Original Paper Int Arch Allergy Immunol 1992;98:355-360
Patricia M.R. e Silva Marco A. Martins Marcia C.R. Lima Alessandra C. Alves Bruno L. Díaz Renato S.B. Cordeiro
Pharmacological Modulation of the Late Eosinophilia Induced by Antigen in Actively Sensitized Rats
Depto de Fisiología e Farmacodinámica, Fundagáo Oswaldo Cruz, Manguinhos, Rio de Janeiro, Brasil
Key Words
Abstract
Eosinophil recruitment Allergy Lipid mediators
The intrathoracic injection of ovalbumin (12 pg/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 PAFacether in the pleural eosinophil accumulation by antigen. Administration of the cyclooxygenase inhibitor indomethacin (2 mg/kg, i.p.), 1 h before the anti gen challenge, failed to modify the 24-hour eosinophilia. In contrast, the dual cyclooxygenase and lipoxygenase inhibitor BW 755C and the more selective in hibitor BW A4C (5 and 10 pg/cavity, i.t.), injected 1 h before the antigen, were effective. Similarly, the PAF-acethcr 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 pg/ cavity). O ur findings suggest that the antigen-induced eosinophil mobilization is dependent on lipoxygenase derivatives and PAF-acether, but not on pros taglandins.
The inflammatory reaction followed by allergen provo cation results in an early phase and a late one, which are related to the timing after the stimulus exposure [1]. The former is believed to be established by vasoactive/bronchoactive mediators with a subsequent increase in the vas cular permeability and airway resistance [2], The latter is characterized by an intense cellular infiltration, mainly eo
sinophils [3]. Since the phenomenon of chemotaxis is one of the proposed mechanisms by which eosinophils may ac cumulate in a tissue [4], some endogenous eosinophil chemotaxins such as eosinophil chemotactic factor of anaphy laxis [5] and mediators such as C5a [6], leukotriene B4 (LTB4) [7] - preferentially PAF-acether [8] - are potential contributors to tissue eosinophil infiltration. We have recently proposed a model of allergic pleurisy in which ovalbumin is injected intrathoracically (i.t.) into
Correspondence to: Dr. Patricia M.R. c Silva Depto de Fisiología c Farmacodinámica Fundaçâo Oswaldo Cruz Av. Brasil, n° 4365 CEP 21045-900 Rio de Janeiro (Brazil)
© 1992S.Kargcr AG,Basel 1018-2438/92/0984-0355 $ 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/26/2018 7:55:06 PM
Introduction
actively sensitized rats [9]. The challenge was followed by a very rapid increase in the vasopermeation and two waves of polymorphonuclear leukocyte accumulation, predom inant neutrophil infiltration at 4 h and a long-lasting eosin ophil accumulation 24 h postantigen [9,10]. Our aim in the present study was to investigate the po tential involvement of lipid mediators in the pleural eosinophilia by ovalbumin. To do so, cyclooxygenase or lipoxy genase inhibitors and PAF-acether antagonists were used, which led us to suggest that arachidonic acid metabolites from the lipoxygenase pathway and PAF-acether, but not prostaglandins, are involved in the pleural eosinophil in flux after the antigen challenge.
romethylphenyl) - 2 - pyrazoline hydrochloride] and BW A4C [N - (3 phenoxycinnamyl) acctohydroxamic acid] were kindly provided by Dr. Salvador Moncada (Wellcome Laboratories, UK) and WEB208613-[4(2 - chlorophenyl) - 9 - methyl - 6H - thieno - 2,2 - f - (1,2,4) - triazolo 4,3 a](l,4) - diazepin - 2 - yl -1 - (4 - morpholinyl) -1 - propanonel by Dr. H. Hcucr(Bochringer-Ingelhcim,FRG).BN52021[3-(l,l-dimethylcthyl) hexahydro-l,4,7b-trihydroxy-8-methyl-9H-l,7a-(epoxy-methanol)lH,6aH-cyclopcnta(c)furo-(2,3-b)furo(3',2':3,4)cyclopenta(l,2-i/)furan - 5,9,12 (4H) - trione] was kindly given by Dr. P. Braquet (Institut Henri Beaufour, France). Statistical Analysis
Data were statistically analyzed by the analysis of variance (ANOVA), followed by the Ncwman-Keuls Student test. In case of comparison between only two groups, the difference in means was analyzed by unpaired Student’s t test, p values of 0.05 or less were considered significant.
