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.     

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.     

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.     

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.     

Effects of transport on constituents of bronchoalveolar lavage fluid from horses.

To determine whether road transport affected pulmonary phagocyte activity, 7 healthy Thoroughbred horses were shipped 1,160 kilometers over 36 hours. Fluid collected by bronchoalveolar lavage (BAL) 12 hours, and 7 and 14 days after transport was analyzed. Results were compared to those from the same horses pre-transport, and 7 non-transported control horses that had BAL performed at the same times as the transported horses. Of cells recovered with BAL the percentage of viable pulmonary alveolar macrophages (PAMs) declined from 90.0 +/- 0.9% pre-transport to 80.0 +/- 3.7% by 2 weeks post transport. Although the ability of PAMs to inhibit the growth of Staphylococcus epidermidis had decreased by 2 weeks post-transport (19.2 +/- 3.7% vs. 8.8 +/- 2.3% inhibition) this could not be attributed to transport as a similar effect occurred in the control group. In contrast, the ability of PAMs to phagocytose sheep erythrocytes labelled with rabbit anti-erythrocyte antibodies increased from 74.0 +/- 8.1% to 92.3 +/- 1.5% by 12 hours post-transport. As all variables were unchanged or only mildly altered following transport, we conclude that this form of transport did not alter the PAM functions we assessed.     

In vitro inhibition of matrix metalloproteinase activity in tracheal epithelial lining fluid from horses with recurrent airway obstruction

OBJECTIVE: To evaluate inhibitory effects of synthetic matrix metalloproteinase (MMP) inhibitors in vitro on gelatinolytic and collagenolytic activities in tracheal epithelial lining fluid (TELF) of horses with recurrent airway obstruction (RAO). ANIMALS: 10 horses with RAO and 5 healthy control horses. PROCEDURES: Substrate-based functional assays, collagen I and gelatin degradation, were used to measure endogenous collagenolytic and gelatinolytic activities in TELF. In vitro inhibition of MMP activity in TELF with 2 chemically modified tetracyclines (CMTs; CMT-3 and CMT-8) and 2 bisphosphonates (BPs; zoledronate and pamidronate) was evaluated. RESULTS: CMT-3, CMT-8, zoledronate, and pamidronate in a dose-dependent manner inhibited TELF type I collagenolytic and gelatinolytic activities, although no complete inhibition of TELF type I collagenolytic and gelatinolytic activities was achieved with the inhibitor concentrations of 25 to 500 microM tested. The CMTs inhibited pathologically induced collagen I degradation more effectively than BPs. Of the tested CMTs, CMT-3 was the most effective inhibitor of gelatinolytic activity, and the efficiency of CMT-3 corresponded with that of the BPs. CONCLUSIONS AND CLINICAL RELEVANCE: An increase in MMP activity in the equine respiratory tract may potentially be inhibited by administration of CMTs or BPs. Distinct synthetic MMP inhibitors may eventually provide an additional means for pharmacologic treatment by decreasing ongoing active tissue destructive inflammation associated with chronic lung disease. The MMP inhibitors such as CMTs and BPs that are targeted to solely inhibit a pathologic increase in MMP activities provide the advantage of minimal adverse effects that are characteristics of other excessively potent MMP inhibitors.     

IgE-bearing cells in bronchoalveolar lavage fluid and allergen-specific IgE levels in sera from RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterized by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of immunoglobulin E antibodies (IgE) in the pathogenesis of RAO is unclear. We hypothesized that the number of cells with receptor-bound IgE in bronchoalveolar lavage fluid (BALF) and IgE levels in serum would be higher in RAO-affected than in healthy horses living in the same environment. Therefore, IgE-positive (+) cells were identified by immunocytochemistry on cytospins from BALF and counted. IgE levels against the mould extracts Aspergillus fumigatus (Asp. f.) and Alternaria alternata (Alt. a.) and the recombinant mould allergen Aspergillus fumigatus 8 (rAsp f 8) were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of seven RAO-affected and 22 clinically healthy mature horses housed in the same conventional stable environment. After correcting for the number of neutrophils, there were no significant differences in IgE+ cells on cytospins from BALF between both groups of horses (5% versus 7%, P > 0.1). Serum IgE levels against the mould extracts were significantly higher in RAO-affected than in clinically healthy horses [median = 119 versus 66 relative ELISA units (REU), P < 0.05]. Furthermore, significantly more RAO-affected than healthy horses had detectable serum IgE against the recombinant allergen rAsp f 8 (4/7 and 3/22, respectively, P < 0.05). Age had no significant effect on BALF cell ratios or on specific serum IgE levels. These results show that high IgE levels against mould antigens are associated with RAO under controlled environmental conditions but ranges of mould-specific serum IgE levels overlapped too much between diseased and clinically healthy animals to be of any diagnostic value. Further studies are needed to assess whether IgE-mediated reactions contribute to the pathogenesis of RAO.     

Efficacy of salmeterol xinafoate in horses with recurrent airway obstruction.

