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Presence of antibodies to native G1 domain of aggrecan core protein in synovial fluids from patients with various joint diseases.

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ARTHRITIS & RHEUMATISM
Vol. 39, No. 12, December 1996, pp 1990-1997
6 1996, American College of Rheumatology
1990
PRESENCE OF ANTIBODIES TO NATIVE G1 DOMAIN OF
AGGRECAN CORE PROTEIN IN SYNOVIAL FLUIDS FROM
PATIENTS WITH VARIOUS JOINT DISEASES
CHRISTOPHER KAROPOULOS, MERRILL J. ROWLEY, MIRNA Z. ILIC,
and CHRISTOPHER J. HANDLEY
Objective. To investigate the occurrence of IgG
antibodies to aggrecan in synovial fluids (SF) from
patients with arthritis and various articular diseases,
and to determine the nature of epitopes present within
aggrecan that react with these antibodies.
Methods. SF samples were reacted with native
aggrecan, reduced and alkylated aggrecan, chondroitin
sulfate, and keratan sulfate, using dot-blots and a novel
enzyme-linked immunosorbent assay (ELISA). The nature of the epitopes present on aggrecan was elucidated
using Western blots and a competitive inhibition
ELISA.
Results. IgG antibodies to aggrecan were found in
>SO% of the SF samples tested. No IgG antibody
reactivity was observed in serum from the same patients. The antibodies appeared to react predominantly
with native aggrecan, and there was no disease specificity. It was shown that the epitopes to these antibodies
were located within the N-terminal region of the core
protein.
Conclusion. This study demonstrates the frequent
occurrence of IgG antibodies to aggrecan in human SF.
The major epitope is located in the G1 domain of the
aggrecan core protein. These IgG antibodies appear to
be produced locally within the synovial cavity, probably
in response to various articular diseases, resulting in
the loss of native aggrecan from articular cartilage.
Christopher Karopoulos, PhD, Merrill J. Rowley, PhD: Monash University, Clayton, Victoria, Australia; Mirna Z. Ilic, PhD,
Christopher J. Handley, DSc: Monash University, Clayton, and La
Trobe University, Bundoora, Victoria, Australia.
Address reprint requests to Christopher J. Handley, DSc,
School of Human Biosciences, La Trobe University, Locked Bag 12,
Carlton South Victoria 3053, Australia.
Submitted for publication December 14, 1995; accepted in
revised form June 24, 1996.
Aggrecan is the major proteoglycan of articular
cartilage. It is trapped as a complex with hyaluronan and
link protein within the collagen network, where its role is
to give articular cartilage the ability to withstand compressive mechanical loads (1). The aggrecan core protein contains 3 globular domains, termed G1, G2, and
G3, which are stabilized by disulfide bonds (3). The G 1
domain, which is responsible for the interaction of
aggrecan with hyaluronan and link protein, is located at
the N-terminal end of the core protein, as is the G2
domain. The G3 domain is at the C-terminal end of the
core protein (2). The core protein of aggrecan also
contains 2 extended regions that are substituted with
either chondroitin sulfate or keratan sulfate glycosaminoglycan (GAG) chains (2).
Studies using animal models of arthritis have
implicated autoimmunity to proteoglycans in the pathophysiology of synovitis. Cell-mediated and humoral immune responses to aggrecan have been reported in
rabbits with experimentally induced synovitis (4-6).
Chronic inflammatory polyarthritis and spondylitis have
been induced in BALB/c mice by repeated injections of
preparations of aggrecan (7-9), although the requirement for intact GAG side chains on the aggrecan
molecule in these experiments remains unclear. These
studies of aggrecan immunity in animals suggest that
autoimmunity to aggrecan could play a direct role in the
development of rheumatoid arthritis (RA) or other
inflammatory joint diseases. T cell reactivity to aggrecan
has been reported in patients with ankylosing spondylitis
(lo), juvenile rheumatoid arthritis ( l l ) , and RA (12).
However, antibodies to aggrecan have rarely been studied, and Glant et a1 (13) found anti-aggrecan antibodies
in only 6 of 197 rheumatoid synovial fluids (SF) tested
during 15 years. In the present study, we demonstrated
the frequent occurrence of IgG antibodies to aggrecan in
ANTIBODIES TO AGGRECAN IN SF
SF, but not serum, from patients with various articular
diseases, and showed that the antibodies recognize a
major conformational epitope on the G1 domain of the
native aggrecan molecule.
PATIENTS AND METHODS
Patients. SF from the knee joint was obtained at the
time of joint aspiration performed for therapeutic purposes or
at arthroscopy. Samples were obtained from 83 patients who
fulfilled the American College of Rheumatology (formerly, the
American Rheumatism Association) criteria for RA (14) and
42 patients with other articular diseases, including 10 with
psoriatic arthritis (PsA), 17 with osteoarthritis, and 15 with
other miscellaneous joint disorders (7 with trauma, 2 with
chondrocalcinosis, 1with gout, 1with unspecified polyarthritis,
1 with monarthritis, 1 with ankylosing spondylitis, 1 with
osteochondritis, and 1 with acute viral arthritis).
Clinical information on the patient group was incomplete, but 62% of the patients with RA and 64% of the patients
with other articular diseases were female. The mean age was
57.7 years (range 22-84) for the RA patients (n = 66) and 52.8
years (range 20-80) for the patients with other articular
diseases. For the 28 RA patients for whom the information was
recorded, disease duration ranged from 3 weeks to 45 years
(mean 16.8 years), and for the 12 patients with other articular
diseases, disease duration ranged from 1 week to 20 years
(mean 4 years). (It should be noted, however, that for most
patients with osteoarthritis, the duration was not recorded.)
Where recorded, the volume of S F ranged from 4 ml to 60 ml
in RA patients (n = 27) and from 4 ml to 100 ml in patients
with other articular diseases (n = 18).
Preparation of aggrecan monomer and link protein.
Articular cartilage (-20 gm) from adult bovine metacarpophalangeal joints was extracted for 48 hours at 4°C with 15 volumes
of 0.05M sodium acetate, pH 6.0, containing 4M guanidinium
chloride and protease inhibitors (15). An A1 aggrecan preparation was prepared by CsCl density gradient centrifugation as
previously described (16). Some of the resulting A1 preparation was recentrifuged to give an A l A l fraction. The remaining A1 fraction was subjected to density gradient centrifugation in the presence of 4M guanidinium chloride, to give a D1
aggrecan preparation (16). The above method was also used to
prepare A l A l D l , AlAlD2, and AlAlD3 fractions from the
A l A l preparations. When analyzed by dot-blot assay using
monoclonal antibody (MAb) 9/30/8-A-4 (to a peptide epitope
in link protein [17]), neither the A l D l nor the A l A l D l
fraction showed any reactivity with the antibody (results not
shown), which indicated that these aggrecan preparations were
free of link protein. Analysis of the A1D3 fraction by sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under reducing conditions, followed by staining with silver,
showed that this Gaction contained only a protein with the
same migration as link protein.
Preparation of chondroitin sulfate and keratan sulfate
from aggrecan. Articular cartilage (-10 gm) from bovine
metacarpophalangeal joints was incubated for 5 hours at 37°C
in 50 ml of a modification of Dulbecco’s modified Eagle’s
1991
medium containing 500 pCi 35S-sulfate (18). The tissue proteoglycans were extracted with 4M guanidinium chloride containing protease inhibitors as described above, and a D1
aggrecan fraction prepared by density gradient centrifugation
In the presence of guanidinium chloride (16).
An aliquot of the D1 fraction was digested with papain
(1 mg papain/4.3 mg aggrecan) for 16 hours at 60°C (16),
followed by treatment with 1M BH,, 0.5M NaOH at 4°C for 72
hours (19). The 35S-labeled GAGS were precipitated with 4
volumes of ethanol at 4°C overnight, collected by centrifugation, and the precipitate was redissolved in 6M urea, 0.05M
sodium acetate, 0.15M NaCl, pH 6.0. The papain digest was
applied to a Sepharose Q column (1.2 X 10 cm; Pharmacia,
Uppsala, Sweden), and the column was washed with 6M urea,
0.05M sodium acetate, 0.15M NaCI, pH 6.0, and then eluted
with a linear 0.15M-1.5M NaCl gradient in the same buffer
(400 ml total volume). Fractions (3.1 ml) were collected and
analyzed for radioactivity. The fractions corresponding to
keratan sulfate and chondroitin sulfate were pooled separately.
Preparation of G1 domain of aggrecan. An aggrecan
complex preparation (Al) containing 52 mg of aggrecan
complex was brought to 10 ml with 0.M Tris acetate buffer,
pH 7.0, and 130 p1 of 10 mg trypsidm1 was added and the
solution incubated overnight at 37°C (20). The G1-link
protein-hyaluronan complex was isolated from the digest as
previously described (21). The G1 domain was separated from
the link protein and hyaluronan by size-exclusion chromatography on a Sepharose CL-6B column (21).
After reduction and alkylation, analysis using dot-blots
(see below) showed that the G1 preparation reacted with MAb
12/21/1-C-6(to a common peptide epitope in both the G1 and
G2 domains of the core protein [21]), 9/30/8-A-4 (to a peptide
epitope present in link protein [17]), and 1/20/5-D-4 (to
oligosaccharides from keratan sulfate consisting of several
repeats of the disulfated disaccharide GlcNAc[6-sulfate] pl-3
Gal[6-sulfate] pl-4 [23]). This fraction did not react with MAb
5/6/3-B-3 (to terminal unsaturated chondroitin-6-sulfate disaccharides [17]), which indicated that the G1 preparation contained mainly G1, with some link protein present.
Chemical and enzymatic treatment of aggrecan, aggrecan domains, and link protein. Aggrecan monomer (AID1 or
AlAlDl), aggrecan G1 preparation, and link protein (AlD3)
were either dissolved or diluted to a concentration of -3
mg/ml with a solution containing 1 mM EDTA and 5 mM
dithiothreitol (D’IT). This solution was heated to 70°C for 1
hour to reduce disulfide bonds, which were then alkylated with
the addition of iodoacetamide to a final concentration of
35 mM.
Aggrecan (2 mg) or G1-link protein-hyaluronan ternary complex was dissolved in 2 ml of 0.1M Tris acetate buffer,
pH 7.0, containing protease inhibitors (24), and 100 pl chondroitinase ABC (2.5 units/ml) and 100 pI keratanase (5
unitdml) were added and the solution incubated overnight at
37°C. The sample was desalted on a Sephadex G-50 column
equilibrated with 0.05M NH,HCO,, pH 8.0, v d freeze dried.
Quantification of aggrecan complex, aggrecan, aggrecan fragments, and link protein. Aggrecan complex and
aggrecan were assayed using the dye (1,9-dirhethylmethylene
blue) binding method of Farndale et a1 (25), adapted for
microtiter plates (26). Whale shark chondruitin sulfate was
used as standard. Aggrecan fragments and link protein were
1992
assayed for total protein content by the Bradford assay (27),
using bovine serum albumin as standard.
Immunolocalization studies. Dot-blots. Dot-blots were
used to assess the purity of various antigen preparations and to
screen SF for antibody reactivity to multiple antigen preparations. Samples of each 5-pl antigen preparation (AlD1 0.175
mg protein/ml; GAGS 4.64 mg dry weight/ml) were spotted
onto marked grids of nitrocellulose membrane and allowed to
dry. After blocking with 5% (weight/volume) skim milk in
phosphate buffered saline (5% SM-PBS) for 2 hours at room
temperature ( B ) ,the membrane was cut into strips and
incubated overnight at 4°C with test SF diluted 150 in 5%
SM-PBS or with MAb 12/21/l-C-6,9/30/8-A-4, 1/20/5-D-4, and
5/6/3-B-3, diluted 1500. Antigens that were reacted with
antibody 12/21/1-C-6 were first reduced and alkylated using
D I T and iodoacetamide, as described above, in order to
expose the epitope. The strips were washed 4 times, and bound
human IgG or mouse MAb was detected with horseradish
peroxidase-conjugated, affinity-purified sheep anti-human
IgG (ychain specific) or anti-mouse Ig antibodies at a dilution
of 1:400 (catalog no. GAH, MAH; Silenus, Hawthorn, Australia) for 2 hours at room temperature. The strips were washed
and then incubated with substrate containing 0.5 mg/ml
4-chloro-l-naphthol, 17% methanol, and 0.005% H,O, in PBS,
pH 7.4.
Western blots. Preparations of chondroitinase ABCand keratanase-treated G1-link protein-hyaluronan ternary
complex (10 pl, containing -3 pg protein/ml) were dissolved
without boiling in SDS sample buffer that did not contain any
reducing agents (29). The presence of SDS in the sample
buffer was found to be sufficient to cause dissociation of the
ternary complex. This preparation was subjected to electrophoresis on SDS-15% polyacrylamide gels and then electrotransferred to nitrocellulose. The resulting blots were probed
with antibodies as described above for dot-blots. Strips were
incubated with SF at a 1:lOO dilution in 5% SM-PBS overnight
at 4°C. In addition, strips were incubated at a dilution of 1:200
with MAb 12/21/1-C-6 and 9/30/8-A-4, which recognize peptide
epitopes present in the G1G2 domain of the aggrecan core
protein and link protein, respectively. Strips that were reacted
with antibody 12/21/1-C-6 were first reduced and alkylated
using D?T and heated for 2 minutes at 1OO"C, to expose the
epitope.
To achieve a stronger signal, some strips were developed using enhanced chemiluminescence (ECL), carried out
with a commercially available ECL kit (Amersham, Amersham, England) according to the manufacturer's instructions.
The SF tested using the ECL development technique were
diluted at 1:600 and the MAb were used at a dilution of
1:2,000. Bound IgG antibodies were detected with the same
anti-human IgG and anti-mouse Ig antibodies described above,
but these were used at a dilution of 15,000 for ECL.
Measurement of IgG antibodies to aggrecan by
enzyme-linked immunosorbent assay (ELISA). Dry nitrocellulose strips were dipped into untreated or reduced and
alkylated preparations of aggrecan (A1AlDl) which contained
-40 mg/ml protein. The strips were allowed to dry, cut into
exactly 1 cm x 0.5 cm pieces, and placed in flat-bottomed
polystyrene microtiter plates (Maxisorp Nunc, Roskilde, Denmark), against the side of the well. These wells were 0.5 cm
deep and contained 300 pl. Uncoated nitrocellulose pieces
were used as no-antigen controls. The plates containing the
KAROPOULOS ET AL
nitrocellulose pieces in each well were blocked overnight at
4°C with 300 pl/well of 5% SM-PBS and then washed 3 times
with 5% SM-PBS with soaking for 10 minutes, using a Titertek
(ICN Biomedical, Costa Mesa, CA) microplate washer.
The optimum dilution of SF for this ELISA was
determined by titrating a positive control SF using doubling
dilutions from an initial dilution of 1 5 0 to a final dilution of
1900. A dilution of 1:600 was chosen as appropriate since at
this dilution, most samples were in the linear range of the
antibody response and gave the best discrimination from the
no-antigen control. SF (100 pl) diluted 1:600 in 5% SM-PBS
was added and incubated overnight at 4°C. The plates were
washed 6 times for 10 minutes each with 5% SM-PBS and
incubated with 200 pl/well of horseradish peroxidaseconjugated, affinity-purified sheep anti-human IgG for 2 hours
at room temperature. The plates were then washed 3 times for
10 minutes each with 5% SM-PBS and then 3 times for 10
minutes each with PBS, before addition of 150 pVwell of
substrate (1 mM 2,2'-azinobis[3-ethylbenzothiazoline-6sulfonic acid] in 0.03M citric acid, 0.04M Na,HPO,, 0.003%
[volume/volume] H,O,, pH 4) and incubation for 10 minutes.
The rate of color production was then determined in a
Bio-Rad 3550 (Syndey, Australia) microplate reader, using the
Kinetic Collector software. For each sample, the rate of change
of optical density obtained in wells containing uncoated nitrocellulose was subtracted from that in wells containing
aggrecan-coated nitrocellulose, to correct for nonspecific binding of antibodies. The use of rate of change of optical density,
rather than optical density at a single time point, allows the use
of a single dilution to compare strongly positive samples, in
which color development takes place very rapidly, and weakly
reactive samples, in which color development is slow. A
positive control SF, arbitrarily defined to contain 100 units/ml
of antibody reactivity, was included in every assay, and the rate
of color development of each sample was expressed as a
percentage of that of the positive control. Samples were tested in
duplicate, and the interassay coefficient of variation for the assay
was 6.3% (n = 6).
Five serum samples were also titrated in an identical
manner to determine the optimum serum dilution for this
ELISA. These included 1 serum sample paired with the
positive control SF (obtained from the same patient). None of
these serum samples showed any reactivity with aggrecan.
ELISA for competitive inhibition of aggrecan. To
further examine the specificity of the IgG reactivity with
aggrecan observed by ELISA, 4 SF that contained high levels
of IgG antibodies to aggrecan were tested in a competitive
inhibition ELISA. These SF were chosen for their strong
anti-aggrecan reactivity by ELISA. SF were either tested
without any inhibitor (negative control using distilled water) or
inhibited with aggrecan monomer (positive control), G1 domain, or link protein preparations. SF from a patient with RA
and a patient with PsA were also tested with reduced and
alkylated aggrecan monomer as the inhibiting antigen, to help
verify that the native molecule is required for correct epitope
expression. The inhibitor or distilled water was added to SF in
a final volume of 200 pl, and maintained at room temperature
for 30 minutes. The samples were then added to the wells of a
microtiter plate containing aggrecan-coated nitrocellulose, and
levels of antibody were measured as for a standard ELISA.
Results are expressed as percent inhibition relative to controls
tested in the absence of inhibitor.
ANTIBODIES TO AGGRECAN IN SF
1993
RA
Untreated A1 D1
Reduced and Alkylated A1 D1
Chondroitin Sulphate
Keratan Sulphate
PA
I
QA
I
I
Untreated A1 D1
Reduced and Alkylated A1 D1
Chondroitin Sulphate
Keratan Sulphate
Figure 1. Dot-blots of native aggrecan, reduced and alkylated aggrecan, chondroitin sulfate, and keratan sulfate,
with synovial fluids from patients with rheumatoid arthritis (RA), psoriatic arthritis (PA), osteoarthritis (OA),
trauma, gout, ankylosing spondylitis, and osteochondritis.
Statistical analysis. The data were analyzed using
Student’s 2-tailed t-test. However, because of possible violations of some of the parametric assumptions of the t-test,
statistical analysis was also carried out by the Mann-Whitney U
test. Correlations were analyzed using Pearson’s product moment and Spearman’s rank correlations.P values less than 0.05
were considered significant. Statistical analysis was performed
on an IBM-compatible PC, using the Complete Statistical
System software package.
RESULTS
IgG antibodies to aggrecan. Detection by dot-blot.
SF samples from 53 patients with RA and 32 patients
with other articular diseases were analyzed, using dotblots, for reactivity to both native aggrecan and aggrecan
that had been reduced and alkylated. Antibody reactivity, graded qualitatively as weak, moderate, or strong,
was detected in 43 of the 85 samples (51%). Figure 1
shows typical patterns obtained in dot-blots from a
number of SF from patients in each of the disease
groups. Reactivity was predominantly with native aggrecan, and very few SF showed any reactivity with the
reduced and alkylated macromolecule. Control experiments using the specific h4Ab showed that native AlD1,
reduced and alkylated AlD1, chondroitin sulfate, and
keratan sulfate bound to nitrocellulose.
Measurement by ELISA. Aggrecan with intact
GAG side chains appeared to bind poorly to polyvinyl
microtiter plates, and hence could not be used in a
conventional ELISA. Although aggrecan digested with
chondroitinase ABC and keratanase bound well to the
plates, as shown by reactivity with MAb 5/6/3-B-3 (to
terminal unsaturated chondroitin-6-sulfate disacchar-
ides) (results not shown), results obtained with human
antibodies using this antigen in a conventional ELISA were
not reproducible. For this reason, levels of IgG antibadies
to aggrecan were measured in a modified ELISA using
aggrecan with intact GAG side chains bound to nitrocellulose. Results were compared with those in a strongly
reactive SF (defined to contain 100 units/ml of antib&)
from a patient with PsA, tested in each assay.
SF tested by ELISA at a dilution of 1:600 contained levels of aggrecan antibodies ranging from 0 to
160 units/ml, and there was good correlation between
the levels of antibodies found by ELISA and the strength
of reactivity by dot-blotting using a dilution of 1:lOO
(Figure 2). In contrast with the reactivity of SF, no
serum samples tested, whether from normal blood donors or from patients, showed reactivity to aggrecan at
any serum dilution. The lack of antibody reactivity in
serum was notable since a paired serum sample obtained
from the positive control at the same time as the SF was
obtained was completely nonreactive, and the level of total
IgG in serum is normally higher than that in SF (30).
The unavailability of SF from the healthy controls made it impossible to define an upper limit of
“normal” for aggrecan IgG antibodies in SF. The range
of antibody levels in SF was similar in each of the disease
groups tested. However, the mean level of 64.7 in the 10
patients with PsA was significantly higher (P < 0.05)
than the mean level of 32.8 in the 83 patients with RA,
and the mean level of 57.8 in the 7 patients with trauma
was nonsignificantly increased compared with the RA
group (Figure 3).
There was no correlation between levels of IgG
KAROPOULOS ET AL
1994
antibodies to aggrecan and disease duration in patients
with RA or in patients with other articular diseases (data
not shown). Also, there was no correlation between
levels of IgG antibodies to aggrecan and age, sex, or
volume of SF in the joint, for the patients with RA or
those with other articular diseases (data not shown).
Epitope mapping of aggrecan IgG antibodies.
The A l D l and A l A l D l aggrecan preparations were
shown Eo produce the same reactivities with SF (results
not shown). To determine whether any of the reactivity
observed with the aggrecan preparations could be attributable to antibodies reactive with chondroitin sulfate
and/or keratan sulfate, SF that reacted with both native
and reduced and alkylated aggrecan were tested for
reactivity to chondroitin sulfate and keratan sulfate by
dot-blotting. No reactivity was observed with any of the
SF tested (Figure 1).
Reactivity to G1 domain, The predominant reactivity of SF with the native preparation of aggrecan
monomer, and the lack of reactivity with the reduced
and alkylated monomer, suggested that the epitope
recognition involved disulfide bonds, and made it likely
that these IgG antibodies were recognizing conformational epitopes in l of the 3 globular regions of the
aggrecan core protein. In 16 SF that contained varying
200
175
f
.a
5
I
0
150
125
.
I
's1
p
43
100
15
0
$
50
25
0
None
Weak
Moderate
Strong
(n = 26)
(n = 16)
(n = 16)
(n = 27)
Aggrecan Antibodies (dot-blot reactivity)
Figure 2. Comparison of IgG antibody reactivity to aggrecan in dot-blots
with levels of IgG antibodies to aggrecan measured by enzyme-linked
immunosorbent assay. Bars represent the mean 2 SEM.
175
n
150
3
a
-2 125
El
I
l
1
o
a
a2
3
loo
15
I
0
a2
L
M
50
P
25
0
RA
PA
OA
Trauma
Others
( n = 83)
( n = 10)
(n = 17)
(n=7)
( n = 8)
Disease Group
Figure 3. Levels of IgG antibodies to aggrecan. determined by
enzyme-linked immunosorbent assay, in synovial fluids from patients
with rheumatoid arthritis (RA), psoriatic arthritis (PA), osteoarthritis
(OA), trauma, and other joint diseases. Bars represent the mean 2
SEM.
levels of IgG antibodies to aggrecan detected by ELISA,
Western blots were performed under nonreducing conditions on a chondroitinase ABC- and keratanasedigested ternary complex of the G1 domain, link protein,
and hyaluronan (Figure 4). For most SF, there was
reactivity with 2 bands, of M , 64 kd and 55 kd, that were
identified as trypsin-generated fragments of the G1
domain of the aggrecan core protein, based on reactivity
with MAb 12/21/1-C-6, which is reactive with the reduced and alkylated G1 domain (21). Notably, there was
no reactivity of SF with the link protein present in the
ternary complex preparation, as evidenced by the finding that reactivity with MAb 9/30/8-A-4 did occur. The
reactivity of MAb 9/30/8-A-4 with a peptide of M , -84
kd indicated the presence of dimers of link protein.
Competitive inhibition of aggrecan IgG antibodies by ELISA. Inhibition assays were performed on 4 SF
that reacted strongly in the ELISA (Figure 5). In each
case, the reactivity to native aggrecan coated on the
nitrocellulose could be totally inhibited with the same
native aggrecan preparation used as inhibitor in solution. Furthermore, the reactivity was also totally inhibited in each case by a purified preparation of the G1
domain, and the inhibition profile was identical to that
1995
ANTIBODIES TO AGGRECAN IN SF
Stds (kDa)
11284-
-64
55
-
53.4-
-
34.9
28.7-
-
20.5
l
I
6
s
!!?Q
. m
-
u
RA
u
OA Trauma
PA
Figure 4. Western blot of the chondroitinase ABC-and keratanase-treated G1-link protein-hyaluronan ternary
complex (each track contained -0.03 pg protein) with monoclonal IgG antibodies 12/21/1-C-6 and 9/30/8-A-4,
and selected synovial fluids from patients with rheumatoid arthritis (RA), psoriatic arthritis (PA), osteoarthritis
(OA), and joint trauma. The strip that was reacted with the antibody 12/21/1-C-6 was first reduced with
dithiothreitol to expose the epitope, as described in Patients and Methods. Stds = standards.
observed with the intact aggrecan monomer. There was
no significant inhibition with native link protein (AlD3)
or reduced and alkylated aggrecan monomer. The latter
findings support the data from the dot-blot experiments
indicating that GAGS were nonreactive with IgG antibodies present in SF.
DISCUSSION
. .
This study demonstrates the frequent occurrence
of IgG antibodies to aggrecan in SF, but not serum, from
patients with various articular diseases. The development of a novel ELISA, using aggrecan bound to
nitrocellulose, has enabled sensitive and reproducible
detection of aggrecan antibodies in human SF. Furthermore, a major epitope recognized by these antibodies
has been identified in the G1 domain of the molecule.
The first globular domain ( G l ) of aggrecan is
located at the N-terminus and is responsible for binding
to hyaluronan and link protein. The G1 domain shares
considerable structural and sequential homology with
link protein, and can be divided into 3 distinct domains:
the immunoglobulin fold, which is responsible for binding to the immunoglobulin fold of link protein, and 2
proteoglycan tandem repeat loops, which are also
present in the G2 domain of aggrecan as well as link
protein (2). Given the similarity in primary and tertiary
Y
I
I
10
1
10
1
Inhibiting Antigen Concentration (pg/ml)
Figure 5. Results of an inhibition enzyme-linked imrnunosorbent
assay carried out with synovial fluids from 1 patient each with
rheumatoid arthritis (RA), psoriatic arthritis (PA), osteoarthritis
(OA), and joint trauma. Inhibiting antigens, i.e., native aggrecan
monomer (@), reduced and alkylated aggrecan monomer (0), native
G I domain (O),
and AID3 fraction containing native link protein (A),
were used at 6 different concentrations. Shaded area represents the range
of inhibition over 5 duplicate assays using distilled water as control.
1996
structure between the G1 domain and link protein, it is
striking that no immunologic cross-reactivity was observed between link protein and the G1 domain of
aggrecan. From our findings, the possibility cannot be
excluded that the G1 and G2 domains share a crossreactive epitope. However, since there is less structural
and amino acid homology between the G1 and G2
domains than between the G1 domain and link protein,
this seems unlikely (2).
Studies analyzing the products of the catabolism
of aggrecan in normal and diseased cartilage have shown
that aggrecan is lost from cartilage by proteolytic cleavage at a specific site between the G1 and G2 domains
of the core protein of this proteoglycan (31). This results
in aggrecan fragments containing the G2 and GAG
attachment regions being rapidly lost from the tissue,
and because they do not contain the G1 domain, they
will not show reactivity with these antibodies to the
native G1 domain. The metabolic fate of the remaining
G1 domain of the aggrecan core protein is not clear;
however, studies have shown that this region can be
detected in SF from patients with RA, indicating that
the G1 domain is lost to the SF (32). It is likely that
this peptide will remain within the extracellular matrix
of cartilage as a complexwith hyaluronan and link protein.
It is possible that in joint trauma, complexes consisting of
native G1, link protein, and hyaluronan will be released
from the tissue as the result of either their becoming
smaller in hydrodynamic size due to proteolytic processing
within the C-terminal region of the aggrecan core protein
(33), fragmentation of the hyaluronan by the action of free
radicals (34), or changes in the collagen network due to the
action of proteases (35).
There have been few previous studies of antibodies to aggrecan in humans, and those that have been
reported have generally had negative findings. In the
study by Glant et a1 (13), aggrecan antibodies were
found in fewer than 4% of rheumatoid SF, and in those
patients, antibodies were detectable only if previously
dissociated from immune complexes. Furthermore, reactivity was enhanced when aggrecan was treated with
hyaluronidase or chondroitinase ABC, suggesting that,
as shown in the present study, the actual epitope recognized is on the protein core, rather than associated with
the GAG side chains.
It is notable that antibodies to aggrecan could not
be reliably measured using either polyvinyl or polystyrene microtiter plates coated directly with aggrecan,
even though aggrecan was shown to be bound to the
plate and was strongly reactive with the mouse MAb
5/6/3-B-3. Since the aggrecan IgG antibodies in SF
KAROPOULOS ET AL
reacted with native but not denatured aggrecan, it is
likely that binding to the plastic influenced the conformation of the molecule, thus accounting for the nonreactivity by conventional ELISA. Similar effects of different substrates have been reported for other antigens
(36), and it is likely that the failure to detect aggrecan
antibodies in SF in previous studies has been due largely
to the unsuitability of conventional ELISAs.
The identification of IgG antibodies to native but
not denatured aggrecan in SF from patients with various
forms of articular damage raises the question of the
origin of these antibodies, and their role in the disease
process. Naturally occurring autoantibodies to many
autoantigens have been detected in both humans and
animals, but their significance remains uncertain. Grabar (37) suggested that they may play a role in homeostasis, and in the removal of tissue debris. The
presence of aggrecan antibodies in SF samples, many of
which had previously been shown to contain antibodies
to collagen, would suggest that such antibodies may
indeed be a normal physiologic response to tissue damage. These antibodies may be produced by B cells within
the joint in response to the appearance of native G1
domain in the synovial cavity, and because they react
with native G1 peptide within the SF or are absorbed to
the surface of cartilage, they may never be released from
the joint cavity.
Indeed, B cells producing antibodies to type I1
collagen have been detected in SF but rarely in serum
(38), and antibodies to type I1 collagen are present in
higher levels in SF than serum (30). However, collagen
antibodies, in contrast to aggrecan antibodies, do sometimes appear in the circulation and do show disease
specificity,being detected more frequently in RA than in
other diseases. If, as has been suggested, immunity to
collagen plays a part in the pathogenesis of RA, it is
likely that the underlying immunologic defect lies in a
failure to down-regulate the normal response to collagen
after tissue damage, rather than an abnormal response
to collagen per se. In the case of antibodies to aggrecan,
there is no information as to whether their production
changes over time; however, these antibodies may be
candidates for markers for the onset of joint disease.
ACKNOWLEDGMENTS
We thank the members of the Departments of Rheumatology at the Royal Melbourne Hospital and the Monash
Medical Centre for providing samples of SF and access to
clinical information. We are grateful to Dr. I. R. Mackay for
valuable discussions.
1997
ANTIBODIES TO AGGRECAN IN SF
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