Veterinary Research Communications, 22 (1998) 493^503 # 1998 Kluwer Academic Publishers. Printed in the Netherlands

RESPONSES OF GUINEA-PIG LUNG PARENCHYMAL STRIPS TO TRACHEOBRONCHIAL LAVAGE FLUID FROM HORSES AFFECTED WITH SUMMER PASTURE-ASSOCIATED OBSTRUCTIVE PULMONARY DISEASE C.S. VENUGOPALAN1*, R.E. BEADLE2, T.L. SEAHORN2 AND E.P. HOLMES1 Departments of 1Veterinary Physiology, Pharmacology and Toxicology and 2 Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA *Correspondence ABSTRACT Venugopalan, C.S., Beadle, R.E., Seahorn, T.L. and Holmes, E.P., 1998. Responses of guinea-pig lung parenchymal strips to tracheobronchial lavage £uid from horses a¡ected with summer pasture-associated obstructive pulmonary disease. Veterinary Research Communications, 22(7), 493^503 The response of parenchymal strips from guinea-pig lungs to tracheobronchial lavage £uid (TBLF) collected from 8 normal horses and from 8 a¡ected with summer pasture-associated obstructive pulmonary disease (SPAOPD) was determined. TBLF was collected during the summer (July) and winter (February) seasons. The serum/TBLF urea nitrogen ratio was used to standardize the mediator concentration in the TBLF. Four strips were used from each guinea-pig. The ¢rst strip did not receive any antagonist and served as the control. The second, third and fourth strips received antagonists of PGE2, LTD4 and PAF, respectively at 10^6 mol/L for 30 min. The tissues were then precontracted with a dose of histamine (10^5 mol/L) and their responses to 1 ml of TBLF were determined. The study showed that TBLF obtained in the summer from una¡ected horses produced a signi¢cantly greater relaxation than that from the a¡ected horses, whereas TBLF obtained in the winter from una¡ected or a¡ected horses did not cause a signi¢cantly di¡erent degree of relaxation. Among the antagonisttreated strips, only those exposed to the PGE2 blocker showed a signi¢cant reduction in the relaxation caused by TBLF obtained in the summer from SPAOPD horses. This suggests that PGE2 is an important mediator present in the summer in the TBLF from horses a¡ected with SPAOPD. Keywords: airway, guinea-pig, horse, in£ammatory mediator, prostaglandin, season, SPAOPD, tracheobronchial lavage £uid Abbreviations: ACh, acetylcholine; CBC, complete blood cell count; CS, clinical score; ED50, e¡ective dose to produce 50% of the maximal response; EDTA, ethylenediaminetetraacetate; EpDRF, epithelium-derived relaxing factor; LTD4, leukotriene D4; NANC, nonadrenergic noncholinergic; PAF, platelet-activating factor; PGE2, prostaglandin E2; SPAOPD, summer pasture-associated obstructive pulmonary disease; TBLF, tracheobronchial lavage £uid

INTRODUCTION Summer pasture-associated obstructive pulmonary disease (SPAOPD), a form of chronic obstructive pulmonary disease (COPD), is a respiratory condition of horses that is prevalent in the Gulf Coast region of the United States (Beadle, 1983). The aetiology and pathogenesis of COPD are not clearly understood, but an allergic 493

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component has been documented (Halliwell et al., 1993). The disease is characterized by airway in£ammation, increased vascular permeability, increased mucus secretion, damage to the airway epithelium and airway obstruction leading to increased pulmonary resistance, decreased dynamic compliance and mild to severe respiratory distress (Buechner-Maxwell, 1993). Chemical mediators of airway in£ammation are released by degranulation of mast cells when sensitized animals are exposed to speci¢c antigens (Woolcock, 1988). Previously, attention has been focused in humans on the mediators leukotriene D4 (LTD4) and platelet-activating factor (PAF) (O'Flaherty, 1987; Barnes et al., 1988). A¡ected animals may counteract the e¡ects of contractile mediators by synthesis and release of agents that mediate relaxation of airways and thereby maintain homeostasis of the respiratory tract. There is evidence that alterations in the biochemical composition of airway secretions occur in various disease conditions, including airway in£ammation and hyperreactivity (Martin, 1986). The airway secretions of a¡ected horses are known to contain active chemical mediators of airway in£ammation and hyperreactivity and also relaxant mediators for airway homeostasis (Buechner-Maxwell, 1993). A positive correlation exists between the intensity of airway hyperreactivity and the ratio of contractile mediators to relaxant mediators in the airway secretions (Watson et al., 1992). Hence, determining the pharmacological e¡ects of tracheobronchial lavage £uid (TBLF) of a¡ected horses would facilitate the assessment of disease status and identi¢cation of the predominant mediators in the TBLF. We hypothesized that the chemical mediators contained in the TBLF from a¡ected horses would be qualitatively and quantitatively di¡erent from those of una¡ected horses. This di¡erence would then produce di¡erent tissue responses that could be used as a diagnostic tool to determine the mediator present in the TBLF. Guinea-pigs were used in the study because they have been used extensively for investigating the pathophysiology of airway diseases. A substantial body of evidence suggests that several aspects of acute airway hyperactivity in man and animals are mimicked by the pulmonary response to antigen in anaesthetized or unanaesthetized guinea-pigs (Drazen, 1977; Popa et al., 1973). Therefore, the primary objective of this in vitro study was to determine the pharmacological e¡ects of TBLF and to compare the responsiveness of lung parenchymal strips to TBLF obtained from una¡ected and SPAOPD-a¡ected horses. A secondary objective was to identify important mediators facilitating the responsiveness of lung strips to TBLF. MATERIALS AND METHODS The study was approved by the Louisiana State University (LSU) Institutional Animal Care and Use Committee. Horses All the horses used were part of a pulmonary studies research herd that is maintained as a closed entity at the LSU School of Veterinary Medicine. The horses in this herd are

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treated with anthelmintic three times per year and vaccinated annually with tetanus toxoid and multivalent (Eastern, Western, and Venezuelan) encephalitis vaccine. Eight horses previously diagnosed as being a¡ected with SPAOPD were used as the a¡ected group. Their ages ranged from 14 to 23 years, with a mean age of 16.9+3.4 years. Eight horses with no history or clinical signs of respiratory disease served as the una¡ected group. Their ages ranged from 6 to 9 years, with a mean age of 7.9+1.1 years. Guinea-pigs American or English shorthair guinea-pigs (450^600 g body weight) were obtained from a USDA licensed dealer and were housed in the vivarium of the Division of Laboratory Animal Medicine of the LSU School of Veterinary Medicine. Clinical scoring On each day of sampling, all the horses were assigned a clinical score (CS) for SPAOPD as determined by the following formula (Seahorn et al., 1997): Medial nostril £are + Lateral nostril £are ööööööööööööööööööö + Abdominal lift = CS 2 The range of scores for each of the three variables comprising CS was 0 to 4. A score of 0 was assigned if the nostril had little movement and if the ventral £ank showed little or no movement. A score of 4 was assigned if the nostril remained maximally £ared throughout the respiratory cycle and for abdominal £attening when the resulting `heavy line' extended cranially to the elbow. The maximum CS was therefore 8. Horses having a total CS of 5.0 or greater were classi¢ed as clinically a¡ected SPAOPD horses. Sampling timetable TBLF was collected twice from each horse during a one-year period. All the horses were being maintained on pasture at the time of each of these samplings. On the day of data collection, each horse was given a clinical score and blood and tracheal lavage samples were collected. The ¢rst collection occurred in July when all the SPAOPDa¡ected horses were exhibiting clinical signs of obstructive pulmonary disease. Each SPAOPD-a¡ected horse had a clinical score 55.0, while the clinical score for each una¡ected horse was 43.0. All a¡ected and una¡ected horses had normal body temperatures, white blood cell counts and plasma ¢brinogen concentrations at the time of this sampling. The second TBLF collection occurred in February, when none of the SPAOPDa¡ected horses was exhibiting obvious clinical signs of obstructive pulmonary disease

