Int. J. Immunopharmac., Vol. 14, No. 5, pp. 847-855, 1992.
0192-0561/92 $5.00 + .00 Pergamon Press Ltd. © 1992 International Society for Immunopharmacology.
Printed in Great Britain.
M O D E L OF B R O N C H I A L A L L E R G I C I N F L A M M A T I O N IN THE B R O W N N O R W A Y RAT. P H A R M A C O L O G I C A L M O D U L A T I O N J. P. T A R A Y R E , M. A L I A G A , M. BARBARA, N. TISSEYRE, S. VIEU a n d J. TISNE-VERSAILLES Centre de Recherche Pierre Fabre, 17 avenue Jean Moulin, 81106 Castres, France
(Received 18 September 1991 and in final form 11 December 1991) Abstract - - Exposure of non-sensitized Brown Norway (BN) rats to a 10%-ovalbumin aerosol induced an increase in the number of neutrophils in the broncho-alveolar lavage (BAL) fluid 3 and 6 h later but with no change in number of cells at 24 h. When the BN rats were actively sensitized (i.m. injection of 10 mg/kg ovalbumin and i.p. injection of killed Bordetella pertussis) and exposed 1 2 - 1 4 days later to a 10%-ovalbumin aerosol there was an increase in the number of eosinophils in the BAL fluid, maximal 2 4 - 48 h after the anaphylactic reaction. The increase in the number of neutrophils in the bronchial lumen 3 and 6 h after the anaphylactic reaction was larger than that obtained in non-specific inflammation and in contrast to this was still present 24 - 48 h after ovalbumin exposure. In passively sensitized BN rats exposed to ovalbumin aerosol, no inflammation appeared in the BAL fluid 24 h after the anaphylactic reaction. Various drugs, administered twice, 5 min and 5 h after the anaphylactic reaction, have been evaluated for their effects on the 24-h inflammation obtained in actively sensitized rats. Dexamethasone acetate (0.08 mg/kg i.p.) and theophylline (50 mg/kg i.p.) decreased the number of eosinophils and neutrophils. Ketotifen fumarate (12.5 mg/kg), cetirizine dihydrochloride (12.5 mg/kg), salbutamol (2 mg/kg), disodium cromoglycate (50 mg/kg) all given intraperitoneally, reduced the number of eosinophils. Tioxamast decreased the number of eosinophils at 12.5 mg/kg i.p. and by the oral route. At higher doses (50 mg/kg i.p.; 150 mg/kg p.o.), it reduced the number of eosinophils and neutrophils. Indomethacin (5 mg/kg), mepyramine maleate (12.5 mg/kg) and atropine sulfate (1 mg/kg) given i.p. were inactive. Thus, 24.h inflammation after an aerosol-induced anaphylactic reaction in actively sensitized BN rats appears a useful model to study the action of the anti-allergic and anti-asthma drugs on a IgE-mediated bronchial inflammation.
Guinea-pigs a n d rats have been used extensively to study the effect o f drugs o n allergic reactions. Guinea-pigs show increased b r o n c h i a l sensitivity following sensitization a n d challenge b u t it is difficult to induce IgE p r o d u c t i o n in this species (FiJgner, 1985). It is easier to produce allergic reaction IgE specific in rats (Mota, 1963; Stechschulte, O r a n g e & Austen, 1970) a n d in particular B r o w n N o r w a y (BN) rats are good IgE producers ( M u r p h y , Brown, Miklos & F i r e m a n , 1974; Pauwels, Bazin, P l a t t e a u & V a n der Straeten, 1979a, b; S m i t h & Petillo, 1976). W e have used the BN rat to study the effects of different types o f antiallergic or a n t i - a s t h m a drugs in a m o d e l o f b r o n c h i a l i n f l a m m a t i o n induced by antigen challenge.
Bronchial anaphylactic sensitization
reaction
after
active
Sensitization. B r o w n N o r w a y rats, 1 7 5 - 2 0 0 g , were sensitized by i.m. injection o f 1 0 m g / k g o v a l b u m i n (Grade V, Sigma) a n d 0.1 m l / r a t by the i.p. r o u t e o f a suspension o f killed Bordetella pertussis (5 x 10 9 o r g a n i s m s / m l ; Difco) (Mota, 1963). Challenge. Twelve to f o u r t e e n days after sensitization, the conscious a n i m a l was placed in a 8-1 circular glass vessel a n d exposed d u r i n g 6 m i n to a n aerosol o f a 1 0 % - o v a l b u m i n solution in 0 . 9 % NaC1. The aerosol was induced by a J o u a n nebulizer p r o d u c i n g dry particles 1 - 3 ~m in d i a m e t e r at a flow rate of 28 m l / h . T h e nebulizer was supplied with c o m p r e s s e d air at a pressure o f 1 bar. In these experimental conditions, the antigen challenge induced a n increase o f respiration rate followed by a slight dyspnea.
EXPERIMENTAL PROCEDURES
Animals Male B N rats (Charles River) were used.
847
848
J. P. TARAVREet al.
Bronchial anaphylactic reaction after passive sensitization The anti-ovalbumin serum was obtained in BN rats 12 days after sensitization according to the conditions described above. The antiserum used induced a 24-h passive cutaneous anaphylaxis in Sprague Dawley rats at 1 / 5 0 0 - 1 / 2 5 0 dilutions (Brocklehurst, 1967). Brown Norway rats, 200 g, were passively sensitized by the i.v. injection in a caudal vein of 1 ml/animal of the above-described anti-ovalbumin serum. Twenty-four hours later, the animals were exposed to an aerosol induced by a 10%-ovatbumin solution in 0.9°70 NaC1 during 6 min under the conditions described above. The animals showed a slight anaphylactic reaction of similar intensity as in the case of active sensitization. Broncho-alveolar lavage (BAL ) At various times after the anaphylactic reaction, the BN rats were killed by i.p. injection of 300 mg/kg sodium pentobarbital (Sanofi). The trachea was then cannulated and the BAL carried out twice with 5 ml of sterile 0.9°7o NaCi. Eighty percent of the fluid injected was recovered on an average. The total leukocytes were counted using a Coulter Counter (Coultronics, ZM model). After spreading on a slide, fixation and staining with M a y Grfinwald-Giemsa, different cell counts were carried out after counting at least 350 cells per slide. Drugs and treatment The following drugs were used: atropine sulfate (Merck), cetirizine dihydrochloride (UCB), ketotifen fumarate (Sandoz), disodium cromoglycate, dexamethasone acetate, indomethacin, mepyramine maleate, salbutamol, anhydrous theophylline (all from Sigma), and tioxamast (PF Medicament) (Tarayre & Bonnaud, 1990). The drugs were administered twice by the i.p. route in 10 mi/kg of 0.9°7o NaC1 (with 1 drop of Tween 80 for insoluble compounds) or by the oral route in water, 5 min and 5 h after the anaphylactic reaction. Controls received the solvent alone under the same experimental conditions. The doses of drugs expressed refer to the form of the product used (base or salt). The doses of indomethacin, mepyramine, salbutamol, theophylline, cromoglycate and tioxamast were the same as those used in a bronchial allergic model in guinea-pigs (Tarayre, Aliaga, Barbara, Tisseyre & Tisne-Versailles 1991c; Tarayre, Aliaga, Barbara, Tisseyre, Vieu & Tisne-Versailles 1991d). Ketotifen
and cetirizine were used at the same dose as mepyramine (12.5 mg/kg). Atropine was given at 1 mg/kg, a dose which decreases the symptoms of the passive anaphylactic shock induced by aerosol in conscious guinea-pigs. Dexamethasone was given at 0.08 mg/kg. Statistical calculations Bonferroni's test or the non-parametric K r u s k a l Wallis and Wilcoxon tests were used.
RESULTS Bronchial inflammation after exposure to an aerosol o f NaCI or o f ovalbumin in actively sensitized or non-sensitized animals The number of mononuclears, neutrophils and eosinophils in the BAL fluid obtained in normal non-sensitized BN rats was, respectively: 290 ___ 17 x 106 ml; 18 _ 4 x 106/ml; 12___ 3 x 106/ml(n = 31). There was no significant difference between cells in the BAL fluid obtained from non-sensitized and sensitized rats exposed to an aerosol of NaCI and those obtained in normal rats (Fig. 1). In non-sensitized rats, exposure to an aerosol of ovalbumin induces, in comparison with animals exposed to isotonic NaCI, a significant increase in the number of neutrophils at 3 h ( x 3.4), which drops down at 6 h ( x 1.9) and is stopped after 24 h [Fig. l(a)]. The number of the other types of leukocytes is not changed. In comparison with sensitized animals submitted to an aerosol of NaCI, rats exposed to antigen show a significant increase in neutrophils at 3 h ( x 7), maximal after 6 h ( x 58) and persisting 24 h ( x 17) and 48 h ( x 12) after the shock [Fig. l(b)]. In comparison with non-sensitized animals [Fig. l(a)], inhalation of ovalbumin induces thus an increase in neutrophils which is greater at 3 h and persists up to 2 4 - 4 8 h. The anaphylactic reaction also induces an increase in the number of eosinophils which is significant at 3 h and maximal at 2 4 - 4 8 h. The number of mononuclear cells in the BAL fluid is not significantly changed after the anaphylactic shock despite a tendency to an increase after 2 4 - 4 8 h. Bronchial inflammation 24 h after a bronchial allergic reaction in passively sensitized animals Following the antigen challenge of passively sensitized rats there was no change in the number of the different categories of leukocytes (Table 1).
Pharmacological Modulation
(a)
3h
6h
24 h
(b)
O
0.50I-
T
6h
24 h
°5°1-
°4°r
°'3°1-~
g 0.40I-
=~ 0.30I0-10r
0,20r 0.10r
0.40
0.40
0.30
0.30
0.20 I-
~o X9
04
3h
j
849
48 h
:I i
0.20 N
:
t
0.1C
0.10
0.01
•
O.Ol
o 0.20I
~
:
0.201
p, ,,, 0-10 I
°1° F O.Olr-
0.01 I-
~ ~
Fig. 1. Number of mononuclear cells (mono), neutrophils (neutro) and eosinophils (eosino) in BAL fluid at various times after exposure to NaCI or ovalbumin aerosol in non sensitized and sensitized BN rats. (a) Non-sensitized animals. (b) Sensitized animals. (F1) 0.9OT0-NaC1 aerosol. (~1) 10o/0-ovalbnmin aerosol. Bars represent S.E.M. 8 - 21 rats per group. (0)/:'
0192-0561/92 $5.00 + .00 Pergamon Press Ltd. © 1992 International Society for Immunopharmacology.
Printed in Great Britain.
M O D E L OF B R O N C H I A L A L L E R G I C I N F L A M M A T I O N IN THE B R O W N N O R W A Y RAT. P H A R M A C O L O G I C A L M O D U L A T I O N J. P. T A R A Y R E , M. A L I A G A , M. BARBARA, N. TISSEYRE, S. VIEU a n d J. TISNE-VERSAILLES Centre de Recherche Pierre Fabre, 17 avenue Jean Moulin, 81106 Castres, France
(Received 18 September 1991 and in final form 11 December 1991) Abstract - - Exposure of non-sensitized Brown Norway (BN) rats to a 10%-ovalbumin aerosol induced an increase in the number of neutrophils in the broncho-alveolar lavage (BAL) fluid 3 and 6 h later but with no change in number of cells at 24 h. When the BN rats were actively sensitized (i.m. injection of 10 mg/kg ovalbumin and i.p. injection of killed Bordetella pertussis) and exposed 1 2 - 1 4 days later to a 10%-ovalbumin aerosol there was an increase in the number of eosinophils in the BAL fluid, maximal 2 4 - 48 h after the anaphylactic reaction. The increase in the number of neutrophils in the bronchial lumen 3 and 6 h after the anaphylactic reaction was larger than that obtained in non-specific inflammation and in contrast to this was still present 24 - 48 h after ovalbumin exposure. In passively sensitized BN rats exposed to ovalbumin aerosol, no inflammation appeared in the BAL fluid 24 h after the anaphylactic reaction. Various drugs, administered twice, 5 min and 5 h after the anaphylactic reaction, have been evaluated for their effects on the 24-h inflammation obtained in actively sensitized rats. Dexamethasone acetate (0.08 mg/kg i.p.) and theophylline (50 mg/kg i.p.) decreased the number of eosinophils and neutrophils. Ketotifen fumarate (12.5 mg/kg), cetirizine dihydrochloride (12.5 mg/kg), salbutamol (2 mg/kg), disodium cromoglycate (50 mg/kg) all given intraperitoneally, reduced the number of eosinophils. Tioxamast decreased the number of eosinophils at 12.5 mg/kg i.p. and by the oral route. At higher doses (50 mg/kg i.p.; 150 mg/kg p.o.), it reduced the number of eosinophils and neutrophils. Indomethacin (5 mg/kg), mepyramine maleate (12.5 mg/kg) and atropine sulfate (1 mg/kg) given i.p. were inactive. Thus, 24.h inflammation after an aerosol-induced anaphylactic reaction in actively sensitized BN rats appears a useful model to study the action of the anti-allergic and anti-asthma drugs on a IgE-mediated bronchial inflammation.
