title: Quantitative Mass Spectrometry To Study Inflammatory Cartilage Degradation and Resulting Interactions with the Complement System.
abstract: Joint diseases are often characterized by inflammatory processes that result in pathological changes in joint tissues, including cartilage degradation and release of components into the synovial fluid. The complement system plays a central role in promoting the inflammation. Because several cartilage proteins are known to interact with complement, causing either activation or inhibition of the system, we aimed to investigate these interactions comprehensively. Bovine cartilage explants were cultured with IL-1α to induce cartilage degradation, followed by incubation with human serum. Label-free selected reaction monitoring mass spectrometry was used to specifically quantify complement proteins interacting with the cartilage explant. In parallel, the time-dependent degradation of cartilage was detected using mass spectrometry analysis (liquid chromatography-tandem mass spectrometry). Complement proteins resulting from activation of the classical, alternative, and terminal pathways were detected on IL-1α-stimulated cartilage at time points when clear alterations in extracellular matrix composition had occurred. Increased levels of the complement activation product C4d, as detected by ELISA in serum after incubation with IL-1α-stimulated cartilage, confirmed the selected reaction monitoring results indicating complement activation. Further, typical activated (cleaved) C3 fragments were detected by Western blotting in extracts of IL-1α-stimulated cartilage. No complement activation was triggered by cartilage cultured in the absence of IL-1α. Components released from IL-1α-stimulated cartilage during culture had an inhibitory effect on complement activation. These were released after a longer incubation period with IL-1α and may represent a feedback reaction to cartilage-triggered complement activation observed after a shorter incubation period.
authors.affiliation: Division of Medical Protein Chemistry, Department of Translational Medicine, Lund University, S-205 02 Malmö, Sweden.
authors.forename: Camilla
authors.identifier: 0000-0001-5487-4270
authors.initials: C
authors.lastname: Melin Fürst
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences Lund, Lund University, S-221 84 Lund, Sweden; and.
authors.forename: Emma
authors.initials: E
authors.lastname: Åhrman
authors.affiliation: Division of Medical Protein Chemistry, Department of Translational Medicine, Lund University, S-205 02 Malmö, Sweden.
authors.forename: Klas
authors.identifier: 0000-0003-0930-2390
authors.initials: K
authors.lastname: Bratteby
authors.affiliation: Department of Immunotechnology, Lund University, S-223 81 Lund, Sweden.
authors.forename: Sofia
authors.identifier: 0000-0002-9896-4064
authors.initials: S
authors.lastname: Waldemarson
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences Lund, Lund University, S-221 84 Lund, Sweden; and.
authors.forename: Johan
authors.identifier: 0000-0002-2889-7169
authors.initials: J
authors.lastname: Malmström
authors.affiliation: Division of Medical Protein Chemistry, Department of Translational Medicine, Lund University, S-205 02 Malmö, Sweden; Anna.Blom@med.lu.se.
authors.forename: Anna M
authors.identifier: 0000-0002-1348-1734
authors.initials: AM
authors.lastname: Blom
mesh_terms: D000818:Animals; D002356:Cartilage / Q000378:metabolism* / Q000473:pathology*; D002417:Cattle; D015935:Complement C4b; D003165:Complement System Proteins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000378:metabolism* / Q000473:pathology; D053582:Interleukin-1alpha / Q000378:metabolism; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000097:blood
chemical_list: D016326:Extracellular Matrix Proteins; D053582:Interleukin-1alpha; D010446:Peptide Fragments; D015935:Complement C4b; C032261:complement C4d; D003165:Complement System Proteins