Annals of the Rheumatic Diseases 1994; 53: 3-5

Immunochemical markers of joint inflammation,skeletal damage and repair: where are we now?

Although the destruction of diarthrodial joints has long beena focus of arthritis research, we still know extremely littleabout the metabolism of joint tissues in vivo and how thismay change in arthritis. The use of skeletal imaging,particularly radiographic analysis, and more recently,magnetic resonance imaging, has provided much insightinto the consequences of the disease process, namely thedestruction of cartilage in rheumatoid arthritis (RA) andosteoarthritis (OA), juxta-articular bone destruction(erosions and osteopenia) in RA, and extensive boneremodelling (sclerosis) in OA. But such analyses tell us littleabout the disease processes that lead to these skeletalchanges.We need to predict outcome of injury to joints that may

cause degenerative changes and joint disease. We haveclinical measures of joint swelling and pain but we must beable to assess joint inflammation in more quantitative waysand the accompanying cartilage and bone damage as itrelates to alterations in the turnover of these tissues longbefore these changes are detectable by more traditionalapproaches. We need to know whether therapy can shutdown cartilage and bone destruction, as well as controllinginflammation. If therapy is to be used to stimulate repairof these tissues which, in the case of articular cartilage, isonly occasionally observed in the adult, we must have waysof detecting and following reparative events in vivo. Thuswe need specific markers of synovial inflammation and ofcartilage and bone synthesis and degradation. Ideally, theseshould be serum/plasma markers. These could haveenormous benefit, if used prognostically, to help decide on

appropriate therapy and in evaluating the capacity oftherapy to control disease activity and promote repair.We are moving towards a realisation of these goals as a

result of ever-growing research on 'markers of skeletalmetabolism', specifically joint and bone disease, but alsoincluding new markers of joint inflammation (table).With the advent of recent new knowledge of the

compositions and metabolism of these tissues, very sensitive

immunoassays have been developed to detect, in bodyfluids, minute amounts of often tissue-specific molecules,the release of which, usually in a degraded state, can bemeasured. These molecular fragments reflect specific rate-limiting events such as bone or cartilage synthesis or

degradation and joint inflammation (fig).'Synovial inflammation in RA and OA can now be

detected by the measurement of hyaluronan or hyaluronicacid (HA). This molecule, which is synthesised by many

cells and tissues, is produced by synovial cells in largeamounts, acting as a boundary lubricant in synovial joints.In RA, its release into the circulation is increased,2 possiblydue to stimulation of synthesis by cytokines such as

interleukin-1 (IL-1) and tumour necrosis factor-o (TNF-a).1 Elevated levels of serum HA in early disease are

indicative of joint inflammation3'4 and the progression oferosive joint diseases.5 The milder, less-pronounced intra-articular inflammation of OA is also reflected by increasedserum hyaluronan, which correlates with joint damage andfunctional capacity.'

Cartilage metabolism is also extremely sensitive tocytokines, which can arrest (TNF-at, IL-1) or stimulate(TGF-P and IGF-1) synthesis of specific cartilagemolecules.7 The chronic inflammation of RA appears todepress synthesis of the cartilage proteoglycan aggrecansystemically since levels of aggrecan degradation productsare inversely related to those of acute phase proteins3 8 9 andTNF-a.9 It appears that impairment of cartilage matrixsynthesis is coupled to increased degradation in thepathogenesis of RA.'° Destruction of articular cartilage isreflected by increased proteoglycan content in synovialfluids and may, like hyaluronan, be prognostic for diseaseoutcome." These degradation products probably includefragments of newly synthesised proteoglycans. These can bedetected by epitopes on chondroitin sulfate chains of theproteoglycan aggrecan'2 '3 which, in the case of epitope 846,resides on the largest and probably intact newly synthesisedmolecules.'2 Their increased detection in arthritic

