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

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.

Secretory leukocyte protease inhibitor, an inhibitor of neutrophil activation, is elevated in serum in human sepsis and experimental endotoxemia.

OBJECTIVES: To document changes in serum secretory leukocyte protease inhibitor (SLPI) in human sepsis and in experimental endotoxemia in vivo. To compare changes in serum SLPI in human sepsis with changes in interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha. To determine whether or not changes in SLPI correlate with the severity of multiple organ dysfunction syndrome as measured by the maximal multiple organ dysfunction score. Finally, because neutrophils have been implicated in tissue injury associated with organ dysfunction, to determine whether recombinant human SLPI blocks activation of isolated human neutrophils. DESIGN: Case-control study and ex-vivo cellular assay. SETTING: Surgical intensive care unit and clinical research center of university hospitals; laboratory of a medical school. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: There was a significant dose-dependent elevation (50.2+/-4.0 ng/mL, p = .01) in plasma SLPI 12 hrs after administration of lipopolysaccharide to seven healthy adults (36.4+/-2.3 ng/mL). Further, serum concentrations of SLPI (132+/-15 ng/mL) were elevated in septic surgical patients compared with healthy controls (43+/-2 ng/mL, p < .01) and nonseptic surgical controls (69+/-10 ng/mL, p = .01). Serum SLPI concentrations correlated (r2 = .71, p < .01) better with organ dysfunction as measured by maximal multiple organ dysfunction score than did serum IL-6 (r2 = .49, p < .01), IL-10 (r2 = .05, p = .22), or TNF-alpha (r2 = .02, p = .44). We found that recombinant human SLPI in vitro inhibits TNF-alpha-induced hydrogen peroxide production by human neutrophils (ID50 = 1-2 microg/mL). CONCLUSIONS: Serum SLPI is elevated in human sepsis and experimental endotoxemia. Maximal concentrations of serum SLPI correlate significantly with maximal multiple organ dysfunction scores in patients with sepsis. Secretory leukocyte protease inhibitor may function to limit ongoing neutrophil-mediated tissue injury associated with organ dysfunction.

Reactive nitrogen intermediates and the pathogenesis of Salmonella and mycobacteria.

Over the past decade, reactive nitrogen intermediates joined reactive oxygen intermediates as a biochemically parallel and functionally non-redundant pathway for mammalian host resistance to many microbial pathogens. The past year has brought a new appreciation that these two pathways are partially redundant, such that each can compensate in part for the absence of the other. In combination, their importance to defense of the murine host is greater than previously appreciated. In addition to direct microbicidal actions, reactive nitrogen intermediates have immunoregulatory effects relevant to the control of infection. Genes have been characterized in Mycobacterium tuberculosis and Salmonella typhimurium that may regulate the ability of pathogens to resist reactive nitrogen and oxygen intermediates produced by activated macrophages.

Lethality of endotoxin in mice genetically deficient in the respiratory burst oxidase, inducible nitric oxide synthase, or both.

Two classes of oxidants are thought to play a critical role in tissue damage in septic shock: reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Particular importance has been ascribed to peroxynitrite, a product arising from the reaction of nitric oxide with superoxide. A major source of ROI is the respiratory burst oxidase of neutrophils, eosinophils, monocytes, and macrophages. A major source of RNI is inducible nitric oxide synthase (iNOS), an enzyme expressed in leukocytes, hepatocytes, vascular smooth muscle cells, endothelium, and cardiac myocytes during inflammation. In previous studies using various mouse models of endotoxic shock, genetic deficiency of iNOS as a sole intervention did not consistently alter survival. Here, using Salmonella typhimurium endotoxic bacterial lipopolysaccharide (LPS) as a sole challenge, genetic deficiency of iNOS was associated with no protection or a reduction in survival, depending on the dose of LPS. Further, no protection from lethality was observed when LPS was injected into mice genetically deficient in the 91 kDa subunit of the respiratory burst oxidase (gp91phox) nor in mice genetically deficient in both gp91phox and iNOS (gp91phox-/-/NOS2-/- mice). For the latter experiments, mice were challenged either with S. typhimurium LPS alone or with inactivated bacille Calmette-Guerin (BCG) followed by Escherichia coli LPS. Deficiency of gp91phox impaired the inflammatory response to inactivated Propionobacterium acnes, rendering survival studies following priming with P. acnes difficult to interpret. Thus, in two models of endotoxic shock, major reductions in the ability to form nitric oxide or superoxide, alone or in combination, failed to improve survival.

Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase.

The two genetically established antimicrobial mechanisms of macrophages are production of reactive oxygen intermediates by phagocyte oxidase (phox) and reactive nitrogen intermediates by inducible nitric oxide synthase (NOS2). Mice doubly deficient in both enzymes (gp91(phox-/-)/NOS2(-/-)) formed massive abscesses containing commensal organisms, mostly enteric bacteria, even when reared under specific pathogen-free conditions with antibiotics. Neither parental strain showed such infections. Thus, phox and NOS2 appear to compensate for each other's deficiency in providing resistance to indigenous bacteria, and no other pathway does so fully. Macrophages from gp91(phox-/-)/NOS2(-/-) mice could not kill virulent Listeria. Their killing of S. typhimurium, E. coli, and attenuated Listeria was markedly diminished but demonstrable, establishing the existence of a mechanism of macrophage antibacterial activity independent of phox and NOS2.

Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay.

To compare antibacterial function in macrophages from mice deficient in the respiratory burst oxidase or inducible nitric oxide synthase, we developed a fluorescence-based microplate assay of bacterial survival. As bacteria grow, they convert a formulation of resazurin termed AlamarBlue from its nonfluorescent oxidized state to its fluorescent reduced state. The time required to achieve a given fluorescence is inversely proportional to the number of viable bacteria present when the dye is added. This relationship allows a precise, accurate assessment of bacterial numbers with greater sensitivity and throughput and at less cost than conventional assays. The assay facilitated quantification of the killing of Escherichia coli by S-nitrosoglutathione and hydrogen peroxide and of Salmonella typhimurium by human neutrophils and mouse macrophages. Mouse macrophages lacking the 91-kDa subunit of the respiratory burst oxidase were deficient in their ability to kill S. typhimurium, while those lacking inducible nitric oxide synthase were unimpaired.

Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase.

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.

A novel antioxidant gene from Mycobacterium tuberculosis.

Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.