Immunological and inflammatory characterisation of three canine cell lines: K1, K6 and DH82

Three canine cell lines, K1, K6 and DH82, derived from canine malignant neo plasms, were characterised. They were examined for expression of surface an tigens, cytokines, neuropeptide receptors, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The growth characteri stics of the cell lines were established and bioassays used to detect produ ction of TNF-alpha, IL-1 and IL-6. In the DH82 cell line, production of TNF -alpha and IL-6 was readily detected. Neither K1 or K6 cell lines produced any measurable amounts of TNF-alpha, IL-1 or IL-6. At a molecular level, us ing reverse transcription-polymerase chain reaction (RT-PCR) to detect spec ific mRNA, the DH82 cell line expressed TNF-alpha, IL-1 and IL-6, whereas t he K1 and K6 cell lines expressed TNF-alpha. Canine IL-5, IL-8 and IL-10 mR NA were detected in the DH82 cell line but only IL-5 and IL-8 mRNA were det ected in the K1 and K6 cell lines. Gelatin zymography was used for the dete ction of MMP-2 and MMP-9 and all three cell lines produced MMP-2 but only t he DH82 cell line produced MMP-9. Reverse zymography was used to detect TIM P-1 and TIMP-2 and all three cell lines produced both proteins. The presenc e of these MMPs and TIMPs was confirmed at a molecular level using RT-PCR. Canine MMP-14 mRNA was detected in all three cell lines. For this investiga tion several genes for canine inflammatory molecules were cloned and sequen ced for molecular detection; these included IL-1, IL-6, IL-8, TNF-alpha, MM P-9, MMP-14 TIMP-1, TIMP-2 and beta-actin. of all the cell surface antigens tested, only CD14 was expressed on the DH82 cell line although CD5 and CD4 5 was partially expressed. The K1 and K6 cell lines were negative for all o f the CD markers tested. K1 and K6 were negative for Neurokinin 1 receptor (NK1-R) but positive for Calcitonin gene related peptide receptor type 1 (C GRP-1R) and Calcitonin gene related peptide receptor component protein (CGR P-RCP). The DH82 cell line expressed neither NK1-R or CGRP-1R; however, it did express CGRP-RCP. Generally the DH82 cell line exhibited considerable s imilarity to canine monocytes, but all three cell lines will be useful as s tandards and for the purification of various immunological and inflammatory mediators in the dog. (C) 2000 Elsevier Science B.V. All rights reserved.