In vitro effects of Actinobacillus pleuropneumoniae on inducible nitric oxide synthase and cyclooxygenase-2 in porcine alveolar macrophages

Objective-To determine the amount of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activity in alveolar macrophages in response to Actinobacillus pleuropneumoniae (APP) by determining nitric oxide (NO) and prostaglandin E-2 (PGE(2)) concentrations. Sample Population-Freshly isolated porcine alveolar macrophages. Procedure-Alveolar macrophages were incubated for 48 hours with APP (1 x 10(4) colony-forming units/mL), interleukin-1beta (IL-1beta; 5 U/mL), tumor necrosis factor-alpha (TNF-alpha; 500 U/mL), interferon-gamma (IFN-gamma; 100 U/mL), or lipopolysaccharide (LPS; 10 mug/mL). In a second experiment, alveolar macrophages were incubated with fresh medium (negative control), APP alone, or APP with 1 of the following: IL-1beta TNF-alpha, or IFN-gamma. In a third experiment, alveolar macrophages were incubated with fresh medium (negative control), LPS (positive control), APP alone, or APP with 1 of the following: an iNOS inhibitor (3.3 muM), a COX-2 inhibitor (10 muM); or both the iNOS and COX-2 inhibitors. Supernatant was obtained at 0, 3, 6, 9, 12, 24, and 48 hours after treatment for determination of NO and PGE(2) production. Results-The addition of APP to alveolar macrophages resulted in significant increases in NO and PGE(2) production. The addition of APP and IFN-gamma synergistically induced NO production. Inhibition of iNOS and COX-2 decreased NO and PGE(2) production, respectively. Conclusions and Clinical Relevance-In vitro activation of alveolar macrophages by APP results in increased production of NO and PGE(2). Nitric oxide and PGE(2) production appears to be largely dependent on iNOS and COX-2 activity. Pharmacologic modulation of NOS and COX-2 activity may represent a therapeutic target for pigs with pleuropneumonia.