Pulmonary Eosinophilia Associated with Increased Airway Responsiveness in Young Racing Horses

Horses are known to acquire small airway disease (SAD), an allergen-induced naturally occurring syndrome of reversible obstructive lung disease accompanied by airway hyperresponsiveness and increased inflammatory cell numbers on bronchoalveolar lavage (BAL). This disorder has received scant attention in young racehorses. The purpose of the present report was to examine the effect of BAL eosinophilia in young racehorses on clinical examination, BAL, hematology, airway responsiveness, and on pulmonary function at rest and after a standardized exercise challenge. Five (3 males, 2 females; age 2.6 ± 0.9 years) with a history of respiratory compromise and BAL eosinophil differential count <5% and 6 controls (4 males, 2 females; age 3.5 ± 1.0 years) training and performing to expectation with normal BAL cell differential (eosinophils < 1%) were studied. Respiratory system clinical examination was performed and expressed as a clinical score. Arterial blood gas measurements, CBC, and pulmonary function testing were performed at rest. Pulmonary mechanics measurements were repeated 1 hour and 20 hours after a standardized treadmill exercise challenge. Incremental histamine inhalation challenge was performed and the concentration of histamine effecting a 35% decrease in dynamic compliance (PC35C_Dyn) was determined. Significant differences were noted between and controls with regard to clinical score ( P = .01), blood eosinophils ( P = .04), BAL cell count ( P = .04), BAL macrophage differential ( P = .04), PC35C_Dyn ( P = .008), and tidal volume and respiratory rate at 20 hours following exercise challenge ( P = .05). We conclude that pulmonary eosinophilia and airway hyperresponsiveness are manifest in some young horses without overt airway obstruction at rest. We speculate that these may be early events in the natural progression of heaves.     

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.     

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.     

Histamine bronchoprovocation does not affect bronchoalveolar lavage fluid cytology, gene expression and protein concentrations of IL-4, IL-8 and IFN-gamma

In diagnosing inflammatory airway disease (IAD) in performance horses, a histamine bronchoprovocation (HBP) test is often performed. In previously published studies, HBP is usually undertaken prior to cytological examination of the bronchoalveolar lavage (BAL) cells. The purpose of this study was to determine if HBP alters (1) the total nucleated cell numbers and distribution in BAL fluid (BALF) and (2) the mRNA and protein concentrations of selected cytokines in BAL cells and BALF, respectively. BALF was initially collected endoscopically from the right middle or diaphragmatic lung lobe in eight healthy young Standardbred horses. Five to six days later, HBP was performed by aerosolization of histamine (8mg) over a 2min period. BALF was again collected within 2-4h of the HBP from the left middle or diaphragmatic lung lobe. In both samples, total and differential WBC counts were obtained. The gene expressions of interleukin-4 (IL-4), IL-8, interferon-gamma (IFN-gamma) and beta-actin in BAL cells were measured using real-time RT-PCR. The cytokine protein concentrations were measured in the BALF using ELISA. HBP was not associated with either a change in the total BAL cell number or in the distribution of the BAL cells. BAL cell expression of IL-4, IL-8 and IFN-gamma, detected in all samples with the exception of IL-4 in one horse (post-HBP), was not altered as a result of HBP. HBP was not associated with a significant change in IL-8 or IFN-gamma concentrations in the BALF. IL-4 protein was undetectable in BALF either prior to or following HBP. We conclude that HBP can precede BALF collection performed within 2-4h of the former without affecting selected parameters analysed in the BAL cells or BALF.     

Chronic exacerbation of equine heaves is associated with an increased expression of interleukin-17 mRNA in bronchoalveolar lavage cells

Recent finding suggests that T-cells may be involved in the pathogenesis of heaves in horses. However, little is known concerning their possible contribution to pulmonary neutrophilia, a characteristic finding in heaves. Interleukin (IL)-17 is a cytokine secreted by activated T-cells that indirectly promotes the maturation, chemotaxis and activation of neutrophils. We therefore hypothesized that IL-17 may be involved in the recruitment of neutrophils into the airways and that its mRNA expression would be increased in bronchoalveolar lavage (BAL) cells of horses with heaves. Heaves susceptible horses (n=4) and control horses (n=4) when in pasture (clinical remission) and after 35 days of continuous exposure to moldy hay were studied. BAL and respiratory mechanics measurements were performed at both time periods. The mRNA expression of IL-17 in BAL was studied using real-time polymerase chain reaction (PCR) and CD3-zeta was used as a marker of T-cell numbers. There was no significant difference in IL-17 mRNA expression between groups of horses while in pasture. However, stabling resulted in an increased expression of IL-17 in all horses with heaves but in none of the control horses. These preliminary results suggest that IL-17 may contribute in the pathogenesis of horses with heaves following chronic antigen challenge.     

