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Epitope-specific recognition of type II collagen by rheumatoid arthritis antibodies is shared with recognition by antibodies that are arthritogenic in collagen-induced arthritis in the mouse.

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ARTHRITIS & RHEUMATISM
Vol. 46, No. 9, September 2002, pp 2339–2348
DOI 10.1002/art.10472
© 2002, American College of Rheumatology
Epitope-Specific Recognition of Type II Collagen by
Rheumatoid Arthritis Antibodies Is Shared With Recognition
by Antibodies That Are Arthritogenic in
Collagen-Induced Arthritis in the Mouse
Harald Burkhardt,1 Tobias Koller,1 Åke Engström,2 Kutty Selva Nandakumar,3 Javier Turnay,4
Hans G. Kraetsch,1 Joachim R. Kalden,1 and Rikard Holmdahl3
Objective. To analyze the fine specificity of IgG
autoantibodies in sera from rheumatoid arthritis (RA)
patients for type II collagen (CII) epitopes that are
arthritogenic in collagen-induced arthritis (CIA), a relevant murine model of RA.
Methods. For enzyme-linked immunosorbent assay (ELISA) analysis of conformation-dependent autoantibody binding, recombinant chimeric collagens that
harbor the respective CII epitopes as an insertion within
the frame of a constant type X collagen triple helix were
constructed. In addition, synthetic peptides mimicking
the native collagen structures were applied for the first
time in the ELISA assessment of humoral CII autoimmunity.
Results. The pathogenicity of IgG responses to
certain CII determinants in CIA was demonstrated by
arthritis development in BALB/c mice upon the combined transfer of 2 mouse monoclonal antibodies specific for precisely mapped conformational CII epitopes
(amino acid residues 359–369 [C1III] and 551–564
[J1]), whereas antibodies to another epitope (F4) were
not arthritogenic. To test whether human autoimmune
responses are similarly directed to these conserved CII
determinants, serum IgG was analyzed. The prevalence
of sera with increased IgG binding to the C1III epitope
was significantly higher in RA compared with sera from
healthy donors or from patients with other rheumatic
conditions, e.g., osteoarthritis (OA), systemic lupus
erythematosus (SLE), or relapsing polychondritis (RP),
whereas levels of antibodies specific for the nonarthritogenic F4 epitope were associated with OA rather than
RA.
Conclusion. Autoimmunity to CII, although detectable in different rheumatic conditions, differs in fine
specificity between distinct disease entities. In RA, in
contrast to degenerative joint disease, RP, and SLE,
autoantibody responses are directed to an evolutionary
conserved CII structure that is also targeted by pathogenic autoimmune responses in murine models of arthritis.
Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 263, project C3), the Bundesministerium für Bildung und
Forschung (project C 2.1, BMBF 01GI9948), the King Gustaf V
80-Year Foundation, the Swedish Association Against Rheumatism,
the Swedish Medical Research Council, and the European Commission (Bio4-98-0479).
1
Harald Burkhardt, MD, Tobias Koller, MD, Hans G.
Kraetsch, MD, Joachim R. Kalden, MD: Friedrich-AlexanderUniversity of Erlangen–Nuremberg, Erlangen, Germany; 2Åke Engström, PhD: Uppsala University, Uppsala, Sweden; 3Kutty Selva
Nandakumar, PhD, Rikard Holmdahl, MD, PhD: Lund University,
Lund, Sweden; 4Javier Turnay, PhD: Universidad Complutense, Madrid, Spain.
Address correspondence and reprint requests to Harald
Burkhardt, MD, Department of Internal Medicine III, FriedrichAlexander-University of Erlangen–Nuremberg, Krankenhausstrasse
12, 91054 Erlangen, Germany. E-mail: harald.burkhardt@med3.imed.
uni-erlangen.de.
Submitted for publication January 14, 2002; accepted in
revised form May 3, 2002.
Cartilage-specific molecules such as type II collagen (CII) have long been suspected to be targets of
pathogenic autoimmune responses in the induction or
perpetuation of joint inflammation in rheumatoid arthritis (RA). The hypothesis of CII-directed autoimmunity
as a relevant pathogenic mechanism in RA is an appealing explanation for the chronicity of the arthritic process
as well as for its fatal consequences for the integrity of
joint cartilage. Several lines of evidence further support
the concept of a CII-driven autoimmune process in RA.
Direct evidence for the arthritogenic potential of CII
autoimmunity is derived from experimental disease
models of collagen-induced arthritis (CIA) in rodents as
well as in nonhuman primates (1). In these models, the
arthritis is initiated by complement-fixing autoantibodies
2339
2340
BURKHARDT ET AL
that bind to CII in the cartilage matrix, and the formation of these antibodies upon immunization is major
histocompatibility complex–restricted and T cell–
dependent (2). In humans, anti–CII IgG in rheumatoid
cartilage and synovium and circulating autoantibodies to
native and denatured CII have been detected in the sera
of RA patients (3–6). Moreover, anti–CII IgG–
producing B cells have been detected in rheumatoid
synovium and synovial fluid (7,8), suggesting an intraarticular antigen-driven immune process.
