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Pharmacological modulation of a model of bronchial inflammation after aerosol-induced active anaphylactic shock in conscious guinea pigs
0192-0561/91 $3.00 + .00 Pergamon Press plc. International Society for lmmunopharmacology.
Int. Z lmmunopharmac., Vol. 13, No. 4, pp. 349-356, 1991. Printed in Great Brilain.
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 OF A M O D E L OF B R O N C H I A L I N F L A M M A T I O N AFTER A E R O S O L - I N D U C E D ACTIVE A N A P H Y L A C T I C SHOCK IN CONSCIOUS G U I N E A PIGS J. P. TARAYRE, M. ALIAGA, M. BARBARA, N. TISSEYRE, S. VIEU and J. TISNE-VERSAILLES Centre de Recherche Pierre Fabre, 17 avenue Jean Moulin, 81106 Castres, France
(Received for publication 7 November 1990)
Abslract - - Twenty-four hours after an active anaphylactic shock induced by inhalation of antigen in conscious guinea pigs sensitized by a large dose of ovalbumin in complete Freund's adjuvant, a noteworthy bronchial inflammation, characterized by increased numbers of neutrophils, mononuclear cells and eosinophils in the bronchoalveolar lavage fluid, was observed. Some drugs administered after the anaphylactic shock were investigated using this model. Disodium cromoglycate primarily reduced the number of mononuclear cells and eosinophils. Dexamethasone and theophylline decreased the number of eosinophils. Salbutamol and mepyramine increased neutrophils. Indomethacin did not give rise to any significant effect. This test appears to be of use for the investigation of anti-inflammatory compounds in the prophylactic treatment of asthma.
In recent years, many works have established that inflammation plays an important part in asthma (review by Morley & Smith, 1989). In clinical pharmacology, as well as in animal models, bronchoalveolar lavage (BAL) permitted the measurement of certain parameters in bronchial inflammation. In clinical pharmacology, bronchoprovocation by inhalation of allergen in atopic asthmatics showed an increase in the number of neutrophils and eosinophils in the BAL fluid during the hours following the challenge (De Monchy, Kauffman, Venge, Koeter, Jansen, Sluiter & De Vries, 1985; Metzger, Richerson, Worden, Monick & Hunninghake, 1985b) and a later increase in the number of macrophages and T-helper lymphocytes (Metzger, Hunninghake & Richerson, 1985a; Metzger, Zavala, Richerson, Moseley, Iwamota, Monick, Sjoerdsma & Hunninghake, 1987). The possible role of delayed hypersensitivity in the inflammatory reactions of asthma is being thoroughly studied (review by Kay, Wardlmaw, Moqbel, Buchanan, Cromwell & Fitzharris, 1987). In guinea pigs various models of bronchial inflammation, after an anaphylactic shock induced by inhalation of antigen, have been described (Iijima, Ishii, Yamauchi, Chad, Kimura, Shimura, Shindoh, Indue, Mue & Takishima, 1987; Coyle, Urwin, Page, Touvay, Villain & Braquet, 1988; Hutson, Church, Clay, Miller & Holgate, 1988).
However, the number of leukocytes in the BAL fluid up to 48 h after the anaphylatic shock only showed an increase in neutrophils and eosinophils. Recently, while sensitizing guinea pigs with a large dose of ovalbumin and complete Freund's adjuvant, we developed a model which allowed us to obtain increased numbers of mononuclear cells in the BAL fluid, in addition to increased numbers of neutrophils and eosinophils 2 4 - 48 h after an active anaphylactic shock by inhalation of antigen (Tarayre, Aliaga, Barbara, Tisseyre, Vieu & TisneVersailles, 1990). Various drugs were investigated using this model in the present work.
EXPERIMENTAL PROCEDURES
Sensitization Male Dunkin-Hartley guinea pigs of average weight 2 5 0 - 2 7 5 g were sensitized by intramuscular injection (1 ml/kg) of 30 mg/kg ovalbumin (Sigma) dissolved in a 50:50 mixture (v/v) of Freund's complete adjuvant (Difco) in 0.9°70 NaCI. Anaphylactic shock Three to six weeks after sensitization, the conscious animal was placed in a circular glass box 26 cm in diameter and 15 cm high. Aerosols were
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induced by a nebuliser (Jouan, Paris, France) producing dry particles 1 - 3 tam in diameter, at a flow rate of 28 ml/h. The nebuliser was supplied with compressed air at a pressure of 1 bar. The animal was exposed for 6 rain to an aerosol of a 5% solution (w/v) of ovalbumin in 0.9% NaC1. In these experimental conditions some animals had reversible convulsions despite continuous exposure to the ovalbumin aerosol. Immediate or delayed mortality was rare. The different groups were homogenized to a maximum as far as the number of guinea pigs having reversible convulsions was concerned. A control group of sensitized animals was exposed for 6 min to an aerosol of saline.
