International Immunopharmacology 1 2001.

13971406
www.elsevier.comrlocaterintimp

Review

Nitric oxide in immunity and inflammation

John W. Coleman )
Department of Pharmacology, Uniersity of Lierpool, Ashton Street, Lierpool L69 3GE, UK

Abstract

Nitric oxide NO. is synthesised by many cell types involved in immunity and inflammation. The principal enzyme
involved is the inducible type-2 isoform of nitric oxide synthase NOS-2., which produces high-level sustained NO
synthesis. NO is important as a toxic defense molecule against infectious organisms. It also regulates the functional activity,
growth and death of many immune and inflammatory cell types including macrophages, T lymphocytes, antigen-presenting
cells, mast cells, neutrophils and natural killer cells. However, the role of NO in nonspecific and specific immunity in vivo
and in immunologically mediated diseases and inflammation is poorly understood. NO does not act through a receptorits
target cell specificity depends on its concentration, its chemical reactivity, the vicinity of target cells and the way that target
cells are programmed to respond. At high concentrations as generated by NOS-2, NO is rapidly oxidised to reactive nitrogen
oxide species RNOS. that mediate most of the immunological effects of NOS-2-derived NO. RNOS can S-nitrosate thiols
to modify key signalling molecules such as kinases and transcription factors. Several key enzymes in mitochondrial
respiration are also inhibited by RNOS and this leads to a depletion of ATP and cellular energy. A combination of these
interactions may explain the multiple actions of NO in the regulation of immune and inflammatory cells. q 2001 Elsevier
Science B.V. All rights reserved.

Keywords: Nitric oxide; Inflammatory cells; Immunity

1. Introduction relaxing agent, a neurotransmitter and an inhibitor of

Nitric oxide NO. is a molecule utilised through-
out the animal kingdom as a signalling or toxic agent
between cells. Generated by many cell types in a
variety of tissues, in mammals it acts as a vascular
Abbreiations: cGMP, cyclic guanosine monophosphate; FAD,
flavin adenine dinucleotide; FMN, flavin mononucleotide; GSH,
reduced glutathione; GS-SG, oxidised glutathione; NK, natural
killer; NO, nitric oxide; NOy
2 , nitrite; NOS, NO synthase types-1,
-2, -3.; Oy y
2 , superoxide anion; ONOO , peroxynitrite; RNOS,
reactive nitrogen oxide species; Th, T helper
)
Tel.: q44-151-794-5551; fax: q44-151-794-5540.
E-mail address: coleman@liv.ac.uk J.W. Coleman..
platelet aggregation w1,2x. In addition to these physio-
logical roles, NO is generated during immune and
inflammatory responseshere its function is less
well defined and more complex w1,2x. It is involved
in innate immunity as a toxic agent towards infec-
tious organisms, but can induce or regulate death and
function of host immune cells, thereby regulating
specific immunity. NO may induce toxic reactions
against other tissues of the host and since it is
generated at high levels in certain types of inflamma-
tion, for example asthma, it has been implicated as a
pro-inflammatory agent. Equally, it may act as an
anti-inflammatory or immunosuppressive agent via
its inhibitory or apoptotic effects on cells.

1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 1 5 6 7 - 5 7 6 9 0 1 . 0 0 0 8 6 - 8
1398 J.W. Colemanr International Immunopharmacology 1 (2001) 13971406

The role of NO in immunity and inflammation 2. Synthesis of nitric oxide

and its mechanisms of action in these processes will
be the subject of this review and the other articles in
this issue of International Immunopharmacology. Be-
fore considering NO in immunity and inflammation,
it is important to understand basic aspects of its
synthesis, chemistry and reactivity with biological
molecules.
NO is synthesised universally from L-arginine and
molecular oxygen by an enzymatic process that
utilises electrons donated by NADPH. The NO syn-
thase NOS. enzymes convert L-arginine to NO and
L-citrulline via the intermediate N-hydroxy-L-ar-
ginine. One molecule of L-arginine produces one

