From the In collagen-induced arthritis, a murine
autoimmune model for rheumatoid arthritis, immunization with native but
not heat-denatured cartilage-specific collagen type II (CII) induces a
B cell response that largely contributes to arthritogenicity.
Previously, we have shown that monoclonal antibodies established from
arthritis prone DBA/1 mice require the triple-helical conformation of
their epitopes for antigen recognition. Here, we present a novel
approach to characterize arthritis-related conformational epitopes by
preparing a panel of 130 chimeric collagen X/CII molecules. The
insertion of a series of CII cassettes into the triple-helical
recombinant collagen X allowed for the first time the identification of
five triple-helical immunodominant domains of 5-11 amino acid length, to which 75% of 36 monoclonal antibodies bound. A consensus motif, "R G hydrophobic," was found in all immunodominant
epitopes. The antibodies were encoded by a certain combination of
V-genes in germline configuration, indicating a role of the consensus
motif in V-gene selection. The immunodominant domains are spread over the entire monomeric CII molecule with no apparent order; however, a
highly organized arrangement became apparent when the CII molecules were displayed in the quarter-staggered assembly within a fibril. This
discrete epitope organization most likely reflects structural constraints that restrict the exposure of CII epitopes on the surface
of heterotypically assembled cartilage fibrils. Thus, our data suggest
a preimmune B cell selection process that is biased by the
accessibility of CII determinants in the intact cartilage tissue.
Rheumatoid arthritis is the most common chronic inflammatory joint
disease in humans. The disease is genetically linked to the
MHC-II1 region (1) and
characterized by relapsing inflammation of synovial tissue and
progressive destruction of cartilage and subchondral bone. The driving
force of this disorder is still obscure. However, immune responses
toward cartilage-specific antigens, particularly B cell responses
against type II collagen (CII), indicate a pathogenic role of
cartilage-specific autoimmunity (2-5).
CII, the predominant collagenous component of cartilage, is one of the
candidate autoantigens potentially fueling tissue-specific immune
reactions in peripheral joints. Immunization with CII is associated
with development of autoimmune arthritis in several species (6-8).
Collagen-induced arthritis (CIA) shares many characteristics with human
rheumatoid arthritis. As most extensively studied in mice, the
development of CIA is strongly associated with certain MHC-II
haplotypes (9, 10), indicating that the model is dependent on T cell
recognition of a restricted set of CII peptides presented by
appropriate MHC molecules (11). Indeed, peptides derived from the same
region of CII (amino acid residues (aa) 256-270) are bound by both DR4
and Aq molecules (10, 12), whose expression is genetically
associated with rheumatoid arthritis and CIA, respectively.
T cell recognition of proteolytically processed CII, however, does not
meet all requirements for the development of arthritis. The induction
of CIA, in fact, is critically dependent on immunization with CII in
its native conformation, i.e. the intact triple-helical structure of
collagen (6, 13). The conformation requirements, as well as the
dependence on functional B cells (14, 15), indicate that autoreactive
CII-specific B cells are crucial to the pathogenesis of CIA. In high
responder mice such as DBA/1 (H2q), for example, there is
apparent lack of negative selection of CII autoreactive B cells. As a
consequence, immunization of DBA/1 mice with heterologous (rat) CII
gives rise to early activation (day 5-9 after immunization) of
IgG-secreting autoreactive B cells; these recognize a set of
immunodominant native structures on the triple-helical moiety of the
autologous CII molecule (14, 16). The autoantibodies are cross-reactive
with CII from various species (i.e. human, chick, and
bovine), but, within the same species, they do not cross-react with any
of the systemically available collagens, for example type I (CI),
despite a homology of 80% at the amino acid level, suggesting that
CI-reactive B cells might have been negatively selected. The pathogenic
potential of CII-specific autoantibodies, in turn, is indicated by the
fact that, after intraperitoneal injection into syngeneic mice, they
bind to articular cartilage (15) and induce synovial inflammation and
even erosive arthritis (15, 17-19).
The aim of the present study was to characterize the dominant target
structures of the B cell response on CII in the initial phase of CIA.