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and therefore had a clinical score 44.0. The clinical score for each una¡ected horse was 42.5 at this sampling time. All a¡ected and una¡ected horses again had normal body temperatures, white blood cell counts and plasma ¢brinogen concentrations at this sampling time. Urea nitrogen determination Blood samples were collected from the external jugular vein into sterile evacuated tubes and serum was collected after clotting. Serum samples were frozen at ^208C and subsequently used for blood urea nitrogen determinations. The urea nitrogen content of serum and tracheobronchial lavage £uid (TBLF) was determined as previously described (Seahorn et al., 1997). Tracheobronchial lavage The horses were bronchodilated using glycopyrrolate (0.0022 mg/kg, i.v.) and sedated using xylazine (0.4 mg/kg, i.v.). Following placement of a nose twitch, a 180 cm613 mm £exible ¢breoptic endoscope was passed through a nasal passage and into the distal trachea, so that its tip lay approximately at the level of the thoracic inlet. A 30 ml aliquot of sterile saline was infused into the distal trachea and proximal bronchi through a transendoscopic polypropylene catheter. Air (5 ml) was used to £ush the catheter and the TBLF was then aspirated immediately. Aspirates were quantitated and ¢ltered through a gauze sponge to remove mucus plugs. They were then centrifuged at 1760g for 10 min at 228C. A sample of TBLF was frozen at ^208C for urea nitrogen determination. Another sample was frozen at ^708C for the in vitro studies. In vitro method The guinea-pigs were killed by stunning and exsanguination. The thoracic cavity was then opened and the trachea and lungs were removed en bloc. Four parenchymal strips of equal size were cut from the lower left and right lobes of the lungs by methods described previously (Drazen and Schneider, 1978). Each tissue preparation was placed in a 10-ml organ bath containing 378C Tyrode's solution and oxygenated continuously with a gas mixture of 95% oxygen and 5% carbon dioxide. One end of the strip was ¢xed to a glass hook in the £oor of the organ bath; the other end was connected to a force transducer (Grass Instruments, Model FT0.03) interfaced to a Grass polygraph. An initial tension of 1 g was applied to each of the parenchymal strips (Venugopalan et al., 1991). After the initial tension was set, the tissues were allowed to remain in the organ bath to equilibrate for at least 30 min. During this time, the Tyrode's solution in the baths was changed at 15-min intervals.

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Experimental protocol Preliminary studies indicated that 1 ml of TBLF was able to induce a response by the guinea-pig parenchymal strips. The strips showed either no response at all or a relaxation to TBLF that had been randomly selected for this preliminary investigation. Since any response by the strip to TBLF was a relaxation, the experimental protocol was modi¢ed to allow determination of relaxation (Venugopalan et al., 1985). For this, the tissues were precontracted with a single dose of histamine (bath concentrations of 10^5 mol/L). This concentration produced a contractile response by the tissues that reached a plateau and maintained that state for about 10 min. As soon as the plateau was reached, 1 ml of the TBLF was added to the bath and the tissue response was noted over 5 min. A relaxation of the tissues to the baseline (precontracted level) was considered to be 100%, any lesser response being reported as a percentage of that maximal relaxation. A correction factor based on the serum/TBLF urea nitrogen concentration ratio was used to standardize the mediator concentrations in the TBLF. Two aspects were investigated using this protocol. First, the relaxation response of the lung strips to every sample collected was determined. This information allowed us to compare the summer and winter samples from the una¡ected and a¡ected horses. The second objective was to identify the predominant chemical mediators in TBLF. For this purpose, four treatment groups were used based on antagonists of the probable mediators. The ¢rst group of strips was not exposed to any antagonist and served as the control (untreated). The second, third and fourth groups of strips were incubated for 30 min with pharmacological antagonists of prostaglandin PGE2 (AH6809), platelet-activating factor PAF (CV-3988) and leukotriene LTD4 (REV-5901), respectively (all agents from Biomol Research Laboratories, PA, USA). Preliminary studies were conducted to select appropriate concentrations of the antagonists. The selected antagonist dose (10^6 mol/L in each case) was able to block the e¡ect of an ED50 dose of the respective agonist on the lung strips. Statistical analyses The data were analysed using a microcomputer-based statistical software program (SAS, 1988). Wilcoxon's signed rank test (Steel and Torrie, 1980a) was used to compare paired summer and winter data for both una¡ected and a¡ected horses. The Mann^ Whitney rank sum test (Steel and Torrie, 1980b) was used to compare the data from a¡ected and una¡ected horses data within each season. A repeated measures analysis of variance procedure with contrasts (SAS, 1988) was used to determine the in£uence of the various pharmacological antagonists on the relaxation produced by the TBLF samples in precontracted guinea-pig lung parenchymal strips. In each case, a comparison was made against the appropriate control sample that did not contain exogenous antagonists. Antagonist data were ranked for each horse by season before the analysis was performed.

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RESULTS The clinical assessment scores for both groups of horses for the summer and winter seasons are illustrated in Figure 1. The median clinical score for SPAOPD horses was greater than that for una¡ected horses in the summer and to a lesser extent in the winter. In addition, the median clinical score for SPAOPD horses in the winter was less than that recorded in the summer for the same horses.

Figure 1. The clinical scores are plotted according to the group in which the horse was included and the season when the data were recorded. Horses in the una¡ected group are identi¢ed by uppercase letters and the horses in the a¡ected group are identi¢ed by lowercase letters. Median values for a¡ected horses were 5.0 in the summer and 3.5 in the winter. Median values for una¡ected horses were 2.0 in the summer and 3.0 in the winter

The samples of TBLF collected from both groups produced either relaxation or no response on the guinea-pig lung parenchymal strips. Figure 2 illustrates the percentage relaxation for each horse in both groups against seasons. The median percentage relaxation was highest with TBLF from the summer una¡ected group and lowest in the summer a¡ected group. In the summer season, the una¡ected and a¡ected groups showed a signi¢cant di¡erence (p50.05) in percentage relaxation. The percentage relaxations induced by the winter samples for the una¡ected and the a¡ected groups were not signi¢cantly di¡erent. There were no signi¢cant di¡erences within the groups

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Figure 2. The percentage maximal relaxation is plotted according to the group in which the horse was included and the season when the data were collected. Horses in the una¡ected group are identi¢ed by uppercase letters and horses in the a¡ected group are identi¢ed by lowercase letters. Boxes indicate the range between the 35th and 75th centiles of the data. A line within the box marks the value of the 50th centile and capped bars indicate the 10th and 90th centile points. S, summer; W, winter. The percentage maximal relaxation values in the summer were signi¢cantly di¡erent between una¡ected and a¡ected horses (p40.05)

due to the di¡erent seasons (a¡ected, summer vs winter p = 0.16; una¡ected, summer vs winter p = 0.55). Statistical analysis of the data generated using antagonists of the presumptive mediators revealed that the PGE2 antagonist signi¢cantly blocked the relaxation response induced by summer TBLF from a¡ected horses (Table I). DISCUSSION The study showed that summer TBLF samples from SPAOPD-a¡ected horses produced a signi¢cantly lower relaxation response than those from una¡ected horses. During the early summer season, horses with SPAOPD spontaneously develop bronchoconstriction when they are allowed to graze on pasture grass (Beadle, 1983). Environmental antigens (allergens in the pasture) are presumed to react with

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TABLE I The results of statistical analyses (p values) of the e¡ect of speci¢c antagonists on samples of TBLF obtained in the summer and winter from control and SPAOPD-a¡ected horses Group Una¡ected (summer) A¡ected (summer) Una¡ected (winter) A¡ected (winter) a

PGE2 antagonist

PAF antagonist

LTD4 antagonist

0.2819 0.006a 0.7294 0.2581

0.1958 0.0998 0.4304 0.0604

0.1191 0.5793 0.3736 0.0562

Signi¢cantly di¡erent from the result in the absence of the antagonist

antibodies in the animal leading to degranulation of mast cells and release of potent chemical mediators. These mediators evoke smooth-muscle contraction, increased vascular permeability and increased secretion of mucus. Chemotactic mediators released by degranulation attract more leukocytes, lymphocytes, mononuclear cells and additional mast cells to the airway epithelium, which leads to epithelial damage and airway hyperreactivity. Epithelial damage leads to further release of contractile mediators and enhanced airway reactivity. Epithelial disruption also releases other chemical agents, including inhibitory prostaglandins, neuropeptides and relaxing factors, to counteract bronchoconstriction and thereby maintain airway homeostasis. Therefore, the chemical agents released into the airway secretions can be broadly classi¢ed as relaxant or contractile agents, based on their ability to induce constriction or dilatation of airways. The response of guinea-pig lung strips to TBLF depends on the net balance of the two opposing forces generated by relaxant and contractile mediators. Thus, the ratio of relaxant to contractile agents in the TBLF will determine the response of the smooth muscle in the airway. TBLF samples from both groups of horses produced relaxation, which suggested an activation of a homeostatic mechanism to maintain airway diameter via relaxants. However, the ability of TBLF from a¡ected horses to produce relaxation was signi¢cantly less than that from una¡ected horses. Presumably, the TBLF from the a¡ected animals had a mediator ratio favouring less relaxation, owing to either increased contractile mediators or decreased relaxant mediators. This decreased relaxation response was re£ected, at least in part, in the higher clinical scores recorded for the a¡ected horses than for the una¡ected horses. The predominant excitatory pathway in airways is through the parasympathetic nervous system. However, recent studies have shown no evidence of increased release of ACh from parasympathetic nerve terminals, even in horses with advanced levels of airway hyperreactivity (LeBlanc et al., 1991). It is possible that horses have a dominant noncholinergic excitatory system or an abundance of contractile mediators other than ACh in the airway secretions, which act directly on airway smooth muscles to produce constriction. The inhibitory pathways, on the other hand, are through sympathetic and