Bronchoalveolar lavage (BAL) Twenty-four hours after anaphylactic shock the guinea pigs were killed by intraperitoneal administration of 300 mg/kg sodium pentobarbital (Sanofi). Their tracheas were then canulated, and the BAL carried out twice with 5 ml of sterile 0.9% NaC1. On average, 80°7o of the fluid administered was recovered and the leukocytes were then counted with a Coulter Counter (Coultronics model ZF). After spreading on a slide, fixation, and staining with M a y - G r i i n w a l d - G i e m s a , the number of mononuclear cells, neutrophils, and eosinophils were determined on at least 350 cells per slide. Treatment Drugs were administered twice by the i.p. route in 10 ml/kg of 0.9% NaC1 (with two drops of Tween 80 for insoluble compounds) 5 min and 5 h after the end of aerosol exposure. Controls received the solvent alone in the same experimental conditions. Disodium cromoglycate was experimented at 12.5, 50 and 300 mg/kg in duplicate. At such doses this compound has an anti-inflammatory effect on zymosan-induced pleurisy in rats (Tarayre, Delhon, Aliaga, Barbara, Bruniquel, Caillol, Puech, Consul & Tisne-Versailles, 1989). Dexamethasone acetate was tested at the high dose of 6.25 mg/kg in duplicate due to the low sensitivity of guinea pigs to corticosteroids (Claman, 1972). Mepyramine maleate was administered at 5 mg/kg in duplicate and anhydrous theophylline at 50 mg/kg in duplicate: these doses led to a 50% drop in mortality induced by an aerosol of histamine and a passive anaphylactic shock by inhalation of antigen in conscious guinea pigs; they also clearly reduced zymosan-induced pleurisy in rats (Tarayre et al., 1989). Salbutamol was administered at 2 mg/kg in duplicate (at this dose the mortality induced by an
aerosol of histamine and by a passive anaphylatic shock by aerosol in conscious guinea pigs was reduced by more than 50%; the pleural exudate induced by PAF-acether in rats was also clearly inhibited at this dose) (Tarayre, Delhon, Aliaga, Bruniquel, Barbara, Puech, Tisne-Versailles & Couzinier, 1987). Likewise, the studied doses of indomethacin (5 mg/kg in duplicate were superior to the DEs0 obtained on the main screening tests of non steroid anti-inflammatory drugs. The six compounds studied were purchased from Sigma (St. Louis, MO).
Statistical calculations Bonferroni's test or the non-parametric Kruskal-Wallis and Wilcoxon tests were used.
RESULTS
Twenty-four hours after exposure to the ovalbumin aerosol, the increase in the number of neutrophils, mononuclear cells and eosinophils in the BAL fluid of the guinea pigs having suffered the anaphylactic shock was always significant in comparison to that of sensitized animals subjected to an aerosol of isotonic NaCI (Figs 1, 2, 3 and 4). Starting from 12.5 mg/kg, in duplicate dose, disodium cromoglycate reduced the number of mononuclear cells (Fig. 1) in proportion to the doses administered. The decreased numbers of eosinophils appeared at 50 mg/kg in duplicate. At 300 mg/kg in duplicate the anti-allergic compound slightly decreased the number of neutrophils. Dexamethasone acetate reduced the number of eosinophils in the bronchial lumen (Fig. 2). At the experimented dose, theophylline lowered the number of eosinophils without having any effect on the other cells (Fig. 3). Salbutamol tested during the same experiment involved an increase in the number of neutrophils. Mepyramine raised the number of neutrophils in the BAL fluid (Fig. 4) while indomethacin did not induce any noteworthy effect (despite a tendency to increase neutrophils). The action of the compounds on the number of various cells in the BAL fluid is summarized in Table 1.
DISCUSSION One hour after the ovalbumin challenge, the number of mononuclear cells had dropped in the bronchoalveolar lavage fluid (Tarayre et al., 1990).
Modulation of a Bronchial Inflammation Model
MONONUCLEARS
NEUTROPHILS
351
EOSINOPHILS
x
2.500_
2.500
0.500
2.000.
2.000
0.400
1.500
1.5OO
0.300
t .000
1.0001
0.200
0.500 l
0.10C
0.500
1231 Fig. 1. Action of disodium cromoglycate on the number of mononuclears, neutrophils and eosinophils in BAL fluid 24 h after an anaphylactic shock. [] 0.9% NaCI aerosol. [] Anaphylactic shock, controls. (1) Disodium cromoglycate 12.5 mg/kg x 2, (2) disodium cromoglycate 50 mg/kg x 2; (3) disodium cromoglycate 300 mg/kg × 2. °°P<0.01: anaphylactic shock control in comparison to isotonic NaCI aerosol. *P<0.05. **P<0.01 : comparison of treated group with anaphylactic shock controls. Bars represent S.E.M.; 20-21 animals per group.
MONONUCLEARS
NEUTROPHILS
,ooo
2.500 2.000
EOSINOPHILS
0.350 0.300
i
1.000.
0.200 1.000 0.500 I
0.I00
O.500 0.050 t
0.100
~
1
Fig. 2. Action of dexamethasone acetate on the number of mononuclears, neutrophils and eosimJplailsfn BAL fluid 24 h after an anaphylactic shock. [] 0.9% NaC1 aerosol. [] Anaphylactic shock, controls. (1) Dexamethasone acetate 6.25 mg/kg x 2. °P<0.05. °°P<0.01: anaphylactic shock control in comparison to isotonic NaC1 aerosol. *P<0.05: comparison of treated group with anaphylactic shock controls. Bars represent S.E.M.; 6 - 7 animals per group. This is probably due mainly to the adhesion of the macrophages to the airways and alveolar epithelium. Thus, activated macrophages might play an essential part in causing the i n f l a m m a t o r y reaction. The number o f neutrophils started increasing in the lavage fluid after 3 h, that of eosinophils after 6 h
and that of mononuclear cells after 24 h. The increase in number of mononuclear cells 2 4 - 4 8 h after the challenge may suggest that certain p h e n o m e n a o f delayed hypersensitivity might play a role at this time. It is likely that the accumulation after 2 4 - 48 h of the three types of leukocytes in the
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J. P. TARAYREet al.
