▪ Abstract At the interface between the innate and adaptive immune systems lies the high-output isoform of nitric oxide synthase (NOS2 or iNOS). This remarkable molecular machine requires at least 17 binding reactions to assemble a functional dimer. Sustained catalysis results from the ability of NOS2 to attach calmodulin without dependence on elevated Ca2+. Expression of NOS2 in macrophages is controlled by cytokines and microbial products, primarily by transcriptional induction. NOS2 has been documented in macrophages from human, horse, cow, goat, sheep, rat, mouse, and chicken. Human NOS2 is most readily observed in monocytes or macrophages from patients with infectious or inflammatory diseases. Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, fungi, protozoa, helminths, and tumor cells. The antimicrobial and cytotoxic actions of NO are enhanced by other macrophage products such as acid, glutathione, cysteine, hydrogen peroxide, or superoxid...

Nitric oxide and macrophage function

This review summarizes recent evidence from knock-out mice on the role of reactive oxygen intermediates and reactive nitrogen intermediates (RNI) in mammalian immunity. Reflections on redundancy in immunity help explain an apparent paradox: the phagocyte oxidase and inducible nitric oxide synthase are each nonredundant, and yet also mutually redundant, in host defense. In combination, the contribution of these two enzymes appears to be greater than previously appreciated. The remainder of this review focuses on a relatively new field, the basis of microbial resistance to RNI. Experimental tuberculosis provides an important example of an extended, dynamic balance between host and pathogen in which RNI play a major role. In diseases such as tuberculosis, a molecular understanding of host-pathogen interactions requires characterization of the defenses used by microbes against RNI, analogous to our understanding of defenses against reactive oxygen intermediates. Genetic and biochemical approaches have identified candidates for RNI-resistance genes in Mycobacterium tuberculosis and other pathogens.

https://www.pnas.org/content/pnas/97/16/8841.full.pdf

Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens

The prototypical xanthine oxidase (XO) inhibitor allopurinol, has been the cornerstone of the clinical management of gout and conditions associated with hyperuricemia for several decades. More recent data indicate that XO also plays an important role in various forms of ischemic and other types of tissue and vascular injuries, inflammatory diseases, and chronic heart failure. Allopurinol and its active metabolite oxypurinol showed considerable promise in the treatment of these conditions both in experimental animals and in small-scale human clinical trials. Although some of the beneficial effects of these compounds may be unrelated to the inhibition of the XO, the encouraging findings rekindled significant interest in the development of additional, novel series of XO inhibitors for various therapeutic indications. Here we present a critical overview of the effects of XO inhibitors in various pathophysiological conditions and also review the various emerging therapeutic strategies offered by this approach.

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Therapeutic Effects of Xanthine Oxidase Inhibitors: Renaissance Half a Century after the Discovery of Allopurinol

Summary: Macrolides have diverse biological activities and an ability to modulate inflammation and immunity in eukaryotes without affecting homeostatic immunity. These properties have led to their long-term use in treating neutrophil-dominated inflammation in diffuse panbronchiolitis, bronchiectasis, rhinosinusitis, and cystic fibrosis. These immunomodulatory activities appear to be polymodal, but evidence suggests that many of these effects are due to inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and nuclear factor kappa B (NF-κB) activation. Macrolides accumulate within cells, suggesting that they may associate with receptors or carriers responsible for the regulation of cell cycle and immunity. A concern is that long-term use of macrolides increases the emergence of antimicrobial resistance. Nonantimicrobial macrolides are now in development as potential immunomodulatory therapies.

Mechanisms of Action and Clinical Application of Macrolides as Immunomodulatory Medications

The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% of bronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2.

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Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis.

We evaluated various biochemical parameters in influenza virus-infected mice and focused on adenosine catabolism in the supernatant of bronchoalveolar lavage fluid (s-BALF), lung tissue, and serum (plasma). The activities of adenosine deaminase (ADA) and xanthine oxidase (XO), which generates O2-, were elevated in the s-BALF, lung tissue homogenate, and serum (plasma). The elevations were most remarkable in s-BALF and in lung tissue: We found a 170-fold increase in ADA activity and a 400-fold increase in XO activity as measured per volume of alveolar lavage fluid. The ratio of activity of XO to activity of xanthine dehydrogenase in s-BALF increased from 0.15 +/- 0.05 (control; no infection) to 1.06 +/- 0.13 on day 6 after viral infection. Increased levels of various adenosine catabolites (i.e., inosine, hypoxanthine, xanthine, and uric acid) in serum and s-BALF were confirmed. We also identified O2- generation from XO in s-BALF obtained on days 6 and 8 after infection, and the generation of O2- was enhanced remarkably in the presence of adenosine. Lastly, treatment with allopurinol (an inhibitor of XO) and with chemically modified superoxide dismutase (a scavenger of O2-) improved the survival rate of influenza virus-infected mice. These results indicate that generation of oxygen-free radicals by XO, coupled with catabolic supply of hypoxanthine from adenosine catabolism, is a pathogenic principle in influenza virus infection in mice and that a therapeutic approach by elimination of oxygen radicals thus seems possible.

