Cytokine Inhibitors Upregulate Extracellular Matrix Anabolism of Human Intervertebral Discs under Alginate Beads and Alginate-Embedded Explant Cultures

Abstract

We investigated the effects of the cytokine inhibitors IL-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor receptor-1 (sTNFR1) on the extracellular matrix metabolism of human intervertebral discs (IVDs) and the roles of IL-1β and TNF in the homeostasis of IVD cells. The 1.2% alginate beads and the explants obtained from 35 human lumbar discs were treated with cytokine inhibitors. Extracellular matrix metabolism was evaluated by proteoglycan (PG) and collagen syntheses and IL-1β, TNF, and IL-6 expressions after three days of culture in the presence or absence of IL-1Ra, sTNFR1, and cycloheximide. Simultaneous treatment with IL-1Ra and sTNFR1 stimulated PG and collagen syntheses in the NP and AF cells and explants. The IL-1β concentration was significantly correlated to the relative increase in PG synthesis in AF explants after simultaneous cytokine inhibitor treatment. The relative increase in PG synthesis induced by simultaneous cytokine treatment was significantly higher in an advanced grade of MRI. Expressions of IL-1β and TNF were upregulated by each cytokine inhibitor, and simultaneous treatment suppressed IL-1β and TNF productions. In conclusion, IL-1Ra and sTNFR1 have the potential to increase PG and collagen synthesis in IVDs. IL-1β and TNF have a feedback pathway to maintain optimal expression, resulting in the control of homeostasis in IVD explants.

Keywords: autocrine; cytokine; intervertebral disc metabolism.

Figure 3

PG and collagen syntheses after treatment with cytokine inhibitors IL-1Ra and sTNFR1 in the alginate bead culture system. A single treatment with IL-1Ra or sTNFR1 did not affect PG synthesis in the both cell types. However, PG synthesis was significantly stimulated by simultaneous treatment with IL-1Ra and sTNFR1 at 0.1 ng/mL in NP cells (a). In AF cells, PG synthesis stimulated by simultaneous treatment showed the similar result (b). Collagen synthesis was significantly upregulated by simultaneous treatment at 0.1 µg/mL in NP cells (c). In AF cells, collagen synthesis was significantly stimulated by a single treatment with 0.1 µg/mL sTNFR1 and simultaneous treatment at 0.1 ng/mL (d). All values are expressed as the mean ± SE. * p < 0.05; ** p < 0.01. Data on PG synthesis and collagen synthesis were obtained from 15 and 9 donors, respectively.

Figure 4

PG synthesis after treatment with cytokine inhibitors IL-1Ra and sTNFR1 in the explant culture system. Simultaneous treatment with IL-1Ra and sTNFR1 at 1.0 ng/mL significantly stimulated PG synthesis in both NP and AF cells. All values are expressed as the mean ± SE. * p < 0.05. Data were obtained from eight donors.

Figure 5

Relationship between the response to cytokine inhibitors and cytokine concentrations or the MRI grade in the explant culture system. (a,b) The concentration of IL-1β was significantly correlated to a relative increase in PG synthesis after simultaneous cytokine inhibitor treatment at 1 μg/mL in AF explants. The concentrations of TNF (c,d) and IL-6 (e,f) showed no relationship with PG synthesis. (g,h) The relative increase in PG synthesis by simultaneous cytokine treatment was significantly higher in an advanced grade of MRI. Both were significantly correlated. Data were obtained from eight donors.

Figure 6

Cytokine levels in condition medium after cytokine inhibitor treatment. (a,b) IL-1β expression was stimulated by a single treatment with IL-1Ra or sTNFR1. sTNFR1 significantly increased IL-1β expression more than IL-1Ra in NP and AF. (c,d) Simultaneous treatment suppressed IL-1β production in both NP and AF. IL-1Ra significantly stimulated TNF expression. Simultaneous treatment did not affect TNF production. All values are expressed as the mean ± SE. * p < 0.05; ** p < 0.01. Data were obtained from eight donors.

Figure 1

Cytokine levels in condition medium after cycloheximide treatment. (a,b) In NP and AF explants, IL-1β was significantly diminished by treatment with cycloheximide. In NP explants, freezing and thawing of cells and following incubation in medium induced the significant release of IL-1β. (c) In NP explants, TNF was significantly diminished by treatment with cycloheximide. (d) In AF explants, cycloheximide and the freezing and thawing of cells decreased TNF. (e) In NP explants, IL-6 showed a similar trend to TNF but did not achieve statistical significance. (f) In AF explants, IL-6 was significantly reduced by freezing and thawing. All values are expressed as the mean ± SE. * p < 0.05; ** p < 0.01. Data were obtained from five donors.

Figure 2

DNA concentration in NP and AF. Data were obtained from six donors.

Figure 7

Alginate beads just made (a), and explant coated with 1.2% alginate (b).