Epsilon Archive for Student Projects

Abstract

Inflammatory airway disease (IAD) is a respiratory disorder that most commonly affects young horses performing at a high level. The clinical signs are often mild but the disorder can cause a marked decrease in performance to a point where the horse has to be taken of training and competing. The aetiology underlying IAD is not yet fully understood. One current theory is that a type I-hypersensitivity reaction due to allergens in the environment is the cause in some horses. The most reliable diagnostic method is a bronchoalveolar lavage (BAL) since it reflects the inflammatory status in the most distal parts of the lungs, which are affected in IAD. Elevated mast cells in the BAL are thus interpreted as reflecting a type I pulmonary hypersensitivity. However, the normal values for mast cell numbers in BAL can vary between laboratory and cell counting methodology.
The first aim of the study is to examine how factors such as the number of cells counted during the cytological evaluation of the BAL-fluid as well as between reader variation affect the final result of the BAL-analysis. For this purpose, BAL cytology slides from 12 horses taken on three occasions were assessed independently by three readers; AE, JP and IL, and intrareader as well as interreader comparisons for all major cell types were made. The results show a marked between reader variation, especially in the evaluation of lymphocytes, macrophages and mast cells where as the evaluation of eosinophils and neutrophils appeared to be more consistent between readers. The number of cells counted during the cytological evaluation of the BAL-fluid for the same reader did not seem to significantly affect the result of the BAL-analysis.
The accompanying aim of this study was, after examination of the repeatability and comparability of assessment of BAL mast cells, to examine the relationship between an increase in mast cells and selected mRNA gene products of proinflammatory cytokines, interleukins (IL)-4 and IL-5 of the Th2 or allergic reaction, and IL-6 in its association with inflammation. Defining such relationships can help to clarify whether the aetiology behind IAD where BAL mast cells are elevated has cytokine changes associated with the type I-hypersensitivity reaction or whether the elevation is associated with a more general inflammatory process where upregulation of IL-6 would be expected. Examination and BAL were performed on 12 horses on three different occasions. Using BAL cell counts from these original assessments, individual horses that had sampling periods with low percent mast cells as well as sampling periods where mast cells were considered high, being at least a two fold increase over the low sampling episode were selected. This resulted in seven horses for which results of real time-PCR mRNA to IL-4, IL-5 and IL-6 from their BAL fluid were compared between sampling episodes of low versus high mast cells. While there was no relationship between an increased level of mast cells and altered regulation of mRNA of IL-4 or IL-5 there was a significant (p=0,022) downregulation of IL-6 mRNA in association with increased BAL mast cell percentage. The horses in this selected group concomitantly had significantly increased neutrophils (p=0,043) when mast cells were low, which was the most probable reason for the unexpected changes to mRNA to IL-6. Nonetheless, the study failed to support the role of mast cells as contributing to the inflammation seen in horses with IAD.