safranine-t and Arthritis--Rheumatoid

In situ hybridization and immunohistochemical techniques were applied to investigate gene expression and extracellular deposition of collagen type II in normal, osteoarthritic and rheumatoid human articular cartilage. Normal cartilage showed an essentially even extracellular distribution of type II collagen with poly- and monoclonal antibodies, while only a few cells were positive for alpha 1(II) collagen mRNA. In situ hybridization of osteoarthritic and rheumatoid cartilage, however, showed strong enhancement of type II collagen gene expression; transcripts were observed predominantly in the upper middle zone of the articular cartilage while the upper layer was mostly negative and correlated with a zone of reduced proteoglycan staining. The elevated mRNA levels frequently coincided with pericellular immunostaining for type II collagen, indicative for enhanced synthesis of the protein. In two samples, however, pericellular loss of collagen type II staining was found despite positive cytoplasmic signals with the alpha 1(II) RNA probe, suggesting enhanced collagen destruction. Control hybridization with a probe for 18S rRNA revealed very few negative cells throughout both normal and arthritic cartilage samples, ruling out major cell necrosis in the specimens investigated. Thus, our observations identify sites of activated type II collagen synthesis in osteoarthritic cartilage that were predicted by previous biochemical studies and support the notion that damaged cartilage attempts to restore matrix by enhanced synthesis of its components.

Topics: Adolescent; Adult; Aged; Arthritis, Rheumatoid; Cartilage, Articular; Collagen; Histocytochemistry; Humans; Immunohistochemistry; In Situ Hybridization; Middle Aged; Osteoarthritis; Phenazines; Proteoglycans; Reference Values; RNA Probes; Staining and Labeling; Tolonium Chloride

Microbiochemical assays of the proteoglycan and collagen content of articular cartilage and synovial lining have been performed on tissue sections taken from rabbits with antigen-induced arthritis. This experimental arthritis is a close analogue of the natural disease-rheumatoid arthritis. Animals were killed at intervals during the first 21 days following induction of arthritis to assess changes in the composition of the extracellular matrices of the synovial lining and articular cartilage during the early development of this experimental lesion. In confirmation of earlier studies these microbiochemical assays revealed a rapid and significant loss of proteoglycan from the articular cartilage. This loss was, however, not uniform but was restricted to the intermediate zone of the cartilage. Over the period studied, there was only a slight loss of proteoglycan from the superficial zone of the cartilage facing the joint cavity. These findings demonstrate that, at least in this model, cartilage proteoglycan loss is not due to the action of proteases present in the synovial fluid. Moreover it suggests that the chondrocytes in the mid-zone of the cartilage are responsive to those signals stimulating proteoglycan breakdown. There was no significant loss of collagen from the cartilage over the time period of this study. The synovial lining from arthritic joints, in contrast, showed a progressive increase in both proteoglycan and collagen.

Topics: Animals; Arthritis, Rheumatoid; Azo Compounds; Cartilage, Articular; Collagen; Disease Models, Animal; Femur; Male; Ovalbumin; Phenazines; Picrates; Proteoglycans; Rabbits; Staining and Labeling

The intensity of safranin 'O' staining is directly proportional to the proteoglycan content in normal cartilage. Safranin 'O' has thus been used to demonstrate any changes that occur in articular disease. In this study, staining patterns obtained using monoclonal antibodies against the major components of cartilage proteoglycan chondroitin sulphate (anti CS) and keratan sulphate (anti KS), have been compared with those obtained with safranin 'O' staining, in both normal and arthritic tissues. In cartilage where safranin 'O' staining was not detectable, the monoclonal antibodies revealed the presence of both keratan and chondroitin sulphate. Thus, safranin 'O' is not a sensitive indicator of proteoglycan content in diseases where glycosaminoglaycan loss from cartilage has been severe.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Arthritis; Arthritis, Rheumatoid; Cartilage, Articular; Chondroitin Sulfates; Extracellular Matrix; Humans; Immunohistochemistry; Keratan Sulfate; Middle Aged; Osteoarthritis; Phenazines; Proteoglycans