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

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 (⅐NO)-dependent mechanism. Herein, SP-A-deficient [SP-A(؊/؊)] and inducible ⅐NO synthasedeficient [iNOS(؊/؊)] mice were infected intranasally with 10 5 or 10 7 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-␥, incubated with SP-A (25 g/ml), and infected with 10 10 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-morpholinosynodiomine⅐HCl (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 ⅐NO donor, prevented this killing. Neither of the reactive oxygen species generated by xanthine oxidase (10 milliunits/ ml, plus 500 M xanthine and 100 M FeCl 3 ), nor ⅐NO generated by 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA NONOate) (100 M) killed mycoplasmas. These data establish that peroxynitrite generation by AMs is necessary for the killing of a pathogen in vitro and in vivo.

Sequential targeted deficiency of SP-A and -D leads to progressive alveolar lipoproteinosis and emphysema

“…Although it is possible compensatory adaptations to SP-A deficiency may obscure an important role in surfactant function, these mice have been useful in confirming a role for SP-A in pulmonary host defense against multiple microbes. SP-A-deficient mice clear pathogens from the lung slowly and have an exaggerated inflammatory response to microbial challenge (2,13,19,20), consistent with numerous in vitro studies (38). Mice deficient in SP-D display progressive postnatal inflammation without evidence for infection (3,18).…”

Peptide methionine sulfoxide reductase fromEscherichia coliandMycobacterium tuberculosisprotects bacteria against oxidative damage from reactive nitrogen intermediates

“…Peroxynitrite is emerging as a major toxin among RNI and ROI, not only toward microbes but also toward mammalian cells (4,(30)(31)(32). Studies of the oxidative damage of proteins by peroxynitrite have focused on nitration of tyrosine residues and, to a lesser extent, oxidation of cysteines.…”

Surfactant Dysfunction in SP-A−/− and iNOS−/− Mice with Mycoplasma Infection

“…As a member of the collectin family of host defense proteins, SP-A also contributes to innate pulmonary immunity (11)(12)(13). We have previously studied M. pulmonis infection in B6 mice and showed that SP-A is necessary for maximal mycoplasma killing (4,14,15) by AM by upregulating the production of NO and reactive oxygen-nitrogen intermediates in these cells (14,15). Both SP-A Ϫ/Ϫ and iNOS Ϫ/Ϫ mice have a decreased ability to clear intratracheally instilled mycoplasmas.…”

“…Mice were bred at the University of Alabama at Birmingham (UAB), and genotype was characterized from tail DNA as described previously (15). C57BL (C57BL/6J-Nos2 tm1Lau ) transgenic mice lacking the gene for iNOS (B6.iNOS Ϫ/Ϫ ) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) (14) and were bred at UAB in autoclaved microisolator cages (Lab Products, Maywood, NJ). Mice of both sexes were used, and animals were maintained in a sterile environment and provided with autoclaved food (Agway, Syracuse, NY) and water ad libitum until being studied at 8-12 wk of age (20-25 g body weight).…”