Quantitative Mass Spectrometry To Study Inflammatory Cartilage Degradation and Resulting Interactions with the Complement System.

Joint diseases are often characterized by inflammatory processes that result in pathological changes in joint tissues, including cartilage degradation and release of components into the synovial fluid. The complement system plays a central role in promoting the inflammation. Because several cartilage proteins are known to interact with complement, causing either activation or inhibition of the system, we aimed to investigate these interactions comprehensively. Bovine cartilage explants were cultured with IL-1α to induce cartilage degradation, followed by incubation with human serum. Label-free selected reaction monitoring mass spectrometry was used to specifically quantify complement proteins interacting with the cartilage explant. In parallel, the time-dependent degradation of cartilage was detected using mass spectrometry analysis (liquid chromatography-tandem mass spectrometry). Complement proteins resulting from activation of the classical, alternative, and terminal pathways were detected on IL-1α-stimulated cartilage at time points when clear alterations in extracellular matrix composition had occurred. Increased levels of the complement activation product C4d, as detected by ELISA in serum after incubation with IL-1α-stimulated cartilage, confirmed the selected reaction monitoring results indicating complement activation. Further, typical activated (cleaved) C3 fragments were detected by Western blotting in extracts of IL-1α-stimulated cartilage. No complement activation was triggered by cartilage cultured in the absence of IL-1α. Components released from IL-1α-stimulated cartilage during culture had an inhibitory effect on complement activation. These were released after a longer incubation period with IL-1α and may represent a feedback reaction to cartilage-triggered complement activation observed after a shorter incubation period.

The C-type lectin of the aggrecan G3 domain activates complement.

Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD) and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP) domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint.