α-Melanocyte-stimulating-hormone (α-MSH) modulates human chondrocyte activation induced by proinflammatory cytokines

Abstract

Background: Alpha-melanocyte-stimulating-hormone (α-MSH) has marked anti-inflammatory potential. Proinflammatory cytokines are critical mediators of the disturbed cartilage homeostasis in osteoarthritis, inhibiting anabolic activities and increasing catabolic activities in chondrocytes. Since human chondrocytes express α-MSH receptors, we evaluated the role of the peptide in modulating chondrocyte production of pro-inflammatory cytokines, matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in response to interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).

Methods: Human articular chondrocytes were obtained from osteoarthritic joint cartilage from subjects undergoing hip routine arthroplasty procedures. The cells were cultured with or without α-MSH in the presence of IL-1β or TNF-α. Cell-free supernatants were collected and cells immediately lysed for RNA purification. Expression of cytokines, MMPs, TIMPs, iNOS was determined by Reverse Transcription Real-time Polymerase Chain Reaction and enzyme-linked immunosorbent assay. Griess reaction was used for NO quantification.

Results: Gene expression and secretion of IL-6, IL-8, MMP-3, MMP-13 were significantly increased in IL-1β or TNF-α-stimulated chondrocytes; α-MSH did not modify the release of IL-6 or IL-8 while the peptide significantly reduced their gene expression on TNF-α-stimulated cells. A significant inhibition of MMP3 gene expression and secretion from IL-1β or TNFα-stimulated chondrocytes was induced by α-MSH. On the other hand, α-MSH did not modify the release of MMP-13 by cytokine-stimulated chondrocyte but significantly decreased gene expression of the molecule on TNF-α-stimulated cells. Detectable amount of TIMP-3 and TIMP-4 were present in the supernatants of resting chondrocytes and a significant increase of TIMP-3 gene expression and release was induced by α-MSH on unstimulated cells. TIMP-3 secretion and gene expression were significantly increased in IL-1β-stimulated chondrocytes and α-MSH down-regulated gene expression but not secretion of the molecule. TIMP-4 gene expression (but not secretion) was moderately induced in IL-1β-stimulated chondrocytes with a down-regulation exerted by α-MSH. IL-1β and TNF-α were potent stimuli for NO production and iNOS gene expression by chondrocytes; no inhibition was induced by α-MSH on cytokine-stimulated NO production, while the peptide significantly reduced gene expression of iNOS.

Conclusions: Our results underscore a potential anti-inflammatory and chondroprotective activity exerted by α-MSH, increasing TIMP-3 gene expression and release on resting cells and down- modulating TNF-α-induced activation of human chondrocytes. However, the discrepancy between the influences exerted by α-MSH on gene expression and protein release as well as the difference in the inhibitory pattern exerted by α-MSH in TNF-α- or IL-1β-stimulated cells leave some uncertainty on the role of the peptide on chondrocyte modulation.

Fig. 1

Effect of α-MSH on IL-6 and IL-8 secretion (a) and gene expression (b) in IL-1β-stimulated chondrocytes. Human primary chondrocytes were incubated for 4 h at 37 °C with 10⁻⁶M α-MSH and further cultured for 40 h in the presence or absence of 10 ng/mL IL-1β. IL-6 and IL-8 concentration in the supernatans (a) and gene expression (b) were then measured. Data from 4 separate experiments (± S.E.M.) are shown. *p < 0.0001 vs Medium

Fig. 2

Effect of α-MSH on IL-6 and IL-8 secretion (a) and gene expression (b) in TNF-α-stimulated chondrocytes. Human primary chondrocytes were incubated for 4 h at 37 °C with 10⁻⁶M α-MSH and further cultured for 40 h in the presence or absence of 10 ng/mL TNF-α. IL-6 and IL-8 concentration in the supernatans (a) and gene expression (b) were then measured. Data from 4 separate experiments (± S.E.M.) are shown. *p < 0.0001 vs Medium; ° p < 0.05 vs TNF-α

Fig. 3

Effect of α-MSH on MMP-3 secretion (a) and gene expression (b) in IL-1β or TNF-α-stimulated chondrocytes. Human primary chondrocytes were incubated for 4 h at 37 °C with 10⁻⁶M α-MSH and further cultured for 40 h in the presence or absence of 10 ng/mL IL-1β or TNF-α. MMP-3 concentration in the supernatans (a) and gene expression (b) were then measured. Data from 4 separate experiments (± S.E.M.) are shown. * p < 0.0001 vs Medium; ^§ p < 0.001 vs IL-1β; ° p < 0.01 vs TNF-α; °° p < 0.001 vs TNF-α

Fig. 4

Effect of α-MSH on MMP-13 secretion (a) and gene expression (b) in IL-1β or TNF-α-stimulated chondrocytes. Human primary chondrocytes were incubated for 4 h at 37 °C with 10⁻⁶M α-MSH and further cultured for 40 h in the presence or absence of 10 ng/mL IL-1β or TNF-α. MMP-13 concentration in the supernatans (a) and gene expression (b) were then measured. Data from 4 separate experiments (± S.E.M.) are shown. * p < 0.0001 vs Medium; °p < 0.05 vs TNF-α

Fig. 5

Effect of α-MSH on TIMP-3 secretion (a) and gene expression (b) in IL-1β or TNF-α-stimulated chondrocytes. Human primary chondrocytes were incubated for 4 h at 37 °C with 10⁻⁶M α-MSH and further cultured for 40 h in the presence or absence of 10 ng/mL IL-1β or TNF-α. TIMP-3 concentration in the supernatans (a) and gene expression (b) were then measured. Data from 4 separate experiments (± S.E.M.) are shown. *P < 0.002 vs Medium; **p < 0.001 vs Medium; ° p < 0.02 vs Medium; °° p < 0.002 vs Medium; ^§ p < 0.05 vs IL-1β

Fig. 6

Effect of α-MSH on TIMP-4 secretion (a) and gene expression (b) in IL-1β or TNF-α-stimulated chondrocytes. Human primary chondrocytes were incubated for 4 h at 37 °C with 10⁻⁶M α-MSH and further cultured for 40 h in the presence or absence of 10 ng/mL IL-1β or TNF-α. TIMP-4 concentration in the supernatans (a) and gene expression (b) were then measured. Data from 4 separate experiments (± S.E.M.) are shown. *p < 0.02 vs Medium; ° P < 0.05 vs IL-1β

Fig. 7

Effect of α-MSH on NO secretion and iNOS gene expression in IL-1β or TNF-α-stimulated chondrocytes. Human primary chondrocytes were incubated for 4 h at 37 °C with 10⁻⁶M α-MSH and further cultured for 40 h in the presence or absence of 10 ng/mL IL-1β or TNF-α. NO concentration in the supernatans (a) and iNOS gene expression (b) were then measured. Data from 4 separate experiments (± S.E.M.) are shown. *p < 0.0001 vs Medium; ^§ p < 0.005 vs IL-1β; °p < 0.005 vs TNF-α