MONONUCLEARS x 2.000
S
EOSINOPHILS
NEUTROPHILS 3. 0 0 0
L 0.350
c
2.500 1.500
0.300 2.000
1,000.
ii
0,250 0.200
1. 500
0.150.
1.000
I
O. 5 0 0 .
0.100.
O. 5 0 0
0.050
Jl 2 I
Fig. 3. Action of theophylline and salbutamol on the number of mononuclears, neutrophils and eosinophils in BAL fluid 24 h after an anaphylactic shock. [] 0.9% NaCI aerosol. [] Anaphylactic shock, controls. (1) Theophylline 50 mg/kg / 2; (2) salbutamol 2 mg/kg x 2. °°P<0.01: anaphylactic shock control in comparison to isotonic NaC1 aerosol. *P<0.05: comparison of treated group with anaphylactic shock controls. Bars represent S.E.M.; 14- 17 animals per group.
MONONUCLEARS
EOSINOPHILS
NEUTROPHILS
3.000 >
2.000
2. 5 0 0
0.250
2.000
0.200
1.500
0.150
1,000
0.1 O0
1.500
1.000
0.500
I
I
,
I
O. 5 0 0
,21
I 112
0.050
I
1
2'
Fig. 4. Action of mepyramine maleate and indomethacin on the number of mononuclears, neutrophils and eosinophils in BAL fluid 24 h after an anaphylactic shock. [] 0,9% NaC1 aerosol. [] Anaphylactic shock, controls. (1) Mepyramine maleate 5 mg/kg x 2; (2) indomethacin 5 mg/kg x 2. °P<0.05. °°P<0.01: anaphylactic shock control in comparison to isotonic NaC1 aerosol. *P<0.05: comparison of treated group with anaphylactic shock controls. Bars represent S.E.M.; 11 - 15 animals per group.
bronchial lumen was a result o f the interactions between various types o f cells t h r o u g h the secretion o f cytokines and interleukins. In our experimental conditions, we have not been able to observe a delayed phase in the anaphylactic shock. We detected a bronchial hyper-reactivity to
inhalation o f histamine 3 - 6 h after the anaphylactic shock but not 2 4 - 48 h after it (Tarayre et al., 1990). In h u m a n s , airway hyper-reactivity seems to depend principally on the delayed phase o f the response to allergens (Cockroft, R u f f i n , Dolovich & Hargreave, 1977; C o c k r o f t , 1983; Hargreave & Dolovich, 1984).
353
Modulation of a Bronchial Inflammation Model Table 1. Summary of the action of the compounds on the number of various cells in the BAL fluid 24 h after the challenge Compound
Eosinophils
Neutrophils
Mononuclears
Cromoglycate
[
0 (slight decrease at high dose)
[
Dexamethasone
]
0
0
Salbutamol
0
l
0
Theophylline
[
0
0
Mepyramine
0
[
0
Indomethacin
0
0
0
I Decrease; [ increase in the number of cells. But in human dual responders to allergens, bronchial hyper-reactivity is already obtained after the immediate response (Thorpe, Steinberg, Bernstein & Murlas, 1987; Durham, Craddock, Cookson & Benson, 1988). Therefore, even if it is difficult to compare with the human situation, bronchial hyperreactivity in our model is a more transitory phenomenon than in the human dual responders to allergens. In our model, airway hyper-reactivity is not correlated with maximal changes in the cell number found in BAL fluid 24 - 48 h after the challenge with ovalbumin. However, the increase at this time of the number of the mononuclear cells, neutrophils and eosinophils in the BAL fluid is of great interest for the study of a bronchial inflammation model, even if the latter cannot pretend to be a model of human asthma. Indeed inhalation of allergen in atopic asthmatics induces an increase in the number of neutrophils and eosinophils in the BAL fluid during the hours following the challenge (De Monchy et al., 1985; Metzger et al., 1985b) and a later increase in number of the macrophages and T-helper lymphocytes (Metzger et al., 1985a; Metzger et al., 1987). As a matter of fact, phenomena of delayed hypersensitivity are a subject of study on asthma of great interest. This has led us to experiment with various drugs on the bronchial inflammatory reaction obtained 24 h after the exposure to ovalbumin. The compounds have been administered after the anaphylactic shock in order to eliminate a possible action on the release of mediators during the anaphylactic shock. An interesting result lies in the decrease in number of mononuclear cells and eosinophils in the BAL fluid under the action of disodium cromoglycate. Such an effect appears at doses of similar magnitudes as
those inhibiting the exudate volume of zymosaninduced pleurisy in rats (Tarayre el al., 1989). Besides, other results have shown that the chromone derivative applied locally at the time of the challenge could diminish contact delayed hypersensitivity to picryl chloride in mice (Tarayre & Lauressergues, 1982) and to various allergens in humans (Meffert, Wischnewski & Gfinther, 1985). In addition to its action on mastocytes (much debated) it is now well established that disodium cromoglycate has an effect on plenty other categories of cells (Kay, Walsh, Moqbel, MacDonald, Nagakura, Carrol & Richerson, 1987; Tsicopoulos, Lassalle, Joseph, Tonnel, Thorel, Dessaint & Capron, 1988). It decreases the number of eosinophils in the BAL fluid of atopic asthmatics (Diaz, Galleguillos, Gonzalez, Pantin & Kay, 1984). Moreover, it reduces the number of eosinophils in the bronchial lumen after PAF-acether inhalation in the guinea pig (Aoki, Boubekeur & Sanjar, 