Effects of equine recombinant interleukin-1α and interleukin-1β on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants

Abstract

Objectives - To evaluate the effects of equine recombinant interleukin-1α (rEqIL-1α) and recombinant interleukin-1β (rEqIL-1β) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture. Sample Population - Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse. Procedure - Expression constructs containing cDNA sequences encoding EqIL-1α and EqIL-1β were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1α or rEqIL-1β treatments (0 to 500 ng/ml). Proteoglycan release was evaluated by 1,9-dimethyl-methylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media. Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan. Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay. Data were collected at 48-hour intervals and normalized by DNA content. Results - Proteoglycan release was induced by rEqIL-1α and rEqIL-1β at concentrations ≥ 0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively. Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations ≥ 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations ≥ 0.1 ng/ml at 2 and 4 days. Conclusions and Clinical Relevance-The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro. These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses.