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. 1999 Apr 27;96(9):4953-8.
doi: 10.1073/pnas.96.9.4953.

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

J Hickman-Davis  1 , J Gibbs-Erwin, J R Lindsey, S Matalon
Affiliations

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

J Hickman-Davis et al. Proc Natl Acad Sci U S A. .

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 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-gamma, incubated with SP-A (25 micrograms/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-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 microM xanthine and 100 microM FeCl3), nor.NO generated by 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA NONOate) (100 microM) killed mycoplasmas. These data establish that peroxynitrite generation by AMs is necessary for the killing of a pathogen in vitro and in vivo.

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Figures

Figure 1
Figure 1
Transgenic SP-A(−/−) mice, mycoplasma-resistant C57BL, and susceptible C3H mice were infected intranasally with 105 cfu of M. pulmonis. Mice were euthanized at 1, 2, 3, or 7 days p.i., and cfu numbers (total recoverable mycoplasmas) were determined on whole-lung homogenates. cfu are graphed on a logarithmic scale. ∗, significant difference from the other two remaining groups at each time point, P < 0.05, #, significant difference from the other two groups at this time point, P < 0.05. Results of quantitative cultures are mean ± SE; n ≥18.
Figure 2
Figure 2
C57BL iNOS(−/−) and control C57BL iNOS(+/+) mice were infected intranasally with 1.5 × 107 cfu per ml M. pulmonis. Mice were euthanized at 1, 2, 3, or 7 days p.i., and cfu numbers (total recoverable mycoplasmas) were determined on whole-lung homogenates. ∗, significant difference between control and experimental conditions at each time point, P < 0.05. Results of quantitative cultures are mean ± SE; n ≥ 18.
Figure 3
Figure 3
Visualization of nitrotyrosine residues and iNOS protein in the lungs of transgenic C57BL iNOS(−/−) and control C57BL iNOS(+/+) mice infected 3 days with 1.5 × 107 cfu per ml M. pulmonis. (A) Nitrotyrosine staining of lungs from resistant C57BL iNOS(+/+) mice. (B) Nitrotyrosine staining from A in the presence of excess nitrotyrosine (10 mM). (C) Nitrotyrosine staining of lungs of C57BL iNOS(−/−) mice. (D) Nitrotyrosine staining from C in the presence of excess nitrotyrosine (10 mM). (E) iNOS staining of BALs from C57BL iNOS(+/+) mice. (F) iNOS staining of BALs C57BL iNOS(−/−) mice. Shown are representative pictures of results, which were reproduced at least twice.
Figure 4
Figure 4
AMs (1 × 105) were activated with 100 units/ml IFN-γ, washed, and incubated with 1,000 units/ml of Cu, ZnSOD. AMs were treated with SP-A (25 μg/ml) or Hepes (5 mM), infected with 1010 cfu of M. pulmonis, and incubated for 0 and 6 h. Results of quantitative cultures are mean ± SE from a total of three experiments with 12–15 data points per group. ∗, significant difference between control and experimental groups at this time point, P <0.05.
Figure 5
Figure 5
M. pulmonis was grown to late logarithmic phase, washed to remove serum, and resuspended in 10 ml of 25 mM Hepes buffer, pH 7.4. Aliquots were taken at 0, 20, 45, 60, and 90 min for determination of cfu. (A) Hepes 25 mM, mycoplasmas alone; SIN-1 1 mM, mycoplasmas + 1 mM SIN-1; SIN-1 200 μM, mycoplasmas + 200 μM SIN-1; SIN-1C, mycoplasmas + 1 mM SIN-1C. (B) cfu vs. ONOO− concentration for the indicated concentrations of SIN-1 as measured by dihydrorhodamine 123 oxidation. (C) Hepes 25 mM, mycoplasmas alone; PAPA 100 μM, mycoplasmas +100 μM PAPANONOate; Cu, ZnSOD 3,000 units/ml, mycoplasmas +1 mM SIN-1 + 3,000 units/ml Cu, ZnSOD.;Cu, ZnSOD 500 units/ml, mycoplasmas +1 mM SIN-1 + 500 units/ml Cu, ZnSOD. ∗, significant difference between control and experimental groups at each time point, P <0.05.
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