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.     

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.     

Clinical findings,bronchoalveolar lavage fluid cytology and matrix metalloproteinase-2 and -9 in canine pulmonary eosinophilia

We characterized clinical and clinicopathological features, and the involvement of gelatinolytic matrix metalloproteinases (MMP-2 and -9) in canine pulmonary eosinophilia (PE). Study material consisted of 20 PE dogs and 16 healthy beagles. All dogs underwent a similar clinical examination and bronchoalveolar lavage (BAL). Analysis for cell count and differential cell count of BAL fluid (BALF), arterial blood gas analysis before and after BAL, and thoracic radiographs before BAL and after treatment were obtained. Twelve dogs were re-evaluated and six relavaged. MMP-2 and MMP-9 in BALF were analysed by zymography, Western immunoblotting and immunocytochemistry.In the PE dogs, BALF, cell count, number and percentage of eosinophils, and numbers of macrophages, lymphocytes, neutrophils, mast cells and epithelial cells were all significantly elevated. Blood eosinophilia was detected in half of the PE dogs. Three PE dogs had mild hypoxaemia. The BAL procedure had an equal effect on PE and healthy dogs' arterial blood gas values. Bronchointerstitial densities were seen in PE dogs' radiographs. Treatment of PE decreased BALF cell count, eosinophil count and percentage and diminished radiographic changes. Gelatinolytic activity was higher in PE dogs' BALF. BALF macrophages and epithelial cells were the principal sources of the MMP-9.     

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.     

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

Thirty-nine horses and 3 ponies underwent a thorough respiratory examination and were grouped as follows: healthy (4 horses and 1 pony); mild chronic pulmonary disease (CPD 11 horses); moderate CPD (16 horses and 1 pony); and severe CPD (8 horses and 1 pony). Bronchoalveolar lavage (BAL) fluid collected from all animals and respiratory secretions (RS) obtained from 39 of these animals were evaluated cytologically and the results were compared. It was concluded that cytological examination of either BAL fluid or RS was useful in diagnosing various equine pulmonary diseases. The only advantage that BAL offered over RS sampling was in cases in which there was no RS available in the trachea. In addition, the severity of the CPD did not always correlate with either RS or BAL cytology.     

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.     

Depletion of pulmonary intravascular macrophages partially inhibits lipopolysaccharide-induced lung inflammation in horses

Horses are unique in their extreme sensitivity to endotoxin-induced cardio-pulmonary shock and mortality. The mechanisms behind increased sensitivity of the horse to endotoxin remain unknown. Pulmonary intravascular macrophages (PIMs) are pro-inflammatory cells occurring in horses. Because the functions of equine PIMs in endotoxemia remain unknown, we studied the role played by equine PIMs in endotoxin-induced pulmonary pathophysiology. We achieved this by using a recently developed protocol to deplete PIMs in order to compare lipopolysaccharide (LPS)-induced pulmonary responses in horses with or without PIMs. Horses treated with gadolinium chloride (GC; 10 mg/kg intravenous) to deplete PIMs or endotoxin-free saline (n = 4) were injected with Escherichia coli LPS (E. coli LPS; 50 ng/kg intravenously) 48 h after GC or saline. Control horses (n = 5) received two injections of endotoxin-free saline at 48 h intervals. All the horses were euthanized 2 h after LPS or saline challenge. Immunohistology for the PIMs showed their reduced numbers in GC-treated horses. The LPS treatment of normal and GC-treated horses increased diastolic and systolic pulmonary arterial pressures at 30 min compared to the saline-treated horses (P < 0.05). However, horses pre-treated with GC did not have an LPS-induced increase in mean pulmonary arterial pressure compared to the LPS-treated horses (P < 0.05). Light and electron microscopic immunocytochemistry detected extensive labeling for LPS in PIMs of LPS-treated horses. Both the LPS-treated groups had more alveolar septal cells positive for TNF-alpha and IL-1beta compared to control horses, which did not receive LPS (P < 0.05). However, GC-treated horses challenged with the LPS showed less IL-1beta-positive cells (P < 0.05). Immuno-electron microscopy localized TNF-alpha and IL-1beta in PIMs. These new data show that PIMs endocytose LPS and contain TNF-alpha and IL-1beta and their depletion partially inhibits LPS-induced pulmonary inflammatory responses.     

Alveolar clearance in horses with chronic obstructive pulmonary disease

OBJECTIVE: To assess sensitivity of scintigraphic alveolar clearance rate as an indicator of alveolar epithelium damage in horses. ANIMALS: 5 healthy horses (group A) and 5 with chronic obstructive pulmonary disease (COPD; group B). PROCEDURE: Horses underwent clearance rate (k [%/min]) determination. Clearance rate of group-B horses was determined after remission of the disease following 2 months at pasture (remission 1), stabling in a controlled environment (remission 2), and during crisis induced by exposure to moldy hay and straw. Methacholine challenge test was performed at each investigation period to determine nonspecific pulmonary airway hyperresponsiveness. Pulmonary function tests (PFT) also were performed, and cell populations in bronchoalveolar lavage (BAL) fluid were determined on another occasion. RESULTS: Group-B horses had significantly faster mean clearance rate during crisis (k = 4.30+/-0.95%/min), compared with that for remission 1(k = 1.98+/-0.55%/min), which did not differ from the rate in group-A horses (k = 1.95+/-0.33%/min). Despite lack of clinical signs of COPD during remission when stabled in a controlled environment, an intermediate value was found (k = 3.20+/-0.72%/min). CONCLUSIONS: This technique allowed grading of lung damage induced by COPD, whereas use of PFT and determination of BAL fluid cell populations failed to differentiate between remission 1 and remission 2. CLINICAL RELEVANCE: Determination of alveolar clearance rate by use of scintigraphy is a sensitive indicator of lung damage. A modified clearance rate was found despite the lack of clinical and functional changes.     

Effect of hyonate administration on pulmonary function and indices of oxidative stress in COPD horses in clinical remission at rest and after exercise

Pulmonary function tests, bronchoalveolar lavages, and venous blood samplings were performed 24 hours before and one hour after a standardized strenous treadmill exercise test on six horses suffering from chronic obstructive pulmonary disease (COPD) in clinical remission. Pulmonary function test consisted of the measurement of the mechanics of breathing and arterial blood gases analyses. During the exercise tests, heart and respiratory rates, and venous plasma lactate, pH and packed cell volume were controlled. Venous blood was analyzed for some markers of the oxidative stress, i.e., hemolysate glutathione redox ratio and oxidized glutathione, and plasma uric acid. BAL was analyzed for cell cytology as well as for markers of oxidative stress, i.e., glutathione redox ratio, and isoprostanes concentration. The procedure was repeated twice, i.e., before and after two IV injections of 40mg hyaluronate administered at seven day intervals. The results were compared to assess a possible influence of the treatment on the pulmonary and systemic inflammatory and/or oxidative status of these horses.Hyaluronate did not influence the pulmonary function test or the physiological parameters recorded during exercise. There was a decrease in the percentage of BAL neutrophils after the treatment, suggesting a possible anti-inflammatory effect at the pulmonary level. As well, the concentration of isoprostanes in the pulmonary epithelium lining fluid was significantly lower after the treatment, which might be either a consequence of the decrease of the BAL neutrophils, or related to a possible antioxidant effect of the treatment. Lastly, the exercise-induced increase in plasma uric acid was significantly less marked after the treatment, which could be due to a systemic antioxidant effect of the hyaluronate.In conclusion, this preliminary study brings some evidence that hyaluronate could have some antiinflammatory and antioxidant effects at the pulmonary—and systemic—level in COPD horses in remission. However, it does not help to understand the possible mechanisms underlying these observations. Further studies are now necessary to better understand its action, to assess the effect of the product on the pulmonary function of horses suffering from other respiratory problems as well as to assess the efficiency of other ways of administration, i.e., nebulization.     

Clinical, radiographic, endoscopic, bronchoalveolar lavage and lung biopsy findings in horses with exercise-induced pulmonary hemorrhage

A clinical study was performed to determine whether clinical, endoscopic, radiographic, bronchoalveolar lavage (BAL) cytological, and pulmonary biopsy findings could be correlated in horses with exercise-induced pulmonary hemorrhage (EIPH) compared with controls. Racing standardbred horses were selected as either EIPH (n = 10) or control (n = 10), based on repeated postexertional endoscopy of the lower airways. Complete physical and respiratory examinations were performed and blood samples were submitted for arterial blood gas analysis, hematologic study, and fibrinogen determination. Bilateral chest radiographs were taken with the horse standing, and a BAL sample was obtained for cytological examination. Lung was biopsied transcutaneously. Weighted scores were calculated for clinical, radiographic, and pulmonary biopsy findings. The conclusion was that only routine physical examination may help the clinician when EIPH is suspected in horses, especially when there are abnormal findings on percussion of the caudodorsal areas of the chest.