1988) and in the delayed phase of the allergen bronchoprovocation in the guinea pig (Hutson, Holgate & Church, 1988) and in the sheep (Abraham, Sielczak, Wanner, Perruchoud, Blinder, Stevenson, Ahmed & Yerger, 1988). But in such experimental models there is no increase in the number of mononuclear cells in the bronchial lumen. Although disodium cromoglycate has been demonstrated as inactive by an experiment on antigenic activation of C D 4 + T-lymphocytes (O'Hehir & Moqbel, 1989) and since contradictory results have been described on interleukin 2 production or effect (Wang & Wang, 1986; Canonica, Scordamaglia, Bagnasco & Ciprandi, 1989; O'Hehir & Moqbel, 1989) it seems that it would be interesting to study its effect on the production by T-helper lymphocytes of different
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interleukins capable of action on eosinophils (IL3, IL5 and Gm-CSF are the most famous ones for the time being) (Owen, Rothenberg, Silberstein, Gasson, Stevens, Austen & Soberman, 1987; Rothenberg, Owen, Silberstein, Soberman, Austen & Stevens, 1987; Lopez, Sanderson, Gamble, Campbell, Young & Vadas, 1988; Saito, Hatake, Dvorak, Leiferman, Donneberg, Arai, Ishizakar & Ishizaka, 1988; Yamaguchi, Hayashi, Sugama, Miura, Kasahara, Kitamura, Torisu, Mita, Tominaga, Takatsu & Suda, 1988). Its effects on the macrophages/eosinophils interactions would also justify a specific study (Dessein, Lee, Elsas, Ravalese, Silberstein, David, Austen & Lewis, 1986; Elsas, Lee, Lenzi & Dessein, 1987). The guinea pig is a species showing little sensitivity to corticosteroid action (Claman, 1972; Giannopoulos & Keichline, 1981). Thus, while in our model dexamethasone does not show any effect on the number of mononuclear cells and neutrophils, both leukocyte categories are highly sensitive to the action of corticosteroids in different immunological and nonimmunological models in rats (Tarayre & Lauressergues, 1980). However, the inhibition by dexamethasone of the number of eosinophils is in agreement with other results obtained in guinea pigs (Aoki et al., 1988; Chand, Hess, Nolan, Diamantis, McGee & Sofia, 1989; Gulbenkian, Fernandez, Kreutner, Minnicozzi, Watnick, Kung & Egan, 1990). In this species, the action of corticosteroids seems to be mainly directed to the production of mediators inducing activation and migration of eosinophils. There are some experimental results in animals and humans which lead to the conclusion that
xanthines, contrary to/32 sympathomimetics, might have a prophylactic effect in the treatment of asthma, independently of their bronchodilating properties. This action might depend on certain antiinflammatory action of these compounds (Page, 1987). However, in many animal models the /32 sympathomimetics have an anti-oedematous action as developed as that of xanthines (Persson, Erjefalt, Grega & Svenj6, 1982; Persson & Svensj6, 1985; Persson, Erjefalt & Andersson, 1986; Tarayre et al., 1987). In our test, theophylline, contrary to salbutamol, reduced the number of eosinophils. Similar results have been described by Aoki et al. (1988) in guinea pigs. Thus, the action difference between xanthines and /32 sympathomimetics, with respect to eosinophils, might be the initial assumption from which we would start to try and differentiate the mechanism of action of these two types of drugs in asthma. Inactivity of mepyramine confirms once again that histamine especially participates in the immediate phase of the anaphylactic shock in guinea pigs. The lack of effect of indomethacin is in favour of the non-participation of the cyclo-oxygenase metabolites in the accumulation of the three types of leukocytes. The results obtained on eosinophils with both these compounds also confirm the works of Aoki et al. (1988) and Gulbenkian et al. (1990). In conclusion, the activity obtained with cromoglycate, dexamethasone and theophylline seems to show that this model might be useful for the study of the anti-inflammatory properties of potential anti-asthma agents.
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PERSSON, C. G. A. & SVENSJO, E. (1985). Vascular responses and their suppression: drugs interfering with venular permeability. In Handbook o f Inflammation, the Pharmacology o f Inflammation (eds Bonta, J. L., Bray, M. A. and Parnham, M. J.), Vol. 5, pp. 6 1 - 81. Elsevier, Amsterdam. ROTHENBERG, M. E., OWEN, W. F., SILBERSTEIN, D. S., SOBERMAN, R. J., AUSTEN, K. F. 8,:; STEVENS, R. L. (1987). Eosinophils cocultured with endothetial cells have increased survival and functional properties. Science, 237, 645 - 647. SA1TO, H., HATAKE, K., DVORAK, A., LE1FERMAN,K. M., DONNEBERG, A. D., ARAI, N., ISHIZAKA,K. & ISHIZAKA,T. (1988). Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins. Proc. natn. Acad. Sci. U.S.A., 85, 2288-2292. TARAYRE, J. P., ALIAGA, M., BARBARA,M., TISSEYRE, N., VIEU, S. & TISNE-VERSAILLES,J, (1990). Model of bronchial hyperreactivity after active anaphylactic shock in conscious guinea pigs. J. Pharmac. Meth., 23, 13- 19. TARAYRE, J. P., DELHON, A., ALIAGA,M., BARBARA,M., BRUNIQUEL,F., CAILLOL,V., PUECH, L., CONSUL,N. & TISNLVERSAILLES, J. (1989). Pharmacological studies on zymosan inflammation in rats and mice. 2 : zymosan-induced pleurisy in rats. Pharmac. Res,, 21, 385- 395. TARAYRE, J. P., DELHON, A., ALIAGA,M., BRUNIQUEL,F., BARBARA,M., PUECH, L., TISNE-VERSAILLES,J. & COUZIN1ER, J. P. (1987). Pharmacological modulation of PAF-acether induced pleurisy in rats. Pharmac. Res., 19, 859- 876. TARAYRE, J. P. 8,: LAURESSERGUES, H. (1980). Comparison of the effect of phenylbutazone, desonide and cyclophosphamide on four types of experimental pleurisy. J. Pharm. Pharmac., 32, 408-412. TARAVRE, J. P. & LAURESSERGUES,H. (1982). Modulation by various locally applied anti-inflammatory and anti-allergic compounds of the immune and non-immune inflammation induced by picryl chloride in mice. Agents Actions, 12, 662 - 670. THORPE, J. E., STEINBERG, D., BERNSTEIN, I. L. 8,:; MURLAS, C. (1987). Bronchial reactivity increases soon after the immediate response in dual-responding asthmatic subjects. Chest, 91, 2 1 - 25. TS1COPOULOS, A., LASSALLE,P., JOSEPH, M., TONNEL, A. B., THOREL, T., DESSAINT,S. P. & CAPRON, A. (1988). Effect of disodium cromoglycate on inflammatory ceils bearing the Fc Epsilon receptor type I1 (Fe~ R I1), Int. J. Immunopharrnac., 10, 227- 236. WANG, S. R. & WANG, S. S. M. (1986). Cellular immunity inhibition by cromolyn sodium. J. Allergy. clin. Immun., 77 1, Pt 2, 217. YAMAGUCHI,Y., HAYASH1,Y., SUGAMA,Y., MIURA,Y., KASAHARA,T., KITAMURA,S., TORISU, M., MITA, S., TOMINAGA, A., TAKATSU, K. &; SUDA, T. (1988). Highly purified murine interleukin 5 (IL-5) stimulates eosinophil function and prolong in vitro survival. J. exp. Med., 167, 1737 - 1742.
Int. Z lmmunopharmac., Vol. 13, No. 4, pp. 349-356, 1991. Printed in Great Brilain.
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 OF A M O D E L OF B R O N C H I A L I N F L A M M A T I O N AFTER A E R O S O L - I N D U C E D ACTIVE A N A P H Y L A C T I C SHOCK IN CONSCIOUS G U I N E A PIGS J. P. TARAYRE, M. ALIAGA, M. BARBARA, N. TISSEYRE, S. VIEU and J. TISNE-VERSAILLES Centre de Recherche Pierre Fabre, 17 avenue Jean Moulin, 81106 Castres, France
(Received for publication 7 November 1990)
Abslract - - Twenty-four hours after an active anaphylactic shock induced by inhalation of antigen in conscious guinea pigs sensitized by a large dose of ovalbumin in complete Freund's adjuvant, a noteworthy bronchial inflammation, characterized by increased numbers of neutrophils, mononuclear cells and eosinophils in the bronchoalveolar lavage fluid, was observed. Some drugs administered after the anaphylactic shock were investigated using this model. Disodium cromoglycate primarily reduced the number of mononuclear cells and eosinophils. Dexamethasone and theophylline decreased the number of eosinophils. Salbutamol and mepyramine increased neutrophils. Indomethacin did not give rise to any significant effect. This test appears to be of use for the investigation of anti-inflammatory compounds in the prophylactic treatment of asthma.
In recent years, many works have established that inflammation plays an important part in asthma (review by Morley & Smith, 1989). In clinical pharmacology, as well as in animal models, bronchoalveolar lavage (BAL) permitted the measurement of certain parameters in bronchial inflammation. In clinical pharmacology, bronchoprovocation by inhalation of allergen in atopic asthmatics showed an increase in the number of neutrophils and eosinophils in the BAL fluid during the hours following the challenge (De Monchy, Kauffman, Venge, Koeter, Jansen, Sluiter & De Vries, 1985; Metzger, Richerson, Worden, Monick & Hunninghake, 1985b) and a later increase in the number of macrophages and T-helper lymphocytes (Metzger, Hunninghake & Richerson, 1985a; Metzger, Zavala, Richerson, Moseley, Iwamota, Monick, Sjoerdsma & Hunninghake, 1987). The possible role of delayed hypersensitivity in the inflammatory reactions of asthma is being thoroughly studied (review by Kay, Wardlmaw, Moqbel, Buchanan, Cromwell & Fitzharris, 1987). In guinea pigs various models of bronchial inflammation, after an anaphylactic shock induced by inhalation of antigen, have been described (Iijima, Ishii, Yamauchi, Chad, Kimura, Shimura, Shindoh, Indue, Mue & Takishima, 1987; Coyle, Urwin, Page, Touvay, Villain & Braquet, 1988; Hutson, Church, Clay, Miller & Holgate, 1988).
However, the number of leukocytes in the BAL fluid up to 48 h after the anaphylatic shock only showed an increase in neutrophils and eosinophils. Recently, while sensitizing guinea pigs with a large dose of ovalbumin and complete Freund's adjuvant, we developed a model which allowed us to obtain increased numbers of mononuclear cells in the BAL fluid, in addition to increased numbers of neutrophils and eosinophils 2 4 - 48 h after an active anaphylactic shock by inhalation of antigen (Tarayre, Aliaga, Barbara, Tisseyre, Vieu & TisneVersailles, 1990). Various drugs were investigated using this model in the present work.
