Cartilage matrix metabolism in osteoarthritis: markers in synovial fluid, serum, and urine

Clin Biochern. Vol. 25, pp. 167-174, 1992 Printed in the USA. All rights reserved.

0009-9120/92 $5.00 + .00 ~?~opyright ¢ 1992 The Canadian Society of Clinical Chemists.

Cartilage Matrix Metabolism in Osteoarthritis: Markers in Synovial Fluid, Serum, and Urine L. STEFAN LOHMANDER, 1 MICHAEL W. LARK, 2 LEIF DAHLBERG, 1 LORI A. WALAKOVITS, 2 and HARALD ROOS 1 1Department of Orthopedics, University Hospital, $22185 Lund, Sweden and the 2Department of Biochemical and Molecular Pathology, Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, USA Osteoarthritis is a major cause of disability and early retirement. Yet we lack the means to diagnose the disease in its early stages or to m o n i t o r the effects of treatment on the target tissue, the joint cartilage. Neither can we identify the disease mechanisms at the tissue or cell level. Current research focuses on the use of markers of cartilage matrix metabolism in body fluids as a means to diagnose and m o n i t o r osteoarthritis. Cartilage proteoglycan, collagen and glycoprotein fragments, as well as proteinases and their inhibitors, are being suggested for this purpose. Structural information on matrix molecule fragments released into body fluids may also help to identify the enzymes active in the destruction of the cartilage, a central issue in osteoarth ritis.

KEY WORDS: osteoarthritis; cartilage; synovial fluid; proteoglycan; collagen; metalloproteinase; stromelysin.

Introduction he joint diseases are a major cause of disability

T and early retirement in the industrialized countries and are, thus, of great socioeconomic significance. Of the joint diseases, osteoarthritis (OA) has by far the greatest prevalence. In the United States OA is responsible for up to thirty times more sickleave days or hospital days than rheumatoid arthritis (1). OA is a slowly progressive disease of multifactorial etiology (Figure 1). The rate of disease progress will vary greatly between different patients, depending on the underlying pathogenetic factors. Consequently, progress from the very early stages to the overt, clinical stages may take from years to decades. The clinical diagnosis of OA should probably not be regarded as a single disease entity, but rather as a final common pathway of joint cartilage failure. The symptoms and radiological presentation in this end-stage are similar, irrespective of the pathogenetic factors. The diagnostic criteria for OA are currently based on the clinical presentation and obligatory radio-

Correspondence: Stefan Lohmander, Department of Orthopedics, University Hospital, S-22185 Lund, Sweden. Manuscript received July 31, 1991; accepted December 2, 1991. CLINICAL BIOCHEMISTRY, VOLUME 25, JUNE 1992

graphic signs (2,3). However, the diagnostic criteria of even the overt stages are disputed (4). Since the radiological diagnosis is usually based on a decreased joint space, it depends on the actual destruction of joint cartilage and will, therefore, be made only late in the disease. We lack routine methods to diagnose preOA or the preradiological stages of OA (Figure 1), a reflection of our lack of techniques to monitor the joint cartilage in vivo. We are thus unable to determine the ongoing disease activity or the prognosis for the patient threatened by joint cartilage destruction. Moreover, we are unable to monitor with any precision or specificity the effect of pharmacological or surgical intervention aimed at retarding or reversing cartilage destruction in OA or other joint diseases. New and improved techniques to diagnose and follow OA need to monitor the in vivo health of the cartilage, r a t h e r t h a n to provide a historical record of past destructive disease. The details of the mechanisms involved in the disease process in OA are not known. Presumably, the pathogenesis is multifactorial, with genetics, joint malalignment, joint overload or trauma, obesity, and aging as some of the known or suspected contributing factors (Figure 1). Even less well known is how these general factors are translated into disease mechanisms at the tissue and cell level. It may also be that the initiation and progression of OA are controlled by different factors. Since, however, changes in the properties of joint cartilage and loss of matrix components are an integral part of the disease process, it can be argued that degradation of cartilage matrix is a key event at some time in the development of OA. During this process, matrix molecules, or fragments thereof, are released to the joint fluid and eventually to other body fluids. These molecules and fragments could be used as markers of cartilage turnover in OA and other joint disease (5-8).

