Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNFalpha expression profile

Abstract

Low back pain is a common and debilitating disorder. Current evidence implicates intervertebral disc (IVD) degeneration and herniation as major causes, although the pathogenesis is poorly understood. While several cytokines have been implicated in the process of IVD degeneration and herniation, investigations have predominately focused on Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFalpha). However, to date no studies have investigated the expression of these cytokines simultaneously in IVD degeneration or herniation, or determined which may be the predominant cytokine associated with these disease states. Using quantitative real time PCR and immunohistochemistry we investigated gene and protein expression for IL-1beta, TNFalpha and their receptors in non-degenerate, degenerate and herniated human IVDs. IL-1beta gene expression was observed in a greater proportion of IVDs than TNFalpha (79% versus 59%). Degenerate and herniated IVDs displayed higher levels of both cytokines than non-degenerate IVDs, although in degenerate IVDs higher levels of IL-1beta gene expression (1,300 copies/100 ng cDNA) were observed compared to those of TNFalpha (250 copies of TNFalpha/100 ng cDNA). Degenerate IVDs showed ten-fold higher IL-1 receptor gene expression compared to non-degenerate IVDs. In addition, 80% of degenerate IVD cells displayed IL-1 receptor immunopositivity compared to only 30% of cells in non-degenerate IVDs. However, no increase in TNF receptor I gene or protein expression was observed in degenerate or herniated IVDs compared to non-degenerate IVDs. We have demonstrated that although both cytokines are produced by human IVD cells, IL-1beta is expressed at higher levels and in more IVDs, particularly in more degenerate IVDs (grades 4 to 12). Importantly, this study has highlighted an increase in gene and protein production for the IL-1 receptor type I but not the TNF receptor type I in degenerate IVDs. The data thus suggest that although both cytokines may be involved in the pathogenesis of IVD degeneration, IL-1 may have a more significant role than TNFalpha, and thus may be a better target for therapeutic intervention.

Figure 1

Absolute gene expression of IL-1β, tumour necrosis factor (TNF)α and their receptors in human intervertebral discs (IVDs). The percentage of disc samples displaying gene expression for the target genes and the copy number/100 ng cDNA expressed within positive samples are given and data is represented as a box and whisker plot. (* = P < 0.05).

Figure 2

Photomicrographs illustrating immunohistochemistry staining for IL-1β, tumour necrosis factor (TNF)α and their receptors in human intervertebral discs. Results for non-degenerate discs (grade 1) are shown in A1 to E1 and result for degenerate discs (grade 12) are shown in A2 to E2: IL-1β (A); IL-1RI (B); TNFα (C); TNF RI (D); IgG controls (E) were all negative. Immunopositivity shows as brown staining. Bars = 570 μm.

Figure 3

Number of cells displaying immunopositivity for IL-1β, tumour necrosis factor (TNF)α and their receptors in human human intervertebral discs. The percentage of cells with immunopositivity is given for IL-1β, IL-1RI, TNFα and TNF RI in the (a) nucleus pulposus, (b) inner annulus fibrosus and (c) outer annulus fibrosus of non-degenerate, degenerate and herniated discs (n = 39). Data are presented as means ± 2 standard error (as a representative of 95% confidence interval). *P < 0.05.