Veterinary Microbiology, 22 (1990) 259-266 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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The Effect of PasteureUa haemolytica C y t o t o x i n on B o v i n e P o l y m o r p h o n u c l e a r L e u k o c y t e s can be A t t e n u a t e d by fl-Adrenoceptor A n t a g o n i s t s P.A.J. HENRICKS*, G.J. BINKHORST, A.A. DRIJVER, H. VAN DER VLIET and F.P. NIJKAMP
Department o[ Pharmacology, Faculty of Pharmacy and Department of Large Animal Medicine, Faculty of Veterinary Medicine, State Universityof Utrecht, Utrecht (The Netherlands) (Accepted 25 September 1989)
ABSTRACT Henricks, P.A.J., Binkhorst, G.J., Drijver, A.A., Van der Vliet, H. and Nijkamp, F.P., 1990. The effect of Pasteurella haemolytica cytotoxin on bovine polymorphonuclear leukocytes can be attenuated by fl-adrenoceptor antagonists. Vet. Microbiol., 22: 259-266. It was investigated whether fl-adrenoceptor antagonists could disturb the interaction between cytotoxin preparations isolated from Pasteurella haemolytica and bovine polymorphonuclear leukocytes (PMNs). The toxicity of the cytotoxin preparation was evaluated by measuring the chemiluminescence response and the viability of the cells after incubation with the cytotoxin. No effect on cell viability was detected when PMNs were incubated with 63 #g cytotoxin per ml while the chemiluminescence response was diminished by approximately 30%. The fl-adrenoceptor antagonists alprenolol (10 -5 M) and propranolol (5X 10-7-5 X 10 -8 M) were able to attenuate this effect of cytotoxin on the chemiluminescence response of PMNs. It seemed unlikely that propranolol and alprenolol diminished the effect of cytotoxin on the chemiluminescence response of PMNs by their fl-adrenoceptor blocking potency because other fl-adrenoceptor antagonists used were without effect. Also, the membrane stabilizing characteristics of the fl-adrenoceptor antagonists used were probably not responsible for the diminished interaction between PMNs and the cytotoxin. Whether fl-adrenoceptor antagonists could be used in vivo to prevent or treat P. haemolytica infections in bovines remains to be examined.
INTRODUCTION
Pasteurella species, especially P. haemolytica type A1, are major bovine respiratory pathogens. The bacterium has been associated with the advanced clinical signs, terminal lesions and fatality of pneumonic pasteurellosis (shipping fever) of cattle (Rehmtulla and Thomson, 1984). The live bacterium is toxic *To whom all correspondence should be addressed: Department of Pharmacology, Faculty of Pharmacy, Catharijnesinge160, 3511 GH Utrecht, The Netherlands.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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P.A.J. HENRICKS ET AL.
for phagocytic cells like alveolar macrophages and polymorphonuclear leukocytes (PMNs) of bovines in vitro (Maheswaran et al., 1980; Berggren et al., 1981 ). The toxic effect is due to the production of a cell-free cytotoxin by actively growing bacteria (Shewen and Wilkie, 1985 ). Biochemical studies have identified the toxin as a heat-labile, oxygen-stable, pH-stable non-dialyzable water-soluble antigenic glycoprotein (Chang et al., 1986b ). This cytotoxin may play a role in the pathogenesis of pneumonic pasteurellosis by diminishing phagocytic cell defense, or indirectly, by causing an inflammatory response mediated by lysosomal constituents released by cytotoxin-exposed leukocytes (Baluyut et al., 1981 ). Bovine respiratory disease is considered to be associated with initial viral infections and/or stressful circumstances (Lopez et al., 1976; Filion et al., 1984). The exact mechanisms by which stress factors influence the host defense system is unknown. Plasma levels of for example cortisol and catecholamines are increased in response to stress (Axelrod and Reisine, 1984; Slocombe et al., 1984). Glucocorticoids have anti-inflammatory and immunosuppressive actions (Roth and Kaeberle, 1981 ) and fl-adrenoceptor agonists depress phagocytic cell activities of PMNs {Nielson, 1987). Interestingly, the incidence of respiratory infections in calves after transport stress was lowered when fl-adrenoceptor antagonists had been given preventively to the animals (Ra§kov~i et al., 1987). A blockade of the immunosuppressive actions of catecholamines could be an explanation for these findings. The hypothesis of our study was that fl-adrenoceptor antagonists could also disturb the interaction between the toxic bacterial components and phagocytic cells. Therefore, it was investigated whether fl-adrenoceptor antagonists were able to prevent the toxic effects of the cytotoxin of P. haemolytica on blood PMNs of calves in vitro. MATERIALSAND METHODS P. haemolytica cytotoxin P. haemolytica type A1 (generously supplied by Dr. N.J.L. Gilmour, Moredun Research Institute, Edinburgh, U.K.) was grown in brain-heart infusion at 37°C on a shaker. The isolation of cytotoxin was performed according to Shewen and Wilkie {1982). Before use, the lyophilized material was reconstituted to the desired concentration in Eagle's minimum essential medium modified with Earle's salts (Eagle's MEM; Flow Laboratories, Irvine, U.K.). A concentration of 2 mg/ml corresponded on a dry weight basis to the concentration before lyophilization. The preparations contained less than 1% endotoxin as determined by using the Limulus amebocyte lysate-5000 Automatic endotoxin detection system (Cape Cod, Woods Hole, MA) at the Department of Medical Microbiology, State University of Utrecht.