EXPERIMENTAL PROCEDURES
Sensitization Male Dunkin-Hartley guinea pigs of average weight 2 5 0 - 2 7 5 g were sensitized by intramuscular injection (1 ml/kg) of 30 mg/kg ovalbumin (Sigma) dissolved in a 50:50 mixture (v/v) of Freund's complete adjuvant (Difco) in 0.9°70 NaCI. Anaphylactic shock Three to six weeks after sensitization, the conscious animal was placed in a circular glass box 26 cm in diameter and 15 cm high. Aerosols were
349
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induced by a nebuliser (Jouan, Paris, France) producing dry particles 1 - 3 tam in diameter, at a flow rate of 28 ml/h. The nebuliser was supplied with compressed air at a pressure of 1 bar. The animal was exposed for 6 rain to an aerosol of a 5% solution (w/v) of ovalbumin in 0.9% NaC1. In these experimental conditions some animals had reversible convulsions despite continuous exposure to the ovalbumin aerosol. Immediate or delayed mortality was rare. The different groups were homogenized to a maximum as far as the number of guinea pigs having reversible convulsions was concerned. A control group of sensitized animals was exposed for 6 min to an aerosol of saline.
Bronchoalveolar lavage (BAL) Twenty-four hours after anaphylactic shock the guinea pigs were killed by intraperitoneal administration of 300 mg/kg sodium pentobarbital (Sanofi). Their tracheas were then canulated, and the BAL carried out twice with 5 ml of sterile 0.9% NaC1. On average, 80°7o of the fluid administered was recovered and the leukocytes were then counted with a Coulter Counter (Coultronics model ZF). After spreading on a slide, fixation, and staining with M a y - G r i i n w a l d - G i e m s a , the number of mononuclear cells, neutrophils, and eosinophils were determined on at least 350 cells per slide. Treatment Drugs were administered twice by the i.p. route in 10 ml/kg of 0.9% NaC1 (with two drops of Tween 80 for insoluble compounds) 5 min and 5 h after the end of aerosol exposure. Controls received the solvent alone in the same experimental conditions. Disodium cromoglycate was experimented at 12.5, 50 and 300 mg/kg in duplicate. At such doses this compound has an anti-inflammatory effect on zymosan-induced pleurisy in rats (Tarayre, Delhon, Aliaga, Barbara, Bruniquel, Caillol, Puech, Consul & Tisne-Versailles, 1989). Dexamethasone acetate was tested at the high dose of 6.25 mg/kg in duplicate due to the low sensitivity of guinea pigs to corticosteroids (Claman, 1972). Mepyramine maleate was administered at 5 mg/kg in duplicate and anhydrous theophylline at 50 mg/kg in duplicate: these doses led to a 50% drop in mortality induced by an aerosol of histamine and a passive anaphylactic shock by inhalation of antigen in conscious guinea pigs; they also clearly reduced zymosan-induced pleurisy in rats (Tarayre et al., 1989). Salbutamol was administered at 2 mg/kg in duplicate (at this dose the mortality induced by an
aerosol of histamine and by a passive anaphylatic shock by aerosol in conscious guinea pigs was reduced by more than 50%; the pleural exudate induced by PAF-acether in rats was also clearly inhibited at this dose) (Tarayre, Delhon, Aliaga, Bruniquel, Barbara, Puech, Tisne-Versailles & Couzinier, 1987). Likewise, the studied doses of indomethacin (5 mg/kg in duplicate were superior to the DEs0 obtained on the main screening tests of non steroid anti-inflammatory drugs. The six compounds studied were purchased from Sigma (St. Louis, MO).
Statistical calculations Bonferroni's test or the non-parametric Kruskal-Wallis and Wilcoxon tests were used.
RESULTS
Twenty-four hours after exposure to the ovalbumin aerosol, the increase in the number of neutrophils, mononuclear cells and eosinophils in the BAL fluid of the guinea pigs having suffered the anaphylactic shock was always significant in comparison to that of sensitized animals subjected to an aerosol of isotonic NaCI (Figs 1, 2, 3 and 4). Starting from 12.5 mg/kg, in duplicate dose, disodium cromoglycate reduced the number of mononuclear cells (Fig. 1) in proportion to the doses administered. The decreased numbers of eosinophils appeared at 50 mg/kg in duplicate. At 300 mg/kg in duplicate the anti-allergic compound slightly decreased the number of neutrophils. Dexamethasone acetate reduced the number of eosinophils in the bronchial lumen (Fig. 2). At the experimented dose, theophylline lowered the number of eosinophils without having any effect on the other cells (Fig. 3). Salbutamol tested during the same experiment involved an increase in the number of neutrophils. Mepyramine raised the number of neutrophils in the BAL fluid (Fig. 4) while indomethacin did not induce any noteworthy effect (despite a tendency to increase neutrophils). The action of the compounds on the number of various cells in the BAL fluid is summarized in Table 1.
DISCUSSION One hour after the ovalbumin challenge, the number of mononuclear cells had dropped in the bronchoalveolar lavage fluid (Tarayre et al., 1990).
Modulation of a Bronchial Inflammation Model
MONONUCLEARS
NEUTROPHILS
351
EOSINOPHILS
x
2.500_
2.500
0.500
2.000.
2.000
0.400
1.500
1.5OO
0.300
t .000
1.0001
0.200
0.500 l
0.10C
0.500
1231 Fig. 1. Action of disodium cromoglycate on the number of mononuclears, neutrophils and eosinophils in BAL fluid 24 h after an anaphylactic shock. [] 0.9% NaCI aerosol. [] Anaphylactic shock, controls. (1) Disodium cromoglycate 12.5 mg/kg x 2, (2) disodium cromoglycate 50 mg/kg x 2; (3) disodium cromoglycate 300 mg/kg × 2. °°P<0.01: anaphylactic shock control in comparison to isotonic NaCI aerosol. *P<0.05. **P<0.01 : comparison of treated group with anaphylactic shock controls. Bars represent S.E.M.; 20-21 animals per group.
MONONUCLEARS
NEUTROPHILS
,ooo
2.500 2.000
EOSINOPHILS
0.350 0.300
i
1.000.
0.200 1.000 0.500 I
0.I00
O.500 0.050 t
0.100
~
1
Fig. 2. Action of dexamethasone acetate on the number of mononuclears, neutrophils and eosimJplailsfn BAL fluid 24 h after an anaphylactic shock. [] 0.9% NaC1 aerosol. [] Anaphylactic shock, controls. (1) Dexamethasone acetate 6.25 mg/kg x 2. °P<0.05. °°P<0.01: anaphylactic shock control in comparison to isotonic NaC1 aerosol. *P<0.05: comparison of treated group with anaphylactic shock controls. Bars represent S.E.M.; 6 - 7 animals per group. This is probably due mainly to the adhesion of the macrophages to the airways and alveolar epithelium. Thus, activated macrophages might play an essential part in causing the i n f l a m m a t o r y reaction. The number o f neutrophils started increasing in the lavage fluid after 3 h, that of eosinophils after 6 h
and that of mononuclear cells after 24 h. The increase in number of mononuclear cells 2 4 - 4 8 h after the challenge may suggest that certain p h e n o m e n a o f delayed hypersensitivity might play a role at this time. It is likely that the accumulation after 2 4 - 48 h of the three types of leukocytes in the
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J. P. TARAYREet al.
MONONUCLEARS x 2.000
S
EOSINOPHILS
NEUTROPHILS 3. 0 0 0
L 0.350
c
2.500 1.500
0.300 2.000
1,000.
ii
0,250 0.200
1. 500
0.150.
1.000
I
O. 5 0 0 .
0.100.
O. 5 0 0
0.050
Jl 2 I
Fig. 3. Action of theophylline and salbutamol on the number of mononuclears, neutrophils and eosinophils in BAL fluid 24 h after an anaphylactic shock. [] 0.9% NaCI aerosol. [] Anaphylactic shock, controls. (1) Theophylline 50 mg/kg / 2; (2) salbutamol 2 mg/kg x 2. °°P<0.01: anaphylactic shock control in comparison to isotonic NaC1 aerosol. *P<0.05: comparison of treated group with anaphylactic shock controls. Bars represent S.E.M.; 14- 17 animals per group.
MONONUCLEARS
EOSINOPHILS
NEUTROPHILS
3.000 >
2.000
2. 5 0 0
0.250
2.000
0.200
1.500
0.150
1,000
0.1 O0
1.500
1.000
0.500
I
I
,
I
O. 5 0 0
,21
I 112
0.050
I
1
2'
Fig. 4. Action of mepyramine maleate and indomethacin on the number of mononuclears, neutrophils and eosinophils in BAL fluid 24 h after an anaphylactic shock. [] 0,9% NaC1 aerosol. [] Anaphylactic shock, controls. (1) Mepyramine maleate 5 mg/kg x 2; (2) indomethacin 5 mg/kg x 2. °P<0.05. °°P<0.01: anaphylactic shock control in comparison to isotonic NaC1 aerosol. *P<0.05: comparison of treated group with anaphylactic shock controls. Bars represent S.E.M.; 11 - 15 animals per group.
bronchial lumen was a result o f the interactions between various types o f cells t h r o u g h the secretion o f cytokines and interleukins. In our experimental conditions, we have not been able to observe a delayed phase in the anaphylactic shock. We detected a bronchial hyper-reactivity to
inhalation o f histamine 3 - 6 h after the anaphylactic shock but not 2 4 - 48 h after it (Tarayre et al., 1990). In h u m a n s , airway hyper-reactivity seems to depend principally on the delayed phase o f the response to allergens (Cockroft, R u f f i n , Dolovich & Hargreave, 1977; C o c k r o f t , 1983; Hargreave & Dolovich, 1984).