In the present study, we focused on the question
of whether the epitope specificity of anti-CII autoantibody formation in RA differs from that in other rheumatic diseases, such as osteoarthritis (OA), relapsing
polychondritis (RP), and systemic lupus erythematosus
(SLE). Recently, a systematic analysis of conformationdependent autoantibody binding in the murine CIA
model in DBA/1 mice revealed a discrete recognition of
sterically privileged sites on collagen fibrils, suggesting
the possible importance of epitope accessibility in the
cartilage matrix for the formation of arthritogenic immune complexes (9). Since recognition of the identified
immunodominant domains is associated with arthritis
development in CIA and since the CII domains are
conserved in their amino acid sequences in mouse, rat,
and human collagen, we have selected these epitopes for
an analysis of the fine specificity of circulating human
anti–CII IgG autoantibodies.
PATIENTS AND METHODS
Arthritis transfer experiments. According to their
previously characterized distinct specificities for conformational epitopes on CII (9), 3 monoclonal antibodies (mAb)
were selected for antibody transfer experiments of experimental arthritis. Whereas mAb CII-C1 (␥2a) recognizes the triplehelical domain amino acids (aa) 359–369 of CII, mAb M2.139
(␥2b) binds to aa 551–564, and mAb CII-F4 (␥2a) binds to aa
926–936 of triple-helical CII. The mAb were purified from
culture supernatants by affinity chromatography on protein G
(GammaBind Plus; Pharmacia, Uppsala, Sweden).
BALB/c mice (The Jackson Laboratory, Bar Harbor,
ME) were bred and used in the Medical Inflammation Unit
animal housing facility in a specific pathogen–free environment as defined at http://net.inflam.lu.se. Groups of mice (n ⫽
7) were injected with a single mAb or with 2 (C1 ⫹ M2.139) or
3 (C1 ⫹ M2.139 ⫹ F4) mAb cocktails intravenously on day 0.
Control mice received phosphate buffered saline (PBS) only.
On day 5, all mice were injected intraperitoneally with lipopolysaccharide (50 ␮g/mouse) from Escherichia coli serotype
O55:B5 (Sigma, St. Louis, MO). Arthritis in each paw was
scored daily on a scale of 0 (low) to 15 (high) for a minimum
of 21 days after antibody transfer.
Study populations. A pilot study on the specific recognition of 5 conformational CII epitopes, originally character-
ized in murine CIA by human serum IgG, was performed using
an enzyme-linked immunosorbent assay (ELISA) with recombinant chimeric collagen molecules (explained below). The
study population consisted of 22 patients with an established
diagnosis of RA (mean ⫾ SD age 56.3 ⫾ 10.5 years), as defined
by the 1987 revised classification criteria of the American
College of Rheumatology (ACR; formerly, the American
Rheumatism Association) (10), and 22 patients with OA
(mean ⫾ SD age 64 ⫾ 10.4 years) according to the ACR
classification criteria for OA of the hip or knee (11,12).
In an extended study, the specific autoantibody response to selected CII epitopes (C1III and J1) was subsequently investigated in patients with 4 distinct rheumatic
diseases: RA (n ⫽ 48, mean ⫾ SD age 49.9 ⫾ 12.3 years, mean
⫾ SD disease duration 10.0 ⫾ 6.9 years, mean ⫾ SD erythrocyte sedimentation rate [ESR] 41.8 ⫾ 20.1 mm/hour), OA (n ⫽
22, age 68.7 ⫾ 11.0 years, ESR 22.2 ⫾ 8.1 mm/hour), RP (n ⫽
26, age 48.8 ⫾ 11.9 years, disease duration 7.2 ⫾ 5.0 years, ESR
45.2 ⫾ 44.9 mm/hour), and SLE (n ⫽ 38, age 42.3 ⫾ 13.3 years,
disease duration 9.32 ⫾ 5.78 years, ESR 25.2 ⫾ 19.81 mm/
hour).
A modified ELISA technique using synthetic triplehelical peptides as antigens (see below) was applied, and all
OA and RA patients were newly recruited to exclude any bias
by preselection of CII-reactive sera from the initial study.
Inclusion into the new RA and OA cohorts also required the
fulfillment of the above-mentioned ACR classification criteria.
The diagnosis of RP was based on the criteria of
Michet et al (13), and the diagnosis of SLE was based on the
ACR 1982 revised criteria (14). In none of the cohorts was any
preselection of patient sera made on the basis of a pretest for
circulating anti–CII IgG or certain clinical characteristics, e.g.,
disease severity or duration. The study protocol was approved
by the review board of the Friedrich-Alexander-University
Erlangen–Nuremberg, and informed consent was obtained
from all individuals before entering the study.
Recombinant collagen constructs. The vector for expression of chimeric collagen molecules has been described
previously and is based on a human type X collagen complementary DNA (cDNA) clone (15). At the position that corresponds to the amino-terminal end of the secreted protein, a
sequence encoding a 6xHis-tag was introduced to facilitate
purification of the recombinant molecules from supernatants
of transfected cells. A human CII cDNA clone (kindly provided by Dr. S. W. Li, Thomas Jefferson University, Philadelphia, PA) was used as a template for polymerase chain reaction
(PCR) amplification of the respective CII fragments. Specific
restriction sites (sites Pst I/Bam HI), which allowed cloning of
these fragments, were introduced via PCR.
The CII fragments were ligated in-frame with type X
collagen cDNA into the Pst I/Bam HI–restricted pUC 57
vector (Fermentas, St. Leon-Rot, Germany), resulting in the
corresponding chimeric CX–CII constructs that were finally
cloned in pRCMV (Promega, Madison, WI). Prior to the
transfection procedure, the chimeric constructs were controlled by in vitro translation and DNA sequencing. The
following CII fragments encoding frequently recognized B cell
epitopes in murine CIA were inserted into the CX frame for
expression of chimeric collagens (Figure 1): C1III (1623–1655
bp, aa 359–369), J1 (2199–2240 bp, aa 551–564), D3 (2607–
2642 bp, aa 687–698), E/F10II (2877–2897 bp, aa 777–783), F4
SHARED B CELL EPITOPES IN CIA AND RA
Figure 1. Applied recombinant chimeric collagen constructs. The
constant type X collagen frame is shown in the shaded area. The
homotrimeric collagenous portion is flanked by noncollagenous (NC)
globular domains at the amino-terminal (NC2) and at the carboxyterminal (NC1). The amino acid sequences of the short type II
collagen (CII) cassettes representing the epitopes under investigation
are depicted in single-letter code. Boldface letters highlight amino acid
residues (aa) that have already been demonstrated to be critical for
binding of murine monoclonal antibodies (9). *The italic letter E, for
glutamic acid, in the control construct indicates the single amino acid
mismatch by which it differs from the J1 construct.
(3326–3356 bp, aa 926–936). In addition, a recombinant collagen molecule was constructed as a control for nonspecific
IgG binding in ELISA by the introduction of a point mutation
into the chimeric construct for the J1 epitope (Figure 1).
The mutation led to a nonconservative amino acid
exchange (R ⬎ E) in the CII epitope J1 (MPGERGAAGIAGPK), rendering it a non–naturally occurring collagen
sequence within the frame of wild-type CX. The mutation
destroyed the binding of J1-specific murine mAb completely
and was not recognized by any of the murine CII-specific mAb
tested.
For the expression of the collagen chimeras, HEK 293
cells were transfected with plasmid DNA constructs using the
calcium phosphate precipitation method described previously
(9). The transfected cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM)/Ham’s F-12, 5% fetal calf serum
(FCS). After 48 hours, the selection was started by supplementation with 800 ␮g/ml G-418 (Gibco, Paisley, UK). Medium
was renewed every 2 days, and collection of supernatants was
started when the G-418–resistant cells reached confluency.
During harvesting, the transfected HEK 293 cells were kept in
FCS-free DMEM/Ham’s F-12 supplemented with ascorbate.
The 6xHis-tagged recombinant collagens in the culture super-
2341
natants were affinity purified on Ni-NTA Superflow agarose
(Qiagen, Hilden, Germany). The purity of the preparations
was controlled by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis using 10% gels. The proper folding of the
recombinant collagen chimeras was tested in ELISA using
mouse mAb that bind in a strict conformation-dependent
manner to the respective CII inserts.
ELISA. The collagen molecules required for the investigation of antibody specificities were extracted from cartilage
preparations (entire CII, see ref. 9), purified as recombinant
proteins from supernatants of transfected cells, or synthesized
as triple-helical polypeptides (as described below). Microtiter
plates (Nunc, Wiesbaden, Germany) were coated overnight
with purified collagens at 4°C and blocked with 2% bovine
serum albumin in PBS for 1 hour. Standardized coating
efficiencies of purified CII and of the recombinant chimeric
collagens were controlled by the immunoreactivity of mouse
mAb specific for distinct conformational epitopes on CII (9).
The following mAb were applied for the standardization
procedures: CII-C1 (epitope specificity C1III), M2.139
(epitope specificity J1), LN2 173 (epitope specificity D3),
CII-E10 (epitope specificity E/F10II), CII-F4 (epitope specificity F4), and the CX-specific mAb X53 (16).
The CII specificities of the human antibodies were
determined by adding the sera diluted in PBS to the collagencoated microtiter wells for 1 hour at room temperature.
Antibody binding was detected using horseradish peroxidase–
conjugated rabbit anti-human IgG (Dianova, Hamburg, Germany) and ABTS as substrate (Boehringer, Mannheim, Germany). A quantitative analysis of specific IgG binding to the
CII epitopes embedded in the recombinant chimeric collagens
required a normalization of the absolute values of absorbance
for antibody interactions with the large CX domain of the
different constructs. In parallel with the determination of IgG
binding to recombinant chimeric collagens containing the
different CII epitopes under investigation, absorbance was also
assessed in microtiter wells coated with the mutated J1 control
construct (Figure 1). Since the control construct shared the CX
frame with all other constructs (accounting for ⬎90% of the
entire sequence), it was used to measure IgG binding to the
constant part, not specific for a particular CII insert in each
sample. The ratio of absorbanceepitope/absorbancecontrol-construct
was determined in triplicate, and the mean value (arbitrary
units [AU]) was used as a parameter of epitope-specific
binding for the statistical analysis.
