Abstract
BackgroundAlpha-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).MethodsHuman 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.ResultsGene 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.ConclusionsOur 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.
Highlights
Effect of pro-inflammatory cytokines on human chondrocytes Of the twelve cytokines evaluated in the supernatants of IL-1β or tumor necrosis factor-α (TNF-α)-stimulated chondrocytes with a qualitative ELISA test (IL-1α, IL-1ß IL-2, IL-4, IL-6, IL-8, IL10, IL-12, IL-17α, IFNγ, TNF-α, GM-CSF), significant levels were observed only for IL-6 and IL-8
Effect of α-Melanocyte-stimulating hormone (α-MSH) on human chondrocytes Pre-treatment of chondrocytes with 10−6M α-MSH did not modify the release of IL-6 or IL-8 by IL-1ß (Fig. 1) or TNF-α (Fig. 2) stimulated chondrocytes
Α-MSH did not modify the release of matrix metalloproteinases (MMPs)-13 by cytokine-stimulated chondrocyte but the peptide caused a decreased gene expression of the molecule that was significant for TNF-α-stimulated cells (Fig. 4)
Summary
The assumed mechanisms involved in chondrocyte dysregulation and/or apoptosis include mechanical stress, age-related functional changes and altered production of pro-inflammatory cytokines, predominantly interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), that induce production of oxygen radicals and proteinases such as matrix metalloproteinases (MMPs) and aggrecanases [2]. These observations suggested that anti-cytokine and anti-oxydant compounds could have chondroprotective effects providing novel therapeutic opportunities for OA treatment [3, 4]. The anti-inflammatory action of the peptide depends primarily on inhibition of cytokine production by target cells, other leukocyte functions, including reactive oxygen intermediate (ROI) production, nitric oxide (NO) generation and release of proteolytic enzymes, are likewise influenced [7,8,9]
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