Enhanced IL-6 transcriptional response to adenosine receptor ligands in horses with lower airway inflammation

Reasons for performing study: Accumulation of extracellular adenosine has been closely associated with human asthmatic responses. However, the relevance of adenosine signalling in equine airways has not previously been investigated. Objectives: To determine the expression of adenosine receptors (AR) in bronchoalveolar lavage (BAL) cells and assess the reactivity of these cells to AR ligands ex vivo, employing IL‐6 as readout of adenosinergic inflammatory signalling. Methods: Eight horses with varying degrees of lower airway inflammation and 10 healthy controls were analysed. Expression of AR‐subtypes in each BAL sample was determined by quantitative RT‐PCR and compared to that in 13 other tissues. Bronchoalveolar lavage cells were stimulated either with the adenosine analogue NECA, CGS‐21680 (A_2AAR selective agonist) or with a combination of NECA and SCH‐58261 (A_2AAR antagonist) and IL‐6 expression assessed. Results: Bronchoalveolar lavage cells predominantly expressed A_2BAR, with lower A_2AAR levels and marginal A₃AR expression; A₁AR was not detected. This pattern was similar to that of PBMCs but different from the other tissues tested. No significant differences in AR expression in BAL cells from both groups were detected, although a trend for decreased A_2BAR in airway‐compromised horses was observed. Treatment of BAL cells with the nonselective agonist NECA upregulated IL‐6 expression in cells from airway‐compromised horses, but levels remained unchanged in control animals. Furthermore, blockage of A_2AAR with SCH‐58261 enhanced IL‐6 mRNA induction by NECA in both groups, with higher levels in airway‐compromised horses; the amplitude of this response correlated with neutrophil count. Conclusions: These results demonstrate the presence of an adenosine/IL‐6 inflammatory axis in the bronchoalveolar milieu of airway‐compromised horses. While A_2BAR is the predominant proinflammatory AR subtype expressed, A_2AAR appears to modulate inflammatory signalling (IL‐6 expression) by adenosine. Potential relevance: This study supports selective AR targeting as a potential therapeutic approach for the modulation of inflammation in the equine lower respiratory tract.     

Evaluation of a risk-screening questionnaire to detect equine lung inflammation: results of a large field study

Reasons for performing study: The diagnosis of equine recurrent airway obstruction (RAO) and inflammatory airway disease (IAD) is based on clinical signs and increased inflammatory cell percentages in the bronchoalveolar lavage (BAL) fluid. Since a BAL is an invasive procedure, a risk‐screening questionnaire (RSQ) would be a valuable screening tool for lung inflammation. Objective: To evaluate the accuracy of a RSQ to detect lower airway inflammation (LAI) in a large population of horses. Methods: A standardised BAL was performed in the field on 167 horses in Alberta, Canada. Horses were separated into 3 categories: 1) BAL normal; 2) BAL mild to moderate LAI (MLAI), and 3) BAL severe LAI (SLAI). The horse owners were asked to complete a RSQ. The RSQ scores were compared to the BAL results to determine the likelihood of a horse having MLAI, SLAI or no LAI. Results: Based on BAL cytology, 28 (17%) horses were normal and 139 (83%) were abnormal, with 110 (66%) showing MLAI and 29 (17%) SLAI. Horses with SLAI and MLAI had a mean RSQ score of 0.95 and 0.70, respectively, compared to 0.60 for normal BAL horses. Horses with SLAI showed more clinical signs than normal and MLAI horses. The sensitivity and negative predictive values of the RSQ for detecting SLAI using a cut‐off score of 0.87, were excellent at 0.90 (95%CI 0.73–0.98) and 0.96 (95%CI 0.82–1.00). Questions on the clinical signs typically found in RAO cases differed significantly between horses with BAL SLAI and those with BAL normal. Conclusions: Prevalence of MLAI was high in this population. Although the RSQ did not allow differentiating normal horses from horses with MLAI, it has a high sensitivity to detect horses with SLAI and is therefore a good screening tool for SLAI.     

Cytokine induction in pulmonary airways of horses with heaves and effect of therapy with inhaled fluticasone propionate

Work in humans and laboratory animals has identified a central role for cytokines and chemokines in development and persistence of lower airway inflammation. The objectives of this study were to determine interleukin (IL)-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha induction in bronchoalveolar lavage (BAL) of control horses and horses with heaves both during remission and exacerbation of the disease, and to determine the effect of therapy with inhaled fluticasone propionate on the cytokine profile of horses with heaves. IL-1 beta and TNF-alpha mRNA expression was significantly higher in horses with heaves after exposure to moldy hay compared to either values obtained during clinical remission or to healthy controls. IL-8 mRNA expression and protein concentrations were significantly higher in horses with heaves than in controls. Both IL-4 and IFN-gamma mRNA expression was increased at various times in heaves-susceptible horses compared to controls. IL-2, IL-5 and IL-10 mRNA expression was not detected in BAL cells of either group. Therapy with inhaled fluticasone propionate after induction of a severe heaves exacerbation resulted in complete resolution of clinical signs, normalization of pulmonary function tests, and significant decrease in BAL neutrophilia. This was associated with a significant decrease in IL-4 mRNA expression and increase in IFN-gamma/IL-4 ratio in horses with heaves. These results demonstrate the clinical efficacy of inhaled fluticasone propionate for the treatment of heaves and suggest a role for cytokines in the development of lower airway inflammation in heaves-susceptible horses.     

Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 2. Evaluation of the diagnostic significance of neutrophil percentage

OBJECTIVE: To determine whether diagnosis of airway inflammation, using cut-off percentages for neutrophils, differs when based on samples from tracheal aspirate (TA) and bronchoalveolar lavage (BAL) collected concomitantly from the same racehorse. DESIGN: Retrospective case series of 48 young Thoroughbred and Standardbred racehorses in race training, but showing poor performance. PROCEDURE TA and BAL samples were collected from all horses 1 to 2 h after high-speed treadmill exercise. Aliquots of the retrieved fluid were cytocentrifuged and smears stained with Diff-Quik. The mean percentage of neutrophils was calculated. Diagnostic cut-off points were set at 20% for TA samples and 5% for BAL samples. Agreement in the interpretations between the two techniques was analysed. RESULTS: In 19 of 51 paired samples (37%) there were differences in diagnostic interpretation between TA and BAL samples. Of these, airway inflammation was indicated only by the TA sample in 13 and only by the BAL in 6. CONCLUSION: TA and BAL samples give important information about different regions of the airway, but neither should be used alone for the diagnosis of inflammation of the entire lung. The limitations of these procedures mean that both samples should be collected when it is desired to cytologically evaluate the entire lower airway.     

Partial divergence of cytokine mRNA expression in bronchial tissues compared to bronchoalveolar lavage cells in horses with recurrent airway obstruction

The aim of this study was to investigate mRNA levels of cytokines in bronchial epithelium in horses with recurrent airway obstruction (RAO) during acute crisis and remission. Additionally, cytokine mRNA levels in endobronchial biopsies and bronchoalveolar lavage (BAL) cells were compared. Seven RAO horses were examined while in respiratory crisis following provocation and again while in remission after 2 months on pasture, during which time six healthy horses on pasture were also examined. Quantitative real-time PCR (RT-PCR) was used to assess mRNA expression for cytokines IL-5, IL-6, IL-8, IL-10, IL-17 and transforming growth factor beta1 (TGF-beta1) in endobronchial biopsies and bronchoalveolar lavage. Expression of IL-8 mRNA was significantly upregulated during crisis in both endobronchial biopsies and BAL cells (p=0.036), while there was a similar trend for upregulation of IL-10 mRNA only in BAL cells that approached significance (p=0.059). Moreover, during crisis the expression of IL-8 mRNA in BAL cells was positively correlated to relative IL-6 mRNA expression (r(s)=0.971, p=0.001) and bronchial epithelial expression of IL-10 and TGF-beta1 mRNA were positively correlated (r(s)=0.943, p=0.005). In comparing the relationship of mRNA expression in BAL to biopsy in individual RAO horses, there was a positive correlation with IL-6 to IL-8 mRNA expression in BAL during respiratory crisis (r(s)=0.971, p=0.001) that also correlated positively with IL-8 expression in biopsies on pasture (r(s)=0.986, p<0.0001 for both). Regarding RAO horses at pasture versus controls neither the cytokine mRNA levels in endobronchial biopsy nor in BAL cells differed significantly. These results further support previous findings that IL-8 mRNA in both BAL cells and bronchial epithelium is upregulated in RAO horses during crisis. However, apart from IL-8, it appears that expression of other cytokines, including IL-5, IL-6, IL-10, IL-17 and TGF-beta1 in bronchial epithelium does not necessarily mirror cytokine expression in BAL cells in individual horses with RAO. Accordingly, examination of markers of inflammation in endobronchial tissue provides complementary but not necessarily identical information to that obtained in BAL cells. Given the potential for repeated sampling over time bronchial biopsy can serve as an invaluable additional tool for investigation of time-dependent changes in inflammatory process in this animal model of asthma.     

