Effect of bacterial lipopolysaccharides on sulfated glycosaminoglycan metabolism and prostaglandin E2 synthesis in equine cartilage explant cultures

Melinda H. MacDonald From the Departments of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine (MacDonald, Stover, Benton), and Division of Statistics (Willits), University of California-Davis, Davis, CA 95616.

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Susan M. Stover From the Departments of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine (MacDonald, Stover, Benton), and Division of Statistics (Willits), University of California-Davis, Davis, CA 95616.

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Neil H. Willits From the Departments of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine (MacDonald, Stover, Benton), and Division of Statistics (Willits), University of California-Davis, Davis, CA 95616.

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Hilary P. Benton From the Departments of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine (MacDonald, Stover, Benton), and Division of Statistics (Willits), University of California-Davis, Davis, CA 95616.

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Summary

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (lps) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of lps from 0 (control) to 100 μg/ml. Glycosaminoglycan (gag) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-di-methylmethylene blue. Newly synthesized gag content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (pge2) synthesis was quantified, using a [3H]pge2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to lps, and explants from 1 horse were used to compare responses to stimulation with lps derived from 2 bacterial sources.

Equine explants cultured with bacterial lps had a dose-dependent decrease in synthesis and increase in release of gag, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of pge2 released into the culture medium in response to incubation with lps.

Comparison of data for gag synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to lps between the 2 species. Equine explants tended to have a greater suppression of gag synthesis in response to incubation with increasing concentrations of lps than did age-corrected bovine samples. However, similar analysis of data on gag release did not indicate any difference in sensitivity between the 2 species for this response. There was no evidence that the presence or absence of serum supplementation or the use of lps derived from different bacterial sources made a significant difference in the response of explants to incubation with lps.

Summary

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (lps) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of lps from 0 (control) to 100 μg/ml. Glycosaminoglycan (gag) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-di-methylmethylene blue. Newly synthesized gag content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (pge2) synthesis was quantified, using a [3H]pge2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to lps, and explants from 1 horse were used to compare responses to stimulation with lps derived from 2 bacterial sources.

Equine explants cultured with bacterial lps had a dose-dependent decrease in synthesis and increase in release of gag, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of pge2 released into the culture medium in response to incubation with lps.

Comparison of data for gag synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to lps between the 2 species. Equine explants tended to have a greater suppression of gag synthesis in response to incubation with increasing concentrations of lps than did age-corrected bovine samples. However, similar analysis of data on gag release did not indicate any difference in sensitivity between the 2 species for this response. There was no evidence that the presence or absence of serum supplementation or the use of lps derived from different bacterial sources made a significant difference in the response of explants to incubation with lps.

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