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Specific antisera to collagen Types I, II and III and proteoglycan were used to investigate the distributions of these molecules in normal human intervertebral discs. Immunofluorescent staining indicated the presence of small amounts of Type III collagen located pericellularly in normal adult intervertebral discs. This finding had not been demonstrated previously by other methods. Similar specimens of intervertebral discs from 17 patients with scoliosis of varying aetiologies were examined, but no evidence was obtained for primary connective tissue defects. Secondary changes, especially marked vascularisation of the inner annulus, were apparent in a number of scoliotic discs, and some of these showed enhanced staining for collagen Type I and proteoglycan, and intercellular matrix staining for Type III collagen.

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.

Collagen Type III, fibronectin and a non-collagenous reticulin component (NCRC) are all secretory products of fibroblasts and other mesenchymal cells. Antisera to human collagen III (isolated from the placenta) human plasma fibronectin, and NCRC (from spleen) have been used to examine possible antigenic relationships between these proteins both by serological studies and by comparison of the distribution of the proteins in various tissues using immunofluorescence. For other comparative purposes, the distribution of collagen and of reticulin in the same tissues was also sought using conventional histological methods. Serologically, the 3 antisera showed distinct specificities. However, when used on sections all 3 antisera gave closely similar staining patterns broadly resembling that of histological "reticulin", although minor differences were noted to be characteristic of the distribution of the respective antigens in certain tissues and in cell monolayers. The results support previous suggestions that histological "reticulin" is not a single entity but is a compound fibrous structure containing collagen Type III, fibronectin and at least 1 additional non-collagenous glycoprotein (NCRC).

Cell-mediated immunity (CMI) to chick type II collagen and its cyanogen bromide (CB) peptides was studied in guinea-pigs using cutaneous delayed hypersensitivity reactions. Responses were largely independent of molecular conformation in animals immunized with either native or denatured collagen, and reactions obtained with CB peptides 8, 9, 10 and 12 suggested that sites in the central regions of collagen chains were recognized in CMI. Antibodies to collagen were detected by haemagglutination and immunofluorescence only in animals immunized with native molecule and not in animals immunized with denatured or CB-digested material. Humoral and CMI responses were similar in that neither recognized the pepsin-labile non-helical regions of the molecule. The responses differed in that humoral reactions were conformation-dependent and type-specific and CMI reactions were not.

Antisera were produced which recognized specifically native type I and type II collagens and proteoglycan. These were used in immunofluorescence studies to investigate the distribution of collagens and proteoglycan in intervertebral discs from adult and newborn pigs. Cervical, thoracic and lumbar discs gave similar staining patterns. In the adult, the outer 1 mm of the annulus fibrosus resembled a perichondrium and was negative for type II collagen. The inner regions of the annulus contained proteoglycan and both types of collagen, but these molecules appeared to have separate distributions. The nucleus showed no staining for type I collagen. Newborn pig discs differed from those of the adult in that type II collagen was restricted to the central notochord and to a narrow zone surrounding it. The newborn annulus was negative for type II collagen but reacted strongly with antibodies to both type I collagen and proteoglycan. It is suggested that during development of the pig annulus fibrosus, cells producing type II collagen may migrate into this area from the central regions.

Sera from 23 patients with chronic low back pain, 20 rheumatoid patients and 16 normal controls were tested for antibodies to collagen types I, II and III, both native and denatured, by haemagglutination. Weak reactions against denatured collagen types I and II were found in 30-40% of the sera. Sera from individuals with rheumatoid arthritis or chronic low back pain behaved similarly, while only one normal serum showed any positive reaction. Reactions to denatured collagen type III and to native collagen of all 3 types were largely negative. Non-antibody serum components were thought to be responsible for these haemagglutination reactions since weakly positive reactions were abolished by cryoprecipitation and could not be confirmed by a solid-phase fluorimetric assay. Using the latter technique sera from 62 rheumatoid patients were screened for antibodies to type II collagen (native and denatured) and only one positive serum found. We conclude that haemagglutination is subject to false positive reactions and that the incidence of anticollagen antibodies in sera from patients with rheumatoid arthritis or chronic low back pain is low.