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

Abstract

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.

Deep Coverage Tissue and Cellular Proteomics Revealed IL-1β Can Independently Induce the Secretion of TNF-Associated Proteins from Human Synoviocytes

“…Among these secretory proteins, CD55, also known as complement decay-accelerating factor, is a specific marker of HS (40). These results are comparable with another proteomics study by Melin et al (6) showing that IL-1a-stimulated bovine cartilage releases intensively more activated C3. In numerous proteomics studies on OA synovial fluid (41)(42)(43), complement components have been deemed primary factors to differentiate OA from the health.…”

“…It is known that synovitis in OA can be initiated by cartilage breakdown, with the subsequent phagocytosis of debris, and activation of synoviocytes, leading to the formation of an inflaming microenvironment and synovial fluid (5). Interestingly, Melin et al (6) recently found with a quantitative secretome analysis that the cartilage itself represents a major inducer of complement proteins upon IL-1a stimulation. Indeed, monocytes/macrophages and dendritic cells are the primary producers of both IL-1 and TNF in the OA joint, but they have a classical self-containing mechanism with NO-induced apoptosis and/or anergy (7).…”

Anti-Vascular Endothelial Growth Factor (VEGF) Antibody Ameliorates Cartilage Degradation in a Rat Model of Chronic Sports Arthritic Injury

“…Joint diseases are typically characterized by inflammatory processes that lead to pathological changes in the articular tissues, including cartilage degradation and the release of components into the synovial fluid [ 15 ]. A number of cytokines appear to induce the expression of VEGF, such as IL-1β, IL-6, prostaglandin E, epidermal growth factor (EGF), and TNF-α [ 16 – 20 ].…”

Advanced hydrogels for the repair of cartilage defects and regeneration

“…Till today, we are unable to achieve engineered cartilage as it is in the human body. Although cartilage has been investigated for many decades, new technologies such as cryo electron microscopy, solid-state NMR and mass spectrometry can be used to deepen our understanding of natural cartilage to reveal the exact components and molecular structure [ 151 , 152 ].…”