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Osteoarthritis (OA) is an imperative ailment in humans as well as in veterinary species especially dogs. Dogs constitute a good biological system to study human diseases because they suffer many diseases analogous to that of humans including OA. Chondrocytes, being the sole cell type in cartilage, are the solitary target of cytokines and other mediators involved in pathogenesis of OA. Among these cytokines, IL-1β and TNFα are two master pro-inflammatory cytokines whereas IL-4 is a major player with anti-inflammatory properties. Taking into consideration the chondrocyte biology and the cytokine hierarchy in OA, the present study was aimed at characterization of chondrocytes to develop a model of inflammatory arthritis where the anti-inflammatory capacity of IL-4 was to be monitored.
Isolated chondrocytes were grown in monolayer (2D) and in alginate-based 3D culture systems. Several morphological, biochemical, and functional features of chondrocytes were examined by immunocytochemistry, western blot and quantitative real time PCR. Attempts were made to generate a chondrocyte cell line by using human telomerase reverse transcriptase. Canine recombinant cytokines (IL-1β and TNFα) were cloned and their purified proteins used as exogenous stimulants in chondrocyte cultures. The expression of typical inflammation markers such as pro-inflammatory cytokines (IL-1β, IL-6, IL-8, GM-CSF and TNFα), enzyme mediators (MMP-3, MMP-13, iNOS, COX-2) and their catabolites (NO, PGE2) was measured. IL-4 was transfected in chondrocytes in both culture systems followed by stimulation with IL-1β and TNFα to study its anti-inflammatory activity. IL-4 protein was detected by Western blot, and an ELISA system was developed for its quantification.
Results show that chondrocyte rapidly lose their characteristic phenotype in serial monolayer culture. This de-differentiation could be reverted by encapsulation of cells in alginate beads. Immortalized chondrocytes were able to produce chondrocyte specific markers in 3D culture but further studies were hampered due to their non-responsiveness to cytokines. Cytokine stimulation of chondrocytes resulted in high expression of all inflammatory markers. Western blot results showed IL-4 as a 17 kDa protein in transduced chondrocytes. The detection level for IL-4 as measured by ELISA was found to be about 3 ng ml-1. The IL-4 expressed in chondrocytes was found to be biologically active since it was capable of inhibiting the selected inflammatory markers in the transduced chondrocytes. Since high expression of STAT6 (signal transducer and activator of transcription 6) was seen almost exclusively in cells expressing IL-4, it is therefore assumed that IL-4 exerts its anti-inflammatory effects through STAT6 signaling. The canine form of STAT6 was identified and partially cloned and sequenced. Avenues are open to further characterize this signal molecule.
These findings altogether provide a solid foundation to validate constructs with cytokine-responsive “intelligent” promoters, which were developed in this laboratory alongside experimental work for this thesis. Thus, using the 3D culture systems described here it was shown that only in the presence of inflammation, the expression of IL-4 is driven in a sophisticated and fine-tuned manner. Thus, combining the above 3D culture system with the application of regulatable delivery of transgenes such as IL-4 or other promising candidate genes will facilitate further development and testing of novel gene therapy strategies against OA.