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

Objective-To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar m acrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses. Procedure-Alveolar macrophages were collected by bronchoalveolar lavage fro m 12 horses and incubated for 6 hours with LPS (0.001 to 10 mu g/ml) or veh icle. Total RNA was extracted and purified. After first-strand cDNA synthes is, mRNA induction was measured, using a polymerase chain reaction (PCR) te chnique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Cultu re medium was assayed for COX-2 and iNOS activity by determining prostaglan din E-2 (PGE(2)) and total nitrite concentrations, respectively. Results-Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar m acrophages. 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 f or iNOS appeared to be less than that for COX-2. Induction of COX-2, but no t 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 macroph ages suggests these enzymes may be important in the pathophysiology of seps is. Pharmacologic modulation of iNOS and COX-2 activity may represent a nov el therapeutic target in the management of endotoxemia in horses.