**Background:** Disc degeneration (DD) is a major cause of lower back pain and is associated with structural changes, metabolic dysregulation, and enhanced production of matrix-degrading enzymes such as MMPs, ADAMTS, and cathepsin D. Previous work identified terminal complement complex (TCC) deposition in human degenerated discs, with TCC levels positively correlating with degeneration grade. In osteoarthritis, complement activation plays a crucial role in pathogenesis through anaphylatoxin generation and TCC deposition, which induce catabolic enzyme expression and inflammatory responses. This study aimed to investigate the cellular consequences of terminal complement activation in disc degeneration and examine the inhibitory effects of complement blockade at the C3 and C5 levels.
**Methods:** IVD tissue biopsies were obtained from 26 patients (16 women/10 men, 63.5 ± 19.5 years old) with DD undergoing lumbar surgery. Tissue was macroscopically separated into annulus fibrosus (AF), nucleus pulposus (NP), and endplate (EP), and cells were enzymatically isolated and expanded to passages 2-4. Cells were stimulated in IVD medium with 10% human serum alone or with 100 µg/mL zymosan (alternative pathway activator) or 0.5 µg/mL cathepsin D, and treated with 5 µM Cp40 (C3 inhibitor) or 1 µM eculizumab (C5 antibody). TCC deposition was quantified via cell-based ELISA after 2 h stimulation. Gene expression of ADAMTS4, MMP1, and COX2 was analyzed by RT-qPCR after 24 h. C3a and C5a concentrations in supernatants were measured by ELISA. MMP1 and PGE2 protein release were quantified by ELISA. For C5 cleavage assays, AF cells were incubated with 20 µg/mL C5 protein under serum-free conditions with or without TNF (10 ng/mL), IL-1β (10 ng/mL), CTSD (0.5 µg/mL), or EP-conditioned medium (pooled from 3 donors). Immunohistochemical staining of TCC and CTSD was performed on paraffin sections of IVD tissue.
**Key Results:** Zymosan significantly increased relative TCC deposition over fourfold compared to 10% HS alone (AF: p < 0.05; NP: p < 0.0001; EP: p < 0.01). Cp40 reduced TCC deposition on AF cells by 40% (p < 0.0001) and EP cells by 30% (p < 0.001). Eculizumab significantly reduced zymosan-mediated TCC deposition to the level of HS alone on all cell types (AF: p < 0.0001; NP: p < 0.05; EP: p < 0.001). C3a generation was increased ~3-fold with zymosan (p < 0.0001), and C5a generation was increased >8-fold (p < 0.0001). Cp40 decreased C3a by 76% (p < 0.0001) and C5a by 95% (p < 0.0001). Eculizumab inhibited C5a generation by 99% (p < 0.0001) but did not affect C3a. Zymosan significantly induced gene expression of ADAMTS4, MMP1, and COX2 compared to HS alone. Eculizumab significantly reduced this induction for ADAMTS4 (p < 0.01), MMP1 (p < 0.01), and COX2 (p < 0.05). Cp40 suppressed ADAMTS4 gene expression (p < 0.01). MMP1 protein release was increased by zymosan (p < 0.001) and reduced by eculizumab (−60%, p < 0.01). PGE2 release was increased ~7-fold by zymosan (p < 0.0001), with Cp40 and eculizumab each reducing it by ~20% (not significant). Direct C5 cleavage was not detected in AF cells alone or with IL-1β or TNF stimulation. However, CTSD significantly enhanced C5a levels (p < 0.01), and EPCM increased C5a generation threefold (p < 0.0001). Immunohistochemical staining showed that tissue samples with strong TCC deposition had higher levels of CTSD-positive cells, while macroscopically non-degenerated controls showed low levels of both.
**Clinical Implications:** This study provides the first evidence that terminal complement activation induces catabolic enzyme expression (ADAMTS4, MMP1, COX2) in human AF cells, suggesting a functional role for complement in disc degeneration progression. The finding that soluble factors from degenerated endplate tissue, including cathepsin D, can directly cleave C5 and activate the terminal complement cascade identifies a potential mechanism for local complement activation in the degenerated disc environment. Complement inhibition at the C5 level with eculizumab effectively reduced catabolic responses, while C3 inhibition with Cp40 showed more limited effects on target gene expression but may have additional therapeutic relevance through C3a-mediated immune cell recruitment. These results suggest that targeting the terminal complement pathway could represent a novel therapeutic approach for attenuating disc degeneration, though further studies are needed to differentiate between C5a- and TCC-mediated effects and to investigate in vivo relevance.