title: 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.
authors.affiliation: Allergy, Clinical Immunology & Rheumatology Unit, Istituto Auxologico Italiano, IRCCS, University of Milan, Piazzale Brescia, 20 - 20149, Milano, Italy. franco.capsoni@unimi.it.
authors.forename: Franco
authors.initials: F
authors.lastname: Capsoni
authors.affiliation: Allergy, Clinical Immunology & Rheumatology Unit, Istituto Auxologico Italiano, IRCCS, University of Milan, Piazzale Brescia, 20 - 20149, Milano, Italy. anna.ongari@unimi.it.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Ongari
authors.affiliation: Center for Preclinical Investigation, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy. caterina.lonati@libero.it.
authors.forename: Caterina
authors.initials: C
authors.lastname: Lonati
authors.affiliation: Traumatology and First Aid Unit, Istituto Ortopedico Galeazzi, IRCCS, Milan, Italy. riccardo.accetta@grupposandonato.it.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Accetta
authors.affiliation: Center for Preclinical Investigation, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy. stefano.gatti@unimi.it.
authors.forename: Stefano
authors.initials: S
authors.lastname: Gatti
authors.affiliation: Center for Preclinical Investigation, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy. annapia.catania@guest.unimi.it.
authors.forename: Anna
authors.initials: A
authors.lastname: Catania
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology*; D053583:Interleukin-1beta / Q000494:pharmacology*; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D015207:Osteoarthritis, Hip / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000893:Anti-Inflammatory Agents; C508606:CXCL8 protein, human; C508600:IL6 protein, human; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; D019714:Tissue Inhibitor of Metalloproteinases; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D020782:Matrix Metalloproteinases