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. 2013;15(6):R213.
doi: 10.1186/ar4408.

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Kate L E Phillips, Neil Chiverton, Anthony L R Michael, Ashley A Cole, Lee M Breakwell, Gail Haddock, Rowena A D Bunning, Alison K Cross, Christine L Le Maitre

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Kate L E Phillips et al. Arthritis Res Ther. 2013.

Abstract

Introduction: The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods: Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results: LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions: Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.

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Figures

Figure 1
Figure 1
Study outline and experimental approaches. Initial low density array (LDA) analysis of cells isolated from human nucleus pulposus (NP) tissue samples identified a diverse cytokine and chemokine expression profile. From these data, 30 targets were selected for further gene expression analysis by real-time qPCR and 4 targets were selected for protein production and localisation analysis by immunohistochemistry. Consideration was given to the frequency at which targets were detected and any differential expression profile observed between non-degenerate and degenerate study groups to select targets for further investigation. Consideration was also given to the detection of receptor mRNA expression (indicating potential for autocrine or paracrine signalling involvement), whether the roles of identified targets had previously been well studied, the identification of novel targets within the intervertebral disc, and links to cytokine and chemokine activity in other arthropathies.
Figure 2
Figure 2
cDNA low density array cytokine and chemokine expression profile of NP cells. Gene expression of 36 cytokines and chemokines was detected in nucleus pulposus cells; 15 of the identified cytokines and chemokines have not been identified previously within the intervertebral disc (IVD). Comparative analysis indicates expression of 16 targets is increased in NP cells derived from degenerate IVDs. ^Increased frequency of mRNA detection in NP cells derived from non-degenerate IVDs compared to degenerate (P <0.05); G, further gene expression analysis performed on this target; P, further protein expression analysis performed on this target.
Figure 3
Figure 3
cDNA low density array LDA cytokine and chemokine expression profile of nucleus pulposus (NP) cells. Gene expression of 36 cytokines and chemokines was detected in NP cells; 15 of the identified cytokines and chemokines have not been identified previously within the intervertebral disc (IVD). Comparative analysis indicates expression of 16 targets is increased in NP cells derived from degenerate IVDs. ^Increased frequency of mRNA detection in NP cells derived from non-degenerate IVDs compared to degenerate (P <0.05); G, further gene expression analysis performed on this target; P, further protein expression analysis performed on this target.
Figure 4
Figure 4
Cytokine-associated gene expression in nucleus pulposus (NP) cells from non-degenerate, degenerate and infiltrated intervertebral discs (IVDs). Relative mRNA quantitation of cytokines and receptors was assessed by real-time q-PCR in cDNA samples from non-degenerate, degenerate and infiltrated study groups. Data shown are detection frequency (ν; pie charts) and relative expression per sample investigated (scatter plots). N-D, non-degenerate study group; D, degenerate study group; I, infiltrated study group; black star, significant difference in detection frequency between study groups (P <0.05); black diamond, significant difference in relative expression levels between study groups (P <0.05). LIFR, leukaemia inhibitory factor receptor.
Figure 5
Figure 5
Chemokine-associated gene expression in nucleus pulposus (NP) cells from non-degenerate, degenerate and infiltrated intervertebral discs (IVDs). Relative mRNA quantitation of chemokines and receptors was assessed by real-time q-PCR in cDNA samples from non-degenerate, degenerate and infiltrated study groups. Data shown are detection frequency (ν; pie charts) and relative expression per sample investigated (scatter plots). N-D, non-degenerate study group; D, degenerate study group; I, infiltrated study group; black star, significant difference in detection frequency between study groups (P <0.05); black diamond, significant difference in relative expression levels between study groups (P <0.05).
Figure 6
Figure 6
IL-16, CCL2, CCL7 and CXCL8 expression in nucleus pulposus (NP) tissue from prolapsed intervertebral discs (IVDs). Representative photomicrographs showing 4-μm paraffin-embedded sections of NP tissue stained for IL-16, CCL2, CCL7 and CXCL8 (A). Abundant IL-16, CCL2, CCL7 and CXCL8 expression was observed in NP cells scattered throughout tissue sections (original magnification ×400, scale bar represents 100 μm). IL-16, CCL2, CCL7 and CXCL8 expression was also observed in immune cell infiltrates (B).
Figure 7
Figure 7
IL-16, CCL2, CCL7 and CXCL8 expression in non-degenerate, degenerate and infiltrated intervertebral discs (IVDs). Immunohistochemical quantification of IL-16, CCL2, CCL7 and CXCL8 expression by NP cells in non-degenerate (ND), degenerate (D) and infiltrated (I) IVDs. Data shown are median values for immunopositivity; black star, significant difference in expression between study groups (P <0.05). IL-16 expression was equivalent across study groups, CCL2, CCL7 and CXCL8 expression was increased in degenerate samples, CCL7 and CXCL8 expression was also increased in infiltrated samples.
Figure 8
Figure 8
CCL2, CCL7 and CXCL8 expression correlates with degenerative tissue changes. Immunohistochemical quantification of CCL2, CCL7 and CXCL8 expression by nucleus pulposus cells. Data shown as are immunopositivity values against histologically determined grade of degeneration. Linear regression analysis was performed to assess correlation between histologically determined grade of degeneration and immunopositivity.
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