Regulation of Neurotrophic Factor Expression in Nerve Cells and Intervertebral Disk Cells by Inflammatory Cytokines, Implications for Pain Pathways

Abstract

Introduction Intervertebral disk (IVD) degeneration and prolapse contribute significantly to low back pain and sciatica cases. Within the IVD, the nucleus pulposus cells are known to produce a number of catabolic cytokines which have been shown to elicit a wide range of effects including increased production of degradation enzymes and decreased production of matrix molecules. Here, we investigate the hypothesis that cytokines induce neurotrophic and angiogenic factors in nucleus pulposus cells and nerve cells and induce neurite outgrowth within nerve cells. Materials and Methods Human NP cells were isolated via collagenase digestion from human IVD obtained from surgical samples. Following expansion in monolayer culture, cells were transferred to alginate bead cultures at P2 and maintained for 2 weeks before stimulation with cytokines to enable re-differentiation to take place. Cells were then stimulated with IL-1 (0.001 to 100 ng/mL), IL-6 (1 to 100 ng/mL), or IL-8 (1 to 100 ng/mL) for 48 hours, RNA extracted and real time PCR performed to investigate expression of nucleus growth factor (NGF), BDNF, NTF3, and VEGF together with the housekeeping genes GAPDH and 18 s. In addition, SHSY-5Y nerve cell line were cultured in the presence of retinoic acid for 7 days before stimulation with IL-1, IL-6, IL-8, or TNF (1 to 100 ng/mL) to investigate the gene expression of NGF, BDNF, and NTF3 together with effects on neurite outgrowth. Results Human NP cells showed a biphasic response to IL-1 in terms of NGF expression with expression decreased at low doses of IL-1 (0.001–0.01 ng/mL) but an increase in NGF expression at high IL-1 doses (1 ng/mL–100 ng/mL) (Fig. 1). A Small but nonsignificant increase in NTF3 was seen following IL-1 stimulation, whereas IL-6 treatment of NP cells showed a 10-fold increase in NTF 3 expression in some patient samples. Within SH-SY5Y cells interestingly, NGF and BDNF expression was not observed but NTF-3 was downregulated by IL-1 and IL-8 treatments and stimulated by IL-6 stimulation. TNF and IL-1 appeared to induce the greatest effects on neurite outgrowth and number of neurites per cell, both of which increased at doses above 1 ng/mL stimulation. VEGF was also upregulated in human NP cells following IL-1 stimulation even at low doses. Conclusion Here, we demonstrate that a number of cytokines which are upregulated during human disk degeneration and prolapse induce a number of neurotrophic factors and angiogenic factors within human NP cells in 3D cultures. A number of cytokines also induce neurotrophic factor expression and neurite outgrowth in a nerve cell line suggesting a role for these cytokines both within the nerve ingrowth seen in an intact disk and intact disks and following prolapse. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared