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Fig. 1. ROI and RNI production in mammalian cells via phox and NOS: parallel but connecting paths. Nitroxyl anion (NO), a one-electron reduction product of nitric oxide (˙NO), is unlikely to arise from ˙NO under physiologic conditions, but is considered by some investigators to be a primary and more toxic product of NOS (91). Reaction of RNI with cysteine sulfhydryls can lead either to S-nitrosylation or to oxidation to the sulfenic acid, as well as to disulfide bond formation (not shown), all of which are potentially reversible. Peroxynitrite anion (OONO) and peroxynitrous acid (OONOH) have distinct patterns of reactivity (92), but for simplicity, the text refers to both as peroxynitrite. OONOH spontaneously decomposes via species resembling the reactive radicals, hydroxyl (OH˙) and/or nitrogen dioxide (˙NO2). When L-arginine is limiting, NOS can produce superoxide along with ˙NO, favoring the formation of peroxynitrite (5).

readily evade them by dispensing with their targets, because the targets are atomic rather than macromolecular, and confer essential chemical functions (2). Instead, pathogens interfer with host cell production of ROI or RNI, catabolize them, or repair their damage (11).

The Limits of Nonredundancy. A cardinal tenet of contemporary research holds that the role and importance of a biochemical pathway are established by the phenotype of its mutant. In effect, the function of a gene product is described by its nonredundancy. However, it does not necessarily follow that apparent redundancy indicates dispensability. A great many null mutations do not yield phenotypes, most likely because the range of conditions tested is narrow (12, 13). Interpretation of redundancy as dispensability is particularly inappropriate for genes engaged in competition with other genomes. To the extent that a well designed defense backs up its most important elements, redundancy can argue for utility. For example, if pathogens are challenged to inhibit production of a given class of host antimicrobial compounds, then the host is challenged to evolve diverse approaches to their synthesis. Moreover, the few dozen pathogens favored by investigators for experiments are a tiny fraction of the hundreds swarming at a host's gates. A given antimicrobial mechanism may be redundant against the pathogens tested but nonredundant against others. Phagocyte antimicrobial mechanisms often work synergistically (14). A phenotype indicating that a given gene product is important for resistance to a pathogen does not imply that other gene products are unimportant in defense against the same pathogen. In short, redundancy and synergy are essential features of the immune system that complicate the interpretation of knock-outs. Recourses include generation of compound knock-outs and diversification of settings in which they are tested. There remains the problem that species with induced deficiencies do not recapitulate key aspects of human immunity. For example, in contrast to humans, mouse neutrophils lack defensins and bacterial permeability increasing factor and respond feebly to tumor necrosis factor and formylated peptides. Mice lack type I CD1 molecules, which are specialized to present particularly hydrophobic microbial antigens, and have not been reported to express granulysin, the only known antibacterial protein of T cells.

Table 1. Antimicrobial products of human phagocytes that are delivered to phagosomes

Product

Neutrophils*

Macrophages

ROI

+

+

RNI

+

+

Myeloperoxidase

+

Lactoferrin

+

Bacterial permeability increasing factor

+

Serprocidins (elastase, cathepsin G, protease 3, azurocidin)

+

Phospholipase A2

+

Cathelicidin

+

Lysozyme

+

Defensins (HNPs) 1, 2, 3, 4

+


Proteins with antimicrobial activity that are predominantly nuclear,cytosolic, or secreted are not listed. Moreover, depletion of (micro)nutrientsfrom phagosomes may be an important antimicrobial mechanism. Forexample, down-regulation of transferrin receptors can starve thepathogen for iron, and Nramp1 is a phagosomal membrane protein thatmay transport iron.

* Antibacterial proteins specific to eosinophils are not included.

Monocytes contain some of the antimicrobial proteins of neutrophils until they differentiate into macrophages.

With these reflections in mind, one approaches the lessons from knock-out mice anticipating that a major effector mechanism in host defense could be found to be both nonredundant and redundant. This proves to be the case for ROI and RNI.

Nonredundant Roles of Phagocyte Oxidase (phox) and Nitric Oxide Synthase 2 (NOS2). Table 1 lists phagocyte products that are microbicidal in vitro and are delivered to phagosomes. From these features it is reasonable to presume that the physiological roles of these products include antimicrobial action. Table 2 lists the five such products that have been shown to play a nonredundant role in mice. Mice whose phagocytes are deficient in elastase or cathepsin G, two of four antimicrobial serprocidins (15), are susceptible to experimental infection with Klebsiella pneumoniae (6), Escherichia coli (16), and Aspergillus fumigatis (75). Myeloperoxidase converts H2O2 into more toxic hypohalites (1); mice lacking myeloperoxidase have increased susceptibility to Candida albicans (17). Mice deficient in the phagocyte oxidase (phox), the major source of pathogen-triggered ROI production (18, 19), are susceptible to several inoculated pathogens. Finally, mice deficient in the high output pathway of nitric oxide production, catalyzed by ˙NO synthase type 2 (NOS2 or iNOS), have a worse course of infection than wild-type mice after inoculation with diverse organisms (20, 21). However, the autotoxic potential of RNI is illustrated by the greater severity of influenza virus pneumonitis (22) and Mycobacterium avium infection (23) in wild-type mice than in NOS2-deficient mice.

An experimental alternative to knock-outs is administration of inhibitors. The major problem is specificity. No phox inhibitors have been reported that are effective and nontoxic in experimental animals. The most potent known inhibitor of phox in vitro, diphenylene iodonium, is 20-fold more potent as an inhibitor of NOS2 (24). In contrast, L-arginine analogs serve as nontoxic, phox-sparing NOS2 inhibitors (25). DeGroote and Fang list reports in which NOS2 inhibitors have exacerbated infection by 80 species of viruses, bacteria, fungi, and protozoa (26).

As for ROI, RNI are critical in host defense not only because they can damage pathogens but also because they are immunoregulatory ( 21). For example, RNI can inhibit G proteins (27), activate or inhibit kinases (28), caspases (29), metalloproteases (106), transcription factors (30), and DNA methyltransferase (31), inhibit lymphocyte proliferation, alter cytokine and prostaglandin (107)



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