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.     

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

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

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.     

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.     

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.     

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.     

In vitro allergy tests compared to intradermal testing in horses with recurrent airway obstruction

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterised 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 IgE-mediated reactions in RAO remains unclear.The aim of the study was to investigate with a serological IgE ELISA test (Allercept?), an in vitro sulfidoleukotriene (sLT) release assay (CAST^®) and with intradermal testing (IDT) whether serum IgE and IgE-mediated reactions against various mould, mite and pollen extracts are associated with RAO. IDT reactions were evaluated at different times in order to detect IgE-mediated immediate type reactions (type I hypersensitivity reactions, 0.5–1 h), immune complex-mediated late type reactions (type III reactions, 4–10 h) and cell-mediated delayed type reactions (type IV hypersensitivity reactions 24–48 h).In the serological test, overall the control horses displayed more positive reactions than the RAO-affected horses but the difference was not significant. Comparison of the measured IgE levels showed that the RAO-affected horses had slightly higher IgE levels against Aspergillus fumigatus than controls (35 and 16 AU, respectively, p < 0.05), but all values were below the cut off (150 AU) of the test. In the sLT release assay, seven positive reactions were observed in the RAO-affected horses and four in the controls but this difference was not significant.A significantly higher proportion of late type IDT reactions was observed in RAO-affected horses compared to controls (25 of 238 possible reactions versus 12 of 238 possible reactions, respectively, p < 0.05). Interestingly, four RAO-affected but none of the control horses reacted with the recombinant mould allergen A. fumigatus 8 (rAsp f 8, p < 0.05), but only late phase and delayed type reactions were observed.In all three tests the majority of the positive reactions was observed with the mite extracts (64%, 74% and 88% of all positive reactions, respectively) but none of the tests showed a significant difference between RAO-affected and control animals. Our findings do not support that IgE-mediated reactions are important in the pathogenesis of RAO. Further studies are needed to investigate whether sensitisation to mite allergens is of clinical relevance in the horse and to understand the role of immune reactions against rAsp f 8.     

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.     

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.     

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.     

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.     

Effect of hay dust extract and cyathostomin antigen stimulation on cytokine expression by PBMC in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is an inflammatory, obstructive airway disease induced by exposure of susceptible horses to inhaled organic dust particles. The immunological process underlying RAO is still unclear. Previous studies have shown that RAO is linked to the Interleukin-4 receptor (IL-4R) gene in one Warmblood family (F1), but not in another (F2). It has also been shown that in F1, but not in F2, RAO is associated with resistance against parasites, suggesting that this association may have an immuno-genetic basis. Therefore, we hypothesized that the T helper (h)1/Th2/regulatory (Treg) cytokine profiles of RAO-associated antigen- and parasite-antigen-stimulated peripheral blood mononuclear cells (PBMC) differ between RAO-affected and healthy horses depending on their genetic background. In our study, PBMC from 17 RAO-affected and 14 healthy control horses of F1 and F2 were stimulated for 24 h with antigens relevant to RAO [hay dust extract (HDE), Aspergillus fumigatus extract (AFE) and lipopolysaccharids (LPS)]; cyathostomin extract (CE) and recombinant cyathostomin antigen (RCA) or with concanavalin A (ConA). Total mRNA levels of IL-4, IL-4R, IL-13, interferon (INF)-γ and IL-10 were examined by qRT-PCR. Stimulation with either HDE or RCA resulted in significant differences in IL-4R mRNA levels between RAO-affected and control horses in F1, but not in F2. For IL-10 mRNA expression, a significant difference between RAO-affected and control horses in F1 but not in F2 was observed only following stimulation with HDE. In contrast to HDE, stimulation with CE resulted in a significant difference of IL-10 mRNA expression level between RAO-affected horses of F2 and healthy horses of F1. No significant differences were detected upon stimulation with any of the other challenge agents. These findings indicate that the immunological response, specifically IL-4R expression, in response to hay dust and cyathostomin antigens, differs between RAO-affected and healthy horses depending on their genetic background. This study shows that analysis of PBMC reveals systemic changes associated with RAO and helps to elucidate immunological pathways involved in this disease.