Characterization of conformation independent antigenic determinants in the triple-helical part of calf and rat collagen

About 20 per cent of the antibodies in rabbit antisera to native calf or rat collagen exhibited affinity for denatured rabbit collagen and could be isolated by immunoadsorption. Such antibodies reacted with the unfolded α1-chain as well as with the α2-chain of collagen. Inhibition experiments suggested that the two kinds of polypeptide chains are not completely equivalent in their antigenic determinants. These determinants were not significantly influenced by a treatment of native collagen with pronase, a procedure known to remove short, non-helical sequences at both ends of the molecule. The results suggested that the antigenic determinants are conformation independent. They are, however, mainly located in the middle region of collagen, having a rather complex conformational structure.

Cyanogen bromide cleavage of collagen did not impair the serologic activity of these determinants but with one exception none of the individual cyanogen bromide peptides possessed the full activity of the entire α-chain. However, most of the peptides could be agglutinated by the antibodies when put onto tanned red cells. Inhibition studies of these agglutination reactions clearly demonstrated that virtually all of the peptides carry unique antigenic determinants, which occasionally are shared by a few other peptides. Additional evidence for heterogeneity was obtained by further cleavage of the cyanogen bromide peptides with proteases. The minimum number of antigenic determinants thus estimated in calf collagen was nine. Evidence is provided that their structure in most cases does not correspond to sequences of the type Gly-Pro-X.

     

Production and specificity of antibodies against the aminoterminal region in type III collagen.

A cross-linked fragment (peptide T1X) with a molecular weight of 13,000 could be isolated from a tryptic digest of insoluble type III collagen of calf skin. Peptide T1X was conjugated on to bovine serum albumin by glutaraldehyde and used for immunization of rabbits. The antisera reacted in passive haemagglutination and radioimmune assay with peptide T1X, type III collagen and its constituent alpha1(III) chain. Little or no reaction was observed with type I collagen and alpha1(I) chain. While rabbit antisera to neutral salt-soluble type III Collagen also showed a strong binding for 125I-labelled peptide T1X much less reaction was observed with antisera to type I collagen. The antigenicity of type III collagen was largely destroyed by pepsin treatment suggesting that it resided in non-helical segments. A fragment of peptide T1X produced by digestion with collagenase retained antigenic activity. The data indicated that the aminoterminal region of type III collagen contains strong antigenic determinants located in a non-helical sequence of about sixteen amino acids. Antibodies to these antigenic determinants were purified and rendered specific for type III collagen by immunoadsorption. The antibodies stained in indirect immunofluorescence tests particularly those regions in various connective tissues which are rich in reticulin fibres. Different staining patterns were observed with antibodies to type I collagen.     

The antigenic properties of some synthetic polyiminoacids: II. The antigenicity of polypeptides related to collagen; peptides containing hydroxyproline and acetyl-hydroxyproline

The antigenic properties of some synthetic polymers containing hydroxyproline and acetyl-hydroxyproline have been tested in guinea-pigs and rabbits by active cutaneous anaphylaxis, delayed skin hypersensitivity reactions, PCA, tanned cell agglutination and fluorescent antibody microscopy.

The antigenic relationships between these polymers, collagen and acetylated collagen have been investigated. The results obtained suggest that acetyl-hydroxyproline is a common antigenic determinant in both acetylated copolymers and acetylated collagen.

Poly-hydroxyproline and poly-acetyl-hydroxyproline were found not to be antigenic in rabbits or guinea-pigs. Rabbit antiserum against acetylated collagen has been used to stain acetylated tissue sections by immunofluorescence. Absorption studies indicate that acetyl-hydroxyproline groups are important antigenic determinants as shown by the considerable decrease in specific fluorescence when the rabbit anti-acetylated collagen is absorbed with a synthetic polymer containing acetyl-hydroxyproline.

The overall results are discussed in terms of the structural similarity existing between collagen and some of the polymers used in this work.

     

Anti-collagen antibodies in sera from rheumatoid arthritis patients.

