© 2011 Nature America, Inc. All rights reserved. Switzerland. Switzerland. Surgery, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. Center, Aurora, Colorado, USA. USA. School of Medicine, Stanford, California, USA. Functional Neuroscience Laboratory, Stanford University School of Medicine, Stanford, California, USA. School, Boston, USA. Massachusetts, Clinic, Cleveland, Ohio, USA. 4 Rheumatology, Stanford University School of Medicine, Stanford, California, USA. 1 gain insight into we osteoarthritis, used mass to spectrometry identify to Seeking that treatments prevent of the development osteoarthritis. The pathogenesis of osteoarthritis is unclear, and there are currently no synovial joints has a key role in the pathogenesis of osteoarthritis. Our findings indicate that dysregulation of complement in (ERK) around chondrocytes in human osteoarthritic cartilage. (MMP13) and with activated extracellular signal-regulated kinase Further, MAC colocalized with matrix metalloprotease 13 induced production of these molecules in cultured chondrocytes. from C5-deficient mice than C5-sufficient mice, and MAC molecules was lower in chondrocytes from destabilized joints deficient mice. Expression of inflammatory and degradative wild-type mice confirmed the results obtained with genetically osteoarthritis. Pharmacological modulation of complement in development of arthritis in three different mouse models of complex (MAC)-mediated arm of complement, is crucial to the show that complement, specifically, the membrane attack (C5), C6 or the complement regulatory CD59a, we Using mice genetically deficient in complement is abnormally high in human osteoarthritic joints. with osteoarthritis, we find that expression and activation of analyses of synovial fluids and membranes from individuals of osteoarthritis. Through proteomic and transcriptomic role for the inflammatory in the pathogenesis in osteoarthritis, its role is unclear ‘wear and tear’ cartilage in synovial joints, has long been viewed as the result of Osteoarthritis, characterized by the breakdown of articular Reuben Gobezie Stuart B Goodman Inyong Hwang D Meegan Larsen Qian Wang osteoarthritis of role a Identification for central complement in nature medicine nature Received 6 April; accepted 3 October; published online 6 November 2011; Center Center for Proteomics and Case Bioinformatics, Western Reserve University School of Medicine, Cleveland, Ohio, USA. Geriatric Research Education and Clinical Centers, Veteran’s Affairs Palo Alto Health Care System, Palo Alto, California, USA. 11 Department Department of Rheumatology, Hospital for Special Surgery, New York, New York, USA. 16 These These authors contributed equally to this work. should Correspondence be addressed to W.H.R. ([email protected]). 1 , 1 2 . Although low-grade is detected , 16 1

, , , Andrew L Rozelle 2 14 advance online publication online advance , , Heidi H Wong 1 , Mary , K Mary Crow , 9 2 , , Tony Wyss-Coray , , James F Crish 6 Department Department of Medicine and Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical 13 Department Department of Medicine, University of Colorado Health Sciences Center, Aurora, Colorado, USA. 7 Rheumatology Rheumatology Unit, Department of Clinical and Experimental Medicine, University of Padova, Padova, Italy. 2– 4 . Here we identify a central 10 1 11 , Department Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California, 2 1 4 , Leonardo , Punzi Leonardo , , V Michael Holers , , , Gurkan Bebek 2 , 16 1 , Christin , M Christin Lepus , 10 , , Steven R Goldring

4 d 7 , o

5 , , Angelo Encarnacion i 12 : , , Susan Y Ritter 1 0

, 13 . 3 1 Rush Rush University Medical Center, Section of Rheumatology, Chicago, Illinois, USA. 0 , , David M Lee found that of the complement system are differentially differentially are system complement the of We proteins that osteoarthritis. found with individuals joints—of synovial that the fluid bathes fluid—the synovial the in expressed aberrantly proteins ment components, we analyzed the expression of encoding encoding genes complement of expression the analyzed we components, ment findings ous with end from individuals ( cartilage in chondrocytes the around and not shown) (data synovium the in MAC of presence the revealed analysis tochemical ( osteoarthritis of phases late at albeit and persists, a of course the lower during osteoarthritis level, the in early joints synovial in occurs activation complement Thus, early oste stage with individuals from fluids synovial in higher ­significantly ( of concentrations the that ( osteoarthritis with MAC were all aberrantly expressed in synovial fluids from individuals central components C3 and C5, and the C5, C7 and C9 components ofthe pathways, B) (factor alternative and ) and (C1s classical the C5b effector complement the prising (com MAC of formation and C5a the of activation mediate that enzymes are which convertases, C5 and C3 the of tion system consists of pathways three distinct that fluids converge at the forma synovial osteoarthritic in proteins ment we fluids previously 10 detected of these 12 expressed differentially comple­ synovial healthy to ( compared osteoarthritic in expressed 15 1 3 Supplementary Table Supplementary 1 , Novartis Novartis Institutes for Biomedical Research, Novartis Pharma, AG, Basel, 8 2 o eemn wehr h snvu i a ore f comple of source a is synovium the whether determine To showed analysis ELISA an results, proteomic our Validating / , , Carla R Scanzello n 11 m . , , Nirmal K Banda 2 12 5 4 Department Department of Immunology, University of Colorado Health Sciences o 3 arthritis than in synovial fluids from healthy individuals. individuals. healthy from fluids synovial in than arthritis 7– - related proteins (those identified in synovial fluid; fluid; synovial in identified (those proteins related 6 9 , , Tamsin M Lindstrom . 9 Department Department of Orthopaedic Surgery, Stanford University 6 , 15 Fig. 1 Fig. 8 & William H Robinson , , Mehrdad Shamloo ). ). Using proteomic less sensitive techniques, - a 3 stage osteoarthritis, consistent with previ stage osteoarthritis, and and , , Jason J Song 12 5 Fig. 1 Fig. Genomic Genomic Medicine Institute, Cleveland , 13 Supplementary Table 1 Supplementary Fig. 1 Fig. , , Joshua M Thurman 2 c Division Division of Immunology and ). Additionally, an immunohis an Additionally, ). 14 - 9) ( 9) b Department Department of Orthopaedic ) and C5b and ) Fig. 1 Fig. 1 , 1 2 , , 2 8 ,

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© 2011 Nature America, Inc. All rights reserved. C5 (C5 complement the ( cascade of nexus the at located is effector C5 the Because requires meniscectomy, which is a risk factor for knee osteoarthritis meniscectomy medial by induced osteoarthritis of model mouse a used we osteoarthritis, by activation. complement excessive to osteoarthritis contributing in role pathogenic a on take may membrane vial ( edly lower, in comparedosteoarthritic to healthy synovial membranes C4 H, factor clusterin, inhibitors complement the encoding transcripts of expression and higher, markedly was C5 and C9 B, factor C4A, C7, effectors ment 1 Fig. ( individuals from healthy the profiles all containing cluster one and stage) end and early (both osteoarthritis with individuals from one profiles expression clusters: the all major containing two cluster revealed clustering hierarchical pervised unsu by Analysis individuals. healthy from and osteoarthritis with Supplementary osteoarthritis. OA, binding; mannose MB, text. blue in shown are inhibitors complement and text, red in shown ( fluid synovial healthy to compared fluid synovial osteoarthritic in expressed differentially proteins complement-related the encoding genes of set the to limited was analysis Our determined). (experimentally osteoarthritis end-stage or early- with individuals from and Omnibus) Expression ( bar, 100 Scale osteoarthritis. with individuals different four from samples in seen that of representative is Staining controls. negative as antibodies hoc post ** cascade. complement the of activation during C3 from generated is that C3a of form ( osteoarthritis early-stage with individuals from and ( arginine des C3a of quantification ( complex. attack membrane MAC, P, D, B, receptor; C5a factors P, C5aR, D, I; B, H and protein; complement I: C4BP,H, C4-binding 1; member inhibitor), G (C1 clade inhibitor, peptidase serpin SERPING1, protease; serine lectin-associated MASP, lectin; mannose-binding binding mannose- MBL, fluid. synovial osteoarthritic in expressed aberrantly as identified inhibitors and effectors complement the denote circles blue-filled 1 Figure s r e t t e l  formation osteophyte loss, cartilage less substantially showed mice Supplementary Table 1 Supplementary e Fig. 1

) Cluster analysis of gene-expression profiles in microarray data sets from synovial membranes from healthy individuals (downloaded from the NCBI NCBI the from (downloaded individuals healthy from membranes synovial from sets data microarray in profiles gene-expression of analysis ) Cluster d a To investigate the role of complement in the pathogenesis of of pathogenesis the in complement of role the investigate To - − sufficient (C5 sufficient , , lacking the ability to secrete the C5 proprotein from cells) . oal, xrsin f rncit ecdn te comple the encoding transcripts of expression Notably, ). e SERPING1 Chemoattraction and

test. ( test. Classical pathway Complement components are aberrantly expressed and activated in human osteoarthritic joints. ( joints. osteoarthritic human in activated and expressed aberrantly are components Complement inflammation Fig. Fig. 1 Anti-MAC Supplementary Supplementary Fig. 1 C4BP d C1q a ) Immunohistochemical staining of MAC in cartilage from individuals with end-stage osteoarthritis. We used isotype-matched isotype-matched We used osteoarthritis. end-stage with individuals from cartilage in MAC of staining ) Immunohistochemical C1r ), ), we in induced surgically C5 osteoarthritis C5aR

Table 1 Table C1s + ) ) wild C5a C4 ). The scale bar represents the fold change in gene expression compared to the reference control. Complement effectors are are effectors Complement control. reference the to compared expression gene in change fold the represents bar scale The ). MB-lectin 1 pathway C2 - CD5 0 binding protein and C1 inhibitor was mark was inhibitor C1 and protein binding MASP-1 MASP-2 . In humans, tearing of the meniscus often often meniscus the of tearing humans, In . MBL - ) in synovial membranes from individuals individuals from membranes synovial in ) C5b C5 C3 9 type mice. Sixteen weeks after surgery, C5 surgery, after weeks Sixteen mice. type C6 C9 C8 C7 P D b ) and the soluble form of MAC (complement effector C5b-9) ( C5b-9) effector (complement MAC of form soluble the ) and ). Our results suggest that the syno Isotype control B H MAC (C5b-9) Alternative pathway I Fig. Fig. 1 e and n = 52) or end-stage osteoarthritis ( osteoarthritis end-stage or = 52) b Supplementary Supplementary C3a des arginine (ng ml–1) 12,000 10,000 2,000 4,000 6,000 8,000 e 0 - Mostly Mostly deficient deficient complement complement 1 Health

2 effectors inhibitors and

1 OA early 6 1 − y ­ ­ ­ ­

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OA early 9 ** OA early 2 wild ( 2d Fig. ovitis induced by medial meniscectomy ( were protected against the ( development MAC of the both of osteoarthritis and component syn integral an C6, in deficient mice the complement is cascade important We in osteoarthritis. found that wild in osteoarthritis of CR2 Administration oarthritis. of C3 and activation protein that C5 fusion inhibits (ref. wild in (ref. C5 to antibody monoclonal izing in C5 the findings (ref. C3 of absence the in even activation proceed C5 to allowing C3, of lack the for compensate factors tion in operate mechanisms that compensatory tion by observa the explained can be osteoarthritis against not protected in deficiency genetic by affected not was model this in osteoarthritis contrast, By C5 than and synovitis development of osteoarthritis in humans Early-stage Early- andend-stageOA Fig. 1 OA early 3 We next determined whether the MAC the whether determined Wenext Not only meniscectomy but also meniscal tearing can lead to the the to lead can tearing meniscal also but meniscectomy only Not

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OA end 9 OA C3 OA end 3 advance online publication online advance OA early 7 (data not shown). The fact that that fact The shown). not (data −

c OA end 2 congenic mouse strain, treatment with a with neutral treatment strain, mouse congenic ) in synovial fluids from healthy individuals ( individuals healthy from fluids synovial ) in Fig. 2 Fig. OA end 4 -

+ OA end 6 type mice ( mice type Fig. 2g Fig. mice ( mice OA early 8 –1 c OA end 7 C5b-9 (ng ml ) 10,000 c a 2,000 4,000 6,000 8,000 ). We also tested the effect of CR2 of effect the tested also We ). ) Schematic of the complement cascade; cascade; complement the of ) Schematic Healthy 6

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© 2011 Nature America, Inc. All rights reserved. mice and at serial time points after medial meniscectomy. * t images, 200 arrowheads indicate areas of cartilage degeneration. Scale bars in the low-magnificationarticular cartilage (top) at images,serial 500time points after medial meniscectomy. front,Representative right front,toluidine-blue–stained left hind, rightsections hind. of the* medial region of stifleleft jointshind; Aaare strideshown; pattern: right front, right hind, left front, leftmice hind;12 weeksCa strideafter pattern:medial meniscectomyright front, ( left front, right hind, left Figurehind; 3 and Cb stride pattern: left spontane developed that osteoarthritis milder the accentuated also DMM ( in phenotype CD59a accentuated mice to the subjected osteoarthritic osteoarthritis of model (DMM) meniscus of medial the role in destabilization the of the complement 500 (top) images, shown in of degeneration the cartilage of sections the of region medial from joints stifle in toluidine-blue–stained degeneration to subjected meniscectomy.medial ( ( C6 from joints ( PBS or CR2-fH ( 750 with intraperitoneally treated in shown ( meniscectomy. medial to subjected mice C5 from joints stifle of region medial the of sections blue–stained toluidine- in degeneration cartilage ( models. mouse different three in osteoarthritis of development the for crucial is acting through its MAC effector arm, Figure 2 nature medicine nature operated mice and test comparing C5 g b d a a ) Representative cartilage degeneration in toluidine-blue–stained sections of sections the of region medial from joints stifle in toluidine-blue–stained degeneration cartilage ) Representative Frequency of use (%) 250 with intravenously treated then and meniscectomy medial to subjected mice wild-type in degeneration cartilage the of ) Quantification degeneration cartilage the of ) Quantification 100 20 40 60 80 0

Fig. Fig. 2i C5 deficiency protects against the progressive development of osteoarthritic joint pathology and gait dysfunction. ( The complement cascade, n a Ca C5 = 6 for C5 ( C5 C5 n + µ = 5 mice per group). ( group). per = 5 mice (12 weeksaftersurgery) – + m. ( + , and C6 and j a n and Cb ) Representative ) Representative c Stride pattern = 5 mice per group). ( group). per = 5 mice n µ + ) Quantification of the cartilage degeneration in C5 = 3 for C5 and C5 m; scale bars in the high-magnification (bottom) images, 200 images, m; (bottom) scale bars in the high-magnification

− Supplementary Fig. Supplementary 2b operated mice; at week 12, advance online publication online advance − mice subjected to medial meniscectomy. ( meniscectomy. medial to subjected mice Aa * C5 − operated mice. Data are the mean – + − and C5 and non-operated mice.

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© 2011 Nature America, Inc. All rights reserved. cellular matrix (ECM) of cartilage to activate complement complement activate to cartilage of (ECM) matrix cellular of components the extra or specific cartilage of ability osteoarthritic in osteoarthritis to tribute joint inflammation severity osteoarthritis the mouse of influence that factors both are which surgery, of time the at age and background genetic their of because probably mice, 4 Fig. C5 whereas ( not did mice synovitis, and loss cartilage C5 however, surgery, after weeks 12 and Eight meniscectomy medial undergone have model DMM the in observed that to similar is latency of period ( meniscectomy medial after weeks 4 and 2 at degeneration cartilage or C5 Time ( gait abnormal an developed mice C5 meniscectomy, medial after weeks Twelve antibody. polyclonal pAb, antibody; monoclonal mAb, significant; not NS, 10 bars, Scale cartilage. osteoarthritic human in colocalization MAC and MMP13 (p-ERK1/2), phospho-ERK2 and phospho-ERK1 ( joints. non-destabilized and destabilized both account ** * experiments. independent two from mice four from results of representative are and triplicates of + s.d. mean the are Data ( C5 from chondrocytes in ( experiments. independent three of representative are and triplicates of ** bar, 50 Scale h. 72 for MAC without or with ( expression COX2 of analysis immunocytochemical and ( expression protein of analysis ELISA ( expression mRNA relative of analysis ** ( osteoarthritis with individuals from fluids synovial in fibromodulin of quantification ( experiments. independent three of values triplicate of mean the are Data PBS. with component hoc post ** controls. negative the EDTA and were PBS controls; positive the were zymosan ( components ECM 20 with ( syn.) (OA synovium or cart.) (OA cartilage osteoarthritic 20 with incubated serum human 67% in MAC) (soluble C5b-9 of ( molecules. catabolic and inflammatory of expression chondrocyte induces MAC and formation, MAC induce 4 Figure s r e t t e l  C5b of formation the induced cartilage osteoarthritic Pulverized n n

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© 2011 Nature America, Inc. All rights reserved. contributes to the development of degenerative diseases, such as as such diseases, age degenerative of development the to contributes which of all effectors, pathology. joint promote complement other and mediators matory the kills matrix produce to them either causes or cells which chondrocytes, on MAC of formation the in results turn in activation Complement unchecked. proceed to tion activa complement permitting in osteoarthritis, tors over inhibitors tissues may contribute to a preponderance local of complement effec comple the trigger may of in joint gene Additionally, expression dysregulation ment cascade. cartilage osteoarthritic in exposed or by released components ECM Cartilage osteoarthritis. of pathogenesis Fos of expression the by inducing to MAC ( ERK2 and ERK1 activated with and MMP13 with colocalized MAC MAC by ( lower also induced is expression whose factors scription ( mice C5 of joints destabilized the mediators from chondrocytes did degradative than and inflammatory these of concentrations ( osteoarthritis against protected joints of lized C5 destabi the from chondrocytes surgery, DMM after weeks Twenty vivo in complement pathogenic osteoarthritis. in signaling amplify to membrane synovial the from duction of complement may thus with synergize complement derived ( effectors complement of expression the cyclo colony 2 (CCL2), ligand cytokines inflammatory (ADAMTSs)), tegrin cartilage encoding genes cytes’ of expression in multiple genes implicated osteoarthritis: chondro the increased MAC Sublytic MAC. of concentrations lytic sub with coated chondrocytes in cultured molecules these encoding of genes Weexpression the examined first in osteoarthritis. enzymes degradative and proinflammatory of expression the induces MAC intact ( morphologically be to seem cartilage osteoarthritic in chondrocytes molecules and catabolic tory proinflamma of expression the acti drive can that MAC pathways signaling sublytic vate whereas death, cell necrotic and lysis cell induces MAC of deposition Extensive cartilage? damage MAC does complement 2 Figs. complement. by activating osteoarthritis of pathophysiology the to contribute may components ECM cartilage nature medicine nature Methods and any associated references are available in the online online at http://www.nature.com/naturemedicine/. the paper the of version in available are references associated any and Methods M disease a as system complement the targeting for rationale a provide findings Our diseases. of list this to added be can osteoarthritis that propose Fig. 4 Fig. ethods We next examined the effect of complement deficiency on the the on deficiency complement of effect the examined next We Our eet idns ugs ta low that suggest findings Recent for the is crucial Here cascade we have complement that the found - related macular degeneration macular related - - oxygenase 2 (ref. (ref. 2 oxygenase Fig. 4 Fig. h Figs. Figs. 1 like and metallopeptidase with thrombospondin type 1 motif 1 motif type thrombospondin with metallopeptidase and like , in situ in expression of these genes in destabilized mouse joints. joints. mouse destabilized in genes these of expression 3 - and and - mediated mediated cell lysis ) findings indicate that MAC is important in mediating mediating in important is MAC that indicate findings ) modifying therapy for osteoarthritis. for therapy modifying Fig. 4 Fig. g - Supplementary Fig. 6 Fig. Supplementary ). mRNA levels of Jun and Fos, proinflammatory tran proinflammatory Fos, and Jun of levels mRNA ). d ( related cartilage damage in osteoarthritis. But how how But osteoarthritis. in damage cartilage related , , Fig. 1 Fig. 4 - h deficient mice, which are deficient in MAC are which mice, deficient and are deficient g and

). In addition, in human osteoarthritic cartilage, cartilage, osteoarthritic human in addition, In ). - erdn enzymes degrading advance online publication online advance d ) and and ) Supplementary Supplementary Fig. 6 26) ( 26) - stimulating factor 1 (CSF1) and CCL5) and CCL5) and 1 (CSF1) factor stimulating 2 9 and stimulate the expression of MMP13 2 Fig. 4d Fig. in vivo in 4 . Because many of MAC the . Because Figs. 2a Figs. 3 0 3 ), kinases that mediate resistance resistance mediate that kinases ), . 1 ( – and Alzheimer’s disease Alzheimer’s and - f Fig. 2e Fig. grade complement activation activation complement grade ). Sublytic MAC also induced induced also MAC Sublytic ). - degrading enzymes, inflam enzymes, degrading 2 17 5 Fig. 4 Fig. , (chemokine (C (chemokine b , 18 and and – ), ), we examined whether , 2 j 2 and and d MP ad disin and (MMPs ); chondrocyte pro chondrocyte ); 3 ), expressed lower lower expressed ), Supplementary Supplementary

27 - - - sufficient sufficient encircled encircled C motif) motif) C , 2 8 3 , were were , 2 . We . ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­

deposition deposition experiments. C.M.L. and J.J.S. performed the immunohistochemical performed the gait analysis studies. Q.W. and H.H.W. performed the conducted the studies of in osteoarthritis mouse models. A.E., andA.L.R. M.S. and Q.W. conducted key studies. Q.W., D.M.A.L.R., H.H.W.Larsen, and W.H.R. andA.L.R. W.H.R. initiated the investigation of complement in osteoarthritis, (V.M.H.); and the Frankenthaler and Kohlberg Foundations (M.K.C.). grant T32 AR007530 (S.Y.R.); National Institutes of Health R01 AR051749 P30 Center Core grant NS069375 (M.S.); US National Institutes of Health training US AR057859 (C.R.S.); National Institute of Neurological Disorders and Stroke MentoredDiseases Scientist Research Clinical Career Development Award K08 Professors and the US National Institute of Musculoskeletal Arthritis, and Skin of Research Rheumatology and the Education Fund, the Association of Specialty Research Fellowship Award, the Atlantic Philanthropies, the American College Award a (A.L.R.); New York Chapter Foundation/MerckArthritis Osteoarthritis a Northern ChapterCalifornia FoundationArthritis Postdoctoral Fellowship Lung, and InstituteBlood Proteomics Center contract N01 HV 28183 (W.H.R.); Merit Award from the Department of Veterans Affairs and the US National Heart, These studies were supported by a Rehabilitation Research and Development Note: Supplementary information is available on the GSE32317. code accession the under Omnibus Expression Gene the in available codes. Accession 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/naturemedicine/. HTML version of the paper at The authors declare competing interests:financial details accompany the full analyzed the data and approved the manuscript.final T.W. input.scientific T.M.L., andA.L.R. W.H.R. wrote the manuscript, and Q.W., C.R.S., provided the C6 provided the antibody to specific C5 and the CR2 stimulation assays using samples provided by S.B.G. V.M.H., J.M.T. and N.K.B. C.M.L., J.J.S.A.L.R., and I.H. performed the and G.B., andR.G. D.M. contributedLee to the analysis of datathese sets. andfluids. C.R.S. M.K.C. performed the gene expression analysis of the synovium, and D.M. performed the Lee ELISA analysis of and osteoarthritic healthy synovial or contributed to the proteomic analysis of synovial fluid. osteoarthritic S.Y.R. analyses of cartilage. J.F.C., G.B., S.Y.R., L.P., andR.G. S.R.G., D.M. conductedLee 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. AUTH A C ckn O

MPETING FINANCIAL INTERESTS FINANCIAL MPETING esn DT Ciia patc. seatrts f h knee. the of Osteoarthritis practice. Clinical D.T. Felson, Cooke, T.D. Significance of immune complex deposits in osteoarthritic cartilage. inosteoarthritic deposits complex immune of Significance T.D. Cooke, A. Corvetta, and immunoglobulins of deposition The O. Ohno, & E.L. innate Bennett, T.D., from Cooke, complements with regulation: T-cell J.P. Atkinson, & C. Kemper, Gobezie, R. C.L. Hill, inflammatory an Osteoarthritis, Pelletier,J.P.,S.B. Abramson, & Martel-Pelletier,J. Osteoarthritis. S.R. Goldring, & M.B. Goldring, J. Rheumatol. J. Rheumatol. trauma. joint acute and osteoarthritis arthritis, rheumatoid by affected cartilage. osteoarthritic in complement immunity. osteoarthritis. and (2007). targets. therapeutic osteoarthritis. knee in loss cartilage and pain to new of selection the for Rheum. Arthritis implication potential disease: (2007). (2006). 841–848 nln, . Lhadr LS Rs fcos o smtmtc ne osteoarthritis knee symptomatic for factors Risk L.S. Lohmander, & M. Englund, Kamekura, S. (2004). meniscectomy. after years twenty-two to fifteen instability. joint knee by induced - C., S.R.G., C., M.K.C., V.M.H.S.R.G., and D.M. edited Lee the manuscript. All authors o O wledgments R R C O et al. et Nat. Rev. Immunol. Rev. Nat.

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© 2011 Nature America, Inc. All rights reserved. ment (12 weeks) starting 1 week after medial meniscectomy. medial after week 1 starting weeks) (12 ment 250 with meniscectomy. We treated 21 medial after week 1 starting weeks) (18 experiment the of end the until week (ref. C5 to specific BB5.1 body 750 with intraperitoneally mice C57Bl/6 osteoarthritis. mouse of Treatment Laboratories. Jackson generations ten for mice C3H/He C6 Laboratories. Jackson from were Hc1H2dH2 C5 congenic A/J studies. DMM C5 the in surgery after studies; we used 20 DMM Cd59a the in surgery after weeks for 12 experiment the continued and 12 ciency: 20 deficiency: C6 20 deficiency: C5 follows: as were surgery after experiment the of duration the and surgery of time the at mice the of age the studies, meniscectomy medial generated the medial meniscectomy medial the generated We of Health. Institutes National US of the guidelines the with accordance in under protocols approved by the Stanford Committee of Animal Research and osteoarthritis. of models mouse Surgical (Invitrogen). antibodies immunofluorescent secondary or Fluor 488–conjugated Fluor 555– Alexa we Alexa used the analysis, For Labs). (Vector Kits ABC Elite VECTASTAIN isotype corresponding the and Signaling) Cell (20G11, phosphor Tyr204 or ERK2 Thr202 on and ylated ERK1 to antibody rabbit (Abcam), MMP13 to body C5b to antibody mouse follows: as were immunofluorescence. and Immunohistochemistry ( s clustering hierarchical unsupervised by profiles expression We transcript genes. for the organized expression average GSE12021) (GEO Omnibus Expression Gene the NCBI the on from array) and (analyzed platform same membranes synovial healthy from data downloaded We GSE32317). (GEO chips 2.0 Plus Human U133 Affymetrix using membranes profiling. Transcriptional C5b and arginine des C3a detect to Complement in synovial fluids. full but no degeneration tears with cartilage for meniscal arthroscopy undergoing undergoing early with patients and from patients joint replacement knee from membranes synovial osteoarthritic obtained we profiling, transcriptional For surgery. replacement joint knee undergoing tis end with patients and imaging, radiographic by or lesions cartilage visible of arthroscopically presence by the 1 year, assessed as early with viduals indi osteoarthritis, knee of symptoms no with individuals healthy from ids flu synovial we obtained analysis, For ELISA the consent. informed subjects’ subjects’ the the obtaining not require did and that Board Review Institutional obtaining Healthcare included that and informed consent as well as under Boards protocols that were approved by Surgery Review the Partners Special for Institutional Hospital the and University Stanford the by approved samples. Human METHODS ONLINE doi:10.1038/nm.2543 o f - t week - w thickness cartilage loss cartilage thickness a r - e control antibodies. For the immunohistochemical analysis, we used used we analysis, immunohistochemical the For antibodies. control - / µ old mice and 16 weeks duration; time course: 20 course: time duration; weeks 16 and mice old c - l g g of CR2 week u 3 - 3 s T18c/nSnJ) and T18c/nSnJ) C5 . . We merged these data sets t e r - / old old mice and 16 weeks duration. We used 15 s o - - We studied human samples under protocols that were were that protocols under samples human studied We - stage knee osteoarthritis with symptoms lasting less than than less lasting symptoms with osteoarthritis knee stage week f - week t fH w Cd59a a 1 r 5 - e - or with PBS once a week until the end of experi end the the until a once PBS week or with old mice and 20 weeks duration; and Cd59a defi Cd59a and duration; weeks 20 and mice old old old mice and continued the experiment for 20 weeks . h 2 t We analyzed RNA from osteoarthritic synovial synovial osteoarthritic from RNA analyzed We 5 m −/− - . week ). - mice were generated as described as generated were mice 38) or an isotype an or 38) We used OptEIA ELISA kits (BD Biosciences) deficient (B10.D2 deficient 3 - - 6 old old wild 9 in synovial fluids. synovial in 9 ; the controls, C3H/He mice, were from from were mice, C3H/He controls, the ; 10 - deficient mice were backcrossed with with backcrossed were mice deficient , 16 e rae 21 treated We 3 4 , - 3 and computed the robust multichip 9 (clone aE11; Dako), rabbit anti rabbit Dako), aE11; (clone 9 5 µ We performed the mouse studies studies mouse the We performed and DMM models DMM and - h g of either the monoclonal anti monoclonal the either of g type type C57Bl/6 mice intravenously t t p : / - / control antibody twice per per twice antibody control - b Hc0H2d - o - stage knee osteoarthritis osteoarthritis knee stage n stage knee osteoarthri knee stage s Primary antibodies antibodies Primary a - i - week . sufficient (B10.D2 sufficient h g - - c T18c/oSnJ) mice T18c/oSnJ) - month week . - j p l wild old 10 / , ~ 16– 3 m - 7 old mice; mice; old - . 1 d old old mice 8 . In the the In . e h - o type type o n - ­ ­ ­ ­ ­ ­ ­ ­ /

II collagenase. We formed sublytic MAC on the surface of the chondro the of surface 0.8 the mixing by on cytes MAC sublytic type and formed We trypsin with collagenase. it II digesting by cartilage osteoarthritic human from chondrocytes. on MAC sublytic of Generation °C, 37 at C5b (Quidel). ELISA measured samples using and tions EDTA the adding added by incubated then reactions Inc.), Wethe stopped (Quidel, Biologicals). serum A (Novus human zymosan normal fibromodulin matrilin or (Sigma), (Pharmacia), Systems) aggrecan 4B (R&D Chondrex), or sepharose (Sigma II added collagen we (Sigma), which to cartilage methods vitro In CatWalk System analysis. Gait summed. were regions six the femoral the of third each in area) surface whole the meaning 3 of score a and area surface the of thirds one meaning 1 of score a (with degeneration cartilage of width × 0–4) of (score degeneration cartilage system scoring described of a version previously a modified using tions of mouse joints with blue. toluidine We degeneration cartilage evaluated osteoarthritis. mouse in degeneration cartilage of Scoring 40. 39. 38. 37. 36. 35. 34. 33. Methods Additional methods. Dunnett’sby a unpaired tailed analyses. Statistical or RNA 18s (2 of those to them normalized and PCR quantitative by joint knee of C5 the from isolated chondrocytes from or cartilage osteoarthritic human from PCR. Quantitative COX2 to antibody goat a with analysis Biotechnology). Cruz PCR (Santa quantitative immunocytochemistry for RNA performed the and isolated amounts, protein of bead sis a for supernatants culture the described harvested as we Chemicals)) (EMD C9 and Chemicals) C8 (EMD (Quidel), (C7 MAC of components complement remaining the of each −

∆ hn Y. Chen, The E.A. Joosten, & G.H. Walenkamp, W.M., Honig, M.A., Marcus, A.F., Gabriel, to antibody monoclonal a of Generation B. Stockinger, & J.D. Lambris, Y., Frei, Holt, D.S. W. Zhou, A.C. Lin, R.A. Irizarry, R. Huber, eie dnrtc el udro auain n idc T1 polarization. Th1 Immunol. induce and maturation undergo cells dendritic derived rat. the in pain CatWalk method: a detailed analysis of behavioral changes after acute inflammatory antibodies. anti-C5 of detection for assay ELISA Probes Cell. Mol. an in application C5 mouse hemoglobinuria. and hemolysis Invest. Clin. J. osteoarthritis. synovial Res. Acids arthritis Nucleic rheumatoid the in profiles expression membrane. mRNA in variances ∆ Ct ). The primers and probes used were from Applied Biosystems. Applied from were used probes and primers The ). opeet ciain assays. activation complement 2 . 3 , we prepared suspensions of pulverized human osteoarthritic osteoarthritic human pulverized of suspensions prepared we ,

t al. et et al. t al. et 37 t al. et t al. et Arthritis Res. Ther. Res. Arthritis post hoc post e nlzd os gi wt te video the with gait mouse analyzed We , 167–176 (2007). 167–176 , t - test and by multiple comparisons one 3 Nat. Med. Nat. deficient and C5 deficient

Targeted deletion of the CD59 gene causes spontaneous intravascular emnl opeet ope Cb9tetd ua monocyte- human C5b-9–treated complex complement Terminal t al. et 9 rdmnn rl fr 5- i rnl shmarpruin injury. ischemia/reperfusion renal in C5b-9 for role Predominant 105 J. Neurosci. Methods Neurosci. J. . ouaig eghg inln cn teut te eeiy of severity the attenuate can signaling hedgehog Modulating dniiain f nr-ru, ne-niiul andgene-specific inter-individual, intra-group, of Identification

1 We isolated RNA from cultured chondrocytes derived derived chondrocytes cultured from RNA isolated We We analyzed cartilage degeneration scores using a two a using scores degeneration cartilage We analyzed µ

Detailed methodology is described in the , 141–149 (1987). 141–149 , , 1363–1371 (2000). 1363–1371 , 31 g ml g umre o Afmti GnCi poe ee data. level probe GeneChip Affymetrix of Summaries test or by a fixed by a or test , e15 (2003). e15 , -

- third of the surface area, a score of 2 meaning two meaning 2 of score a area, surface the of third 15 medial and tibial and medial −1 , 1421–1425 (2009). 1421–1425 , of C5b6 (EMD Chemicals) with 10 10 with Chemicals) (EMD C5b6 of

Blood 10 - sufficient mice. Wemice. sufficient mRNA measured levels , R98 (2008). R98 ,

163 98 - , 442–449 (2001). 442–449 , effect ANOVA.effect , 9–16 (2007). 9–16 , - medial condyles. The scores for for scores The condyles. medial Using previously described described previously Using We isolated chondrocytes chondrocytes Weisolated - array (Millipore) analy (Millipore) array - way way ANOVA followed nature medicine nature We sec stained Supplementary - 4 based Noldus Noldus based - 0 9 concentra 9 . After 72 h, h, 72 After . 1 µ 0 : depth of : depth g ml g β u. J. Eur. - actin actin −1 of of - 3 - ­ ­ ­ ­ -