P. HAEMOLYTICA CYTOTOXIN CAN BE ATTENUATED BY fl-ADRENOCEPTOR ANTAGONISTS
261
PMNs suspensions Peripheral blood samples were obtained from 6-15-week-old Friesian-Holstein calves by jugular vein punction and collected in heparinized tubes. PMNs were isolated by lysis of contaminating red blood cells and differential density centrifugation (Henricks et al., 1987). The final cell preparations contained 5 X 106 P M N s / m l Eagle's MEM.
fl-Adrenoceptor antagonists 1-Alprenolol tartrate, pindolol and oxprenolol were purchased from Sigma Chemical Company, St. Louis, MO. The following drugs were kindly supplied as gifts: timolol maleate (Merck, Sharp and Dohme, Haarlem, The Netherlands), practolol, ICI 118.551 hydrochloride and propranolol hydrochloride (Imperial Chemical Industries, Macclesfield, U.K. ).
Incubation of PMNs with cytotoxin and/or fl-adrenoceptor antagonists PMNs (1 X 106 cells) were incubated with or without cytotoxin (0-250 #g/ ml) a n d / o r fl-adrenoceptor antagonists (0.1-10 ttM) in a total volume of 0.4 ml Eagle's M E M for 0-60 min at 37°C. The PMNs were mixed with the fladrenoceptor antagonists and incubated for 10 min at room temperature before addition of other reagents. The PMNs were pelleted after incubation and resuspended in Eagle's M E M for measurement of viability and generation of chemiluminescence. Viability was determined by trypan blue exclusion.
Generation of chemiluminescence Generation of chemiluminescence by P M N s (1 X 106 cells) was measured in a luminometer (type 1251 LKB Wallac, Turku, Finland) in the presence of 0.5 m M lucigenin (bis-N-methylacridinium nitrate; Sigma Chemical Company, St. Louis, MO ) in a total volume of 0.5 ml. Zymosan (2.5 mg/ml; Sigma Chemical Company, St. Louis, MO), preopsonized in 100% pooled bovine serum, was used as stimulus. Chemiluminescence was determined every 60 sec over a 45 min period.
Statistical analysis Grouped data are expressed as the m e a n + s t a n d a r d error of the mean (s.e.m.). Student's t-test and Wilcoxon's rank sum test were applied for statistical evaluation of the results. P values exceeding 0.05 were considered not significant. RESULTS
Effect of cytotoxin on PMNs chemiluminescence response and cell viability P M N s stimulated with opsonized zymosan exhibited a moderate increase in chemiluminescence during the initial 15-25 min whereafter there was a pro-
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gressive decrease in the amount of light emitted. In our system, the cells produced 128 + 11 mV peak chemiluminescence (n = 13; range 50-230 mV). The amount of chemiluminescence generated was dose-dependently reduced when the PMNs had been incubated with different amounts of cytotoxin (Fig. 1; Table 1), which is in agreement with previous observations (Chang et al., 1986a). Incubating PMNs with 63 ~g cytotoxin/ml for 30 min at 37 ° C resulted in 85 2 12 mV peak chemiluminescence (n= 13; range 30-190 mV) generated. 250
~200 E 0 150
100
50
10
20
30
Time (min)
40
Fig. 1. Generation of chemiluminescence by P M N s (1 × 106 cells) incubated for 30 min with 0 ( • ) , 31 ( • ), 63 ( A ), 125 ( [ ] ) or 250 ( O ) ltg cytotoxin/ml upon stimulation with opsonized zymosan. The generation of chemiluminescence was measured every 60 s over a 45 min period in a luminometer in the presence of 0.5 m M lucigenin. Results shown are representative of four separate experiments. TABLE1 Viability and peak chemiluminescence response upon stimulation with zymosan of P M N s incubated with various amounts of cytotoxin for 30 min at 37 ° C ( n = 4)
Control 16 # g / m l 31 # g / m l 63 #g/ml 125 #g/ml 250#g/ml
cytotoxin cytotoxin cytotoxin cytotoxin cytotoxin
% Viability
% Chemiluminescence
98 ± 1 96 ± 1 96+ 1 95 -+ 2 79 _+21 61 ±21
100 101 +_ 4 99+_ 7 71 -+ 81 50 -+ 111 21 -+ 71
1p < 0.05 as compared to control values.
P. HAEMOLYTICA CYTOTOXIN CAN BE ATTENUATEDBYfl-ADRENOCEPTORANTAGONISTS
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The decrease in chemiluminescence generated was also dependent on the time period by which the cells had been incubated with the cytotoxin. Chemiluminescence generated by PMNs was diminished by approximately 10, 15, 30 and 50% when the cells were incubated with 63/~g cytotoxin/ml for respectively 5, 10, 30 or 60 min at 37 ° C. Table 1 also shows the effect of P. haemolytica cytotoxin on the viability of bovine PMNs as determined by trypan blue exclusion. When PMNs had been incubated with less than 63/tg cytotoxin/ml for 30 min no effects on the viability of the cells were observed (Table 1). Diminished viabilities were observed when higher amounts were used ( > 125 zg cytotoxin/ml; Table 1 ). Viability of PMNs was also more depressed when PMNs were incubated with cytotoxin for longer periods of time as compared to an incubation period of 30 min.
Effect of fl-adrenoceptor antagonists on cytotoxin-induced changes in the chemiluminescence response of PMNs PMNs were incubated with 63 ]lg cytotoxin/ml in the presence and absence of selective and non-selective fl-adrenoceptor antagonists for 30 min. No fladrenoceptor antagonists in the concentrations used had any effects on the chemiluminescence response of control PMNs (data not shown). The reducAlprenolol
'°80]°1 ~ 40 100
Oxprenolol
Pindolol
Practolol
Propranolol
Timolol
ICI 118,551
-~ 60
-~
Q.
40
40
0.10.51 510
0.10.51 5 1 0
0.1051 5 1 0 Concentration (juM)
Fig. 2. Peak chemiluminescence response of P M N s (% of control) upon stimulation with opsonized zymosan (n = 4-7 ). The phagocytic cells had been incubated for 30 min with 63 ttg cytotoxin per ml in the presence of 10-7-10 -5 M p-adrenoceptor antagonists before stimulation. The generation of chemiluminescence was measured every 60 s over a 45 min period in a luminometer in the presence of 0.5 m M lucigenin. All data were significantly diminished ( P < 0.05) as compared to control values except * P > 0.05 and **P > 0.10. N.D. = not done.
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tion induced by cytotoxin of the chemiluminescence generated by PMNs could not be prevented by the fl-adrenoceptor antagonists oxprenolol, pindolol, timolol, practolol or ICI 118.551 (Fig. 2). However, propranolol in concentrations between 5 × 10 -7 and 5 × 10 -6 M and alprenolol in a concentration of 10 -~ M were able to attenuate the effect of cytotoxin on the chemiluminescence response of PMNs (Fig. 2). DISCUSSION Results from the present study have confirmed previous observations related to the leukotoxic effects ofP. haemolytica cytotoxin (Shewen and Wilkie, 1982; Chang et al., 1986a). It was found that the cytotoxin was able to diminish the chemiluminescence response of bovine PMNs in vitro dose-dependently. Chemiluminescence generated by PMNs correlates with their metabolic activation and is used as an assay to study phagocytic cellular function (Allen et al., 1972; Easmon et al., 1980). Lucigenin was used as an amplifying agent in this study and lucigenin-dependent chemiluminescence reflects the degree of superoxide generation by PMNs undergoing activation (Stevens and Hong, 1984). The chemiluminescence assay is found to be the most sensitive method to study the effect of cytotoxin on phagocytic cell function (Chang and Renshaw, 1986). No effect on cell viability was detected when PMNs were incubated with 63 #g cytotoxin per ml while the chemiluminescence response was diminished by approximately 30%. The toxic effect of P. haemolytica cytotoxin on phagocytic cells of bovines may be an important pathogenic factor in the early stage of P. haemolyticainduced pneumonia. Stress factors are thought to contribute to the facilitation of the clinical pattern (Filion et al., 1984; Rehmtulla and Thomson, 1984; Slocombe et al., 1984). Administration of fl-adrenoceptor antagonists to animals lowers the incidence of respiratory infections in calves after transportation (Ra~kov~i et al., 1987). This could be due to a blockade of the impairment of phagocytic cell activity induced by enhanced levels of catecholamines or to fladrenoceptor antagonists-induced changes in the membrane of the cells resulting in a diminished interaction between the cytotoxin and phagocytic cells. To our knowledge, no data are available in the literature about the mechanism of interaction between P. haemolytica cytotoxin and phagocytic cells. It was found in this study that the non-selective fl-adrenoceptor antagonists propranolol (5X 10-7-5X 10 -6 M) and alprenolol (10 -5 M) were able to neutralize the effect of cytotoxin on the chemiluminescence response of PMNs. The other fl-adrenoceptor antagonists used in this study did not influence the effect of cytotoxin. Also, propranolol in a concentration of 10 -6 M was without effect on the detrimental action of cytotoxin. Therefore it seems unlikely that propranolol influences the effect of cytotoxin on PMNs due to its fl-adrenoceptor blocking potency.