Synthesis of triple-helical collagen peptides. Triplehelical collagen peptides were synthesized according to the
method described by Grab et al (17). The sequence of the 2
triple-helical peptides containing either the C1III or the J1
epitope is shown in Figure 6. A triple C-terminal anchor was
obtained by assembling the peptide NH2-Lys(Dde)-Lys(Dde)Tyr(tBu)-Gly on Wang resin (p-benzyloxybenzyl alcohol resin). After removing the Dde protecting groups by treating the
peptide resin 3 times with 2% hydrazine in N,Ndimethylformamide for 3 minutes, each of the 3 strands in the
triple helix was assembled in parallel using the 3 free amino
groups on the anchor peptide still on the synthesis resin. The
peptide resin was treated with acid to release the 3-stranded
peptides from the resin and to remove the side chain protecting groups. Following precipitation, the peptide was washed in
2342
BURKHARDT ET AL
Figure 2. a, Arthritis induction in 4-month-old BALB/c mice by intravenous injection of
monoclonal antibody (mAb) cocktail. Lipopolysaccharide was given intraperitoneally as a secondary immune stimulus. Typical clinical signs of joint inflammation in the mAb (M2.139 ⫹
CII-C1)–injected mouse (right) compared with the phosphate buffered saline–injected control
(left) are shown. b, Inhibition of mAb-induced arthritis by the CII-F4 mAb. Different concentrations of 2 or 3 mAb cocktails were given, and arthritis was scored daily for 3 weeks. Each group
consisted of 7 mice. Values are the mean area under the arthritis curve (AUC).
diethyl ether. The 3-stranded peptides were used without
further purification.
Matrix-assisted laser desorption ionization–time-offlight (MALDI-TOF) mass spectrometry analysis was performed on tryptic peptide fragments. The expected mass-tocharge ratio of the fragments was found in the mass spectra.
Peptide synthesis was performed in an ABI 430 peptide
synthesizer (Applied Biosystems, Foster City, CA) using
9-fluorenylmethoxycarbonyl (FMOC) chemistry with activation of amino acid derivatives with HBTU. For MALDI-TOF
analysis, a Kratos Kompakt IV mass spectrometer was used
(Kratos Analytical, Manchester, UK). Amino acid derivatives
were obtained from Alexis Biochemicals (Grünberg, Germany) or Novabiochem (Laufelfingen, Switzerland). FMOCGly-Wang resin and FMOC-Lys(-1[4,4-dimethyl-2,6dioxocyclohexylidene]-1-ethyl) were purchased from
Novabiochem.
Circular dichroism measurements. Far-ultraviolet circular dichroism spectra were recorded between 180 and 250
nm in a J-715 spectropolarimeter (Jasco, Easton, MD)
equipped with an RTE-111 thermostat (Thermo Neslab, Portsmouth, NH) using 0.01-cm optical pathlength thermostatized
cuvettes and a peptide concentration of 0.07 mM in PBS. The
spectra of triple-helical peptides dissolved in 0.1M acetic acid
were almost identical to those obtained in PBS. Ellipticity is
expressed as the mean residue weight molar ellipticity (␪MRW)
in degrees ⫻ cm2 ⫻ dmole⫺1 units.
Statistical analysis. For the evaluation of statistical
differences in immunoreactivity and clinical parameters between the different cohorts (RA, OA, RP, SLE, and healthy
subjects), analysis of variance (ANOVA) and the MannWhitney U-test were applied.
RESULTS
Transfer of arthritis by mouse mAb with specificity for conformational epitopes on CII. To assess the
arthritogenicity of mAb that bind to 3 well-characterized
conformational CII determinants frequently targeted by
autoantibody responses in murine CIA, antibody transfer experiments were performed by systemic administration of mAb with the respective epitope specificities C1,
J1, and F4. Whereas the intravenous injection of any
single mAb at doses of up to 4.5 mg did not result in
arthritis development in recipient BALB/c mice (results
not shown) as in PBS-injected controls, the combined
application of CII-C1 and CII-J1 antibodies, however,
led to severe joint inflammation (Figure 2a). A pronounced amelioration of arthritis severity was detectable
when CII-F4 antibodies were added to the cocktail of
CII-specific mAb. The resulting reduction of the area
under the curve of arthritis scores (Figure 2b), was
maintained even at high doses of the antibody cocktail
(18 mg) applied in the transfer experiments. Thus, a
pure dilution of the arthritogenic CII-C1/J1 combination
SHARED B CELL EPITOPES IN CIA AND RA
in the triple mixture cannot account for the ameliorating
effect of the CII-F4 mAb. Accordingly, the results
suggest, in addition to the arthritis-promoting function
of the CII-C1 and CII-J1 antibodies, a protective role of
the CII-F4 antibody in the model of anti-CII antibody–
induced arthritis.
