Colloquium Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens Carl Nathan* and Michael U. Shiloh Department of Microbiology and Immunology and Department of Medicine, Weill Cornell Medical College and Program in Immunology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021 This review summarizes recent evidence from knock-out mice on recognition, the process by which one genome identifies another, the role of reactive oxygen intermediates and reactive nitrogen affords some of the most impressive examples of specificity in intermediates (RNI) in mammalian immunity. Reflections on redun- biology. When it comes to actually inactivating DNA, however, dancy in immunity help explain an apparent paradox: the phago- specificity is hard to preserve, because the reactions by which cyte oxidase and inducible nitric oxide synthase are each nonre- DNA is built, copied, guarded, and repaired are extensively dundant, and yet also mutually redundant, in host defense. In shared among organisms. combination, the contribution of these two enzymes appears to be Perhaps the most striking example of nonspecificity in the greater than previously appreciated. The remainder of this review immune system is its reliance on the production of chemically focuses on a relatively new field, the basis of microbial resistance reactive micromolecules that do not discriminate the genomic to RNI. Experimental tuberculosis provides an important example source of their chemical targets (Fig. 1). Reactive oxygen of an extended, dynamic balance between host and pathogen in intermediates (ROI) (1) and reactive nitrogen intermediates which RNI play a major role. In diseases such as tuberculosis, a (RNI) (2) can damage DNA and several chemical moieties on molecular understanding of host–pathogen interactions requires which its propagation and protection depend, including Fe-S characterization of the defenses used by microbes against RNI, clusters, tyrosyl radicals, hemes, sulfhydryls, thioethers, and analogous to our understanding of defenses against reactive alkenes. ROI are produced by all aerobic cells and RNI by many. oxygen intermediates. Genetic and biochemical approaches have High output production of ROI is the specialty of mammalian identified candidates for RNI-resistance genes in Mycobacterium phagocytes, with polymorphonuclear leukocytes in the lead and tuberculosis and other pathogens. immunologically activated macrophages capable of generating about 1͞3–1͞2 as much. High output production of RNI is his overview begins by rationalizing the prominence in attainable by many mammalian cells in response to appropriate Timmunity of chemically reactive micromolecules whose inflammatory stimuli, macrophages considerably outpacing polymorphonuclear leukocytes (2). Macrophages have the op- widespread interactions contrast with the exquisite specificity . ⅐ of B and T cell receptors, the signature proteins of the immune portunity to produce superoxide (O2) and nitric oxide ( NO) in system. Next we consider the limitations of asking knock-out nearly equimolar amounts and thus can be prolific generators of mice to tell us what is important in host defense. Interpretation their joint and particularly destructive product, peroxynitrite (OONOϪ) (see Fig. 1 legend) (3–5). RNI of dietary origin are of knock-outs is often based on the premise that the impor- COLLOQUIUM tance of a gene is defined by its nonredundancy. The short- put to use as antimicrobial agents in gastric juice, a key com- comings of this premise can be particularly problematic in ponent of the innate immune system of epithelium (6). immunity. Recent evidence from knock-out mice is weighed, It seems puzzling that evolution has entrenched defenses then measured against a disease of commanding interest, with the potential to damage uninfected cells. However, from tuberculosis. The foregoing analysis prompts a key question: Is the perspective of the whole organism, it is both a privilege and there an enzymatic basis of resistance to reactive nitrogen a necessity of multicellularity to lose parts to save the whole. intermediates (RNI), as there is to reactive oxygen interme- As abscess formation illustrates in mammals, phagocytes are diates (ROI)? [The term “RNI” refers to oxidation states and quick to sacrifice themselves and any volume of host tissue to adducts of the nitrogenous products of nitric oxide synthases, prevent microbial metastasis. Plants meet the same need with ⅐ Ϫ ranging from nitric oxide ( NO) to nitrate (NO3 ), that arise the hypersensitivity reaction, a phagocyte-free version of Ϫ ⅐ Ϫ in physiological environments, including NO , NO2,NO2 , abscess formation that depends on production of ROI and Ϫ N2O3,N2O4, S-nitrosothiols, peroxynitrite (OONO ), and RNI (7–10). dinitrosyl-iron complexes. The term “ROI” refers to interme- A downside of highly specific recognition as a pillar of the diate reduction products of O en route to water, namely, immune response is that a microbe can sometimes escape . 