Material and Methods
Wistar rats of both sexes, weighing 150-200 g, from the Oswaldo Cruz Breeding (Rio de Janeiro), were used. The animals were sensi tized subcutaneously with a mixture (0.2 ml) of 50 pg ovalbumin and 5 mg of aluminium hydroxide. Fourteen days later, the ovalbumin (12 ,ug/0.1 ml/cavity) was i.t. administered and after different time inter vals the animals were sacrificed with an overdose of ether. The pleu ral cavity was opened, rinsed with 3 ml of heparinized saline (10 IU/ ml) and the recovered volume evaluated in a graduated syringe. Nonsensitized rats in which ovalbumin was injected i.t. were used as nega tive controls. Cell Analysis
Total leukocytes were counted in a Coulter counter ZBI and ex pressed as millions of cells/cavity. The differential analyses were per formed in smears prepared in a cytocentrifuge (Incibras) and colored with May-Grunwald-Gicmsa stain. Pleurisy by PAF-Acether and Leukotriene B 4
This reaction was produced by the i.t. injection of PAF-acether (1 gg/cavity) diluted with sterile 0.9% NaCl (saline) containing 0.01% BSA. LTB4 (1 gg/cavity) was diluted with saline. Control animals re ceived the same volume of the vehicle. The evaluation of exudation and leukocyte counts was performed as described above. Drug Treatment
The steroidal anti-inflammatory agent dexamethasone (5 and 10 pg/site), the dual inhibitor of cyclooxygenase and lipoxygenase BW 755C (5 and 10 pg/cavity) and the lipoxygenase inhibitor BW A4C (5 and 10 pg/site) were topically administered 1 h before ovalbumin. The cyclooxygenase inhibitor indomethacin (2 mg/kg) and the anti-PAF antagonists BN 52021 or WEB 2086 (20 mg/kg) were administered intraperitoneally 1 h before the antigen challenge. Drugs
Ovalbumin was purchased from Biochemika Fluka (Switzerland) and PAF-acether (1 - O - hexadecyl - 2 - acetyl - sn -glyceryl - 3 - phosphorylcholine) from Bachcm (Switzerland). Dexamethasone was pur chased from Merck, Sharp &Dohmc (USA) and indomethacin and LTB4fromSigmaChemicalCo.(USA).BW755C[3-amino-l-(3-trifluo-
356
Results
As illustrated in figure 1, the i.t. injection of ovalbumin (12 pg/cavity) into actively sensitized rats induced a marked eosinophil accumulation, first noted at 24 h (fig. 1). The antigen challenge also caused an early in crease in the pleural neutrophil counts from 0.32 ±0.01 (mean ±SEM ; n = 20) to 14.20± 1.04 x 106/cavity (n = 19, p
Patricia M.R. e Silva Marco A. Martins Marcia C.R. Lima Alessandra C. Alves Bruno L. Díaz Renato S.B. Cordeiro
Pharmacological Modulation of the Late Eosinophilia Induced by Antigen in Actively Sensitized Rats
Depto de Fisiología e Farmacodinámica, Fundagáo Oswaldo Cruz, Manguinhos, Rio de Janeiro, Brasil
Key Words
Abstract
Eosinophil recruitment Allergy Lipid mediators
The intrathoracic injection of ovalbumin (12 pg/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 PAFacether in the pleural eosinophil accumulation by antigen. Administration of the cyclooxygenase inhibitor indomethacin (2 mg/kg, i.p.), 1 h before the anti gen challenge, failed to modify the 24-hour eosinophilia. In contrast, the dual cyclooxygenase and lipoxygenase inhibitor BW 755C and the more selective in hibitor BW A4C (5 and 10 pg/cavity, i.t.), injected 1 h before the antigen, were effective. Similarly, the PAF-acethcr 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 pg/ cavity). O ur findings suggest that the antigen-induced eosinophil mobilization is dependent on lipoxygenase derivatives and PAF-acether, but not on pros taglandins.
The inflammatory reaction followed by allergen provo cation results in an early phase and a late one, which are related to the timing after the stimulus exposure [1]. The former is believed to be established by vasoactive/bronchoactive mediators with a subsequent increase in the vas cular permeability and airway resistance [2], The latter is characterized by an intense cellular infiltration, mainly eo
sinophils [3]. Since the phenomenon of chemotaxis is one of the proposed mechanisms by which eosinophils may ac cumulate in a tissue [4], some endogenous eosinophil chemotaxins such as eosinophil chemotactic factor of anaphy laxis [5] and mediators such as C5a [6], leukotriene B4 (LTB4) [7] - preferentially PAF-acether [8] - are potential contributors to tissue eosinophil infiltration. We have recently proposed a model of allergic pleurisy in which ovalbumin is injected intrathoracically (i.t.) into
Correspondence to: Dr. Patricia M.R. c Silva Depto de Fisiología c Farmacodinámica Fundaçâo Oswaldo Cruz Av. Brasil, n° 4365 CEP 21045-900 Rio de Janeiro (Brazil)
© 1992S.Kargcr AG,Basel 1018-2438/92/0984-0355 $ 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/26/2018 7:55:06 PM
Introduction
actively sensitized rats [9]. The challenge was followed by a very rapid increase in the vasopermeation and two waves of polymorphonuclear leukocyte accumulation, predom inant neutrophil infiltration at 4 h and a long-lasting eosin ophil accumulation 24 h postantigen [9,10]. Our aim in the present study was to investigate the po tential involvement of lipid mediators in the pleural eosinophilia by ovalbumin. To do so, cyclooxygenase or lipoxy genase inhibitors and PAF-acether antagonists were used, which led us to suggest that arachidonic acid metabolites from the lipoxygenase pathway and PAF-acether, but not prostaglandins, are involved in the pleural eosinophil in flux after the antigen challenge.
romethylphenyl) - 2 - pyrazoline hydrochloride] and BW A4C [N - (3 phenoxycinnamyl) acctohydroxamic acid] were kindly provided by Dr. Salvador Moncada (Wellcome Laboratories, UK) and WEB208613-[4(2 - chlorophenyl) - 9 - methyl - 6H - thieno - 2,2 - f - (1,2,4) - triazolo 4,3 a](l,4) - diazepin - 2 - yl -1 - (4 - morpholinyl) -1 - propanonel by Dr. H. Hcucr(Bochringer-Ingelhcim,FRG).BN52021[3-(l,l-dimethylcthyl) hexahydro-l,4,7b-trihydroxy-8-methyl-9H-l,7a-(epoxy-methanol)lH,6aH-cyclopcnta(c)furo-(2,3-b)furo(3',2':3,4)cyclopenta(l,2-i/)furan - 5,9,12 (4H) - trione] was kindly given by Dr. P. Braquet (Institut Henri Beaufour, France). Statistical Analysis
Data were statistically analyzed by the analysis of variance (ANOVA), followed by the Ncwman-Keuls Student test. In case of comparison between only two groups, the difference in means was analyzed by unpaired Student’s t test, p values of 0.05 or less were considered significant.
Material and Methods
Wistar rats of both sexes, weighing 150-200 g, from the Oswaldo Cruz Breeding (Rio de Janeiro), were used. The animals were sensi tized subcutaneously with a mixture (0.2 ml) of 50 pg ovalbumin and 5 mg of aluminium hydroxide. Fourteen days later, the ovalbumin (12 ,ug/0.1 ml/cavity) was i.t. administered and after different time inter vals the animals were sacrificed with an overdose of ether. The pleu ral cavity was opened, rinsed with 3 ml of heparinized saline (10 IU/ ml) and the recovered volume evaluated in a graduated syringe. Nonsensitized rats in which ovalbumin was injected i.t. were used as nega tive controls. Cell Analysis
Total leukocytes were counted in a Coulter counter ZBI and ex pressed as millions of cells/cavity. The differential analyses were per formed in smears prepared in a cytocentrifuge (Incibras) and colored with May-Grunwald-Gicmsa stain. Pleurisy by PAF-Acether and Leukotriene B 4
This reaction was produced by the i.t. injection of PAF-acether (1 gg/cavity) diluted with sterile 0.9% NaCl (saline) containing 0.01% BSA. LTB4 (1 gg/cavity) was diluted with saline. Control animals re ceived the same volume of the vehicle. The evaluation of exudation and leukocyte counts was performed as described above. Drug Treatment
The steroidal anti-inflammatory agent dexamethasone (5 and 10 pg/site), the dual inhibitor of cyclooxygenase and lipoxygenase BW 755C (5 and 10 pg/cavity) and the lipoxygenase inhibitor BW A4C (5 and 10 pg/site) were topically administered 1 h before ovalbumin. The cyclooxygenase inhibitor indomethacin (2 mg/kg) and the anti-PAF antagonists BN 52021 or WEB 2086 (20 mg/kg) were administered intraperitoneally 1 h before the antigen challenge. Drugs
Ovalbumin was purchased from Biochemika Fluka (Switzerland) and PAF-acether (1 - O - hexadecyl - 2 - acetyl - sn -glyceryl - 3 - phosphorylcholine) from Bachcm (Switzerland). Dexamethasone was pur chased from Merck, Sharp &Dohmc (USA) and indomethacin and LTB4fromSigmaChemicalCo.(USA).BW755C[3-amino-l-(3-trifluo-
356
Results
As illustrated in figure 1, the i.t. injection of ovalbumin (12 pg/cavity) into actively sensitized rats induced a marked eosinophil accumulation, first noted at 24 h (fig. 1). The antigen challenge also caused an early in crease in the pleural neutrophil counts from 0.32 ±0.01 (mean ±SEM ; n = 20) to 14.20± 1.04 x 106/cavity (n = 19, p