353
Modulation of a Bronchial Inflammation Model Table 1. Summary of the action of the compounds on the number of various cells in the BAL fluid 24 h after the challenge Compound
Eosinophils
Neutrophils
Mononuclears
Cromoglycate
[
0 (slight decrease at high dose)
[
Dexamethasone
]
0
0
Salbutamol
0
l
0
Theophylline
[
0
0
Mepyramine
0
[
0
Indomethacin
0
0
0
I Decrease; [ increase in the number of cells. But in human dual responders to allergens, bronchial hyper-reactivity is already obtained after the immediate response (Thorpe, Steinberg, Bernstein & Murlas, 1987; Durham, Craddock, Cookson & Benson, 1988). Therefore, even if it is difficult to compare with the human situation, bronchial hyperreactivity in our model is a more transitory phenomenon than in the human dual responders to allergens. In our model, airway hyper-reactivity is not correlated with maximal changes in the cell number found in BAL fluid 24 - 48 h after the challenge with ovalbumin. However, the increase at this time of the number of the mononuclear cells, neutrophils and eosinophils in the BAL fluid is of great interest for the study of a bronchial inflammation model, even if the latter cannot pretend to be a model of human asthma. Indeed inhalation of allergen in atopic asthmatics induces an increase in the number of neutrophils and eosinophils in the BAL fluid during the hours following the challenge (De Monchy et al., 1985; Metzger et al., 1985b) and a later increase in number of the macrophages and T-helper lymphocytes (Metzger et al., 1985a; Metzger et al., 1987). As a matter of fact, phenomena of delayed hypersensitivity are a subject of study on asthma of great interest. This has led us to experiment with various drugs on the bronchial inflammatory reaction obtained 24 h after the exposure to ovalbumin. The compounds have been administered after the anaphylactic shock in order to eliminate a possible action on the release of mediators during the anaphylactic shock. An interesting result lies in the decrease in number of mononuclear cells and eosinophils in the BAL fluid under the action of disodium cromoglycate. Such an effect appears at doses of similar magnitudes as
those inhibiting the exudate volume of zymosaninduced pleurisy in rats (Tarayre el al., 1989). Besides, other results have shown that the chromone derivative applied locally at the time of the challenge could diminish contact delayed hypersensitivity to picryl chloride in mice (Tarayre & Lauressergues, 1982) and to various allergens in humans (Meffert, Wischnewski & Gfinther, 1985). In addition to its action on mastocytes (much debated) it is now well established that disodium cromoglycate has an effect on plenty other categories of cells (Kay, Walsh, Moqbel, MacDonald, Nagakura, Carrol & Richerson, 1987; Tsicopoulos, Lassalle, Joseph, Tonnel, Thorel, Dessaint & Capron, 1988). It decreases the number of eosinophils in the BAL fluid of atopic asthmatics (Diaz, Galleguillos, Gonzalez, Pantin & Kay, 1984). Moreover, it reduces the number of eosinophils in the bronchial lumen after PAF-acether inhalation in the guinea pig (Aoki, Boubekeur & Sanjar, 1988) and in the delayed phase of the allergen bronchoprovocation in the guinea pig (Hutson, Holgate & Church, 1988) and in the sheep (Abraham, Sielczak, Wanner, Perruchoud, Blinder, Stevenson, Ahmed & Yerger, 1988). But in such experimental models there is no increase in the number of mononuclear cells in the bronchial lumen. Although disodium cromoglycate has been demonstrated as inactive by an experiment on antigenic activation of C D 4 + T-lymphocytes (O'Hehir & Moqbel, 1989) and since contradictory results have been described on interleukin 2 production or effect (Wang & Wang, 1986; Canonica, Scordamaglia, Bagnasco & Ciprandi, 1989; O'Hehir & Moqbel, 1989) it seems that it would be interesting to study its effect on the production by T-helper lymphocytes of different
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J. P. TARAYREet al.
interleukins capable of action on eosinophils (IL3, IL5 and Gm-CSF are the most famous ones for the time being) (Owen, Rothenberg, Silberstein, Gasson, Stevens, Austen & Soberman, 1987; Rothenberg, Owen, Silberstein, Soberman, Austen & Stevens, 1987; Lopez, Sanderson, Gamble, Campbell, Young & Vadas, 1988; Saito, Hatake, Dvorak, Leiferman, Donneberg, Arai, Ishizakar & Ishizaka, 1988; Yamaguchi, Hayashi, Sugama, Miura, Kasahara, Kitamura, Torisu, Mita, Tominaga, Takatsu & Suda, 1988). Its effects on the macrophages/eosinophils interactions would also justify a specific study (Dessein, Lee, Elsas, Ravalese, Silberstein, David, Austen & Lewis, 1986; Elsas, Lee, Lenzi & Dessein, 1987). The guinea pig is a species showing little sensitivity to corticosteroid action (Claman, 1972; Giannopoulos & Keichline, 1981). Thus, while in our model dexamethasone does not show any effect on the number of mononuclear cells and neutrophils, both leukocyte categories are highly sensitive to the action of corticosteroids in different immunological and nonimmunological models in rats (Tarayre & Lauressergues, 1980). However, the inhibition by dexamethasone of the number of eosinophils is in agreement with other results obtained in guinea pigs (Aoki et al., 1988; Chand, Hess, Nolan, Diamantis, McGee & Sofia, 1989; Gulbenkian, Fernandez, Kreutner, Minnicozzi, Watnick, Kung & Egan, 1990). In this species, the action of corticosteroids seems to be mainly directed to the production of mediators inducing activation and migration of eosinophils. There are some experimental results in animals and humans which lead to the conclusion that
xanthines, contrary to/32 sympathomimetics, might have a prophylactic effect in the treatment of asthma, independently of their bronchodilating properties. This action might depend on certain antiinflammatory action of these compounds (Page, 1987). However, in many animal models the /32 sympathomimetics have an anti-oedematous action as developed as that of xanthines (Persson, Erjefalt, Grega & Svenj6, 1982; Persson & Svensj6, 1985; Persson, Erjefalt & Andersson, 1986; Tarayre et al., 1987). In our test, theophylline, contrary to salbutamol, reduced the number of eosinophils. Similar results have been described by Aoki et al. (1988) in guinea pigs. Thus, the action difference between xanthines and /32 sympathomimetics, with respect to eosinophils, might be the initial assumption from which we would start to try and differentiate the mechanism of action of these two types of drugs in asthma. Inactivity of mepyramine confirms once again that histamine especially participates in the immediate phase of the anaphylactic shock in guinea pigs. The lack of effect of indomethacin is in favour of the non-participation of the cyclo-oxygenase metabolites in the accumulation of the three types of leukocytes. The results obtained on eosinophils with both these compounds also confirm the works of Aoki et al. (1988) and Gulbenkian et al. (1990). In conclusion, the activity obtained with cromoglycate, dexamethasone and theophylline seems to show that this model might be useful for the study of the anti-inflammatory properties of potential anti-asthma agents.
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