2 superoxide (O2) hydrogen peroxide (H2O2), and hydroxyl recognition by altering a molecular feature that flags it, such as radical (OH⅐), and reactive products of these with halides and the order of monomers in its polymers. Conversely, the advan- amines.] Answers are beginning to emerge for both microbes tage of using ROI and RNI for defense is that a microbe cannot and mammalian cells. Here we survey findings in microbes, in an effort to help lay the groundwork for molecular control over This paper was presented at the National Academy of Sciences colloquium ‘‘Virulence and one important facet of host–pathogen relationships. Defense in Host–Pathogen Interactions: Common Features Between Plants and Animals,’’ held December 9–11, 1999, at the Arnold and Mabel Beckman Center in Irvine, CA. Chemically Reactive Micromolecules in Host Defense Abbreviations: NOS, nitric oxide synthase; phox, phagocyte oxidase; ROI, reactive oxygen The Uses of Nonspecificity. Immunity is the ability of the largest intermediates; RNI, reactive nitrogen intermediates; SOD, superoxide dismutase; STM, inherited genome in a body to restrict the replication of the signature-tagged transposon mutagenesis. smaller, noninherited genomes in the same body. Immune *To whom reprint requests should be addressed. E-mail: [email protected]. PNAS ͉ August 1, 2000 ͉ vol. 97 ͉ no. 16 ͉ 8841–8848 Downloaded by guest on September 27, 2021 Table 1. Antimicrobial products of human phagocytes that are delivered to phagosomes Product Neutrophils* Macrophages† ROI ϩϩ RNI ϩϩ Myeloperoxidase ϩϪ Lactoferrin ϩϪ Bacterial permeability increasing factor ϩϪ Serprocidins (elastase, cathepsin G, ϩϪ protease 3, azurocidin) Phospholipase A2 ϩϪ Cathelicidin ϩϪ Lysozyme ϩϪ Defensins (HNPs) 1, 2, 3, 4 ϩϪ Fig. 1. ROI and RNI production in mammalian cells via phox and NOS: parallel Proteins with antimicrobial activity that are predominantly nuclear, cyto- but connecting paths. Nitroxyl anion (NOϪ), a one-electron reduction product ⅐ ⅐ solic, or secreted are not listed. Moreover, depletion of (micro)nutrients from of nitric oxide ( NO), is unlikely to arise from NO under physiologic conditions, phagosomes may be an important antimicrobial mechanism. For example, but is considered by some investigators to be a primary and more toxic product down-regulation of transferrin receptors can starve the pathogen for iron, of NOS (91). Reaction of RNI with cysteine sulfhydryls can lead either to and Nramp1 is a phagosomal membrane protein that may transport iron. S-nitrosylation or to oxidation to the sulfenic acid, as well as to disulfide bond *Antibacterial proteins specific to eosinophils are not included. formation (not shown), all of which are potentially reversible. Peroxynitrite † Ϫ Monocytes contain some of the antimicrobial proteins of neutrophils until anion (OONO ) and peroxynitrous acid (OONOH) have distinct patterns of they differentiate into macrophages. reactivity (92), but for simplicity, the text refers to both as peroxynitrite. OONOH spontaneously decomposes via species resembling the reactive rad- ⅐ ⅐ gens, and have not been reported to express granulysin, the only icals, hydroxyl (OH ) and͞or nitrogen dioxide ( NO2). When L-arginine is lim- . ⅐ known antibacterial protein of T cells. iting, NOS can produce superoxide (O2) along with NO, favoring the forma- tion of peroxynitrite (5). With these reflections in mind, one approaches the lessons from knock-out mice anticipating that a major effector mecha- readily evade them by dispensing with their targets, because the nism in host defense could be found to be both nonredundant targets are atomic rather than macromolecular, and confer and redundant. This proves to be the case for ROI and RNI. essential chemical functions (2). Instead, pathogens interfere with host cell production of ROI or RNI, catabolize them, or Nonredundant Roles of Phagocyte Oxidase (phox) and Nitric Oxide repair their damage (11). Synthase 2 (NOS2). Table 1 lists phagocyte products that are microbicidal in vitro and are delivered to phagosomes. From The Limits of Nonredundancy. A cardinal tenet of contemporary these features it is reasonable to presume that the physiological research holds that the role and importance of a biochemical roles of these products include antimicrobial action. Table 2 lists pathway are established by the phenotype of its mutant. In effect, the five such products that have been shown to play a nonre- the function