Effects of certain antiarthritic agents on the synthesis of type II collagen and glycosaminoglycans in rat chondrosarcoma cultures.

Cartilage destruction is a characteristic feature of osteoarthritis. Treatment with certain nonsteroidal anti-inflammatory drugs could exacerbate cartilage destruction by impairing the synthesis of cartilage matrix proteins, type II collagen and proteoglycan. In order to monitor the changes occurring in cartilage collagen synthesis, we developed a type II collagen specific ELISA. The effects of antiarthritic agents on type II collagen and glycosaminoglycan synthesis were examined in rat chondrosarcoma cultures. Drugs were added to the monolayer cultures and 4 days later the total type II collagen, as determined by the type II collagen ELISA, and glycosaminoglycan content, as measured by dimethyl-methylene blue dye binding assay, was measured. All drugs except tiaprofenic acid decreased type II collagen synthesis by at least 40% at 100 micrograms/ml. Tiaprofenic acid at 1 microgram/ml increased type II collagen content by 54% of the controls. Glycosaminoglycan synthesis was decreased by acetylsalicylic acid, diclofenac and tiaprofenac acid, at 50 micrograms/ml or above. Indomethacin, naproxen and dexamethasone had no effect. Interestingly, tenidap stimulated the glycosaminoglycan synthesis by 32% at 100 micrograms/ml. We show that the combination of chondrosarcoma cultures, type II collagen specific ELISA and dimethylmethylene blue dye binding assay serves as a useful model for screening the effects of agents capable of modulating type II collagen and glycosaminoglycan synthesis.

Production of type II collagen specific monoclonal antibodies.

Immunoassays are used for the specific measurement of type II collagen, a major cartilage protein, which is lost in osteoarthritic joints. Poor immunogenicity and species dependent immune response to type II collagen make it difficult to obtain specific antibodies required for immunoassay development. In addition, type II collagen antibodies exhibit reactivity to structurally dissimilar antigens such as actin, myoglobin, thyroglobulin and ssDNA, complicating the isolation of specific antibodies. It is therefore necessary to characterize the antibody reactivity against both noncollagenous antigens and different collagen types. In this study, immune response to type II collagen was improved by conjugation to carrier proteins, KLH and BSA. Hybridomas were generated by fusions of lymphocytes derived from lymph nodes or spleens with X63-653-Ag8 myeloma cells. Compared to spleens, the utilization of lymph nodes as a source of lymphocytes resulted in a 23% higher number of hybridomas secreting type II collagen antibodies. Hybridomas secreting polyreactive antibodies were identified based on their reactivity to thyroglobulin and eliminated. Extensive testing of the remaining monoclonal antibodies with other structurally dissimilar antigens and various types of collagen for reactivity, allowed us to isolate specific monoclonal antibodies to type II collagen. We emphasize the importance of characterization of the reactivity of type II collagen monoclonal antibodies before employing them for immunoassays.

Increased collagen synthesis and the kinetic characteristics of prolyl hydroxylase in tissues of rabbits with experimental arteriosclerosis.

Increased aortic and liver prolyl hydroxylase activity has been suggested as an early biochemical indicator of the fibrotic changes which occur in rabbits with injury induced arteriosclerosis. Daily administration of epinephrine (0.025-0.050 mg/kg, i.v.) and thyroxine (0.050 mg/kg, i.p.) to rabbits for 3 weeks produced aortic fibrous plaques with a 4-fold increase in aortic prolyl hydroxylase and also a 5-fold increase in liver prolyl hydroxylase. Histopathologically, the livers of these rabbits show subcapsular areas of necrosis. When total prolyl hydroxylase related antigen was measured. the increase in liver prolyl hydroxylase activity accounted for only a small portion of the total prolyl hydroxylase antigen. However, in the aorta a majority of the increase in antigen is due to the increased amount of enzyme. DNA content per aorta was unchanged and RNA content increased in the aortic tissue of the arteriosclerotic rabbits. However DNA and RNA levels increased 60% in the livers of arteriosclerotic rabbits. In vitro incorporation of radioactively labeled proline into collagenase digestable protein was at least 2-fold greater in aorta and liver minces from arteriosclerotic rabbits. Michaelis--Menten kinetic parameters were obtained for the liver prolyl hydroxylase purified by affinity chromatography from arteriosclerotic rabbits. The Km for the enzyme from treated animals was not significantly different from control. However, the Vmax of the enzyme purified from diseased liver was 4-fold greater when compared to controls.