Human IVD tissue was collected from patients undergoing lumbar spinal surgery for DDD or from cadavers (within 18 hours of death) with the consent of the patients or their relatives and the approval of the Central Manchester, Bury, Rochdale, Salford and Trafford Research ethics committees. Tissue was processed for cell extraction, and representative samples of all tissues containing intact AF and NP regions were formalin-fixed and paraffin-embedded, and sections were histologically graded as previously reported 41 . Graded tissue was given a score between 0 and 12, with 0 to 3 being classified as nondegenerative, 4 to 7 being classified as mildly degenerative and 8 to 12 being classified as severely degenerative. Nondegenerative IVDs were collected from three cadavers (mean donor age, 47 years; age range, 37 to 57 years), and histologically degenerative IVDs were collected from two patients who had undergone surgery for DDD and from one cadaver (mean donor age, 50 years; age range, 29 to 66 years) (see Table 1 for details).

Paraffin-embedded AF and NP tissue obtained from a cohort of nine individuals, including patient and postmortem samples (mean age, 54 years; age range, 34 to 79 years) (Table 2), were processed for immunohistochemical analysis as previously reported 50 51 , with articular cartilage tissue used as a positive control. Briefly, mounted sections were dewaxed in xylene and treated with trypsin (Invitrogen, Paisley, UK) for antigen retrieval, and endogenous peroxidase activity was blocked by using hydrogen peroxide (Fisher Scientific UK Ltd, Loughborough, UK). Samples were blocked with either 10% wt/vol rabbit serum with bovine serum albumin (BSA) (Sigma, Poole, UK) or 10% wt/vol donkey serum with BSA (Sigma, Poole, UK) and incubated at room temperature for 1 hour with primary antibodies for IL-4 receptor α (IL-4Rα) (1:300 dilution, catalogue no. MAB230; R&D Systems, Abingdon, UK), IL-2 receptor-γ (IL-2Rγ) (1:10 dilution, catalogue no. MAB284; R&D Systems, Abingdon, UK) or IL-13 receptor α₁ (IL-13Rα₁) (1:25 dilution, catalogue no. AF152; R&D Systems, Abingdon, UK). After being washed with Tris-buffered saline, samples were incubated at room temperature for 1 hour with biotinylated secondary antibodies (1:400 dilution of either rabbit anti-mouse, catalogue no. E0464; Dako, Ely, UK; or donkey anti-goat, catalogue no. SC-2042; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and their binding was visualised using the streptavidin-biotin complex (Dako, Ely, UK) technique with 3,3'-diaminobenzidine tetrahydrochloride solution (Sigma, Poole, UK). Sections were counterstained with haematoxylin (Surgipath Europe Ltd, Peterborough, UK).

AF tissue was separated from the IVD within 24 hours of death or surgical removal and finely minced prior to enzymatic digestion as previously reported 18 . AF cells were cultured in standard medium (Dulbecco's modified Eagle's medium with 4.5 g/L glucose, GlutaMAX™ and pyruvate (Gibco, Invitrogen, Paisley, UK) containing 50 μg/mL ascorbic acid, 250 ng/mL amphotericin, 100 U/mL penicillin, 100 μg/mL streptomycin (Invitrogen) and 10% foetal calf serum (Invitrogen, Paisley, UK) and expanded in monolayers with medium changes every 2 or 3 days. Subconfluent AF cells with passage numbers ≤6 were trypsinised (Invitrogen, Paisley, UK), seeded onto untreated silicone membrane BioFlex culture plates (Flexcell International, Hillsborough, NC, USA) at a density of 1 × 10⁵ cells/mL in 2 mL of standard medium and allowed to adhere for 48 hours (passage numbers >6 have been found to influence cell behaviour (J.A. Hoyland, unpublished data)). Media were changed to serum-free media 15 to 17 hours prior to the application of CTS.

AF cells in serum-free media adhered to Bioflex culture plates were treated with or without the IL-1 receptor antagonist (IL-1Ra) (0.1 μg/mL) (catalogue no. 280-RA; R&D Systems) or a blocking antibody to the IL-4 receptor (IL-4RAb) (10 μg/mL) (catalogue no. MAB230; R&D Systems) 10 minutes prior to the application of CTS.

Using the Flexcell Tension™ FX-4000™ System, a CTS previously shown to alter AF cell function consisting of a 10% strain at 1.0-Hz frequency for 20 minutes was delivered to the base of the silicone membranes within the Bioflex culture plates, and consequently to the AF cells that adhered to these membranes, using computer-controlled negative pressure as previously described in detail 18 . The loading regime was chosen to be within the physiological range, with IVD tissue strains estimated to range from 1% to 25% during complex motions while compressed with a load physiologically similar to walking 52 53 and 1.0-Hz estimated to be the frequency of locomotion 54 . Unstimulated AF cells that adhered to Bioflex plates served as controls. Mechanically stimulated or unstimulated cytokine inhibitor-treated and untreated AF cells were incubated at 37°C with 5% CO₂ for either 0 (baseline control), 1 hour (nondegenerative AF cells) or 24 hours (degenerative AF cells) postload, and total RNA was extracted. The time points for mRNA analysis were chosen on the basis of previously published observations. Of the time points investigated (0 (baseline control), 1, 3 or 24 hours after application of 1.0-Hz CTS), gene expression was altered at 1 and 24 hours in AF cells derived from nondegenerative and degenerative IVDs, respectively 18 .

Cell viability was assessed using the Trypan blue (0.4%; Sigma) exclusion assay as previously reported 18 .

Total RNA was extracted from each BioFlex culture plate well using TRIzol™ reagent (Invitrogen) according to the manufacturer's instructions, and samples were treated with DNase I (Ambion, Austin, TX, USA). RNA quality and quantity were determined using the Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), and 500 ng of RNA were reverse-transcribed using the High Capacity 'cDNA' Reverse Transcription Kit (Applied Biosystems, Warrington, UK). A quantitative real-time polymerase chain reaction assay was performed in triplicate using TaqMan Universal PCR Master Mix (Applied Biosystems) with primers and probes for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aggrecan and type I collagen for degenerative AF cells, and MMP3 and a disintegrin and metalloproteinase with a thrombospondin type 1, motif 4 (ADAMTS4) for nondegenerative AF cells using previously published sequences and concentrations 18 . The genes were chosen for analysis on the basis of previously published observations whereby, from among a panel of genes investigated (aggrecan, types I and II collagen, MMP3, MMP9, MMP13 and ADAMTS4), only MMP3 and ADAMTS4 expression and aggrecan and type I collagen gene expression were altered in 1.0-Hz CTS-treated nondegenerative and degenerative AF cells, respectively 18 . Data were analysed using the 2^-ΔΔC _T method 18 55 and normalised to the endogenous control gene GAPDH and unloaded baseline controls.

The nonparametric data as determined by the Shapiro-Wilke test were analysed using the Mann-Whitney U test.

Immunopositivity was seen for IL-4R in both AF and NP cells from all nondegenerative and degenerative samples and was predominantly localised intracellularly throughout the cytoplasm and nucleus (Figures 1a and 1d). IL-2Rγ immunopositivity was also seen in both AF and NP cells from all nondegenerative and degenerative samples, with localisation occurring intracellularly but exclusively to the cytoplasm (Figures 1b and 1e). IL-13R immunopositivity was seen only in the positive control articular chondrocyte sample (Figure 1g), with no immunopositive cells in the AF or NP tissues of the nine samples investigated (Figures 1c and 1f).

AF cells isolated from nondegenerative and degenerative IVDs remained viable (>90%) throughout the culture period, with cell viability unaffected by either mechanical stimulation or cytokine inhibitor treatment. Unloaded controls showed no significant change in gene expression for any of the genes investigated at any of the time points analysed.

When stimulated at 1.0-Hz CTS, AF cells derived from nondegenerative tissue responded with a decrease in MMP3 and ADAMTS4 relative gene expression, suggesting a shift towards a less catabolic phenotype. This reduced catabolic response following 1.0-Hz CTS appears to be IL-1- and IL-4-dependent, as treatment with the cytokine inhibitors IL-1Ra or IL-4RAb inhibited the load-induced decrease in ADAMTS4 gene expression and caused an increase in MMP3 gene expression.

Unexpectedly, treatment of AF cells derived from nondegenerative tissue with IL-1Ra caused an increase in the baseline level of MMP3 gene expression prior to load. MMP activity has previously been shown to be inhibited following treatment of IVD tissue with IL-1Ra 59 , suggesting that the unexpected IL-1Ra-induced increase in MMP3 gene expression observed in this study might correlate negatively with enzyme activity. This potential phenomenon should be investigated further because increased MMP gene expression in IVD cells caused by inhibited enzyme activity due to IL-1Ra treatment could pose a concern with regard to IL-1Ra as a potential treatment for DDD. Although enzyme activity may be regarded as the more biologically significant factor, elevated levels of MMP mRNA (with the potential for protein translation) following IL-1Ra treatment could be detrimental to IVD tissue homeostasis.

Previous studies have reported modulation of increased IL-1 β-dependent MMP gene expression following treatment with IL-1Ra 60 ; however, the response of MMP gene expression to IL-1Ra treatment in the absence of IL-1 agonist has never been reported until now. Our data indicate that, in the absence of an agonist, treatment with IL-1Ra upregulates MMP3 gene expression by a currently undefined mechanism. It is also worth noting that treatment with IL-4RAb had no effect on the baseline gene expression level of MMP3 in AF cells derived from nondegenerative tissue, suggesting that the maintenance of baseline MMP3 gene expression occurs independently of IL-4. Importantly, although baseline MMP3 gene expression was upregulated following treatment with IL-1Ra, exposure of the cells to 1.0-Hz CTS caused a further significant increase in gene expression. This finding is opposite to that observed in untreated mechanically stimulated cells in which CTS caused a decrease in MMP3 gene expression. Such results implicate IL-1 in the CTS-induced decreased catabolic response observed in nondegenerative AF cells.

Treatment of AF cells derived from nondegenerative tissue with either cytokine inhibitor IL-1Ra or IL-4RAb had no effect on the baseline gene expression level of ADAMTS4, suggesting that the maintenance of ADAMTS4 baseline gene expression occurs independently of both IL-1 and IL-4. This observation is in contrast to IL-1Ra-dependent MMP3 baseline gene expression. However, the CTS-induced downregulation of ADAMTS4 gene expression in AF cells derived from nondegenerative tissue does appear to be cytokine-dependent, with cytokine inhibitors of both IL-1 and IL-4 preventing the decrease in gene expression following mechanical stimulation.

Although the expression of IL-4 has previously been reported in the IVD 46 , expression of its receptors has not yet been described. Here we report, for the first time, and albeit in a small number of samples, immunopositivity for the IL-4 receptor subunits IL-4Rα and IL-2Rγ and immunonegativity for the receptor subunit IL-13Rα₁ in human IVD cells. During IL-4R activation, IL-4 first binds IL-4Rα, leading to the recruitment of a second receptor subunit, predominantly IL-2Rγ in cells of haematopoietic origin 61 (termed 'type I IL-4R') and IL-13Rα₁ in cells of nonhaematopoietic origin 62 63 64 (termed 'type II IL-4R'). It is therefore surprising that IL-4 appears to signal through type I IL-4R (IL-4Rα/IL-2Rγ heterodimer) in human IVD cells and not through type II IL-4R (IL-4Rα/IL-13Rα₁ heterodimer) as reported in other cartilaginous tissues such as articular cartilage 65 . The downstream signalling pathways activated during IL-4 signalling are IL-4R type-dependent, with differential Janus kinase (JAK) phosphorylation occurring between activated receptor types (phosphorylated JAK1/3 66 67 and JAK2/tyrosine kinase 2 (Tyk2) 65 68 69 following IL-4R types I and II activation, respectively) suggesting that IL-4-dependent mechanotransduction could differ between IVD cells and cells from other cartilaginous tissues.

In addition to IL-4, IL-1 also appears to be necessary for human AF cell mechanotransduction following 1.0-Hz CTS, with similar alterations and inhibitions to the CTS-induced decreases in MMP3 and ADAMTS4 gene expression, respectively. IL-1 β has previously been shown to be involved in human bone cell mechanotransduction, where an autocrine/paracrine IL-1 β-induced release of prostaglandin E₂ is suggested to precede the 0.33-Hz CTS-induced hyperpolarisation response 35 . IL-1 β has also been implicated in osteoarthritic (but not nonosteoarthritic) chondrocyte mechanotransduction, where the pretreatment of cells with neutralising antibodies to IL-1 β prevented the CTS-induced depolarisation response, suggesting altered mechanotransduction in chondrocytes derived from degenerative tissue 40 . Although in our study it is not clear which subtype of IL-1 is involved in AF cell mechanotransduction, information from other studies of connective tissue mechanical loading suggests IL-1 β as the primary candidate 35 40 . Although the involvement of IL-1 in disc cell mechanotransduction has not previously been reported, the expression of IL-1 (IL-1α and IL-1 β), its antagonist (IL-1Ra) and its receptor (IL-1 receptor, type I) have been reported in both nondegenerative and degenerative IVDs, with the IL-1 agonists, but not the IL-1 antagonist, increasing with the severity of degeneration 42 . Furthermore, evidence from a study using rats suggests that potential interactions exist between the IL-1 and CTS signalling pathways in AF cells as demonstrated by the partial inhibition of IL-1 β-induced catabolic gene expression with the addition of 6% CTS treatment 44 . Thus, in addition to the role of IL-1 as a catabolic factor implicated in DDD, our findings support the concept that it may play a role in transducing physiological mechanical stimuli leading to tissue remodelling.

When AF cells derived from degenerative tissue were subjected to 1.0-Hz CTS, the observed response, reduced relative gene expression of the matrix proteins aggrecan and type I collagen, did not appear to involve either IL-1 or IL-4 as demonstrated by the inability of the cytokine inhibitors IL-1Ra and IL-4RAb to prevent the CTS-induced changes in gene expression. Although pretreatment with either cytokine inhibitor had no effect on the baseline gene expression level of aggrecan in AF cells derived from degenerative tissue, type I collagen baseline gene expression was reduced in these cells. This reduced type I collagen baseline gene expression might be expected following IL-4Rab treatment, as inhibition of chondroprotective IL-4 could be predicted to have a reduced anabolic or even catabolic effect. However, the fact that reduced type I collagen baseline gene expression followed treatment with IL-1Ra is an unexpected observation. These data therefore suggest that treatment of human AF cells derived from both nondegenerative and degenerative tissue with cytokine inhibitors appears to have a different effect from that previously observed in the presence of agonists.

Although treatment with either cytokine inhibitor caused a decrease in type I collagen baseline gene expression, exposure of the cells to 1.0-Hz CTS led to a further decrease in gene expression, suggesting that while IL-1 may be involved in basal collagen expression, neither IL-1 nor IL-4 is necessary for this mechanotransduction response. This lack of cytokine-dependent mechanotransduction was also observed in the regulation of aggrecan, where treatment of degenerative AF cells with either cytokine inhibitor IL-1Ra or IL-4RAb had no effect on baseline or 1.0-Hz CTS-induced downregulation of aggrecan gene expression.

Anabolic and catabolic gene expression is known to occur through the involvement of different transcription factors (for example, SRY (sex-determining region Y)-box 9, or SOX9, and nuclear factor κ-light chain enhancer of activated B cells (NFκB) for matrix protein and matrix-degrading enzyme gene regulation, respectively), with specific transcription factors regulated via specific signalling pathways (for example, IL-1-dependent NFκB regulation). Thus, differences in the regulation of matrix protein and matrix-degrading enzyme gene expression between AF cells derived from degenerative and nondegenerative tissue, respectively, could be due to differences in cytokine-dependent transcription factor activation brought about by the differential cytokine involvement observed with 1.0-Hz CTS between nondegenerative and degenerative AF cells.

This lack of involvement of IL-1 and IL-4 in the mechanoresponse of degenerative cells indicates that an altered mechanotransduction pathway may be in operation. Although cells from osteoarthritic cartilage have also been shown to signal through an altered mechanotransduction pathway in response to CTS 40 , the alteration in signalling occurred through the involvement of an additional cytokine, namely IL-1β, and not through the loss of cytokine signalling shown to be necessary for mechanotransduction to occur in the absence of disease as reported here. These changes in signalling pathways indicate further differences in mechanobiology between these cartilaginous cell types.

Interestingly, NP cells derived from degenerative tissue have also been shown to utilise an altered mechanotransduction pathway following stimulation with HP. Le Maitre et al. 29 reported that the HP-induced decrease in aggrecan gene expression occurred via an RGD-recognising integrin pathway in NP cells derived from nondegenerative, but not degenerative, tissue, suggesting that disc cell mechanotransduction becomes altered with degeneration, which is in agreement with the results of this study.