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.

We generated congenic surfactant protein A (SP-A)-deficient (SP-A[-/-]) mice on the mycoplasma resistant C57BL/6 background (B6.SP-A[-/-]) and characterized their response to mycoplasma infection in comparison to C57BL/6 (B6) mice. B6.SP-A(-/-) mice infected with 10(6) colony-forming units (cfu) of Mycoplasma pulmonis had significantly higher bacterial lung loads than B6 mice at 72 h postinfection (p.i.). At the higher infection dose of 10(7), B6.SP-A(-/-) mice had significantly higher lung cfu at 24 h; however, no difference in mycoplasma cfu was observed between B6 and B6.SP-A(-/-) mice at 48 and 72 h p.i. We found that uninfected B6 mice had lower bronchoalveolar lavage nitrite (NO(2)(-)) and nitrate (NO(3)(-)) levels as compared with B6.SP-A(-/-) mice. On the other hand, infection of B6 mice with mycoplasmas resulted in significantly higher bronchoalveolar lavage NO(2)(-) and NO(3)(-) as compared with B6.SP-A(-/-) mice. These data indicate that SP-A may help regulate NO production in response to a specific stimulus, i.e., suppression of NO in the absence of bacteria and increased NO in the presence of bacteria. These data indicate that the contribution of SP-A to mycoplasma killing may be limited to lower doses of pathogens.

Current evidence suggests that host defense in respiratory mycoplasmosis is dependent on both innate and humoral immunity. To further delineate the roles of innate and adaptive immunity in antimycoplasmal defenses, we intranasally infected C3H/HeSnJ-scid/scid (C3H-SCID), C3H/HeSnJ (C3H), C57BL/6J-scid/scid (C57-SCID), and C57BL/6N (C57BL) mice with Mycoplasma pulmonis and at 14 and 21 days postinfection performed quantitative cultures of lungs and spleens, quantification of lung lesions, and histopathologic assessments of all other major organs. We found that numbers of mycoplasmas in lungs were associated with genetic background (C3H susceptible, C57BL resistant) rather than functional state of adaptive immunity, indicating that innate immunity is the main contributor to antimycoplasmal defense of the lungs. Extrapulmonary dissemination of mycoplasmas with colonization of spleens and histologic lesions in multiple organs was a common occurrence in all mice. The absence of adaptive immune responses in severe combined immunodeficient (SCID) mice resulted in increased mycoplasmal colonization of spleens and lesions in extrapulmonary sites, particularly spleens, hearts, and joints, and also reduced lung lesion severity. The transfer of anti-M. pulmonis serum to infected C3H-SCID mice prevented extrapulmonary infection and disease, while the severity of lung lesions was restored by transfer of naive spleen cells to infected C3H-SCID mice. Collectively, our results strongly support the conclusions that innate immunity provides antimycoplasmal defense of the lungs and humoral immunity has the major role in defense against systemic dissemination of mycoplasmal infection, but cellular immune responses may be important in exacerbation of mycoplasmal lung disease.

Indirect evidence suggests that innate immune mechanisms involving alveolar macrophages (AMs) are of major importance in antimycoplasmal defense. We compared the effects of AM depletion on intrapulmonary killing of Mycoplasma pulmonis during the early phase of infection in mycoplasma-resistant C57BL/6NCr (C57BL) and mycoplasma-susceptible C3H/HeNCr (C3H) mice. More than 80% of AMs were depleted in both strains of mice by intratracheal insufflation of liposome-encapsulated dichloromethylene bisphosphonate (L-Cl 2 MBP), compared to no significant AM depletion in either strain following insufflation of liposomeencapsulated phosphate-buffered saline (L-PBS), PBS alone, or no treatment. AM-depleted (L-Cl 2 MBP) and control (L-PBS) mice were infected intranasally with 10 5 CFU of M. pulmonis UAB CT, and their lungs were quantitatively cultured to assess intrapulmonary killing at 0, 8, 12, and 48 h postinfection. AM depletion exacerbated the infection in C57BL mice by reducing killing of the organism to a level comparable to that in C3H mice without AM depletion. In contrast, AM depletion did not alter killing in C3H mice. These results directly identify the AM as the main effector cell in early pulmonary antimycoplasmal defense and suggest that differences in mycoplasmal killing by AMs may explain the resistance of C57BL mice and the susceptibility of C3H mice to mycoplasmal infection.