Lack of clinical efficacy of a phosphodiesterase-4 inhibitor for treatment of heaves in horses

Phosphodiesterase-4 (PDE 4) enzyme inhibitors have been shown to have anti-inflammatory properties in various animal disease processes and therefore could be effective drugs for the treatment of equine airway diseases. The purpose of this study was to evaluate the efficacy and adverse effects of the PDE 4 inhibitor L-826,141 in horses with heaves. In a blinded parallel design, horses with heaves exposed daily to moldy hay were given a placebo for 14 days and then administered either L-826,141 (n = 6; loading dose of 1 mg/kg IV followed by 0.5 mg/kg IV q48h) or dexamethasone (n = 6; 0.04 mg/kg IV q24h) from days 15 to 29 (study 1). Pulmonary function and bronchoalveolar (BAL) cytology were evaluated weekly from baseline (day 0) to 29 days. In study 2, horses were treated with L-826,141 (1.0 mg/kg IV q24h) for 8 days. Although ex vivo lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha and LTB4 production by fresh blood were inhibited up to 90% after repeated administrations of L-826,141, this treatment failed to improve lung function. In contrast, dexamethasone (positive control) treatment resulted in significant improvement in lung mechanics and airway function in all horses. Neither drug had a significant effect on BAL total cell counts and differential cytology. Administration of the PDE 4 inhibitor L-826,141 for up to 14 days to horses with heaves was not associated with an improvement in airway function or inflammation. These findings suggest that the PDE 4 enzyme is not a key mediator of lung inflammation in heaves.     

Stabling is associated with airway inflammation in young Arabian horses

We examined the effect of stabling on upper and lower airway inflammation in 14 yearling Arabian horses that had been at pasture since birth. Horses were divided into 2 groups of 7. One group was stabled for 3 months and the other remained at pasture. The groups were then switched over for another 3 months. The nasopharynx, guttural pouches and trachea were examined endoscopically and bronchoalveolar lavage performed every month. An upper airway inflammation score was devised based on the magnitude of pharyngeal lymphoid hyperplasia and guttural pouch inflammation. During stabling this score remained constant, whereas it decreased during the 3 months at pasture. Stabling was also associated with a higher number and percentage of neutrophils in bronchoalveolar lavage fluid and with a smaller percentage of lymphocytes. There was no correlation between upper airway inflammation score and bronchoalveolar lavage cytology. During a nasal occlusion test, dorsal displacement of the soft palate occurred more times in stabled than in pastured horses, but this was heavily biased by the results from one animal. We conclude that stabling is associated with inflammation of both the upper and lower airway of young horses.     

