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From the Joint Diseases Laboratory,*
Shriners Hospital
for Children, Division of Surgical Research, Department of Surgery,
McGill University, Montreal, Quebec, Canada, and Orthopedic Research
Laboratories,
Montefiore Hospital Medical
Center, Bronx Medical Center, Bronx, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In cartilage matrix, aggrecan binds to hyaluronan via the G1 globular domain (hyaluronic acid binding region). A protein called link protein (LP),4,13 which shares some structural homology with the G1 domain,14,15 stabilizes this binding. LP as well as G1 binds to hyaluronan and they bind to each other. We recently showed that the T cells of patients with juvenile rheumatoid arthritis frequently respond to LP, unlike the T cells of nonarthritic controls, in whom such responses are uncommon.16 In the present study, we show that LP, purified from bovine cartilage, can produce a persistent, erosive, inflammatory polyarthritis when injected repeatedly into BALB/c mice. This immunity involves recognition of a predominant T-cell epitope and B-cell epitopes located in three separate domains. These observations indicate that the immunity to LP is able to induce an erosive inflammatory arthritis and may be of importance in the pathogenesis of these joint diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (6 to 8 weeks old, 17 to 20 g) were obtained from Charles River Canada (St. Constant, Quebec, Canada).
Reagents and Culture Media
The following reagents were used: cesium chloride (Kodak Chemicals, Rochester, NY); guanidine hydrochloride, iodoacetamide, phenylmethylsulfonyl fluoride, pepstatin A, and ethylene diamine tetraacetic acid (Sigma Chemical Co., St. Louis, MO); and Freund's complete adjuvant and incomplete Freund's adjuvant (Difco Laboratories, Detroit, MI). The "complete culture medium" (CM) used for lymphocyte cultures was RPMI 1640 (Life Technologies, Inc., Grand Island, NY), supplemented with 5 x 10-5 mol/L 2-mercaptoethanol (Serva Chemie, Heidelberg, Germany), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/L L-glutamine, and 1% nonessential amino acids (Life Technologies). In T-cell proliferation assays, purified protein derivative of tuberculin (Statens Serum Institute, Copenhagen, Denmark) and concanavalin A were used as controls at final concentrations of 10 and 5 µg/ml, respectively. We prepared T-cell growth factors from supernatants of concanavalin A-stimulated spleen cells. Briefly, spleen cells from BALB/c mice were cultured in CM supplemented with 0.1% fresh autologous serum (one spleen per 10 ml of medium) and concanavalin A (5 µg/ml). The cultures were incubated in a humidified incubator with a constant gas flow of 5% CO2 in air. After 24 hours, the cultures were pooled and centrifuged at 900 x g for 10 minutes. The supernatants were collected and stored in aliquots at -20°C until use.
Synthetic Peptides of LP
The synthetic peptides of LP covered the full-length bovine LP
sequence, and each peptide overlapped the next by 5 to 10 amino acids
(Figure 1)
. They were synthesized using
standard 9-fluorenylmethoxycarbonyl chemistry on a solid-phase peptide
synthesizer (model 431A; Applied Biosystems, Foster City, CA). Crude
peptides were purified by reverse-phase chromatography (Prep-10
Aquapore C8 column, Applied Biosystems) using an acetonitrile gradient
in 0.1% trifluoroacetic acid. Peptides B1, B2, and B3 have a cysteine
at their N terminus followed by three glycine residues as spacers.
