Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes K.L.E.

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Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes K.L.E. Phillips, K. Cullen, N. Chiverton, A.L.R. Michael, A.A. Cole, L.M. Breakwell, G. Haddock, R.A.D. Bunning, A.K. Cross, C.L. Le Maitre Osteoarthritis and Cartilage Volume 23, Issue 7, Pages 1165-1177 (July 2015) DOI: 10.1016/j.joca.2015.02.017 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 CD4, CCR1, CXCR1 and CXCR2 Expression in Human NP Tissue. Representative photomicrographs showing 4 μm paraffin embedded sections of NP tissue stained for CD4, CCR1, CXCR1 and CXCR2. A) Abundant CD4, CCR1, CXCR1 and CXCR2 expression was observed in NP cells scattered throughout tissue sections. B) CD4, CXCR1 and CXCR2 expression was also seen by immune cell infiltrates (where present) in tissue sections, whilst expression was not observed in immune cell infiltrates for CCR1. Scale bars indicated A: 100 μm; B: 60 μm. Osteoarthritis and Cartilage 2015 23, 1165-1177DOI: (10.1016/j.joca.2015.02.017) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Immunohistochemical (IHC) quantification of CD4, CCR1, CXCR1 and CXCR2 Expression in Non-Degenerate (ND), Degenerate (D) and Infiltrated (I) IVDs. A) Linear Regression of percentage immunopositivity for CD4, CCR1, CXCR1 and CXCR2 expression by NP cells against histologically determined grade of degeneration, tissues containing infiltrated cells shown with hollow circles whist those with no infiltrating cells shown with solid circles. Non-parametric linear regression analysis of the full data set regardless of infiltrated status shown with line indicating NP cell CXCR2 immunopositivity increases concordant to histologically determined degenerative tissue changes n = 30 (P < 0.05). B) IHC quantification of CD4, CCR1, CXCR1 and CXCR2 expression by NP cells in non-degenerate (ND) (n = 3), degenerate (D) (n = 16) and infiltrated (I) (n = 11) IVDs. NP cell CD4 expression was increased in degenerate and infiltrated samples, CCR1, CXCR1 and CXCR2 expression was not statistically different between study groups. Data shown are scatter plots indicating the immunopositivity within each NP sample*, significant difference in expression between study groups (P < 0.05). Osteoarthritis and Cartilage 2015 23, 1165-1177DOI: (10.1016/j.joca.2015.02.017) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 IL-1β Stimulated Expression of Aggrecan, MMP-3 and MMP-13 mRNA in Primary Human NP Cultures. IL-1β stimulated a bi-phasic dose dependent aggrecan mRNA expression profile and up-regulated MMP-3 and MMP-13 mRNA expression in NP cultures. Data shown as geometric mean of relative target gene expression normalised to two internal reference genes (GAPDH and 18S) and untreated controls ± 95% confidence intervals. * indicates significant alteration in mRNA expression profile compared to unstimulated control (P < 0.05). Individual patient responses for Aggrecan, MMP 3 and MMP 13 mRNA expression following stimulation with 0 and 100 ng/ml IL-1, IL-16, CCL2, CCL3, CCL7 and CXCL8. Each dot plotted shows target gene expression normalised to two internal reference genes (GAPDH and 18S) and the individual patients 0 ng/ml. Gene expression for aggrecan following IL-1 stimulation performed in 5 patient whilst all other genes and stimulations performed in 3 independent IVD samples from 3 different patients. Osteoarthritis and Cartilage 2015 23, 1165-1177DOI: (10.1016/j.joca.2015.02.017) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 IL-1β Stimulated Expression of IL-1β, IL-16, CCL2, CCL3, CCL7 and CXCL8 mRNA in Primary Human NP Cultures. Basal expression of IL-16, CCL2, CCL7 and CXCL8 but not IL-1β or CCL3 was recorded in NP cultures. IL-1β stimulation down regulated IL-16 mRNA expression and up-regulated CCL2, CCL7 and CXCL8 mRNA expression. IL-1β stimulation induced expression of IL-1β and CCL3 mRNA. Data shown as relative target gene expression normalised to two internal reference genes (GAPDH and 18S) and untreated controls ± 95% confidence intervals. * indicates significant alteration in mRNA expression profile compared to unstimulated control (P < 0.05). Individual patient responses for IL-16, CCL2, CCL7 and CXCL8 mRNA expression following stimulation with 0 and 100 ng/ml IL-1, IL-16, CCL2 (CCL2 mRNA only), CCL3, CCL7 and CXCL8 (CCL2 mRNA only). Each dot plotted shows target gene expression normalised to two internal reference genes (GAPDH and 18S) and the individual patient's 0 ng/ml. No mRNA expression for IL-1 and CCL3 was observed in NP cells stimulated with IL-16, CCL2, CCL3, CCL7 or CXCL8 (not shown as negative). Gene expression for IL-16 following IL-1 stimulation performed in 5 patients, CCL7 in 4 patients whilst all other genes and stimulations performed in 3 independent IVD samples from 3 different patients. Osteoarthritis and Cartilage 2015 23, 1165-1177DOI: (10.1016/j.joca.2015.02.017) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 IL-1β Stimulated Cytokine Release from Primary Human NP Cultures. IL-1β stimulated release of IL-4, IL-6, IL-10, TNF-α, IFN-γ and CSF2 from NP cultures. Data shown is cytokine concentration in culture supernatant following IL-1β stimulation for each replicate with each patient represented as separate colour, median value for each patient shown * indicates significant increase in cytokine concentration compared to unstimulated control (P < 0.05). Osteoarthritis and Cartilage 2015 23, 1165-1177DOI: (10.1016/j.joca.2015.02.017) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 IL-1β Stimulated Chemokine Release from Primary Human NP Cultures. IL-1β stimulated release of CCL2, CCL3, CCL4, CCL5, CCL7, CXCL1, CXCL8, CXCL9 and CXCL10 from NP cultures. Data shown is cytokine concentration in culture supernatant following IL-1β stimulation for each replicate with each patient represented as separate colour, median value for each patient shown * indicates significant increase in cytokine concentration compared to unstimulated control (P < 0.05). Osteoarthritis and Cartilage 2015 23, 1165-1177DOI: (10.1016/j.joca.2015.02.017) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions