INTRODUCTION

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Keywords: neutrophils; Th2 cells; cytokines; asthma; inflammation

There is a general belief that asthma is a condition unique to humans. However, heaves is a common disease of horses that shares many features of human asthma, including lower airway inflammation, reversible airflow obstruction, and bronchial hyperresponsiveness (1). Heaves can be induced in susceptible horses by the inhalation of dust from moldy hay and the airway obstruction is reversible when horses are maintained on pasture (1, 4). Similar to asthma, heaves can be controlled by the administration of corticosteroids and bronchodilators. Heaves develops over a period of years and offers a unique model to study the respiratory system under chronic inflammatory conditions.

Extrinsic asthma is an allergic response to environmental allergens, and a similar immunologic mechanism may be implicated in heaves. The positive passive cutaneous anaphylaxis test observed using sera from horses with heaves (5) and the elevated levels of IgE in serum and bronchoalveolar lavage (BAL) of affected horses are supportive of the involvement of a type I hypersensitivity reaction in the airway response to exposure to moldy hay (6).

Th2-type lymphocytes, mast cells and eosinophils are considered the primary effector cells leading to allergic airway inflammation (9, 10). However, the individual roles of these cells and their mediators in events leading to the morphologic and functional changes of the asthmatic lung are uncertain (11). Heaves, unlike asthma, is characterized by a neutrophil recruitment into airway secretions after antigenic stimulation (12). There is also evidence that neutrophils may be implicated in some cases of asthma as blood neutrophils are activated and infiltrate the airway of chronic asthmatics, and indeed they have been reported to be the predominant cells in the airway secretions of patients with acute asthma (13).

The purpose of this study was to determine whether horses with heaves present a polarized cytokine expression in the bronchoalveolar (BAL) cells and to compare this cytokine profile with horses without respiratory disease. We reasoned that the study would shed light on the nature of the inflammatory process in the equine airway in heaves and that the results might have relevance to these subjects with asthma whose airway inflammation is predominantly neutrophilic. Another important feature of the study is that the changes found are a response to a prolonged exposure to the offending agents.

	METHODS

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All experimental procedures were performed in accordance with the guidelines of the Canadian Council for Animal Care and were approved by the Animal Care Committee of the Faculty of Veterinary Medicine of the Université de Montréal.

Horses

Twelve horses weighing 400 to 500 kg were used for the study. Horses with heaves (n = 7, 14.8 ± 0.9 yr of age, mean ± SEM) had a history of chronic respiratory diseases and abnormal respiratory mechanics measurements and a > 5% neutrophils in bronchoalveolar fluid (BAL). Control horses (n = 5, 9.6 ± 2.7 yr of age) had no history or clinical signs of respiratory diseases and had < 5% neutrophils in BAL. A physical examination and upper airway endoscopy were performed prior to the study to exclude the presence of concomitant medical conditions. The degree of respiratory distress of each horse was determined using a respiratory score based on a visual analog scale from 1 to 100 and related to the excursion of abdominal muscles and distension of the nostrils during quiet breathing.

Horses were fed with dry Timothy hay and sweet feed twice a day and bedded on straw. No changes had occurred in the environment of horses for > 3 mo prior to the study with the exception of one horse, which had been recruited only 3 wk before the study. No historic information was available for this latter horse. Animals were therefore exposed for a prolonged period ranging from at least 3 wk to 3 mo. No treatments were administered to horses for at least 3 wk prior to the study.

Bronchoalveolar Lavage

Bronchoalveolar lavages were performed under sedation using a fibroptic endoscope, as previously described (18). The BAL fluid was collected in siliconized glass vessels and kept on ice. It was then filtered through sterile gauze, centrifuged (1,500 rpm at 5° C for 5 min) and the cell pellets were resuspended in RPMI 1640 culture medium (Sigma-Aldrich Canada, Oakville, ON, Canada). BAL fluid was analyzed within 1 h of collection.

The total cell counts were determined on a fresh specimen of lavage fluid using a hemacytometer. The differential cell counts were determined on a cytospin slide that was prepared with a Cytospin model II (Shandon Southern Instruments, Sewickley, PA) and stained with a Wright Giemsa stain. At least 400 cells in each specimen were counted under oil-immersion microscopy.

Cytologic Preparations for Immunocytochemistry and in situ Hybridization

Cytospin slides were prepared on poly-L-lysine-coated glass slides, fixed in 4% paraformaldehyde for 30 min, and washed with PBS (twice for 5 min each time) prior to processing. In situ hybridization was performed as previously described (19, 20) using antisense and sense riboprobes prepared from cDNAs coding for equine IL-4, IL-5 mRNA (pEQUIL4 and pEQUIL5zeo were kindly provided by Dr. Horohov, Louisiana State University, Baton Rouge, LA), and IFN- mRNA (kindly provided by Dr. Antczak, Cornell University, Ithaca, NY).

Slides were coded, and positive cells were counted blindly using magnification ×40 with an eyepiece graticule. For negative controls cytospins were hybridized with sense probes or pretreated with RNase prior to the application of probes.

Data Analysis

Differences between groups were determined using the Mann-Whitney U test. Differences were considered significant when p values were less than 0.05. Data are expressed as means ± 1 SEM.

	RESULTS

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Horses

Horses with heaves had evidence of respiratory distress at the time of the study as indicated by the significant (p = 0.006) differences in respiratory scores in diseased horses (42 ± 9.2) and control horses (3.0 ± 3.0).

Bronchoalveolar Lavage Leukocytes

The total volume of BAL recovered was smaller in horses with heaves (median, 125 ml) than in control horses (median, 300 ml). There were no significant differences in total cell recovery in horses with heaves (median 2.6 × 10⁶/ml) compared with control horses (median, 7.3 × 10⁶/ml). Neutrophils predominated in BAL of horses with heaves, whereas lymphocytes and macrophages were the predominant cells in BAL from control horses (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Differential cell cytology in BAL cells of horses with heaves (n = 7) and control horses (n = 5). Asterisks indicate significantly (p < 0.05) different between groups.

Cytokine mRNA Expression

Positive hybridization for IL-4, IL-5, and IFN- mRNA was observed in the BAL cells of all horses. The number of cells expressing IL-4 and IL-5 mRNA was significantly greater in BAL cells of horses with heaves, whereas the number of cells expressing IFN- was greater in control horses (Figure 2). No hybridization was present in the specimens that were incubated with sense probes or in the specimens that were pretreated with RNAse.

View larger version (10K): [in this window] [in a new window]   Figure 2.   Number of total cells per thousand expressing mRNA for IL-4, IL-5, and IFN- in BAL cells of horses with heaves (n = 7) and control horses (n = 5). Asterisks indicate significantly (p < 0.05) different between groups.

	DICUSSION

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The increased number of cells expressing mRNA for IL-4 and IL-5 and a decreased expression of IFN- in the present study suggests that inflammatory cells in lungs of horses with heaves display a predominant Th2-type cytokine profile. This finding is in agreement with the hypothesis that heaves is an allergic condition similar to that of human asthma. Although a polarization of T helper cells into predominantly Th1 and Th2 subsets has not been thoroughly investigated in horses, the finding of the present study is consistent with the Th2 cytokine profile (increased expression of IL-4 mRNA and a decreased expression IL-2 mRNA) observed with in vitro culture of equine CD4+ T cells after chronic allergen stimulation (21). The presence of cytokine mRNA in sufficient copy number to be detected by in situ hybridization provides evidence of gene expression, but it does not prove active synthesis of the relevant protein. At the time of this writing, there were no monoclonal antibodies available for quantitative measurement of the equine IL-4, IL-5, and IFN- production.

IL-4 promotes the development and the growth of Th2 cell phenotype and is essential for the induction of B-cell isotype switching to IgE antibody production (22). The increase in IL-4 mRNA expression in heaves is consistent with the elevated levels of antigen-specific IgE in serum and BAL of horses with heaves (6). The results of IL-5 mRNA expression in the present study are less clear. An increased expression of IL-5 mRNA is usually associated with tissue eosinophil migration, which is not a common feature in heaves. Eosinophil migration into lung tissue results from a complex sequence of cumulative events involving cytokines, including IL-5, chemokines, and various inflammatory molecules (23). The presence of IL-5 within the airway lumen may not be sufficient by itself to induce local eosinophilia, as suggested by the results of a study in a murine model, showing that circulating but not local IL-5 results in pulmonary eosinophilia (24). It is also possible that IL-5 mRNA expression may not have been associated with a protein production in the present study, as it has occurred in human bronchitis (25). The recent finding that horses with heaves have a high level of NF-B consisting mainly of truncated p65 homodimers, rather than classic p65-p50 heterodimers, may contribute to the lack of BAL eosinophilia in heaves (26), as loss of the p50 locus in knockout mice ablates the eosinophilic airway response (27). The importance of NF-B p65 homodimers to equine eosinophil chemoattractant synthesis, however, remains to be determined. Clearly, further studies are required before the dissociation between IL-5 mRNA expression and tissue eosinophilia observed in heaves is understood.

When susceptible horses are exposed to moldy hay, a delay of 5 h is expected before the initiation of neutrophil recruitment in lung tissues and the appearance of clinical signs of heaves (12). This finding is reminiscent of the late allergic response (LAR) in asthmatic subjects after allergen challenge, which is characterized by recruitment, activation, and tissue infiltration of primarily eosinophils, but also neutrophils and lymphocytes (14, 28). Although a number of potent chemotactic mediators of neutrophils such IL-8 and leukotriene B₄ may be present in airway secretions (28, 29), recent finding suggests that T lymphocytes play a key role in the modulation of the LAR and tissue neutrophilia. Little is known concerning the mechanistic relationship between T lymphocytes and neutrophils, but T-lymphocyte cytokines such as IL-13 and IL-17 may play a central role in the selective recruitment of neutrophils into the airways via the release of C-X-C chemokines by bronchial epithelial cells (30).

Airway obstruction in asthma is not completely reversible, presumably because of inflammation-induced structural changes of the airways (33). Although the pathophysiologic role of eosinophils in asthma is well documented, it has been suggested that neutrophils also contribute to airway remodeling and to development of nonreversible airway obstruction (34). In chronic asthmatics, neutrophils are activated and infiltrate the airways several hours after antigen challenge and neutrophils are the predominant inflammatory leukocytes in airway secretions during severe asthma (13, 35, 36). The finding that heaves and severe asthma share many structural changes such as epithelial detachment and regeneration, goblet cell hyperplasia, and hyperplasia of the bronchial smooth muscles, further supports a role of neutrophils in airway remodeling (33, 37, 38). Neutrophils are capable of releasing mediators such as elastase, free oxygen radicals, and leukotriene B4, which may cause exacerbation of obstructive airway disease via tissue damage and airway gland hypersecretion (32, 39). It is now clear that neutrophils also have the capacity to produce a number of inflammatory cytokines and chemokines that may contribute to the modulation of airway inflammation in asthma (40). Although there is support for a role for neutrophils in airway inflammation and hypersecretion in asthma, their exact contribution to the airway pathology is poorly understood.

The finding in the present study of a predominant Th2 cytokine profile in airway cells associated with airway neutrophilia, but not with eosinophilia, suggests that this animal model of asthma may be useful in the study of the mechanisms implicated in the regulation of allergen-induced neutrophil migration.