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Dependence on O2- generation by xanthine oxidase of pathogenesis of influenza virus infection in mice.

Erythromycin (EM) is an antibiotic with potent antiinflammatory effects that is used for treating chronic lower respiratory tract infections. It has been shown that free radicals, such as the superoxide anion and nitric oxide (NO), are pathogenic molecules in viral disease. Much attention has been given to a critical role of NO in the pathologic events of various inflammatory diseases. In the present study, we evaluated the effects of EM on influenza-virus-induced pneumonia in mice infected with a lethal dose of influenza virus A/Kumamoto/Y5/67 (H2N2). The administration of EM at a dose of 3.3 mg/kg/d (intraperitoneally, from Days 1 to 6 after infection), significantly improved the survival rate of mice infected with influenza virus, and the survival rate of the virus-infected mice at Day 20 after infection increased in a dose-dependent fashion with EM administered to the animals, from 14% among controls to 42% among animals given EM at 1.0 mg/kg/d and 57% among those given EM at 3.3 mg/kg/d. The inductio...

https://www.atsjournals.org/doi/pdf/10.1164/ajrccm.157.3.9703098

Therapeutic effect of erythromycin on influenza virus-induced lung injury in mice

Susceptibility of three different strains of Mycobacterium avium complex (MAC), i.e., one strain of M. avium (Mino) and two strains of M. intracellulare (31F093T and KUMS 9007), to nitric oxide (NO) generated by rat alveolar macrophages (M phi) or NO generated chemically by acidification of NO2- was examined in vitro. We also investigated the effects of NO on phagocytosis and superoxide anion (O2-) generation by M phi. The intracellular growth of M. avium Mino was significantly suppressed by NO generated by gamma interferon (IFN-gamma)-stimulated M phi, whereas that of two strains of M. intracellulare (31F093T and KUMS 9007) was not. M. avium Mino was also more susceptible to NO generated chemically by acidification of NO2- than the two M. intracellulare strains. In L-arginine (1 mM)-containing medium, NO release from the M phi assessed by measuring NO2- increased as the concentration of IFN-gamma increased. The enhancing potential of IFN-gamma for NO release became more pronounced when M phi were infected with 31F093T, an NO-resistant strain. A large amount of NO generated by IFN-gamma-stimulated M phi suppressed both phagocytosis and O2- generation by the M phi, especially after infection of the M phi with strain 31F093T. These results indicate that the intracellular growth of MAC is not always inhibited by NO generated by immunologically activated M phi; rather, NO generation induced by infection with an NO-resistant MAC strain suppresses phagocytosis of the M phi, which may allow extracellular spreading of such NO-resistant mycobacteria. Therefore, the pathogenic potential of MAC may be partly attributed to its resistance to NO.

Resistance to nitric oxide in Mycobacterium avium complex and its implication in pathogenesis.

The antimicrobial action of .NO against Cryptococcus neoformans was investigated by using imidazolineoxyl N-oxide, which we recently reported removes .NO via oxidation (T. Akaike, M. Yoshida, Y. Miyamoto, K. Sato, M. Kohno, K. Sasamoto, K. Miyazaki, S. Ueda, and H. Maeda, Biochemistry 32:827-832, 1993). No appreciable fungicidal activity was observed in neutral .NO solutions. Imidazolineoxyl N-oxide induced or enhanced fungicidal action in neutral or acidic .NO solutions, respectively. Our results provide convincing evidence that .NO is not a microbicidal molecular species.

Pronounced enhancement of .NO-dependent antimicrobial action by an .NO-oxidizing agent, imidazolineoxyl N-oxide.

To clarify the intracellular killing mechanisms of alveolar macrophages against Mycobacterium avium complex, effects of cytokines on O2- and NO2- production from normal and BCG-induced alveolar macrophages were studied. Intracellular growth of M. avium complex was inhibited in the alveolar macrophages stimulated by TNF, but not IFN. Enhancement of O2- production by normal alveolar macrophages stimulated by cytokines, was associated with the inhibition of intracellular growth of M. avium complex. However, when NO2- production by the alveolar macrophages was enhanced by the stimulating of IFN or IFN+TNF, in the presence L-arginine in the culture medium, their defense activity against M. avium complex decreased.

T Doi

Papers

Dependence on O2- generation by xanthine oxidase of pathogenesis of influenza virus infection in mice.

Therapeutic effect of erythromycin on influenza virus-induced lung injury in mice

Resistance to nitric oxide in Mycobacterium avium complex and its implication in pathogenesis.

Pronounced enhancement of .NO-dependent antimicrobial action by an .NO-oxidizing agent, imidazolineoxyl N-oxide.

Intracellular killing mechanisms of alveolar macrophages against Mycobacterium avium complex