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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 Vgenes 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-II 1 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 cartilagespecific autoimmunity (2-5). CII, the predominant collagenous component of cartilage, is one of the candidate autoantigens potentially fueling tissuespecific 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 A q molecules (10, 12), whose expression is genetically associated with rheumatoid arthritis and CIA, respectively.T cell recognition of proteolytically pro...

Type X collagen is a short-chain, network-forming collagen found in hypertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human ␣1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT1080. Stable clones were obtained secreting recombinant human type X collagen (hrColX) in amounts of 50 g/ml with ␣1(X)-chains of apparent molecular mass of 75 kDa. Pepsin digestion converted the native protein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion. Polyclonal antibodies prepared against purified hrColX reacted specifically with type X collagen in sections of human fetal growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at increasing temperatures indicated triple helical molecules with a reduced melting temperature of 31°C as a result of partial underhydroxylation. Ultrastructural analysis of hrColX by rotary shadowing demonstrated rodlike molecules with a length of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even after reduction and heat denaturation, and gave rise to monomers of 18 -20 kDa after treatment with trichloroacetic acid. The NC-1 domains prepared by collagenase digestion comigrated with NC-1 domains prepared as recombinant protein in HEK293 cells, both in the multimeric and monomeric form. These studies demonstrate the potential of the pCMVsis expression system to produce recombinant triple helical type X collagens in amounts sufficient for further studies on its structural and functional domains.Type X collagen is a short chain collagen with a triple helical portion half the length of fibril-forming collagens, flanked by globular, noncollagenous (NC) 1 domains at the amino terminus (NC-2) and the carboxyl terminus (NC-1) (for reviews, see Refs. 1-3). It is primarily expressed in hypertrophic cartilage of epiphyseal growth plates of long bones, ribs, and vertebrae (4 -6), but also in bone fracture callus (7) and in osteoarthritic cartilage (8 -10). A substantial body of information on the structure and molecular assembly of type X collagen is available from studies on type X collagen isolated from hypertrophic cartilage (6,7,(11)(12)(13)(14)(15) and from cell cultures of chicken, rabbit, and bovine hypertrophic chondrocytes (16 -20). Electron microscopic studies indicate that type X collagen molecules form fine pericellular filaments in vivo in association with type II collagen (21), or assemble into a hexagonal meshwork in vitro (22). Rotary shadowing data show that type X collagen molecules aggregate primarily through their COOH-terminal, nontri...

Little is known about matrix biochemistry and cell differentiation patterns in chondrogenic neoplasms. This is the first description of the focal expression of collagen type X by neoplastic chondrocytes in situ and its incorporation into the extracellular matrix of cartilaginous tumors. This shows that neoplastic chondrocytes have the potential to undergo the full program of cell differentiation, including hypertrophy, comparable to their physiological counterparts in the growth plate. However, only in benign osteochondromas was a zonal expression of type X collagen found similar to that observed in the growth plate, where the cells immediately above the ossification frontier are selectively positive for type X collagen. In enchondromas and chondrosarcomas, the expression was randomly distributed within the tumors. Surprisingly, in less differentiated chondrosarcomas with spindle-shaped cells and non-cartilaginous extracellular matrix, exceptional expression of collagen type X was observed, which indicates potential uncoupling of collagen type X expression from the differentiated chondrocytic phenotype in neoplastic chondrocytes in vivo.

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