Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1β and TNFα expression profile

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 (TNFα). 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-1β, TNFα and their receptors in non-degenerate, degenerate and herniated human IVDs. IL-1β gene expression was observed in a greater proportion of IVDs than TNFα (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-1β gene expression (1,300 copies/100 ng cDNA) were observed compared to those of TNFα (250 copies of TNFα/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-1β 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 TNFα, and thus may be a better target for therapeutic intervention

The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

In this study, we investigated the hypotheses that in human intervertebral disc (IVD) degeneration there is local production of the cytokine IL-1, and that this locally produced cytokine can induce the cellular and matrix changes of IVD degeneration. Immunohistochemistry was used to localize five members of the IL-1 family (IL-1α, IL-1β, IL-1Ra (IL-1 receptor antagonist), IL-1RI (IL-1 receptor, type I), and ICE (IL-1β-converting enzyme)) in non-degenerate and degenerate human IVDs. In addition, cells derived from non-degenerate and degenerate human IVDs were challenged with IL-1 agonists and the response was investigated using real-time PCR for a number of matrix-degrading enzymes, matrix proteins, and members of the IL-1 family. This study has shown that native disc cells from non-degenerate and degenerate discs produced the IL-1 agonists, antagonist, the active receptor, and IL-1β-converting enzyme. In addition, immunopositivity for these proteins, with the exception of IL-1Ra, increased with severity of degeneration. We have also shown that IL-1 treatment of human IVD cells resulted in increased gene expression for the matrix-degrading enzymes (MMP 3 (matrix metalloproteinase 3), MMP 13 (matrix metalloproteinase 13), and ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs)) and a decrease in the gene expression for matrix genes (aggrecan, collagen II, collagen I, and SOX6). In conclusion we have shown that IL-1 is produced in the degenerate IVD. It is synthesized by native disc cells, and treatment of human disc cells with IL-1 induces an imbalance between catabolic and anabolic events, responses that represent the changes seen during disc degeneration. Therefore, inhibiting IL-1 could be an important therapeutic target for preventing and reversing disc degeneration

Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration

Current evidence implicates intervertebral disc degeneration as a major cause of low back pain, although its pathogenesis is poorly understood. Numerous characteristic features of disc degeneration mimic those seen during ageing but appear to occur at an accelerated rate. We hypothesised that this is due to accelerated cellular senescence, which causes fundamental changes in the ability of disc cells to maintain the intervertebral disc (IVD) matrix, thus leading to IVD degeneration. Cells isolated from non-degenerate and degenerate human tissue were assessed for mean telomere length, senescence-associated β-galactosidase (SA-β-gal), and replicative potential. Expression of P16INK4A (increased in cellular senescence) was also investigated in IVD tissue by means of immunohistochemistry. RNA from tissue and cultured cells was used for real-time polymerase chain reaction analysis for matrix metalloproteinase-13, ADAMTS 5 (a disintegrin and metalloprotease with thrombospondin motifs 5), and P16INK4A. Mean telomere length decreased with age in cells from non-degenerate tissue and also decreased with progressive stages of degeneration. In non-degenerate discs, there was an age-related increase in cellular expression of P16INK4A. Cells from degenerate discs (even from young patients) exhibited increased expression of P16INK4A, increased SA-β-gal staining, and a decrease in replicative potential. Importantly, there was a positive correlation between P16INK4A and matrix-degrading enzyme gene expression. Our findings indicate that disc cell senescence occurs in vivo and is accelerated in IVD degeneration. Furthermore, the senescent phenotype is associated with increased catabolism, implicating cellular senescence in the pathogenesis of IVD degeneration

Studies of human intervertebral disc cell function in a constrained in vitro tissue culture system

STUDY DESIGN : This is a laboratory-based study examining a novel in vitro culture system for intervertebral disc tissue. OBJECTIVES : Address the hypothesis that "the novel culture system will preserve intervertebral disc tissue matrix and cell function and prevent cellular apoptosis for periods up to 21 days." SUMMARY OF BACKGROUND DATA : Studies of cell function in human intervertebral disc tissue are scarce. In vivo study of human intervertebral disc cells remains impracticable; in situ molecular biology in histologic sections lacks a dynamic dimension; and as for in vitro studies, cell culture often lacks physiologic relevance and explant cultures are subject to loss of tissue integrity and altered cell behavior. There is a biologic and therapeutic need for a satisfactory explant culture system for studying human intervertebral disc tissue in a controlled environment. METHODS : Samples of human intervertebral disc tissue, obtained at surgery, were examined for a number of tissue and cell parameters immediately after excision (controls) and following culture of tissue samples either in a plastic ring or unconstrained in tissue culture medium for up to 3 weeks. Data were compared between cultured tissue and controls. RESULTS : By comparison with control tissue, unconstrained explants swelled, tissue structure was disturbed, and there were profound changes in cell function. By contrast, tissue cultured in plastic rings maintained tissue structure, and after 3 weeks, the cellular parameters were the same as in controls. CONCLUSIONS : This is the first reported system to preserve cell function of human discal explants for long periods in tissue culture. It will be a useful tool for a wide range of investigations of intervertebral disc biology that have not hitherto been possible.</p

Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc.

The histological and biochemical changes that occur in the extracellular matrix of the intervertebral disc (IVD) during ageing and degeneration have been investigated extensively. However, the mechanisms behind these changes are not fully understood. A number of studies have suggested the involvement of matrix metalloproteinases (MMPs) and ADAMTS in IVD degeneration, but few have localized the site of production of these enzymes to the cells of the degenerate disc. This study uses immunohistochemical techniques to localize and quantify the production of degrading enzymes (MMPs 1, 3, and 13, and ADAMTS 4) and their inhibitors (TIMPS 1, 2, and 3) within non-degenerate and degenerate discs of varying severity of degeneration. In all discs investigated, the cells that produced the enzymes and their inhibitors were the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus (AF), with little immunopositivity in the outer AF. Non-degenerate discs showed low numbers of cells expressing the degradative enzymes MMP 1 and ADAMTS 4, suggesting a role for these enzymes in normal homeostasis. No MMP 3 or MMP 13 immunopositivity was observed in non-degenerate discs. In degenerate discs, the number of cells immunopositive for MMPs 1, 3, 13 and ADAMTS 4 increased with the severity of degeneration. This increase in degrading enzymes was also accompanied by increases in the number of cells immunopositive for TIMPs 1 and 2 but not TIMP 3. This study highlights that although the expression of a number of MMPs increases with degeneration, this is accompanied by an increase in their inhibitors. However, the increase in the number of cells immunoreactive for ADAMTS 4 with increasing degeneration was not paralleled by a rise in its inhibitor TIMP 3. This finding indicates that the aggrecanases, rather then the MMPs, are a possible therapeutic target for the inhibition of disc degeneration

Correlation of senescent phenotype with expression of matrix-degrading enzymes

<p><b>Copyright information:</b></p><p>Taken from "Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration"</p><p>http://arthritis-research.com/content/9/3/R45</p><p>Arthritis Research & Therapy 2007;9(3):R45-R45.</p><p>Published online 11 May 2007</p><p>PMCID:PMC2206356.</p><p></p> Correlation of and gene expression in human intervertebral disc (IVD) cells. Spearman rank correlation < 0.05. Correlation of and gene expression in human IVD cells. Spearman rank correlation < 0.05. ADAMTS 5, a disintegrin and metalloprotease with thrombospondin motifs 5; MMP-13, matrix metalloproteinase-13

Absolute gene expression of IL-1β, tumour necrosis factor (TNF)α and their receptors in human intervertebral discs (IVDs)

<p><b>Copyright information:</b></p><p>Taken from "Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1β and TNFα expression profile"</p><p>http://arthritis-research.com/content/9/4/R77</p><p>Arthritis Research & Therapy 2007;9(4):R77-R77.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC2206382.</p><p></p> 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)

Number of cells displaying immunopositivity for IL-1β, tumour necrosis factor (TNF)α and their receptors in human human intervertebral discs

<p><b>Copyright information:</b></p><p>Taken from "Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1β and TNFα expression profile"</p><p>http://arthritis-research.com/content/9/4/R77</p><p>Arthritis Research & Therapy 2007;9(4):R77-R77.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC2206382.</p><p></p> The percentage of cells with immunopositivity is given for IL-1β, IL-1RI, TNFα and TNF RI in the nucleus pulposus, inner annulus fibrosus and outer annulus fibrosus of non-degenerate, degenerate and herniated discs (= 39). Data are presented as means ± 2 standard error (as a representative of 95% confidence interval). *< 0.05

Photomicrographs illustrating immunohistochemistry staining for IL-1β, tumour necrosis factor (TNF)α and their receptors in human intervertebral discs

<p><b>Copyright information:</b></p><p>Taken from "Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1β and TNFα expression profile"</p><p>http://arthritis-research.com/content/9/4/R77</p><p>Arthritis Research & Therapy 2007;9(4):R77-R77.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC2206382.</p><p></p> 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