Guinea-pigs a n d rats have been used extensively to study the effect o f drugs o n allergic reactions. Guinea-pigs show increased b r o n c h i a l sensitivity following sensitization a n d challenge b u t it is difficult to induce IgE p r o d u c t i o n in this species (FiJgner, 1985). It is easier to produce allergic reaction IgE specific in rats (Mota, 1963; Stechschulte, O r a n g e & Austen, 1970) a n d in particular B r o w n N o r w a y (BN) rats are good IgE producers ( M u r p h y , Brown, Miklos & F i r e m a n , 1974; Pauwels, Bazin, P l a t t e a u & V a n der Straeten, 1979a, b; S m i t h & Petillo, 1976). W e have used the BN rat to study the effects of different types o f antiallergic or a n t i - a s t h m a drugs in a m o d e l o f b r o n c h i a l i n f l a m m a t i o n induced by antigen challenge.
Bronchial anaphylactic sensitization
reaction
after
active
Sensitization. B r o w n N o r w a y rats, 1 7 5 - 2 0 0 g , were sensitized by i.m. injection o f 1 0 m g / k g o v a l b u m i n (Grade V, Sigma) a n d 0.1 m l / r a t by the i.p. r o u t e o f a suspension o f killed Bordetella pertussis (5 x 10 9 o r g a n i s m s / m l ; Difco) (Mota, 1963). Challenge. Twelve to f o u r t e e n days after sensitization, the conscious a n i m a l was placed in a 8-1 circular glass vessel a n d exposed d u r i n g 6 m i n to a n aerosol o f a 1 0 % - o v a l b u m i n solution in 0 . 9 % NaC1. The aerosol was induced by a J o u a n nebulizer p r o d u c i n g dry particles 1 - 3 ~m in d i a m e t e r at a flow rate of 28 m l / h . T h e nebulizer was supplied with c o m p r e s s e d air at a pressure o f 1 bar. In these experimental conditions, the antigen challenge induced a n increase o f respiration rate followed by a slight dyspnea.
EXPERIMENTAL PROCEDURES
Animals Male B N rats (Charles River) were used.
847
848
J. P. TARAVREet al.
Bronchial anaphylactic reaction after passive sensitization The anti-ovalbumin serum was obtained in BN rats 12 days after sensitization according to the conditions described above. The antiserum used induced a 24-h passive cutaneous anaphylaxis in Sprague Dawley rats at 1 / 5 0 0 - 1 / 2 5 0 dilutions (Brocklehurst, 1967). Brown Norway rats, 200 g, were passively sensitized by the i.v. injection in a caudal vein of 1 ml/animal of the above-described anti-ovalbumin serum. Twenty-four hours later, the animals were exposed to an aerosol induced by a 10%-ovatbumin solution in 0.9°70 NaC1 during 6 min under the conditions described above. The animals showed a slight anaphylactic reaction of similar intensity as in the case of active sensitization. Broncho-alveolar lavage (BAL ) At various times after the anaphylactic reaction, the BN rats were killed by i.p. injection of 300 mg/kg sodium pentobarbital (Sanofi). The trachea was then cannulated and the BAL carried out twice with 5 ml of sterile 0.9°7o NaCi. Eighty percent of the fluid injected was recovered on an average. The total leukocytes were counted using a Coulter Counter (Coultronics, ZM model). After spreading on a slide, fixation and staining with M a y Grfinwald-Giemsa, different cell counts were carried out after counting at least 350 cells per slide. Drugs and treatment The following drugs were used: atropine sulfate (Merck), cetirizine dihydrochloride (UCB), ketotifen fumarate (Sandoz), disodium cromoglycate, dexamethasone acetate, indomethacin, mepyramine maleate, salbutamol, anhydrous theophylline (all from Sigma), and tioxamast (PF Medicament) (Tarayre & Bonnaud, 1990). The drugs were administered twice by the i.p. route in 10 mi/kg of 0.9°7o NaC1 (with 1 drop of Tween 80 for insoluble compounds) or by the oral route in water, 5 min and 5 h after the anaphylactic reaction. Controls received the solvent alone under the same experimental conditions. The doses of drugs expressed refer to the form of the product used (base or salt). The doses of indomethacin, mepyramine, salbutamol, theophylline, cromoglycate and tioxamast were the same as those used in a bronchial allergic model in guinea-pigs (Tarayre, Aliaga, Barbara, Tisseyre & Tisne-Versailles 1991c; Tarayre, Aliaga, Barbara, Tisseyre, Vieu & Tisne-Versailles 1991d). Ketotifen
and cetirizine were used at the same dose as mepyramine (12.5 mg/kg). Atropine was given at 1 mg/kg, a dose which decreases the symptoms of the passive anaphylactic shock induced by aerosol in conscious guinea-pigs. Dexamethasone was given at 0.08 mg/kg. Statistical calculations Bonferroni's test or the non-parametric K r u s k a l Wallis and Wilcoxon tests were used.
RESULTS Bronchial inflammation after exposure to an aerosol o f NaCI or o f ovalbumin in actively sensitized or non-sensitized animals The number of mononuclears, neutrophils and eosinophils in the BAL fluid obtained in normal non-sensitized BN rats was, respectively: 290 ___ 17 x 106 ml; 18 _ 4 x 106/ml; 12___ 3 x 106/ml(n = 31). There was no significant difference between cells in the BAL fluid obtained from non-sensitized and sensitized rats exposed to an aerosol of NaCI and those obtained in normal rats (Fig. 1). In non-sensitized rats, exposure to an aerosol of ovalbumin induces, in comparison with animals exposed to isotonic NaCI, a significant increase in the number of neutrophils at 3 h ( x 3.4), which drops down at 6 h ( x 1.9) and is stopped after 24 h [Fig. l(a)]. The number of the other types of leukocytes is not changed. In comparison with sensitized animals submitted to an aerosol of NaCI, rats exposed to antigen show a significant increase in neutrophils at 3 h ( x 7), maximal after 6 h ( x 58) and persisting 24 h ( x 17) and 48 h ( x 12) after the shock [Fig. l(b)]. In comparison with non-sensitized animals [Fig. l(a)], inhalation of ovalbumin induces thus an increase in neutrophils which is greater at 3 h and persists up to 2 4 - 4 8 h. The anaphylactic reaction also induces an increase in the number of eosinophils which is significant at 3 h and maximal at 2 4 - 4 8 h. The number of mononuclear cells in the BAL fluid is not significantly changed after the anaphylactic shock despite a tendency to an increase after 2 4 - 4 8 h. Bronchial inflammation 24 h after a bronchial allergic reaction in passively sensitized animals Following the antigen challenge of passively sensitized rats there was no change in the number of the different categories of leukocytes (Table 1).
Pharmacological Modulation
(a)
3h
6h
24 h
(b)
O
0.50I-
T
6h
24 h
°5°1-
°4°r
°'3°1-~
g 0.40I-
=~ 0.30I0-10r
0,20r 0.10r
0.40
0.40
0.30
0.30
0.20 I-
~o X9
04
3h
j
849
48 h
:I i
0.20 N
:
t
0.1C
0.10
0.01
•
O.Ol
o 0.20I
~
:
0.201
p, ,,, 0-10 I
°1° F O.Olr-
0.01 I-
~ ~
Fig. 1. Number of mononuclear cells (mono), neutrophils (neutro) and eosinophils (eosino) in BAL fluid at various times after exposure to NaCI or ovalbumin aerosol in non sensitized and sensitized BN rats. (a) Non-sensitized animals. (b) Sensitized animals. (F1) 0.9OT0-NaC1 aerosol. (~1) 10o/0-ovalbnmin aerosol. Bars represent S.E.M. 8 - 21 rats per group. (0)/:'
