Induction of an anti-human type II collagen response by monoclonal anti-idiotypic antibody

Several distinct epitopes on human type II collagen were defined by using mAb. The presence of species-specific and species-nonspecific (common) epitopes was thus clarified. Anti-idiotypic mAb (Ab2) was developed against one of the antibodies (Ab1) reactive with species-specific epitopes. Thus Ab2 was demonstrated to recognize an idiotope expressed on the Ag-binding site (paratope) of Ab1, since the binding of Ab1 to human type II collagen was blocked by Ab2, and the binding of Ab2 to Ab1 was inhibited by soluble human type II collagen, but not by murine and bovine type II collagens. DBA/1 mice immunized with Ab2 coupled to keyhole limpet hemocyanin produced an antibody (Ab3) specifically reactive with human type II collagen. It was also demonstrated that Ab3 expressed an idiotype similar to that of Ab1. These findings indicate that anti-idiotypic antibody directed against mAb to human type II collagen mimics a species-specific epitope on human type II collagen. The anti-idiotypic antibody bearing internal image of type II collagen will open the way to isolation of the arthritogenic epitope on type II collagen.     

Immunohistochemical study of collagens of the extracellular matrix in cartilage of Sepia officinalis

We used various anti-collagen antibodies to perform indirect immunofluorescent staining of cartilage sections from cuttlefish (S. officinalis). On ultrathin sections and collagen fibril preparations from the same tissue, we performed immunostaining with colloidal gold. The extracellular matrix (ECM) of S. officinalis cartilage reacted intensely and homogeneously with an antibody directed against type I-like collagen isolated from the cartilage of cuttlefish and with anti-rat type V collagen antibody. A weak reaction was observed with anti-fish and anti-chicken type I collagen antibodies, while no reaction was observed with anti-rat type I and anti calf type II collagen antibodies. Anti-chicken type II, anti calf type IX and type XI collagen antibodies reacted weakly with ECM, while stained cell bodies and cell processes reacted more intensely. A similar pattern of reaction was observed on cartilage section and isolated collagen fibrils prepared for electron microscopy. These findings suggest that ECM of cuttlefish cartilage may be composed of molecules similar to the type I, type V, type IX and type XI collagen molecules of vertebrates. Cephalopods have evolved a cartilage of structure and macromolecular organisation similar to that of vertebrate cartilage. However, the main molecular components of S. officinalis cartilage--type I-like and type V collagens--differ from those of vertebrate cartilage. We suggest that this type I-like collagen can be considered an initial step toward the evolution of type II collagen typical of vertebrates.     

Type II collagen-induced arthritis in mice. IV. Variations in immunogenetic regulation provide evidence for multiple arthritogenic epitopes on the collagen molecule

Type II collagen from six mammalian species was investigated for the capacity to induce an immune response and collagen-induced arthritis (CIA) in C57/B10 congenic mouse strains. H-2q haplotype mice were susceptible to chick, bovine, deer, rat, and human type II collagen, but were resistant to arthritis induced by porcine type II collagen. H-2r haplotype mice only developed CIA in response to bovine, deer, and porcine collagen. High antibody responses in the absence of disease, directed against a specific type II collagen, were observed in many independent haplotypes. The cross-reactive capacity of different antisera to the various collagen species was studied. The data support the existence of two arthritogenic and multiple nonarthritogenic epitopes on the type II collagen molecule.     

Characterisation of a monoclonal antibody against native human type I collagen

A monoclonal antibody against a pepsin-soluble mammalian type I collagen has been produced. This antibody, subclass IgG1, kappa, was specific for type I collagen and did not cross-react with a range of other collagen types or connective tissue proteins. The epitope recognized by the antibody was dependent upon an intact triple-helical structure for the collagen, and was shown by rotary shadowing and by immunoblotting of collagenase-derived fragments to be near the C-terminal of the pepsin-soluble collagen. Although the antibody had a low affinity, with Kd = 4 x 10(-7) M, it could be used for immunohistology of tissue sections and for studies of collagen produced by cells in culture. The antibody, which was raised against human collagen, also recognized type I collagens from certain other species, including calf, pig, sheep, goat and dog.     

Subepithelial type II collagen deposition during embryonic chick limb development

Summary Type II collagen is a major component of hyaline cartilage but recent studies have demonstrated the presence of this protein in a variety of interfaces that separate epithelia from mesenchyme, particularly in early stages of embryonic chick development. In the present study an immunohistochemical analysis of the distribution of type II collagen was performed on closely staged wing buds of early chick embryo. This report describes how using two different monoclonal antibodies against type II collagen and the peroxidase or fluorescence staining technique reveals that deposition of type II collagen at the ectoderm-mesenchyme interface occurs in the proximal part of the limb coincidentally with the appearance of this protein in the proximal core region, where chondrogenesis begins (stage 25). Then the staining in the subepithelial region spreads distallly with time, following the progression of the formation of cartilage rudiments. At about 7 days of development type II collagen is present under the apical ectoderm ridge and surrounds completely the wing bud underneath the epithelium. At the same time, another antibody directed against the cartilage-specific proteoglycan core protein only stains the chondrogenic central core of the limb and not the subepithelium. Although type II collagen and cartilage-specific proteoglycan are closely associated in the cartilage, the observations presented here suggest that the deposition of these proteins can be regulated independently during limb formation. The role of type II collagen at the epithelium-mesenchyme interface during limb formation is still to be determined.     

Characterization of a murine type IIB procollagen-specific antibody

Type II collagen is the major collagenous component of the cartilage extracellular matrix; formation of a covalently cross-linked type II collagen network provides cartilage with important tensile properties. The Col2a1 gene is encoded by 54 exons, of which exon 2 is subject to alternative splicing, resulting in different isoforms named IIA, IIB, IIC and IID. The two major procollagen protein isoforms are type IIA and type IIB procollagen. Type IIA procollagen mRNA contains exon 2 and is generated predominantly by chondroprogenitor cells and other non-cartilaginous tissues. Differentiated chondrocytes generate type IIB procollagen, devoid of exon 2. Although type IIA procollagen is produced in certain non-collagenous tissues during development, this developmentally-regulated alternative splicing switch to type IIB procollagen is restricted to cartilage cells. Though a much studied and characterized molecule, the importance of the various type II collagen protein isoforms in cartilage development and homeostasis is still not completely understood. Effective antibodies against specific epitopes of these isoforms can be useful tools to decipher function. However, most type II collagen antibodies to date recognize either all isoforms or the IIA procollagen isoform. To specifically identify the murine type IIB procollagen, we have generated a rabbit antibody (termed IIBN) directed to a peptide sequence that spans the murine exon 1–3 peptide junction. Characterization of the affinity-purified antibody by western blotting of collagens extracted from wild type murine cartilage or cartilage from Col2a1^+ ex2 knock-in mice (which generates predominantly the type IIA procollagen isoform) demonstrated that the IIBN antibody is specific to the type IIB procollagen isoform. IIBN antibody was also able to detect the native type IIB procollagen in the hypertrophic chondrocytes of the wild type growth plate, but not in those of the Col2a1^+ ex2 homozygous knock-in mice, by both immunofluorescence and immunohistochemical studies. Thus the IIBN antibody will permit an in-depth characterization of the distribution of IIB procollagen isoform in mouse skeletal tissues. In addition, this antibody will be an important reagent for characterizing mutant type II collagen phenotypes and for monitoring type II procollagen processing and trafficking.     

Widespread distribution of type II collagen during embryonic chick development

Type II collagen is a major component of hyaline cartilage, and has been suggested to be causally involved in promoting chondrogenesis during embryonic development. In the present study we have performed an immunohistochemical analysis of the distribution of type II collagen during several early stages of embryonic chick development. Unexpectedly, we have found that type II collagen is widely distributed in a temporally and spatially regulated fashion in basement membranes throughout the trunk of the embryo at stages 14 through 19, including regions with no apparent relationship to chondrogenesis. Immunohistochemical staining with two different monoclonal antibodies against type II collagen, as well as with an affinity-purified polyclonal antibody, is detectable in the basement membranes of the neural tube, notochord, auditory vesicle, dorsal/lateral surface ectoderm, lateral/ventral gut endoderm, mesonephric duct, and basal surface of the splanchnic mesoderm subjacent to the dorsal aorta, and at the interface between the epimyocardium and endocardium of the developing heart. In contrast, immunoreactive type IX collagen is detectable only in the perinotochordal sheath in the trunk of the embryo at these stages of development. Thus type II collagen is much more widely distributed during early development than previously thought, and may be fulfilling some as yet undefined function, unrelated to chondrogenesis, during early embryogenesis.     

A monoclonal anti-type II collagen antibody with cross-reactive anti-Ig activity specific for the F(ab')2 fragment

An IgG2a hybridoma antibody (BC-10) was obtained by a myeloma fusion with lymphocytes from B10.RIII mice immunized against native bovine type II collagen. This anti-collagen monoclonal exhibited extensive cross-reactivity with several type II collagen species. BC-10 was found to have self-associating properties, but not the specificity of a typical IgG rheumatoid factor, inasmuch as this mAb bound to F(ab')2 fragments of itself and of normal mouse IgG. Self binding was inhibited by the association of BC-10 with type II collagen, and inhibition assays indicated that antibodies with the capacity to inhibit BC-10 binding to collagen were present in the sera from B10.RIII arthritic mice, but not from DBA/1 LacJ arthritic mice. Joint inflammation and histopathologic features consistent with arthritis were observed in mice injected with the BC-10 hybridoma.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.     

Immunoidentification of type XII collagen in embryonic tissues

We have generated a monoclonal antibody against a synthetic peptide whose sequence was derived from the nucleotide sequence of a cDNA encoding alpha 1(XII) collagen. The antibody, 75d7, has been used to identify the alpha 1(XII) chain on immunoblots of SDS-PAGE tendon extracts as a 220-kD polypeptide, under reducing conditions. Amino-terminal amino acid sequence analysis of an immunopurified cyanogen bromide fragment of type XII collagen from embryonic chick tendons gave a single sequence identical to that predicted from the cDNA, thus confirming that the antibody recognizes the type XII protein. Immunofluorescence studies with the antibody demonstrate that type XII collagen is localized in type I-containing dense connective tissue structures such as tendons, ligaments, perichondrium, and periosteum. With these data, taken together with previous results showing that a portion of the sequence domains of type XII collagen is similar to domains of type IX, a nonfibrillar collagen associated with cross-striated fibrils in cartilage, we suggest that types IX and XII collagens are members of a distinct class of extracellular matrix proteins found in association with quarter-staggered collagen fibrils.     

Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.     

Antibodies Specific for Carbamylated Proteins Precede the Onset of Clinical Symptoms in Mice with Collagen Induced Arthritis

Objective

The immune response to post-translationally modified antigens is a key characteristic of rheumatoid arthritis. Carbamylation is such a posttranslational modification. Recently, we demonstrated that autoantibodies recognizing carbamylated proteins are present in sera of rheumatoid arthritis. The molecular mechanisms underlying the break of tolerance and hence the induction of anti-CarP antibody responses are unknown as well as their appearance in mouse models for systemic arthritis. Therefore we analyzed their appearance in the mouse collagen-induced arthritis model.

Methods

collagen induced arthritis was induced by immunization with type II collagen in complete Freund''s adjuvant. Arthritis severity was monitored by clinical scoring and anti-CarP antibody levels were determined by ELISA.

Results

Anti-CarP antibodies were detectable in mice with collagen induced arthritis. We did not detect ACPA in mice with collagen induced arthritis. The specificity of the antibodies for carbamylated proteins was confirmed by inhibition assays and immunoblotting. Injection with complete Freund''s adjuvant without type II collagen could also induce anti-CarP antibodies, however, in mice with arthritis, the anti-CarP antibody response was stronger and developed more rapidly. The onset of collagen induced arthritis was preceded by an increase of anti-CarP IgG2a levels in the serum.

Conclusion

In mice with collagen induced arthritis we did not observe an immune response against citrullinated antigens, but we did observe an immune response against carbamylated antigens. This anti-CarP response already appeared before disease onset, indicating that collagen induced arthritis can be used as an in vivo model to study anti-CarP antibodies. Our data also indicate that the tolerance to carbamylated proteins, in contrast to the response to citrullinated proteins, is easily broken and that arthritis boosts the immune response against these proteins. The anti-CarP response in mice with CIA can be used as a model for immune responses to post-translationally modified proteins.     

Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg²⁺ or Mn²⁺ but not Ca²⁺. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ~110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.     

Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.     

Cellular heterogeneity in cultured human chondrocytes identified by antibodies specific for alpha 2(XI) collagen chains

Collagen type XI is a component of hyaline cartilage consisting of alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains; with 5-10% of the total collagen content, it is a minor but significant component next to type II collagen, but its function and precise localization in cartilaginous tissues is still unclear. Owing to the homology of the alpha 3(XI) and alpha 1(II) collagen chains, attempts to prepare specific antibodies to native type XI collagen have been unsuccessful in the past. In this study, we report on the preparation and use for immunohistochemistry of a polyclonal antibody specific for alpha 2(XI) denatured collagen chains. The antibody was prepared by immunization with the isolated alpha 2(XI) chain and reacts neither with native type XI collagen nor type I, II, V, or IX by ELISA or immunoblotting, nor with alpha 1(XI) or alpha 3(XI), but with alpha 2(XI) chains. Using this antibody, it was possible to specifically localize alpha 2(XI) in cartilage by pretreating tissue sections with 6 M urea. In double immunofluorescence staining experiments, the distribution of alpha 2(XI) as indicative for type XI collagen in fetal bovine and human cartilage was compared with that of type II collagen, using a monoclonal antibody to alpha 1(II). Type XI collagen was found throughout the matrix of hyaline cartilage. However, owing to cross-reactivity of the monoclonal anti-alpha 1(II) with alpha 3(XI), both antibodies produced the same staining pattern. Cellular heterogeneity was, however, detected in monolayer cultures of human chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibody against chicken type IX collagen: preparation, characterization, and recognition of the intact form of type IX collagen secreted by chondrocytes

A series of monoclonal antibodies was prepared against the pepsin-resistant fragment of type IX collagen designated HMW. One of these antibodies (called 2C2) was selected for further analysis. Antibody 2C2 showed no cross-reactivity with other collagen types by inhibition enzyme-linked immunosorbent assays. It recognized an epitope present in native HMW, but failed to recognize any of the three chains of HMW fractionated after denaturation followed by reduction and alkylation of interchain disulfide bridges. Electron microscopic observations after rotary shadowing showed that the location of the epitope for antibody 2C2 was close to the carboxy-terminus of HMW. Immunofluorescent staining of sections of embryonic and adult cartilage with antibody 2C2 after removal of proteoglycans by testicular hyaluronidase digestion showed that type IX collagen is distributed throughout the cartilage matrix, and is not present in other connective tissues or skeletal muscle. The intact type IX collagen molecule, which was secreted by a suspension culture of freshly isolated embryonic chick chondrocytes, was recognized by rotary shadowing in the presence of antibody 2C2 after first precipitating the procollagens from the culture medium with ammonium sulfate (30%). Two different collagenous molecules were present in the precipitate: a longer molecule of type II procollagen (average length, 335 nm) with both amino- and carboxy-propeptides still remaining uncleaved, and a shorter molecule (average length, 190 nm) which was identified as type IX collagen. Antibody 2C2 consistently bound to the shorter molecules at a site located 136 nm from a distinctive knob at one end of the molecule, and did not bind to any specific site on the type II procollagen molecules. The structure of the intact type IX collagen molecule with the location of both collagenous and noncollagenous domains was as predicted after converting the nucleotide sequence of a cDNA clone encoding for one of the chains of type IX collagen to an amino acid sequence (Ninomiya, Y., and B. R. Olsen, 1984, Proc. Natl. Acad. Sci. USA, 81:3014-3018).     

Histochemical and immunohistochemical studies on the articular disk of the temporomandibular joint in rats

The effects of surgically induced malocclusion upon the articular disk of the temporomandibular joint in adult male rats were investigated histochemically and immunohistochemically using antibody against type II collagen. In the intermediate portion of the articular disk, the number of collagen fibers and chondrocytes was much less and the collagen fibers became more wavy, rough and irregular in surgically operated rats than in control rats. Clusters of chondrocytes were found in some experimental rats. The present experiments revealed that surgically induced malocclusion causes noticeable changes in collagen fibers and chondrocytes within the extracellular matrix of the articular disk of the temporomandibular joint.     

Involvement of HMGB1 and HMGB2 proteins in exogenous DNA integration reaction into the genome of HeLa S3 cells

Electrophoretic and Western blot studies were conducted on collagen fractions extracted from Sepia officinalis (cuttlefish) cartilage using a modified salt precipitation method developed for the isolation of vertebrate collagens. The antibodies used had been raised in rabbit against the following types of collagen: Sepia I-like; fish I; human I; chicken I, II, and IX; rat V; and calf IX and XI. The main finding was that various types of collagen are present in Sepia cartilage, as they are in vertebrate hyaline cartilage. However, the main component of Sepia cartilage is a heterochain collagen similar to vertebrate type I, and this is associated with minor forms similar to type V/XI and type IX. The cephalopod type I-like heterochain collagen can be considered a first step toward the evolutionary development of a collagen analogous to the typical collagen of vertebrate cartilage (type II homochain). The type V/XI collagen present in molluscs, and indeed all phyla from the Porifera upwards, may represent an ancestral collagen molecule conserved relatively unchanged throughout evolution. Type IX-like collagen seems to be essential for the formation of cartilaginous tissue.     

Attenuation of collagen arthritis and modulation of delayed-type hypersensitivity by type II collagen reactive T-cell lines

T-cell lines were established from the lymph node cells of syngeneic Louvain (LOU) rats previously immunized with native chick type II collagen (CII) emulsified in incomplete Freund's adjuvant. The CII lines proliferated in vitro to type II collagen but not to type I collagen, ovalbumin (OV), or PPD. Control lines, developed from LOU rats immunized with OV emulsified in complete Freund's adjuvant, were OV specific because they did not respond to other antigens in vitro. CII line cells could adoptively transfer delayed-type hypersensitivity (DTH) but did not induce IgG antibody production to collagen. Moreover, the intravenous administration of 2 X 10(7) CII line cells prevented the subsequent induction of collagen arthritis following immunization and suppressed DTH to collagen without affecting antibody responses in the recipients. Spleen cells, but not sera, from these resistant rats decreased CII line reactivity in vitro. OV or irradiated CII lines had no effect on clinical or immunologic parameters in this model. These findings demonstrate protection from arthritis afforded by T-cell line transfer and suggest that the phenomenon results from down-regulation of the recipients' cellular immunity to collagen.     

Catalytic properties of lysyl hydroxylase from cells synthesizing genetically different collagen types.

Crude preparations of lysyl hydroxylase were extracted from chick-embryo tendons synthesizing exclusively type I collagen, chick-embryo sterna synthesizing exclusively type II collagen and HT-1080 sarcoma cells synthesizing exclusively type IV collagen. No differences were found in the Km values for Fe2+, 2-oxoglutarate and ascorbate between these three enzymes preparations. Similarly no differences were found in the Km values for type I and type II protocollagens and the rate at which type IV protocollagen is hydroxylated between these enzyme preparations. The extent to which type I protocollagen could be hydroxylated by the three enzymes was likewise identical. These data strongly argue against the existence of collagen-type-specific lysyl hydroxylase isoenzymes.