Differentiation of viable, apoptotic and necrotic cells in bronchoalveolar lavage fluid of normal horses and horses with recurrent airway obstruction

Proinflammatoric cytokines are released extracellularly during necrosis. These lead to inflammation and destruction of surrounding tissues. The aim of this study was to compare the number of viable, apoptotic and necrotic cells in the bronchoalveolar lavage fluid (BALF) of normal horses and horses with recurrent airway obstruction (RAO) and to determine if fluorescence microscopy is a reliable method for this examination. A group of six normal horses and a group of ten horses with RAO were examined. Samples were assessed using annexin-V and propidium iodide immunofluorescence assay and examined by fluorescence microscopy (16 horses) and flow cytometry (nine of 16 horses). We found no significant differences in percentages of apoptotic and viable cells between both groups. The number of necrotic cells was significantly increased in horses with RAO counted by fluorescence microscopy. Cells with high granularity and macrophages had a significantly higher percentage of necrotic cells than lymphocytes. There was a good agreement between both methods. No significant differences were detected. The correlation between both methods is significant. Higher amounts of necrotic cells in the bronchial lumina of horses with RAO could be a reason for tissue damage and continuous lung tissue inflammation. Fluorescence microscopy was applicable for examination of BALF. Therapy should be aimed at the reduction of necrotic cells in the bronchial lumina. Further studies are required to find ways to reduce inflammatory cell infiltration and necrosis in bronchial lumina.     

Increased adenosine concentration in bronchoalveolar lavage fluid of horses with lower airway inflammation

Several reports have suggested a role for adenosine in the pathogenesis of chronic airway conditions and this has led to new therapeutic strategies to limit airway inflammation. In this study, detectable levels of adenosine in bronchoalveolar lavage (BAL) samples from 11 horses with non-infectious lower-airway inflammation and 14 healthy controls are reported, with significantly higher values in horses with airway inflammation. Although these increased levels did not correlate with changes in neutrophil percentage in BAL, a positive association between adenosine levels and signs of lower airway inflammation (clinical score) was observed. These novel findings support the hypothesis that adenosine may contribute to bronchoconstriction and also act as a pro-inflammatory mediator in the bronchoalveolar milieu of horses with airway inflammation. Further investigation of this axis could lead to new approaches for the treatment of highly prevalent lower airway inflammatory conditions in the horse.     

Time-dependent changes of cytokines mRNA in bronchoalveolar lavage fluid from symptomatic recurrent airway obstruction-affected horses

During an 18 day test, we measured the cytokine mRNA expression (Interleukin-1beta [IL-1beta], Interleukin-8 [IL-8], Interferon-gamma [IFN-gamma], Tumor Necrosis Factor-alpha [TNF-alpha]) of cells from bronchoalveolar lavage fluid [BALF] in five horses previously diagnosed with RAO, before and during challenge exposure, and after the desensitization phase which involved dexamethasone treatment and environmental modification. Simultaneously, the same cytokine mRNA expression of cells from BALF in four asymptomatic RAO-affected horses maintained outdoors was analyzed. An evident respiratory distress was observed in the challenge group within 3 days, with a significant overexpression of IL-8 and TNF-alpha mRNA on the ninth day. The pharmacological and environmental desensitization provided a down regulation of all the cytokines. No statistical modification characterized the cytokine kinetics of the asymptomatic horses maintained outdoors. A comparison for each time point of the cytokines between the exposed and unexposed horses showed no significant differences. The study suggested that a standardized exposure protocol and sampling time in experimental studies of RAO is mandatory for a correct comparison of the results obtained by different Authors. However, the absence of significant changes between the exposed and unexposed horses could depend on the lack of the sample uniformity since the evolution of the disease represents a continuum from a healthy to a pathological condition.     

Effects of pentoxifylline on pulmonary function and results of cytologic examination of bronchoalveolar lavage fluid in horses with recurrent airway obstruction

OBJECTIVES: To determine the effects of pentoxifylline (PTX) administration on lung function and results of cytologic examination of bronchoalveolar lavage fluid in horses affected by recurrent airway obstruction (RAO). ANIMALS: 10 RAO-affected horses. PROCEDURES: 6 horses were orally administered PTX (16 g) mixed with corn syrup, and 4 horses were administered corn syrup alone, twice daily for 14 days. Pulmonary function was evaluated before administration (day 0) and on days 8 and 15. Bronchoalveolar lavage (BAL) was performed on days 0 and 15. Reversibility of airway obstruction was assessed by measuring pulmonary function before and after administration of atropine (0.02 mg/kg, IV). Serum concentration of PTX was measured in 4 horses 30 minutes and 2 and 4 hours after administration of PTX on days 1, 2, 3, 7 and 14. RESULTS: Administration of PTX to BAO-affected horses resulted in a decrease in elastance value on day 8 and on elastance and resistance (RL) values on days 8 and 15. Results for cytologic examination of BAL fluid obtained on day 15 did not differ significantly, compared with values for day 0. Values of RL decreased in all horses following administration of atropine. When mixed in corn syrup and administered orally, PTX was poorly absorbed in horses, and there was noticeable variation in serum PTX concentrations over time and among horses. CONCLUSIONS AND CLINICAL RELEVANCE: Based on these results, it can be concluded that administration of PTX at high doses improved respiratory function of RAO-affected horses maintained in an unfavorable environment.     

Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction.

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis-cell migration chamber. The number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAO-affected horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P = 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAOaffected horses.     

Effects of the bronchoalveolar lavage procedure on lung function in horses with clinical exacerbation of recurrent airway obstruction

OBJECTIVE: To evaluate whether bronchoalveolar lavage (BAL) alters respiratory mechanics of horses with recurrent airway obstruction (ie, heaves) over a 48-hour period. ANIMALS: 6 horses affected with heaves. PROCEDURES: Horses were subjected to a complete BAL procedure, which included sedation with xylazine and butorphanol, intratracheal administration of lidocaine, and instillation and aspiration of two 250-mL boluses of saline (0.9% NaCl) solution through an endoscope (study 1). To evaluate the effects of saline solution, horses were subjected to the same procedure without saline solution instillation and aspiration (study 2). Lastly, the endoscope was similarly introduced into the lower airways, without sedation or saline instillation and aspiration (study 3). Respiratory mechanics were performed at baseline (time 0) and at 3, 6, 12, 24, and 48 hours after each procedure. RESULTS: In study 1, BAL induced a significant decrease in pulmonary resistance lasting up to 6 hours. This may have resulted from clearance of mucus in large airways. We also observed a significant increase in lung elastance and transpulmonary pressure at 12 hours after BAL in all 3 studies, which may be attributed to a circadian effect. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that the temporal effects of BAL procedures on lung mechanics should be taken into account when designing research protocols involving horses with heaves. Future studies should address the immediate effects of BAL on lung function.     

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.     

Pulmonary function,airway cytology and bronchoalveolar lavage fluid drug concentration after aerosol administration of cefquinome to horses

The administration of antibiotics by aerosol to horses suffering from respiratory infections may partially circumvent the limitations of antimicrobial therapy, e.g. large injection volumes, low bioavailability and risk of diarrhoea. Only injectable formulations are available currently and usually contain other substances that could irritate the mucosa and induce coughing and bronchospasm. In addition, the quality of the aerosol, particularly in terms of the delivery of antibiotics to the deep parts of the lung, is unknown. Although used under field conditions, cefquinome delivered by aerosol has never been studied in horses. This study examined the safety of cefquinome injectable solution, administered by aerosol at a dose of 225 mg/inhalation to 7 healthy horses, by assessing (1) pulmonary function before and 15 min after a single inhalation, at the first day (Day 1) and the fifth day (Day 5) of a 5 day period treatment; and (2) the inflammatory status of the lung, i.e. percentage neutrophils and myeloperoxidase concentration, based on bronchoalveolar lavage (BAL) at D1 and D5. In addition, cefquinome concentrations were measured in bronchoalveolar lavage fluid after aerosol, intravenous (i.v.) and intramuscular (i.m.) administrations. A single aerosol of cefquinome injectable solution did not induce any immediate nor delayed pulmonary side effects in healthy horses and produced cefquinome concentrations in bronchoalveolar lavage (BAL) within 30 min that were higher than the minimal inhibitory concentration of the main equine respiratory pathogens. These results should stimulate further studies, especially in horses suffering from bronchial hyper‐reactivity. Aerosol delivery of antibiotics may well have a role in equine therapeutics.     

Arachidonate metabolites in bronchoalveolar lavage fluid from horses with and without COPD.

Arachidonate metabolites were measured in bronchoalveolar lavage fluid (BALF) from horses with (N = 4) and without (N = 7) chronic obstructive pulmonary disease (COPD). Prostaglandin (PG) D2, leukotriene (LT) B4 and LTC4 were present in highest concentrations in BALF from clinically normal horses. Concentrations of PGE2 and PGF were significantly higher in BALF from horses with COPD than in BALF from normal horses, but no differences were detected in thromboxane B2, 6-keto-PGF1 alpha, PGD2, LTB4 or LTC4.     

Correlation and discriminant analysis between clinical, endoscopic, thoracic X-ray and bronchoalveolar lavage fluid cytology scores, for staging horses with recurrent airway obstruction (RAO)

As recurrent airway obstruction (RAO) is progressive and as medical history is frequently unknown by owners, it's important to suggest a score model to characterize RAO stages for a more accurate diagnosis and treatment. The authors correlated clinical (CS), endoscopic (ES), thoracic X-ray (XRS) and bronchoalveolar lavage fluid (BALFS) scores in horses with RAO, in an attempt to establish relevance of each factor's contribution for the characterization of RAO stages and to suggest a staging method. Thirty horses with RAO and ten healthy controls were studied. Pearson correlation coefficients were determined between CS, ES, XRS and BALFS. Only significant correlation coefficients (>0.60) were considered. One way variance analyses were used to compare the two groups. A discriminant analysis model was adjusted on the RAO staging method suggested. There was a significant correlation coefficient between the CS cough, nostril flare and abdominal lift, all the mucus ES (0.61-0.84), the XRS interstitial pattern, bronchial radiopacity and thickening and tracheal thickening (0.67-0.78) and the BALFS neutrophil percentages (0.63-0.84). These variables (e.g., cough) which presented a significant correlation coefficient were considered relevant and chosen for a score model to characterize RAO stages. The ten healthy controls were attributed stage 0 and the 30 RAO horses were attributed stages 1 (4 horses), 2 (7 horses), 3 (10 horses) and 4 (9 horses). There was also a significant correlation coefficient between all the relevant variables and the RAO stage (0.61-0.89). Furthermore, discriminant analysis of the RAO staging method showed 92.5% of original grouped cases and 85.0% of cross-validated grouped cases correctly classified, having confirmed major contribution of the same variables that had significant correlation coefficients. Even though further confirmation by lung functional testing is desirable, the significant correlation between relevant variables and RAO stage and the results of RAO staging discriminant analysis support the suggestion of our score model for the characterization of RAO stages.     

Immunostimulation of bronchoalveolar lavage cells from recurrent airway obstruction-affected horses by different CpG-classes bound to gelatin nanoparticles

Recurrent airway obstruction (RAO) in horses has become a common problem in stabled horses in industrialized countries and deserves new therapeutic strategies. CpG-oligodeoxynucleotides (CpG-ODNs) were developed as effective immunostimulating agents to induce a Th2/Th1 shift. These agents showed a beneficial therapeutic effect in allergic diseases with predominant Th2 immunoresponse. CpG-ODN delivery by gelatin nanoparticles (GNPs) resulted in enhanced cellular uptake in murine and human in vitro studies and was a starting point for the present trial. The aim of this study was to identify an optimal stimulating CpG motif in horses with regard to species specificity on equine bronchoalveolar lavage (BAL) cells, in terms of a possible specific immunomodulation effect (Th2/Th1 shift) by used CpG-ODN. Accordingly, GNPs were evaluated as a delivery system to improve CpG-ODN immunostimulation in equine BAL cells. BAL fluid (BALF) was obtained from seven horses with moderate RAO and from four healthy horses and was subsequently incubated with five different CpG-ODN sequences (from A-, B- and C-class) and one ODN without any CpG motif. Release of three key cytokines (IL-4, IL-10 and IFN-γ) was quantified by ELISA to detect an allergy mediated Th2 immunoresponse (IL-4) as well as a proinflammatory Th1 response (IFN-γ). Due to its specific anti-inflammatory and anti-allergic effects, IL-10 was considered as a beneficial agent in pathophysiology of RAO. Results showed a significant upregulation of IL-10 and IFN-γ on the one hand and a downregulation of IL-4 on the other hand in RAO affected horses. Cell cultures from healthy horses had a significantly stronger response in cytokine release to all the applied stimuli in contrast to RAO derived cells. Comparing all five CpG sequences, A-class 2216 significantly showed the highest immunomodulatory effects on equine BALF cells and, hence, was chosen for follow-up preliminary clinical studies.     

Coughing,mucus accumulation,airway obstruction,and airway inflammation in control horses and horses affected with recurrent airway obstruction

OBJECTIVE: To investigate relationships between cough frequency and mucus accumulation, airway obstruction, and airway inflammation and to determine effects of dexamethasone on coughing and mucus score. ANIMALS: 13 horses with recurrent airway obstruction (RAO) and 6 control horses. PROCEDURE: 6 RAO-affected and 6 control horses were stabled for 3 days. Coughing was counted for 4 hours before and on each day horses were stabled. Before and on day 3 of stabling, tracheal mucus accumulation was scored, airway obstruction was assessed via maximal change in pleural pressure (deltaPpl(max)), and airway inflammation was evaluated by use of cytologic examination of bronchoalveolar lavage fluid (BALF). Effects of dexamethasone (0.1 mg/kg, IV, q 24 h for 7 days) were determined in 12 RAO-affected horses. RESULTS: To assess frequency, coughing had to be counted for 1 hour. In RAO-affected horses, stabling was associated with increases in cough frequency, mucus score, and deltaPpl(max). Control horses coughed transiently when first stabled. In RAO-affected horses, coughing was correlated with deltaPpl(max), mucus score, and airway inflammation and was a sensitive and specific indicator of deltaPpl(max) > 6 cm H2O, mucus score > 1.0, and > 100 neutrophils/microL and > 20% neutrophils in BALF Dexamethasone reduced cough frequency, mucus score, and deltaPpl(max), but BALF neutrophil count remained increased. CONCLUSIONS AND CLINICAL RELEVANCE: Because of its sporadic nature, coughing cannot be assessed accurately by counting during brief periods. In RAO-affected horses, coughing is an indicator of airway inflammation and obstruction. Corticosteroid treatment reduces cough frequency concurrently with reductions in deltaPpl(max) and mucus accumulation in RAO-affected horses.     

Evaluation of cytokine mRNA expression in bronchoalveolar lavage cells from horses with inflammatory airway disease

Inflammatory airway disease (IAD) is a common disorder of performance horses and is associated with poor performance and accumulation of mucus and inflammatory cells in lower airway secretions. Horses with IAD frequently have increased relative counts of neutrophils in bronchoalveolar lavage fluid (BALF); less commonly relative counts of eosinophils and/or mast cells may be increased. The aetiopathogenesis of IAD is unknown and may involve innate and/or acquired immune responses to various factors including respirable dust constituents, micro-organisms, noxious gases and unconditioned air. The molecular pathways and role of the immune system in the pathogenesis of IAD remain poorly defined and it is unknown whether polarised T cell responses occur in the disease, as have been reported to occur in equine recurrent airway obstruction and asthma in humans. Elucidating cytokine responses that develop in horses with IAD may allow a greater understanding of the possible aetiopathological pathway(s) involved and could contribute to development of novel treatments. We compared the mRNA expression of tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-2, IL-4, IL-8, IL-13, IL-17 and IL-23 in cell pellets extracted from BALF of horses with IAD (n=21) and horses free of respiratory tract disease (n=17). Horses with IAD had significantly increased levels of TNF-α, IL-1β and IL-23 mRNA; no significant differences in the other cytokine mRNAs were detected. The results of this study indicate that IAD of horses is associated with increased mRNA expression of pro-inflammatory cytokines in BALF cells, which may reflect stimulation of the innate immune responses to inhaled antigens. There was no evidence of a polarised T-cell cytokine response suggesting hypersensitivity responses may not be involved in the aetiopathogenesis of IAD.     

Lower shedding of strongylid eggs by Warmblood horses with recurrent airway obstruction compared to unrelated healthy horses

An association between equine recurrent airway obstruction (RAO) and increased resistance to intestinal parasites has been demonstrated in descendants of an RAO-affected stallion. It was hypothesised that members of another high-incidence RAO family (F) and unrelated RAO-affected Warmblood horses (UA) would shed fewer strongylid eggs than unrelated RAO-unaffected pasture mates (PM) under the same environmental conditions. Faecal worm egg counts were performed on faecal samples (63 F, 86 UA, 149 PM) and classified into three categories: 0, 1-100 and >100 eggs per gram. While results for F did not differ from PM, UA were 2.5-times less likely to shed strongylid eggs than PM. RAO-affected Warmblood horses may be more resistant to strongylid nematodes than unrelated unaffected pasture mates and a family history of RAO does not necessarily confer protection against helminth infections.     

Cardiovascular effects of acute pulmonary obstruction in horses with recurrent airway obstruction

BACKGROUND: Recurrent airway obstruction (RAO) is common in horses. Although pulmonary artery (PA) pressure increases during RAO, cardiac function in horses with RAO has received limited attention. HYPOTHESIS: The purpose of this study was to noninvasively determine the cardiovascular effects of acute pulmonary obstruction (APO) in horses with RAO and their reversibility. ANIMALS: Five geldings with RAO, inducible by exposure to moldy hay, were studied. METHODS: Pulmonary mechanics, echocardiography, serum troponin I concentrations, arterial blood gases, and hematocrit were obtained before and after 7 days of APO. Heart rate, PA diameter and flow characteristics, right and left ventricular luminal dimensions and wall thicknesses, global cardiac performance, and evidence of myocardial damage were evaluated. Pulmonary mechanics and echocardiography were reevaluated during remission. RRESULTS: Severe, transient APO did not induce chronic cor pulmonale in horses, because cardiac anatomy and function were normal between episodes. An acute episode of APO produced anatomical and functional cardiac changes in both the right and left heart (including increased PA diameter, abnormal septal motion, and decreased left ventricular diameter and estimated stroke volume), possibly because of the development of pulmonary hypertension, without apparent myocardial damage. The decrease in stroke volume was offset by the increase in heart rate. CONCLUSIONS AND CLINICAL IMPORTANCE: With APO of 7 days' duration, cardiovascular abnormalities and the functional airway changes that produce them are reversible when the offending allergens are removed.