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Proteoglycan epitopes as potential markers of normal and pathologic cartilage metabolism.

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Chondrocytes in normal hyaline cartilages are
actively involved in the metabolism of proteoglycans
throughout the lifetime of the tissue (1). This includes
anabolic processes (synthesis and secretion), organizational processes (aggregation in the extracellular
matrix), and catabolic processes (degradation and subsequent loss from the tissue). Even though the mechanisms involved in these processes are poorly understood, it is assumed that the chondrocytes normally
regulate them in response to various environmental
factors, such as hormones, nutrients, mechanical load,
or biochemical signals from other cells such as
interleukin-I. It is also assumed that, under normal
conditions, the dynamic balance of these processes
ultimately determines the concentration of proteoglycans in the immediate vicinity of the chondrocyte and,
therefore, the mechanical properties (resilience and
deformation in response to compressive load) of the
cell’s local matrix (2). There is also evidence that, in
many forms of arthritis, the metabolism of proteoglycans is significantly altered, frequently with accelerated catabolism and, eventually, loss of the entire
matrix (3).
For this discussion, the catabolic processes are
particularly important. In cartilage explant cultures,
more than 90% of the proteoglycans lost from the
matrix appear as partially degraded, large products in
the rnedium (4). Thus, only a small portion of the
From the National Institute of Dental Research, National
Institutes of Health, Bone Research Branch, Bethesda, Maryland.
Vincent C. Hascall, PhD; Tibor T. Glant, MD (current
address: Institute of Anatomy, Histology, and Embryology, University Medical School, Debrecen, Hungary).
Address reprint requests to Vincent C. Hascall, PhD,
NIDR, NIH, Bone Research Branch, Bethesda, MD 20892.
Arthritis and Rheumatism, Vol. 30,No. 5 (May 1987)
proteoglycans removed from the matrix are taken up
by the resident chondrocytes for total degradation via
lysosomal pathways. It is now clear that a significant
proportion of the proteoglycans removed from cartilage in vivo are also lost from the matrix as large
fragments, which diffuse into the synovial fluid and,
eventually, into circulation. The studies discussed
below utilized specific immunologic probes for cartilage proteoglycans to identify and partially characterize some of these degradation products in both synovial fluid and serum. They provide preliminary
evidence that the concentration of these products
can potentially be used to assess normal and abnormal catabolism of cartilage proteoglycans, as well
as the response of cartilage to clinical intervention,
in diseases involving chronic or acute inflammation
of joints.
Specific polyclonal antisera against cartilage
proteoglycans , developed in Heineggrd’s laboratory,
have been used in a series of studies of synovial fluid
samples from humans (5,6) and from dogs used in an
experimental model for osteoarthritis (OA) (7). The
sensitivity and specificity of the enzyme-linked immunosorbent assay procedures, combined with
greater access to clinical samples of synovial fluid,
make screening of larger patient populations and longitudinal studies of patients more feasible. The Scandinavian studies illustrate this. For example, Saxne et
a1 ( 5 ) screened synovial fluids from 109 patients with
various degrees and types of inflamed synovial tissue,
and they concluded that cartilage proteoglycan antigens: (a) were greatly increased above normal in
synovial fluids from patients with reactive arthritis; (b)
were significantly decreased as the severity of rheumatoid arthritis (RA) increased, probably as a result of
net loss of cartilage tissue; and (c) were significantly
decreased during the very early stages of inflammation
in RA patients, compared with similar degrees and
duration of inflammation in patients with reactive
arthritis. Transient synovitis and septic arthritis of the
hip in children also cause highly elevated proteoglycan
antigen contents in synovial fluid (8).
Longitudinal studies of a small population of
patients showed that decreased inflammation as a
result of hydrocortisone treatment or spontaneous
remission led to significantly decreased contents of
proteoglycan antigens in synovial fluid (6). Furthermore, comparison of paired fluids from control and
operated knees in the dog OA model (OA induced by
severing the cruciate ligament) showed significantly
higher proteoglycan antigen in the affected joint (7).
These longitudinal studies clearly demonstrate the
potential of this screening approach for learning more
about cartilage diseases that involve inflammation or
tissue damage.
