Carl Nathan

Nitric oxide as a secretory product of mammalian cells.

Evolution has resorted to nitric oxide (NO), a tiny, reactive radical gas, to mediate both servoregulatory and cytotoxic functions. This article reviews how different forms of nitric oxide synthase help confer specificity and diversity on the effects of this remarkable signaling molecule.— Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 6: 3051‐3064; 1992.

Nitric oxide synthases: Roles, tolls, and controls

Carl Nathan and Qiao-wen Xie Beatrice and Samuel Seaver Laboratory Department of Medicine Cornell University Medical College New York, New York 10021 Imagine it’s 1985. You’ve joined an environmentally con- scious friend at a sidewalk cafe. Your companion is irked that traffic has fouled the air with nitric oxide (NO). Be- cause your imagination was piqued by a paper in the Pro- ceedings of the National Academy of Sciences reporting inorganic nitrite production by activated macrophages (StuehrandMarietta, 1985),youcounteryourfriend’scom- plaint with a prediction that the next ten years will bring forth evidence that NO is produced in slime molds, locusts, beetles, horseshoe crabs, mollusks, chickens, mice, rats, cows, and humans (Eiphick et al., 1993; Geiperin, 1994; Lee et al., 1994; Nathan and Xie, 1994; Werner-Feimayer et al., 1994). if so, your friend retorts, this would be no more than eukaryotic smog, a waste product of L-arginine metabolism. You concede that L-arginine-derived NO and its oxidation products will be excreted in people’s saliva, breath, and urine, but you insist NO is an autacoid, not a byproduct. Its physiologic roles will be at least as protean as those discovered for corticosteroids in the 194Os- 1980s and eicosanoids in the 196Os-198Os- ail three the products of hemecontaining oxygenases. You speculate that NO will regulate the following: tran- scription factor activation; translation of mRNAs controi- ling Fe metabolism; mutagenesis; apoptosis; giycolysis and mitochondriai electron transport; protein acyiation; deoxynucieotide synthesis; fusion of myObiaSt8; adhesion of platelets and neutrophiis; proliferation of myeloid pro- genitors T cells, keratinocytes, and tumor ceils; release of pituitary hormones; the tone of bronchi and sphincters; the contractions of

Points of control in inflammation

Inflammation is a complex set of interactions among soluble factors and cells that can arise in any tissue in response to traumatic, infectious, post-ischaemic, toxic or autoimmune injury. The process normally leads to recovery from infection and to healing, However, if targeted destruction and assisted repair are not properly phased, inflammation can lead to persistent tissue damage by leukocytes, lymphocytes or collagen. Inflammation may be considered in terms of its checkpoints, where binary or higher-order signals drive each commitment to escalate, go signals trigger stop signals, and molecules responsible for mediating the inflammatory response also suppress it, depending on timing and context. The non-inflammatory state does not arise passively from an absence of inflammatory stimuli; rather, maintenance of health requires the positive actions of specific gene products to suppress reactions to potentially inflammatory stimuli that do not warrant a full response.

Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase

Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death.

Role of nitric oxide synthesis in macrophage antimicrobial activity.

Research over the past 5 years has demonstrated that immunologic activation of mouse macrophages induces the activity of nitric oxide synthase, which oxidizes a guanidino nitrogen of L-arginine, yielding citrulline and the reactive radical, nitric oxide. A review of the biochemistry and immunologic regulation of this pathway in macrophages provides a backdrop against which to evaluate its effector functions. Reports published in the past 2 years suggest that synthesis of NO mediates much of the antimicrobial activity of mouse macrophages against some fungal, helminthic, protozoal and bacterial pathogens.

Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages Insights into the Phagosomal Environment

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon γ– and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and β-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of σE-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogens cell envelope.

Inducible nitric oxide synthase: what difference does it make?

Fire sweeps through the brush. In its aftermath, dormant seeds of chaparral, savanna, heath, and scrub begin to germinate in response to a “go” signal in the smoke. Even though smokesoaked water kills the seeds, in diluted form it triggers their development. The chemical cues are nitrogen oxides (1). This lesson of death and life in the field mirrors comparable events within us, where reactive nitrogen intermediates (RNI) 1 deliver both deathand life-promoting messages. As described in Michel and Feron’s introduction to this series (2), RNI include not only nitric oxide (NO), the primary reactive product of nitric oxide synthases (NOSs), but also those species resulting from NO’s rapid oxidation, reduction, or adduction in physiologic milieus, such as NO 2 , NO 2 2 , N 2 O 3 , N 2 O 4 , S -nitrosothiols, and peroxynitrite (OONO 2 ). In mammals, there is a rough correspondence between toxic and homeostatic functions of NO and its production in large and small quantities, respectively. The high-output path of NO production is the hallmark of the second isoform of NOS to be cloned, NOS2. NOS2 was named “iNOS” (3) to connote its independence of elevated intracellular Ca 2 1 , the distinguishing biochemical feature primarily responsible for conferring the capacity of this isoform for more sustained catalysis than typically exercised either by nNOS (NOS1) or eNOS (NOS3) (4). Because iNOS is expressed in most cells only after induction by immunologic and inflammatory stimuli, the “i” doubles for “inducible.” 5 yr after mouse iNOS cDNA was cloned (3, 5, 6), and 2 yr after the NOS2 gene was disrupted in mice through homologous recombination (7–9), it is timely to take stock: What does iNOS contribute to mammalian pathophysiology? The complexity of this question has elicited multiple responses addressed to different facets of an answer (e.g., references 10–15). The approach of this Perspective is to focus on lessons emerging from iNOS “knock-out” mice. The compound phenotype of these mice (Table I) invites prediction, the limitations of pathophysiologic analysis through gene disruption deserve reflection, and the bottom line demands inspection: In what light does this new knowledge cast iNOS as a potential therapeutic target?

Beyond oxidative stress: an immunologist's guide to reactive oxygen species

Reactive oxygen species (ROS) react preferentially with certain atoms to modulate functions ranging from cell homeostasis to cell death. Molecular actions include both inhibition and activation of proteins, mutagenesis of DNA and activation of gene transcription. Cellular actions include promotion or suppression of inflammation, immunity and carcinogenesis. ROS help the host to compete against microorganisms and are also involved in intermicrobial competition. ROS chemistry and their pleiotropy make them difficult to localize, to quantify and to manipulate — challenges we must overcome to translate ROS biology into medical advances.

The high-output nitric oxide pathway: role and regulation.

Nitric oxide synthase (NOS) catalyzes the production of nitric oxide (NO), a short‐lived radical gas with physiological or pathophysiological roles in nearly every organ system. The inducible NO synthase (iNOS) is a high‐output isoform compared to the two constitutive NOSs. The iNOS from murine macrophages tightly binds calmodulin as a subunit, and its activity is not dependent on exogenous calmodulin or elevated calcium. This iNOS is induced at the transcriptional level by bacterial lipopolysaccharide (LPS) and interferon‐γ. The promoter region of the murine iNOS gene contains at least 24 oligonucleotide motifs corresponding to elements involved in the binding of transcription factors in the promoters of other cytokine‐inducible genes. Nuclear factor NF‐xB/c‐rel, interacting with cycloheximide‐sensitive protein(s) and binding to the NF‐xBd site in the iNOS promoter, controls the induction of iNOS by LPS. However, iNOS is also regulated posttranscriptionally. Complex regulation of iNOS at multiple levels may reflect the dual role of iNOS in host defense and autotoxicity. J. Leukoc. Biol. 56: 576–582; 1994.

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 others 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.