Similarity of antigelatin factor and cold insoluble globulin.

Antigelatin factor, a protein capable of complexing denatured collagen, was separated from human serum by adsorption onto immobilized collagen. Antiserum raised against the material binding to denatured collagen permitted the development of a radioassay for the determination of antigelatin factor in which the complex of antigelatin factor and denatured 125I-labeled collagen is precipitated with this antiserum. Further purification of antigelatin factor was achieved by chromatography on DEAE-cellulose yielding an electrophoretically homogeneous protein. Its migration rate in dodecyl sulfate-polyacrylamide gel electrophoresis was identical with that of cold insoluble globulin (molecular weight approx. 440 000) prepared from human plasma by a published procedure amended by DEAE-cellulose chromatography. Reduction of disulfide bonds yielded subunits of molecular weight approx. 220 000, indistinguishable from those of cold insoluble globulin. The amino acid composition of both proteins was very similar. Immunological identity of both proteins was demonstrated by gel diffusion against monospecific anti-cold insoluble globulin antiserum. Closely related binding curves were obtained if denatured 125I-labeled collagen was reacted with increasing amounts of either cold insoluble globulin or antigelatin factor and the complexes formed were precipitated with anti-cold insoluble globulin antiserum. In addition, antigelatin factor and cold insoluble globulin mediated the fixation of denatured 125I-labeled collagen to trypsinized macrophages in the same way. Therefore, it is concluded that antigelatin factor and cold insoluble globulin are identical or very closely related proteins.

Identification of the sites in collagen alpha-chains that bind serum anti-gelatin factor (cold-insoluble globulin).

Anti-gelatin factor was prepared from guinea-pig and human serum by affinity chromatography on denatured type-I collagen. As shown previously, this component is related to cold-insoluble globulin. It reacted with 125I-labelled denatured collagen, and the reaction could be inhibited by preincubation with unlabelled collagenous components. In the inhibition assay comparable activities were observed for native and denatured type-I, -II, -III and -IV collagens. There was also no difference in reactivity between collagens of different species. The reactive sites in the collagen alpha-chains were located by inhibition assays on distinct CNBr- and collagenase-derived peptides. The results obtained with fragments from alpha1(I)-, alpha2- and alpha1(II)-chains indicate that the most active region is located between positions 643 and 819 of the alpha1-chain. Lower activities were found for other regions of collagen and may indicate that the factor has the potential to interact with several sites in the alpha-chains. The present data agree with observations by Kleinman, McGoodwin & Klebe [Biochem. Biophys. Res. Commun. (1976) 72, 426-432] on the specificity of a serum factor promoting the attachment of fibroblasts to collagen.

Dependence of the antibody response of guinea-pigs to collagen on the type of adjuvant and on the conformation of the antigen.

The immunological response to collagen of guinea-pigs is strongly dependent on the conformation of the antigen and on the type of adjuvant. Freund's complete adjuvant facilitated excellent delayed hypersensitivity skin reactions to native (triple helical conformation) as well as denatured (random coil conformation) collagen. Immunization of guinea-pigs with collagen in this adjuvant gave rise to very low levels of antibody to native collagen and failed to induce antibodies to denatured collagen. Use of Freund's incomplete adjuvant resulted in excellent antibody responses to native collagen, but it did not induce antibodies to denatured collagen. Animals injected with collagen and Freund's incomplete adjuvant were not sensitized for cell-mediated immunological reactions. The antibodies to collagen were specific with regard to collagen from various species but displayed different degrees of cross-reactivities depending on the species of collagen used for immunization. They were specific for the triple helical conformation of the collagen molecule.

Specific suppression of delayed hypersensitivity skin reactions to collagen in guinea-pigs after immunization with collagen and Freund's incomplete adjuvant.

Partial suppression of cutaneous delayed hypersensitivity reactions to collagen in guinea-pigs was induced by pre-immunization with collagen and FIA. This suppression is specific since: (a) pretreatment with OA and FIA or FIA alone did not cause suppression of skin reactions to collagen; (b) suppression was observed only if the collagen used for pretreatment was from the same species as that employed for sensitization for delayed hypersensitivity reactions; and (c) animals with depressed skin reactivity to collagen reacted normally to PPD. The suppression is not mediated by inducible, circulating antibodies to collagen since: (a) antibody titres measured by passive haemagglutination did not correlate with the degree of suppression; (b) suppression was observed with collagen in random coil conformation which sensitizes guinea-pigs for delayed hypersensitivity skin reaction but does not induce antibodies to denatured collagen; (c) best suppression was obtained if the animals were pretreated with collagen and FIA 3 days before the sensitizing injection; and (d) passively transferred antibody from animals with suppressed skin reactivity did not suppress skin reactivity of animals made hypersensitive to collagen by injection of collagen and FCA.

The relation between the cell-mediated immunological response and the induction of circulating antibodies to collagen in guinea-pigs.

Cutaneous delayed hypersensitivity reactions to collagen in guinea-pigs were partially but specifically suppressed if the animals had been pretreated with collagen and Freund's incomplete adjuvant. Such animals responded normally to skin-reactive factor prepared with ovalbumin. Lymphoid cells from animals with normal delayed hypersensitivity to collagen functioned normally in animals with suppressed skin reactivity. Cells from animals with suppressed delayed hypersensitivity were specifically, functionally impaired since they transferred delayed hypersensitivity into neutral recipients efficiently for PPD but not for collagen. Suppression could be induced in Cy-treated animals, and it persisted for at least 143 days. It is concluded that guinea-pigs with depressed delayed hypersensitivity to collagen are functionally impaired with respect to those T cells normally generated by induction of delayed hypersensitivity.

Recongnition by guinea-pig peritoneal exudate cells of conformationally different states of the collagen molecule.

Guinea-pig peritoneal exudate cells were tested in vitro in the presence or absence of specific antiserum to native collagen for their capacity to discriminate between native and denatured collagens of various species. Adherent exudate cells bound denatured collagens, regardless of the origin of the collagen or the presence of serum. The binding was reduced if the cells were pretreated with trypsin. Recovery of binding was mediated by a normal serum component resembling an IgM antibody to denatured collagen. In the presence of normal serum, native collagen was only marginally bound, apparently in a non-specific manner. Uptake of native heterologous collagens was greatly increased in the presence of specific antiserum to native collagen with specificity of binding reflecting the type of collagen. Binding of denatured and native collagen occur via independent mechanisms.

Immunohistochemical evidence for the presence of collagen type III in human arterial walls, arterial thrombi, and in leukocytes, incubated with collagen in vitro.

Sections of arterial walls and of thrombi and smears of leukocytes previously incubated in vitro with collagen type III were examined by immunohistochemical technique for the presence of collagen types I, II and III. In arterial walls collagen type III was detected immediately underlaying the endothelial cell layer and in the tissue between tunica elastica interna and adventitia. Collagen type I was not shown in the subendothelial layer. Fresh thrombi contained occasionally collagen, but only of type III. This was associated with leukocytes. Leukocytes were capable in vitro to associate and/or phagocytose collagen type III and this could be visualized immunohistochemically. The data show that collagen type III in vivo may play a crucial role in the initiation of thrombus formation.