Inflammation in OA. Osteoarthritis. Crystals found in synovial fluid. Calcium-containing crystals in OA 10/28/2013. I have no disclosures

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1 Dublin Academic Medical Centre Dublin Academic Medical Centre How Do Calcium Crystals Induce Inflammation and Contribute to Osteoarthritis I have no disclosures Geraldine McCarthy MD, FRCPI Clinical Professor of Medicine DAMC 2009 Oct 28,2013 DAMC 2009 Osteoarthritis Most common form of arthropathy Increasing prevalence as population ages Complex etiology (obesity, genetics, trauma, inflammation) Involves all joint components No known drug slows or reverses pathologic process in OA Great need for drug discovery in OA Inflammation in OA Sohn et el Crystals found in synovial fluid Monosodium urate monohydrate (MSU) Calcium pyrophosphate dihydrate (CPP) Basic calcium phosphate (hydroxyapatite, octacalcium phosphate) BCP/AF M Calcium-containing crystals in OA SF studies demonstrate calcium crystals in 30-60% of unselected OA patients BCP crystals found in 100% and CPP in 20% of knee and hip cartilages removed at joint replacement Correlates with clinical symptoms and histological grade of disease Symptom modification by colchicine in OA of the knee 1

2 Once in a blue moon, crystals are considered in an osteoarthritis talk! Crystals contribute to cartilage damage through multiple mechanisms They directly elicit catabolic cytokines and enzymes from cartilage and synovium. They produce inflammatory responses. They cause damage by mechanically eroding tissue. Pathophysiology of Inflammation and Cartilage Degeneration (CPP) Inflammatory host response to CPP crystals Phagocytosis of crystals by neutrophils results in release of lysosomal enzymes and cell-derived chemotactic factors Phagocytosis by synovial-lining cells leads to cell proliferation and release of prostaglandins, cytokines and matrix metalloproteases capable of matrix degradation EULAR Proposed Classification of the Main Clinical Phenotypes of CPP Crystal Deposition Asymptomatic CPPD (fortuitous imaging finding) Osteoarthritis with CPPD Acute CPP crystal arthritis, self-limiting synovitis ( pseudogout ) Chronic CPP crystal inflammatory arthritis (chronic oligoarthritis or polyarthritis with inflammatory symptoms and signs. DDx RA) (Not specified pseudoneuropathic, pseudo PMR) Whelan BR et al Rheumatology 2008;47:

3 Physical characteristics of CPP Small crystals more inflammatory than large ones Monoclinic (elongated rod-like) more inflammatory than triclinic ( squat rhomboid or cuboid) Triclinic final most stable form May result from mechanical effects (eg greater ease of phagocytosis) or greater surface area/weight presented for protein or membrane interaction Calcium crystals from OA cartilage CPP crystals extracted from cartilage samples from patients undergoing joint replacement Primary chondrocyte and synovial fibroblast culture CPP elicited increase in NO, MMP-13 and PGE2 Suggests contribution to cartilage degradation and synovitis in OA L i BCP crystals Identifiable in the majority of OA joints Partially carbonate substituted hydroxyapatite, octacalcium phosphate Identification difficult (ultramicroscopic) Present in 100% cartilages from joints undergoing joint replacement (Fuerst M et al) Generated locally within the joint Associated with more severe radiographic joint degeneration and larger joint effusions compared with joints without crystals Rosenthal AK et al Arthritis Rheum 2008 The synovium in OA Major site of gross and microscopic inflammatory change Hyperplasia of synovial lining cells with infiltration of inflammatory cells, especially macrophages Early cartilage degradation events may drive synovial inflammation Activation of innate immunity (patternrecognition receptors responding to PAMPS/DAMPS Animal studies BCP crystals injected into murine knee joints Synovial inflammation with synovial macrophage infiltration,persisted at day 30 Cartilage degradation (by loss of proteoglycan staining by Safranin-O,concomitant expression of VDIPEN epitopes) Increased chondrocyte apoptosis. EA HK et al.plos One. 2013; 8(2): e Animal studies (cont.) Lapine OA ( partial lateral meniscectomy, section fibular ligaments) IA injections 1 mg or 10 mg CPPD, weekly for 8 weeks Resulted in more severe OA than in meniscectomized but non-injected knees Fam AG et al Arthritis Rheum 1995;38:

4 COX1 Expression/control COX2 Expression/control BCP crystals in vitro Induce mitogenesis, activation of NF-κB, AP-1, PKC, MAPK Upregulate matrix metalloproteinase (MMP) production (MMP-1,3,8,9 and 13), downregulate TIMPs. Stimulate COX 1 and 2 and prostaglandin E production Induce nitric oxide (NO) production. Stimulate cytokine production:incl. IL-1β,IL-18 MMP-1 protein secretion human fibroblasts COX-1 mrna Expression COX2 mrna Expression Untreated BCP4 BCP7 BCP24 BCP32 PMA 0 Untreated BCP1 BCP4 BCP7 BCP24 BCP32 n=3, p<0.05 vs untreated n=3, p<0.05 vs Untreated 4

5 PGE2 pg/ug/control BCP crystal PGE 2 induction Control B4 B8 B24 B32 Hamilton JA et al Arthritis Res 2001 n=4, p<0.05 vs control BCP Crystals Lysosome Cytokines in OA/innate immunity PGE 2 COX-2 COX-1 PGH 2 EP4 PGE 2 - mpges1 + PGI 2 PGE 2 camp + PGE 2 + EP2 MMP-3 MMP MMP-13 Synovial inflammation IL-1b expression increased in OA synovium Upregulates MMPs, aggecanases Suppresses ECM biosynthesis Induces inflammatory mediators and cell infiltration IL-18 expression increased in OA synovium Inhibits aggrecan synthesis Induces PG production Molloy ES et al Ann Rheum Dis 2008;67: SIGNAL 1 NLRP3 SIGNAL 2 Pore-forming toxins ATP Crystals BCP Crystals induce IL-1β production in the absence of LPS K + Efflux Lysosomal Rupture Cathepsin release ROS generation Adapted from Bryant and Fitzgerald, Trends in Cell Biology (2009) 5

6 mrna Fold Change & S100A9 BCP injection into joint increases serum levels of S100A9 (Narayan et al, 2011) and S100A9 are damage-associated molecules Found in high amounts in synovial fluid of OA patients Endogenous activators of TLR4 Exhibit TLR-4 dependent catabolic effect on human chondrocytes (Schelbergen RF et al, 2012) Ctl BCP (50 μg/ml) Sup Sup + + BCP - + Piceatannol (50 μm) Piceatannol (100 μm) Supernatant BCP crystals induce can serve as Signal 1 in vitro Ctl BCP (50 μg/ml) BCP Piceatannol (50 μm) Piceatannol (100 μm) Sup Sup Supernatant BCP Piceatannol (50μM) Piceatannol (100μM) LY (50μM) PD98059 (100μM) is induced downstream of: Syk, PI3K, ERK and BCP induce IL-1β production SIGNAL 1 BCP Crystals S100A 8 SY K PI3K SIGNAL 2 Caspase 1 activation S100A 8 Pro-IL-1β, Pro-IL-18 TLR4 mil-1β/18 Pro-IL-1β, Pro-IL-18 6

7 How calcium crystals contribute to OA Hope for the future? Bone Hypertrophic Synovium lining Degenerate cartilage PG MMP CYTOKINES BCP CPPD Chondrocyte Fibroblast-like synoviocyte (FLS) What needs to be done We need a reliable and accurate bed-side test for synovial fluid BCP crystals Need further understanding of the biological effects of calcium crystals Need stronger link with OA scientific community as opportunities are missed Need to understand that the complexity OA dictates necessity for heterogeneity of treatment Acknowledgements Supported by: a research grant for the Immunology Research Centre from Science Foundation Ireland and a HRB Health Research Award 7

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