Witter et a1 (9), in their study published elsewhere in this issue of Arthritis and Rheumatism, have
taken a different approach. They used 2 monospecific
polyclonal immune sera, which recognize different
spectra of epitopes in the core protein, and 1
monoclonal antibody, which recognizes keratan sulfate on protein core fragments with many keratan
sulfate chains, to study a smaller set of patients with
different forms of arthritis. The primary purposes
were: (a) to define more precisely the properties of the
cartilage proteoglycan degradation products in the
synovial fluid samples to learn more about the mechanism of proteoglycan degradation; and (b) to determine if different subsets of arthritis might generate
distinctly different degradation products. Thus, the
studies at this stage were not designed for screening
large patient populations or for longitudinal analyses.
However, such studies may eventually define better
parameters for larger scale investigations.
A series of studies by Thonar and collaborators
(10-14) takes advantage of the essential “uniqueness”
of keratan sulfate as a marker for cartilage proteoglycans, and its concentration in serum as a potential
monitor for cartilage metabolism. These investigators
suggest that more than 95% of the mass of this
glycosaminoglycan is present in cartilaginous tissues
(10). They used a monoclonal antibody (1/20/5-D-4),
which recognizes highly sulfated, sequential blocks of
3-4 disaccharide-repeating units of keratan sulfate
(15,16), to demonstrate that 136 normal adults or
patients with no apparent cartilage disease* had a
mean keratan sulfate epitope level of 268 2 133 ng/ml
of serum (10). The serum-derived antigenic molecules
bound to DEAE and showed similar size distribution
on molecular sieve chromatography as that of the
single keratan sulfate chains isolated from cartilage.
Since the epitope is found only in highly sulfated
blocks on the keratan sulfate chains, this approach
may or may not provide an accurate measure of
keratan sulfate concentration in serum. It is possible,
for example, that the extent of keratan sulfate sulfation
may vary considerably from person to person, and this
could contribute, in part, to the wide range of values
observed in normal subjects (53-1,009 ng/ml) (12).
Interestingly, if the keratan sulfate epitope that
was recognized in the synovial fluid by monoclonal
antibody AN9P1 (used by Witter et a1 [9]), which is on
large fragments, is represented in the population of
serum keratan sulfate chains recognized by monoclonal antibody 1/20/5-D-4 (used by Thonar et a1 [lo]),
then considerable further degradation occurs during
movement from the synovial fluid compartment to the
serum compartment. It should be emphasized that
even though both of these monoclonal antibodies are
specific for keratan sulfate, the assay for the AN9Pl
antibody measures primarily, or only, products with
multiple keratan sulfate chains on a single peptide,
whereas the 1/20/5-D-4 antibody recognizes single
keratan sulfate chains as well. Thus, the fragments in
serum that are recognized by the latter antibody would
not have been detected by the antibody used in Witter
and coworkers’ study.
The potential utility of serum keratan sulfate
measurements for clinical screening was demonstrated
when 43 OA patients were shown to have a significantly higher mean epitope concentration (381 + 107
ng of keratan sulfate/ml of serum) than was found in a
representative population of 45 normal subjects (25 1 2
78 ng/ml) (12). The large range hinders the usefulness
of the assay as a diagnostic test for OA or its subsets;
however, longitudinal studies, in which each patient
provides his or her own baseline, show that day-to-day
variation is much smaller. This indicates that the large
range observed is inherent in the test population and
* Three patients with corneal macular dystrophy had no
detectable serum keratan sulfate epitope and no apparent cartilage
abnormalities. In most patients with this inherited disorder, sulfate
cannot be added to the keratan sulfate proteoglycan in the cornea,
and, as implied by the absence of keratan sulfate epitope in their
sera, keratan sulfate does not seem to fully sulfate on their cartilage
proteoglycans (1 I).
not in the assay per se. It would be interesting to
determine in prospective studies whether those apparently normal individuals with significantly higher-thanaverage levels of serum keratan sulfate epitope are at
greater risk for subsequent development of OA.
Two additional, preliminary studies by Thonar’s group demonstrate that the serum keratan sulfate
epitope concentration can reflect cartilage proteoglycan metabolism. First, patients undergoing localized chymopapain treatment to reduce swelling from
herniated discs showed a transient, highly elevated
serum concentration of keratan sulfate epitope after
treatment, whereas patients undergoing laminectomy
did not (13). Second, 9 of 10 patients undergoing
surgery to remove chondrosarcomas had a significant
decrease (into the normal range) in circulating keratan
sulfate epitope after removal of the tumor. The one
exception was a patient who was receiving radiation
therapy as well (14).
In conclusion, these studies using immunologic
probes demonstrate: (a) that synovial fluid and serum
contain specific antigens derived from cartilage
proteoglycans; (b) that the concentrations of these
antigens are related to cartilage proteoglycan metabolism and can reflect normal conditions, pathologic
conditions, or the response of the tissue to clinical
intervention; and (c) that a better description of the
nature of the degradation products in synovial fluid
and serum may help define mechanisms involved in
these processes in normal and diseased cartilage.
Future advances will occur as the nature of the
epitopes are better defined, as more monoclonal antibodies to additional cartilage proteoglycan epitopes
are developed, and as longitudinal studies define more
precisely the changes that occur in these parameters
during the course of cartilage diseases and their
Handley CJ, McQuillan DJ, Campbell MA, Bolis S:
Steady state metabolism in cartilage explants, Articular
Cartilage Biochemistry. Edited by KE Kuettner, R
Schleyerbach, VC Hascall. New York, Raven Press,
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Maroudas A, Katz EP, Wachtel EJ, Mizrahi J, Soudry
M: Physicochemical properties and functional behavior
of normal and osteoarthritic human cartilage, Articular
Cartilage Biochemistry. Edited by KE Kuettner, R
Schleyerbach, VC Hascall. New York, Raven Press,
1986, pp 31 1-329
Muir H: Current and future trends in articular cartilage
research and osteoarth,ritis, Articular Cartilage Biochemistry. Edited by KE Kuettner, R Schleyerbach, VC
Hascall. New York, Raven Press, 1986, pp 423-440
4. Hascall VC, Morales TI, Hascall GK, Handley CJ,
McQuillan DJ: Biosynthesis and turnover of proteoglycans in organ culture of bovine articular cartilage. J
Rheumatol (suppl) 11:45-52, 1983
5. Saxne T, Heinegkd D, Wollheim FA, Pettersson H:
Difference in cartilage proteoglycan level in synovial
fluid in early rheumatoid arthritis and reactive arthritis.
Lancet II:127-128, 1985
6. Saxne T, Heineggrd D, Wollheim FA: Therapeutic effects on cartilage metabolism in arthritis as measured by
release of proteoglycan structures into the synovial
fluid. Ann Rheum Dis 45:491497, 1986
7. Heinegkd D, Inerot S, Wieslander J , Lindblad G: A
method for the quantification of cartilage proteoglycan
structures liberated to the synovial fluid during developing degenerative joint disease. Scand J Clin Invest
45:421-427, 1985
8. Lohmander LS, Wingstrand H, HeinegArd D: Transient
synovitis of the hip in the child: increased levels of
proteoglycan fragments in joint fluid. J Orthop Res (in
9. Witter J, Roughley PJ, Webber C, Roberts N, Keystone
E, Poole AR: The immunologic detection and characterization of cartilage proteoglycan degradation in synovial
fluids of patients with arthritis. Arthritis Rheum 30:
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Pachman LM, Glickman P, Katt R, Huff J, Kuettner
KE: Quantification of keratan sulfate in blood as a
marker of cartilage catabolism. Arthritis Rheum 28:
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Maldonado B, Hassell J , Hewitt AT, Stark WJ, Stock L ,
Kuettner KE, Klintworth GK: Absence of normal
keratan sulfate in the blood of patients with macular
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12. Thonar EJ-MA, Schnitzer TJ, Kuettner KE: Quantification of keratan sulfate in blood as a marker of cartilage
catabolism. J Rheumatol (in press)
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Sinkora G, Layfer LF, Thonar EJ-MA: Keratan sulfate
release following chemonucleolysis (abstract). Orthop
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14. Kliner DJ, Gorski JP, Thonar EJ-MA: Keratan sulfate
levels in sera of patients bearing cartilage tumors. Cancer (in press)
15. Caterson B, Christner JE, Baker JR: Identification of a
monoclonal antibody that specifically recognizes corneal
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cartilage proteoglycan. J Biol Chem 258:8848-8854, 1983
16. Mehmet H , Scudder P, Tang PW, Hounsell E F ,
Caterson B, Feizi T: The antigenic determinants recognized by three monoclonal antibodies to keratan sulphate involve sulphated hepta- or larger oligosaccharides
of the poly(N-acetyl-lactosamine) series. Eur J Biochem
157:385-391, 1986
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potential, markers, proteoglycans, pathologic, metabolico, epitopes, norman, cartilage
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