gms | German Medical Science

Objectives: Formation of terminal complement complex (TCC), an activation product of the complement system that triggers inflammation and induces cell lysis, has been identified in human intervertebral discs from patients with disc degeneration (DD) and was shown to positively correlate with the grade of degeneration [1], (Teixeira et al. Eur Spine J. 2019). This indicates that the TCC might play a role in disc degeneration and complement inhibition might be a new therapeutic approach for DD. Therefore, the effect of the TCC inhibitor eculizumab, a monoclonal antibody against complement factor C5 that is already in clinical use in other complement-mediated diseases, was investigated in vitro in human intervertebral disc cell cultures.

Methods: Anulus fibrosus (AF), nucleus pulposus (NP) and endplate (EP) cells were enzymatically isolated from human disc tissue biopsies from DD and scoliosis patients (DD: n=6, age 52±9; scoliosis: n=5, age 16.5±3.5) and cultured separately. Cells were stimulated with 10% human serum (HS) with and without addition of zymosan (Zym; 100 µg/mL) and eculizumab (Ecu; 1 µM). Cells incubated with serum-free medium served as control. After 2 h, TCC deposition on disc cells was analyzed via a cell-based ELISA. For gene expression analysis, AF cells were stimulated accordingly for 24 h. Further, standardized tissue explants of AF, NP and EP were stimulated with 30% HS alone or supplemented as described above. Subsequently TCC deposition was analyzed by immunohistochemistry. Statistics: one-way ANOVA.

Results and Conclusion: Addition of 100 µg/mL zymosan, known to activate the alternative complement pathway, significantly increased TCC deposition on disc cells in comparison to 10% HS alone in median (AF: 2.5-fold, P<0.0001; NP: 1.7-fold, P=0.0271; EP: 1.7-fold, P=0.0311). Supplementation of 1 µM eculizumab completely abolished zymosan-mediated increase of TCC deposition ([10% HS + Zym vs. 10% HS + Zym + Ecu]: AF: 0.5-fold, P=0.0229; NP: 0.7-fold, P=0.0146; EP: 0.5-fold, P=0.0279). First investigations in 3D tissue culture could confirm these observations by immunohistochemical staining of TCC. Gene expression analyses of AF cells indicate a zymosan-mediated upregulation of MMP1 (24-fold) and MMP3 (5-fold). This effect could be partially inhibited by eculizumab for MMP1 (by 70% of the median, P=0,0942) but not for MMP3 (3-fold upregulation relative to control).

This preliminary in vitro data shows effective inhibition of TCC deposition on disc cells and reduction of TCC-induced MMP1 expression by eculizumab. Using intervertebral disc cells and tissue cultures, the impact of eculizumab on TCC-mediated inflammatory response and other molecules involved in matrix turnover will be further addressed to study the therapeutic potential.

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