exudation and the increase in the number of mononuclear cells, neutrophils and eosinophils observed 6 h after PAF-acether. Nevertheless, antagonism of PAF-acether may not be relevant to the inhibition observed in the present model of allergic pleurisy, as the inhibition was refractory to three distinct PAF-acether receptor antagonists. In contrast, like azelastine, the histamine H1 receptor antagonist meclizine and the dual histamine and serotonin receptor antagonist cyproheptadine blocked antigen-induced exudation and failed to interfere with cell influx. We conclude that the anti-exudatory activity of oral azelastine on antigen-induced pleurisy is consistent with it exerting direct effects against vasoactive amines, but is not related to an effect against leucocyte infiltration nor to its ability to inhibit PAF-acether.

Interactions between local inflammatory and systemic haematological effects of PAF-acether in the rat.

Intraplantar injections of PAF-acether into the rat paw (0.025-16 micrograms/paw) were followed by a bell-shaped dose-response curve for oedema. In parallel to the ascending phase (0.25-2 micrograms/paw) a significant leukopenia was observed whereas thrombocytopenia, haemoconcentration and leukocytosis were seen during the descending phase (4-16 micrograms/paw) as was also observed after the i.v. injection of PAF-acether. I.v. injections of PAF-acether inhibited dose dependently the oedema induced by PAF-acether itself but were inactive against serotonin. Dexamethasone (0.1 mg/kg) inhibited the PAF-acether-induced thrombocytopenia and haemoconcentration but failed to modify the leukocytosis whereas aspirin (100 mg/kg) and indomethacin (1.0 mg/kg) inhibited only the haemoconcentration. The PAF-acether antagonist 48740 RP blocked the systemic haematological alterations and restored the auto-reduced oedema induced by a high dose of PAF-acether (16 micrograms/paw) but was unable to inhibit the paw oedema induced by PAF-acether. It is suggested that haemoconcentration, thrombocytopenia and leukocytosis are independent phenomena associated with the presence of PAF-acether in the bloodstream, these haematological changes and the local oedema induced by PAF-acether involve distinct mechanisms and the auto-inhibitory property of PAF-acether is not restricted to in vitro situations, but extends to local inflammation.

Pharmacological Modulation of the Late Eosinophilia Induced by Antigen in Actively Sensitized Rats

The intrathoracic injection of ovalbumin (12 micrograms/cavity) into actively sensitized rats led to a long-lasting eosinophil recruitment, which appeared 24 h after stimulation. In this study, pharmacological antagonists were used in order to evaluate the potential involvement of arachidonic acid metabolites and PAF-acether in the pleural eosinophil accumulation by antigen. Administration of the cyclooxygenase inhibitor indomethacin (2 mg/kg, i.p.), 1 h before the antigen challenge, failed to modify the 24-hour eosinophilia. In contrast, the dual cyclooxygenase and lipoxygenase inhibitor BW 755C and the more selective inhibitor BW A4C (5 and 10 micrograms/cavity, i.t.), injected 1 h before the antigen, were effective. Similarly, the PAF-acether antagonists BN 52021 and WEB 2086 (20 mg/kg, i.p.) abrogated the eosinophil accumulation, which was also sensitive to the topical treatment with the glucocorticoid dexamethasone (5 and 10 micrograms/cavity). Our findings suggest that the antigen-induced eosinophil mobilization is dependent on lipoxygenase derivatives and PAF-acether, but not on prostaglandins.

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 μg/cavity) into animals actively sensitized 14 days before. Successive daily i.t. injections of PAF (μg/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 fig/cavity) also inhibited the allergic pleurisy, whereas the noneosinophilotactic substances histamine (200 μg/cavity) and serotonin (100 μg/cavity) were inactive. Furthermore, drugs that share the ability to impair PAF‐induced eo‐ sinophilia, 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.

Pro-inflammatory activity of enterolobin: a haemolytic protein purified from seeds of the Brazilian tree Enterolobium contortisiliquum

The pro-inflammatory activity of enterolobin, a haemolytic protein from Enterolobium contortisiliquum seeds, was investigated. In doses ranging from 1 to 20 micrograms/site, enterolobin induced a dose-dependent paw oedema and pleurisy in rats. The effect was apparent after 15 min, peaked at 6 hr and decreased 24 hr after enterolobin was administered. One hour after the intrathoracic injection of enterolobin, the total leukocyte content of the pleural cavity increased significantly, mainly due to mononuclear and neutrophil accumulation. At 24 hr, although the number of mononuclear and neutrophil cells tended to decrease, a great rise in eosinophil counts was noted. Intraperitoneal treatment with the dual lipoxygenase and cyclooxygenase blockers, BW 755c (25 mg/kg) and NDGA (50 mg/kg) or the corticosteroid dexamethasone (0.1 mg/kg) inhibited enterolobin-induced paw oedema by 35, 38 and 47% respectively, whereas indomethacin (2 mg/kg) was inactive. The H1 antagonist, meclizine (25 mg/kg), was also effective against enterolobin oedema while the PAF-antagonists WEB 2086 and PCA 4248 (20 mg/kg) did not modify the reaction. It was concluded that enterolobin is a potent inducer of pleural exudation, cellular infiltration and paw oedema. Furthermore, enterolobin-induced oedema is partially dependent on lipoxygenase metabolites and histamine, while PAF and prostaglandins did not seem to be important in this reaction.

Intererence of the PAF receptor antagonist, PCA 4248, with the rat pleurisy evoked by inflammatory mediators or allergen

This study investigated the effect of the platelet-activating factor (PAF) receptor antagonist, PCA 4248, on the rat pleurisy caused by PAF, serotonin, bradykinin, histamine or allergen. The pleurisy was assessed by measuring liquid extravasation and leucocyte infiltration. Oral pretreatment with PCA 4248 (2.5-20 mg/kg) completely inhibited the pleural exudation caused by intrathoracic (i.t.) injection of PAF (1 microgram/cavity) (ED50 = 6.1 mg/kg), partially (42% reduction) the one induced by serotonin (100 micrograms/cavity), but was inactive against histamine (200 micrograms/cavity) or bradykinin (50 micrograms/cavity). PCA 4248 blocked the increase in the number of neutrophils, eosinophils and mononuclear cells observed 6 h after the i.t. injection of PAF, as well as the selective eosinophil accumulation noted 24 h later. In actively sensitized rats, PCA 4248 (20 mg/kg) failed to modify the increase in the total leucocyte counts noted 4 h after ovalbumin (12 micrograms/cavity), but dose dependently inhibited the pleural exudation observed within 1 h and the late eosinophil infiltration noted 24 h post-antigen. These observations led us to suggest that PCA 4248 is a potent PAF antagonist with anti-serotoninergic properties. Its interference with exudation and eosinophil infiltration caused by allergen is consistent with the interpretation that PCA 4248 may be useful in the management of allergic dysfunctions.

Suppression by cetirizine of pleurisy triggered by antigen in actively sensitized rats

The efficacy of cetirizine in comparison with meclizine, another piperazine H1 receptor antagonist, in rat pleurisy caused by allergen or autacoid was investigated. Sensitization was achieved by subcutaneous injection of a mixture of ovalbumin and aluminium hydroxide. Fourteen days later, the animals were challenged with an intrathoracic injection of ovalbumin (12 micrograms/cavity), which caused drastic mast cell degranulation, followed by pleural oedema and leucocyte influx. Cetirizine and meclizine (2.5-30 mg/kg i.p.), 1 h before challenge, inhibited the exudatory response evoked by antigen, under conditions where neutrophil and eosinophil accumulation was affected only by the former. When administered intrathoracically 22 h after allergen, i.e. using a curative approach, cetirizine (15 micrograms/cavity) drastically reduced the pleural eosinophilia noted 24 h post-challenge, indicating that this drug can reverse an already established eosinophilia. Cetirizine (15 mg/kg i.p.) also restored, to about 39% (P < 0.001), the number of uninjured mast cells recovered from the pleural cavity following allergen stimulation. In normal rats, cetirizine (5-15 micrograms/cavity) completely inhibited the pleural exudation elicited by histamine and only partially the exudation caused by 5-hydroxytryptamine or bradykinin, but was quite inactive against platelet-activating factor. We conclude that the pleural exudation triggered by allergen, vasoactive amines or bradykinin is clearly sensitive to cetirizine. In addition, the ability of the drug to interfere with pleural neutrophil or eosinophil mobilization and mast cell degranulation seems not to be associated with its ability to block the histamine H1 receptor.

Role of the IgE-Mediated System in Eosinophil Recruitment Triggered by Two Consecutive Cycles of Sensitisation and Challenge in Rats

In this study, we postulated that repeated cycles of IgE passive sensitisation and antigen challenge may play a role in up-regulating eosinophil response in allergic conditions. Antigen-mediated stimulation of the pleural cavity of rats passively sensitised with a single injection of IgE anti-DNP resulted in mast cell degranulation, increase in vascular permeability and mild neutrophilia, but no pleural eosinophilia. In contrast, a second cycle of sensitisation and challenge, performed within 7 days, showed a marked eosinophilia in parallel with a lower plasma leakage and comparable neutrophilia. The eosinophilic phenomenon was not reproduced when (1) IgE sensitisation or antigen challenge was omitted in the first cycle, or (2) the first cycle was replaced by either a histamine and 5-HT dual challenge or a PAF challenge. Furthermore, we found an increase in eotaxin levels in animals subjected to two rather than one cycle of sensitisation and challenge. Treatment with the PAF receptor antagonist BN 52021 or with the lipoxygenase inhibitor zileuton, but not mast cell granule depletion, prevented the allergen-evoked eosinophil accumulation in rechallenged animals. Our results indicate that repeated cycles of IgE-driven inflammation may lead to eosinophil accumulation in a mechanism dependent on eotaxin, PAF and leukotrienes.

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.