Antibodies specific for human or murine Toll-like receptors detect canine leukocytes by flow cytometry

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns on microbes, and ligand recognition results in production of pro-inflammatory cytokines, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. The purpose of this study was to test antibodies specific for human or murine TLR2, 3, 4, 5 and 9 for their use in dogs. Twenty-two antibodies were assessed for recognition of canine monocytes (Mo), granulocytes (Gr) or lymphocytes (Ly) by flow cytometry, and results were compared with isotype-matched controls and other antibodies against the same TLR.Nine monoclonal antibodies detected canine leukocyte subpopulations. Antibodies TL2.1 and TL2.3 (specific for human TLR2), HTA125 (specific for human TLR4), as well as 85B152.5 and 19D759.2 (specific for human TLR5) recognized Mo and Gr but not Ly without permeabilization, and putatively cross-react with canine TLR2, 4 and 5, respectively. Antibodies 40C1285.6 and TLR3.7 (specific for human TLR3) as well as 26C593.2 and 5G5 (specific for human and murine TLR9) recognized Mo, Gr and Ly after their permeabilization and putatively cross-react with canine TLR3 and 9, respectively, inside the cell. None of these nine antibodies recognized paraformaldehyde-treated (4%) canine leukocytes but all except 40C1285.6, TLR3.7 and 5G5 recognized methanol-fixed cells, suggesting that they might be useful also in immunohistochemistry.