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 E2 (PGE2) concentrations.


METHODS
Freshly isolated porcine alveolar macrophages.


METHODS
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 (TNFalpha; 500 U/mL), interferon-gamma (IFN-gamma, 100 U/mL), or lipopolysaccharide (LPS; 10 microg/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 microM), a COX-2 inhibitor (10 microM); 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-gamma synergistically induced NO production. Inhibition of iNOS and COX-2 decreased NO and PGE2 production, respectively.


CONCLUSIONS
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.
Cho
Wan-Seob
WS
Department of Veterinary Pathology, College of Veterinary Medicine, Building No. 85-608, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu 151-742, Seoul, Republic of Korea.
Chae
Chanhee
C
D016977:Actinobacillus pleuropneumoniae / Q000201:enzymology* / Q000378:metabolism*; D000818:Animals; D003124:Colorimetry; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D016676:Macrophages, Alveolar / Q000737:chemistry / Q000201:enzymology*; D009569:Nitric Oxide / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D013552:Swine
D007527:Isoenzymes; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone