J Korean Neurosurg Soc.  2014 May;55(5):237-243. 10.3340/jkns.2014.55.5.237.

Matrix Degradative Enzymes and Their Inhibitors during Annular Inflammation: Initial Step of Symptomatic Intervertebral Disc Degeneration

• Kim JH
• Park JH
• Moon HJ
• Kwon TH
• Park YK

• KMID: 2018027
• DOI: http://doi.org/10.3340/jkns.2014.55.5.237

Abstract

OBJECTIVE
Symptomatic disc degeneration develops from inflammatory reactions in the annulus fibrosus (AF). Although inflammatory mediators during annular inflammation have been studied, the roles of matrix metalloproteinases (MMPs) and their inhibitors have not been fully elucidated. In this study, we evaluated the production of MMPs and tissue inhibitors of metalloproteinase (TIMPs) during annular inflammation using an in vitro co-culture system. We also examined the effect of notochordal cells on annular inflammation.
METHODS
Human AF (hAF) pellet was co-cultured for 48 hours with phorbol myristate acetate-stimulated macrophage-like THP-1 cells. hAF pellet and conditioned media (CM) from co-cultured cells were assayed for MMPs, TIMPs, and insulin-like growth factor (IGF)-1 levels using real-time reverse-transcriptase polymerase chain reaction and enzyem-linked immunosorbent assay. To evaluate whether notochordal cells affected MMPs or TIMPs production on annular inflammation, hAF co-cultured with notochordal cells from adult New Zealand White rabbits, were assayed.
RESULTS
MMP-1, -3, -9; and TIMP-1 levels were significantly increased in CM of hAF co-cultured with macrophage-like cells compared with hAF alone, whereas TIMP-2 and IGF-1 levels were significantly decreased (p<0.05). After macrophage exposure, hAF produced significantly more MMP-1 and -3 and less TIMP-1 and -2. Interleukin-1beta stimulation enhanced MMP-1 and -3 levels, and significantly diminished TIMP-2 levels. Co-culturing with rabbit notochordal cells did not significantly influence MMPs and TIMPs production or COL1A2 gene expression.
CONCLUSION
Our results indicate that macrophage-like cells evoke annular degeneration through the regulation of major degradative enzymes and their inhibitors, produced by hAF, suggesting that the selective regulation of these enzymes provides future targets for symptomatic disc degeneration therapy.

Keyword

Annular inflammation; Symptomatic intervertebral disc degeneration; Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; IGF-1; Notochordal cells

MeSH Terms

Adult
Coculture Techniques
Culture Media, Conditioned
Gene Expression
Humans
Inflammation*
Insulin-Like Growth Factor I
Interleukin-1beta
Intervertebral Disc Degeneration*
Macrophages
Matrix Metalloproteinases
Myristic Acid
Notochord
Polymerase Chain Reaction
Rabbits
Tissue Inhibitor of Metalloproteinase-1
Tissue Inhibitor of Metalloproteinase-2
Culture Media, Conditioned
Insulin-Like Growth Factor I
Interleukin-1beta
Matrix Metalloproteinases
Myristic Acid
Tissue Inhibitor of Metalloproteinase-1
Tissue Inhibitor of Metalloproteinase-2

Figure

• 

  Fig. 1 Schematic representation of the in vitro co-culture experiment. hAF : human annulus fibrosus pellet (2×105/well), rNC : rabbit notochordal cell clusters (2×104/well), M : activated macrophage-like THP-1 cells (1×105/well), hAF(M) : previously macrophage-exposed hAF pellet, hAF(rNC-M) : previously rNC cell and macrophage exposed hAF pellets, PMA : 160 nM phorbol myristate acetate.

• 

  Fig. 2 MMPs, TIMPs, and IGF-1 production from co-cultured cells. The hAF pellet was co-cultured with macrophage-like cells for 48 h. The levels of MMP-1 (A), -3 (B), -9 (E), and TIMP-1 (C) were increased significantly in the conditioned media (CM) of hAF pellets co-cultured with macrophage-like cells (hAF-M) compared with hAF pellets alone (hAF), whereas the levels of TIMP-2 (D) and IGF-1 (F) were decreased. The data were presented as mean (five individual experiments, n=14-18 in each group, 95% confidence interval). MMPs : matrix metalloproteinases, TIMPs : tissue inhibitors of metalloproteinases, IGF-1 : insulin-like growth factor, hAF : human annulus fibrosus, M : macrophage-like cells.

• 

  Fig. 3 Stimulation of nemotic hAF pellet by 1 ng/mL interleukin (IL)-1β. To evaluate the MMPs (A and B) and TIMPs (C and D) production levels in the hAF pellet with IL-1β stimulation, naïve hAF pellets (hAF) and nemotic hAF pellets (hAF(M)) were cultured with 1 ng/mL recombinant human IL-1β for 48 h. After macrophage exposure, hAF produced significantly higher levels of MMP-1 and -3 and lower levels of TIMP-1 and -2. Stimulation with 1 ng/mL IL-1β enhanced the levels of MMP-1 and -3 production significantly from hAF pellets and decreased the level of TIMP-2 significantly (p<0.05). The data were presented as mean (five individual experiments, n=14-18 in each group, 95% confidence interval). hAF : human annulus fibrosus, MMPs : matrix metalloproteinases, TIMPs : tissue inhibitors of metalloproteinases.

• 

  Fig. 4 hAF pellet expression of MMP-3 (A) and TIMP-1 (B) after co-culture. Gene expression was analyzed using real-time RT-PCR. MMP-3 expression was increased significantly by macrophage exposure (hAF(M)), and decreased significantly after co-culture with rNC cells and macrophages (hAF(rNC-M)) compared with hAF(M). TIMP-1 expression was decreased significantly in hAF(M) compared with hAF, and increased in hAF(rNC-M) compared with hAF(M). The data were presented as mean (three individual experiments, n=6-8 in each group, triplicated data, 95% confidence interval). hAF : human annulus fibrosus, MMPs : matrix metalloproteinases, TIMPs : tissue inhibitors of metalloproteinases, RT-PCR : reverse transcriptase polymerase chain reaction.

• 

  Fig. 5 MMPs (A and B) and TIMPs (C and D) production levels in hAF pellets after co-culture with rNC cells and macrophages. Co-culturing with rNC (hAF(rNC-M)) tended to decrease the levels of MMP-3 and TIMP-1, but did not affect the production of MMP-1 or TIMP-2 in the hAF pellet with macrophages (hAF(M)). The data were presented as mean (five different experiments, n=14-18 in each group, 95% confidence interval). MMPs : matrix metalloproteinases, TIMPs : tissue inhibitors of metalloproteinases, hAF : human annulus fibrosus.

• 

  Fig. 6 hAF pellet COL1A2 expression after co-culture. Gene expression was assessed by real-time RT-PCR. COL1A2 expression by hAF was not influenced by macrophage exposure (hAF(M)), 1 ng/mL interleukin (IL)-1β stimulation, or rNC cell treatment (hAF(rNC-M)) in 48-h cultures. The data were presented as mean (three individual experiments, n=6-8, triplicated data, 95% confidence interval). hAF : human annulus fibrosus, RT-PCR : reverse transcriptase polymerase chain reaction.

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