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Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration.

Millward-Sadler SJ, Costello PW, Freemont AJ, Hoyland JA - Arthritis Res. Ther. (2009)

Bottom Line: IL-1beta and IL-1Ra expressions were significantly upregulated by TNF-alpha, whereas IL-1alpha and IL-1R1 were unchanged.The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix.These results support the hypothesis that, while TNF-alpha may be an important initiating factor in matrix degeneration, IL-1beta plays a greater role in established pathological degradation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tissue Injury and Repair Group, School of Clinical and Laboratory Sciences, Faculty of Human and Medical Sciences, University of Manchester, Manchester M13 9PT, UK. Jane.Sadler@manchester.ac.uk

ABSTRACT

Introduction: The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture.

Methods: Cells isolated from normal and degenerate nucleus pulposus regions of human intervertebral discs were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-alpha or IL-1beta for 48 hours prior to RNA extraction. Quantitative real-time polymerase chain reaction was used to assess the effect of TNF-alpha or IL-beta stimulation on the expression of matrix metalloproteinase (MMP)-3, -9 and -13, TNF-alpha, TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2), IL-1alpha, IL-1beta, IL-1 receptor 1 (IL-1R1) and IL-1 receptor antagonist (IL-1Ra).

Results: MMP-3 and MMP-9 gene expressions were upregulated to a greater level by IL-1beta than TNF-alpha. MMP-13 was upregulated by each cytokine to a similar extent. TNF-alpha and TNF-R2 expressions were upregulated by both TNF-alpha and IL-beta, whereas TNF-R1 expression was not significantly affected by either cytokine. IL-1beta and IL-1Ra expressions were significantly upregulated by TNF-alpha, whereas IL-1alpha and IL-1R1 were unchanged.

Conclusions: TNF-alpha does not induce MMP expression to the same degree as stimulation by IL-1beta, but it does act to upregulate IL-1beta expression as well as TNF-alpha and TNF-R2. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results support the hypothesis that, while TNF-alpha may be an important initiating factor in matrix degeneration, IL-1beta plays a greater role in established pathological degradation.

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Effect of cytokine stimulation on tumour necrosis factor-alpha (TNF-α) and TNF receptor gene expression in normal or degenerate nucleus pulposus (NP) cells. Normal or degenerate NP cells were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-α (a) or IL-1β (b). Quantitative real-time polymerase chain reaction was used to analyse the effect of cytokine stimulation on TNF-α, TNF receptor 1 (TNF-R1) and TNF receptor 2 (TNF-R2) gene expression All samples are relative to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and normalised back to untreated controls. Results are given as mean ± standard error of the mean (n = 3). *P ≤ 0.05 when compared with untreated controls.
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Figure 3: Effect of cytokine stimulation on tumour necrosis factor-alpha (TNF-α) and TNF receptor gene expression in normal or degenerate nucleus pulposus (NP) cells. Normal or degenerate NP cells were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-α (a) or IL-1β (b). Quantitative real-time polymerase chain reaction was used to analyse the effect of cytokine stimulation on TNF-α, TNF receptor 1 (TNF-R1) and TNF receptor 2 (TNF-R2) gene expression All samples are relative to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and normalised back to untreated controls. Results are given as mean ± standard error of the mean (n = 3). *P ≤ 0.05 when compared with untreated controls.

Mentions: The addition of recombinant TNF-α for 48 hours resulted in a significant upregulation of TNF-α and TNF-R2 genes in both normal and degenerate NP cells (Figure 3a). TNF-R1 mRNA was upregulated by TNF-α in normal NP cells but downregulated in degenerate samples (Figure 3a). Neither of these changes in mRNA expression was statistically significant, although the difference between the upregulation in normal cells and the downregulation in degenerate cells did reach significance (P = 0.05). TNF-R2 mRNA was upregulated to a greater extent in normal than in degenerate samples (P = 0.05).


Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration.

