Surfactant protein A mediates mycoplasmacidal activity of alveolar macrophages by production of peroxynitrite

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Abstract
We have previously shown that surfactant protein A (SP-A) mediates in vitro killing of mycoplasmas by alveolar macrophages (AMs) from resistant C57BL/6 mice through a nitric oxide (⋅NO)-dependent mechanism. Herein, SP-A-deficient [SP-A(−/−)] and inducible ⋅NO synthase-deficient [iNOS(−/−)] mice were infected intranasally with 105 or 107 colony-forming units of Mycoplasma pulmonis. SP-A(−/−) mice were as susceptible to mycoplasmal infection as highly susceptible C3H/He mice, and far more susceptible than resistant C57BL/6 mice. iNOS(−/−) mice had significantly greater numbers of mycoplasmas and severity of lung lesions than iNOS(+/+) controls. In vitro, AMs isolated from C57BL/6 mice, activated with IFN-γ, incubated with SP-A (25 μg/ml), and infected with 1010 colony-forming units of M. pulmonis, killed mycoplasmas within 6 h. Mycoplasmal killing was abrogated by 1,000 units/ml of copper-zinc superoxide dismutase. In the absence of AMs, incubation of M. pulmonis with the peroxynitrite generator 3-morpholinosynodiomine⋅HCl (SIN-1) effected complete killing of mycoplasmas by 90 min in a dose-dependent manner. Addition of copper-zinc superoxide dismutase (3,000 units/ml), which converts SIN-1 to a ⋅NO donor, prevented this killing. Neither of the reactive oxygen species generated by xanthine oxidase (10 milliunits/ml, plus 500 μM xanthine and 100 μM FeCl3), nor ⋅NO generated by 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA NONOate) (100 μM) killed mycoplasmas. These data establish that peroxynitrite generation by AMs is necessary for the killing of a pathogen in vitro and in vivo.