Upregulation of immunoproteasomes by nitric oxide: potential antioxidative mechanism in endothelial cells.

Nitric oxide (*NO) was shown to stimulate the proteasomal function and the ubiquitin-proteasome pathway and to ameliorate endothelial apoptotic signaling induced by oxidants. Understanding the regulatory mechanisms by which *NO stimulates proteasomes and affords cytoprotection in endothelial cells has therapeutic implications, as many vascular diseases are characterized by a deficiency in *NO. Here we report that *NO/cGMP/cAMP-induced immunoproteasome subunit expression is responsible for the increased proteasomal activities. Cells pretreated with protein kinase G and protein kinase A inhibitors markedly attenuated *NO-dependent proteasome activation. Results show that the *NO/cGMP/cAMP signaling mechanism enhanced the phosphorylation of the transcription factor cAMP-response element-binding protein, elevated the cAMP-response element-promoter activity and induced the expression of immunoproteasomal subunits (LMP2 and LMP7). *NO-dependent proteasomal activity was abrogated in cells transfected with antisense LMP2 and LMP7 oligonucleotides. Lower levels of LMP2 and LMP7 were detected in aorta of iNOS(-/-) mice compared to wild-type controls, suggesting that endogenous production of *NO is important in the basal regulation of immunoproteasome. The *NO/cGMP/cAMP signaling pathway mitigates transferrin-iron-mediated oxidative stress and apoptosis through induction of immunoproteasomes. These results provide new insights on the regulatory mechanisms by which the *NO-mediated immunoproteasome signaling pathway affords cytoprotection in endothelial cells.

Cyclophosphamide decreases nitrotyrosine formation and inhibits nitric oxide production by alveolar macrophages in mycoplasmosis.

We previously reported that congenic C57BL/6 inducible nitric oxide synthase(-/-) (iNOS(-/-)) mice infected with Mycoplasma pulmonis developed higher bacterial numbers and lung lesion scores than C57BL/6 iNOS(+/+) controls but had similar lung nitrotyrosine levels. The present studies investigated the role of inflammatory cells in nitrotyrosine formation during mycoplasmal infection. iNOS(+/+) and iNOS(-/-) mice were injected with cyclophosphamide (CYP) and inoculated with 10(7) CFU of M. pulmonis. CYP pretreatment of M. pulmonis-infected iNOS(+/+) and iNOS(-/-) mice reduced polymorphonuclear cells (PMNs) within bronchoalveolar lavages (BALs) by 88 and 72%, respectively, and whole-lung myeloperoxidase levels by 80 and 78%, respectively, at 72 h postinfection but did not alter the number of alveolar macrophages (AMs) in BALs. CYP treatment also significantly decreased nitrate and nitrite (NOx) levels in BALs and plasma of infected iNOS(+/+) mice, whereas neither CYP nor mycoplasmal infection altered NOx in iNOS(-/-) mice. CYP reduced lung nitrotyrosine levels in both iNOS(+/+) and iNOS(-/-) mice to uninfected-control levels as shown by immunohistochemical staining and enzyme-linked immunosorbent assay and inhibited mycoplasmal killing by iNOS(+/+) mice in vivo. CYP inhibited the production of gamma interferon-inducible NOx by iNOS(+/+) AMs in vitro but did not alter the number of iNOS-positive AMs, as detected by immunocytochemistry. In addition, AMs from CYP-treated iNOS(+/+) mice had significantly decreased ability to kill mycoplasmas in vitro. These results demonstrate that reactive species generated by inflammatory cells as well as PMN myeloperoxidase are important contributors to nitrotyrosine formation during mycoplasmal infection and that treatment with CYP decreases NO* production by AMs and inhibits mycoplasmal killing.

Lung surfactant and reactive oxygen-nitrogen species: antimicrobial activity and host-pathogen interactions.

Surfactant protein (SP) A and SP-D are members of the collectin superfamily. They are widely distributed within the lung, are capable of antigen recognition, and can discern self versus nonself. SPs recognize bacteria, fungi, and viruses by binding mannose and N-acetylglucosamine residues on microbial cell walls. SP-A has been shown to stimulate the respiratory burst as well as nitric oxide synthase expression by alveolar macrophages. Although nitric oxide (NO.) is a well-recognized microbicidal product of macrophages, the mechanism(s) by which NO. contributes to host defense remains undefined. The purpose of this symposium was to present current research pertaining to the specific role of SPs and reactive oxygen-nitrogen species in innate immunity. The symposium focused on the mechanisms of NO*-mediated toxicity for bacterial, human, and animal models of SP-A- and NO.-mediated pathogen killing, microbial defense mechanisms against reactive oxygen-nitrogen species, specific examples and signaling pathways involved in the SP-A-mediated killing of pulmonary pathogens, the structure and binding of SP-A and SP-D to bacterial targets, and the immunoregulatory functions of SP-A.

Implications of mouse genotype for phenotype.

Genetic engineering of inbred mice offers the capability to separate interactions of multiple genes that control host resistance to pathogens. An understanding of the negative impact that genetic variability of mouse substrains can have on data is necessary for the design of experiments to dissect out complicated gene interactions.

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

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.

Surfactant protein A mediates mycoplasmacidal activity of alveolar macrophages.

Mycoplasma pneumoniae is a leading cause of pneumonia and exacerbates other respiratory diseases in humans. We investigated the potential role of surfactant protein (SP) A in antimycoplasmal defense using alveolar macrophages (AMs) from C57BL/6NCr (C57BL) mice, which are highly resistant to infections of Mycoplasma pulmonis. C57BL AMs, activated with interferon (IFN)-gamma and incubated with SP-A (25 micrograms/ml) at 37 degrees C, produced significant amounts of nitric oxide (.NO; nitrate and nitrite production = 1.1 microM.h-1.10(5) AMs-1) and effected an 83% decrease in mycoplasma colony-forming units (CFUs) by 6 h postinfection. Preincubation of AMs with the inducible nitric oxide synthase inhibitor NG-monomethyl-L-arginine abolished .NO production and SP-A-mediated killing of mycoplasmas. No decrease in CFUs was seen when IFN-gamma-activated macrophages were infected with mycoplasmas in the absence of SP-A despite significant .NO production (nitrate and nitrite production = 0.6 microM.h-1.10(5) AMs-1). These results demonstrate that SP-A mediates killing of mycoplasmas by AMs, possibly through an .NO-dependent mechanism.

Depletion of alveolar macrophages exacerbates respiratory mycoplasmosis in mycoplasma-resistant C57BL mice but not mycoplasma-susceptible C3H mice.

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-Cl2MBP), compared to no significant AM depletion in either strain following insufflation of liposome-encapsulated phosphate-buffered saline (L-PBS), PBS alone, or no treatment. AM-depleted (L-Cl2MBP) 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.