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Monoclonal antibodies that detect biochemical markers of arthritis in humans.

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Number 5, May 1995, pp 6 5 5 6 5 9
0 1995, American College of Rheumatology
Objective. To evaluate the potential of using
monoclonal antibodies (MAb) 3-B-3(-) and 7-D-4 to
detect biochemical markers of altered cartilage metabolism in human arthritides.
Methods. Fifty-five samples of normal articular
cartilage (subjects’ age range 18 weeks of gestation to 83
years of age) and 89 samples of arthritic cartilage
(patients’ age range 20-81 years) were collected, and their
proteoglycans were extracted and analyzed for the presence of the epitopes recognized by MAb 3-B-3 and 7-D-4.
Results. Native 3-B-3(-) mimotope was expressed at a high incidence in proteoglycans extracted
from the cartilage of patients with most of the arthritic
diseases examined (osteoarthritis, juvenile rheumatoid
arthritis, rheumatoid arthritis, avascular necrosis, and
degenerative meniscal tears). Its expression in normal
cartilage specimens was very low or absent, occurring
mainly in the young, skeletally immature individuals. In
contrast, expression of the 7-D-4 epitope was more
variable in patients with different arthritides and was
also frequently found in normal cartilage specimens.
Immunohistochemical analyses with both 3-B-3( -) and
7-D-4 showed strong focal positive staining in superficial
Previously published in abstract form: Slater RR, Caterson
B, Lachiewicz PF, Bayliss MT. Trans Orthop Res SOC17:277,1992.
Supported by NIH grant AR-40364, the Arthritis and Rheumatism Council of Great Britain, and Orthopaedic Research and
Education Foundation grant 90-545. Dr. Slater received the 1993
AOA-Zimmer Annual Travel Award for Orthopaedic Residents for
this work.
Robert R. Slater, Jr., MD, Paul F. Lachiewicz, MD, Bruce
Caterson, PhD: University of North Carolina, Chapel Hill; Michael
T. Bayliss, PhD: The Kennedy Institute of Rheumatology, London,
England; Denise M. Visco, PhD: Miles Research Center, West
Haven, Connecticut (current address: Merck & Co., Rahway, New
Address reprint requests to Bruce Caterson, PhD, NorfleetRaney Professor of Research in Orthopaedics, CB# 7055, BurnettWomack Building, The University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599-7055.
Submitted for publication June 14, 1994; accepted in revised form November 21, 1994.
areas, where cartilage degeneration, remodeling, and
repair were greatest.
Conclusion. The biochemical markers recognized
by MAb 3-B-3(-) and 7-D-4 are indicative of altered
proteoglycan synthesis and metabolism in human articular cartilage. The data suggest that in human cartilage,
the 3-B-3(-) epitope might be a better marker of
biochemical changes than the 7-D-4 epitope.
Osteoarthritis (OA) is a common disease of
diarthrodial joints, and next to cardiovascular disease,
it is the second leading cause of disability in patients
>50 years old in the United States (1). Other arthritides such as rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), and the arthritis that follows
avascular necrosis (AVN) are less common. Many of
the changes that occur in arthritic joints are well
characterized clinically as well as at a macroscopic
and microscopic level (2), but the diagnosis is generally based upon clinical and radiographic changes that
occur in the later stages of the disease. Currently,
there are no diagnostic methods available for detecting
the biochemical compositional changes in arthritic
cartilage from patients during early stages of disease.
However, the potential for measuring cartilage metabolites in body fluids (serum, synovial fluid) of patients
with arthritis has been investigated and reviewed (3).
We have developed and identified monoclonal
antibodies (MAb) that recognize subtle biochemical
differences present in the extracellular matrix of articular cartilage from arthritic joints, so-called “markers” of arthritis (4). Previous reports from our laboratories have demonstrated the power and usefulness of
MAb technology in detecting these markers as well as
their correlation with disease progression in a canine
model (5-7) and a guinea pig model (8) of OA. The
biochemical characterization of the native 3-B-3(-)
epitope (a mimotope [9]) has been described previously (5-7,lO). This antibody recognizes atypical
structures at the nonreducing terminal of the chondroitin sulfate (CS) glycosaminoglycan side chains of
the proteoglycans (see Figure 1). Previous work (5-7)
has also identified a second antibody (7-D-4) that
recognizes atypical structures (sulfation patterns) in
native CS glycosaminoglycans of proteoglycans from
OA dog cartilage. In addition, Rizkalla et a1 (11)
recently described an antibody (denoted 846) that also
recognizes an atypical epitope in native CS that is
increased in human OA cartilage. All these antibodies
against native CS structures-3-B-3(-),
7-D-4, and
different epitopes that appear to be
associated with 6-sulfated isomers of CS (ref. 11, and
Caterson B, Griffin JP: unpublished observations).