For this purpose, epitope mapping was performed on a collection of B
cell hybridomas isolated from lymph nodes and spleens of
arthritis-prone DBA/1 mice on day 9-11 following immunization (14,
20). This collection of mAbs is well characterized in terms of binding
to intact articular cartilage, arthritogenic potential, and
representation of immunodominant epitopes (16, 17, 20). Since all
antibodies bind in a conformation-dependent manner, attempts to map
epitopes by synthetic peptides or purified enzymatic collagen fragments
have achieved rather limited results (21). Therefore, we have
established a method that allows the construction of recombinant
homotrimeric collagen chimeras; this approach is based on the
mutagenesis of the Monoclonal Antibodies--
The hybridomas obtained during the
primary response to immunization with CII were produced as follows. On
day 0, DBA/1 mice (8-12 weeks old) were immunized in the right footpad
with 50 µg of chicken or rat CII emulsified in complete Freund's
adjuvans (Difco, Detroit, MI); 9-11 days later, cells were obtained
from the popliteal lymph nodes that drain the injection site and were fused with the SP2/0 myeloma cell line, obtaining the clones E1, E5,
E10, E8, D7, D8, A12, C2, D3 (day 9 after immunization) and the clones
F4, F10, CB239-340, and H2O6 (day 11). For the characterization of the
mAbs, some clones were derived from DBA/1 mice undergoing a secondary
response; the immunization in this case proceeded as follows. On day 0, 50 µg of chicken or rat CII emulsified in complete Freund's adjuvant
were injected at the base of the tail; booster doses (50 µg of
chicken or rat CII emulsified in IFA) were administered either
intraperitoneally, in which case the spleens were removed and fused 21 days afterward obtaining the clones C1 and M2 76-332; or in the right
footpad, in which case the popliteal lymph nodes were removed and fused
11 days after booster, obtaining the clones LN2 7-423. Each fusion was
seeded into five 96-well plates, screened with anti- In Vivo Experiments with CII-specific Monoclonal
Antibodies--
Female, 8-15-week-old DBA/1J mice were used. Anti-CII
mAbs (D3, C2, and E8) and a syngeneic control IgG2a mAb (F3) were
purified, biotinylated, dialyzed against PBS, and sterile filtered (14, 23). The IgG content was determined spectrophotometrically (absorbance at 280 nm divided by 1.5) (15); 500 µg of biotinylated mAbs were
administered intraperitoneally as described previously (15).
Immunohistochemical Techniques--
Mice were sacrificed 96 h after injection of the biotinylated mAbs. The demineralized tissue
specimens were frozen in isopentane prechilled with liquid nitrogen,
and kept at Vector Construction--
The vectors for the expression of the
chimeric collagen molecules were based on a human collagen X cDNA
clone (26) propagated in Bluescript (New England Biolabs, Beverly, MA)
and a human CII cDNA clone (kindly provided by Dr. S. W. Li
and Prof. D. J. Prockop, Jefferson University, Philadelphia, PA).
The CII cDNA was used as template for the PCR amplification of
parts of the CII sequence. Specific restriction sites (NcoI,
PstI, and BamHI), which allowed cloning of these
fragments, were introduced via the PCR primers. Very short CII
fragments (30-50 bp) were directly generated from annealed
complementary synthetic oligonucleotides, which were subsequently
filled with nucleotides by Klenow DNA polymerase (27). The CII
fragments and the collagen X cDNA clone were digested with the
respective restriction enzymes (NcoI/BamHI or
PstI/BamHI), and the corresponding DNA fragments
were purified by agarose electrophoresis. The CII fragments were
ligated in frame into the collagen X cDNA, resulting in the
corresponding chimeric CX-CII constructs in Bluescript. For the
expression in eukaryotic cells, these chimeric CX-CII constructs were
cloned in the pRCMV vector (Promega, Madison, WI). Prior to the
transfection procedure, the chimeric constructs were controlled by
in vitro translation and DNA sequencing (28).
HEK 293 Cell Culturing and Transfection--
HEK 293 cells,
maintained in Dulbecco's modified Eagle's medium (DMEM/F-12), were
seeded on a 10-cm plate (1 × 106 cells/plate) and
grown overnight. Transfection was performed with 10 µg of the plasmid
DNA construct by the calcium phosphate precipitation method. The cells
were incubated for 24 h with a mixture of DNA and calcium
phosphate in 10 ml of DMEM containing 10% FCS. This mixture was
replaced by DMEM/F-12, 5% FCS; after 48 h, the selection was
started by supplementation with 800 µg/ml G-418 (Life Technologies,
Inc.). The medium was renewed every 2 days; the collection of
supernatants was started when the G-418 resistant cells reached
confluence. During harvesting, the transfected HEK 293 cells were kept
in FCS-free DMEM/F-12 supplemented with ascorbate.
Immunoblot--
Protein was precipitated from 1 ml of
supernatant by the addition of 139 µl of Triton X-100 (1% in
H2O) and 250 µl of trichloroacetic acid (55% in
H2O). The precipitate was dissolved in 40 µl of sample buffer and immediately subjected to SDS-polyacrylamide gel
electrophoresis using 10% gels. The separated proteins were
subsequently electroblotted to a nitrocellulose sheet (Schleicher & Schuell, Dassel, Germany) using the semidry blot technique. The blots
were blocked with 3% BSA in PBS and subsequently probed with a mAb X53
raised against recombinant collagen X (Ref. 29; generous gift of Dr. I. Girkontaite, Institute of Experimental Medicine, Erlangen, Germany).
Specific binding was detected using horseradish peroxidase-conjugated
rabbit anti-mouse IgG (Dianova, Hamburg, Germany) and ECL detection
(Amersham, Braunschweig, Germany).
ELISA--
Serum-free cell culture supernatants from transfected
HEK 293 cells were directly used for coating of ELISA plates (Nunc, Wiesbaden, Germany). Media from non-transfected HEK 293 cells, as well
as supernatants from HEK 293 cells transfected with the cDNA of
wild-type collagens X or II, were identically processed for control.
After coating, the wells were blocked with 2% BSA in PBS for 1 h
and subsequently incubated with the mAbs diluted to 10-100 µg/ml for
1 h. The specific binding was detected using horseradish
peroxidase-conjugated rabbit anti-mouse IgG (Dianova) and
2,2-azino-di-[3-ethylbenzthiazoline sulfonate] diammonium salt as
substrate (ABTS tablets, Boehringer, Mannheim, Germany). The coating
efficiency of the different chimeras was standardized by the
immunoreactivity of anti-collagen X-specific mAb X53. Some ELISA
results (see Table I) are presented in a digital scoring system (+ and
Department of Internal Medicine III,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1(X) collagen chain, allowing the presentation of
epitopes in triple-helical conformation. This enables to characterize
precisely the conformation-dependent binding of 36 mAbs and
to identify the immunodominant B cell epitopes of CIA in DBA/1 mice,
giving insight into the structural basis of autoimmune recognition of
the most abundant collagen type in the joints.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
FC secondary
antibody, and cloned at least twice before expanding the cells into
bulk cultures. Supernatants were purified on protein A-Sepharose
(Pharmacia, Uppsala, Sweden), dialyzed against phosphate-buffered
saline (PBS), and filtered. The mAbs were of the IgG1, IgG2a, and IgG2b
isotype (exception: D8 (IgG3)). For details on the individual mAbs, see Refs. 14, 20, 22, and 23.
70 °C until cryosectioned. Sections of the joints (6 µm thick) were fixed in cold acetone for 5 min. After washing in PBS,
the endogenous peroxidase was depleted by washing in 0.3%
H2O2 in PBS for 15 min. After additional washings in PBS, the sections were incubated for 30 min in a humid atmosphere at room temperature with 50 µl of rat mAbs specific for
mouse MHC-II (I-A) (MASO53, Seralab, Sussex, United Kingdom), CD11b
(C3bi receptor) (MASO34, Seralab), or CD4 (hybridoma H129.19 (24)).
Peroxidase-conjugated rabbit anti-rat Ig diluted in PBS-BSA were used
as secondary antibodies. Binding of biotin-labeled primary antibodies
(anti-CII IgG) was detected by incubation with avidin-biotin-peroxidase complexes (25). The reaction was developed with 3-amino-9-ethylcarbazol containing H2O2. All sections were
counterstained with Mayer's hematoxylin.
). ELISA values for chimeric constructs with anti-CII mAbs were
considered negative if they did not exceed 20% of the negative control
(mAb reactivity with recombinant wild type collagen X). A prerequisite
for classifying binding experiments as negative was always a positive
ELISA result with the anti-CX mAb. The positive results usually
exceeded controls by a factor of 10-15.
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RESULTS |
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Antibody Specificity for Collagen II
The CII-specific B cell hybridomas, derived from DBA/1 mice during
the primary or secondary response upon immunization with rat or chick
CII, produced 45 mAbs that bound with similar avidity to CII from
chicken, mouse, rat, and human, as determined by ELISA (data not
shown); they did not cross-react with ubiquitously expressed collagens
such as type I (Fig. 1A). Two
mAbs (D3 and C2), however, cross-reacted with native collagen XI (data
not shown), which contains one 1 (II) chain as a subunit of
its heterotrimeric structure (35). A representative example (mAb F4)
for antibody specificity is shown in Fig. 1A.
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Antibody Specificity for the Native Conformation of Collagen II
All mAbs bound in a conformation-dependent manner to CII, i.e. they required the triple helix of the epitope, as shown in a representative ELISA with native and heat-denatured CII for mAb D3 (Fig. 1B). The binding ability of mAb D3 was lost after denaturation. The control antibodies, i.e. the isotype-matched mAb B1, recognizing a linear collagen epitope (Fig. 1B), and a CII-specific polyclonal rabbit antibody, did bind to CII under all conditions tested (results not shown). Heat-denatured CII was exposed to antibodies at 37 °C, and not at room temperature, to prevent refolding of the collagen triple helix.
In Vivo Effects of the CII-specific Monoclonal Antibodies
Antibody Binding-- The intraperitoneal injection of the biotinylated mAbs E8, D3, and C2, but not that of the control mAb F3 (which does not bind to CII), led to antibody binding to the articular cartilage. In the case of the mAb D3, the binding could be considered specific because blocked by an anti-idiotypic antiserum in vivo (36). Single staining with avidin-peroxidase confirmed the binding of the biotin-conjugated anti-CII mAbs, leaving the synovia unstained, as expected (data not shown).
Induction of Synovial Inflammation-- The intraperitoneal administration of the CII-specific mAbs led to an inflammatory response in the synovial membrane (Fig. 2), consisting of hyperplasia and inflammatory infiltration. The inflammatory infiltrate included lymphocyte-like cells positive for the T helper cell marker CD4 (Fig. 2A) and macrophage-like cells expressing CD11b (C3bi receptor) and MHC-II (Fig. 2, B and C, respectively). No staining of the cartilage matrix or signs of inflammatory responses were detected after systemic administration of the biotin-conjugated control mAb F3 (results not shown).
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Characterization of the Recombinant Chimeric Collagen Molecules
For precise mapping of the triple-helical epitopes, chimeric
collagen constructs were made using the frame of the human full-length 1(X) cDNA to insert different fragments of the human
1(II)
cDNA. For this purpose, a 295-bp-long
1(X) fragment flanked by
the unique restriction sites PstI and BamHI, or a
338-bp-long cassette generated by restriction with NcoI and
BamHI, was replaced by PCR-amplified
1(II) inserts of
varying length. Initially, constructs were generated that contained
larger
1(II) inserts (up to 900 bp), to cover the entire length of
the CII molecule with a few recombinant collagen molecules.
Subsequently, a total of 130 recombinant chimeras were constructed to
localize the B cell epitopes precisely on different recombinant
chimeric collagen molecules containing
1(II) inserts with only small
sequence overlaps. To maintain stable triple helices, the recombinant
collagen chimeras were constructed without interfering with the
Gly-X-Y sequence. Collagen X was chosen, since it
is a homotrimeric molecule and none of the mAbs cross-reacted with this
collagen type (Fig. 1A). Recombinant chimeric collagen II/X
molecules were produced by transfection of HEK 293 cells with chimeric
constructs and expression under the cytomegalovirus promoter. The
Western blot in Fig. 3, developed with
the collagen X-specific mAb X53, demonstrates the differences in
electrophoretic mobility of the recombinant collagen X chimeras depending on the varying length of
1(II) inserts (bp positions as in
Ref 37).