Anti-cartilage antibodies, demonstrable by immunofluorescence, were found in 3.3% of rheumatoid arthritis patients. In most of these patients antibodies to type II collagen were detected. In specificity studies on these anti-collagen antibodies, they appeared to be type specific, showing no reaction with collagen types I and III. Denatured type II collagen reacted much less well than native type II, but isolated peptides from different regions of the collagen molecule were differentiated by individual sera. Removal of the glycoside side chains from native type II collagen had no effect on its antigenicity. The findings suggest that these patients produce highly specific antibodies which react with the triple helix of type II collagen.     

Depolymerisation of Human and Bovine Polymeric Collagen Fibrils

Bovine and human polymeric collagen fibrils were incubated at 37°C over a range of pH with a crude lysosomal enzyme preparation obtained from rat liver. Electron microscopic analysis demonstrated depolymerisation of the fibrils over the pH range 3.5 to 7.5 with the cleavage of the fibrils into short segments, followed by unwinding of the protofibrils and cleavage of the protofibrils into protofibrillar particles. The depolymerisation of polymeric collagen to soluble products was followed by hydroxyproline analysis; the pH optimum for this process was approxi- mately 4.0. The soluble depolymerisation products of bovine polymeric collagen fibrils digested at pH 4.0 were found to be native tropocollagen-like molecules which rapidly aggregated at physiological pH to form intermolecularly cross-linked native type fibrils. No soluble hydroxyproline containing products were produced by this enzyme system at physiological pH, although the cleavage of polymeric collagen fibrils could be observed at the electronmicroscopic level. Polymeric collagen fibrils obtained from human ischaemic skin exhibited a similar fragmentation pattern to the in vitro depolymerisation of polymeric collagen fibrils.     

An Attempt to Characterize the Lupus Erythematosus Cell Antigen

Particulars are given of immunization procedures carried out in rabbits in an unsuccessful attempt to produce lupus factor.

Experiments on the substrate specificity and inhibition of the lupus reaction are described.

From the results it is inferred that the antigenic determinants involved in the lupus reaction lie in the `backbone' configuration of native nucleo-proteins, both nucleohistone and most probably nucleoprotamine.

     

Conformation dependence of antigenic determinants on the collagen molecule

Three different types of antigenic determinants were demonstrated in soluble collagen with the aid of rat, rabbit and chicken antisera to native collagen. Helical antigenic determinants which require an intact triple-helical structure of the molecule are mainly recognized by rat antisera. Renaturation of the serologically inactive unfolded polypeptide chains (denatured collagen) is accompanied by a significant recovery of serological activity. Central antigenic determinants which are probably located in the same regions of amino acid sequence are less accessible in the native antigen and become exposed upon denaturation. Equal titres for both types of determinants are found in chicken antisera. Immunization with denatured collagen, however, revealed a response restricted to the central type. Isolated antibodies specific for terminal non-helical antigenic determinants, as yet only known to occur in rabbit antisera, reacted equally well with native collagen, the unfolded polypeptide chains and with small cyanogen bromide peptides. Independence of conformation is therefore suggested for these antigenic structures.     

Independent appearance of anti-thymocyte and anti-RNA antibodies in NZB/NZW F1 mice.

Mouse antibodies to soluble bovine skin (type I) collagen react with determinants which are located in the rigid triple-helical portion of the antigen and become destroyed upon unfolding the molecule. Helical antigenic determinants are dependent on the genuine chain assembly, e.g. alpha[1(I)]2alpha2. Artefactual triplehelical structures of the composition [alpha1(I)]3 or [alpha2]3 or a genetically distinct type II collagen from cartilage showed no or only weak cross-reactivity. Pepsin treatment of type I collagen known to remove short, non-helical sequences at both ends of the molecule had virtually no effect on antigenicity and immunogenic activity. A radioimmunoassay failed to detect antibodies in three congenic resistant mouse strains immunized with denatured type I collagen. These strains had been previously classified as high or low responders to native type I collagen. Agglutination titres vs denatured collagen culd already be demonstrated in nonimmune sera. The agglutinating activity was labile against heating at 56 degrees and could not be increased by immunization. Two out of five inbred strains showed a high response against pepsin-dissolved bovine type II collagen with the chain composition [alpha1(II)]3. Lack of correlation in the responder state to both collagen types indicated control by different immune response genes. Antibodies to type II collagen also reacted against triple-helical antigenic determinants and showed neglible cross-reaction with type I collagen.     

The antigenicity of some synthetic poly-iminoacids: I. Antigenic properties of poly-L-proline

The antigenic properties of a group of synthetic poly-iminoacids have been tested in guinea-pigs and rabbits by active cutaneous anaphylaxis, delayed skin hypersensitivity reactions, passive cutaneous anaphylaxis, (PCA), tanned cell agglutination and agglutination-inhibition. The antigenicity of a linear homopolymer, poly-L-proline, has been demonstrated in guinea-pigs. This polymer exists in two forms differing only in their secondary structure, Form I having a right-handed helix and Form II a left-handed helix. That both forms are antigenic and yet non-cross-reacting emphasizes the importance of secondary structure in this system.Comparison of polyprolines of different molecular weights suggests the presence of more than one antigenic determinant; a polymer of an average molecular weight of 1500 was antigenically deficient compared with a polyproline of a molecular weight of 17,000.No cross-reactions were detected between anti-polyproline or anti-polyproline-glycine and collagen or its derivatives.The possible structure of the antigenic determinants involved is discussed.     

Rat parietal yolk sac basement membrane. An investigation of the antigenic determinants using a radioimmunoassay.

Previous studies have shown that there is microscopic and biochemical evidence that rat parietal yolk sac synthesizes basement membrane (type IV) collagen; this study shows that a radioimmunoassay may be used for the detection of type IV collagen in such biosynthetic systems. Rat parietal yolk sacs incubated in medium containing (14C) proline either with or without alphaalpha-dipyridyl produced either unhydroxylated or hydroxylated (14C)collagen. The immunological reactivity of these two preparations was investigated using antibodies to bovine type IV collagen in a radioimmunoassay which demonstrated that the hydroxylated (14C)collagen preparation had a considerably higher level of antigenicity than the unhydroxylated (14C)collagen. Hydroxylated rat type IV (14C)collagen which had been reduced and alkylated was intermediate in antigenicity between hydroxylated and unhydroxylated material. These findings suggest that there are antigenic determinants which depend upon hydroxylation of the collagen molecule, and others dependent upon intact disulphide bonds. In addition, various levels of pepsin extracted unlabelled calf anterior lens capsule collagen caused inhibition of antibody binding to (14C)collagen. Rat type IV (14C)collagen which had been digested with collagenase was inactive in the radioimmunoassay, while pepsin digestion caused no reduction in antigenicity. These findings suggest that the antiserum is directed towards the collagenous part of the molecule and may be a useful tool in the detection of biosynthesized basement membrane collagen.     

Immunogenicity and specificity of collagen: V. Demonstration of three different antigenic determinants on calf collagen

Eighty rabbits were immunized with native or denatured acid-soluble calf skin collagen. Fifty-five antisera showed haemagglutination titres higher than 1:64. These sera were investigated with calf skin collagen, pepsin-treated calf skin collagen and rabbit skin collagen, used as coating antigens and inhibitors. Three serologically distinct collagen-antibody fractions could be demonstrated: a general, non-species specific fraction reacting with all three antigens, a species specific fraction active with the two calf preparations, and an antibody fraction to pepsin-labile structures of collagen reacting only with untreated calf collagen. The antibody fractions occur in the antisera either alone or as mixtures. The titres of distinct antibody fractions present in the mixed sera were determined by haemagglutination—inhibition experiments.

Antibodies to pepsin-labile structures could be equally well inhibited by collagen peptides obtained after trypsin or collagenase treatment. A ten-fold higher amount of peptides as compared with undergraded antigen was necessary to obtain the same inhibition effect. The results were compared with previously reported peptide inhibition experiments performed with the other two types of antibodies.

     

The antigenic and immunosuppressive properties of normal and antilymphocytic equine IgG subfractions

The antigenic and immunosuppressive properties of normal and antilympho-cytic equine globulin subfractions were investigated in the rat model in an attempt to increase the efficacy of prolonged ALG therapy by limiting the immunogenic stimulus of `inactive' subfractions.