Markers forjoint metabolism

Marker Primary indication Osteoarthritis Rheumatoid arthritisSerum SF Serum SF

SynoviumHyaluronan (hyaluronic acid, hyaluronate, HA) Synovitis, HA synthesis is stimulated by IL-i t ND t t

and TNF-aCartilage

C-propeptide of type II collagen Synthesis of cartilage type II collagen t t t t

Type II collagen a chain fragments Degradation of cartilage type II collagen Studies in progressProteoglycan aggrecan:(a) keratan sulphate Synthesis/degradation of aggrecan t t I t

(b) core protein Degradation t t(c) intact chondroitin sulphate epitopes for example, 846, 3B3 Probably synthesis t t tl* tCartilage oligomeric protein (COMP) Synthesis and/or degradation? ND t tCartilage matrix protein Synthesis and/or degradation? only present in ND ND t ND

non-articular cartilagesBoneBone sialoprotein Synthesis/degradation Studies in progressOsteocalcin Synthesis t t t3-hydroxypyridinium (pyridinoline and Degradation urine only urine onlydeoxypyridinoline) cross-links

ND = not determinedSF = synovial fluid* = acute disease only

3

on Decem

ber 11, 2021 by guest. Protected by copyright.

http://ard.bmj.com

/A

nn Rheum

Dis: first published as 10.1136/ard.53.1.3 on 1 January 1994. D

ownloaded from

Poole

Removal in LIVER& degradation

Removalin K0

NON4.ARTICUL,APA PIl LAG f

iJ'rachel& Dronchusr twsai. et.

Pyridinoline arnc* eoxypyridinonir

Sross linksK plte .iai,

-r:)

; Hyaluronan

,ros! iri.846e2p

LYMPHATICS,pitopePKRPQ^,MP (Partial degradation)

K 2'C'0M

... ..

JIDNEs

I

U ine deoxypyridinohine-ollagen cross links

Release of skeletal marker molecules and their degradation products from hyaline cartilages, bone and synovium into synovialfluid, blood and theirclearance by liver and kidney. The collagen cross-links (pyridinoline and deoxytyridinoline), C-propeptide of type IIprocollagen (CPII), keratan sulphate(KS) of the cartilage proteoglycan aggrecan, and cartilage oligomeric protein (COMP) and the 148 kD proteinfrom non-articular cartilages are showntogether with other molecules.

cartilages'2 and very much increased contents in synovialfluids over levels in blood'0 suggest that increased synthesisfollowed by degradation of newly synthesised molecules isa key feature ofboth these diseases. These markers may alsobe useful in detecting decreased synthesis, such as thatassociated with joint immobilisation.'4

Cartilage collagen synthesis is reflected by the release ofthe C-propeptide of type II procollagen into serum indevelopment,'5 and synovial body fluids in arthritis.'6Degradation of type II collagen can now also be measured. 17These markers are now available for studies in arthritis.The 145 kDa cartilage matrix protein is absent from

articular cartilages and therefore provides the opportunityto study selectively systemic cartilage metabolism.'Moreover, the systemic osteoporosis of RA and the severejuxta-articular osteopenia, a consequence of enhanced boneresorption resulting from an imbalance in RA, can bemeasured by immunoassayl9-21 or high performance liquidchromatography22 of the urinary hydroxypyridinium cross-links, derived from cartilage and bone matrix collagens.Even skeletal changes in OA are detectable by elevatedcross-links in urine.22 The dominance of bone mass meansthat these cross-links are probably mainly derived from thistissue. New immunoassays should soon be able to permitcollagen cross-link detection in blood.

In contrast, bone synthesis is reflected by the release ofosteocalcin, synthesised by osteoblasts. Serum osteocalcin ishardly changed in RA,' probably due to the suppression ofsynthesis by inflammation. But in OA, the extensivehypertrophy and juxta-articular bone turnover isaccompanied by elevated serum osteocalcin (A R Poole, MIonescu, A Swan and P Dieppe, manuscript inpreparation).

Degeneration of cartilages, such as frequently results frompost-traumatic injuries, can eventually lead to clinically-

defined OA. Research at the University of Lund in Sweden,headed by Drs Dick Heinegard, Frank Wolheim, StefanLohmander and Tore Saxne, together with the work of DrsEugene Thonar and Klaus Kuettner in Chicago, haspioneered many developments in this field. The early workof Dick Heinegard's group was the first to useimmunoassays to study cartilage marker molecules.23 TheLund group drew attention to alterations in cartilageturnover in RA" and following traumatic injuries toarticular tissues such as cruciate ligaments and menisci.24 25In this issue of the journal they describe a study of thecartilage oligomeric protein (COMP),26 a member of thethrombospondin family, first identified by Fife and Brandt27and cloned by Heinegard's group.2" Like aggrecan, thismolecule is significantly elevated in synovial fluid aftertraumatic joint injury. The elevations of both aggrecan andCOMP persist, albeit often at lower levels, and are acharacteristic feature of post-traumatic OA. The reportedelevations in serum aggrecan in OA,29 30 although notdetected by others,8 10 may also reflect differences incartilage metabolism that predispose to OA, rather thanbeing a consequence of disease.COMP is interesting since its synthesis may be differently

regulated to that of other cartilage molecules, such as typeII collagen and aggrecan. Whereas in inflammation,cytokines such as, IL-1 and TNFo8, can inhibit synthesis ofcartilage aggrecan and collagens,7 8-10 COMP synthesis maybe preferentially stimulated by the cytokine TGF-P (Dr AD Recklies, Joint Diseases Laboratory, Shriners Hospital,Montreal, personal communications), which is released inincreased amounts in inflammation. Moreover, and moreimportantly, COMP levels in serum may reflect those insynovial fluids.3' In RA, levels of COMP are elevated inpatients with rapidly progressive erosive joint disease overthose with much slower erosive disease.32 This may result

-O.N-M.04

4 on D

ecember 11, 2021 by guest. P

rotected by copyright.http://ard.bm

j.com/

Ann R

heum D

is: first published as 10.1136/ard.53.1.3 on 1 January 1994. Dow

nloaded from

Immunochemical markers ofjoint inflammation, skeletal damage and repair: where are we now?

directly from the elevated levels ofTGF-3 in these inflamedjoints and the lack of inhibition of synthesis by cytokinessuch as IL- I and TNF-ot.The development and use of these new immunological

assays to identify 'markers' of joint metabolism and diseaseis still in its infancy. They hold much promise in studyingskeletal development. In arthritis research, we need torigorously assess these markers by their use in carefully-controlled clinical trials. We must study sub-groupscharacterised by clearly recognisable differences in diseaseactivity. Establishment of the molecular bases of theirproduction, correlations with well-defined clinicalparameters and with each other are essential if we are tounderstand their true significance and explore their valuefully. Too little has so far been done in these areas. Toomuch emphasis has been placed in earlier work on themeasurement of levels of single markers, often with littleregard to clinical changes and to each other, and to themolecular mechanisms regulating their release.

But their use is already helping us to a betterunderstanding of the pathobiology of these diseases in vivo.The bone markers have been shown to have considerablepotential in the study of osteoporosis and metastasis tobone. Other markers may be of considerable benefit inidentifying more appropriate therapy, especially in earlydisease. As in other areas of clinical research, they shouldhelp us not only evaluate the effectiveness of therapy butprovide a basis for the development of new therapies,designed to control inflammation and damage to skeletaltissues, stimulate repair, and overall, help us achieveimproved management of these diseases.

I would like to acknowledge the many major contributions made by mycollaborators in this research, in particular Professor Paul Dieppe. The author'swork in this area has been funded by the Shriners of North America, MedicalResearch Council of Canada, Pharmacia Diagnostics, Pfizer Central Researchand Monsanto Corporation.

Joint Diseases Laboratory, A ROBIN POOLEShriners Hospitalfor Crippled Children, andDivision of Surgical Research,Department of Surgery, McGill University,1529 CedarAvenue, Montreal, Quebec H3G 1A6, Canada

1 Poole A R, Dieppe P. Biological markers in rheumatoid arthritis. Sem ArthRheum 1994; (in press).

2 Engstrom-Laurent A, Hallgren R. Circulating hyaluronate in rheumatoidarthritis: relationship to inflammatory activity and the effect ofcorticosteroid therapy. Ann Rheum Dis 1985; 44: 83-88.

3 Poole A R, Witter J, Roberts N, Piccolo F, Brandt R, Paquin J, Baron M.Inflammation and cartilage metabolism in rheumatoid arthritis. Studies ofblood markers hyaluronic acid, orosomucoid and keratan sulfate. ArthritisRheum 1990; 33: 790-9.

4 Engstrom-Laurent A, Hallgren R. Circulating hyaluronic acid levels vary withphysical activity in healthy subjects and in rheumatoid arthritis patients.Relationship to synovitis mass and moming stiffness. Arthritis Rheuni 1987;30: 1333-8.

5 Paimela L, Heiskanen A, Kurki P, Helve T, Leirisalo-Repo M. Serumhyaluronate level as a predictor of radiologic progression in earlyrheumatoid arthritis. Arthritis Rheum 1991; 34: 815-21.

6 Goldberg R L, Huff J P, Ienz M E, Glicksman P, Katz R, Thonar E J-MA. Elevated plasma levels of hyaluronate in patients with osteoarthritis andrheumatoid arthritis. Arthritis Rheuni 1991; 34: 799-807.

7 Poole A R. Cartilage in health and disease. Arthritis and allied conditions.A textbook of rheumatology, 12th ed. In: McCarty, Koopman W, eds.Philadelphia: Lea and Febiger, 1993: 279-323.

8 Spector T D, Woodward L, Hall G M, et al. Keratan sulphate in rheumatoidarthritis, osteoarthritis and in inflammatory diseases. Ann Rheuinpi Dis 1992;51: 1134-7.

9 Manincourt D-H, Triki R, Fukuda K, Devogelaer J-P, Nagant deDeuxchaisnes C, Thonar E J-M A. Levels of circulating tumor necrosisfactor ot and interleukin-6 in patients with rheumatoid arthritis.Relationship to serum levels of hyaluronan and antigenic keratan sulfate.Arthritis Rheumn 1993; 36: 490-9.

10 Poole A R, Ionescu M, Swan A, Dieppe P. Changes in cartilage metabolismin arthritis are reflected by altered serum and synovial fluid levels ofglycosaminoglycan epitopes on fragments of the cartilage proteoglycanaggrecan: implications for pathogenesis. J Clin Invest (in press).

11 Saxne T, Wolheim F A, Pettersson H, Heinegard D. Proteoglycanconcentration in synovial fluid: predictor of future cartilage destruction inrheumatoid arthritis. BMJ 1987; 295: 1147-8.

12 Rizkalla G, Reiner A, Bogoch E, Poole A R. Studies of the articular cartilageproteoglycan aggrecan in health and osteoarthritis: evidence for molecularheterogeneity and extensive molecular changes in disease. J Clini Invest1992;90: 2268-77.

13 Caterson B G, Mahmoodian F, Sorrell J M, et al. Modulation of nativechondrotin sulphate structure in tissue development and in disease. J CellScience 1990; 97: 411-7.

14 Ratcliffe A, Shuretv W, Caterson B. The quantitation of a native chondroitinsulfate epitope in synovial fluid lavages and articular cartilage from canineexperimental osteoarthritis and disease atrophy. Arthritis Rheumn 1993; 36:543-51.

15 Carey D E, Alini M, Piccolo F, et al. An immunoassay for the C-propeptideof type II collagen in human serum: changes during growth. (In press).

16 Shinmei M, Ito K, Matsuyama S, Yoshihara Y, Matsuzawa K. Joint fluidcarboxy-terminal type II procollagen peptide as a marker of cartilagecollagen biosynthesis. Osteoarthritis Cartilage 1993; 1: 121-8.

17 Hollander A P, Heathfield T F, Webber C, et al. Increased damage to typeII collagen in osteoarthritic articular cartilage detected by a newvimmunoassay. J Clin Invest, (submitted).

18 Saxne T, Heinegard D. Involvement of nonarticular cartilage, asdemonstrated by release of a cartilage specific protein in rheumatoidarthritis. Arthritis Rheunn 1989; 32: 1080-6.

19 Robins S P, Stewart P, Astbury C, Bird H A. Measurement of the cross-linking compound, pyridinoline, in urine as an index of collagendegradation in joint diseases. Ann Rheumn Dis 1986; 45: 969-73.

20 Hanson D A, Weis M A E, Bollen A-M, Maslan S L, Singer F R, Eyre DR. A specific immunoassay for monitoring human bone resorption:quantitation of type I collagen cross-linked N-telopeptides in urine. J BonieMin Res 1992; 7: 1251-8.

21 Seyedin S M, Kung V T, Daniloff Y N, et al. Immunoassav for urinarypyridinoline: the new marker of bone resorption. J Bone Mimi Res 1993; 8:635-41.

22 Seibel M J, Duncan A, Robins S P. Urinary hydroxy-pyridinium cross linksprovide indices of cartilage and bone involvement in arthritic diseases. 7Rheuniatol 1989; 16: 964-70.

23 Heinegard D, Inerot S, Wieslander J, Lindblad G. A method for thequantification of cartilage proteoglycan structures liberated to the synovialfluid during developing degenerative joint disease. Scand 37 Clini Lab Invest1985;45:421-7.

24 Lohmander L S, Dahlberg L, Ryd L, Heinegard D. Increased levels ofproteoglycan fragments in knee joint fluid after injury. Arthritis Rheum1989; 32: 1434-42.

25 Dahlberg L, Ryd L, Heineggrd D, Lohmander L S. Proteoglycan fragmentsin joint fluid. Influence of arthrosis and inflammation. Acta Orthop Scand1992; 63: 417-23.

26 Lohmander L S, Saxne T, Heinegard D K. Release of cartilage oligomericprotein (COMP) into joint fluid after knee injury and in osteoarthritis. AnnRheum Dis 1994; 53: 8-13.

27 Fife R S and Brandt K D. Identification of a high-molecular weight(>400,000) protein in hyaline cartilage. Biochimni Biophys Acta 1994; 802:506-14.

28 Oldberg A, Antonsson P, Lindblom K, Heinegard D. COMP (cartilageoligomeric matrix protein) is structurally related to the thrombospondins.7 Biol Chemn 1992; 267: 22346-50.

29 Thonar E-J, Lenz M E, Klintworth G K, et al. Quantification of keratansulfate in blood as a marker of cartilage metabolism. Arthritis Rheumn 1985;28: 1367-76.

30 Mehraban F, Finegen C, Moskowitz R W. Serum keratan sulfate.Quantitative and qualitative comparisons in inflammatory versusnoninflammatory arthritides. Arthritis Rheum 1991; 34: 383-92.

31 Saxne T, Glennas A, Kivien T K, Melby K, Heinegard D. Release of cartilagemacromolecules into the synovial fluid in patients with acute and prolongedphases of reactive arthritis. Arthritis Rheum 1993; 36: 20-25.

32 Forslind K, Eberhardt K, Jonsson A, Saxne T. Increased serumconcentrations of cartilage oligomeric protein. A prognostic marker in earlyrheumatoid arthritis. BrJ7 Rheuniatol 1992; 31: 593-8.

5 on D

ecember 11, 2021 by guest. P

rotected by copyright.http://ard.bm

j.com/

Ann R

heum D

is: first published as 10.1136/ard.53.1.3 on 1 January 1994. Dow

nloaded from