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.     

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.     

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.     

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.     

Influence of environmental and genetic factors on allergen-specific immunoglobulin-E levels in sera from Lipizzan horses.

To investigate whether allergen-specific IgE production is influenced by environmental and genetic factors, IgE levels against 2 mould extracts (Alternaria alternata [Alt a] and Aspergillus fumigatus [Asp f]) and against recombinant (r) rAlt a 1, rAsp f 7 and rAsp f 8 were determined by ELISA in sera from 448 Lipizzan horses living in 6 studfarms. Statistical evaluation showed a significant effect of studfarm-specific environment on IgE levels against the different allergens, but genetic factors also influenced allergen-specific IgE production: an heritability of 0.33 was found for IgE levels against the 2 mould extracts and of 0.21 for rAsp f 8-specific IgE. Heritability estimates for rAlt a 1- and rAsp f 7-specific IgE were negligible. Investigations for a possible association between Major Histocompatibility Complex (MHC) class I antigens and specific IgE levels were carried out. The most consistent significant association was found between the equine leucocyte antigen (ELA) A8 and undetectable IgE titres against rAsp f 7 and rAsp f 8. Significant ELA associations were also demonstrated between ELA A1 and higher specific IgE levels, between ELA A14 and lower IgE levels against the mould extracts and in one studfarm between ELA Be27 and lower Aspergillus-specific IgE levels.     

Effect of transportation stress on bronchoalveolar lavage fluid analysis in female horses

Four bronchoalveolar lavages were performed sequentially on 9 control and 8 transport-stressed female horses. Alterations in results of fluid cytologic analyses, microbial content, and phagocyte function of recovered pulmonary macrophages in all horses were determined. Seemingly, absolute and relative increase in the number of inflammatory cells detected in the second bronchoalveolar lavage fluid of control horses was the result of irritation of the first lavage. This increased response was not observed in transport-stressed horses until 5 days after transport (third lavage; 10 days after initial lavage). Seemingly, delayed inflammatory response was the result of the transport stress. Microbial content and macrophage function were not significantly different between the 2 groups (P greater than 0.05).     

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.     

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.     

Effects of lung site and fluid volume on results of bronchoalveolar lavage fluid analysis in horses.

Bronchoalveolar lavage (BAL) fluid was analyzed in healthy horses, using different lavage fluid volumes and lung sites. The only significant difference in the cellular composition of BAL fluid between the right and left lungs was the mast cell numbers, which were significantly higher in the left lung. Total cell count ranged from 34 to 330 cells/microliter for the right lung and 43 to 330 cells/microliter for the left lung. Percentage of neutrophils ranged from 1 to 7% in the right lung and 1 to 5% in the left lung. The small-volume (50 ml) lavage had a greater percentage of neutrophils and a lesser percentage of mast cells in the large-volume (350 ml) lavage. Statistical difference in the composition of BAL fluid recovered was not detected between the 3 sequential 100-ml lavages and a single 300-ml lavage, except that macrophages were significantly higher in the 3 sequential 100-ml lavages. Values for BAL fluid analysis in healthy horses have varied considerably and this variation is from a failure to adhere to any standard technique for volume of fluid infused.     

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.     

IgE ELISA using antisera derived from epsilon chain antigenic peptides detects allergen-specific IgE in allergic horses

Equine disease with an allergic etiology is common. Environmental antigens most often implicated as allergens in horses include molds, dusty hay, grass pollen, hay dust mites, and insect saliva. Although intradermal testing with allergen is a useful diagnostic tool for some species, skin testing frequently produces false positive results in horses. Allergen deprivation as a diagnostic tool is often impossible and at best it is ineffective at diagnosing the specific allergic reactivity. Synthesis of IgE after exposure to allergen is the instigator of the allergic process. While IgE exerts its effect after binding strongly to mast cell Fc receptors, the presence of free IgE in the serum can be used to quantify and determine the allergen specificity of the allergic disease. A lack of widely available reagents for detection of equine IgE has limited this approach in horses. We have used the nucleotide sequence of equine IgE to prepare a peptide-based immunogen to elicit equine epsilon chain-specific antisera. Selection of peptides was based on antigenic attributes of the deduced amino acid sequence of the equine epsilon chain. Six peptides were selected for conjugation to carrier molecules and rabbit immunization. Of these, one peptide elicited antisera that was successfully used in enzyme linked immunosorbant assay (ELISA) to screen horse serum from 64 allergic horses for allergen-specific IgE. Twenty-four of the 64 horses showed positive reactivity to one or more of the following allergens: grass, grain mill dust, mosquito, and horsefly. This study demonstrates the usefulness of peptide-based immunogens for development of antisera to rare or difficult to purify antigens such as IgE. Resultant antisera has great usefulness in diagnostic assays for equine allergy and as a research tool.     

Total IgE and allergen-specific IgE and IgG antibody levels in sera of atopic dermatitis affected and non-affected Labrador- and Golden retrievers

Canine atopic dermatitis (CAD) is an allergic skin disease associated with IgE and IgG antibodies (Ab) to environmental allergens. The aim of this study was to determine which other factors influence serum Ab levels in CAD-affected and non-affected dogs as this has only been poorly investigated in dogs so far. Total and allergen-specific IgE levels and Dermatophagoides farinae (DF)-specific IgG1 and IgG4 were measured by ELISA in sera of 145 CAD-affected and 271 non-affected Labrador- and Golden retrievers. A multivariable logistic regression analysis including the factors age, breed, gender, castration, clinical CAD status and allergen-specific immunotherapy (ASIT) was performed. Golden retrievers had more frequently total (OR=1.87, 95% CI=1.26-2.87, p<0.01) and specific IgE levels above the threshold value than Labrador retrievers, suggesting that genetic factors influence IgE levels in dogs. Castration was generally associated with low Ab levels (OR=0.43-0.65, p<0.05). Surprisingly, dogs with CAD did not have increased odds for high IgE against any of the allergens tested. ASIT with DF was associated with high DF-specific IgG1 (OR=4.32, 95% CI 1.46-12.8, p<0.01) but was not associated with DF-specific IgG4 or decreased IgE levels. Further studies are needed to understand the role of allergen-specific IgE in CAD and of IgG1 in ASIT.     

Factors affecting allergen-specific IgE serum levels in cats

Pruritic skin diseases are common in cats and demand rigorous diagnostic workup for finding an underlying etiology. Measurement of a serum allergen-specific IgE in a pruritic cat is often used to make or confirm the diagnosis of a skin hypersensitivity disease, although current evidence suggests that elevated allergen-specific IgE do not always correlate with a clinical disease and vice versa. The aim of the study was to to assess the possible influence of age, deworming status, lifestyle, flea treatment, and gender on allergen-specific IgE levels and to evaluate the reliability of IgE testing in predicting the final diagnosis of a pruritic cat. For this purpose sera of 179 cats with pruritus of different causes and 20 healthy cats were evaluated for allergen-specific IgE against environmental, food and flea allergens using the Fc-epsilon receptor based enzyme-linked immunosorbent assay (ELISA) test. The results of the study showed positive correlation between age, outdoor life style, absence of deworming, absence of flea control measures and levels of allergen-specific IgE. Gender and living area (urban versus rural) did not seem to affect the formation of allergen-specific IgE. According to these findings, evaluating allergen-specific IgE levels, is not a reliable test to diagnose hypersensitivity to food or environmental allergens in cats. On the contrary, this test can be successfully used for diagnosing feline flea bite hypersensitivity.     

Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport

An enzyme-linked immunosorbent assay (ELISA) was developed for equine surfactant proteins SP-A and SP-D in bronchoalveolar lavage fluid (BALF). Anti-equine SP-A or SP-D monoclonal antibodies (mAb) were produced by hybridoma technology, purified by the antibody purification reagent, and analysed by Western blotting analysis. The immunoreaction (two-site sandwich ELISA) with a mAb, peroxidase-labelled mAb and BALF sample was carried out simultaneously and analytical recovery and precision were assayed. Six mAb for SP-A and four mAb for SP-D were successfully cloned in limiting dilution to monoclonality. These mAb were reacted with equine SP-A or SP-D on Western blotting analysis. For SP-A, a combination of solid-phase TA08 and horseradish peroxidase (HRP)-conjugated WA28 was found to be more sensitive than other combinations, gave a good dose response and was capable of measuring 0.78 to 100 ng of protein/ml. For SP-D, a combination of solid-phase TD13 and HRP-conjugated WD19 was found to be more sensitive than other combinations, had a good dose response and was capable of measuring 0.78 to 200 ng of protein/ml. The assay was used to determine the effect of 41 hours of road transport on the concentrations of SP-A and SP-D in the BALF of 30 horses. The concentrations of SP-A and SP-D decreased by 55 per cent and 36 per cent, respectively, decreases similar to the decrease in phosphatidylglycerol concentration previously reported by the authors.     

Presumed lupus erythematosus cells identified in bronchoalveolar lavage fluid from a Mexican Hairless dog

A neutered male Mexican Hairless dog was presented for generalized weight loss and weakness. Initial laboratory testing and diagnostic imaging revealed thrombocytopenia and an interstitial to miliary lung pattern affecting all lung fields. Mild joint effusion was found on physical examination affecting the stifle, tarsal, carpal, and elbow joints. Examination of synovial fluid demonstrated an inflammatory polyarthropathy in 3 joints. Cytocentrifuged and direct preparations of the bronchoalveolar lavage (BAL) fluid sample were made and cells consistent with lupus erythematosus (LE) cells and ragocytes were found. Based on these findings, the anti‐nuclear antibody (ANA) titer was determined as 1:640. A clinical diagnosis of systemic LE was made based on the satisfaction of 2 major criteria (thrombocytopenia and inflammatory polyarthritis), 4 minor criteria (central nervous system signs, lymphadenopathy, fever of unknown origin, and pleuritis), positive ANA titer, and the identification of presumed LE cells in BAL fluid. This case report highlights a novel finding of LE cells in respiratory secretions and provides a review of diagnostic criteria of systemic LE.     

Effects of antitussive agents administered before bronchoalveolar lavage in horses

OBJECTIVE: To determine whether treatment of horses with antitussive agents before bronchoalveolar lavage (BAL) reduces the frequency and intensity of the cough reflex during BAL. ANIMALS: 8 healthy horses. PROCEDURE: Standard BAL was performed on each horse weekly for 6 weeks. Detomidine was used as a general sedative, and various antitussive agents were evaluated for their suitability to suppress undesirable coughing. Treatments administered prior to BAL consisted of saline (0.9% NaCl) solution (control treatment), codeine, butorphanol tartrate, glycopyrrolate, lidocaine hydrochloride (final concentration, 0.33%), and lidocaine hydrochloride at a final concentration of 0.66% (lidocaine 0.66%). Frequency and intensity of coughing were digitally recorded throughout the BAL procedure. The volume of BAL fluid collected was measured, and the fluid was cytologically examined to assess potential effects of the medications on composition. RESULTS: Coughing frequency was significantly reduced after intratracheal administration of lidocaine 0.66%. Moreover, intratracheal administration of lidocaine 0.66% or IV administration of butorphanol resulted in a significant reduction in the intensity of coughing episodes. All other treatments failed to significantly suppress coughing frequency and intensity, compared with results for the saline treatment. Glycopyrrolate caused obvious adverse clinical effects. Treatments did not influence the volume of BAL fluid collected nor composition of the fluid. CONCLUSIONS AND CLINICAL RELEVANCE: Intratracheal administration of lidocaine (final concentration, 0.66%) proved to be the most reliable method to reduce frequency and intensity of coughing in horses during BAL.