Apoptotic effects of tamoxifen on leukocytes from horse peripheral blood and bronchoalveolar lavage fluid

A reduction in inflammatory cell apoptosis is an important concept in the maintenance of inflammation and a potential target for the resolution of inflammation in many inflammatory diseases. Dysregulation of apoptosis has been implicated in a range of diseases, including tumors, neurodegenerative disorders and autoimmunity, and may also be implicated in allergic asthma. In horses, recurrent airway obstruction (RAO) is an asthma-like condition that is characterized increased survival neutrophil bronchial. Tamoxifen is a synthetic, non-steroidal, anti-estrogen agent that is widely used for treating all stages of breast cancer and has been approved for the prevention of breast cancer in high-risk women. The observed efficacy of tamoxifen has been attributed to both growth arrest and the induction of apoptosis. Therefore, the aim of our study was to evaluate the ability of tamoxifen to induce apoptosis in vitro in granulocytic cells from peripheral blood and in mononuclear cells from bronchoalveolar lavage fluid (BALF) in horses. Flow cytometry using commercial AnnexinV-FITC and propidium iodide was used to quantify early and late apoptotic leukocytes, respectively. The results showed a significant increase in early apoptosis in peripheral blood and bronchial granulocytic cells treated with tamoxifen. The rate of early apoptosis of mononuclear cells from blood and BALF when incubated with tamoxifen was significantly lower compared with granulocytic cells. We did not observe a direct effect of tamoxifen on late apoptosis in any of the in vitro assays in the cell types used here. These results indicate that the apoptotic mechanisms under these experimental conditions would affect only blood and BALF granulocytic cells, particularly in early apoptosis. Finally, further in vitro and in vivo studies are needed to better understand apoptotic mechanisms because tamoxifen could be used to treat chronic, inflammatory pathologies associated with granulocytes and allergic diseases, such as asthma or equine RAO.     

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.     

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.     

Antioxidant and inflammatory responses of healthy horses and horses affected by recurrent airway obstruction to inhaled ozone

REASONS FOR PERFORMING STUDY: Inhaled ozone can induce oxidative injury and airway inflammation. Horses affected by recurrent airway obstruction (RAO) have a decreased pulmonary antioxidant capacity, which may render them more susceptible to oxidative challenge. It is currently unknown whether RAO-affected horses are more susceptible to oxidative stress than those unaffected by RAO. OBJECTIVES: To determine whether ozone exposure induces greater oxidative stress and airway inflammation in RAO-affected horses in remission than in healthy horses. METHODS: Seven healthy control horses and 7 RAO-affected horses were exposed to 0.8 ppm ozone for 2 h at rest. RESULTS: At baseline, bronchoalveolar lavage fluid (BALF) ascorbic acid concentrations were lower in RAO-affected horses than healthy controls. Ozone appeared to preferentially oxidise glutathione rather than ascorbic acid 6 h after exposure. Individual healthy and RAO-affected horses demonstrated oxidation of BALF glutathione after ozone exposure. Overall, RAO-affected horses did not demonstrate increased oxidative stress following ozone exposure, compared with healthy horses. Ozone did not induce significant airway inflammation in either group. CONCLUSIONS: RAO-affected horses in remission are not more sensitive to ozone despite a decreased pulmonary antioxidant capacity. Sensitivity to ozone appears to be independent of initial pulmonary antioxidant status. POTENTIAL RELEVANCE: Horses with high susceptibility to oxidative stress may benefit from antioxidant supplementation.     

Assessment of leukotriene B4 production in leukocytes from horses with recurrent airway obstruction

OBJECTIVE: To determine the ex vivo leukotriene (LT) biosynthesis in peripheral blood neutrophils (PBNs) and inflammatory cells in bronchoalveolar lavage fluid (BALF) obtained from horses affected with recurrent airway obstruction (RAO). ANIMALS: 6 RAO-affected and 6 control horses. PROCEDURES: Before and 6, 24, and 48 hours after stabling, disease severity was determined subjectively by clinical and mucus scores and measurement of the maximal change in pleural pressure (deltaPpl(max)); PBNs were isolated and BALF samples were examined cytologically. The PBN and BALF cells were activated with a calcium ionophore in the presence of arachidonic acid, and production of LTC4 and LTB4 was measured per 10(6) cells. RESULTS: Clinical and mucus scores and deltaPpl(max) increased during stabling in RAO-affected horses, but not in control horses. In neutrophils and BALF cells from both groups, production of LTB4 exceeded that of LTC4. At all times, LTB4 production by PBNs was less in RAO-affected horses than it was in control horses. Before stabling, LTB4 production by cells in BALF was low in RAO-affected horses, but increased considerably after 6 hours of stabling. This increase coincided with the migration of neutrophils into the airways. In control horses, production of LTB4 did not change during stabling. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested increased production of LTB4 in airways of RAO-affected horses, compared with control horses, that may contribute to the infiltration of neutrophils into the lungs and the sustained inflammation associated with RAO.     

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.     

Mast cells and IgE-bearing cells in lungs of RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterised by small airway inflammation and obstruction following exposure of susceptible horses to mouldy hay and straw. The aim of the present study was to investigate whether lung tissue from horses with RAO contains higher numbers of IgE-protein positive (+) cells and mast cells compared to controls after mouldy hay challenge. Furthermore, mast cell subtypes in lung tissue were investigated. IgE+ cells were detected in most lung tissue samples but no significant differences between RAO-affected and control horses were found. In the wall of the bronchi and bronchioli of both RAO-affected and control horses, mainly chymase+ mast cells (MC(C)) were present (85% in the bronchial wall and 77% in the wall of the bronchioli), while 73% of the mast cells (MC) around blood vessels were tryptase+ mast cells (MC(T)). No double stained MCs were detected. RAO-affected horses had significantly more MC(C) than controls in the wall of the bronchi (median=7.6 and 1.7 cell/mm(2), respectively, P< or =0.05). They also showed a tendency for more MC(C) in the wall of the bronchioli than controls (median=21 and 2.9 cells/mm(2), respectively, P=0.07) but there were no differences in MC(T) numbers. The data suggest an involvement of MC(C) in the pathogenesis of RAO. Independently of the clinical diagnosis, there was a significant relationship between high MC(C) numbers in the bronchial wall and lung fibrosis, suggesting that these MC(C) may be involved in tissue remodelling. Furthermore, high MC(C) numbers were also associated with increased infiltration with lymphocytes and neutrophils.     

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.     

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.     

Time-dependent alterations in gene expression of interleukin-8 in the bronchial epithelium of horses with recurrent airway obstruction

OBJECTIVE: To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF). ANIMALS: 8 RAO-susceptible horses and 9 control horses. PROCEDURE: In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expression of IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and Toll-like receptor 4 (TLR4) in epithelium and IL-8, IL-17, and TLR4 in BALF cells was measured. In experiment 2, bronchial biopsy specimens were evaluated for IL-8 immunoreactivity. RESULTS: In RAO-susceptible horses after 14 days of challenge exposure, there was a 3- and 10-fold increase in gene expression of IL-8 for epithelial and BALF cells and an increase in IL-8 immunoreactivity in epithelial cells. Challenge exposure failed to alter gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 in epithelial cells of any horses at any time point. During challenge exposure, gene expression of BALF cell IL-17 was downregulated in control horses (day 1) and upregulated in RAO-affected horses (day 35). CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial-derived IL-8 may promote airway neutrophilia, but the inciting stimulus is unlikely to be IL-17 because upregulation of this gene is subsequent to that of IL-8 in epithelial cells.     

Abnormalities in lung surfactant in horses clinically affected with recurrent airway obstruction (RAO)

Background: Abnormalities in lung surfactant are well described in human respiratory diseases including asthma, but are poorly described in horses. Hypothesis: Lung surfactant is abnormal in horses with clinical signs of recurrent airway obstruction (RAO). Animals: Six healthy horses and 5 horses with RAO. Methods: Bronchoalveolar lavage fluid (BALF) was obtained from all horses by standard procedures. Cell‐free BALF was separated into crude surfactant pellets (CSP) and supernatant via ultracentrifugation. Phospholipid and protein content was analyzed from both of these fractions. Phospholipid composition of CSP was determined using high‐performance liquid chromatography with an evaporative light scatter detector. Surface tension of CSP was measured with a pulsating bubble surfactometer. Results: Compared with healthy horses, surfactant from RAO‐affected horses was characterized by significantly decreased phospholipid content in total surfactant (median; range: 23.2; 14.7–62.2 μg/mL BALF versus 172; 111–267 μg/mL BALF, P= .0062) and CSP (20.2; 6.4–48.9 μL/mL BALF versus 155; 94.4–248 μg/mL BALF, P= .0062), and a significantly lower percentage of phosphatidylglycerol (PG) (4.5; 3.6–5.6% versus 6.6; 4.1–7.6%, P= .028). Furthermore, the ratio between the percentages of phosphatidylcholine and PG was significantly higher in RAO‐affected horses than in healthy horses (20.9; 16.6: 25.9 versus 13.9; 11.8–22.8, P= .045). Conclusions and Clinical Importance: This study demonstrates that surfactant from RAO‐affected horses is abnormal. Further studies are needed to determine if these abnormalities are related to an increased tendency for bronchoconstriction and to a decreased ability to clear airway mucus in RAO‐affected horses.     

Tamoxifen induces apoptotic neutrophil efferocytosis in horses

Macrophages and neutrophils are important cellular components in the process of acute inflammation and its subsequent resolution, and evidence increasingly suggests that they play important functions during the resolution of chronic, adaptive inflammatory processes. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neutrophils, and improvement in animals’ clinical status. The aim of this study was to describe if TX induces in vitro efferocytosis of neutrophils by alveolar macrophages. Efferocytosis assay, myeloperoxidase (MPO) detection and translocation phosphatidylserine (PS) were performed on neutrophils isolated from peripheral blood samples from five healthy horses. In in vitro samples from heathy horses, TX treatment increases the phenomenon of efferocytosis of peripheral neutrophils by alveolar macrophages. Similar increases in supernatant MPO concentration and PS translocation were observed in TX-treated neutrophils, compared to control cells. In conclusion, these results confirm that tamoxifen has a direct effect on equine peripheral blood neutrophils, through stimulation of the engulfment of apoptotic neutrophils by alveolar macrophages.     

Randomized, controlled study of inhaled fluticasone propionate, oral administration of prednisone, and environmental management of horses with recurrent airway obstruction

OBJECTIVE: To determine whether administration of glucocorticoids provides additional benefits to environmental management of horses with recurrent airway obstruction (RAO). ANIMALS: 28 horses with RAO. PROCEDURE: Horses were classified as having mild, moderate, or severe RAO. Within each category, horses were randomly assigned to receive inhaled fluticasone propionate, inhaled control substance, or oral administration of prednisone. During the 4-week study, horses were maintained outdoors and fed a pelleted feed. Clinical scores, pulmonary function, results of cytologic examination of bronchoalveolar lavage fluid (BALF), and adrenal gland function were determined before and 2 and 4 weeks after initiation of treatment. RESULTS: Clinical score and pulmonary function of all RAO-affected horses improved during the treatment period. After 4 weeks, clinical scores and pulmonary function of horses treated with a glucocorticoid were not different from those for the control treatment. In horses with severe RAO, treatment with fluticasone for 2 weeks resulted in significantly greater improvement in pulmonary function, compared with pulmonary function after treatment with prednisone or the control substance. Treatment with a glucocorticoid for 4 weeks and a low-dust environment did not have any effect on cellular content of BALF Treatment with prednisone for 2 weeks resulted in a significant decrease in serum cortisol concentration, compared with concentrations after administration of fluticasone or the control substance. CONCLUSIONS AND CLINICAL RELEVANCE: Environmental management is the most important factor in the treatment of horses with RAO. Early treatment with inhaled fluticasone can help accelerate recovery of horses with severe RAO.     

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.     

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.     

The effect of adding oral dexamethasone to feed alterations on the airway cell inflammatory gene expression in stabled horses affected with recurrent airway obstruction

BACKGROUND: Chemokine expression in airway epithelium and bronchoalveolar lavage fluid (BALF) cells of horses with recurrent airway obstruction (RAO) is increased. HYPOTHESIS: For RAO-affected horses that are stabled and fed a pelleted ration, the addition of oral dexamethasone further improves pulmonary function and reduces inflammatory gene expression in pulmonary cells. ANIMALS: Twelve RAO-affected horses. METHODS: In a randomized cross-over experiment, the effect of feeding pellets in lieu of hay to stabled, RAO-affected horses was compared with the effect of feeding pellets and administering a 21-day decreasing dose regimen of oral dexamethasone on the expression (by kinetic polymerase chain reaction) of interleukin-8 (IL-8), chemokine (C-X-C motif) ligand 2 (CXCL2), IL-1beta, IL-6, and beta-actin in the BALF cells and of IL-8, CXCL2, 2 IL-1 receptor (IL-1R2), Toll-like receptor 4 (TLR4), and glyceraldehyde 3-phosphate dehydrogenase in the bronchial epithelium 2 days after the final dose. RESULTS: Both treatments reduced airway neutrophilia and breathing efforts but the addition of dexamethasone was associated with fewer treatment failures. Compared with feed changes alone, dexamethasone administration further reduced the expression of IL-8, CXCL2, and IL-1beta in the BALF cells 3.3-, 2.5-, and 4.7-fold, respectively. In the airway epithelium, both treatments were equally efficacious in reducing the expression of IL-8 and CXCL2 expression relative to pretreatment values, but either treatment failed to alter the expression of IL-1R2 and TLR4. CONCLUSIONS AND CLINICAL IMPORTANCE: For a rapid and consistent improvement in pulmonary function and a reduction in inflammatory gene expression of the BALF cells, a decreasing dose of oral dexamethasone in combination with feed alterations is more efficacious for horses that must remain stabled.     

Effect of potential therapeutic agents in reducing oxidative stress in pulmonary tissues of recurrent airway obstruction‐affected and clinically healthy horses

Reasons for performing study: To determine and compare the reactive oxygen and nitrogen species (ROS and RNS) in pulmonary tissues of horses affected with recurrent airway obstruction (RAO) and clinically healthy horses, and to evaluate the effectiveness of potential therapeutic agents in reducing ROS and RNS in the tissues of these horses. Objectives: We hypothesised that RAO‐affected horses would have high levels of reactive species and that the test agents would reduce them. The objectives were as follows: 1) to determine the level of ROS and RNS in pulmonary tissues (bronchial and arterial rings) of RAO‐affected and clinically healthy horses; and 2) to determine the ability of pentoxifylline, pyrrolidine‐dithiocarbamate and a combined use of endothelin A and B receptor antagonists (BQ123 and BQ788, respectively) in reducing reactive species. Methods: Arterial and bronchial rings were collected from the diaphragmatic lung lobe of each horse immediately after euthanasia. The levels of ROS and RNS were measured in control tissues and those incubated with test agents, using an electron paramagnetic resonance instrument. Results: The levels of ROS and RNS were significantly greater in arterial and bronchial tissues of RAO‐affected than of clinically healthy horses. Pentoxifylline and endothelin antagonists reduced both ROS and RNS in tissues from RAO‐affected horses. Basal levels of reactive species in clinically healthy horses were not affected by these agents. No difference in the level of reactive species was observed between arterial and bronchial tissues. Conclusions: Horses affected by RAO had higher ROS and RNS than clinically healthy horses. Pentoxifylline and endothelin antagonists effectively reduced ROS and RNS in pulmonary tissues of RAO‐affected horses. Potential relevance: The study suggested a potential use for pentoxifylline and endothelin antagonists in treating RAO‐affected horses. As endothelin is involved in physiological functions, therapeutic use of its antagonists is cautioned.     

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.