gms | German Medical Science

Objectives: The complement system is activated by cartilage trauma, leading to the generation of anaphylatoxins C3a and C5a by cleavage of C3 and C5. This might contribute to the progression of posttraumatic osteoarthritis (PTOA). The anaphylatoxin receptors C3aR and C5aR1 were found to be involved in osteogenic differentiation (OD) of mesenchymal stem cells. To elucidate the role of anaphylatoxins in OA pathogenesis, we investigated the ability of human articular chondrocytes (hAC) to cleave C5 under pathophysiologic conditions, as well as the link between C3aR and C5aR1 expression and cartilage calcification.

Methods: Macroscopically intact (OARSI ≤1) and highly degenerated (OARSI ≥ 3) human cartilage was obtained from donors undergoing knee joint replacement (n=28). Cartilage was either used for qPCR or histological analysis (IHC or Alizarin Red S staining). Intact cartilage was used to isolate hAC. For the cleavage assay, C5 was incubated for 4 hours w/ or w/o hAC and in presence or absence of cartilage homogenizate (HG). C5a was assessed by ELISA. C3aR and C5aR1 expression of hAC was analysed by flow cytometry and IHC. For OD, hAC were cultured for 21 days in differentiation medium. Alizarin Red S staining was applied to assess matrix mineralisation. Statistical analysis: Student's t-test and one-way ANOVA.

Results and conclusion: We found by C5a ELISA that hAC can cleave C5 (P=0.0437), which was further enhanced by addition of HG (+26%; P=0.055). Gene expression of C3 and C5 did not differ between intact and degenerated cartilage. However, both anaphylatoxin receptors were significantly higher expressed in degenerated cartilage. On mRNA level, C3aR was upregulated by 4-fold (P=0.043) and C5aR1 by 4.1-fold (P=0.044). In addition, IHC staining confirmed that the number of C3aR- and C5aR1-positive cells was increased by 73% (P=0.0006) and 31% (P=0.0004), respectively, as compared to intact tissue. We could also show that the protein expression of C3aR (r=0.68; P=0.04) and C5aR1 (r=0.55; P=0.103) was positively associated with the calcification of cartilage, examined by Alizarin Red S staining. In line with this, the expression of C3aR (r=0.61; P=0.063) and C5aR1 (r=0.71; P=0.021) on hAC correlated positively with the Alizarin Red S staining intensity after in vitro OD.

Our study demonstrated that HG amplified C5a generation by hAC, suggesting that a posttraumatic environment (e.g., extracellular matrix debris) may contribute to the complement activation. We could also draw a connection between the enhanced expression of anaphylatoxin receptors in degenerated cartilage and calcification. Considering our previous results showing increased calcification during OD of hAC in presence of C5a, we conclude that both amplified C5a generation after trauma and increased expression of C3aR and C5aR1 might contribute to cartilage calcification. Targeting anaphylatoxin generation and receptors might be a promising strategy to reduce cartilage degeneration due to calcification during OA progression.