Myeloperoxidase concentration in bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction

The aim of this work was to measure the myeloperoxidase (MPO) concentration in bronchoalveolar lavage (BAL) fluid collected from horses with recurrent airway obstruction (RAO), both in crisis and in remission, as well as from healthy horses. Seven horses with RAO were exposed to moldy hay until the maximum change in pleural pressure was greater than 1.5 kPa. At that point, BAL was performed, and the total cell counts and percentages in the fluid were immediately determined. To measure the MPO concentration in BAL-fluid supernatant, we used a specific enzyme-linked immunosorbent assay with polyclonal antibodies against equine MPO. The tests were repeated on the horses with RAO after they had spent 2 mo on pasture. Six healthy horses serving as controls underwent the same tests. The absolute and relative neutrophil counts and the MPO concentration in the BAL fluid were significantly greater in the horses with an RAO crisis than in the control horses. After 2 mo on pasture, the horses that had been in RAO crisis were clinically normal, and their neutrophil counts and MPO levels in BAL fluid had significantly decreased; during remission their neutrophil counts were not significantly different from those in the healthy horses, but their MPO concentration remained significantly higher. This study showed that determining the MPO concentration in a horse's BAL fluid is technically possible and that during remission from RAO the concentration remains higher than normal. Thus, MPO may be a marker of neutrophil presence and activation in the lower airways.     

Pulmonary epithelial lining fluid and plasma ascorbic acid concentrations in horses affected by recurrent airway obstruction

OBJECTIVE: To determine the pulmonary epithelial lining fluid (ELF) concentrations and degree of oxidation of ascorbic acid in horses affected by recurrent airway obstruction (RAO) in the presence and absence of neutrophilic airway inflammation. ANIMALS: 6 RAO-affected horses and 8 healthy control horses. PROCEDURE: Nonenzymatic antioxidant concentrations were determined in RBC, plasma, and ELF samples of control horses and RAO-affected horses in the presence and absence of airway inflammation. RESULTS: ELF ascorbic acid concentration was decreased in RAO-affected horses with airway inflammation (median, 0.06 mmol/L; 25th and 75th percentiles, 0.0 and 0.4 mmol/L), compared with RAO-affected horses without airway inflammation (1.0 mmol/L; 0.7 and 1.5 mmol/L) and control horses (2.2 mmol/L; 1.4 and 2.2 mmol/L). Epithelial lining fluid ascorbic acid remained significantly lower in RAO-affected horses without airway inflammation than in control horses. Moreover, the ELF ascorbic acid redox ratio (ie, ratio of the concentrations of dehydroascorbate to total ascorbic acid) was higher in RAO-affected horses with airway inflammation (median, 0.85; 25th and 75th percentiles, 0.25 and 1.00), compared with RAO-affected horses without airway inflammation (0.04; 0.02 and 0.22). The number of neutrophils in bronchoalveolar lavage fluid was inversely related to the ELF ascorbic acid concentration (r = -0.81) and positively correlated with the ascorbic acid redox ratio (r = 0.65). CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophilic inflammation in horses affected by RAO is associated with a reduction in the ELF ascorbic acid pool. Nutritional supplementation with ascorbic acid derivatives in horses affected by RAO is an area for further investigation.     

Effect of nutritional antioxidant supplementation on systemic and pulmonary antioxidant status,airway inflammation and lung function in heaves-affected horses

An oxidant/antioxidant imbalance in favour of oxidants has been identified as playing a decisive role in the pathogenesis of chronic inflammatory airway diseases. Nutritional antioxidant supplementation might reduce oxidative damage by enhancement of the antioxidant defence, thereby modulating inflammatory processes. In a placebo-controlled, blind study, it was tested whether a dietary antioxidant supplement administered for 4 weeks would improve lung function and reduce airway inflammation in heaves-affected horses. Eight horses in clinical remission of heaves were investigated at rest and after a standardised exercise test before and after treatment with an antioxidant supplement (consisting of a mixture of natural antioxidants including vitamins E and C and selenium from a variety of sources) or placebo (oatfeed pellets without additive). Pulmonary function and exercise tolerance were monitored; systemic and pulmonary lining fluid uric acid, glutathione and 8-epi-PGF(2alpha) were analysed, and bronchoalveolar lavage (BAL) cytology and inflammatory scoring of the airways were performed. The antioxidant treatment significantly improved exercise tolerance and significantly reduced endoscopic inflammatory score. Plasma uric acid concentrations were significantly reduced, suggesting downregulation of the xanthine-dehydrogenase and xanthine-oxydase pathway. Haemolysate glutathione showed a nonsignificant trend to increase, while plasma 8-epi-PGF(2alpha) remained unchanged. Pulmonary markers and BAL cytology were not significantly affected by antioxidant supplementation. The present study suggests that the antioxidant supplement tested modulated oxidant/antioxidant balance and airway inflammation of heaves-affected horses.     

Expression of thymic stromal lymphopoietin in equine recurrent airway obstruction

Thymic stromal lymphopoietin (TSLP) is a cytokine involved in lymphocyte development. In humans and mice, TSLP drives the differentiation of T helper 2 (Th2) cells and the development of allergic inflammation. The equine TSLP gene has been previously identified and characterized, but its role in the pathogenesis of equine allergic diseases is not known. Our objective was to assess the expression of TSLP in bronchoalveolar lavage (BAL) cells and in primary bronchial epithelial cells (BEC) isolated from horses with recurrent airway obstruction (RAO). RNA was isolated from BAL cells sampled from clinical cases of RAO (n=8) and from control horses (n=12). Furthermore, BAL samples were taken from an additional group of 8 RAO-susceptible and 8 control horses when on pasture (remission) and after 30 days of exposure to moldy hay (exacerbation). In order to study epithelial cells as a potential source of TSLP, cultures of primary bronchial epithelial cells (BEC) were established from 6 RAO-affected and 6 healthy horses and stimulated in vitro with hay dust solution (HDS). Expression of TSLP mRNA was assessed by quantitative real-time RT-PCR (qPCR). Clinical RAO-cases had higher TSLP expression in BAL than control horses (p<0.05). In an experimental group of horses there was no difference between healthy and susceptible horses in remission, whereas after 30-day experimental exposure to moldy hay, all susceptible horses upregulated TSLP expression in BAL (p=0.008, average 6.36-fold increase), whereas in healthy horses there was no significant increase in TSLP expression. BEC generated both from healthy and RAO-affected horses strongly upregulated TSLP expression after 6 h stimulation with HDS, which identifies epithelial cells as potential sources of TSLP in RAO. Finding of increased TSLP expression by BAL cells of RAO-affected horses is in agreement with the contribution of Th2-driven allergic inflammation in the pathogenesis of RAO.     

Comparison of bronchoalveolar lavage and respiratory secretion cytology in horses with histologically diagnosed pulmonary disease

Equine bronchoalveolar lavage (BAL) fluid collected from 70 horses and respiratory secretions (RS) obtained from 61 of these horses were evaluated cytologically and grouped according to the histological diagnosis of the lungs from which they were obtained. The histological categories included: normal lung (8 horses); pulmonary eosinophilic infiltration (9 horses); interstitial pneumonia (5 horses); pulmonary hemorrhage (5 horses); and mild (12 horses), moderate (7 horses) and severe (24 horses) chronic small airway disease. In horses with pulmonary disease, all BAL samples and all but one RS sample differed cytologically to those obtained from normal horses; however, the type and severity of the pulmonary disease could not always be determined using either BAL or RS cytology. There was a positive association between the percentage of neutrophils in BAL and the neutrophil scores in RS specimens; there was no positive association between other cell types.     

Clinical signs, evaluation of bronchoalveolar lavage fluid, and assessment of pulmonary function in horses with inflammatory respiratory disease

OBJECTIVE: To evaluate the association among clinical signs, results of cytologic evaluation of bronchoalveolar lavage (BAL) fluid, and measures of pulmonary function in horses with inflammatory respiratory disease. ANIMALS: 9 healthy horses, 5 horses with inflammatory airway disease (IAD), and 9 horses with chronic obstructive pulmonary disease (COPD). PROCEDURES: Clinical examination, lung function tests, and BAL were performed on each horse. RESULTS: Standard lung mechanics of horses with exacerbated COPD differed significantly from those of healthy horses; however, there were few differences among horses with IAD, horses with COPD during remission, and healthy horses. Most variables for forced expiration (FE) in horses with COPD or IAD differed significantly from those for healthy horses. Results of clinical examination had low to moderate sensitivity and predictive values for a diagnosis of COPD (range, 67 to 80%). Results of FE tests had high sensitivity, specificity, and predictive values for a diagnosis of COPD (79 to 100%), and results of standard lung mechanics tests had low sensitivity and predictive values (22 to 69%). Percentage of neutrophils in BAL fluid was highly sensitive (100%) but moderately specific (64%) for a diagnosis of COPD. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical examination is moderately accurate for establishing a diagnosis of COPD. Forced expiration tests can specifically detect early signs of airway obstruction in horses with COPD and IAD that may otherwise be inapparent. Cytologic evaluation of BAL fluid allows early detection of inflammatory respiratory disease, but it is not specific for COPD.     

IL-4, IL-5 and IFN-gamma mRNA expression in pulmonary lymphocytes in equine heaves

Heaves is a common condition of horses of cold climate that is characterized by small airway inflammation and obstruction following exposure of susceptible horses to moldy hay and straw. It has been shown that helper T lymphocytes (Th) orchestrate the inflammatory response in asthma and in various animal models of allergic lung diseases by the release of Th2-type cytokines. Results of previous studies indicate that a predominant expression of Th2-type response by airway cells may also be present in heaves. To evaluate the temporal mRNA expression of Th1 (IFN-gamma) and Th2 (IL-4, IL-5) type cytokines in heaves and their relationship to clinical disease, we studied the pulmonary mechanics and cytokine mRNA expression (IL-4, IL-5 and IFN-gamma) in bronchoalveolar lavage lymphocytes of horses with heaves (n=6) and control (n=6) before and after 24h and 9 days of continuous natural inhalation challenge. Starting 24h after challenge horses with heaves, but not control horses, had a significant increase in pulmonary elastance and the number of lymphocytes expressing mRNA for IL-4 and IL-5. These changes were further increased at 9 days, at which time the number of cells positive for IFN-gamma mRNA was decreased. In this study we have shown that BAL lymphocytes of horses with heaves during clinical exacerbation have a predominant Th2-type cytokine response and that this response coincides in time with the presence of airway obstruction.     

Use of a protected catheter brush for culture of the lower respiratory tract in horses with small airway disease.

A protected catheter brush introduced by fiberoptic bronchoscopy was used to sample the tracheai and bronchial mucosa in 28 horses with small airway disease. Tracheal and bronchial brushings were examined for the presence of fungi, aerobic and anaerobic bacteria, and a cytoiogical evaluation was also done on fluid collected by the bronchoalveolar lavage (BAL) technique. Microorganisms (bacteria and fungi) were isolated more often in tracheal brushings (53.6%) than in bronchial brushings (10.7%). Anaerobic bacteria were not isolated. Results of this study indicate that fiberoptic bronchoscopy using a protected catheter brush is an easy and practical technique to obtain minimally contaminated samples for isolation of microorganisms from the lower respiratory tract of horses. However, no association was observed between isolation of high numbers of microorganisms from the bronchi and severity of small airway disease.     

What's in a name? Inflammatory airway disease in racehorses in training

The term 'inflammatory airway disease' (IAD) is often used to describe the syndrome of lower airway inflammation that frequently affects young racehorses in training around the world. In practice, this inflammation is generally diagnosed using a combination of endoscopic tracheal examination, including grading of amounts of mucus present and tracheal wash sampling. However, a recent consensus statement from the American College of Veterinary Internal Medicine concluded that bronchoalveolar lavage (BAL) sampling, rather than tracheal wash (TW) sampling, is required for cytological diagnosis of IAD and that tracheal mucus is not an essential criterion. However, as BAL is a relatively invasive procedure that is not commonly used on racing yards, this definition can only be applied routinely to a biased referral population. In contrast, many practitioners continue to diagnose IAD using endoscopic tracheal examination and sampling. We argue that, rather than restricting the use of the term IAD to phenotypes diagnosed by BAL, it is important to distinguish in the literature between airway inflammation diagnosed by BAL and that identified in the field using TW sampling. We suggest the use of the term brIAD for the former and trIAD for the latter. It is essential that we continue to endeavour to improve our understanding of the aetiology, pathogenesis and clinical relevance of airway inflammation identified in racehorses in training using tracheal examination and sampling. Future studies should focus on investigations of the component signs of airway inflammation.     

Temporal regulation of cytokine mRNA expression in equine recurrent airway obstruction

Acute and chronic inflammation of the airway remains an important health problem for equids. "Heaves" or recurrent airway obstruction (RAO) remains one of the most commonly diagnosed conditions affecting the lung of older horses in Europe and the United States. The typical clinical signs of RAO include non-productive coughing, serous nasal discharge, labored expiratory effort, and flaring of the nostrils. Auscultation of the lungs of the affected horse often reveals abnormal respiratory sounds, described as crackles and wheezes, throughout the area of the lung field. These clinical signs occur secondary to an inflammatory response that results in bronchospasm, excessive mucus production and airway obstruction. This inflammatory response is characterized by the presence of excessive mucus and inflammatory cells, primarily neutrophils, in the small airways. Most evidence suggests that RAO is the result of a pulmonary hypersensitivity to inhaled antigens. Exposure of affected horses to hay dust, pollens, and mold spores leads to neutrophil accumulation in the lung and bronchospasm. The identification of allergen-specific IgE in bronchoalveolar lavage (BAL) fluid and sera of affected horses supports the involvement of a late phase, IgE-mediated, hypersensitivity reaction in the pathogenesis of equine RAO. The production of IgE antibodies is regulated by the cytokines IL-4 and IL-13. Using a quantitative PCR method we have reported that horses with RAO exhibit a modified Type 2 cytokine response characterized by the production of IL-4 and IL-13 mRNA, but not IL-5 mRNA in BAL cells. Interferon-gamma mRNA was also elevated, suggesting a mixed response. While these results are consistent with equine RAO being the result of an aberrant Type 2 cytokine response to inhaled allergens, others have failed to find any evidence of elevated Type 2 cytokine mRNA in BAL from horses with "heaves". It is likely that these disparate results could be the result of differences in the clinical stage of the affected animals or the timing of sample collection. Here, we report a diverse pattern of cytokine gene expression when sampling a group of affected horses over a period of time.     

Lung function and airway cytologic profiles in horses with recurrent airway obstruction maintained in low-dust environments

BACKGROUND: The effects of long-term environmental management on airway obstruction and inflammation in horses with recurrent airway obstruction (RAO) are unknown. HYPOTHESIS: Horses with RAO maintained in low-dust environments have persistent airway obstruction and neutrophilic inflammation. ANIMALS: Study horses were treated for RAO and then maintained in low-dust environments with no medical management. Horses were classified into 3 groups by years after diagnosis: 1 year (time 1, n = 9), 2-3 years (time 2, n = 7), and 5-6 years (time 3, n = 8). The comparison groups were age-matched healthy horses. METHODS: In this cross-sectional study, a clinical examination was performed, and the clinical score was calculated. Standard lung function, forced expiratory maneuvers, and the cytology of bronchoalveolar lavage fluid (BALF) were evaluated. RESULTS: The clinical scores of the RAO horses were higher than those of the non-RAO horses at time 2 (P = .018). Standard lung function data were not different between the groups at any time point. The forced expiratory flow between 75-95% of exhaled vital capacity was lower in RAO horses than in non-RAO horses at all time points (P < .02), indicating persistent peripheral airway obstruction. Cytologic evaluation of BALF revealed no difference in total nucleated cell numbers or differential cell counts between RAO and non-RAO horses at any time point. CONCLUSIONS AND CLINICAL IMPORTANCE: The peripheral airway obstruction detected in horses with RAO maintained in low-dust environments likely is due to irreversible airway remodeling but is not associated with cytologic evidence of airway inflammation.