Review
Natural killer cells and nitric oxide

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Abstract

Natural killer (NK) cells and nitric oxide (NO) are both important components of the natural or innate immune response. NK cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by MHC antigens. They are abundant in blood, spleen, liver and lungs and are distinct from both T and B lymphocytes in their circulation patterns, profile of surface antigens, receptor repertoire and the way in which they discriminate between self and non-self. Uniquely, NK cells express receptors that can recognize and discriminate between normal and altered MHC class I determinants. NK cell cytotoxic activity is strongly induced by cytokines such as IL-2 and IL-12, and this activation is associated with synthesis of NO. Inhibitors of NO synthesis impair NK cell-mediated target cell killing, demonstrating a role for NO in NK cell function. Furthermore, NO itself can regulate NK cell activation. In this article, evidence that NO is a mediator of NK cell-mediated target cell killing, and that NO is a regulator of NK cell activation will be reviewed. Results of NO synthase gene deletion studies will be discussed, and rodent and human NK cells will be compared.

Introduction

Nitric oxide (NO) is a critical molecule in host defense to infection. Its role in immunity has been highly conserved over the course of evolution, thus supporting the hypothesis of the common evolutionary origin of the natural immune and inflammatory response [1], [2] NO synthesis is a necessary component of non-specific defense mechanisms against several pathogens, including bacteria, viruses, parasites, and fungi as well as neoplastic cells [1], [3], [4], [5]. Moreover, production of NO in tumor cells has been associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis [6].

NO production has been recorded in birds, molluscs, horseshoe crabs, insects, protozoa, and slime molds [1]. Thus, evolution appears to have selected a non-specific cytotoxic molecule such as NO because it is small and lipophilic, so that few microbes could block its entry, and because its prime chemical targets (sulphydryls and iron) are so central in biochemistry that few microbes or even animal cells could mutate to a fully resistant state.

The basis of NO-mediated cytotoxicity depends on the combination of NO with metal-containing moieties in key enzymes of the mitochondrial respiratory chain leading to the inhibition of mitochondrial electron transport, inhibition of aconitase activity affecting iron metabolism, and inhibition of ribonucleotide reductase, the rate-limiting step in DNA synthesis [7], [8]. NO can also damage DNA directly by deamination of purine and pyrimidine bases resulting in mutations and strand breaks [9]. In addition, NO-induced DNA damage activates the nuclear poly (ADP-ribose) polymerase accompanied by nicotinamide adenine dinucleotide and adenosine-triphosphate depletion leading to cell death by energy deprivation [10]. Initially, macrophages were thought to be the only source of cytotoxic NO, but it is now apparent that other cell types, such as NK cells, hepatocytes, pulmonary epithelial cells, keratinocytes and endothelial cells, also produce NO that contributes to innate immunity [4].

NO may also modulate several immunological events in control of inflammation and tissue damage induced by infection, including immune cell adherence and function, cellular proliferation, and cytokine production [11]. Moreover, NO exerts immunomodulatory effects as well as apoptosis of several immunocompetent cells, such as thymocytes, lymphocytes, and macrophages, and may play a role in many clinically important inflammatory and autoimmune diseases (see other articles in this issue). A key role for NO has also been demonstrated in tumor-induced immunosuppression, either directly by NO-producing tumor cells or indirectly via induction of iNOS in tumor-infiltrating macrophages [12].

Section snippets

NO: linking innate and acquired immunity

Protective immunity results from the complementary contributions of two fundamental systems: innate immune system and acquired immune system. These separate but interdependent pathways work in harmony to identify, contain and eliminate harmful agents. Their interplay is critical for successful detection and elimination of infectious pathogens and tumors and the early innate immune response can influence subsequent acquired immunity. Cells that are capable of guarding sites of pathogen entry and

NK cells

Natural killer (NK) cells, a major component of innate immunity, play an important role in host resistance to infections from bacteria, viruses, parasites, and Listeria [18], [19], [20], [21]. NK cells are involved in immune surveillance of tumors and rejection of transplanted organs [18], [22], [23]. They can also influence the adaptive immune system and direct the pattern of T responses [24], [25]. The functions of NK cells, as well as their maturation and differentiation, are regulated by

Role of nitric oxide in rodent NK cell activation and cytotoxic functions against tumor target cells

In recent years, increasing evidence has supported a role for nitric oxide in NK cell activation. Park et al. [70] reported that NK and LAK cytotoxicity could be enhanced in a dose-dependent way by addition of exogenous arginine, suggesting a role for arginine metabolism in the lytic events mediated by these cells as it occurs in activated macrophages.

In 1994, our group firstly demonstrated that rat NK cell-mediated cytotoxicity is directly dependent upon concentration of l-arginine in the

Role of nitric oxide in rodent IL-2-activated NK cells and LAK cells

IL-2 is a major cytokine leading to increased NK cell-mediated cytotoxicity [31], [32], [33], [80]. Nevertheless, the mechanisms underlying the enhanced cytotoxic ability of IL-2-activated NK cells are not fully understood. Hibbs et al. [81] reported increased levels of NO synthesis in patients with cancer after IL-2 administration. Both in vivo and in vitro IL-2 treatments that markedly stimulated NK cell tumoricidal activity were correlated with increased NO production determined as nitrite

How is NO produced in rodent NK cells—cNOS or iNOS?

We recently examined whether IL-2-induced NO release by rat NK cells was due to an induction of iNOS or cNOS activity using enzyme activity assays based on the conversion of l-arginine to l-citrulline. IL-2 significantly increased citrulline generation in either peripheral blood or spleen NK cells (Fig. 1) [91]. This event was attributed to iNOS rather than cNOS activity since it was not affected by the absence of Ca/CAM or the presence of the CAM antagonist W13. Moreover, IL-2-induced iNOS

NO in rodent NK-dependent immunity to viruses/parasites

iNOS-dependent NO generation may also be involved in NK cell-mediated resistance against viruses and parasites. Tay and Welsh [95] reported that the antiviral effector mechanisms by which NK cells control murine cytomegalovirus infection in liver are abrogated by in vivo administration of the NOS inhibitor l-NMMA. In 1997, Zhang et al. [96] examined the ability of IL-12 and IL-18 to induce the production of IFN-γ and NO by murine peritoneal exudates cells (PEC) and to stimulate the

NO in human NK cell activity and functions

Although NO is important in rodent NK-mediated immune responses, its involvement in the human peripheral blood NK cell system is still unclear. In 1995, Xiao et al. [83] provided evidence that the human NK cell effector mechanism causing target cytolysis had a requirement for l-arginine. Moreover, l-arginine addition to the culture medium enhanced NK cell activity in a dose-dependent manner. The stimulatory effect of l-arginine was accompanied by an increase in NO formation as determined by

NO and NK cells in pregnancy

A consistent body of literature indicates that in human, mouse, and rat pregnancy, maternal NK cells accumulate and differentiate at implantation sites. These cells, termed as uterine NK cells, express iNOS and develop cytolytic molecules such as perforin and granzymes during differentiation in situ. The results of studies aiming to investigate expression of the iNOS gene in the pregnant mouse uterus showed that the enzyme was localised to mouse uterine leukocytes including mast-cells,

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