Analysis of the fine specificity of circulating
anti–CII IgG autoantibodies with recombinant chimeric
collagen molecules. To investigate whether circulating
human anti-CII antibodies share specificities with murine IgG autoantibodies that arise early in experimental
arthritis (9) and are capable of transferring disease to
naive recipient mice (Figure 2), a systematic ELISA
study was performed on patients’ sera. Recombinant
chimeric collagen molecules that harbor the relevant CII
domains C1III, J1, D3, E/F10II, and F4 in a native
conformation were used as antigens for the ELISA
analysis. In preliminary titration experiments with some
selected patient sera, concentration-dependent specific
IgG binding to the C1III epitope in comparison with the
recombinant control construct could be demonstrated at
antibody dilutions of up to 1:480 (data not shown). For
a comparative study of CII epitope–specific IgG in OA
and RA sera, all assays were performed at a standard
dilution of 1:60, and the results were expressed as the
ratio of absorbanceepitope/absorbancecontrol-construct for
the statistical analysis as described.
Two chimeric constructs containing the J1 and
E/F10II epitopes were not recognized to any significant
extent in any of the sera tested (Figure 3). Since IgG
binding to the J1 and E/F10II constructs did not considerably differ from the respective controls, resulting in
AU values that rarely exceeded 1.2, immunoreactivity to
these constructs was regarded as negative and was used
for the definition of a threshold (in AU) above which an
ELISA result with one of the other epitopes (C1III, F4,
D3) was considered positive. Accordingly, this threshold
is defined as the mean ⫹ 2SD of all J1- and E/F10IIspecific ELISA results (in AU) and is equivalent to 1.2.
It is indicated by the horizontal line in Figures 3a–c. The
cutoff value for negative immunoreactivity was further
validated by analysis of sera from 10 age-matched control patients without inflammatory or degenerative joint
diseases (7 women, 3 men, mean ⫾ SD age 61 ⫾ 12
years) without circulating anti–CII IgG. The range of
absorbance obtained with these 10 control sera for
binding to either the control construct or the different
epitopes (C1III, J1, E/F10II, F4, D3) was between 0.07
and 0.10 (not shown), resulting in AU values below the
threshold of 1.2 (mean 1.0, range 0.7–1.2) in all epitopespecific ELISAs.
Autoantibody binding to the D3 epitope was
2343
Figure 3. Differences in fine specificity of type II collagen (CII)–
directed immune responses between rheumatoid arthritis (RA) and
osteoarthritis (OA). The scatterplots show the results in sera from
patients with OA and RA as assessed with an epitope-specific enzymelinked immunosorbent assay (ELISA) using recombinant chimeric
collagens for the detection of CII autoantibodies. Arbitrary units (AU)
are given as a quantitative measure of the ELISA results. a, IgG
binding to the C1III epitope (amino acids 359–369) was increased in
the RA compared with the OA population, indicating an RA-specific
immunorecognition of this CII domain. b, Autoantibody formation
with specificity for the F4 epitope was detectable at an elevated level
in OA sera compared with RA (P ⬍ 0.02). c, Immunoreactivity to the
D3 epitope (amino acids 687–698), the J1 epitope (amino acids
551–564), and the E10/F10 epitope (amino acids 777–783) does not
differ between both cohorts. Horizontal lines show the mean ⫹2SD (in
AU) of all J1- and E/F10-specific ELISA results.
detectable in RA and OA sera. The results of parallel
measurements using epitope-specific ELISAs revealed a
statistically significant increase in circulating D3-specific
IgG above the threshold for negative immunoreactivity
according to the above definition (P ⬍ 0.01 for comparison of all data presented in Figure 3c). However, the
levels of D3-specific IgG did not differ between the OA
and RA sera, indicating a lack of relationship between
circulating IgG with D3 specificity and the underlying
disease condition in the joints.
The C1III epitope was preferentially recognized
by IgG autoantibodies from RA patients. In this respect,
statistically significant quantitative differences became
apparent in comparison with the ELISA results obtained
with the OA-derived antibodies (P ⬍ 0.01) (Figure 3a).
In contrast, an increased ELISA reactivity with the F4
epitope was detectable in OA sera (P ⬍ 0.02) (Figure 3b).
Thus, the results demonstrate binding of human
IgG autoantibodies to target structures on CII (epitopes
2344
C1III, D3, and F4) that are conserved across species
barriers. Moreover, the results depicted in Figure 3
pinpoint differences in epitope specificity of circulating
CII autoantibodies in the sera of patients, depending on
the underlying disease, with a preferential recognition of
the C1III epitope in RA. In this respect, it is noteworthy
that all 22 OA and RA sera that were included in this
initial investigation contained CII-specific IgG as measured under identical ELISA conditions in microtiter
plates coated with the entire native CII (data not
shown).
This initial approach to analysis of the fine specificity of CII-directed autoantibody responses in RA and
OA sera was subsequently extended, leading to the
inclusion of SLE and RP patients as additional control
cohorts in order to test the hypothesis on the RA
specificity of C1III epitope–directed autoantibody responses. However, in the extended study, a novel technology was introduced for the assessment of CII
epitope–specific IgG that circumvents some disadvantages of the recombinant collagen approach (see Discussion).
Assessment of C1III-specific autoantibodies with
triple-helical synthetic peptides. Triple-helical peptides
containing the C1III or J1 epitope sequences were synthesized using a methodology for solid-phase assembly
of branched triple-helical peptides as described previously (17). These epitopes were selected because they
had been found to be arthritogenic in BALB/c mice.
Three nascent peptide chains were carboxyterminal linked to 1 N␣ amino and 2 N␧ amino groups of
lysine residues and stabilized by 7 GPP* repeats. Triplehelical conformation of the synthetic peptides was assessed by circular dichroism spectroscopy, as shown for
the C1III peptide in Figure 4a. At low temperatures, the
␣1(II) 359–369 peptide exhibited typical features of a
collagen-like conformation, such as a large negative
␪MRW at ␭ ⫽ 198 nm and a positive ␪MRW at ␭ ⫽ 225 nm.
Identical circular dichroism spectroscopic results were
obtained for the ␣1(II) 551–564 peptide (J1) (data not
shown).
Further evidence for the native conformation of
the synthetic peptides is provided in Figure 4b, showing
their immunorecognition by collagen-specific antibodies
in ELISA. The mouse mAb CII-C1 (C1III specific) and
M2.139 (J1 specific) recognize the entire CII in a strict
conformation-dependent manner, as demonstrated by a
lack of binding to heat-denatured collagen. Both antibodies also bound to the specific synthetic triple-helical
peptides that contained the sequence of their corresponding epitopes. The titration curves for recognition
BURKHARDT ET AL
Figure 4. a, Circular dichroism (CD) spectra of the synthetic collagen
peptide containing the C1III sequence (amino acids 359–369). Spectra
were recorded at 10°C and 85°C. At low temperatures, the ␣1(II)
359–369–containing peptide exhibits typical features of a collagen-like
conformation, e.g., a large negative (␪) molar ellipticity at ␭ ⫽ 198 nm
and a positive ␪ at ␭ ⫽ 225 nm. The CD characteristics of a
triple-helical molecule are lost upon heat denaturation. b, Mouse
monoclonal antibodies (mAb) CII-C1 and M2.139 bind in a
conformation-dependent manner to type II collagen (CII) and recognize the synthetic collagen mimetics in enzyme-linked immunosorbent
assay (ELISA). Binding of the mAb CII-C1 and M2.139 to the
branched synthetic collagen peptides ␣1(II) 359–369 and ␣1(II) 551–
564 are shown at different antibody dilutions in ELISA. Identical
titration curves obtained with native CII or the respective synthetic
peptides indicate that the collagen-mimetic molecules contain a perfect image of the conformational epitopes. In contrast, the mAb do not
bind to heat-denatured CII.
SHARED B CELL EPITOPES IN CIA AND RA
Figure 5. Titration of serum IgG binding to the synthetic C1III collagen
peptide (␣1[II] 359–369) in enzyme-linked immunosorbent assay
(ELISA). The sera were from 6 rheumatoid arthritis patients (P1–P6).
ELISA reactivity (absorbance at 405 nm) could be detected in samples
P4–P6, even at higher serum dilutions in phosphate buffered saline,
whereas P1–P3 remained negative.
of the synthetic triple-helical peptides were identical to
those obtained with the entire native CII after purification from cartilage for both mAb. These results provide
clear experimental evidence for the adequate representation of native collagen structures by the synthetic
collagen mimetic molecules.
The results of an ELISA of 6 patient sera using
the synthetic C1III epitope are shown in Figure 5. The
titration curves of IgG binding show that the peptide is
indeed recognized in 3 of the patients, whereas the
remaining 3 were negative.
Detection of IgG serum autoantibodies with specificity for triple-helical synthetic peptides. Sera from
RA (n ⫽ 48), OA (n ⫽ 24), RP (n ⫽ 24), and SLE (n ⫽
38) patients were assessed at a standard dilution of 1:100
in PBS for CII-directed autoantibodies with specificity
for the J1 and C1III epitopes using ELISA plates coated
with synthetic triple-helical peptides containing either
the C1III epitope or the J1 epitope. Sera from patients
with no clinical signs of a rheumatic disease were
assessed in parallel with healthy donor control samples
(n ⫽ 19, 14 women, 5 men, age mean ⫾ SD 63.1 ⫾ 11.1
years). The prevalence of positive ELISA results (data
not shown) for the binding capacity of serum IgG to the
2345
entire native CII differed between the cohorts under
investigation: 66.7% in RA (32 positive of 48 tested),
62.5% in OA (15 of 24), 96% in RP (23 of 24), 31.6% in
SLE (12 of 38), and 5.3% in healthy subjects (1 of 19).
As shown in Figure 6, enhanced binding of IgG
autoantibodies to the ␣1(II) 359–369 peptide was detectable in the RA sera. This result on the preferential
recognition of the C1III epitope by RA-derived autoantibodies was statistically highly significant compared with
data obtained with serum samples from all other cohorts
(P ⬍ 0.0001 by ANOVA). However, a slight increase in
IgG binding to C1III epitope-coated microtiter wells
above the level measured in the OA and SLE sera was
also detectable in sera from RP patients. Assays for the
J1 epitope showed slightly increased absorbance values
with RA and RP sera (P ⬍ 0.001 by ANOVA), but the
absolute measures and the quantitative differences in
the respective OA and SLE controls remained rather
low. A comparison of all ELISA results obtained with
the 2 synthetic CII peptides in the different cohorts
revealed a selective epitope recognition in association
with a given disease entity exclusively for the dominance
of C1III-specific IgG in the RA sera. Thus, the investigations with the synthetic collagen mimetics confirmed
the initial results obtained with the recombinant chimeric collagens (see above) on fine specificity of antiCII autoantibodies.
DISCUSSION
In addition to cartilage-specific proteins, ubiquitously expressed proteins have been suggested as candidate autoantigens in the pathogenesis of RA. Thus,
strong evidence for a potential role of glucose-6phosphate isomerase (G6PI) has recently been provided
by the arthritogenic potential of anti-G6PI IgG in a
murine arthritis model and by the detection of elevated
anti-G6PI autoantibodies in RA sera and synovial fluids
in up to 64% of the samples tested (18). However, RA,
as defined by the commonly accepted classification
criteria, is a rather heterogenous disease, and several
different pathways may result in similar clinical manifestations, as reflected by the animal models that can be
induced by distinct provocations. Several lines of evidence suggest a critical role of genetically linked autoimmunity to cartilage-specific CII in arthritic joint destruction. CII-specific B cells in the inflamed synovium
and in synovial fluid have been shown to produce IgG
autoantibodies that can bind to native collagen structures, indicating an intraarticular antigen-driven immune process in RA (7,8,19). The locally produced
2346
BURKHARDT ET AL
Figure 6. Binding of serum IgG to enzyme-linked immunosorbent assay plates coated with synthetic collagen peptides (structure is shown with amino
acid residues in one-letter code) that contain either the C1III epitope (␣1[II] 359–369) or the J1 epitope (␣1[II] 551–564). Sera were from patients
with rheumatoid arthritis (RA), osteoarthritis (OA), relapsing polychondritis (RP), systemic lupus erythematosus (SLE), and normal healthy donors.
Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent
the median. P values show significant differences in the absorbance at 405 nm, by Mann-Whitney U test. Absorbance values for binding of circulating
IgG to the C1III epitope were significantly elevated in RA patients compared with all other disease entities. In contrast, the absorbance values
measured for J1-specific IgG binding in serum samples from all cohorts evaluated remained at the detection limit. Significant epitope-specific
differences in IgG binding were only detectable in the RA cohort with the preferential recognition of the C1III-containing peptide (ⴱ). Ahx ⫽
aminohexanoic acid, P* ⫽ hydroxyproline.
autoantibodies may be largely consumed in the joint by
complex formation with the cartilage matrix (20),
thereby giving rise to activation of the complement
cascade. Consistent with antibody escape into the circulation, CII-specific IgG is also detectable in the sera of
RA patients, although at a lower frequency compared
with the joint compartment (21). Therefore, the serum
titers of CII-specific autoantibodies might reflect, at
least to a certain extent, the vigor of the local immune
response.
Consequently, several studies have been performed to detect correlations between circulating antiCII antibodies and clinical parameters in RA patients
(3–6). However, formation of autoantibody to CII also
occurs in other rheumatic diseases, e.g., in RP (22), SLE
(5), and degenerative joint disease. Although immunoreactivity to denatured collagen seems to prevail in OA
sera, autoantibodies to triple-helical CII are also detect-
able (23). It therefore appears rather difficult to decide
whether the occurrence of anti-CII autoantibodies plays
a role in the pathogenesis of RA or is merely a result of
the exposure of destroyed cartilage. More direct experimental evidence of a pathogenic potential has been
demonstrated by the ability of human and murine
CII-specific autoantibodies to transfer arthritis to a
normal mouse (21,24). However, the fine specificity of
the arthritogenic anti-CII autoantibodies has remained
obscure. In this respect, the arthritis transfer experiments and the ELISA analysis of CII epitope-specific
human IgG in the present investigation provide the first
experimental evidence for the pathogenicity of autoantibody formation directed to an evolutionary conserved
conformational determinant that is located between aa
359–369 of the CII triple helix.
Our results revealed that the CII region that is an
immunodominant target of arthritogenic murine B cell
SHARED B CELL EPITOPES IN CIA AND RA
responses in CIA is also recognized by human IgG
autoantibodies in RA sera. Moreover, the prevalence of
IgG autoantibodies binding to this particular conformational epitope in RA clearly varies from other rheumatic
conditions, suggesting disease-related differences in fine
specificity of CII-directed autoimmunity. The intriguing
hypothesis that differences in B cell epitope specificities
might be linked to distinct functional aspects of humoral
autoimmune responses is further supported by the predominant IgG binding to the CII region aa 926–936 (F4
epitope) measured in OA compared with RA sera. Thus,
a mouse mAb (CII-F4) with specificity for this region
exhibited a remarkable disease-ameliorating effect in
arthritis transfer experiments in combination with 2
arthritogenic CII-specific mouse mAb. Although the
underlying mechanism of the protective effect of mAb
CII-F4 is presently unknown, at least 2 hypothetical
modes of action can be envisioned. One possibility is the
induction of antiidiotypic immunoregulatory responses
directed to cross-reactive idiotopes on anti-CII antibodies (25). Alternatively, a steric hindrance of proteolytic
degradation of cartilage collagen in the N-telopeptide
region close to the crosslinks in the fibril by stromelysin
could account for the arthritis-ameliorating effect of the
F4 mAb, because the F4 epitope colocalizes in the
quaternary collagen structure with the stromelysin cleavage sites on adjacent molecules (9,26).
For elucidation of disease-associated qualitative
differences in fine specificities of CII-directed humoral
autoimmunity, a novel technology was introduced in the
present investigation. In this approach, recombinant
chimeric collagens were replaced by small synthetic
collagen-mimetic peptides that were synthesized according to a protocol described by Grab et al (17) for the
assessment of conformation-dependent epitope recognition in ELISA. The assay conditions have the advantage
that they do not require additional control for the
exclusion of false-positive results that may arise from
antibody recognition of the type X collagen frame
surrounding the CII epitope inserts in the recombinant
chimeric collagens, and it was also found to mimic the
antibody recognition of native CII. The application of
the new ELISA technology confirmed the role of the
C1III epitope as a preferential target of CII-directed IgG
autoantibody responses in RA sera, whereas J1-specific
IgG remained at the limit of detectability. Remarkably,
the levels of C1III-directed autoantibody binding in RA
not only greatly exceeded the values determined in OA
and SLE sera, but also the ELISA results obtained with
the RP samples, although inflammatory cartilage destruction and CII autoimmunity are common features in
2347
both disease conditions. However, the primary target
tissue of the destructive autoimmune process in RP is
the extraarticular cartilage, e.g., in the ear, nose, and
respiratory tract, whereas the joint is only occasionally
affected by nonerosive inflammation.
In RP, the involvement of extraarticular cartilage
is accompanied by autoimmune responses to matrilin 1,
a cartilage matrix component that is not expressed in
joint cartilage, suggesting its potential importance as the
major autoantigen (27). Accordingly, CII autoantibody
formation in this condition might arise as a secondary
phenomenon during epitope spreading, thus lacking the
epitope specificity that is required for the induction of
erosive joint inflammation. In rodents and, as shown by
the results of our study, in humans as well, autoantibody
responses with C1III fine specificity are associated with
an inflammatory disease leading to the destruction of
hyaline cartilage in the joints. The autoantibodies are
directed to a collagen structure that is conserved in
evolution; it is localized on the cyanogen bromide fragment 11 of CII that has previously been shown to possess
all the structural requirements of an arthritogenic immunogen for the induction of CIA in DBA/1 mice
(28,29).
The systematic assessment of sera from patients
with different rheumatic diseases for immunoreactivity
to CII epitopes that were originally characterized in an
experimental arthritis model in rodents revealed striking
differences in fine specificities of the immune response
that usually remain invisible with detection systems that
use the entire antigen. Two of the epitopes under
investigation (J1 and E/F10II) were not recognized by
human autoantibodies despite the conserved amino acid
sequence in human CII and were detected as major
epitopes in the mouse CIA model. A possible explanation is that sera obtained from CIA mice are obtained
during the priming response and during the early acute
phase of arthritis, phases of disease that are not possible
to address in humans.
The focus of this pilot study on well-defined
collagen structures relevant to the pathogenesis of murine experimental arthritis implies that the recognition
of additional epitopes in human anti-CII autoimmunity
during early events of the disease or the spreading to
other CII determinants in the course of the disease
cannot be excluded. However, the new technical approaches using recombinant collagen chimeras and synthetic triple-helical collagen mimetics also offer promising strategies for future prospective investigations of
larger patient populations to answer these yetunresolved questions of CII-directed autoimmunity in
2348
BURKHARDT ET AL
the pathogenesis of inflammatory joint disease. Therefore, this first comparative study on epitope specificity of
circulating anti-CII autoantibodies in different rheumatic diseases demonstrates the potential of the approach to uncover hidden associations between clinical
conditions and immunorecognition that may relate to
the underlying pathogenic mechanisms and should encourage further work on the fine specificity of jointspecific autoimmunity.
ACKNOWLEDGMENTS
The authors are grateful to Eva Bauer and Carlos
Palestro for excellent technical assistance.
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