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

Abstract

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 E2 (PGE2) concentrations. Sample Population - Freshly isolated porcine alveolar macrophages. Procedure - Alveolar macrophages were incubated for 48 hours with APP (1 × 104 colony-forming units/mL), interleukin-1β (IL-1β; 5 U/mL), tumor necrosis factor-α (TNF-α; 500 U/mL), interferon-γ (IFN-γ; 100 U/mL), or lipopolysaccharide (LPS; 10 μg/mL). In a second experiment, alveolar macrophages were incubated with fresh medium (negative control), APP alone, or APP with 1 of the following: IL-1β, TNF-α, or IFN-γ. 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 (3.3μM), a COX-2 inhibitor (10μM); 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 PGE2 production. Results - The addition of APP to alveolar macrophages resulted in significant increases in NO and PGE2 production. The addition of APP and IFN-γ synergistically induced NO production. Inhibition of iNOS and COX-2 decreased NO and PGE2 production, respectively. Conclusions and Clinical Relevance - In vitro activation of alveolar macrophages by APP results in increased production of NO and PGE2. Nitric oxide and PGE2 production appears to be largely dependent on iNOS and COX-2 activity. Pharmacologic modulation of iNOS and COX-2 activity may represent a therapeutic target for pigs with pleuropneumonia.