Effects of equine recombinant interleukin-1alpha and interleukin-1beta on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants.
OBJECTIVE
To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture.


METHODS
Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse.


METHODS
Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta 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-1alpha or rEqIL-1beta treatments 10 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-1alpha and rEqIL-1beta at concentrations > or =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 > or = 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days.


CONCLUSIONS
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.
Takafuji
Vivian A
VA
Department of Large Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg 24061-0442, USA.
McIlwraith
C Wayne
CW
Howard
Rick D
RD
D000818:Animals; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015232:Dinoprostone / Q000096:biosynthesis*; D006736:Horses / Q000378:metabolism*; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D008751:Methylene Blue / Q000031:analogs & derivatives* / Q000737:chemistry; D011509:Proteoglycans / Q000378:metabolism*; D011897:Random Allocation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes
D007375:Interleukin-1; D011509:Proteoglycans; D011994:Recombinant Proteins; D013431:Sulfates; D013462:Sulfur Radioisotopes; D004247:DNA; D015232:Dinoprostone; D008751:Methylene Blue; C016401:1,9-dimethylmethylene blue