Millward-Sadler SJ, Costello PW, Freemont AJ, Hoyland JA - Arthritis Res. Ther. (2009)

Effect of cytokine stimulation on tumour necrosis factor-alpha (TNF-α) and TNF receptor gene expression in normal or degenerate nucleus pulposus (NP) cells. Normal or degenerate NP cells were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-α (a) or IL-1β (b). Quantitative real-time polymerase chain reaction was used to analyse the effect of cytokine stimulation on TNF-α, TNF receptor 1 (TNF-R1) and TNF receptor 2 (TNF-R2) gene expression All samples are relative to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and normalised back to untreated controls. Results are given as mean ± standard error of the mean (n = 3). *P ≤ 0.05 when compared with untreated controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2714110&req=5

Figure 3: Effect of cytokine stimulation on tumour necrosis factor-alpha (TNF-α) and TNF receptor gene expression in normal or degenerate nucleus pulposus (NP) cells. Normal or degenerate NP cells were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-α (a) or IL-1β (b). Quantitative real-time polymerase chain reaction was used to analyse the effect of cytokine stimulation on TNF-α, TNF receptor 1 (TNF-R1) and TNF receptor 2 (TNF-R2) gene expression All samples are relative to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and normalised back to untreated controls. Results are given as mean ± standard error of the mean (n = 3). *P ≤ 0.05 when compared with untreated controls.
Mentions: The addition of recombinant TNF-α for 48 hours resulted in a significant upregulation of TNF-α and TNF-R2 genes in both normal and degenerate NP cells (Figure 3a). TNF-R1 mRNA was upregulated by TNF-α in normal NP cells but downregulated in degenerate samples (Figure 3a). Neither of these changes in mRNA expression was statistically significant, although the difference between the upregulation in normal cells and the downregulation in degenerate cells did reach significance (P = 0.05). TNF-R2 mRNA was upregulated to a greater extent in normal than in degenerate samples (P = 0.05).

Bottom Line: IL-1beta and IL-1Ra expressions were significantly upregulated by TNF-alpha, whereas IL-1alpha and IL-1R1 were unchanged.The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix.These results support the hypothesis that, while TNF-alpha may be an important initiating factor in matrix degeneration, IL-1beta plays a greater role in established pathological degradation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tissue Injury and Repair Group, School of Clinical and Laboratory Sciences, Faculty of Human and Medical Sciences, University of Manchester, Manchester M13 9PT, UK. Jane.Sadler@manchester.ac.uk

ABSTRACT

Introduction: The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture.

Methods: Cells isolated from normal and degenerate nucleus pulposus regions of human intervertebral discs were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-alpha or IL-1beta for 48 hours prior to RNA extraction. Quantitative real-time polymerase chain reaction was used to assess the effect of TNF-alpha or IL-beta stimulation on the expression of matrix metalloproteinase (MMP)-3, -9 and -13, TNF-alpha, TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2), IL-1alpha, IL-1beta, IL-1 receptor 1 (IL-1R1) and IL-1 receptor antagonist (IL-1Ra).

Results: MMP-3 and MMP-9 gene expressions were upregulated to a greater level by IL-1beta than TNF-alpha. MMP-13 was upregulated by each cytokine to a similar extent. TNF-alpha and TNF-R2 expressions were upregulated by both TNF-alpha and IL-beta, whereas TNF-R1 expression was not significantly affected by either cytokine. IL-1beta and IL-1Ra expressions were significantly upregulated by TNF-alpha, whereas IL-1alpha and IL-1R1 were unchanged.

Conclusions: TNF-alpha does not induce MMP expression to the same degree as stimulation by IL-1beta, but it does act to upregulate IL-1beta expression as well as TNF-alpha and TNF-R2. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results support the hypothesis that, while TNF-alpha may be an important initiating factor in matrix degeneration, IL-1beta plays a greater role in established pathological degradation.

Show MeSH
Related in: MedlinePlus