In this paper, we describe studies determining
the relative occurrence of proteoglycans containing
the 3-B-3(-) and 7-D-4 epitopes in extracts of human
articular cartilage obtained from patients with various
arthritic diseases.
Tissue sources. Proteoglycans were extracted from 55
samples of normal human articular cartilage obtained from
donors whose ages ranged from 18 weeks of gestation (a
spontaneously aborted fetus) to 83 years. Most samples of
normal cartilage were obtained during surgery or within 24
hours postmortem. The cartilage from the older patients was
thinner and yellower than that of the younger patients, but
looked narmal for age, and there were no intraarticular OA
lesions in any of the specimens.
Eikhty-nine pathologic samples of articular cartilage
were obtained from the hips and knees (and 1 first metatarsophalangealjoint) of patients with a variety of diagnoses: 53
from OA patients ages 57-81 years; 21 from RA patients ages
56-62 years; 2 from JRA patients ages 22 and 29 years; 7
from AVN patients ages 34-70 years; and 6 from patients
with meniscal tears ages 20-43 years.
Cartilage samples were analyzed for 3-B-3(-) and
7-D-4 epitope at 2 different institutions. Specimens from the
London area hospitals were analyzed at the Kennedy Institute of Rheumatology (27 normal, 16 OA, 19 RA, and 1
JRA). The remainder were collected and analyzed at the
University of North Carolina. Each cartilage sample was
analyzed without knowledge of the patient’s diagnosis, after
which hospital charts and surgeons’ notes were reviewed to
determine the nature and source of the tissue. Histologic
analyses of specimens obtained at joint arthroplasty were
performed by the hospital pathology department and their
reports were reviewed independently. Clinical diagnoses
were made by professional personnel at hospitals in both the
UK and US.
Proteoglycan extraction and immunochemical analysis. Specimens were frozen immediately at -20°C until
further analysis. Human articular cartilage was subjected to
16p microsectioning at -20°C to ensure efficient extraction
of the cartilage proteoglycans (12). Proteoglycans were
rapidly and quantitatively extracted for 4 hours at 50°C, with
constant agitation, using 4M guanidine HC1, 0.001M EDTA
0.5M Tris acetate, pH 8.0, containing 10 mg/ml dithiothreitol, and then were alkylated with the addition of 30 mdml
sodium iodoacetate (13). After extraction, residues were
washed with distilled water (dH,O) and digested with papain
(1 mg/ml papain solution in 0.05M sodium acetate, 0.025M
EDTA, pH 5.6, for 16 hours), dialyzed against dH20, and
analyzed for uronic acid. The extraction yield varied for
different tissues and patient ages, ranging from 82% to 97%
of the total uronic acid.
After extraction, the reduced extracts were dialyzed
against dH,O, lyophilized, and reconstituted in 100 pl of 8M
urea, 1 mM Na,SO, containing 0.04M Tris HCl, pH 6.8
(composite gel sample buffer), and the proteoglycan concentration in these samples was determined by uronic acid
assay. Proteoglycans were then separated from other macromolecules in the cartilage extracts, using composite agarosel
polyacrylamide gel electrophoresis (6,13,14), and electrophoretically transferred to nitrocellulose (Schleicher and
Schuell, Keene, NH) before immunolocation analyses with
MAb 3-B-3 and 7-D-4 (5,6,13). Bound primary antibody was
detected using an alkaline phosphatase-conjugatedgoat antimouse second antibody kit (ProtoBlot Western Blot AP
systems kit; Promega, Madison, WI) in accordance with the
manufacturer’s instructions. For some experimental conditions the electrophoretically separated proteoglycans on
nitrocellulose transfers were pretreated with chondroitinase
ABC (0.1 units reconstituted in 10 ml 0.1% bovine serum
albumin, 0.1M Tris acetate, pH 8.0, for 1 hour at 37°C).
Pretreatment of the nitrocellulose sheets with chondroitinase
facilitates detection by 3-B-3 of immunoreactive unsaturated
chondroitin-6-sulfate “stubs” that remain attached to the
core protein after chondroitinase digestion (6,8,10). Immunolocation with antibody 3-B-3 without enzyme pretreatment
detects only the native atypical nonreducing terminal structure that serves as a biochemical “marker’’ of OA (-,lo).
Immunohistochemical analyses were performed on specimens of OA cartilage obtained from 10 different patients at
the time of joint arthroplasty and analyzed using procedures
described previously (7).
In this study, immunolocation with MAb 3-B-3
was used under 2 distinct experimental conditions.
After predigestion of the electrophoretically separated
proteoglycans with chondroitinase, 3-B-3( +) was applied. Under these conditions, this MAb recognizes its “enzyme-generated’’ epitope containing an
immunoreactive, nonreducing-terminal, unsaturated,
6-sulfated CS disaccharide attached to the proteoglycan core protein (see Figure I). This immunolocation
procedure is used as a general method of identifying
proteoglycans containing chondroitin-6-sulfate (4-6).
Second, in immunolocation analyses with MAb 3-B-3
without chondroitinase pretreatment, 3-B-3( -) was
used to identify a “native” mimotope (9) that occurs at
the nonreducing terminal of some of the CS chains
(4-7,lO). This native structure is called a mimotope
because it “mimics” the original epitope that was used
to generate antibody 3-B-3. The difference between
the 3-B-3 epitope and its mimotope is that the epitope
has a nonreducing-terminal unsaturated glucuronic
acid adjacent to 6-sulfated N-acetylgalactosamine,
whereas the mimotope has a saturated glucuronic acid
as its terminal structure (5,lO).
Figure 2 shows a representative immunoblot of
proteoglycans extracted from cartilage of normal and
OA patients using 3-B-3 with and without chondroitinase pretreatment. Both the normal and the OA cartilage extracts show strong immunostaining for all 3
cartilage proteoglycan subpopulations when they have
been pretreated with chondroitinase prior to immunolocation (3-B-3[ +J in Figure 2).
363 (-1
Figure 1. Schematic diagram of a proteoglycan (PG) monomer and
the binding sites of the monoclonal antibodies used in this study. To
the left side of the core protein are depicted chondroitin sulfate (CS)
glycosaminoglycans before chondroitinase pretreatment and the
sites of the 3-B-3(-) mimotope and 7-D-4 epitope. To the right side
of the core protein are depicted the CS oligosaccharide “stubs”
containing the 3-B-3(+) epitope that are produced after chondroitinase pretreatment. KS = keratan sulfate; HA = hyaluronan; G1,
G2, and G3 = the 3 globular protein domains of the PG monomer.
Figure 2. Immunolocation analyses performed on proteoglycans
extracted from normal and osteoarthritic (OA) articular cartilage.
Normal cartilage was obtained from the knee of a 61-year-old
woman undergoing above-the-knee amputation for vascular disease.
OA cartilage was obtained immediately postmortem from the knee
of a 64-year-old man who had clinical and radiographic evidence of
knee OA. Immunolocation was performed using antibody 3-B-3,
with (+) or without (-) chondroitinase pretreatment. Positive
immunolocation analysis with 3-B-3( -) is indicative of subtle biochemical changes in the chondroitin sulfate glycosaminoglycans
extracted from arthritic tissue. The electrophoretic migration of
large (L) and small (S) proteoglycan subpopulation standards (electrophoresed simultaneously) is shown to the right.
Several electrophoretic subpopulations of 6sulfated CS proteoglycans occur in extracts of human
articular cartilage, the slowest-migrating band corresponding to the newly synthesized proteoglycan subpopulation (14). In contrast, positive immunostaining
with 3-B-3( -) occurs in only a relatively homogeneous
slower-migrating large proteoglycan subpopulation
(Figure 2), indicating that it is expressed in newly
synthesized proteoglycans.
A total of 53 cartilage extracts from OA patients
were analyzed in a similar manner. Expression of
3-B-3(-) mimotope occurred in 51 of 53 of the OA
patients. Cartilage extracts from patients with different
arthritides were also examined for the expression of
3-B-3( -) mimotope. Cartilage extracts from 2 patients
with JRA both showed expression, while analysis of 21
RA patient extracts showed a much lower frequency
of expression of 3-B-3(-) epitope, occurring in only 13
of the 21 samples tested. In contrast, the epitope
occurred in 6 of 7 of the extracts from patients who
had had AVN and in all of the extracts from patients
who had sustained a degenerative meniscal tear.
Analysis of 55 normal cartilage extracts indicated that in general (48 of the 5 3 , expression of the
3-B-3(-) mimotope was absent. Three of the 7 normal
cartilage extracts that showed positive 3-B-3( -) were
Toluidlne Blue
Safranin 0
383 (-)
Figure 3. Histochemical and immunohistochemical analysis of a specimen of osteoarthritic cartilage. Toluidine blue and
Safranin 0 stains show generalized loss of proteoglycans in the superficial zone, with fibrillation and cartilage breakdown.
Positive immunostaining with 3-B-3(-) and 7-D-4 is most pronounced in the superficial zones (arrows). Reactivity with
7-D-4 is stronger and occurs in association with chondrocytes throughout the depth of the cartilage (circle).
from patients ages 11-18 years old, and 1 was from a
spontaneously aborted fetus at 18 weeks of gestation.
In 2 other cases in which 3-B-3(-) expression was
observed, reexamination of the surgeon’s notes indicated that these cartilage specimens were obtained
from “apparently normal” samples taken from arthritic joints adjacent to OA lesions.
Our analysis of the expression of the 7-D-4
epitope in patients with different arthritides and normal cartilage indicated that its expression was more
variable compared with that of the 3-B-3(-) mimotope. Analysis of 37 cartilage extracts from OA patients showed that the 7-D-4 epitope was expressed in
22 samples. Furthermore, 7-D-4 epitope expression
occurred in 2 of the 3 cartilage extracts from patients
with RA or JRA, and expression was much less
frequent in the proteoglycans extracted from the cartilage of patients who either had AVN or had torn a
meniscus (2 of 5 and 2 of 6, respectively). In contrast
to the 3-B-3(-) data described above, 15 of the 28
extracts from normal cartilage samples showed expression of the 7-D-4 epitope.
Immunohistochemical and histochemical analyses were performed on tissue sections obtained from
10 patients undergoing joint arthroplasty for OA. Figure 3 shows an example of the results obtained.
Routine histologic stains (toluidine blue or Safranin 0
stains) showed generalized loss of proteoglycan in the
superficial zone, associated with fibrillation and cartilage breakdown. In contrast, expression of the 3-B3(-) and 7-D-4 epitopes occurred predominantly in the
most superficial regions, where the major losses of
proteoglycan had occurred. Reactivity with 7-D-4 was
stronger than that of 3-B-3(-) and tended to occur
around chondrocytes found throughout the depth of
the cartilage. This immunostaining pattern is very
similar to that seen with cartilage sections obtained
from the Pond-Nuki dog model of OA (7).
In this study, we have demonstrated the power
of monoclonal antibody technology to detect subtle
changes in the biochemistry of cartilage proteoglycans
extracted from arthritic versus normal human tissue.
The presence of the native 3-B-3( -) mimotope, recognized by MAb 3-B-3, was most prevalent in cartilage
extracts from patients with OA, AVN, and meniscal
tears compared with those with RA or JRA. However,
this difference does not necessarily detract from the
utility of using 3-B-3( -) to detect metabolic changes in
this disease subset. All of the rheumatoid specimens
were obtained at total joint arthroplasty and many had
superimposed degenerative changes. The absence of
3-B-3(-) mimotope in 8 of the 21 samples may have
been due to the advanced disease state that resulted in
excessive loss of proteoglycans from the tissue.
In contrast, our analysis showed that expression of the 7-D-4 epitope in CS glycosaminoglycans of
proteoglycans isolated from human arthritic versus
normal tissue was less discriminative than was the
expression of the 3-B-3(-) mimotope. These findings
are different from those observed in the Pond-Nuki
canine model of OA, where both 3-B-3(-) and 7-D-4
have proven useful for monitoring subtle changes in
the biochemistry of proteoglycans from OA versus
normal cartilage (5-7). Recent studies (1 1) from another laboratory using a related MAb 846 have also
shown that subtle changes occurring in CS glycosaminoglycans (expression of 846 epitope) also occur in
proteoglycans isolated from human arthritic cartilage
when compared with normal tissue. Collectively,
these data suggest that both the expression of the
3-B-3(-) mimotope and the 846 epitope in proteoglycans from human cartilage may prove useful for diagnosing and monitoring the progression of arthritis in
Data from our present study suggest that expression of the 3-B-3( -) marker reflects proteoglycan
synthesis by the articular chondrocytes, in an attempt
to repair and replace proteoglycans lost during the
disease progression. Several lines of evidence support
this conclusion. The 3-B-3(-) mimotope is found in a
single, relatively homogeneous proteoglycan subpopulation with the same electrophoretic mobility as the
newly synthesized proteoglycans (6,14). Immunohistochemical analyses in this study also demonstrated
positive immunostaining for the 3-B-3( -) mimotope in
the most superficial cartilage layers, where proteoglycan loss is most evident. This suggests that proteoglycans containing this marker were being synthesized in
an attempt to replace these losses. In addition, our
observation that the 3-B-3(-) mimotope is expressed
in normal cartilage during the adolescent growth
phase, at a time when the articular tissues are growing
and remodeling, also supports its expression in new
proteoglycan synthesis. Similar conclusions have been
made in studies using MAb 846 (11).
Special thanks are extended to Karen Slater and
Barbara Nail for their assistance with the preparation of this
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