Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory and destructive joint disorder that affects tens of millions of people worldwide. Normal healthy joints maintain a balance between the synthesis of extracellular matrix (ECM) molecules and the proteolytic degradation of damaged ones. In the case of RA, this balance is shifted toward matrix destruction due to increased production of cleavage enzymes and the presence of (autoimmune) immunoglobulins resulting from an inflammation induced immune response. Herein we demonstrate that a polyclonal antibody against the proteoglycan biglycan (BG) causes tissue destruction that may be analogous to that of RA affected tissues. The effect of the antibody is more potent than harsh chemical and/or enzymatic treatments designed to mimic arthritis-like fibril de-polymerization. In RA cases, the immune response to inflammation causes synovial fibroblasts, monocytes and macrophages to produce cytokines and secrete matrix remodeling enzymes, whereas B cells are stimulated to produce immunoglobulins. The specific antigen that causes the RA immune response has not yet been identified, although possible candidates have been proposed, including collagen types I and II, and proteoglycans (PG's) such as biglycan. We speculate that the initiation of RA associated tissue destruction in vivo may involve a similar non-enzymatic decomposition of collagen fibrils via the immunoglobulins themselves that we observe here ex vivo.

Competing interests: Author Joseph Orgel is a section editor and member of the editorial board of PLoS ONE. The Journal had no part or influence in the research reported or in the writing of the originally submitted manuscript. A patent application has been made that pertains to data reported in this manuscript. #12/419,689: “THIN FIBRIL COLLAGEN MATERIAL AND METHOD FOR MAKING THIN FIBRIL COLLAGEN FROM NATIVE COLLAGEN FIBERS.” This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Funding: Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract No. W-31-109-ENG-38. BioCAT is a National Institutes of Health-supported Research Center RR-08630. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Center for Research Resources or the National Institutes of Health. This work was also supported by the National Science Foundation (Grant #MCB-0644015 CAREER). This material is based on the work supported by, or in part by, the US Army Research Laboratory and the US Army Research Office under contract/grant number W911NF 09-1-0378. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

PG's, such as the small leucine rich repeat proteins (sLRRP's) decorin and biglycan, are essential for stabilization of fibril-bundle structures [11] and for conveying compression resistance together with hyaluronan and aggrecan. Here we report that biglycan bound type II collagen fibril-bundles (or fibers, also known as thick fibrils of 30–50 nm diameter) are decomposed into discrete fibrils (also known as thin-fibrils of ∼10–15 nm diameter) through the action of anti-biglycan antibody, even in the absence of cells, enzymes, other antibodies and in the presence of enzyme inhibitors. This antibody induced process results in the breakdown of notochord and the appearance of thin-fibrils in cartilage samples.

The synovium, or synovial membrane, is a thin sheet of vascularized mesenchymal tissue that surrounds the joint cavity and produces synovial fluid, which is responsible for joint lubrication and chondrocyte nutrition, since avascular cartilage is impermeable to oxygen and nutrients in mature joints [10] . The ECM of synovium is composed of collagen fibrils (types I, III, and V, type II is not present) of relatively small diameter (30 nm) with 67 nm periodicity and thin filaments (10 nm) of collagen type VI (with 100 nm periodicity), which are integrated with hyaluronan, fibronectin, and fibrillin, providing tissue permeability and structural integrity [10] .

The major parts of the joint which are affected most prominently in RA, are articular cartilage and the synovium. Collagen type II fibrils are major structural elements of the cartilage ECM and the cartilage-like notochord of cartilaginous fishes. They form 67 nm periodic fibrils and fibers, with the participation of proteoglycans (PG's), which bind collagen by their core protein and regulate collagen fiber diameter through their anionic glycosaminoglycan (AGAG) chains. These collagen-PG interactions are also essential for fiber/fibril stability and determine a number of their mechanical properties [1] – [5] . In cartilage, collagen type II also aggregates with other collagen types (I, V, IX and XI) [6] and PG's to form complex fibrillar meshworks, in contrast to the simple arrangement in notochord. Although the type II collagen fibrils themselves are indistinguishable between the two tissues [7] – [9] . Aggrecan in complex with hyaluronan is embedded within these meshworks and accumulates a substantial amount of water (70% of cartilage mass) by their highly negative charge.

Results and Discussion

Antibody induced tissue decomposition Native fibrils of lamprey notochord do not show any detectable difference between its collagen type II fibers and those seen from the tissues of mammals [21] (FIG. 1). They have the same diameter of about 35 nm and typical positive staining pattern, although there are some differences in cellular and proteoglycan content, as well as tissue architecture. Lamprey notochord appears to have a very specific cell distribution (layered, outside of the bands of extracelluar material), whereas cartilage cells are embedded in a collagen meshwork and can be seen throughout the whole tissue. The PG content of lamprey notochord is fairly simple containing only biglycan type I and II (which are very close in sequence to the bovine or human biglycans, and hence structurally related to decorin and fibromodulin), in contrast to the mammalian cartilage which has several types of PG's and glycoproteins present [22], [23]. TEM images of lamprey tissues treated with the biglycan antibodies showed (FIGS. 1–2) a striking difference from the native fibers: 1) much thinner collagen fibers about 11 nm in diameter are present through-out the samples or more accurately, there was a notable absence of native thick-fibrils (Tables S1 and S2) and 2) the ECM became significantly less well ordered. The antibody treated notochord (FIG. 1B) after treatment was very delicate and easy to deform, unable to withstand even minimal strain (<5%) without its destruction. Mammalian tissues were not affected so dramatically (FIGS. 1 and 4), but they also showed the appearance of thin fibrils and disrupted fibers in the presence of the antibody, not present in the control (pre-treated) sections in such quantities (FIG. 1). The difference in the relative effect on the two animal tissues (cartilage vs. notochord) is presumably due to the mammalian cartilage possessing a wider repertoire of sLRRP's that are either completely or partially resistant to the effects of the anti-biglycan antibody (see below). Lamprey notochord with only biglycan is therefore more susceptible to any putative effects of its antibody. Regardless of the difference in scale of effect, the same unexpected observation was made between the tissues: Significant and rapid non-enzymatic decomposition of collagen fibrils occurs in the presence of the anti-biglycan antibody. Large BG core protein sized structures observed in TEM of incubating solution with presence of gold particles. Biochemical analysis shows significantly elevated levels of BG in this solution relative to non-ab containing controls (see SI methods S1, Tables S3 and S4, and FIG. 5). We suggest, that anti-biglycan antibodies attach to biglycan core-proteins on the surface of thick-fibrils and that this interaction disrupts the bonding between the core-protein and the collagen molecules comprising the fibrils. Even though GAG bridges remain intact, the loosening of the biglycan core-protein – collagen interaction ‘unties the string’ that holds the thick-fibrils together (FIG. 3). This further implies that ‘thick-fibrils’ are in fact bundles of thin fibrils (i.e. the ‘fibril bundles’ discussed throughout this study). Whereas a ‘thin-fibril’ [24] is an irreducible collagen fibril (without enzymatic digestion or mechanical force) formed from closely packed collagen molecules and held together through collagen-collagen interactions such as lysine-hydroxylysine bonds.

Controls In order to test the hypothesis that disruption of the biglycan core-protein – collagen interaction accounts for the fibril-debundling, lamprey notochord samples were treated with Guanidine hydrochloride and ABC lyase respectively as a positive control. Guanidine hydrochloride causes protein denaturation, therefore its action would mimic the hypothesized action of the biglycan antibody, albeit in a more caustic and less specific manner. TEM images of Guanidine treated notochord and cartilage illustrated the same type of degradation of collagen fibrils, although the incubation time had to be much longer (24 h instead of 1 h for the antibody) to achieve a similar degree of decomposition. In contrast, ABC lyase removes the AGAG chain from the protein core and should produce the similar results albeit via a different structural mechanism. Thin fibrils in both notochord and articular cartilage samples were seen in TEM images after this treatment, although damage was relatively mild in comparison to the antibody-mediated decomposition. In addition to these chemical methods of tissue degradation, mechanical degradation was also examined. Friction was applied to native lamprey notochord tissues and the results of this damage were analyzed by TEM and compared with the other experiments. Mechanical impact, used for this study, may correspond to damage of the articular cartilage due to trauma or normal wear. The presence of thin fibrils were observed (absence of regular 30–50 nm fibrils) in certain areas, which had higher load, although some parts of tissue still had normal architecture in comparison to undamaged, non-treated control samples. Finally, significant tissue degradation (and biglycan release) was observed in the presence of protease inhibitors but not in the presence of alternative antibodies such as anti-collagen (see SI methods S1).

Notochord structure: differences in scale of the effect of anti-biglycan on lamprey vs. mammalian tissues Lamprey notochord is a cartilage-like tissue that spans the length of the chordate back, located beneath and parallel to the central nervous system between the brain and tail. Although it is the main axial skeleton at the embryonic stage, the notochord is replaced by the vertebral column in most vertebrates. However, in some chordates it remains into adulthood (e.g., lamprey, lungfish, sturgeon, and some sharks). The mature notochord contains a soft cellular inner part, surrounded by protective fibrous sheath, composed of three layers: inner basal lamina, thick collagenous (cartilage-like) layer, and elastic filamentous membrane [25]. The composition of collagenous part of lamprey notochord is rather simple compared to articular cartilage, which makes it very convenient to study. It has two main collagen type II fiber orientations: circular (perpendicular to the main body axis) and longitudinal (parallel to the main axis of the body). Longitudinally organized fibers are located at the outer layer and are the most prevalent. The fibrillar architecture is supported by the lamprey biglycan. Although the organization of articular cartilage and lamprey notochord seem to be different, the structure of collagen type II fibrils in both of them is indistinguishable [25] and their sequence is highly homologous. Variations in the different level of damage (thin/normal collagen type II fibrils), obvious in TEM images of mammalian and lamprey tissues, can be explained by differences in the molecular composition of these tissues that in turn influences the tissue architecture. Lamprey notochord contains primarily collagen type II and biglycan. Human and bovine articular cartilage contain biglycan, fibromodulin, decorin, and other ECM molecules, which regulate fibrillogenesis, fiber diameter, support fibers, and give the tissue specific mechanical properties. Cartilage and meniscus contain more biglycan than decorin and the ratio changes from zone to zone. The superficial zone contains about 32% of decorin and 38% of biglycan of all PG content, the inner deep zone contains about 23% of decorin and 53% of biglycan of all PGs, and middle zone has 28% of decorin and 52% of biglycan of all PG's [26]. As a result these tissues can be expected to be more resistant to short duration (1–2 hours) antibody treatment. Due to the presence of decorin, which seems to be stable in the presence of the antibody (this antibody has higher affinity to biglycan, than decorin). But longer exposure of cartilage tissues to biglycan antibodies has a stronger effect on cartilage collagen fiber disassociation, presumably because the antibodies need more time to penetrate to deeper layers where biglycan is predominant.

Antibody concentration and effect Although our observations are ex vivo, it is worth noting that human autoimmune disease antibody concentration levels have been reported in body fluids at levels similar to or higher than we have applied in our experiments [27]–[29], although we also observed this effect in lower starting concentrations (see SI methods S1). What is more, the observed fibril decomposition permeated whole tissue samples immersed in the antibody containing vials. The concentration of the antibody in the tissue interior must have been substantially lower than in the surrounding solution, and yet, still caused tissue degradation. That the effect was observed in so short a period of time (in notochord) is also significant. Given that the concentration of the antibody used may be physiologically relevant, the nature of its disruptive affect may be the same in vivo as our ex vivo experiments, be that through competitive bond disruption or steric repulsion or both. However, as FIG. 3 indicates, it is quite unlikely that more than one antibody molecule may locate to one biglycan core proteins epitope, making the steric repulsion consideration less likely. Especially in light of published studies that indicate sLRRP's conformations are not easily given to significant changes to their arching structure but do seem to be sensitive to the environments effect on their inner-core to collagen fibril hydrogen bonding network [1], [4]. The fact that we observed some sensitivity of tissues to certain salts in buffer systems (see SI methods S1and PBS) and that simple denaturing conditions (see guanidine hydrochloride GHCL results) were less effective than the application of the antibody may be considered collaborating evidence in favor of the interaction interference effect of the anti-biglycan antibody. It has long been known that pH causes collagen fiber bundles to disassociate into smaller species [30], [31], the latter reference speculating that the sLRRP proteoglycans are sensitive to both salt and pH conditions. That we observed this affect at natural pH in a phosphate based buffer system would seem to collaborate these prior considerations. That we did not observe fibril-bundle degradation in TBS controls and poorer destruction of fibril bundles in TBS via GHCL or enzyme catalysis, suggests a novel effect of the anti-biglycan antibody against type II collagen fiber bundles. That the thin-fibrils were not further degraded but remained indefinitely stable rules out the possibility of a general or collagen specific proteinase. The significantly elevated levels of biglycan detected in the anti-biglycan solution in which the tissue were incubated relative to controls without the anti-antibody, supports the hypothesis that anti-biglycan causes the disassociation of biglycan from thick-fibrils.

Collagen fibril MMP cleavage site and collagenase-interaction domain Collagen fibrils are assembled in such way that the MMP collagenase cleavage-site is protected by the C-telopeptide in folded conformation [32]–[34]. This folded C-telopeptide corresponds to the X3 ridge in type I collagen, and appears to have an analogous structure in type II collagen as observed from AFM and X-ray diffraction data [32], [33]. The C-telopeptide is usually cross-linked covalently with neighboring collagen molecules, that helps makes its conformation very stable. In order to gain access to the MMP cleavage-site, other proteases have to cleave the C-telopeptide first. This process is very slow with MMP1 alone due to a very low number of available cleavage sites (presumably at the fibril tips were collagen packing is looser). Our results show that the biglycan antibody can induce the process of collagen fiber decomposition in cartilage-like tissues. Even a short, one-hour exposure of lamprey notochord to biglycan antibody completely altered the structure of the collagen matrix. In fact there were only few areas in the treated notochord that were not altered dramatically (fibrils disassociated, but stayed close and parallel to each other) and therefore were capable of yielding diffraction patterns (FIG. 2). Even these ‘less affected’ tissue area's showed significant structural changes and most importantly, almost uniform fibrillar decomposition. Our preliminarily data (not shown) of enzymatic digestion of antibody incubated notochord showed that the tissue was decomposed at least a factor of 2 times faster than the controls (at 4°C, compared with MMP1, trypsin and pepsin), indicating that previously exposed cleavage sites were becoming exposed due to the action of the antibody on BG (i.e because fibril-bundles are being disrupted, yielding their constituent thin-fibrils and biglycan, see FIGS. 3 and 5).