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nonadrenergic inhibitory innervation, as well as through inhibitory mediators such as epithelium-derived relaxing factor (EpDRF) and inhibitory prostanoids (Olson et al., 1989; Yu et al., 1993). The present study showed that PGE2, an inhibitory prostanoid, in the TBLF of a¡ected horses signi¢cantly facilitated relaxation of parenchymal strips, whereas relaxation produced by TBLF from una¡ected horses was not due to PGE2. This suggests that the relaxation in una¡ected horses may be mediated predominatly through another substance, perhaps through EpDRF, whose major role in airway relaxation has been documented (Olson et al., 1989; Yu et al., 1993). Even though the relaxation produced by TBLF of a¡ected horses was signi¢cantly blocked by the PGE2 antagonist, the blockade was not complete, which indicates that other relaxant mediator(s) may also participate in the homeostasis. The role of PGE2 in airway secretions in a¡ected horses is equivocal. Gray and coworkers (1992) reported a reduction in inhibitory prostaglandin production by epithelial strips taken from horses with `heaves'. On the other hand, Watson and colleagues (1990, 1992) observed signi¢cantly increased levels of PGE2 in the bronchoalveolar lavage £uids from horses with COPD. A similar situation has also been observed in other species. In allergen-stimulated sheep, Dworski and colleagues (1989) noted increased levels of PGE2 in bronchoalveolar lavage £uids. The present study indicated an important role for PGE2 in maintaining airway homeostasis, perhaps by countering bronchoconstriction by contractile mediators. Henson and colleagues (1982) suggested a role for increased levels of PGE2 in airway in£ammation through vasodilatation and neutrophil migration. The net e¡ect of contraction or relaxation can be changed by decreasing or increasing the amounts of contractile or relaxant mediators. This study has shown that the TBLF of a¡ected horses contains PGE2, but the reduced relaxation observed suggests that the TBLF probably contained contractile mediators as well. Well-known contractile mediators include ACh, histamine, PAF and LTD4. Preliminary studies in our laboratory using atropine did not support further investigation of ACh as an important mediator. Histamine was not investigated because, in preliminary studies, TBLF did not produce a contraction of guinea-pig parenchymal strips, which are highly sensitive to histamine. Antagonists of PAF and LTD4 revealed that these agents independently produced a substantial, but not a signi¢cant (p = 0.06) blockade, suggesting that PAF and LTD4 may be involved in the net response of decreased relaxation observed with the winter samples from the a¡ected horses. The relaxation, although less than that produced by TBLF from una¡ected horses, was signi¢cantly blocked in the summer samples of a¡ected horses by the PGE2 antagonist, suggesting that PGE2 was an important relaxant mediator in a¡ected horses, regardless of the presence of other relaxant mediators. In this study, TBLF was collected only once each during the summer and winter seasons. It is important to know that the ratio of contractile to relaxant mediators in TBLF does not remain constant, but seems to change as the environmental conditions change.

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ACKNOWLEDGEMENTS The authors thank The Equine Veterinary Research Program of the Louisiana State University School of Veterinary Medicine for providing ¢nancial support for this research. They also thank Mr Frank Garza and Dr Alvaro Celedone for their technical assistance in collecting tracheobronchial lavage £uid. REFERENCES Barnes, P.J., Chung, K.F. and Page, C.P., 1988. In£ammatory mediators of asthma. Pharmacological Reviews, 40, 49^84. Beadle, R.E., 1983. Summer pasture-associated obstructive pulmonary disease. In: N.E. Robinson (ed.), Current Therapy in Equine Medicine, (W.B. Saunders, Philadelphia), 512^516 Buechner-Maxwell, V., 1993. Airway hyperresponsiveness. Equine respiratory disorders. The Compendium, 15, 1379^1389 Drazen, J.M., 1977. Pulmonary physiologic abnormalities in animal models of acute asthma. In: L.M. Lichtenstein and K.F. Austen (eds), Asthma: Physiology, Immunopharmacology and Treatment, (Academic Press, New York), 249^262 Drazen, J.M. and Schneider, W.M., 1978. Comparative responses of tracheal spirals and parenchymal strips to histamine and carbachol in vitro. Journal of Clinical Investigation, 61, 1441^1447 Dworski, R., Sheller, J.R., Wickersham, N.E., Oates, J.A., Brigham, K.L., Roberts, L.J. and Fitzgerald, G.A., 1989. Allergen-stimulated release of mediators into sheep bronchoalveolar lavage £uid. E¡ect of cyclooxygenase inhibition. American Review of Respiratory Diseases, 139, 46^51 Gray, P.R., Derksen, F.J., Broadstone, R.V., Robinson, N.E. and Peters-Goden, M., 1992. Decreased airway mucosal prostaglandin E2 production during airway obstruction in an animal model of asthma. American Review of Respiratory Diseases, 146, 586^591 Halliwell, R.E., McGorum, B.C., Irving, P. and Dixon, P.M., 1993. Local and systemic antibody production in horses a¡ected with chronic obstructive pulmonary disease. Veterinary Immunology and Immunopathology, 38, 201^215 Henson, P.M., Larsen, G.L., Webster, R.O., Mitchell, B.C., Goins, A.J. and Henson, J.E., 1982. Pulmonary microvascular alterations and injury induced by complement fragments: Synergistic e¡ect of complement activation, neutrophil sequestration and prostaglandins. Annals of New York Academy of Science, 384, 287^300 LeBlanc, P.H., Broadstone, R.V., Derksen, F.J. and Robinson, N.E., 1991. In vitro responses of distal airways in horses with recurrent airway obstruction. American Journal of Veterinary Research, 52, 999^ 1003. Martin, M.G., 1986. Pharmacology of airway secretions. Pharmacological Reviews, 38, 273^287 O'Flaherty, J.T., 1987. Platelet-activating factor: mechanism of cellular activation. In: F. Snyder (ed.), Platelet Activating Factor and Related Lipid Mediators, (Plenum, New York), 283^297 Olson, L.E., Perkowski, S.Z., Mason, D.E. and Muir, W.W. III., 1989. Epithelium and mucosa-dependent relaxation and contraction of normal equine trachealis muscle in vitro. American Journal of Veterinary Research, 50, 1720^1724 Popa, V., Douglas, J.S. and Bouhuys, A., 1973. Airway responses to histamine, acetylcholine and propranolol in anaphylactic hypersensitivity in guinea-pigs. Journal of Allergy and Clinical Immunology, 51, 344^356 SAS, 1988. SAS/STAT User's Guide, Release 6.03 edition, (SAS Institute Inc., Cary, NC) Seahorn, T.L., Beadle, R.E., McGorum, B.C. and Marley, C.L., 1997. Quanti¢cation of antigen-speci¢c antibody concentrations in tracheal lavage £uid of horses with summer pasture-associated obstructive pulmonary disease. American Journal of Veterinary Research, 58, 1408^1411 Steel, R.G.D. and Torrie, J.H., 1980a. Wilcoxon's signed rank test. In: Principles and Procedures of Statistics: Biometrical Approach, 2nd edn, (McGraw-Hill, New York), 539^540 Steel, R.G.D. and Torrie, J.H., 1980b. The Wilcoxon^Mann^Whitney two-sample test. In: Principles and Procedures of Statistics: Biometrical Approach, 2nd edn, (McGraw-Hill, New York), 542^543

503 Venugopalan, C.S., Holmes, E.P., Fucci, V. and Guthrie, A.J., 1991. Stimulation frequency-dependent nonadrenergic noncholinergic airway responses of the guinea-pig. Journal of Applied Physiology, 70, 1006^1010 Venugopalan, C.S., O'Rourke, Y.M. and Tucker, T.A., 1985. Broncho-relaxing activity of lithium in vitro. Journal of Pharmaceutical Sciences, 74, 1120^1122 Yu, M., Robinson, N.E., Wang, Z. and Derksen, F.J., 1993. Independent modulation of horse airway smooth muscle by epithelium and prostanoids. Respiratory Physiology, 93, 279^288 Watson, E.D., Mair, T.S. and Sweeney, C.R., 1990. Immunoreactive prostaglandin production by equine monocytes and alveolar macrophages and concentrations in bronchoalveolar lavage £uid. Research in Veterinary Sciences, 49, 88^91 Watson, E.D., Sweeney, C.R. and Steensma, K.A., 1992. Arachidonate metabolites in bronchoalveolar lavage £uid from horses with and without COPD. Equine Veterinary Journal, 24, 379^381 Woolcock, A.J., 1988. Asthma ^ what are the important experiments? American Review of Respiratory Diseases, 138, 730^744 (Accepted: 9 July 1998)