title: Identification of Novel Chondroprotective Mediators in Resolving Inflammatory Exudates.
abstract: We hypothesized that exudates collected at the beginning of the resolution phase of inflammation might be enriched for tissue protective molecules; thus an integrated cellular and molecular approach was applied to identify novel chondroprotective bioactions. Exudates were collected 6 h (inflammatory) and 24 h (resolving) following carrageenan-induced pleurisy in rats. The resolving exudate was subjected to gel filtration chromatography followed by proteomics, identifying 61 proteins. Fractions were added to C28/I2 chondrocytes, grown in micromasses, ions with or without IL-1β or osteoarthritic synovial fluids for 48 h. Three proteins were selected from the proteomic analysis, α1-antitrypsin (AAT), hemopexin (HX), and gelsolin (GSN), and tested against catabolic stimulation for their effects on glycosaminoglycan deposition as assessed by Alcian blue staining, and gene expression of key anabolic proteins by real-time PCR. In an in vivo model of inflammatory arthritis, cartilage integrity was determined histologically 48 h after intra-articular injection of AAT or GSN. The resolving exudate displayed protective activities on chondrocytes, using multiple readouts: these effects were retained in low m.w. fractions of the exudate (46.7% increase in glycosaminoglycan deposition; ∼20% upregulation of COL2A1 and aggrecan mRNA expression), which reversed the effect of IL-1β. Exogenous administration of HX, GSN, or AAT abrogated the effects of IL-1β and osteoarthritic synovial fluids on anabolic gene expression and increased glycosaminoglycan deposition. Intra-articular injection of AAT or GSN protected cartilage integrity in mice with inflammatory arthritis. In summary, the strategy for identification of novel chondroprotective activities in resolving exudates identified HX, GSN and AAT as potential leads for new drug discovery programs.
authors.affiliation: William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and m.kaneva@qmul.ac.uk m.perretti@qmul.ac.uk.
authors.forename: Magdalena K
authors.initials: MK
authors.lastname: Kaneva
authors.affiliation: William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and.
authors.forename: Karin V
authors.initials: KV
authors.lastname: Greco
authors.affiliation: William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and.
authors.forename: Sarah E
authors.identifier: 0000-0003-4819-7132
authors.initials: SE
authors.lastname: Headland
authors.affiliation: William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and.
authors.forename: Trinidad
authors.initials: T
authors.lastname: Montero-Melendez
authors.affiliation: UCB Celltech, Slough SL1 3WE, United Kingdom.
authors.forename: Prashant
authors.identifier: 0000-0003-1696-0068
authors.initials: P
authors.lastname: Mori
authors.affiliation: UCB Celltech, Slough SL1 3WE, United Kingdom.
authors.forename: Kevin
authors.identifier: 0000-0002-6782-6611
authors.initials: K
authors.lastname: Greenslade
authors.affiliation: William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and.
authors.forename: Costantino
authors.identifier: 0000-0003-1326-5051
authors.initials: C
authors.lastname: Pitzalis
authors.affiliation: UCB Celltech, Slough SL1 3WE, United Kingdom.
authors.forename: Adrian
authors.identifier: 0000-0001-5560-300X
authors.initials: A
authors.lastname: Moore
authors.affiliation: William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom; and m.kaneva@qmul.ac.uk m.perretti@qmul.ac.uk.
authors.forename: Mauro
authors.identifier: 0000-0003-2068-3331
authors.initials: M
authors.lastname: Perretti
mesh_terms: D000818:Animals; D001169:Arthritis, Experimental / Q000473:pathology*; D019902:Chondrocytes / Q000187:drug effects*; D004195:Disease Models, Animal; D005122:Exudates and Transudates / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D020370:Osteoarthritis, Knee / Q000473:pathology; D010998:Pleurisy / Q000276:immunology*; D040901:Proteomics; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction