Endotoxin induction of nitric oxide synthase and cyclooxygenase-2 in equine alveolar macrophages

R. A. Hammond From the Department of Biochemical Pharmacology, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ (Hammond, Hannon, Flower); the Department of Pharmacology, Royal Veterinary College Field Station, Hawkshead Lane, Hatfield Herts AL9 7TA (Frean, Armstrong); and the Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Rd. Cambridge CB3OES (Bryant), United Kingdom.

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R. Hannon From the Department of Biochemical Pharmacology, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ (Hammond, Hannon, Flower); the Department of Pharmacology, Royal Veterinary College Field Station, Hawkshead Lane, Hatfield Herts AL9 7TA (Frean, Armstrong); and the Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Rd. Cambridge CB3OES (Bryant), United Kingdom.

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S. P. Frean From the Department of Biochemical Pharmacology, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ (Hammond, Hannon, Flower); the Department of Pharmacology, Royal Veterinary College Field Station, Hawkshead Lane, Hatfield Herts AL9 7TA (Frean, Armstrong); and the Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Rd. Cambridge CB3OES (Bryant), United Kingdom.

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S. J. Armstrong From the Department of Biochemical Pharmacology, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ (Hammond, Hannon, Flower); the Department of Pharmacology, Royal Veterinary College Field Station, Hawkshead Lane, Hatfield Herts AL9 7TA (Frean, Armstrong); and the Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Rd. Cambridge CB3OES (Bryant), United Kingdom.

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R. J. Flower From the Department of Biochemical Pharmacology, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ (Hammond, Hannon, Flower); the Department of Pharmacology, Royal Veterinary College Field Station, Hawkshead Lane, Hatfield Herts AL9 7TA (Frean, Armstrong); and the Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Rd. Cambridge CB3OES (Bryant), United Kingdom.

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C. E. Bryant From the Department of Biochemical Pharmacology, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ (Hammond, Hannon, Flower); the Department of Pharmacology, Royal Veterinary College Field Station, Hawkshead Lane, Hatfield Herts AL9 7TA (Frean, Armstrong); and the Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Rd. Cambridge CB3OES (Bryant), United Kingdom.

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Abstract

Objective

To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS).

Sample Population

Alveolar macrophages obtained from 12 horses.

Procedure

Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 µg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively.

Results

Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent.

Conclusion

Lipopolysaccharide induces COX-2 and iNOS in equine macrophages.

Clinical Relevance

The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses. (Am J Vet Res 1999;60:426-431)

Abstract

Objective

To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS).

Sample Population

Alveolar macrophages obtained from 12 horses.

Procedure

Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 µg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively.

Results

Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent.

Conclusion

Lipopolysaccharide induces COX-2 and iNOS in equine macrophages.

Clinical Relevance

The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses. (Am J Vet Res 1999;60:426-431)

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