Endotoxin-induced production of interleukin 6 by equine peritoneal macrophages in vitro

Debra Deem Morris From the Departments of Large Animal Medicine (Morris, Crowe, Moore) and Physiology and Pharmacology (Morris, Moore), College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7385; and the Department of Surgery (Moldawer), Cornell University Medical College, New York, NY 10021.

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Natalie Crowe From the Departments of Large Animal Medicine (Morris, Crowe, Moore) and Physiology and Pharmacology (Morris, Moore), College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7385; and the Department of Surgery (Moldawer), Cornell University Medical College, New York, NY 10021.

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James N. Moore From the Departments of Large Animal Medicine (Morris, Crowe, Moore) and Physiology and Pharmacology (Morris, Moore), College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7385; and the Department of Surgery (Moldawer), Cornell University Medical College, New York, NY 10021.

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Lyle L. Moldawer From the Departments of Large Animal Medicine (Morris, Crowe, Moore) and Physiology and Pharmacology (Morris, Moore), College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7385; and the Department of Surgery (Moldawer), Cornell University Medical College, New York, NY 10021.

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Summary

A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (il-6) in vitro in response to endotoxin. Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages. Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, or 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5). Culture medium supernatants were collected after 3, 6, 12, and 24 hours’ incubation and were frozen at −70 C until assayed for il-6 activity. Supernatant il-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B 13.29 clone B.9, which is dependent on il-6 for survival.

Results indicated that equine peritoneal macrophages produce il-6 in vitro and that supernatant medium il-6 activity was significantly (P < 0.05) increased by exposure to endotoxin. Significant (P < 0.05) time and treatment effects on macrophage il-6 production were apparent. The il-6 activity peaked at 6 or 12 hours’ incubation, then remained high through 24 hours’ incubation, regardless of endotoxin exposure. Medium il-6 activity during 3 and 6 hours’ incubation was significantly (P < 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak il-6 activity was similar among all endotoxin concentrations. Endotoxin concentration did not have an effect on medium il-6 activity from macrophages exposed to endotoxin for 12 or 24 hours.

Summary

A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (il-6) in vitro in response to endotoxin. Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages. Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, or 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5). Culture medium supernatants were collected after 3, 6, 12, and 24 hours’ incubation and were frozen at −70 C until assayed for il-6 activity. Supernatant il-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B 13.29 clone B.9, which is dependent on il-6 for survival.

Results indicated that equine peritoneal macrophages produce il-6 in vitro and that supernatant medium il-6 activity was significantly (P < 0.05) increased by exposure to endotoxin. Significant (P < 0.05) time and treatment effects on macrophage il-6 production were apparent. The il-6 activity peaked at 6 or 12 hours’ incubation, then remained high through 24 hours’ incubation, regardless of endotoxin exposure. Medium il-6 activity during 3 and 6 hours’ incubation was significantly (P < 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak il-6 activity was similar among all endotoxin concentrations. Endotoxin concentration did not have an effect on medium il-6 activity from macrophages exposed to endotoxin for 12 or 24 hours.

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