Nitric oxide production and signaling in inflammation.

Abstract

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. It possesses cytotoxic properties that are aimed against pathogenic microbes, but it can also have damaging effects on host tissues. NO reacts with soluble guanylate cyclase to form cyclic guanosine monophosphate (cGMP), which mediates many of the effects of NO. NO can also interact with molecular oxygen and superoxide anion to produce reactive nitrogen species that can modify various cellular functions. These indirect effects of NO have a significant role in inflammation, where NO is produced in high amounts by inducible nitric oxide synthase (iNOS) and reactive oxygen species are synthesized by activated inflammatory cells. The present review deals with NO production and signaling in inflammation, especially in relation to human neutrophils and eosinophils.

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Current Drug Targets - Inflammation & Allergy, 2005, 4, 471-479471
1568-010X/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.
Nitric Oxide Production and Signaling in Inflammation
Riku Korhonen, Aleksi Lahti, Hannu Kankaanranta and Eeva Moilanen*
The Immunopharmacology Research Group, University of Tampere Medical School and Research Unit, Tampere
University Hospital, Tampere, Finland
Abstract: Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. It possesses cytotoxic properties that are aimed
against pathogenic microbes, but it can also have damaging effects on host tissues. NO reacts with soluble guanylate cyclase to form cyclic
guanosine monophosphate (cGMP), which mediates many of the effects of NO. NO can also interact with molecular oxygen and superoxide anion to
produce reactive nitrogen species that can modify various cellular functions. These indirect effects of NO have a significant role in inflammation,
where NO is produced in high amounts by inducible nitric oxide synthase (iNOS) and reactive oxygen species are synthesized by activated
inflammatory cells. The present review deals with NO production and signaling in inflammation, especially in relation to human neutrophils and
eosinophils.
INTRODUCTION
Since its discovery in 1987 [1,2], nitric oxide (NO) has been a target
of intensive research and drug development. NO is a gaseous signaling
molecule that regulates various physiological and pathophysiological
responses in the human body. These include circulation and blood
pressure, platelet function, host defense, and neurotransmission in central
nervous system and in peripheral nerves.
The role of NO in host defense and immune responses was started to
be understood at the second half of the 1980s. Stuehr and Marletta
reported that mouse macrophages produce nitrite and nitrate in response to
bacterial lipopolysaccharide [3], and a compound with the reactivity of
NO proved to be an intermediate in the process [4]. Hibbs and co-workers
were able to identify NO as an effector molecule in macrophage-
mediated cytotoxicity [5,6]. Since then, a number of reports on the effects
of NO in inflammatory responses have been published. High levels of NO
are produced in response to inflammatory stimuli and mediate
proinflammatory and destructive effects. However, like most other
inflammatory mediators, NO has also protective effects in some
inflammatory responses.
The physiological and pathophysiological role of NO in the respiratory
system has been reviewed recently [7,8]. NO acts as a neurotransmitter in
NANC nerves and regulates smooth muscle tone. NO is also involved in
the host defense in bronchial epithelium, and it acts as an inflammatory
mediator in pathological states. Inhaled NO may have clinical implications
in certain conditions as a bronchodilator and vasodilator, and NOS
inhibitors are believed to be of benefit in inflammatory lung diseases. In
addition, exhaled NO can be measured as a marker of asthma and other
inflammatory lung diseases. In the present review, we will discuss the
regulation of NO production in response to inflammatory stimuli as well as
the targets of NO, especially in neutrophilic and eosinophilic inflammation.
BIOSYNTHESIS OF NO
NO is synthesized from L-arginine in a reaction catalyzed by a family
of nitric oxide synthase (NOS) enzymes. Active NOS is a tetramer formed
by two NOS proteins and two calmodulin molecules. Conversion of L-
arginine to NO and L-citrulline requires also NADPH and O2 as co-
substrates and (6R)-tetrahydrobiopterin (BH4), FAD, FMN and iron
protoporphyrin IX (haem) as co-factors [9-11].
Three different NOS isoforms have been characterized. The neuronal
NOS (nNOS, NOS I) is predominantly expressed in neurons in brain and
peripheral nervous system [12]. Endothelial NOS (eNOS, NOS III) is
mainly expressed in endothelial cells [13]. Both nNOS and eNOS are
constitutively expressed and are inactive in resting cells. Increase in free
intracellular calcium concentration ([Ca2+]i) stabilizes the binding of
calmodulin to eNOS and nNOS, and activates the enzyme to produce NO.
Stimuli that increase the [Ca2+]i (eg. acetylcholine in endothelial cells)
trigger the production of NO, and when the [Ca2+]i decreases, the NO
production ceases. That regulation makes NO production by constitutively
expressed NOSs transient and short lasting [9-11].
The third isoform of the NOS family is the inducible NOS (iNOS, NOS
II). No iNOS expression is found in most resting cells. Exposure to
*Address correspondence to this author at the Immunopharmacology Research
Group, Medical School, FIN-33014 University of Tampere, Finland; Fax: + 358 3
3551 8082; E-mail: eeva.moilanen@uta.fi
microbial products, such as lipopolysaccharide (LPS) and dsRNA or
proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis
factor-α (TNF-α) and interferon-γ (IFN-γ) induces the expression of
iNOS gene in various inflammatory and tissue cells. Binding of calmodulin
to iNOS is tight even at low [Ca2+]i, and therefore, iNOS is also called as a
calcium-independent NOS, and it can constantly produce high levels of
NO for prolonged periods [11,14,15].
REGULATION OF iNOS EXPRESSION
Although iNOS expression has been shown in various mouse and human
cells, there are marked cell type and species specific differences in the
responsiveness of iNOS expression to different stimuli [14-16]. For
instance, responses in human cells seem to be quite different from those in
mouse cells, which have been widely used in the studies on iNOS
expression. Many mouse cells readily express iNOS in response to LPS or
to a single cytokine, whereas human cells usually require a combination of
different cytokines for detectable iNOS expression and NO synthesis.
Furthermore, iNOS is expressed at high levels in activated murine
macrophages, but it has been difficult to induce iNOS in human monocytes
/ macrophages in vitro, although iNOS expression in macrophages in
inflamed human tissues has been shown in ex vivo studies [17-19].
Regulation of Murine iNOS Transcription
Transcription of mouse iNOS gene is regulated by ~1 kb promoter
[20,21], which contains putative binding sites for a number of transcription
factors. In vivo footprinting assay revealed LPS-induced binding to
octamer (Oct), E-box, nuclear factor kappa B (NF-κB) and interferon-
stimulated response element (ISRE) sites [22].
The mouse iNOS promoter contains two NF-κB elements, which are
critical for iNOS expression as site directed mutagenesis of either of these
sites significantly reduces promoter activity, and chemical inhibitors of
NF-κB prevent iNOS expression and NO production [23-26].
IFN-γ is an efficient enhancer of iNOS expression in most cells. iNOS
expression is seriously impaired in macrophages from mice deficient of
signal transducer and activator of transcription 1 (Stat1) [27], IFN
regulatory factor-1 (IRF-1) [28] or IFN consensus sequence binding
protein (ICSBP) [29], demonstrating the importance of IFN-responsive
transcription factors in iNOS expression. iNOS promoter contains a
functional GAS site which binds Stat1 [30], and an ISRE site which binds a
complex of IRF-1 and ICSBP [31,32].
Next to a proximal NF-κB site is an Oct site, which is also required for
iNOS promoter activity. Oct site binds members of the POU family of
transcription factors (Oct-1, Brn-3a, Brn-3b) and high mobility group
proteins (HMG-I(Y)) [33-37]. HMG-I(Y) alone cannot promote
transcription, but it augments and stabilizes the p50/p65 binding to NF-κB
site [35]. Additional transcription factors which have been shown to
regulate mouse iNOS promoter activity include Epithelium-specific Ets
(ESE-1) [38] and CAAT/enhancer binding protein beta (C/EBPβ) [39].
The role of activator protein 1 (AP-1) family of transcription factors in the
regulation of murine iNOS transcription is unclear. Existence of both
positive and negative regulatory AP-1 sites has been reported [40,41], but
the composition of AP-1 which binds to these sites is not known. Over-
expression of c-fos, fosB and c-jun suppresses iNOS promoter activity
[41]. Stat3 [42], Elk-3 [43] and upstream stimulatory factors 1 and 2 (USF-
1 and USF-2) [44] have been shown to negatively regulate iNOS
transcription.

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472 Current Drug Targets - Inflammation & Allergy, 2005, Vol. 4, No. 4Korhonen et al.
Regulation of Human iNOS Transcription
The promoter of the human iNOS gene is rather different from the
murine iNOS promoter. Deletion analysis of the human iNOS 5’flanking
region indicates presence of regulatory elements on the length of 16 kb
[45]. There is conflicting evidence about the presence of regulatory
elements on the first 4.7 kb of the human promoter. In some studies,
induction by cytokines was reported when promoter constructs containing
the first 3.2 kb [46] and 1 kb [47] were used. However, other reports
observed only basal activity but no induction by cytokines with constructs
containing first 1 - 4.7 kb of the promoter [45,48-51].
Human iNOS promoter is activated by NF-κB. A proximal NF-κB site
seems to be important in controlling transcription of human iNOS
[50,52,53], although lack of functionality for this site has also been
reported [51]. Several other functional NF-κB sites have been found
further upstream of the promoter [53,54].
IFN-γ is an important cytokine for iNOS expression in human cells like
in murine cells (see above). However, the IFN-γ -inducible factors that
regulate the activity of human iNOS promoter are less well-characterized
than those that regulate the mouse promoter. Two functional GAS sites
have been found. The upstream site contains overlapping NF-κB and Stat1
binding sites, and binding of both of these factors to this site is required for
promoter activity [55].
Additional factors shown to regulate human iNOS transcription
include C/EPBβ [56] and Krüppel-like factor 6 (KLF6) [57]. Both positive
and negative regulatory AP-1 sites have been found in the human iNOS
promoter [58-60], and the activity of the human promoter is suppressed by
overexpressing c-jun and c-fos [61]. Human iNOS promoter contains also
a negative regulatory element, which binds NF-κB repressing factor, that
is a constitutively expressed silencer that acts as a suppressor of basal
transcription [62].
The inter-species differences in the promoters mostly explain the
differences in the inducibility of iNOS expression between human and
murine cells [48]. However, activation of NF-κB and Stat1 pathways
seems to be important for both human and mouse iNOS transcription.
Post-Transcriptional Regulation of iNOS Expression
Recent evidence supports the idea that regulation of iNOS mRNA
stability is an important means to regulate iNOS expression. In
unstimulated cells, nuclear run-on assays show continuous iNOS
transcription, and human iNOS promoter constructs have basal activity.
However, no iNOS mRNA or protein can be detected in these cells,
suggesting that the iNOS mRNA is highly unstable in unstimulated cells
[45,49,63]. The basal activity of the human iNOS promoter is suppressed,
and its inducibility is increased if the luciferase construct also contains the
3’-untranslated region (UTR) of the human iNOS mRNA [64]. Both
human and murine 3’-UTRs contain AU-rich elements (AREs) [65,66],
which are characterized by the presence of AUUUA pentamers or
UUAUUUAUU nonamers. AREs have been shown to control mRNA
stability and translation of many transiently expressed cytokines and
growth factors [67].
There is only fragmentary data on factors / mechanisms that regulate
iNOS mRNA stability. Treatment with inhibitor of translation,
cycloheximide, stabilizes mouse iNOS mRNA, suggesting that stability of
iNOS mRNA is regulated by factors, which require de novo protein
synthesis, or that the stability is coupled to translation [68-70]. It seems that
the 3’-UTR ARE sequences of human iNOS mRNA alone are not
sufficient for destabilization, but additional elements in the 3’-UTR are
required [71]. 3’-UTR of iNOS mRNA binds an mRNA stability regulating
protein HuR [71]. Down-regulation of HuR expression results in reduced
iNOS expression and NO production without reduction in promoter
activity. This suggests that HuR stabilizes iNOS mRNA [71].
Protein kinase C δ (PKC δ) [72] and c-Jun N-terminal kinase (JNK) [73]
have been implicated in the regulation of iNOS mRNA stability. Reduced
iNOS mRNA stability has also been observed after treatment with
transforming growth factor–β (TGF-β) [74], dexamethasone [75], 8-
bromo-cyclic guanosine monophosphate (cGMP) [76] and intracellular
calcium elevating agents [77]. Increased iNOS mRNA stability has been
observed after treatment with forskolin (activates adenylate cyclase) or
dibutyryl cyclic adenosine monophosphate (cAMP) (membrane permeable
cAMP analog) [78,79], following BH4 [80] or the β-adrenergic agonist,
isoproterenol [81].
Interestingly, it was recently shown that in addition to serving as a
substrate of NO biosynthesis, L-arginine also controls iNOS expression.
The intracellular L-arginine concentration required for maximal NO
production is higher than the Km of L-arginine for iNOS. Reduction of
intracellular L-arginine concentrations by overexpression of arginase or
depletion of extracellular L-arginine reduces iNOS mRNA translation due
to inactivation of eukaryotic initiation factor2α, an important factor in
initiation of translation [82,83].
Signaling Pathways Regulating iNOS Expression
Various signal transduction pathways have been suggested to regulate
iNOS expression. The importance of pathways leading to the activation of
transcription factors NF-κB and Stat1 have been discussed above. cAMP
activating compounds can both enhance [78,84-86] and inhibit [86,87]
cytokine induced iNOS expression. Use of PKC activating phorbol esters
or PKC inhibitors have mainly suggested a positive role for PKC in
cytokine induced iNOS expression [88-90], but also a negative role has
been reported [91,92]. This may reflect the observations that different
PKC isoforms may have opposite effects [88,89,93].
Although iNOS activity is independent of the [Ca2+]i, changes in
[Ca2+]i regulate iNOS expression. Both stimulation [94,95] and inhibition
[77,96]of iNOS expression by [Ca2+]i elevating agents have been
reported. Interestingly, the effect of [Ca2+]i seems to depend on the extent
of inducing stimulus. At low LPS concentrations, increase in [Ca2+]i
stimulates iNOS expression, whereas at high LPS concentrations, increase
in [Ca2+]i inhibits iNOS expression [97].
The role of the mitogen-activated protein kinases in the regulation of
iNOS expression has been investigated intensively. Extracellular signal-
regulated kinase 1 and 2 have been shown to up-regulate [98-101] or to
have no role [102,103] in iNOS expression. Also p38 MAP kinase has
been reported to up-regulate [101,104,105], down-regulate, [106-108] or
to have no role [109,110] in iNOS expression. Activation of JNK up-
regulates iNOS expression [73,111,112].
NO: MOLECULAR MECHANISMS OF ACTION
NO is a reactive molecule that has a variety of effects depending on
the relative concentrations of NO and the surrounding milieu in which NO
is produced. There are both direct effects of NO that are mediated by the
NO molecule itself, and indirect effects of NO that are mediated by
reactive nitrogen species produced by the interaction of NO with
superoxide anion or with oxygen. cGMP that is produced by the interaction
of NO with soluble guanylate cyclase, mediates many of the physiological
effects of NO, and it is also an important example of the direct effects of
NO.
The molecular mechanisms that mediate the biological activities of
NO can be divided into three categories. Firstly, NO reacts readily with
transition metals, such as iron, copper and zinc. These metals are
abundantly present in prosthetic groups of enzymes and other proteins, and
by that mechanism, NO regulates the activity of various enzymes.
Secondly, NO is able to induce the formation of S-nitrosothiols from
cysteine residues in a reaction called S-nitrosylation. Nitrosylation has
been shown to modify the activity of several proteins involved in cellular
regulatory mechanisms [113]. Thirdly, NO reacts very quickly with
superoxide anion (O2-), resulting in the formation of peroxynitrite (ONOO-).
Peroxynitrite is a nitrating agent and a powerful oxidant that is able to
modify proteins, lipids and nucleic acids.
The first mechanism represents direct effects of NO and the two latter
mechanisms are referred as indirect effects of NO. At low concentrations
of NO (< 1uM), the direct effects of NO predominate, whereas at higher
concentrations (> 1uM), the indirect effects become more important [114]
(Fig. 1).
NO Signaling through Transition Metals
Due to its chemical structure, NO can act as an electron donor in
chemical reactions, and thereby is prone to react with transition metals
resulting in the formation of metal-nitrosyl complexes. In biological
systems, important transition metals that react with NO are iron, copper
and zinc.
One of the main targets in NO signaling is soluble guanylate cyclase
(sGC). sGC is an enzyme containing a heme structure with ferrous iron,
and it converts GTP to an important intracellular signaling molecule
cGMP. The basal activity of sGC is low, but it is rapidly activated by even
low concentrations of NO (10-100nM). NO binds directly to the heme in
sGS to form a ferrous-nitrosyl-heme complex resulting in changes in the
porphyrin ring structure. This leads to the activation of sGC and 400-500-
fold increase in the rate of cGMP synthesis [114,115].

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Fig. (1). NO production and targets of NO-mediated signaling. Activation of a cell surface receptor by its agonists, such as acetylcholine in endothelial cells or glutamate
in neurons, leads to an increase in the intracellular free calcium concentration ([Ca2+]i ) that subsequently results in the activation of NO production by eNOS or nNOS.
Inflammatory signals, such as bacterial products or proinflammatory cytokines, induce the expression of iNOS that is able to synthesize NO independently of changes in
[Ca2+]i. Effects of NO are mediated mainly via three routes. Firstly, NO can react directly with transition metals (e.g. iron, copper and zinc) found in catalytic sites in enzymes
like guanylate cyclase. This leads to formation of metal-nitrosyl complexes, and it may result in changes in the catalytic activity of the target enzyme. Secondly, NO can react
with cysteinyl residues of proteins forming nitrosothiols in a reaction called S-nitrosylation. S-nitrosylation may alter the activity of proteins in a similar manner as
phosphorylation, and it is a reversible event. Thirdly, a reaction between NO and superoxide anion results in a formation of peroxynitrite (ONOO-) that is a strong oxidant
and nitrating agent that is able to modify proteins, lipids and nucleic acids.
The principal mediator of cGMP signals is cGMP dependent protein
kinase (PKG). PKG is a serine/threonine kinase that is activated upon
binding of cGMP. Two types of PKG have been characterized. PKG I is a
cytosolic enzyme that is ubiquitously expressed with particularly high
expression levels in cerebellum, platelets and smooth muscle cells. PKG I
has various targets which are related to smooth muscle relaxation and to
platelet and neutrophil activation. [116,117]. PKG I deficient mice show
vascular, intestinal and erectile dysfunction [118,119], which stresses the
mediator role of cGMP and PKG I in the NO-induced smooth muscle
relaxation. PKG II is a membrane-bound protein that is expressed in
various tissues but not in the cardiovascular system. It is related to
intestinal secretory functions, and PKG II knockout resulted in intestinal
secretory defects and dwarfism [120].
Other factors that convey cGMP mediated signaling are cyclic nucleotide
gated channels (CNG), cAMP dependent protein kinase (PKA) and
phosphodiesterases. CNGs are voltage gated cation channels that are
involved in the processing of visual information in retina [121,122].
Because the cyclic nucleotide binding domains of PKG and PKA have
significant homology, cGMP is able to activate PKA, although with a 50-
fold lower selectivity than cAMP [116,117]. Fourth target of cGMP is
phosphodiesterases (PDEs), which catalyze the conversion / inactivation
of cAMP or cGMP to 5’AMP and 5’GMP, respectively. Different families
of PDEs are either regulated (stimulated or inhibited) by cGMP or they
target cGMP. PDE 5 is specific for cGMP, and selective inhibitors of this
enzyme are widely used in the treatment of erectile dysfunction. PDE 5
inhibitors, such as sildenafil, enhance cGMP levels and by that mechanism,
augment NO-induced vasodilatation in penile vessels [116,117,123].
Another well-documented target of direct NO regulated signaling is
cytochrome c oxidase (CcO). CcO is the terminal enzyme of the electron
transport chain, which is responsible for the synthesis of ATP in
mitochondria. CcO contains two heme moieties and two Cu2+ centers. NO
has been found to inhibit CcO function in a reversible manner, thereby
hindering mitochondrial respiration [124,125]. Another example is
catalase, which is a ferric heme containing protein responsible for the
metabolism of hydrogen peroxidase. NO reacts with the ferric moiety
resulting in the inhibition of catalase function. This leads to the increased
intracellular concentrations of hydrogen peroxide, and may contribute to
the cytotoxicity of NO [126].
NO Signaling through S-Nitrosylation
S-nitrosylation of proteins has been recognized as an important
mechanism that regulates the functions of the target proteins, and it has
been even compared to protein phosphorylation [113]. In aqueous
solutions, NO reacts readily with molecular oxygen (O2) forming
dinitrogen trioxide (N2O3) in a process called as NO autoxidation. N2O3 is
decomposed rapidly to nitrosonium ion (NO+) and nitrite. Nitrosonium ion
is responsible for the nitrosylation of thiols, secondary amines and
phenolics. Rate of autoxidation is mainly dependent on the concentrations
of NO and oxygen, and it is dramatically accelerated within lipid
membranes. This means that the rate of formation of N2O3 is high at the
site of NO synthesis. This stresses the importance of the distance between
the site of NO synthesis and target molecules and subcellular environment
and conditions in NO signaling [113].
Although nitrosylation of proteins is a chemical reaction, there seems
to be specificity, which is a requirement for a proper signaling mechanism.
For example, p21ras, a kinase involved in the MAP kinase cascade
activation, is a target for NO-based signaling. p21ras contains four cysteine
residues, but only cysteine 118 is nitrosylated resulting in the activation
[127]. Another example is coxsackievirus protease A2 that is inhibited by
nitrosylation of cysteine 110 [128]. The group of cellular target proteins
that are regulated by S-nitrosylation is increasing, and includes
transcription factors, kinases involved in signaling cascades, caspases, ion
channels and metabolic protein as shown in Table 1.
NO Signaling through Peroxynitrite
Reaction between NO and superoxide anion (O2-) forms ONOO- that
is a reactive molecule able to nitrate and oxidize proteins, lipid, and
nucleotides. The reaction between NO and superoxide anion is very rapid,
the rate constant of the reaction being about three times grater than the
rate of superoxide decomposition by superoxide dismutase (SOD). The
rate of ONOO- production is strongly dependent on the presence of NO
and superoxide, and ONOO- formation is favored in an environment
containing equivalent amounts of NO and superoxide. Sources of
superoxide production are mainly considered to be mitochondria and
immune cells (macrophages and granulocytes), and the synthesis of both
NO and superoxide is increased in inflammation [114].
Excessive peroxynitrite formation leads to nitrated proteins, inhibition
of mitochondrial respiration, depletion of cellular energetics, DNA
damage, apoptosis and necrotic cell death, resulting in cellular/tissue injury
[114,129] (See Table 2). Nitrotyrosine has been used as a marker of
peroxynitrite formation and tissue injury. Tyrosine nitration is becoming
increasingly recognized also as a functionally significant protein
modification. Nitration of proteins and enzymes modulates catalytic
activity, cell signaling and cytoskeletal organization. [129-131]. It is of
interest that also the activity of iNOS is regulated by ONOO- –mediated
nitration. Nitration of iNOS decreases catalytic activity, and this may be a
regulatory mechanism in patients with sepsis [132]. NO can also nitrate
tyrosine residues within proteins without formation of ONOO-. For
example, NO has been shown to nitrate tyrosine residues and thereby

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474 Current Drug Targets - Inflammation & Allergy, 2005, Vol. 4, No. 4 Korhonen et al.
Table 1. Modulation of Protein Functions by S-Nitrosylation
Protein Target EffectRef.
Transcription factors
OxyRS-nitrosylation Cys199 or Cys208 activation[170]
SoxR nitrosylation of [2Fe-2S] clusters activation[171]
Ace-1 formation of S-nitrosothiols or disulfides inhibition[172]
NF-kB S-nitrosylation of p50 (Cys62)inhibition of DNA binding[151,152]
AP-1 S-nitrosylation of c-Jun Cys272 and c-Fos Cys54
S-glutathionylation Cys269
(DNA binding domain)
inhibition of DNA binding[162,163]
Kinases
p21ras
S-nitrosylation of Cys118 activation[127,228]
JNK
p60c-Src
S-nitrosylation of Cys116inhibition [229]
S-nitrosylation, S-S bond formationinhibition [230]
EGFR tyrosine kinaseinhibition [231]
Caspases
Caspases 3 and 9inhibition [232,233]
Ion channels
Ryanodine receptorsS-nitrosylation of Cys3635desensitization[234]
NMDA receptorS-nitrosylation of Cys399inhibition[235]
Metabolic proteins
Creatine kinase inhibition[236]
Ornithine kinaseinhibition[237]
Table 2. Effects of Oxidation and Nitration Reactions Induced by Peroxynitrite
ProteinEffectRef.
Oxidation
DNA breakage, base modification, activation of PARP[238-240]
ThiolsGSH oxidation[241]
Lipidsperoxidation[242]
Mitochondrial enzymesinhibition-depletion in cellular energetics [243]
Nitration
Prostaglandin I2 synthase
Manganese SOD
inhibition, reduction in PGI2 dependent vasorelaxation
inhibition, transplant rejection
[244,245]
[246]
Cyclooxygenase 1 inhibition [133]
Cyclooxygenase 2 inhibition[134]
Ribonucleotide reductaseinhibition [247]
inhibit activity of enzymes cyclooxygenase-1 [133] and cyclooxygenase-2
[134].
NO AND REGULATION OF IMMUNE RESPONSE
The research that led to the discovery of NO as a signaling factor in
biological systems was conducted largely in the field of cardiovascular
research. Still, the connection between NO and immune system was
observed already in the early days of NO research, when NO was found
to be the mediator of macrophage cytotoxicity [6]. Since then, the
conception of the role of NO in immune system has broaden vastly. NO
has been found to be involved in a number of regulatory functions in
inflammation. These include infection control, regulation of signaling
cascades and transcription factors, regulation of vascular responses, and
regulation of leukocyte rolling, migration, cytokine production,
proliferation and apoptosis [14,135-137]. Inhibitors of NO synthesis,
especially selective iNOS inhibitors have been shown to be anti-
inflammatory in various forms of experimentally induced inflammation,
such as arthritis and colitis [135,137-139], and selective iNOS inhibitors
are under development for treatment of inflammatory diseases.
NO AND INFECTION CONTROL
Exposure of inflammatory and tissue cells to bacterial products such
as LPS, lipoteichoic acid (LTA), peptidoglycan, or bacterial DNA, or to
intact bacteria, induces iNOS expression and enhanced NO production. In
these conditions, NO produced by iNOS, partly through formation of
peroxynitrite, is a cytotoxic molecule serving as a killing mechanism
against invading microbes [140-144]. The importance of iNOS expression
and NO formation in the control of infectious agents is well-established in
rodents. To date, data gathered from studies with iNOS gene-deficient
mice strongly suggest that NO has a central role in fighting viral, bacterial
and protozoa infections in mice [14,144].
The role of iNOS expression in antimicrobial reactions in humans is
not as clear, and may be less important than in rodents. There is
nonetheless evidence that iNOS expression also participates in anti-
microbial defense in humans. Increased iNOS expression has been
observed in patients with urinary tract infection, tuberculosis, malaria and
sepsis [141,145-147], and human neutrophils are found to produce NO-
derived oxidants through myeloperoxidase [148]. In addition, human

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macrophages are found to induce iNOS expression and NO formation also
in aseptic inflammation, e.g. in rheumatoid arthritis and aseptic loosening
of joint implants [18,19]. In these conditions, the cytotoxic effects of NO
that were originally aimed against invading microbes are focused against
host tissues and involved in the pathogenesis of inflammation and tissue
destruction.
REGULATION OF INFLAMMATORY TRANSCRIPTION
FACTORS BY NO
Cellular responses to oxidative and nitrosative stress are often
regulated at the level of transcription [149]. Both prokaryotic and
eukaryotic cells have transcription factors that are regulated by NO.
NF-κ κ κ κB Pathway
The family of NF-κB/Rel transcription factors has a central role in the
regulation of inflammatory responses in mammalian cells including T and
B cell proliferation, expression of cytokines and adhesion molecules, as
well as regulation of apoptosis. NF-κB activation is involved in several
pathological states in humans, such as asthma, rheumatoid arthritis, and
inflammatory bowel disease [150]. Activation of NF-κB is essential for
iNOS expression (see above), and NO regulates NF-κB at various points in
its activation cascade. NO has been shown to inhibit the activity of NF-κB
by S-nitrosylation of p50 subunit [151,152]. In addition, NO has been
found to positively modulate NF-κB activation by affecting signaling
cascades regulating NF-κB activation. One target is p21ras, which is
activated by oxidative stress and leads to an increase in NF-κB activity.
p21ras is activated by NO through S-nitrosylation of cysteine 118 residue
[127,153,154]. Dissociation of IκB from NF-κB leads to activation of NF-
κB, and it is regulated by enzyme IKK. Transcription and degradation of
IKK has been reported to be modulated by NO resulting in reduced
activation of NF-κB [155,156], whereas low concentrations of NO seem to
have an opposite effect [157]. In vivo studies show that NO up-regulates
NF-κB activation in situations like hemorrhagic shock and reperfusion-
injury [158,159].
AP-1 Pathway
AP-1 is a heterodimeric transcription factor consisting of c-fos and c-
jun subunits. NO regulates AP-1 activity in biological systems but the
effects seem to be cell type specific and concentration dependent. NO has
been reported to up-regulate the expression of c-fos by a cGMP
-dependent mechanism [160,161]. NO was also found to enhance AP-1
dependent gene expression through activation of JNK [159]. In addition,
inhibition of AP-1 dependent transcription by S-nitrosylation of c-fos and
c-jun subunits has been reported [162,163].
Jak-STAT Pathway
The members of Janus family of tyrosine kinases (Jak1, Jak2, Jak3,
Tyk2) are expressed ubiquitously, and are involved in the regulation of a
number of cellular functions. They convey extracellular signals from
cellular surface to Stat proteins, which in turn activate transcriptional
responses within the cell, leading to adaptation to altered extracellular
environment [164,165]. NO has been found to inhibit Jak2 and Jak3
activities in kinase assays [166]. Accordingly, NO produced by activated
alveolar macrophages and myeloid suppressor cells inhibited T-cell
proliferation, and it was due to NO-dependent inhibition of Jak1 and Jak3
[167,168]. However, iNOS derived NO was found to enhance Stat3
dependent expression of IL-6 and G-CSF in mice suffering from
hemorrhagic shock [158].
Bacterial Transcription Factors
OxyR is a bacterial transcriptional activator in E.coli that has been shown
to be activated by NO through S-nitrosylation of cysteine residues
[169,170]. Another bacterial activator of transcription, SoxR is activated
by NO through nitrosylation of [2Fe-2S] clusters within the protein [171].
The activation of these transcription factors leads to protective response to
nitrosative and oxidative conditions in bacteria. In yeast, activity of a
metal-responsive transcription factor Ace1 was found to be attenuated by
NO, possibly through formation of S-nitrosothiols or disulfides [172].
NO AND NEUTROPHILS
There are interspecies differences in the synthesis of NO by
neutrophils. Rodent neutrophils are known to produce NO through iNOS
pathway in response to inflammatory stimuli, such as LPS, TNF-α and
IFN-γ [173,174]. Inflammatory stimuli known to induce iNOS in rat
neutrophils do not have the same action in human neutrophils, at least
under in vitro conditions, and enzymatic production of NO in human
neutrophils remains controversial. However, expression of iNOS is likely
to occur in certain situations in vivo, because neutrophils isolated from the
oral cavity or from urine from patients with urinary track infections were
reported to express iNOS [146,175]. In addition, it was shown that
bacterial infection or treatment with cytokines resulted in iNOS expression
and nitration of ingested bacteria in human neutrophils [146,176]. In
contrast, many researchers have not found NO synthesis in human
neutrophils [177-181]. We found that even though activated neutrophils
did not produce NO, they converted N-hydroxy-L-arginine to nitrite,
nitrate and citrulline [181]. Interestingly, enzyme myeloperoxide, which is
present in neutrophils, has been shown to produce nitrating oxidants and
nitrotyrosine from hydrogen peroxide and nitrite [182].
In addition to direct antimicrobial effects, NO also regulates
neutrophil functions. In in vitro studies, NO / NO-donors have been shown
to inhibit degranulation, leukotriene production, superoxide anion
generation and chemotactic movement in activated neutrophils [183,184].
NO has also shown to regulate leukocyte recruitment into the
inflammatory focus. Endogenous NO as well as NO-releasing compounds
attenuate leukocyte rolling and adhesion to activated endothelium [185-
187]. The underlying mechanism is not known, but it has been related to
antioxidant mechanisms of NO [188] and may be dependent on cGMP
[187]. NO is also shown to down regulate adhesion molecules that mediate
the interaction between leukocytes and endothelium, examples being P-
selectin [189], E-selectin [190] and intracellular adhesion molecule-1
(ICAM-1) [185,191].
The role of NO on leukocyte recruitment in vivo is controversial.
Mice deficient in eNOS and nNOS were found to have enhanced
leukocyte migration in thioglycollate-induced peritonitis, while iNOS
deficient mice showed neutrophil infiltration comparable to that seen in
wild type mice [189]. iNOS knockout mice have been reported to have
markedly increased neutrophil activity and showed exacerbated
inflammation and tissue injury in experimental colitis [192,193]. In
contrast, selective inhibition of iNOS has been reported to reduce
neutrophil infiltration and tissue injury in trinitrobenzene sulfonic acid
-induced colitis [194,195]. In addition to colitis, NO has been found to
regulate leukocyte accumulation in pathological states, such as allergic
lung inflammation [196,197], septic lung injury [198] and myocardial
infarction [159].
NO AND EOSINOPHILS
The human bronchial epithelial lining expresses iNOS and produces
NO [199], and endogenous NO can be detected in exhaled air [200,201].
The role of continuous NO production in lung epithelial cells is not clear,
but it may be involved in maintaining mucosal defense mechanisms in
lungs. Patients with asthma show markedly elevated levels of NO in
exhaled air [200,202,203], and they have enhanced iNOS expression and
nitrated protein residues in airway epithelium and inflammatory cells [17].
Exhaled NO levels correlate to clinical symptoms of asthma, to sputum
eosinophilia, and to markers of eosinophil activation [203,208].
NO production in human eosinophils is controversial. Arock et al.,
reported that eosinophils produce NO when activated by IgE-
immunocomplexes through CD23 receptors [209]. Purified eosinophils and
human eosinophilic leukemia cell line EOL-3 were found to produce NO
and to express iNOS that was highly identical to macrophage iNOS [210].
However, there are also reports suggesting that eosinophils do not express
iNOS [211,212]. Eosinophils may produce reactive nitrogen species also
by a NOS-independent manner through eosinophil peroxidase activity
[213,214].
NO seems to regulate eosinophil accumulation into the site of allergic
inflammation by its effects on adhesion molecules, migration and
apoptosis. NO and NO donors have been reported to inhibit eosinophil
adhesion and migration in vitro [215-217]. However, in animal models of
allergic airway inflammation, iNOS derived NO was associated with
accumulation of eosinophils and exacerbation of lung inflammation.
Accordingly, iNOS deficient mice showed decreased eosinophil
accumulation in experimentally induced allergic lung inflammation. This
was associated with increased production of Th1 cytokine, IFNγ, and
depletion of IFNγ led to enhanced eosinophil accumulation in lung tissue
and more severe inflammation [218,219]. Interestingly, iNOS knockout
mice were more susceptible to pulmonary fibrosis than wild-type mice
[220]. This may, however, be rather due to altered neutrophil than
eosinophil functions or survival [221,222]. Interestingly, a recent report
showed that NO-releasing budesonide was more potent than budesonide in