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The conservation of triple helicity of recombinant collagen was controlled by resistance to trypsin/chymotrypsin digestion up to a melting temperature of ~43 °C (results not shown).
Characterization of the B Cell Epitopes
One hundred and thirty different chimeric collagen constructs were expressed in HEK 293 cells. Cell culture medium was coated to ELISA plates and coating efficiency controlled by a specific anti-CX mAb prior to the mAb testing. From the original 45 hybridomas, 9 mAbs showed high background reactivity and were not further investigated. For the remaining 36 mAbs, epitopes were mapped by constructing a series of overlapping fragments. After testing these fragments by ELISA, the mAb binding sites were thereby narrowed down to stretches of 5-14 aa within the CII triple helix. Table I summarizes the binding properties of six representative mAbs to a selected number of chimeras.
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The minimal CII-specific sequences required for recognition are highlighted in Table I. These binding regions for representative mAbs were defined as epitopes C1III, J1, D3, E/F10I, E/F10II, and F4. Remarkably, the epitopes E/F10I and E/F10II differed only by 1 aa. An even subtler microdiversity in antibody recognition could be detected in the region of the C1III epitope, as revealed by the reactivity of the mAbs C1, LN 2.7, and CB300 (Fig. 4). Three overlapping, but nevertheless clearly distinct epitopes (C1I, C1II, and C1III) were identified between bp positions 1623 and 1655 (aa 359-369).
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The extensive mapping strategy using the entire set of recombinant chimeras was applied on each of the 36 mAbs; a summary of the results is given in Fig. 6 and Table II, which also contains the information about the V-gene usage of the CII-specific B cells (20). From the data of Table II, it is evident that the epitopes presented in Table I and Fig. 4 are indeed dominant targets of the B cell response, since each was recognized by more than one mAb.
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The binding regions of mAbs recognized by only one antibody (Table II) were not systematically mapped; however, the localization of their epitopes was confined to regions shorter than 50 aa with a few exceptions (M2 136, M2 191, and LN2; Ref. 15). These singly recognized epitopes turned out to spread along the entire triple helix and could be localized on all cyanogen bromide fragments (CB) of CII.
The specificities of the remaining 27 mAbs were clustered to eight major epitopes (Table II) and localized in five distinct regions (C1I-III, J1, D3, E/F10I-II, and F4) on the CB11, CB10, and CB9. These immunodominant regions were not only characterized by binding of several antibodies to one epitope, but also by clustering of overlapping, yet distinct epitopes. This was most impressive in the dominant region within the CB11 fragment (C1I-III). Within this 11 aa stretch (aa 359-369), three different epitopes were recognized by as many as 15 mAbs, i.e. 41.7% of the entire collection. Two overlapping epitopes differing only by 1 aa were also localized on CB10 (aa 776-783), in close vicinity to the collagenase cleavage site (aa 775; Refs. 38 and 39). The aa sequences characteristic of the immunodominant regions are shown in Fig. 5A.
The sequence data revealed subtle differences in epitope specificity, as recognized by the mAb binding to the dominant region C1I-III in CB11. The minimal requirements for antibody recognition (C1I epitope) were determined by 5 aa residues (ARGLT), the two other epitopes (C1I and C1II) were dependent on one or two additional turns of Gly-X-Y along the triple helix. A similar fine specificity was indicated by the critical dependence of anti-CII mAb binding on one leucine residue, whose absence determined the difference between epitopes E/F10I and E/F10II. However, despite such subtle differences, all dominant epitopes shared a consensus motif R G hydrophobic, as indicated in Fig. 5A.
For the understanding of specific collagen binding, however, one has to
consider not only the linear sequences, but also the array of aa side
chains on the surface of the triple helix. While the glycine residues
remain hidden in the axis of the homotrimeric triple helix and do not
contribute to mAb binding, the residues in the X and
Y positions are exposed on the cylindrical surface of the
molecule. This structural arrangement is schematically presented for
the epitope E/F10II in Fig. 5B, based on a
previously described model (40). Although the primary sequence of the
E/F10II epitope differs from the corresponding region on
the 1(I) chain of collagen I by only one conservative aa exchange,
the triple-helical arrangement reveals considerable differences in the
array of aa side chains on surface between the homotrimeric CII and the
heterotrimeric CI.
New aspects on the peculiarity of the CII epitope localization and their accessibility to antibodies became apparent when the epitopes were depicted within the 4.40 D-staggered arrangement of CII molecules in the microfibril (Fig. 7). Interestingly, the epitopes E/F10I-II and F4 on one CII molecule were located in the immediate vicinity of J1 and D3 on the adjacent molecule, close to the borders between gap and overlap zones. Thus, the distribution of the immunodominant domains exhibited a higher degree of order within the quarternary arrangement of collagen; this arrangement exists in intact cartilage tissue but obviously not in the monomeric CII molecule used as arthritogen. Remarkably, the F4/D3 epitope cluster was located close to sites of cross-link formation (Fig. 7B), while E/F10I-II/J1 was adjacent to the collagenase cleavage site, suggesting a higher accessibility of these regions to enzymes and antibodies (see "Discussion").
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DISCUSSION |
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The mapping and characterization of the arthritogenic epitopes of
CII, necessary to understand the link between B cell immunity and
disease, has revealed so far a number of linear epitopes covering the
entire sequence of the immunodominant CNBr-fragment 11 (1(II)CB11) (41, 42). However, since CII epitopes require a triple-helical conformation to be arthritogenic, and since they are difficult to
reveal due to the limited accuracy of rotary shadowing (± 30 aa) (21),
a novel strategy was developed in the present study, based on the
insertion of a series of CII cassettes into triple-helical collagen X. The use of a eukaryotic expression system resulted in successful
presentation of chimeric collagens in triple-helical conformation. In
addition to proving relevant for unraveling B cell epitopes, the
present method can find widespread applicability for the elucidation of
other conformation-dependent ligand interactions with
extracellular matrix components, for example that of collagen with
integrin (40), decorin (43), or fibromodulin (44).
The set of mAbs investigated in the present study is well characterized for their pathogenic importance, i.e. for their capacity of inducing synovial inflammation; indeed, CIA is reversed by administration of an anti-idiotypic mAb (C1C3) detecting a cross-reactive idiotype shared by mAbs C1, C2, and E10 (45). Competitive ELISA assays document as well that a substantial part of antibodies in the sera of arthritic mice binds to the same epitopes (46).
While single B cell epitopes proved to spread along the entire triple
helix of CII, the statistical analysis uncovered binding clusters in
five distinct domains. Most remarkable is the binding of 41.7% of the
entire mAb collection to a 11-aa-long stretch within the CB11
(ARGLTGRPGDA). Within this major region, there was clear
microheterogeneity, since the fine mapping for the minimal epitope
requirements revealed not less than three distinct epitopes of varying
length (5-11 aa) starting at a conserved N terminus. A similar
microheterogeneity distinguished an epitope localized in the
CNBr-fragment 10 (1(II) CB10), close to the collagenase cleavage
site. The present results confirm therefore that immunodominance of CII
is limited to a few discrete domains on the 1014-aa-long triple-helical
moiety. Notably, the most frequently recognized epitope, localized on
the CNBr fragment
1(II) CB11, was previously identified as the
predominant target of B cell response in DBA/1 mice (13, 16).
Accordingly, only immunization with renatured CIICB11, but not with
other CNBr fragments, induces experimental arthritis (13). Hence, the
CIICB11 contains structures of crucial importance for the induction of
autoimmune arthritis in DBA/1 mice; the present data indicate that the
triple helix between aa 359 and 369 is one of these structures, namely
the immunodominant B cell epitope. It is likewise remarkable that the
1(II) CB11 also harbors the dominant T cell determinants (aa
256-270) for recognition of CII (11, 47).
Although the five dominant B cell epitopes are spread over a considerable distance on the monomeric CII molecule, their distribution is unlikely to be random. In cartilage, CII molecules assemble in a multimeric, quarter-staggered fibrillar array. On the basis of the current model for packing of molecules in native fibrils (48, 49), the calculation of the position of the single epitopes in the quarternary structure of collagen revealed a spatially ordered distribution of the immunodominant domains (Fig. 7A). In this multimeric structure, the E/F10 and J1 epitopes, as well as the D3 and F4 epitopes, coalesce in clusters within two distinct narrow regions (corresponding to a length of <20 aa) on neighboring triple helices. Thus, the repetitive D period of 66 nm length of the collagen microfibril is likely subdivided into three segments, proportional in size at a ratio of approximately 2:1:2 by the three loci of B cell epitope clusters (E/F10/J1, C1I-III, and D3/F4; Fig. 7A).
In intact cartilage, the microfibril structure is even more complex, due to the heterotypic interactions of CII with other extracellular matrix molecules, e.g. collagen IX (50, 51), collagen XI (52), decorin (43), or fibromodulin (44). Such interactions determine structural constraints on the accessibility of collagen for antibody recognition; the spatially ordered distribution of immunodominant B cell epitopes may thus result from such constraints. In the heterotypic fibrils, the collagen surface is largely masked by other extracellular matrix molecules. The immunodominant epitopes are, however, apparently localized in less densely coated regions of the CII molecules and may be thus accessible to the early antibody response characteristic of the initial phase of CIA.
Peculiarities of B cell epitope localization in the heterotypic fibril emerge when the relative molecular arrangements of collagen IX and XI to CII are reconstructed on the basis of a model that takes into account the precise localizations of intermolecular cross-links and the constant dimensions of the rigid triple-helical molecules (50-52). Although the contribution of decorin and fibromodulin cannot be estimated due to the lack of information on their arrangement, all B cell epitopes appear to localize either within the gap region (C1I-III), less densely packed than the overlap region, or at the borders of the gap region (E/F10I+II/J1 and D3/F4) (Fig. 7A). In the heterotypic arrangement, the telopeptide regions of collagen II and XI, as well as three of four non-collagenous domains of collagen IX, form clusters to the same regions of the D period on CII to which the dominant B cell epitopes have been mapped (Fig. 7B). The relative flexibility of non-collagenous domains of collagen may thus uncover triple-helical domains in their close vicinity, especially upon mechanical stress in the fibrillar network during movement.
The in vivo binding of the mAbs to normal cartilage did not require further unmasking procedures (Ref. 15 and Fig. 2A); thus, the B cell epitopes appear more easily accessible than other CII regions. In the case of the two clusters that reside close to the gap region of the collagen fibril, the triple helices in their immediate vicinity are sufficiently exposed to permit enzymatic attack during normal matrix metabolism. The E/F10I epitope maps in fact to position aa 776, which is directly adjacent to the unique collagen cleavage site at aa 775 (38, 39). The F4 epitope, in turn, starts at aa 932 in the immediate vicinity of the lysine residue in position 930, which is one of the two hydroxylysines in CII triple helices (aa 87 and 930) involved in intermolecular cross-linking (49). The aldehyde-derived cross-links require the precedent action of the enzyme lysyl oxidase (53) on adjacent (hydroxy)lysines in the neighboring telopeptide regions (as indicated in Fig. 7B), indicating that this site is accessible in the native fibril. Thus, the colocalization of immunodominant B cell epitopes with sites of selective enzymatic modification suggests a structural peculiarity that may permit distinction from the repetitive Gly-X-Y surrounding.
The immunodominant B cell epitopes share a consensus motif R G hydrophobic. This conserved sequence, however, does not define all requirements for specific binding, since there is no cross-reactivity between mAbs that have been mapped to different immunodominant regions. Thus, the aa residues flanking the consensus are also important for specific interaction with the complementarity determining regions of the mAbs. The importance of the flanking sequences may be due to the homotrimeric assembly of CII (as depicted in Fig. 5B), which leads to considerable changes of the triple-helical surface as a result of only one aa exchange in the primary sequence. Antibody recognition of microdiverse structures on the cylindrical collagen surface may therefore circumvent cross-reactivity among different members of the highly homologous collagen family.