Matrix metabolism in joint cartilage The physiological turnover of adult joint cartilage matrix is slow (9), with average half-lives of matrix 167

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Figure 1 -- Osteoarthritis progresses slowly from subclinical and preradiologic stages to radiologically overt, symptomatic disease. The rate of progress varies greatly between different patients, and in some joints remission and healing may take place. The pathogenesis is multifactorial and some known or suspected factors are illustrated. Adapted from ref. 6. components in the order of months and years. The turnover of different matrix components is, however, not homogeneous; for example, pools of matrix proteoglycans are metabolized at different rates (10). Little or nothing is known about the turnover rates of the more recently characterized components of the cartilage matrix, such as the minor collagens, the cartilage matrix proteins, or the small proteoglycans (11). Experimental evidence suggests, however, that the general mechanisms of matrix degradation are similar in both normal and stimulated cartilage turnover (12). The chondrocytes seem to play an important role in both the physiological metabolism of cartilage matrix in development and growth and in the pathological degradation that occurs in joint disease. Degradative enzymes derived from synovial cells may also be of consequence in certain conditions, especially in inflammatory joint disease. Nevertheless, the role of enzymes derived from leukocytes in the matrix degradation of arthritis may be questioned since joint destruction is not inhibited in neutropenic, arthritic animals, nor in arthritic animals genetically deficient in neutrophil elastase or cathepsin G (13,14). The degradative activity of chondrocytes (and fibroblasts) is greatly s t i m u l a t e d by cytokines such as interleukin-1 or tumor necrosis factor released from cells of the synovium or perhaps by the chondrocytes themselves, and this has been suggested to be an important disease mechanism in inflammatory joint disease (15-18). However, the significance of this signal pathway for the stimulation of cartilage destruction in OA has not been conclusively demonstrated. The complexity of the interaction between the chondrocytes, cytokines, and growth factors is further illustrated by the fact that transforming growth factor-beta and insulin-like growth factor-1 are effective antagonists of interleukin-1 action on cartilage (19,20). The primary cleavage of cartilage matrix molecules is extracellular and is mediated by proteinases, most of which are probably released by the chondrocytes themselves. A central role in cartilage 168

matrix degradation has been proposed for the metalloproteinase family with stromelysin-1, collagenase, and gelatinase as its most prominent members (21-24). These Zn 2÷ and Ca 2+ requiring endopeptidases are closely related, and exhibit extensive sequence similarity. They are secreted in a latent proenzyme form, are activated extracellularly, and the active forms are inhibited by strong binding to tissue inhibitors of metalloproteinases (TIMPs) or to alpha-2-macroglobulin. Several levels of regulation of cartilage matrix degradation therefore exist: enzyme gene expression, secretion of proenzyme, extracellular activation of proenzyme, and inhibition of activated enzyme by TIMPs and other inhibitors. TIMP-1 is also synthesized by the chondrocytes (25). The potential thus exists for a very precise and local regulation of proteinase activity in the cartilage matrix. The extracellular activation of the latent proforms of metalloproteinases can take place by several different pathways, in which a cascade involving plasminogen activators and plasmin may play a major role in the tissues (24). Recent investigations suggest that TIMPs can inhibit metalloproteinase activity not only at the level of the activated enzyme, but also at the level of proenzyme activation (26). Net activity of the metalloproteinases is, consequently, a result of the balance between the enzymes and their specific inhibitors. An increase in metalloproteinase activity may, therefore, be caused by either an increase in the amount of activated enzyme or by a decrease in the amount of available inhibitor, or both. Interestingly, such perturbations in the equilibrium between enzyme and inhibitors have been shown to occur in osteoarthritic cartilage and in metastatic cells (21,27). Collagenase primarily cleaves the native triple helix of Types II and X cartilage collagens, while gelatinase cleaves denatured collagen, as well as Types IV and V collagen. Stromelysin-1, on the other hand, has a broader specificity and cleaves not only the core protein of the large cartilage proteoglycan, but also Types II and IX collagen and other components of the cartilage matrix (28,29). The chondrocyte thus produces a range of proteinases that can degrade most or all of the structural components of the cartilage matrix. The number of matrix molecules with known primary structure is rapidly increasing, and now includes the human large aggregating proteoglycan (30); consequently, sequence analysis of the matrix molecule fragments released from osteoarthritic cartilage by proteinase action may help to identify the responsible enzymes (Figure 2).

