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

Vivian A. TakafujiOrthopedic Research Laboratory, Department of Large Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061-0442.

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C. Wayne McIlwraithOrthopaedic Research Center, College of Veterinary Medicine, Colorado State University, Ft. Collins, CO 80523.

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Rick D. HowardOrthopedic Research Laboratory, Department of Large Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061-0442.

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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-dimethylmethylene 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. (Am J Vet Res 2002; 63:551–558)

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-dimethylmethylene 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. (Am J Vet Res 2002; 63:551–558)