In this study, the majority of B cell hybridomas was derived during the course of the primary response to CII. V-gene analyses of the hybridomas revealed that the antibodies are germline-encoded and exhibit a recurrent usage of single genes from the VHJ558, VHX24, and VK21 families (20). Moreover, there was even sharing of V-genes, i.e. the same V-gene was found in hybridomas binding to different epitopes on the same antigen (VK 21c is used for recognition of C1I, D3, and F4; Table II). This heavily biased V-gene combination (Table II and 20) indicates that conserved germline structures in the V-regions are important for CII binding (22). Thus, the recurrent V-gene usage may reflect selection of B cell populations based on antigen-specific structural constraints. It should be noted that immunization of DBA/1 mice with CII induces an IgG-switched response which is not preceded by a typical IgM surge (20); this constellation, reminiscent of a recall rather than a primary response, suggests that selection of CII-autoreactive B cells might occur in the naive animal prior to immunization. The identified triple-helical consensus motif R G hydrophobic may thus represent a conserved repetitive recognition structure for B cells; this structure may be critically involved in V-gene selection for autoantibodies relevant to the pathogenesis of CIA.
The spatially ordered distribution of the dominant epitopes containing the R G hydrophobic motif is reminiscent of the B cell epitope organization on paracrystalline surface structures of pathogens. In the case of the virus envelope glycoprotein VSV-G, for example, the contact of mature B cells with antigen in an optimal rigid repetitive epitope organization (in the absence of cognate T cell help) does not necessarily lead to anergy or apoptosis, but rather to activation of B cells via optimal Ig receptor cross-linking (54). It is therefore an intriguing possibility that the early occurrence in arthritis-prone DBA/1 mice of V-gene-selected B cells secreting anti-CII IgG are connected to the repetitiveness of determinants in discrete accessible sites of collagen fibrils in cartilage; this may result in positive selection rather than deletion of autoreactive B cells. B cells, in turn, could be exposed to CII in the bone marrow during cartilage resorption in enchondral bone formation; once left the bone marrow, these B cells may encounter cognate T cell help in the periphery during exposure to antigen. This sequence of events may shape a pre-activated pool of clonally selected CII-specific B cells, present already in naive animals. Upon various provocations, such as immunization with CII, this preshaped pool could be recruited in arthritogenic immune responses; this may occur on one hand by secretion of anti-CII IgG, and on the other hand by lowering the threshold for engagement of autoreactive T cells, due to the efficiency of activated B cells in antigen presentation (55, 56). Although it cannot be formally excluded that the selection process of CII specific B cells is initiated by the immunization and governed by requirements for T cell help, we suggest a preimmune impact that is biased by the accessibility of CII determinants in the intact cartilage tissue. This selection may explain why autoantibodies bind to intact cartilage in vivo, and challenges the concept that CII is an immunoprivileged self protein.
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. K. Kühn, Max-Planck Institute for Biochemistry, Martinsried for critical comments and expert advice and to Dr. E. Palombo-Kinne for reading the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Medical Research Council, the Swedish Association against Rheumatism, and the King Gustav V's 80-years, Axson Johnson, Nanna Swartz, Åke Wiberg, Åsterlund, Kock, and Anna-Greta Craaford Foundations (all to R. H.) and by Deutsche Forschungsgemeinschaft Grant SFB 263 (Project C3) (to H. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Internal
Medicine III, University of Erlangen-Nürnberg, Krankenhausstr. 12, D-91054 Erlangen, Germany. Fax: 49-9131-856341; E-mail:
harald.burkhardt{at}med3.med.uni-erlangen.de.
1 The abbreviations used are: MHC, major histocompatibility complex; mAb, monoclonal antibody; CII, collagen type II; CI, collagen type I; CIA, collagen-induced arthritis; HEK, human embryonic kidney; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; ELISA, enzyme-linked immunosorbent assay; bp, base pair(s); D period, fibril period of collagen.
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