Release of matrix fragments from joint cartilage Fragments of cartilage matrix molecules that result from enzyme action are either taken up by the chondrocytes and further degraded by lysosomal enzymes or are lost to the joint fluid by diffusion (Figures 2 and 3). It is possible that, in addition, matrix CLINICAL BIOCHEMISTRY,VOLUME 25, JUNE 1992

MARKERS OF CARTILAGE DEGRADATION IN OSTEOARTHRITIS Hyaluronan

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Figure 2 -- Joint cartilage proteoglycan aggregate is degraded by metalloproteinase and other enzymes in the matrix. The released fragments are free to diffuse into the synovial fluid. Such fragments can be isolated and their structure determined. This may help to identify the enzymes responsible for the cartilage matrix degradation. Adapted from ref. 5. Abbreviations of human cartilage proteoglycan core protein domains (30): G1, G2, G3 - globular domains 1 to 3; C S 1 , C S 2 - - chondroitin sulfate rich domains 1 and 2; KS -- keratan sulfate rich domain; IGD -- interglobular domain. L i n k - - cartilage link protein. Branched sidechains represent N-linked oligosaccharides, short sidechains represent keratan sulfate and O-linked oligosaccharides, long sidechains represent chondroitin sulfate chains. components are lost from the tissue by diffusion, independent of enzyme action (31,32). Fragments of matrix molecules released to the joint fluid may be taken up by, and further degraded in, the synovial tissue cells, or they may be removed by bulk flow with the synovial fluid to the lymph circulation (33) as shown in Figure 3. The half-life of proteoglycan and hyaluronan in the synovial fluid compartment has been estimated to be about 12 to 18 h [(34), JRE Fraser, personal communication]. Tissue fragments and collagen fibrils can be phagocytosed by cells of the joint fluid (35). A substantial proportion of matrix molecule fragments removed by the lymphatic circulation are eliminated, or at least further degraded, in the regional lymph nodes (33,36). The majority of the remaining fragments t h a t reach the blood stream are removed from the circulation within minutes, most likely by the liver cells (37-40). The collagen cross-links, however, apparently survive in the circulation and are found enriched in urine (41,42). It should be noted, however, t h a t only a small proportion (< 10%) of the body cartilage mass is located in the joints, the rest being in ribs, airways, intervertebral disks, etc. (43). In addition, the concentration of a marker in joint fluid or serum cannot be CLINICAL BIOCHEMISTRY, VOLUME 25, JUNE 1992

Figure 3 -- Cartilage matrix fragments released to joint fluid are mainly eliminated via the lymphatic circulation. A large proportion of the fragments are captured and metabolized in the regional lymph nodes. Fragments that reach the blood circulation are often rapidly eliminated via liver uptake and metabolism. Adapted from refs. 6 and 8. interpreted in a q u a n t i t a t i v e fashion unless the clearance rate for that fragment is also known (44). Since the clearance rate of synovial fluid components from this compartment has been shown to vary in different joint diseases (45), and the rate of elimination in lymph nodes and liver can also be presumed to vary, great caution should be exercised when attempting quantitative interpretation of data on cartilage markers in body fluids. A further factor complicating the i n t e r p r e t a t i o n of i m m u n o a s s a y data on cartilage matrix fragments in body fluids is the influence of subtle, m e t a b o l i c a l l y induced, changes in the structure of the epitopes recognized by the antibodies used in the assays. Such changes may strongly affect antibody affinity, with little change in the total amount of the fragment in question (46). Moreover, fragments of closely related structure may be eliminated from the circulation at different rates (40). Assay of markers of cartilage matrix turnover in osteoarthritis MARKERS

In the wide sense, markers of cartilage turnover in OA can be identified within several domains: cytokines, growth factors, proenzymes, active proteinases, proteinase inhibitors, fragments of matrix molecules produced by enzymes, serum autoantibodies to cartilage components, and matrix molecules synthesized as an adaptive response to matrix degradation. Sensitive and specific assays are now available for several molecular species within each of these groups: A . C y t o k i n e s can be determined in synovial fluid (47-50). Growth factors may play a role in the re169

LOHMANDER, LARK, DAHLBERG, WALAKOVITS, AND ROOS

generation of cartilage observed in animal and human OA (51,52). Our knowledge of the role of these factors in the development and growth of cartilage and bone is rapidly increasing (53). B. Proteinases and their inhibitors can be assayed, either by enzyme activity or by enzyme protein content (21,54-57). C. Matrix components and their fragments can be assayed in the form of glycosaminoglycans (58,59), hyaluronan (60-62), keratan sulfate (63-65), different forms of chondroitin sulfate (66), proteoglycans (67-72), matrix proteins (73), collagen crosslinks (41,42) and collagen propeptides (74,75). D. Serum antibodies to cartilage collagen and chondrocyte membrane proteins have been detected in joint disease (76-79). E. The availability of monoclonal antibodies to specific structures on the chondroitin sulfate chain allows the assay of proteoglycan subpopulations synthesized in increased amounts as a response to joint disease (66). COMPARTMENTS

The interpretation of data obtained with any of the above assays will depend on the compartment chosen for sampling: a joint fluid sample will reflect the condition of the cartilage contained within that joint, while both serum and urine samples will provide an integrated measure of cartilage turnover in all joints and potentially all body cartilage. Although serum samples are easy to obtain and may be simpler to interpret because of more straightforward clearance calculations (44), marker concentrations are lower in serum than in synovial fluid by several orders of magnitude. Additionally, many joint cartilage markers may be variably eliminated and further degraded en route to the blood circulation, and they are also diluted by markers from healthy joints and non-articular cartilage (Figure 3). As an exception to this rule, the collagen crosslinks survive endogeneous metabolism and are concentrated in urine. In fact, the levels of type II collagen hydroxypyridinium cross-link in joint fluid are below the detection limit for current HPLCbased methods and are less than 1% of urine levels (D. Eyre and L.S. Lohmander, unpublished). Several factors favour efforts focusing on synovial fluid samples in the initial stages of marker investigations. When promising markers have been identified in joint fluid, they should be sought in more accessible body fluid compartments, such as serum. Above all, efforts should be made to the correlation of marker levels in several compartments, and the assay of more than one marker in the same sample are important objectives. STRATIFICATION All reports on markers of cartilage turnover in body fluids demonstrate a considerable range of values between individual patients within each diag170

nostic group. While a significant portion of this variability is certainly due to "biological" variation between individuals, it is also clear that many patient groups are heterogeneous, and the criteria for inclusion or exclusion are often ill-defined. Several studies have shown that stratification of the patients within each diagnostic group will decrease the scatter of the data (64,71,72,80,81). For example, proteoglycan fragments in knee-joint fluid after injury greatly decrease in concentration within the first six months after trauma, and a plateau is reached after six months. Recent joint t r a u m a will greatly influence synovial fluid marker concentrations. In addition, the type and severity of the injury influence the temporal patterns of matrix fragment release into the joint fluid; the patterns of proteoglycan fragment concentrations in joint fluid differ between meniscus injury and cruciate ligament injury (Figure 4). Stratification of OA patients, according to disease stage, further demonstrates an inverse relation between OA stage (as estimated by arthroscopy and radiology) and joint fluid proteoglycan fragment concentration; cases with advanced radiological changes show lower concentrations than early cases with only mild joint cartilage damage. This again illustrates the importance of careful stratification of patients included in marker studies. MODELS

Important information on the disease mechanisms of OA has been gained by using a variety of animal models, reflecting different aspects of the h u m a n disease (7). For example, naturally occurring OA in monkeys and mice has been used as a model of human primary OA (82,83), while surgically induced Proteoglycan Fragments vs. Time after Injury 300

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MARKERS OF CARTILAGE DEGRADATION IN OSTEOARTHRITIS

lesions of the ligament or meniscus, or blunt t r a u m a of the knee, have been used in the rabbit or dog to model h u m a n post-traumatic OA (84-88).

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In human studies, we need to select subgroups of patients to decrease scatter and to simplify the interpretation of marker data. The subpopulation of patients with post-traumatic OA of the knee offers an attractive model for the general disease. It has the distinct advantage of being common, we can identify the beginning of the disease process by the time of trauma, we can provide an early and precise diagnosis of the injury through arthroscopy, and we can follow the disease from its early stages in prospective studies in a high-risk group. Finally, the disease progress is comparatively rapid, and these patients reach a radiological stage of the disease 1 5 20 years earlier in life than patients with primary OA (8). Patients with a recent joint t r a u m a thus provide an important starting point for prospective studies on OA and markers of cartilage turnover (72). These patients will also carry the same risk for primary OA, which may influence the course of the secondary OA in individual patients (89). Another human model of considerable interest is the familial OA due to a single base defect in the gene for cartilage collagen type II (90). This defect results in the synthesis of abnormal type II collagen chains (91) and is associated with early onset OA (92). It remains to be shown whether such genetic abnormalities are responsible for a significant proportion of human OA. Investigations on animal post-traumatic OA have demonstrated early and dramatic changes in joint cartilage composition and metabolism, showing the advantages of being able to monitor the earliest stages of the disease before secondary cascade phenomena complicate the picture (88,93). Indeed, the concentration of proteoglycan fragments in synovial fluid increases within a few hours after knee injury in the h u m a n (72) as shown in Figure 5. Interestingly, the concentration of stromelysin in joint fluid also increases shortly after injury, but the maximum level is not reached until several weeks later. The concentrations of both of these markers of cartilage matrix turnover decrease again after about three months, but remain significantly increased over control values for many years after the injury. Further work is needed to determine changes in the balance between matrix fragments, proteinases, and their inhibitors in joint fluid in the early stages of OA. Possibly, the patterns of release of various types of matrix molecule fragments from the joint cartilage may also vary at different stages of the disease (Figure 2). With available technology for the isolation, purification and sequencing of small amounts of peptides it is now possible to determine the N-terminal sequences of proteolytic fragments of cartilage proteoglycans (Figure 2) in small amounts of joint fluid CLINICAL BIOCHEMISTRY, VOLUME 25, J U N E 1992

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Figure 5 -- Proteoglycan fragment and stromelysin concentration in synovial fluid after injury to meniscus or cruciate ligament of the knee (n = 402), diagnosis by arthroscopy, one sample per patient. Logarithmic time scale, symbols represent average, bars represent SE. Upper shaded area represents average proteoglycan concentration in normal control group -+SE, lower shaded area average stromelysin concentration in normal control group -+SE. Proteoglycan fragments in joint fluid were determined by immunoassay (ref. 68). Stromelysin in joint fluid was determined by immunoassay (ref. 56 and 57). (94). Such information should help to determine whether enzymes such as stromelysin-1 are involved in the destruction of cartilage, a central issue in OA. This will enable a rational search for procedures to retard or inhibit the joint destruction in OA. Conclusions Research in this area is still in the early stages of identifying useful m a r k e r s and suitable disease models. The results of retrospective case-control studies have generated hypotheses that can be prospectively tested. Such longitudinal, prospective studies will be necessary to answer questions on the relevance of cartilage markers for the monitoring and prediction of disease outcome. At the same time, current work gives us a better understanding of the disease mechanisms involved in OA at the cell and tissue levels. This offers the means for improved diagnostic and prognostic tools in OA, and a basis for the rational development of treatment with the aim of retarding the cartilage destruction.

Acknowledgements The helpful cooperation of the staff at the Department of Orthopedics at Lund University Hospital is gratefully acknowledged, as is the expert technical assistance of Chris Ebner and Elisaveta Trigueiros. Supported by grants from the Swedish Medical Research Council, the King Gustaf V 80-Year Birthday Fund, the Ax:son Johnson, the Kock, the Zoega, and the Osterlund Foundations, and the Medical Faculty of Lund University. 171

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