P. HAEMOLYTICA CYTOTOXIN CAN BE ATTENUATED BY fl-ADRENOCEPTOR ANTAGONISTS
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fl-Adrenoceptor antagonists are able to induce a perturbation of the lipid membrane and extent a membrane stabilizing effect due to their lipophilic properties. The membrane stabilizing characteristics of some fl-adrenoceptor antagonists changes the physico-chemical properties of the cell surface and could alter membrane receptor properties as a consequence of distortion of the microenvironment. Propranolol is relatively more potent as pindolol, timolol and practolol to influence lipid membranes (Kerry et al., 1984; Ondria~ et al., 1987). Higher amounts of alprenolol were needed to modify the effect of cytotoxin on the chemiluminescence response of P M N s as compared to propranolol although alprenolol has a similar lipophilicity as propranolol (Ondria§ et al., 1987; IJzerman et al., 1987). Also, oxprenolol possesses membrane stabilizing activity (Reynolds ~ n d Prasad, 1982) but did not neutralize the effect of cytotoxin on the chemiluminescence response of P M N s . Therefore, the action of some fl-adrenoceptor antagonists resulting in a diminished effect of the cytotoxin of P. haemolytica on P M N s can not be explained by the lipophilic properties of the fl-adrenoceptor antagonists. W h e t h e r fl-adrenoceptor antagonists could be used in vivo to prevent or treat the detrimental effects of P. haemolytica cytotoxin in the lungs of bovines remains to be investigated. ACKNOWLEDGMENTS The authors t h a n k Dr. N.J,L. Gilmour (Moredun Research Institute, Edinburgh) for supplying the Pasteurella haemolytica A1 strain, Mr. Rob van der Pol (Department of Veterinary Bacteriology, Utrecht) for handling the bacterial strain and Ms. Gerien de Zeeuw (Department of Medical Microbiology, Utrecht) for carrying out the endotoxin determinations.
REFERENCES Allen, R.C., Sternholm, R.L. and Steele, R.H., 1972. Evidence for the generation of an electronic excitation state (s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem. Biophys. Res. Commun., 47: 679-684. Axelrod, J. and Reisine, T.D., 1984. Stress hormones: their interaction and regulation. Science, 224: 452-459. Baluyut, C.S., Simonson, R.R., Bemrick, W.J. and Maheswaran, S.K., 1981. Interaction of PasteureUa haemolytica with bovine neutrophils: identification and partial characterization of a cytotoxin. Am. J. Vet. Res., 42: 1920-1926. Berggren, K.A., Baluyut, C.S., Simonson, R.R., Bemrick, W.J. and Maheswaran, S.K., 1981. Cytotoxic effects of Pasteurella haemolytica on bovine neutrophils. Am. J. Vet. Res., 42: 13831388. Chang, Y.-F. and Renshaw, H.W., 1986. Pasteurella haemolytica leukotoxin: comparison of ~lChromium-release, trypan blue exclusion, and luminol-dependent chemiluminescence-inhibition assays for sensitivity in detecting leukotoxin activity. Am. J. Vet. Res., 47: 134-138. Chang, Y.-F., Renshaw, H.W., Martens, R.J. and Livingston, C.W., 1986a. Pasteurella haemoly-
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tica leukotoxin: chemiluminescent responses of peripheral blood leukocytes from several different mammalian species to leukotoxin- and opsonin-treated living and killed PasteureUa haemolytica and Staphylococcus aureus. Am. J. Vet. Res., 47: 67-74. Chang, Y.-F., Renshaw, H.W. and Richards, A.B., 1986b. PasteureUa haemolytica leukotoxin: physicochemical characteristics and susceptibility of leukotoxin to enzymatic treatment. Am. J. Vet. Res., 47: 716-723. Easmon, C.S., Cole, P.J., Williams, A.J. and Hastings, M., 1980. The measurement of opsonic and phagocytic function by luminol-dependent chemiluminescence. Immunology, 41: 67-74. Filion, I.G., Willson, P.J., Bielefeldt-Ohmann, H., Babiuk, L.A. and Thomson, R.G., 1984. The possible role of stress in the induction of pneumonic pasteurellosis. Can. J. Comp. Med., 48: 268-274. Henricks, P.A.J., Binkhorst, G.J. and Nijkamp, F.P., 1987. Stress diminishes infiltration and oxygen metabolism of phagocytic cells in calves. Inflammation, 11: 427-437. Kerry, R., Scrutton, M.C. and Wallis, R.B., 1984. fl-Adrenoceptor antagonists and human platelets: relationship of effects to lipid solubility. Blochem. Pharmacol., 33: 2615-2622. Lopez, A., Thompson, R.G. and Savan, M., 1976. The pulmonary clearance of PasteureUa haemolytica in calves infected with bovine parainfluenza-3 virus. Can. J. Comp. Med., 40: 385391. Maheswaran, S.K., Berggren, K.A., Simonson, R.R., Ward, G.E. and Muscoplat, C.C., 1980. Kinetics of interaction and fate of PasteureUa haemolytica in bovine alveolar macrophages. Infect. Immun., 30: 254-262. Nielson, C.P., 1987. fl-Adrenergic modulation of the polymorphonuclear leukocyte respiratory burst is dependent upon the mechanism of cell activation. J. Immunol., 139: 2392-2397. Ondria~, K., Sta~ko, A., JanSinovd, V. and Balgav:~, P., 1987. Comparison of the effect of eleven fl-adrenoceptor blocking drugs in perturbating lipid membrane: an ESR spectroscopy study. Mol. Pharmacol., 31: 97-102. Ra~kov~i, H., Celeda, L., Lavick);, J., Van~ek, J., Urbanov~, Z., K~eSek, J., P ~ o r s k ~ , Z., Elis, J. and Krej~, I., 1987. Pharmacologic interventions to antagonize stress-induced immune consequences. In: B.D. Jankovid, B.M. Markovid and N.H. Spector (Editors), Neuroimmune Interactions: Proceedings of the Second International Workshop on Neuroimmunomodulation. Ann, N.Y. Acad. Sci., 496: 436-446. Rehmtulla, A.J. and Thomson, R.G., 1984. A review of the lesions in shipping fever of cattle. Can. Vet. J., 22: 1-8. Reynolds, E.F. and Prasad, A.B., 1982. Martindale. The Extra Pharmacopoeia, 28th Edn. The Pharmaceutical Press, London, pp. 1324-1352. Roth, J.A. and Kaeberle, M.L., 1981. Effects of in vivo dexamethasone administration on in vitro bovine polymorphonuclear leukocyte function. Infect. Immun., 33: 434-441. Shewen, P.A. and Wilkie, B.N., 1982. Cytotoxin of Pasteurella haemolytica acting on bovine leukocytes. Infect. Immun., 35: 91-94. Shewen, P.A. and Wilkie, B.N., 1985. Evidence for the Pasteurella haemolytica cytotoxin as a product of actively growing bacteria. Am. J. Vet. Res., 46:1212-1214. Slocombe, R.F., Derksen, F.J., Robinson, N.E., Trapp, A., Gupta, A. and Newman, J.P., 1984. Interactions of cold stress and PasteureUa haemolytica in the pathogenesis of pneumonic pasteurellosis in calves: method of induction and hematologic and pathologic changes. Am. J. Vet. Res., 45: 1757-1763. Stevens, P. and Hong, D., 1984. The role of myeloperoxidase and superoxide anion in the luminoland lucigenin-dependent chemiluminescence of human neutrophils. Microchem. J., 30: 135146. IJzerman, A.P., Nagesser, A. and Garritsen, A., 1987. The membrane stabilizing activity of fladrenoceptor ligands. Quantitative evaluation of the interaction of phenoxypropanolamines with the [3H ] batrachotoxinin A 20-~-benzoate binding site on voltage-sensitive sodium channels in rat brain. Biochem. Pharmacol., 36: 4239-4244.
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The Effect of PasteureUa haemolytica C y t o t o x i n on B o v i n e P o l y m o r p h o n u c l e a r L e u k o c y t e s can be A t t e n u a t e d by fl-Adrenoceptor A n t a g o n i s t s P.A.J. HENRICKS*, G.J. BINKHORST, A.A. DRIJVER, H. VAN DER VLIET and F.P. NIJKAMP
Department o[ Pharmacology, Faculty of Pharmacy and Department of Large Animal Medicine, Faculty of Veterinary Medicine, State Universityof Utrecht, Utrecht (The Netherlands) (Accepted 25 September 1989)
ABSTRACT Henricks, P.A.J., Binkhorst, G.J., Drijver, A.A., Van der Vliet, H. and Nijkamp, F.P., 1990. The effect of Pasteurella haemolytica cytotoxin on bovine polymorphonuclear leukocytes can be attenuated by fl-adrenoceptor antagonists. Vet. Microbiol., 22: 259-266. It was investigated whether fl-adrenoceptor antagonists could disturb the interaction between cytotoxin preparations isolated from Pasteurella haemolytica and bovine polymorphonuclear leukocytes (PMNs). The toxicity of the cytotoxin preparation was evaluated by measuring the chemiluminescence response and the viability of the cells after incubation with the cytotoxin. No effect on cell viability was detected when PMNs were incubated with 63 #g cytotoxin per ml while the chemiluminescence response was diminished by approximately 30%. The fl-adrenoceptor antagonists alprenolol (10 -5 M) and propranolol (5X 10-7-5 X 10 -8 M) were able to attenuate this effect of cytotoxin on the chemiluminescence response of PMNs. It seemed unlikely that propranolol and alprenolol diminished the effect of cytotoxin on the chemiluminescence response of PMNs by their fl-adrenoceptor blocking potency because other fl-adrenoceptor antagonists used were without effect. Also, the membrane stabilizing characteristics of the fl-adrenoceptor antagonists used were probably not responsible for the diminished interaction between PMNs and the cytotoxin. Whether fl-adrenoceptor antagonists could be used in vivo to prevent or treat P. haemolytica infections in bovines remains to be examined.
INTRODUCTION
Pasteurella species, especially P. haemolytica type A1, are major bovine respiratory pathogens. The bacterium has been associated with the advanced clinical signs, terminal lesions and fatality of pneumonic pasteurellosis (shipping fever) of cattle (Rehmtulla and Thomson, 1984). The live bacterium is toxic *To whom all correspondence should be addressed: Department of Pharmacology, Faculty of Pharmacy, Catharijnesinge160, 3511 GH Utrecht, The Netherlands.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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P.A.J. HENRICKS ET AL.
for phagocytic cells like alveolar macrophages and polymorphonuclear leukocytes (PMNs) of bovines in vitro (Maheswaran et al., 1980; Berggren et al., 1981 ). The toxic effect is due to the production of a cell-free cytotoxin by actively growing bacteria (Shewen and Wilkie, 1985 ). Biochemical studies have identified the toxin as a heat-labile, oxygen-stable, pH-stable non-dialyzable water-soluble antigenic glycoprotein (Chang et al., 1986b ). This cytotoxin may play a role in the pathogenesis of pneumonic pasteurellosis by diminishing phagocytic cell defense, or indirectly, by causing an inflammatory response mediated by lysosomal constituents released by cytotoxin-exposed leukocytes (Baluyut et al., 1981 ). Bovine respiratory disease is considered to be associated with initial viral infections and/or stressful circumstances (Lopez et al., 1976; Filion et al., 1984). The exact mechanisms by which stress factors influence the host defense system is unknown. Plasma levels of for example cortisol and catecholamines are increased in response to stress (Axelrod and Reisine, 1984; Slocombe et al., 1984). Glucocorticoids have anti-inflammatory and immunosuppressive actions (Roth and Kaeberle, 1981 ) and fl-adrenoceptor agonists depress phagocytic cell activities of PMNs {Nielson, 1987). Interestingly, the incidence of respiratory infections in calves after transport stress was lowered when fl-adrenoceptor antagonists had been given preventively to the animals (Ra§kov~i et al., 1987). A blockade of the immunosuppressive actions of catecholamines could be an explanation for these findings. The hypothesis of our study was that fl-adrenoceptor antagonists could also disturb the interaction between the toxic bacterial components and phagocytic cells. Therefore, it was investigated whether fl-adrenoceptor antagonists were able to prevent the toxic effects of the cytotoxin of P. haemolytica on blood PMNs of calves in vitro. MATERIALSAND METHODS P. haemolytica cytotoxin P. haemolytica type A1 (generously supplied by Dr. N.J.L. Gilmour, Moredun Research Institute, Edinburgh, U.K.) was grown in brain-heart infusion at 37°C on a shaker. The isolation of cytotoxin was performed according to Shewen and Wilkie {1982). Before use, the lyophilized material was reconstituted to the desired concentration in Eagle's minimum essential medium modified with Earle's salts (Eagle's MEM; Flow Laboratories, Irvine, U.K.). A concentration of 2 mg/ml corresponded on a dry weight basis to the concentration before lyophilization. The preparations contained less than 1% endotoxin as determined by using the Limulus amebocyte lysate-5000 Automatic endotoxin detection system (Cape Cod, Woods Hole, MA) at the Department of Medical Microbiology, State University of Utrecht.
P. HAEMOLYTICA CYTOTOXIN CAN BE ATTENUATED BY fl-ADRENOCEPTOR ANTAGONISTS
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PMNs suspensions Peripheral blood samples were obtained from 6-15-week-old Friesian-Holstein calves by jugular vein punction and collected in heparinized tubes. PMNs were isolated by lysis of contaminating red blood cells and differential density centrifugation (Henricks et al., 1987). The final cell preparations contained 5 X 106 P M N s / m l Eagle's MEM.
fl-Adrenoceptor antagonists 1-Alprenolol tartrate, pindolol and oxprenolol were purchased from Sigma Chemical Company, St. Louis, MO. The following drugs were kindly supplied as gifts: timolol maleate (Merck, Sharp and Dohme, Haarlem, The Netherlands), practolol, ICI 118.551 hydrochloride and propranolol hydrochloride (Imperial Chemical Industries, Macclesfield, U.K. ).
Incubation of PMNs with cytotoxin and/or fl-adrenoceptor antagonists PMNs (1 X 106 cells) were incubated with or without cytotoxin (0-250 #g/ ml) a n d / o r fl-adrenoceptor antagonists (0.1-10 ttM) in a total volume of 0.4 ml Eagle's M E M for 0-60 min at 37°C. The PMNs were mixed with the fladrenoceptor antagonists and incubated for 10 min at room temperature before addition of other reagents. The PMNs were pelleted after incubation and resuspended in Eagle's M E M for measurement of viability and generation of chemiluminescence. Viability was determined by trypan blue exclusion.
Generation of chemiluminescence Generation of chemiluminescence by P M N s (1 X 106 cells) was measured in a luminometer (type 1251 LKB Wallac, Turku, Finland) in the presence of 0.5 m M lucigenin (bis-N-methylacridinium nitrate; Sigma Chemical Company, St. Louis, MO ) in a total volume of 0.5 ml. Zymosan (2.5 mg/ml; Sigma Chemical Company, St. Louis, MO), preopsonized in 100% pooled bovine serum, was used as stimulus. Chemiluminescence was determined every 60 sec over a 45 min period.
Statistical analysis Grouped data are expressed as the m e a n + s t a n d a r d error of the mean (s.e.m.). Student's t-test and Wilcoxon's rank sum test were applied for statistical evaluation of the results. P values exceeding 0.05 were considered not significant. RESULTS
Effect of cytotoxin on PMNs chemiluminescence response and cell viability P M N s stimulated with opsonized zymosan exhibited a moderate increase in chemiluminescence during the initial 15-25 min whereafter there was a pro-
262
P.A.J. HENRICKS ET AL.
gressive decrease in the amount of light emitted. In our system, the cells produced 128 + 11 mV peak chemiluminescence (n = 13; range 50-230 mV). The amount of chemiluminescence generated was dose-dependently reduced when the PMNs had been incubated with different amounts of cytotoxin (Fig. 1; Table 1), which is in agreement with previous observations (Chang et al., 1986a). Incubating PMNs with 63 ~g cytotoxin/ml for 30 min at 37 ° C resulted in 85 2 12 mV peak chemiluminescence (n= 13; range 30-190 mV) generated. 250
~200 E 0 150
100
50
10
20
30
Time (min)
40
Fig. 1. Generation of chemiluminescence by P M N s (1 × 106 cells) incubated for 30 min with 0 ( • ) , 31 ( • ), 63 ( A ), 125 ( [ ] ) or 250 ( O ) ltg cytotoxin/ml upon stimulation with opsonized zymosan. The generation of chemiluminescence was measured every 60 s over a 45 min period in a luminometer in the presence of 0.5 m M lucigenin. Results shown are representative of four separate experiments. TABLE1 Viability and peak chemiluminescence response upon stimulation with zymosan of P M N s incubated with various amounts of cytotoxin for 30 min at 37 ° C ( n = 4)
Control 16 # g / m l 31 # g / m l 63 #g/ml 125 #g/ml 250#g/ml
cytotoxin cytotoxin cytotoxin cytotoxin cytotoxin
% Viability
% Chemiluminescence
98 ± 1 96 ± 1 96+ 1 95 -+ 2 79 _+21 61 ±21
100 101 +_ 4 99+_ 7 71 -+ 81 50 -+ 111 21 -+ 71
1p < 0.05 as compared to control values.
P. HAEMOLYTICA CYTOTOXIN CAN BE ATTENUATEDBYfl-ADRENOCEPTORANTAGONISTS
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The decrease in chemiluminescence generated was also dependent on the time period by which the cells had been incubated with the cytotoxin. Chemiluminescence generated by PMNs was diminished by approximately 10, 15, 30 and 50% when the cells were incubated with 63/~g cytotoxin/ml for respectively 5, 10, 30 or 60 min at 37 ° C. Table 1 also shows the effect of P. haemolytica cytotoxin on the viability of bovine PMNs as determined by trypan blue exclusion. When PMNs had been incubated with less than 63/tg cytotoxin/ml for 30 min no effects on the viability of the cells were observed (Table 1). Diminished viabilities were observed when higher amounts were used ( > 125 zg cytotoxin/ml; Table 1 ). Viability of PMNs was also more depressed when PMNs were incubated with cytotoxin for longer periods of time as compared to an incubation period of 30 min.
Effect of fl-adrenoceptor antagonists on cytotoxin-induced changes in the chemiluminescence response of PMNs PMNs were incubated with 63 ]lg cytotoxin/ml in the presence and absence of selective and non-selective fl-adrenoceptor antagonists for 30 min. No fladrenoceptor antagonists in the concentrations used had any effects on the chemiluminescence response of control PMNs (data not shown). The reducAlprenolol
'°80]°1 ~ 40 100
Oxprenolol
Pindolol
Practolol
Propranolol
Timolol
ICI 118,551
-~ 60
-~
Q.
40
40
0.10.51 510
0.10.51 5 1 0
0.1051 5 1 0 Concentration (juM)
Fig. 2. Peak chemiluminescence response of P M N s (% of control) upon stimulation with opsonized zymosan (n = 4-7 ). The phagocytic cells had been incubated for 30 min with 63 ttg cytotoxin per ml in the presence of 10-7-10 -5 M p-adrenoceptor antagonists before stimulation. The generation of chemiluminescence was measured every 60 s over a 45 min period in a luminometer in the presence of 0.5 m M lucigenin. All data were significantly diminished ( P < 0.05) as compared to control values except * P > 0.05 and **P > 0.10. N.D. = not done.
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P.A.J. HENRICKS ET AL.
tion induced by cytotoxin of the chemiluminescence generated by PMNs could not be prevented by the fl-adrenoceptor antagonists oxprenolol, pindolol, timolol, practolol or ICI 118.551 (Fig. 2). However, propranolol in concentrations between 5 × 10 -7 and 5 × 10 -6 M and alprenolol in a concentration of 10 -~ M were able to attenuate the effect of cytotoxin on the chemiluminescence response of PMNs (Fig. 2). DISCUSSION Results from the present study have confirmed previous observations related to the leukotoxic effects ofP. haemolytica cytotoxin (Shewen and Wilkie, 1982; Chang et al., 1986a). It was found that the cytotoxin was able to diminish the chemiluminescence response of bovine PMNs in vitro dose-dependently. Chemiluminescence generated by PMNs correlates with their metabolic activation and is used as an assay to study phagocytic cellular function (Allen et al., 1972; Easmon et al., 1980). Lucigenin was used as an amplifying agent in this study and lucigenin-dependent chemiluminescence reflects the degree of superoxide generation by PMNs undergoing activation (Stevens and Hong, 1984). The chemiluminescence assay is found to be the most sensitive method to study the effect of cytotoxin on phagocytic cell function (Chang and Renshaw, 1986). No effect on cell viability was detected when PMNs were incubated with 63 #g cytotoxin per ml while the chemiluminescence response was diminished by approximately 30%. The toxic effect of P. haemolytica cytotoxin on phagocytic cells of bovines may be an important pathogenic factor in the early stage of P. haemolyticainduced pneumonia. Stress factors are thought to contribute to the facilitation of the clinical pattern (Filion et al., 1984; Rehmtulla and Thomson, 1984; Slocombe et al., 1984). Administration of fl-adrenoceptor antagonists to animals lowers the incidence of respiratory infections in calves after transportation (Ra~kov~i et al., 1987). This could be due to a blockade of the impairment of phagocytic cell activity induced by enhanced levels of catecholamines or to fladrenoceptor antagonists-induced changes in the membrane of the cells resulting in a diminished interaction between the cytotoxin and phagocytic cells. To our knowledge, no data are available in the literature about the mechanism of interaction between P. haemolytica cytotoxin and phagocytic cells. It was found in this study that the non-selective fl-adrenoceptor antagonists propranolol (5X 10-7-5X 10 -6 M) and alprenolol (10 -5 M) were able to neutralize the effect of cytotoxin on the chemiluminescence response of PMNs. The other fl-adrenoceptor antagonists used in this study did not influence the effect of cytotoxin. Also, propranolol in a concentration of 10 -6 M was without effect on the detrimental action of cytotoxin. Therefore it seems unlikely that propranolol influences the effect of cytotoxin on PMNs due to its fl-adrenoceptor blocking potency.
P. HAEMOLYTICA CYTOTOXIN CAN BE ATTENUATED BY fl-ADRENOCEPTOR ANTAGONISTS
265
fl-Adrenoceptor antagonists are able to induce a perturbation of the lipid membrane and extent a membrane stabilizing effect due to their lipophilic properties. The membrane stabilizing characteristics of some fl-adrenoceptor antagonists changes the physico-chemical properties of the cell surface and could alter membrane receptor properties as a consequence of distortion of the microenvironment. Propranolol is relatively more potent as pindolol, timolol and practolol to influence lipid membranes (Kerry et al., 1984; Ondria~ et al., 1987). Higher amounts of alprenolol were needed to modify the effect of cytotoxin on the chemiluminescence response of P M N s as compared to propranolol although alprenolol has a similar lipophilicity as propranolol (Ondria§ et al., 1987; IJzerman et al., 1987). Also, oxprenolol possesses membrane stabilizing activity (Reynolds ~ n d Prasad, 1982) but did not neutralize the effect of cytotoxin on the chemiluminescence response of P M N s . Therefore, the action of some fl-adrenoceptor antagonists resulting in a diminished effect of the cytotoxin of P. haemolytica on P M N s can not be explained by the lipophilic properties of the fl-adrenoceptor antagonists. W h e t h e r fl-adrenoceptor antagonists could be used in vivo to prevent or treat the detrimental effects of P. haemolytica cytotoxin in the lungs of bovines remains to be investigated. ACKNOWLEDGMENTS The authors t h a n k Dr. N.J,L. Gilmour (Moredun Research Institute, Edinburgh) for supplying the Pasteurella haemolytica A1 strain, Mr. Rob van der Pol (Department of Veterinary Bacteriology, Utrecht) for handling the bacterial strain and Ms. Gerien de Zeeuw (Department of Medical Microbiology, Utrecht) for carrying out the endotoxin determinations.
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