Chromatographic separation of equine IgG components yielded an electro-phoretically slow gamma 2 fraction consisting of IgG2a and IgG2b and a more heterogeneous fast gamma 1 subfraction. Immunosuppression resulting from the administration of isolated subfractions was measured by the response to alum-BSA and skin allograft survival. Antigenicity was determined by a variety of immunological procedures. The immunosuppressive character of the ALG was confined to the gamma 2 fraction, however this fraction also proved antigenic in our system.

The administration of normal equine IgG subfractions in combination with Freund's complete adjuvant resulted in the demonstration of antigenic differences between the fast and slow IgG components.

     

Immunoglobulin allotype in the rat: localization of the specificity to the light chain

An immunoglobulin allotypic antigen has been described in the rat. This antigenic specificity, called RI-1, is present on the IgG and IgM of the DA strain and is recognized by antisera raised in the Lewis strain. The anti-DA rat allotypic antisera were capable of precipitating ¹²⁵I-labelled DA rat IgG and of agglutinating sheep erythrocytes coated with sub-agglutinating amounts of DA rat anti-sheep erythrocyte antibody.

Inhibition studies with sub-units of DA rat IgG in the microprecipitin assay showed the RI-1 specificity to be on light chains but not on heavy chains of IgG.

     

Shwartzman reaction in endotoxin-resistant rabbits induced by heterologous endotoxin

Moderate resistance to Escherichia coli and Serratia marcescens endotoxin was induced in rabbits.

The E. coli endotoxin-resistant rabbits did not respond with a Shwartzman reaction if the skin preparation was carried out by E. coli endotoxin and the provocation by Serratia endotoxin. However, the same animals showed definite haemorrhagic reactions if their skin was prepared by endotoxin from other serological types, e.g. Serratia or Salmonella typhi endotoxin.

Serratia endotoxin resistant rabbits did not display the Shwartzman reaction if the skin preparation was carried out by Serratia endotoxin. On the contrary, they displayed marked Shwartzman reactions at the sites of the skin preparation with E. coli endotoxin. The provocation of this haemorrhagic reaction could be carried out either by E. coli or by Serratia endotoxin.

The above results point to the possibility of the role of an immunological specificity in the skin preparation of the Shwartzman phenomenon. No such specificity could be demonstrated with regards to the intravenous provocation of the reaction.

The immunospecificity (serological specificity) in the preparation of the Shwartzman reaction is limited, because it disappears if a higher degree of resistance is established. The immunospecificity in resistance or in sensitivity is dose-dependent. By raising the preparing or provoking doses, the Shwartzman reaction can be induced by heterologous or homologous endotoxins in rabbits that have a higher degree of resistance. The possible cause is discussed.

     

Naturally occurring anti-tissue antibodies in rat sera

Seventy per cent of normal rat sera have been shown to contain heat labile serum component(s) active against various rat organ homogenates as demonstrated by haemolytic complement fixation and passive haemagglutination tests. The main antigenic activity in rat liver has been found in the mitochondrial fractions.

It was also demonstrated by the indirect fluorescent antibody technique that both guinea-pig complement and high molecular weight rat globulins were fixed to rat organ sections.

Chemotactic activity has also been observed with rat serum and rat liver mitochondria and it is suggested that these naturally occurring antibodies may be implicated in the removal of tissue breakdown products.

     

The structural basis of cell-mediated immunological reactions of collagen: Characteristics of cutaneous delayed hypersensitivity reactions in specifically sensitized guinea-pigs

Cell-mediated immunological properties of collagen were studied in guinea-pigs employing cutaneous delayed hypersensitivity reactions. The animals were sensitized by a single injection of highly purified native collagen in Freund's complete adjuvant (FCA). Reactivity could be induced with 150 μg of calf collagen. Maximal reactivity was obtained 20 days after sensitization and persisted for more than 3 months. Histologically, the reactions displayed the typical features of delayed reactions with infiltration of predominantly mononuclear cells. No reactivity was induced in animals injected with FCA alone, with collagen in Freund's incomplete adjuvant (FIA), or with guinea-pig collagen in Freund's complete adjuvant. Reactivity was impaired when carrageenan was injected intraperitoneally at the time of challenge. Cyanogen bromide digested collagen was still reactive in sensitization as well as in elicitation. The reaction was found to be species specific in the sense that maximal reactions were obtained when the challenging collagen was from the same species as the sensitizing preparation. In vitro, denatured rat collagen was found to inhibit the migration of macrophages from specifically sensitized animals. By alterations of the immunization schedule antibodies, reactive with collagen in a haemagglutination system, could be induced.

The system lends itself to a comparative investigation of the structural requirements on a natural protein for the induction of the cell-mediated and the humoral immune response.

     

Studies on the types of immune responses to synthetic antigens in guinea-pigs

The immune response in guinea-pigs to linear and multichain copolymers of two, three or four different amino acids including tyrosine, glutamic acid, alanine and lysine, was studied.

Delayed-type response was favoured in the case of preparations of relatively low molecular weight, containing only tyrosine and glutamic acid in their potential antigenic specificity determinants. Delayed sensitivity to one of these preparations was passively transferred with lymphoid cells.

Antibody response resulted from the immunization of animals with preparations of relatively high molecular weight and with a more heterogeneous chemical composition. The antibody formation was intensified and accelerated, when p-azobenzenearsonate conjugates of the polypeptides were used as antigens.

Variations in the immunizing dose had little or no effect on the nature of the reactions. A response of exclusively delayed type could not be intensified by repeated immunizations. The presence of mycobacteria in the immunizing injection was essential for the elicitation of the response to multichain, but not to linear copolymers.

     

The selective inactivation of influenza virus haemagglutinin by pyridine

Summary Pyridine has been shown to selectively inactivate the haemagglutinating activity of influenza virus. V-antigen and neuraminidase activity were hardly affected and the virions maintained their basic integrity, although some change in morphology was evident. The pyridine treated virus particles obtained were thus unlike the products of virus degradation reported with other solvents or biological agents. Production of multivalent neuraminidase components with no haemagglutinating activity provided the first unequivocal demonstration that neuraminidase does not contribute to the haemagglutinating activity of native virions. The pyridine treated virus retained its antigenic ability although its antigenicity was less than that of the normal virus.     

The structural basis of cell-mediated immunological reactions of collagen: Recognition by the cutaneous delayed hypersensitivity reaction in guinea-pigs of conformational alternations of rat and calf skin collagen

Guinea-pigs were sensitized for delayed cutaneous hypersensitivity reactions to native and thermally denatured rat and calf skin collagens. Native and denatured collagens sensitized the animals to a comparable degree. However, the immune mechanism of the animals differentiated the purely conformational alterations of the molecule, even across the species barrier. Denaturation was accompanied by loss of immunogenic information as well as by exposure of new determinants, not recognizable in the native molecule. The sera of these animals were examined for the presence of antibodies reactive with native collagen in a passive haemagglutination system. Low levels of such antibodies were found in animals sensitized with native collagen and no antibodies reactive with native collagen in animals sensitized with denatured molecules although the latter group of guinea-pigs responded well with delayed skin reactions to native collagen.     

Thermal Stability of Mineralized and Demineralized Dentin: A Differential Scanning Calorimetric Study

The purpose of this study was to determine the difference, if any, in the thermal stability of collagen in mineralized and demineralized dentine compared to that in unmineralized tissues, using differential scanning calorimetry, DSC. Human tooth dentin blocks, about 1×1×2 mm in size, were used in this study. Some dentin blocks were demineralized using a Plank Rychlo solution; others, using EDTA solution. The mineralized dentin showed an exothermic peak at about 310°C and the combustion of organic materials was completed at about 450°C. For the demineralized dentin, the combustion was completed at higher temperature range and showed a strong exothermic peak at about 470°C. An exotherm at the temperature between 450°C and 470°C was also observed in DSC pattern of native type I collagen from calf skin and rat tail tendon. DSC pattern of rat tail collagen showed a close similarity to that of the demineralized dentin. Statistically, the same heat flow value was obtained both from the mineralized dentin and the demineralized dentin and from the native type I collagen.

These findings indicated that the thermal stability of collagen in dentin is lower than collagen in uncalcified connective tissue. It is suggested that in calcified collagen, the apatite crystallites may have intruded into spaces of the crosslinks of intra- and inter-fibrils, and in so doing, destroyed the crosslinks.