Other peptides, namely peptide Y1, Y2, Y5, Y6, and Y18, have an
additional glycine at the C terminus (Figure 1)
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LP and the proteoglycan aggrecan were extracted with 4 mol/L guanidine hydrochloride from adult bovine nasal cartilage and purified as described earlier.13,17 Briefly, the proteoglycan aggregate fraction A1 was isolated by equilibrium density gradient centrifugation under associative conditions. This contains LP, aggrecan, and hyaluronan. It was then subjected to equilibrium density gradient centrifugation under dissociative conditions to produce an A1D1 fraction (proteoglycan aggrecan) and an A1D3 fraction. The A1D3 fraction was chromatographed on Sepharose CL-6B in 4 mol/L guanidine hydrochloride and 50 mmol/L Tris-HCl, pH 7.3. Fractions were collected and monitored at 280 nm for protein and by the 1,9-dimethylmethylene blue assay for sulfated glycosaminoglycan.18 The peak fractions containing protein were dialysed and further analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with silver staining and by immunoblotting (see below) for LP and for the glycosaminoglycan keratan sulfate (KS) of aggrecan and G1 domain.11 Fractions containing LP, free of detectable KS and G1, were used as the antigen. LP was also isolated from trypsin-treated bovine aggrecan aggregate fraction (A1A1).19 The protein content was determined by the Lowry assay.20
Gel Electrophoresis and Western Blotting
The primary contaminant of LP is the G1 globular domain of aggrecan. This has recently been shown to contain KS,21 which is immunodetectable using the monoclonal antibody AN9P1.11 Alternatively, G1 can also be demonstrated by specific monoclonal (1C6) or polyclonal antibodies. In an effort to identify any G1 contamination of the LP, preparations were electrophoresed on a 7.5% SDS-polyacrylamide gel electrophoresis minigel (Bio-Rad Laboratories, Richmond, CA) under reducing conditions. They were silver stained for protein. Alternatively, after electrophoresis, the proteins were electrotransferred (60 V, 300 mA) for 60 minutes in 50 mmol/L Tris-glycine with 20% methanol onto a nitrocellulose membrane. The nitrocellulose membrane was immunoblotted with the mouse monoclonal antibody 8A4 (immunoglobulin (Ig) G2b) to LP,22 with the monoclonal antibody AN9P1 (IgG2a) specific for KS chains,23 and with aggrecan G1-specific mouse monoclonal antibody 1C624 or polyclonal rabbit anti-bovine G1 antiserum that was prepared in our laboratory according to the method described elsewhere.25 Briefly, the membrane was blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 hour, and individual lanes were cut out. The lanes were incubated with the different antibodies for 1 hour at room temperature with shaking, followed by three washes in PBS-0.1% Tween 20 for 5 minutes. Goat anti-mouse IgG (Cedarlane Laboratories, Hornby, Ontario) conjugated with alkaline phosphatase was then incubated with the membranes at room temperature for 1 hour followed by three washes in PBS-0.1% Tween 20. Specific antibody binding was visualized by addition of the insoluble alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Bio-Rad).
Induction of Arthritis
Arthritis was induced in BALB/c mice using a protocol described earlier for aggrecan8 but with some modifications. Briefly, mice were immunized with 25 µg of adult nasal bovine LP per intraperitoneal injection. LP was injected in an emulsion of 50 µl of 50 mmol/L Tris-NaCl buffer, with 50 µl of Freund's complete adjuvant (day 0), or with 50 µl of incomplete Freund's adjuvant on days 15, 43, 71, and 99. The injections were stopped when arthritis appeared but continued for the nonarthritic mice up to a maximum of five injections. Injected mice were examined for clinical signs of arthritis every 2nd day as previously described.10 Briefly, joints of front and rear paws and ankles were examined for swelling, redness, and limitation of movement. Severity scores were determined on a scale of 0 to 4 for each front paw or rear paw/ankle as follows: grade 1, mild redness and swelling in one or more joints; grade 2, moderate redness and swelling in one or more joints; grade 3, severe redness and swelling involving the whole paw; and grade 4, grade 3 plus loss of movement in the affected joint. The sum of arthritic scores in all four paws was taken as the severity score of the mouse. Thus, the maximum possible score was 16.
Histology
Fore and hind paws, ankles, knees, and spines were fixed in 10% buffered formalin, decalcified, and stained with hematoxylin and eosin by standard techniques.
Antigen-Specific T-Cell Lines
Long-term culture of specific T-helper cell lines was established essentially as described.26 Briefly, BALB/c mice were inoculated with 60 µg of protein or peptide emulsified in Freund's complete adjuvant in the hind footpads of one foot. Seven to 9 days later, the enlarged draining lymph nodes were removed, and a single cell suspension was prepared. The lymphocytes were incubated in CM containing 1% fresh mouse serum in the presence of antigen for 3 days. The activated T lymphoblasts were then separated by gradient centrifugation and propagated in CM containing 15% T-cell growth factor with restimulation every 10 to 15 days.
T-Cell Proliferation Assay
T cell lines (3 to 4 x 104 cells/well) and irradiated (4000 rads) syngeneic spleen cells (1 x 106/well), as a source of antigen-presenting cells, were co-cultured with the optimal dose of LP (20 µg/ml) or peptides (20 µg/ml) in 200 µl of CM in round-bottomed microtiter plates (Nunc, Naperville, IL). After 48 hours, the cultures were labeled with tritiated thymidine (0.2 µCi/well; Amersham Corp., Arlington Heights, IL) and harvested 12 to 16 hours later with a Titertek multiple harvester (Flow Laboratories, McLean, VA). Cell-associated radioactivity was determined by ß-scintillation counting.
Enzyme-Linked Immunosorbent Assays (ELISAs) for Determining Antibody Titer to LP and Specificity
Blood samples were collected by retro-orbital bleeding. The sera were used for a standard ELISA. Briefly, LP (50 ng per well) or each individual LP peptide was diluted to 20 µg/ml in 0.1 mol/L carbonate buffer, pH 9.2, and 50 µl (1 µg) was added to each well of an Immulon-2 flat-bottom microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA). After 24 hours at 4°C, the plates were washed three times with PBS containing 0.1% v/v Tween-20 (PBS-Tween, Sigma). Noncoated sites were blocked with 150 µl/well of 1% (w/v) casein (Fisher Scientific Inc., Springfield, NJ) in PBS for 1 hour at room temperature. The plates were washed three times with PBS-Tween. Then 50 µl serum (diluted to 1:200) from LP-hyperimmunized BALB/c mice were added to individual wells. After 90 minutes at room temperature, the plates were washed three times with PBS-Tween. Alkaline phosphatase-conjugated goat anti-mouse Ig diluted at 1:1000 in PBS-Tween was added at 50 µl/well. After 1 hour at room temperature, the plates were washed three times with PBS-Tween. Finally, 50 µl of freshly prepared alkaline phosphatase substrate (disodium p-nitrophenol phosphate, Sigma) at 0.5 mg/ml in 8.9 mmol/L diethanolamine and 0.25 mmol/L MgCl2, pH 9.8, was added to each well for 20 to 30 minutes at room temperature. The absorbencies were measured at 405 nm on an ELx808 plate reader (Bio-Tek Instruments, Inc., Winooski, VT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To ensure that LP did not contain the G1 domain of aggrecan or any
other molecules, purified LP was electrophoresed on an SDS
acrylamide gel under reducing conditions and silver stained.
Figure 2A
shows that the characteristic
45- and 49-kd subunits of LP were detected with silver staining. A very
faint sharp doublet of higher molecular weight is shown in the LP
preparation (Figure 2A)
. These additional band(s) in Figure 2A
are not
likely due to a contamination of G1 domain, because G1 bands are
usually broad and faint, as shown in Figure 2, D through F
, because of
varying glycosylation. Moreover, Western blot analysis, with the
LP-specific monoclonal antibody 8A4 (Figure 2B)
, KS epitope-specific
monoclonal antibody AN9P1 (Figure 2C)
, aggrecan G1-specific monoclonal
antibody 1C624
(Figure 2E)
, or polyclonal rabbit
anti-bovine G1 antiserum (Figure 2F)
, further demonstrated the absence
of contaminating proteoglycan or G1 domain. Therefore, there was no
evidence for the presence of high molecular weight G1 domain in our LP
preparation.
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BALB/c mice injected with bovine LP developed an erosive
inflammatory arthritis (Figures 3 and 4)
. In half of mice
(n = 10/21), bovine LP produced a clinically and
histologically defined arthritis (Figure 4)
that was of moderate
severity (mean 4.5) and located in the front and rear paws and ankles
(Figure 4)
. Bovine LP isolated from trypsin-digested proteoglycan
aggregate, which lacks the first 13 N-terminal residues,27
produced a lower incidence of arthritis (n =
4/22, Figure 3
). No arthritis developed in animals immunized with
adjuvant only (n = 0/10). The arthritis produced
by bovine LP had a mean onset of 88 days and reached its highest
incidence at days 100 to 120. The joint swelling persisted in some
animals up to at least day 145 when observations were discontinued
because of ethical limitations. In general, the arthritis was less
severe than the arthritis induced by the aggrecan G1
domain11
but presented in a similar manner.
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Examination of all paws, ankles, knees, and spines of mice
injected with LP within 2 weeks of onset of arthritis revealed that the
swelling, redness, and deformity observed in affected paws and ankles
always involved extensive mononuclear and polymorphonuclear leukocyte
infiltration. In the paws, this was seen around the tarsal and
metatarsal joints as well as carpal, metacarpal, and phalangeal joints
(Figure 4D)
. This resulted in pannus formation and synovitis (Figure 4, B and C)
. An extensive erosive destruction of cartilage and bone was
observed in association with a pronounced synovitis with pannus
formation 85 days after LP immunization (Figure 5, a and b)
. Polymorphonuclear leukocytes
were commonly seen in joint cavities and synovia but were absent from
the joints of control animals. Synovitis and joint destruction were
never observed in the control animals (Figure 4A)
. Spines all appeared
normal in mice injected with LP (data not shown). There was, therefore,
no evidence of the spondylitis that we have observed in mice injected
with the G1 globular domain of bovine aggrecan (after removal of
KS)11
or with human fetal aggrecan (after removal of
chondroitin sulfate).8
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We were unsuccessful in generating T-cell lines to
purified LP. However, five T-cell lines, three raised to a peptide
mixture covering the whole LP and two to peptide Y15, were generated
from different animals as described in Materials and Methods. All of
the T-cell lines were of the CD3+, CD4+, and
CD8- phenotype (data not shown) and responded well to
native LP, but did not respond to native or KS chain-depleted G1
domain of aggrecan or type II collagen. Table 1
shows the results of T-cell
proliferation assays against native LP and the overlapping synthetic LP
peptides (Figure 1)
. All of the T-cell lines responded to peptide Y15,
but not to any other LP peptides (Table 1)
. We have thus identified
a single T-cell epitope of LP located within residues 266 to 290 and
shown that this epitope can be actively processed and presented by
antigen-presenting cells using native LP.
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To identify immunogenic region(s) and arthritis-related B-cell
epitope(s) in LP, sera were collected from LP-immunized BALB/c mice at
an early stage in immunization before the onset of arthritis (day 29)
and also on the day when clinical symptoms appeared (day 58 to 113).
These sera were assessed by an ELISA against LP, as well as by using a
set of synthetic LP peptides. BSA was used as a control. All of the
sera (n = 21) showed binding to native LP,
whereas none bound to BSA, the control protein. There were three main
regions recognized, represented by peptides covering residues 1 to 36,
186 to 230, and 286 to 310 (Figure 6)
.
Residues 1 to 36 (peptides B1, B2, and B3) constitute the N-terminal
end of the molecule, which is the most variable region of LP. Removal
of amino acids 1 to 13 by trypsin from this area results in reduced
arthritis incidence (Figure 3)
. In contrast, residues 186 to 230
(peptides Y12 and Y13) and 286 to 310 (peptide Y16) are located within
the most conserved regions, around the second and third cysteines of
loops B and B' of LP (Figure 7)
.
Both regions are homologous in humans,28
cows (T. M.
Hering, J. Kollar, T. D. Huynh, L. J. Sandell, unpublished
data), and rats.29
The mouse LP sequence is not yet
published.
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. Although the response to peptide B3
(residues 29 to 36) is higher in the arthritic group, this difference
was not quite significant (P = 0.0573) by the
Mann-Whitney U test (Figure 8)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
determined both clinically and histologically (Figures 4 and 5)
. There
was no histological evidence of spondylitis (data not shown), which is
observed on immunization with the keratanase-treated G1
domain.11
The LP arthritis is milder but persistent and is
thereby distinguished from proteoglycan and G1 domain-induced
polyarthritis in BALB/c mice. Although our LP preparation may have been
contaminated with an amount of the aggrecan G1 protein too small to
detect even with our very sensitive methods, contamination is very
unlikely, because the arthritogenic ability of the bovine G1 is wholly
dependent on the removal of KS chains from G1 protein,11
and our LP was never treated in this way. Moreover, there was also no
spondylitis with LP.
We have identified at least one major T-cell epitope in bovine LP
(Table 1)
located at residues 266 to 290 of bovine LP
(NDGAQIAKVGQIFAAWKLLGYDRCD) using LP- or peptide Y15-specific T-cell
lines. It is premature to conclude that this is the predominant T-cell
epitope, because we were unable to isolate T-cell lines and clones
using native LP and the peptides that we used overlap by five amino
acid residues, whereas typical T-cell determinants consist of eight to
nine residues. One important feature of T cell-recognizing epitope 266
to 290 is their cross-reactivity to native LP, indicating that peptide
266 to 290 contains a functional T-cell epitope, especially because the
antigen-presenting cells from mice can process and present native LP to
these T-cell lines (Table 1)
. It remains to be established whether the
T-cell response to epitope 266 to 290 is involved in the induction of
arthritis.
In human arthritis, the immune system is probably exposed to the Gl globular domain and LP, because molecules such as aggrecan and LP are released from cartilage and are detectable by immunoassay in synovial fluid.1 Our recent studies also demonstrate that cellular immunity to human and bovine LP, either as the native protein or in the form of synthetic polypeptides, is frequently observed in patients with juvenile rheumatoid arthritis, compared with nonarthritic age and sex-matched controls. Moreover, in these patients, there was a significant correlation between cellular immunity to LP and clinically measured disease activity.16
Although the present antibody analyses have some limitations in that we
used bound peptides, and this may preclude detection of epitopes
through masking of peptide binding site(s), we have never encountered
this problem in studies of antibodies to peptides and their known
proteins. Our study of antibodies in these mice did, however, reveal
some associations with arthritis induction. Using this approach whereby
peptides were used, we found that the antibodies in sera from
LP-hyperimmunized mice reacted mainly with three domains of LP. One was
located at the N terminus, the most variable region of LP at residues 1
to 36. The other two domains were located at corresponding regions of
loops B and B', at residues 186 to 230 and 286 to 310, respectively
(Figures 6 and 8)
. Both are located in the most conserved part of the
LP molecule. The sequences of these two regions show complete homology
in human,28
cow (T. M. Hering et al, unpublished
data), and rat LP.29
The sequence of mouse LP is not yet
available. Moreover, both regions include a AGWLSDGSVQYPI motif that is
repeated twice in LP and four times in proteoglycan aggrecan core
protein, and is also shared by the proteoglycans versican, neurocan,
the glial hyaluronan-binding protein, and the hyaluronan receptor
CD44.31
Thus, cross-reactivity may occur with these
proteins, but this remains to be established.
That the humoral response against N-terminal peptides 1 to 19 was
significantly higher in arthritic than in nonarthritic mice (Figure 8)
suggests that humoral immunity to this N-terminal epitope may be
associated with the pathogenesis of LP-induced arthritis. The
observation that LP isolated from aggrecan aggregate treated with
trypsin, which lacks 13 residues of the N-terminal
domain,32
has a reduced capacity to induce arthritis
(Figure 2)
also points to the importance of this domain and to a role
for humoral immunity to this region in disease induction.
Amino-terminal peptides up to 30 residues in length are preferentially
removed by extracellular cleavage of LP in proteoglycan aggregates by a
variety of proteases.32
In vivo, this N-terminal
segment is lost from human cartilage.32
Hence, these
peptides are probably released from articular cartilage during the
normal process of turnover and aging, leading to their uptake and
recognition by cells of the immune system, such as monocytes and B
cells.33
We have also observed that human LP can also
induce arthritis in BALB/c mice and that the antibodies that bind to
bovine LP residues 1 to 15 and 23 to 36 often cross-reacted strongly
with the human LP peptides from the same region (Y. Zhang and A.
R. Poole, unpublished data). In summary, although we are limited by our
existing ELISA system, being aware that only linear epitopes have been
identified, which may not represent all such epitopes, we believe that
these B-cell epitope analyses are of value in identifying immunogenic
domains of LP in BALB/c mice. There are two oligosaccharide attachment
sites in the N terminus of LP. They may influence antibody recognition
and may be important in inducing arthritis. Studies are planned to
determine the predominance of antibodies to LP epitopes in immunity to
whole LP. We are currently interested to see whether an immunization of
BALB/c mice with these T- and B-cell epitope peptides will lead to
induction of arthritis.
These collective observations draw attention to the fact that LP, as well as other cartilage molecules that are capable of inducing a rheumatoid-like arthritis in mice, may also serve as an autoantigen in patients with rheumatoid arthritis. Of equal interest, LP is not entirely restricted to cartilage in its distribution in the body, although hyaline cartilage is the principal tissue in which it is concentrated. We and others have shown previously that it is also present in the aortic media,34,35 the central nervous system, and the sclera,33 where it may serve to stabilize the binding of aggregating proteoglycans to hyaluronic acid found in these tissues. Thus, as suggested previously,34 some extra-articular complications of rheumatic disease36 may involve and be caused by immunity to matrix LP. Our study draws further attention to the capacity of different cartilage antigens to induce a persistent inflammatory erosive polyarthritis in experimental animals. Hence, these different cartilage molecules may act as autoantigens in human arthritis.
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Acknowledgements |
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Footnotes |
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Address all correspondence and reprint requests to Dr. Yiping Zhang, Joint Diseases Laboratory, Shriners Hospital for Children, 1529 Cedar Avenue, Montreal, Quebec, H3G 1A6, Canada. E-mail: yzhang@shriners.mcgill.ca.
Accepted for publication July 18, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain of the nicotinic acetylcholine receptor (Torpedo californica). Eur J Immunol 1988, 18:551-557[Medline]
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–
–
, n =
10) were immunized with the same amount of
adjuvant without antigen.
) and were assessed for their binding to BSA,
LP, and individual LP synthetic peptides using an ELISA. The results
are expressed as arbitrary units, calculated as experimental optical
density value divided by the background optical density value.
Statistical significance between arthritic and nonarthritic animals was
assessed by Mann-Whitney U test. The P
value with significant difference between the arthritic and
nonarthritic groups is shown.