AbstractsBiology & Animal Science

Abstract

Background: The synovial membrane (SM) is part of every diarthrosis and potential hub in the pathogenesis of osteoarthritis (OA). Pro-inflammatory cytokines, e.g. tumor necrosis factor α (TNFα), which are detectable in osteoarthritic joints, might induce the synthesis of pro-inflammatory and catabolic mediators, e.g. interleukin 6 (IL-6), in synovial fibroblasts (SF), leading to a vicious circle that could accelerate cartilage degradation and chondrocyte apoptosis. The effects of intra-articular cytokines on SF are not completely understood. Hence, it was the aim of this dissertation to characterize the interplay between TNFα and the anti-inflammatory IL-10 in SF to decide whether IL-10 might modulate or block its catabolic effects. Methods: Primary human SF were isolated from knee joints of OA-affected donors, cultured, expanded and stimulated with TNFα, IL-10 or TNFα + IL-10 for 24 h. Gene expression of IL-6, IL-10, matrix metalloproteinases-1 & -3 (MMP-1; MMP-3) was investigated via RTD-PCR. Protein synthesis of the same mediators as well as type I collagen, CD44 and β_1 integrin was de-termined using flow cytometry (FC), immunofluorescence labeling (IF) and western blot (WB). IL-10 adenoviral transduction was performed to simulate a possible OA treatment. The transduction effects were measured using RTD-PCR and investigation of IL-10 protein secretion via an enzyme-linked immunosorbent assay (ELISA). Finally, the permanent K4IM cell line was established as a possible substitute for cultured human SF in future experiments. Results: Cultured SF were activated by TNFα and TNFα + IL-10, increasing their gene expression of IL-6, IL-10, MMP-1 & -3. Investigation of the protein synthesis via IF showed results consistent with RTD-PCR, while FC revealed distinct differences. WB demonstrated varied stimulation effects on the synthesis of extracellular matrix proteins and matrix receptors. The adenoviral transduction was successful. The gene expression of IL-10 was greatly elevated. Effects on the gene expression of IL-6, MMP-1 and MMP-3 were statistically not significant. Reactions in the K4IM cell line were largely congruent with those observed in cultured human SF. Conclusion: Human SF in culture are strongly activated by TNFα and TNFα + IL-10, increasing the gene expression and protein synthesis of pro-inflammatory and catabolic mediators. These findings illuminate the role of SF in the pathogenesis of OA, suggesting them as a potential future therapeutic target, even though IL-10 overexpression alone appears to be insufficient for an effective OA therapy. The K4IM cell line could be a suitable substitute for cultured human SF in future stimulation experiments. Hintergrund: Die Synovialmembran (SM) ist Bestandteil jeder Diarthrose und potentieller Knotenpunkt in der Arthrose-Pathogenese. Pro-inflammatorische Zytokine, insbesondere Tumornekrosefaktor α (TNFα), die in arthrotischen Gelenken in erhöhter Konzentration nachweisbar sind, könnten die Synthese zusätzlicher pro-inflammatorischer und kataboler…