gms | German Medical Science

Objectives: The formation of the terminal complement complex (TCC), a complement system activation product that acts as an inflammatory trigger and induces cell lysis, was previously identified in degenerated disc tissues and it was shown to correlate with the degree of degeneration (Teixeira et al. Eur Spine J. 2020). However, it is unclear which molecular factors play a role in complement activation during disc degeneration (DD). Therefore, here we have investigated possible triggers of TCC formation in the context of DD.

Methods: Disc tissue biopsies were collected from adolescent idiopathic scoliosis (AIS, n=8, age 16±3) and DD (n=11, age 56±15) patients with ethical approval and informed consent. Standardized tissue punches from nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) were separately cultured. Isolated cells were analyzed by flow cytometry and gene expression. Cells and tissues were stimulated with medium containing 5% human serum alone or supplemented with interleukin-1β (IL-1β, 10 ng/mL), cathepsin-D (0.5 μg/mL) or zymosan (100 μg/mL). Serum-free medium cultures were used as control. In cell cultures, TCC deposition on the cell membrane and soluble CC formation were determined by ELISA. In tissues cultures, TCC and CD59 formation were analyzed by immunohistochemistry. Statistics: Kruskal-Wallis in cell cultures, one-way ANOVA in explant cultures.

Results and Conclusion: In isolated cells, the expression of CD46, CD55 and CD59 significantly increased with culture (p<0.05). However, no differences were found in response to proinflammatory/degenerative stimuli, neither for scoliosis nor DD patients. In tissue cultures, compared to non-treated tissues, IL-1β stimulation led to lower percentage of TCC+ cells in AF and EP (p<0.05), whereas the presence of cathepsin-D significantly increased TCC formation in NP (p<0.01). The percentage of CD59+ cells significantly increased in AF and NP after stimulation with cathepsin-D and zymosan (p<0.05).

Overall, the data suggest that complement activation and TCC formation can be induced in vitro as long as disc cells are kept in their native tissue environment. Interestingly, the presence of IL-1β, a pro-inflammatory molecule, led to less TCC formation in AF and EP. Although strong TCC deposition may be a degeneration-associated event, proinflammatory conditions may influence cell lysis or TCC sublytic effects via a negative feedback mechanism. Nevertheless, TCC formation was shown to be triggered by cathepsin-D, an important player in osteoarthritis, but poorly described in DD. Moreover, the effect of TCC in sublytic concentrations is being investigated in the context of IVD degeneration. Although further mechanistic studies are ongoing, overall, these results suggest a functional relevance of IL-1β and cathepsin-D in modulating TCC formation, suggesting that TCC may be a possible new target for DD therapy.

Acknowledgment: Funded by the German Research Foundation (DFG, NE_549/6-1, BR_919/12-1).