Flow cytometric analysis of equine bronchoalveolar lavage fluid cells in horses with and without severe equine asthma

Abstract

Severe equine asthma (SEA) is a common, debilitating lower airway inflammatory disorder of older horses. Alveolar macrophages (AMs) survey inhaled particulates from barn sources causing them to switch from an anti-inflammatory to a proinflammatory phenotype, resulting in neutrophil recruitment to the lung. This proinflammatory switch may contribute to the development and prolongation of SEA. Validated antibodies to identify the cells involved in the pathogenesis of SEA are lacking. In this study, monoclonal antibodies against CD90, CD163, and CD206 were tested for reactivity with equine leukocytes by immunocytochemistry and flow cytometry. A multi-color flow cytometric assay was developed to identify leukocytes in equine bronchoalveolar lavage fluid (BALF). Four control and 4 SEA-susceptible horses had BALF collected before and after a 48-hour moldy hay challenge. Antibodies against CD90 uniquely labeled equine neutrophils, and antibodies against CD163 and CD206 identified equine macrophages. Postchallenge AM surface expression of CD163 increased in both groups of horses, but the increase was statistically significant in only the SEA-susceptible group ( P = .02). The surface expression of CD206 on AMs increased significantly in the SEA-susceptible group ( P = .03) but was unchanged in the control group ( P = .5). Increased expression of CD163 and CD206 during exacerbation of SEA suggested an association between AM phenotype and lung inflammation. However, functions of AMs in the pathogenesis of SEA remain to be elucidated.

Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences

“…The phenotypes of BAL Mph and PBMC monocytes were further characterized according to their expression of the classical monocyte/Mph marker CD14 (lipopolysaccharide co-receptor ( 17 , 18 ),), and CD16 (FcγRIIIa), which has been described and used as a non-classical monocyte marker ( 12 , 17 ). Staining of mannose receptor CD206 and scavenger receptor CD163, other markers of equine BAL Mph ( 11 , 30 ), was performed on some BAL samples and the majority of the putative Mph (70-90% of DH24A - NL) were positive for both, CD14 and CD206 or CD14 and CD163 (data not shown). Due to less interference with Mph autofluorescence in FITC and PE (compare ( 11 )) with the available conjugates for CD14 antibodies compared to CD206 or CD163, and the indication of regulation of the latter two surface molecules ( 11 ), we selected CD14 as the main marker for Mph identification and quantification in our analyses (compare Figures 2C, E ).…”

“… a used for ex vivo analysis; b used for in vitro analysis, c conjugated in-house according to the manufacturer’s instructions, d ms mouse, f the DH24A antibody binds CD90 in dogs, but it is unclear if CD90 is also the target on equine granulocytes labelled by DH24A ( 11 , 16 ), g the polyclonal anti-horse IgG (H+L) antibody binds all equine immunoglobulin isotypes by the heavy (H) or light chains (L); Staining with this antibody is labelled ‘Ig’ in the results. DyL: DyLight™, AF: AlexaFluor™.…”

“… f the DH24A antibody binds CD90 in dogs, but it is unclear if CD90 is also the target on equine granulocytes labelled by DH24A ( 11 , 16 ), …”

“…For the investigation of immune responses in the lung, the analysis of cells from that compartment is most beneficial and has been utilized in various approaches using equine BALF samples ( 5 – 7 , 9 , 11 ). However, the mixed leukocyte population in BALF and the established differences between healthy and asthmatic horses hamper exact interpretation in many bulk-analysis-based approaches.…”

“…However, the mixed leukocyte population in BALF and the established differences between healthy and asthmatic horses hamper exact interpretation in many bulk-analysis-based approaches. Accordingly, single-cell-based approaches are more informative and have been performed in more recent studies after the improved availability of new methodology and the development or confirmation of equine-specific antibodies ( 11 – 16 ). Recent reports established several leukocyte phenotype markers for equine BAL cells and PBMC, allowing for in-depth phenotypic characterization of equine leukocytes to elucidate the composition of mixed cell populations ( 11 , 12 , 16 – 18 ).…”