OBJECTIVE: To determine the onset, magnitude, and duration of bronchodilation after administration of aerosolized salmeterol xinafoate in horses with recurrent airway obstruction. DESIGN: Randomized controlled study ANIMALS: 6 horses with recurrent airway obstruction. Procedure Horses received aerosolized salmeterol (210 microg) or no treatment, using a crossover design. Salmeterol was administered, using a mask designed for aerosol delivery in horses. Subjective rating of airway obstruction (RAO), maximal change in pleural pressure (deltaPplmax), and pulmonary resistance (RL) were determined at baseline; 5, 15, and 30 minutes; and 1, 2, 4, 6, 8, 10, and 12 hours after administration of salmeterol and in horses that did not receive treatment. RESULTS: The deltaPpl and RL were improved 15 minutes through 6 hours after administration of salmeterol, compared with values obtained from horses receiving no treatment. The RAO was improved 15 minutes through 2 hours after administration of salmeterol. The maximal response to salmeterol was evident 30 to 60 minutes after administration and was characterized by a 59 + 19% decrease in deltaPpl and a 56 +/- 13% decrease in RL. The deltaPpl and RL were not different from baseline values 8 hours after salmeterol administration. CONCLUSIONS AND CLINICAL RELEVANCE: Duration of action of salmeterol in these horses was approximately 6 hours. Maximal bronchodilation was somewhat delayed (30 to 60 minutes), and the magnitude of response was similar to that of short-acting beta2-adrenergic agonists. Salmeterol provides moderately sustained bronchodilation in horses with recurrent airway obstruction and may be an effective drug for long-term control of this condition.     

Efficacy of oral and intravenous dexamethasone in horses with recurrent airway obstruction

REASONS FOR PERFORMING STUDY: Although the efficacy of dexamethasone for the treatment of recurrent airway obstruction (RAO) has been documented, the speed of onset of effect and duration of action are unknown, as is the efficacy of orally administered dexamethasone with or without fasting. OBJECTIVES: To document the time of onset of effect and duration of action of a dexamethasone solution i.v. or orally with and without fasting. METHODS: Protocol 1 used 8 RAO-affected horses with airway obstruction in a crossover design experiment that compared the effect of i.v. saline and dexamethasone (0.1 mg/kg bwt) on pulmonary function over 4 h. Protocol 2 used 6 similar horses to compare, in a crossover design, the effects of dexamethasone i.v. (0.1 mg/kg bwt), dexamethasone per os (0.164 mg/kg bwt) with and without prior fasting, and dexamethasone per os (0.082 mg/kg) with fasting. RESULTS: Dexamethasone i.v. caused significant improvement in lung function within 2 h with a peak effect at 4-6 h. Dexamethasone per os was effective within 6 h with peak effect at 24 h at a dose of 0.164 mg/kg bwt prior to feeding. The duration of effect was, for all dexamethasone treatments, statistically significant for 30 h when compared to saline and tended to have a longer duration of effect when used orally. Dexamethasone per os at a dose of 0.164 mg/kg bwt to fed horses had mean effects comparable to dexamethasone at a dose of 0.082 mg/kg bwt per os given to fasted horses, indicating that feeding decreases bioavailability. CONCLUSIONS: Dexamethasone administered i.v. has a rapid onset of action in RAO-affected horses. Oral administration of a bioequivalent dose of the same solution to fasted horses is as effective as i.v. administration and tends to have longer duration of action. Fasting horses before oral administration of dexamethasone improves the efficacy of treatment. POTENTIAL RELEVANCE: Oral administration to fasted horses of a dexamethasone solution intended for i.v. use provides an effective treatment for RAO-affected animals.     

Evaluation of leukotriene biosynthetic capacity in lung tissues from horses with recurrent airway obstruction

OBJECTIVE: To evaluate leukotriene (LT) biosynthetic capacity in lung tissue from healthy horses and horses with recurrent airway obstruction (RAO). SAMPLE POPULATION: Lung parenchyma and airway specimens from 8 RAO-affected and 5 healthy horses. PROCEDURE: Horses were stabled for > or = 72 hours. Blood was drawn before euthanasia, after which lung specimens were collected. Tissue strips from small airways and parenchyma were incubated in organ baths with the precursor LTA4 or stimulated with calcium ionophore A23187 or the tripeptide N-formyl-Met-Leu-Phe (fMLP), with or without exogenous arachidonic acid, in the presence of isolated blood neutrophils. RESULTS: Stabling induced typical clinical signs of airway obstruction in RAO-affected horses but not control horses. When lung parenchyma or airway specimens from both groups of horses were incubated with calcium ionophore, with or without arachidonic acid, they did not form LT. In contrast, addition of LTA4 to both tissues resulted in conversion to LTB4, although concentrations of LTC4 were negligible in airways and parenchymal strips from healthy and RAO-affected horses. Incubation of airway and parenchymal strips with suspensions of autologous neutrophils did not influence formation of LT stimulated by calcium ionophore or fMLP, with or without exogenous arachidonic acid. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that lung parenchyma and airway tissues themselves are not of substantial importance for LT formation in the lungs, although these tissues possessed some LTA4 hydrolase activity, enabling LTB4 formation. It may be speculated that LTB4 originates primarily from neutrophils and may play a role in the inflammatory events of RAO.     

Indices of oxidative stress in blood and pulmonary epithelium lining fluid in horses suffering from recurrent airway obstruction.

To test the hypothesis that reactive oxygen species could be associated to the lower airway disorders occurring in horses suffering from recurrent airway obstruction (RAO), indices of oxidative stress were studied in blood and pulmonary epithelium lining fluid in 5 RAO horses either in clinical remission or 24 h after the onset of a crisis of bronchospasm and in 5 healthy horses. Venous blood and bronchoalveolar lavage fluid (BALF) samples were collected and analysed for reduced glutathione (GSH), oxidised glutathione (GSSG), total glutathione (TGSH), glutathione redox ratio (GRR) in blood haemolysate and pulmonary epithelium lining fluid (PELF). The haemolysate concentrations of GSH, GSSG, TGSH and GRR were similar in the 3 groups. The PELF glutathione status was significantly different in the RAO horses in acute crisis compared to healthy horses, indicating the occurrence of an oxidative stress. When RAO horses were in crisis their GSH and TGSH remained unchanged but their GSSG and GRR were significantly increased compared to the remission. These results support the hypothesis that oxidative stress is associated with lower airway disorders occurring in horses suffering from RAO.     

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.