Fig. 1. Nitric oxide NO. production and signalling. Nitric oxide is generated either by the constitutively expressed enzymes NOS-1 and
NOS-3 or the induced enzyme NOS-2. NOS-1 and NOS-3 are activated in response to physiological stimuli that trigger an intracellular
Ca2q signal; they produce NO rapidly and transiently at low levels. NOS-2 is not expressed in resting cells but is induced by immunological
stimuli such as bacterial lipopolysaccharide LPS. or cytokines such as IL-1, TNF-a or IFN-g . After a lag period corresponding to mRNA
and protein synthesis, NOS-2 produces NO at sustained high levels. The actions of NO are direct at low concentrations but indirect at high
concentrations. Low-level NO produced by NOS-1 and NOS-3 interacts directly with the iron atom in the heme group of guanylyl cyclase to
activate the enzyme to produce cGMP, which then triggers a rapid transient cellular response. High concentrations of NO generated by
NOS-2 are unstable and are rapidly oxidised under aerobic conditions to reactive nitrogen oxide species RNOS. with the general formula
NO x . RNOS nitrosate the thiol group in glutathione to produce S-nitrosoglutathione GS-NO. or thiol groups in proteins to generate
proteinS-NO. Such nitrosations inhibit the activity of many proteins including mitochondrial enzymes and transcription factors, and
produce long-term cellular effects. Under conditions of high NO combined with high oxidative stress, superoxide Oy 2 interacts with NO to
.
produce the highly toxic peroxynitrite anion OONOy. .
 J.W. Colemanr International Immunopharmacology 1 (2001) 13971406
molecule of NO, the nitrogen atom of the latter
deriving from a terminal guanidino group of the
arginine side chain.
There are three types of NOS. Two of these are
constitutively expressed while the other is expressed
only in activated cells. One constitutive form was
originally characterised in neurons and was therefore
known as neuronal NOS or nNOS, while the other,
originally characterised in endothelial cells, was
known as endothelial NOS or eNOS. Now that these
two NOS isoforms have been fully genetically char-
acterised and also found to be distributed more widely
than originally thought, they have been renamed
NOS-1 and NOS-3, respectively w3x. The third type
of NOS is not expressed in resting cells, but is
synthesised upon cell activation. Originally described
in mouse macrophages w4,5x, this inducible form of
NOS is known either as iNOS or NOS-2 w35x.
The constitutive forms of NOS NOS-1 and NOS-
3. are activated in response to a calcium signal
generated for example by the arrival of an action
potential at a nerve ending, or activation of endothe-
lial cell receptors by acetylcholine Fig. 1.. Enzyme
activation occurs rapidly and transiently, according
to the kinetics of the calcium signal. Production of
NO is equally transient, providing a rapid pulse-like
signal to the responding cell e.g. neuron or smooth
muscle cell. w1,2x. NOS-2, on the other hand, is not
activated by a calcium signal but is continuously
active once expressed. Its expression is induced by
several agents including bacterial lipopolysaccharide
or cytokines such as interferon-g , IL-1 or TNF-a
Fig. 1.. There is a lag phase of several hours
between cell activation and NO production, reflect-
ing the time taken for mRNA and protein synthesis
w35x. In contrast to NOS-1 and NOS-3, NOS-2
generates high concentrations of NO n rather than p
molar. and this level of synthesis is sustained for
hours or days or longer, depending on how long the
enzyme is present in the cells or tissue Fig. 1..
The three isoforms of NOS show approximately
50% primary amino acid sequence homology to one
another, but across mammalian species each isoform
is highly conserved approximately 90% homology.
w2,3,6x. The NOS enzymes are large and complex
proteins comprising homodimers of a 130133 kDa
NOS-2 and NOS-3. or 160 kDa NOS-1. polypep-
tide w6x. Essentially, each enzyme molecule com-
1399
prises two halvesa C-terminal electron generating
reductase-type region, and an N-terminal oxidase-
type region. In combining oxidase and reductase
regions, NOS are unique among mammalian en-
zymes. The C terminal portion of NOS shows a high
degree of homology to cytochrome P450 reductase,
and like this homologue incorporates binding sites
for the cofactors NADPH, flavin adenine dinucleo-
tide FAD. and flavin mononucleotide FMN.. The
enzyme generates electrons from NADPH that are
shunted via FAD and FMN towards the N-terminal
NO-generating region of the enzyme. The N-terminal
region incorporates a heme moiety that is associated
with the substrate-binding site. All NOS incorporate
a calmodulin-binding site in their midregion. Activa-
tion of NOS-1 and NOS-3 is triggered by calcium
binding to calmodulin, whereas in NOS-2 calmod-
ulin is tightly bound regardless of calcium levels.
This explains the calcium responsiveness of NOS-1
and NOS-3 and the unstimulated continuous activity
of NOS-2 w16x.
3. Biological chemistry of nitric oxide
Nitric oxide is a simple diatomic molecule whose
physico-chemical and biological properties are deter-
mined by its small size 30 Da., absence of charge
and its single unpaired electron. NO is a gas under
atmospheric conditions but a solute within cells and
tissues. Its solubility and diffusion properties resem-
ble closely those of oxygen. It is readily diffusible in
body fluids and tissues and freely crosses cell mem-
branes. Its lone outer electron renders it a radical and
therefore chemically reactive, but for a radical it is
relatively stableit does not react with itself and
has a physiological half-life of seconds to minutes
depending on its concentration and immediate chem-
ical environment w79x.
At high concentrations, as produced by the in-
ducible form of NOS NOS-2., and under aerobic
conditions, NO is rapidly oxidised to reactive nitro-
gen oxide species RNOS. with the generic formula
NO x Fig. 1.. Under gaseous conditions the RNOS
formed are nitrogen dioxide NO 2 ., dinitrogen triox-
ide N2 O 3 . and trinitrogen tetraoxide N2 O4 . but in
aqueous solution, and in a biological systems, N2 O 3
is the major oxidative product w8x. RNOS are unsta-
1400 J.W. Colemanr International Immunopharmacology 1 (2001) 13971406
ble and rapidly nitrosate thiols or amines, or are
hydrolysed and excreted as nitrite NOy 2
. w8x. At
higher concentrations, RNOS induce cell toxicity by
nitrosating DNA and tyrosine residues, and inducing
lipid peroxidation w8,9x. The chemical oxidation of
NO occurs non-enzymatically, and the reaction dy-
namics are such that the chemical half-life of NO is
inversely proportional to its concentrationfor ex-
ample at 1 mM its half-life is less than 1 s, whereas
at 1 m M its half-life is 1015 min w8x. Hence, as NO
diffuses away from its source, its half-life is ex-
tended as its concentration declines.
Under conditions of combined nitrosative and ox-
idative stress, when both NO and the superoxide
anion Oy 2
. are formed at high concentrations, these
two radicals interact to generate the highly reactive
oxidant peroxynitrite anion ONOOy. Fig. 1.. Per-
oxynitrite is thought to mediate many of the most
severe toxic effects of NO w7,8x.
The reduced form of the cysteine-containing
tripeptide glutathione GSH. is the major thiol in
cells and plays an important anti-oxidant or detoxi-
cant role against RNOS, as well as reactive oxygen
species and reactive chemical metabolites w10,11x.
RNOS nitrosate GSH to generate S-nitrosoglutath-
ione GS-NO., which then acts as a reservoir of
available NO and GSH. In the presence of other
thiols, for example on proteins, NO is displaced from
GS-NO to generate the oxidised form of glutathione
GS-SG. and an S-nitrosated second thiol e.g. pro-
teinS-NO. Fig. 1.. In this manner NO can be
shunted from one molecule to anothera process
called transnitrosationthe effect of which is to
provide storage and transport of NO, thereby pro-
longing its availability and range w10,11x. For exam-
ple, transnitrosation may be involved in NO transport
towards the cell nucleus where it can influence tran-
scription factor activity or even directly interact with
DNA w11x. Likewise, GS-NO can act as a reservoir
of GSH that can be transferred to other thiols by
S-gluthiolation w10,11x. As described by Kroncke et
al. w11x in this issue, different cell types differ in
their capacity to maintain and replenish GSH in the
face of nitrosative stress. For example, unlike lym-
phoma cells, mastocyte and fibroblast cell lines are
able to maintain cellular GSH levels in the face of
short or long-term exposure to NO, by continued
enzymatic resynthesis and recycling w11x. These lat-
ter cell types are therefore better able to defend
themselves against NO-induced shift of cellular re-
dox potential towards a more oxidative state. In
conclusion, GSH acts as a chemical sponge to soak
up NO in cells, but as it becomes saturated other
thiols, particularly thiolated proteins, become impor-
tant targets for nitrosation.
4. Nitric oxide in immunity
NO plays several roles in immunityas a toxic
agent towards infectious organisms w1214x, an in-
ducer or suppressor of apoptosis w11x or an im-
munoregulator w1522x. Basic considerations point to
NOS-2 as the likely NOS isoform involved in the
immune response. Because the immune system is
activated in response to infection, any associated NO
response would develop in parallel, over days or
weeks rather than within a fraction of a second as in
physiological responses. Also, for NO to be effective
as a toxic or immune regulatory mediator it needs to
be generated at high levels for a sustained period of
time. Therefore, the high output sustained NO pro-
duction by NOS-2 rather than the ApulsatileB NO
production by the constitutive NOS enzymes ex-
pected, and this is indeed largely supported by the
experimental evidence w2,1118x. Many cells express
NOS-2, including fibroblasts, endothelial and epithe-
lial cells, keratinocytes and chondrocytes w15,16x,
monocytesrmacrophages w4,5,17x, antigen-present-
ing cells w18x, and natural killer NK. cells w19x.
However, the question of whether or not T lympho-
cytes w18,2023x, mast cells w2427x and neutrophils
w28x produce immunologically significant NO is still
contentious.
In addition to being produced by a wide range of
immune and inflammatory cell types, NO exerts
regulatory effects on these cells. In most cases the
effect of NO is inhibitory on cell function or growth,
but in certain circumstances it can be anti-apoptotic
w11x. Macrophage-derived NO was first shown to
inhibit T lymphocyte proliferation in rodent mixed
lymphocyte cultures and in vivo models of T cell-
mediated immunity reviewed in Refs. w15,18x.. Sub-
sequent studies in mice have claimed that NO is
preferentially produced by the Th1 subset of T helper
lymphocytes w20x and selectively inhibits Th1 re-
 J.W. Colemanr International Immunopharmacology 1 (2001) 13971406
sponses and production of the Th1 cytokine IFN-g
w21x. These findings led to the speculation that Th1
cell-derived NO could down-regulate Th1 cell re-
sponses in an autocrine manner and at the same time
promote Th2 responses, possibly leading to enhanced
production of IgE and promotion of IgE-mediated
diseases such as asthma w22x. However, these find-
ings and conclusions have since been seriously ques-
tioned w18,23,29,30x. For example, Thuring
et al. w23x
found no evidence of NO production by mouse
lymphocytes or T cell clones. Likewise van der Veen
et al. w18,29x found that mouse Th1 cells do not
express NOS-2 and also that NO inhibits prolifera-
tion of mouse Th1 and Th2 cells equally without
inhibiting production of IFN-g and other cytokines.
Another study found that NO inhibits production of
both Th1 and Th2 cytokines by human T lympho-
cytes w30x. Although clearly much is yet to be re-
solved in this area, van der Veen et al. w18,29x argue
that NO can inhibit Th cell proliferation indepen-
dently of cytokine expression, at a later stage in the
growth response, probably at the G1rS transition in
the cell cycle. NO inhibits T cell growth at concen-
trations lower than those that induce apoptosis, and T
cells are highly sensitive to small changes in NO
levels, suggesting that NO might act as an onroff
switch for proliferation w18x.
NO exerts immunosuppressive effects in vivo
since NOS-2 gene knockout mice have enhanced
Th1 responses to Leishmania infection w21x and ex-
acerbated severity of autoimmune encephalitis w31x.
Interestingly, superoxide anion Oy 2
. generated by
macrophages overcomes the T cell anti-proliferative
effect of NO w32x. Since NO interacts with Oy 2 to
produce peroxynitrite ONOOy. , clearly its effect is
direct or through RNOS rather than through toxic
peroxynitrite formation w18,32x.
As reviewed in other articles in this issue, NO
exerts multiple regulatory actions on immune cells.
For example, NO is a mediator of NK cell killing of
target cells and regulates NK cell function w19x; it
inhibits activation of mast cells w24,25x and can
enhance or inhibit neutrophil activation depending
on its concentration w28x. In conclusion, we are just
beginning to understand the role of NO in the regula-
tion of the immune response and a great deal of
work remains to be done before we understand this
and the mechanisms involved more fully.
1401
5. Nitric oxide in inflammation and asthma
As reviewed elsewhere in this issue w17x, NO is
generated at high levels during human inflammatory
reactions such as asthma w33x and, as in the immune
response, the principal NOS isotype involved is
NOS-2 w34x. Although the majority of mediators
generated during inflammation, such as IFN-g , TNF-
a , leukotrienes and most prostaglandins, are pro-in-
flammatory, others such as the cyclopentanone
prostaglandins are anti-inflammatory w35x. Therefore,
any mediator generated during inflammation cannot
be accused of guilt by association. Certainly the role
of NO in inflammation is uncertain. The picture may
be further complicated by NO exerting a combina-
tion of toxic, regulatory, apoptotic and anti-apoptotic
effects on different cell types at different stages of
the inflammatory process. The role of NO in contact
allergen-induced skin inflammation is likewise com-
plexNO is pro-inflammatory at low concentrations
by inducing vasodilatation and the recruitment of
neutrophils, whereas at high concentrations it down-
regulates adhesion molecules, suppresses activation
and induces apoptosis of inflammatory cells w36x.
The first event in IgE-mediated inflammatory dis-
eases such as asthma is the activation of mast cells
by antigen. Mast cells release histamine that, in
synergy with other mediators, dilates and perme-
abilises the blood vessels, leading to increased blood
supply to the tissues, raised temperature, edema and
infiltration of white blood cells. Mast cells also
release several cytokines, such as TNF-a w37x, that
may promote the later phases of inflammation by
recruiting other inflammatory cell types. It has been
known for some time that NO inhibits mast cell
activation w38x and more recently NO has been re-
ported to mediate the inhibitory effects of IFN-g on
mast cells in mixed cell populations w26,39x.
Several studies show NO inhibits mast cell activa-
tion and mast cell-dependent inflammatory processes
in vivo. Administration of NOS inhibitors to rats
enhances mucosal mast cell degranulation and gut
epithelial permeability w40x, and induces degranula-
tion of perivascular mast cells and adherence of
leukocytes to the vascular endothelium w41x. Further-
more, administration of the nitric oxide donor sper-
mine-NO to rats inhibited in vivo mast cell degranu-
lation, granulocyte adhesion to blood vessels and
1402 J.W. Colemanr International Immunopharmacology 1 (2001) 13971406
rolling, and microvascular leakage of albumin w42x.
In contrast, a NOS inhibitor suppressed antigen-in-
duced microvascular leakage in guinea pig lung in
vivo w43x, although this reaction may not have been
mast cell-dependent. Overall, the weight of evidence
from animal models indicates that NO suppresses
mast cell activation in vitro and in vivo, suggesting
that NO is anti-inflammatory at least in the acute
mast cell-dependent phase of the response. This may
be of physiological significance since NO is gener-
ated at high levels in the lungs of asthmatic patients
by NOS-2 w33,34x.
In human asthma NO may be beneficial by virtue
of its capacity to dilate airway smooth muscle, prob-
ably reflecting the natural function of NOS-1 in the
lung w44x, and as mentioned above, may inhibit mast
cell activation w2427x. On the other hand, NO causes
dilation and permeation of blood vessels and this
may contribute to airway edema w45x. Although glu-
cocorticoids are an effective treatment for asthma
and inhibit NO synthesis in asthmatic lungs w46x, this
does not necessarily imply a causative link between
NO and the disease. Overall, the role of NO in
human asthma as pro- or anti-inflammatory is unre-
solved w17x.
In mouse models of asthma, NOS-2 is up-regu-
lated upon aerosol challenge with allergen w47,48x.
Airway responsiveness to methacholine challenge is
reduced in mice genetically deleted of NOS-1 but
not NOS-2 or -3 w47x, whereas in another study
inflammatory responses including recruitment of
eosinophils, microvascular leakage, edema and air-
way occlusion were all reduced in NOS-2 knockout
mice w48x. IFN-g but not IL-4 or IL-5 production
was increased in NOS-2 knockout mice w48x, sup-
porting the idea w22x that NO produced by NOS-2
might be pro-inflammatory in the lung by selective
inhibition of IFN-g production.
6. Regulation of cell death by nitric oxide
As described in two articles in this issue w11,49x,
NO regulates death of immune cells, either through
induction or inhibition of apoptosis, or by necrosis.
In addition, NO can mediate killing of tissue-specific
cells in immunologically mediated diseases. For ex-
ample, NO production by macrophage NOS-2 is
responsible for the killing of b-islet cells in a rodent
model of autoimmune diabetes w50x. These cells enter
into the early stages of apoptosis, but subsequent
energy and ATP depletion of the cells by NO
switches the cells to necrotic cell death w11,51x. NO
regulates death of immune cells in vivo. In the
mouse thymus, production of NOS-2-dependent NO
leads to apoptotic cell death of an immature sub-
population of thymic T lymphocytes, whereas a sec-
ond mature subset is rendered resistant by NO to
dexamethasone-induced apoptosis w51x. In general,
low concentrations of NO appear to be anti-apoptotic
whereas higher concentrations induce cell death ei-
ther by apoptosis or necrosis; at higher or sustained
concentrations of NO the switch from apoptosis to
necrosis may reflect depletion of cellular energy
w11,4951x. Even so, whether NO induces or inhibits
apoptosis is highly dependent on cell type. For ex-
ample, macrophages, pancreatic cells and thymo-
cytes are highly susceptible to NO-induced apoptosis
w49x. As described by Kim et al. w49x, RNOS such as
N2 O 3 and peroxynitrite can directly modify DNA by
nitration of guanine and can induce single strand
DNA breaks. These can have down-stream conse-
quences such as up-regulation of the tumor suppres-
sor p53 or activation of nuclear polyADP-ri-
bose.polymerase, which can then lead to apoptosis.
Furthermore, NO can disrupt the zinc finger motifs
of DNA repair enzymes w11x. In apoptosis, signalling
through cell surface molecules such as fas, triggers a
family of cysteine proteases known as caspases that
target other caspase family members, diverse pro-
teases and nucleases, and this ultimately leads to
degradation of DNA. Multiple kinases that act as
intermediates in receptor-mediated activation of cas-
pases may be targets in NO-induced apoptosis w52x.
7. Nitric oxide target selectivityrelevance to im-
munity
NO is unusual as a signalling molecule since it
has no cell surface receptor but enters cells indis-
criminately. Its biological selectivity depends on: 1.
its concentration and reactivity with other molecules,
2. the proximity of target cells, and 3. the way in
which the target cell is programmed to respond.
Another important question is how the source cell
 J.W. Colemanr International Immunopharmacology 1 (2001) 13971406
protects itself from NO. Bearing in mind that the
half-life of NO increases markedly as its concentra-
tion decreases w8x. Its effects, which are dependent
on high concentrations such as toxicity mediated by
RNOS, will be most evident close to its source of
production. NO will reach lower concentrations fur-
ther from its source and here it could exert direct
effects such as activation of guanylyl cyclase, even
when derived from NOS-2. In physiological systems,
in which NO is generated at low concentrations in a
pulsatile fashion by NOS-1 or NOS-3, the target cell
for example a neuron or smooth muscle cellsis
very close to the source cell. Here the radius of NO
activity is very much less than one cell diameter.
Cells that produce NO by NOS-1 or -3 have a
very ingenious way of protecting themselves from
NO. The concentrations of intracellular calcium ions
that activate NOS-1 and NOS-3 also inhibit guanylyl
cyclase w53x. Therefore, cells in which calcium-de-
pendent constitutive NOS is activated cannot re-
spond to the NO so produced. One would expect a
similar principle to apply to cells that produce high
level NO through NOS-2. If high NOS-2 expressing
cells were inhibited or killed by NO, then they would
certainly not be able to perform their function effec-
tively. NOS-2 expressing cells probably utilise GSH
w10,11x as their defense against NO. As reviewed by
Kroncke et al. w11x, different cell types differ in their
capacity to maintain and replenish GSH. It would
certainly be of interest to establish any relationship
between the capacity of immune and inflammatory
cells to produce NO and to defend themselves against
it, by GSH or any other mechanism. As a general
rule, as in physiological systems, one would expect
high NO producing immune cells not to respond to
NO and highly NO responsive cells not to produce
NO. This principle may be borne out by T lympho-
cytes whose growth is inhibited by NO but are not
significant producers of NO w18,23x. Likewise, mast
cells are highly sensitive to NO w2427,39x but, at
least in this authors laboratory, do not produce it
w26,27x.
8. Nitric oxide signalling in the immune system
Biological signalling by NO can be classified into
direct and indirect actions Fig. 1. and it is almost
1403
certainly the latter that are most important in the
immune system.
The direct actions of NO occur at low concentra-
tions, as generated by the constitutive nitric oxide
synthases NOS-1 and NOS-3. Here, NO is not read-
ily oxidised and interacts directly with positively
charged metal ions in proteins, for example the iron
atom in the heme moiety of hemoglobin, myoglobin,
guanylyl cyclase, cytochrome P450 and NOS itself
w1,2,9x. This interaction leads to enzyme activation in
the case of guanylyl cyclase, and inhibition in the
case of cytochrome P450 or NOS. In physiological
systems, for example in smooth muscle cells, neu-
rones and platelets, activation of guanylyl cyclase by
NO yields cyclic guanosine monophosphate cGMP.
that in turn activates cGMP-dependent protein ki-
nases that mediate relaxation, neuronal transmission
and inhibition of platelet aggregation, respectively
Fig. 1..
Several points argue that direct signalling by NO
through cGMP is unlikely to be involved in immune
regulation.
1. The Afast-responseB associated with cGMP is
inconsistent with the types of response important in
immunity and inflammationfor example, expres-
sion of surface molecules and cytokines, cell growth
and differentiation and apoptosisall of which oc-
cur within hours or days rather than fractions of
seconds.
2. In immune and inflammatory cells, surface
molecule and cytokine expression, cell growth and
cell differentiation are triggered by multiple sig-
nalling pathways involving cytokine receptors, ki-
nases and transcription factors, but generally these
do not involve cGMP.
3. Cells that are important in immunity and
inflammation, such as macrophages, fibroblasts, neu-
trophils and epithelial cells, express NOS-2 rather
than the constitutive forms of NOS. NO produced by
NOS-2 at high levels is very rapidly oxidised to
RNOS.
In view of these considerations and experimental
support w1118x, signalling between immune and
inflammatory cells by NO is largely indirect through
NOS-2-derived NO and the subsequently formed
RNOS Fig. 1.. As discussed above, RNOS such as
N2 O 3 are unstable and rapidly nitrosate thiols or
amines w8x. Once intracellular GSH is saturated then
1404 J.W. Colemanr International Immunopharmacology 1 (2001) 13971406
RNOS will S-nitrosate proteins and this can lead to
changes in protein and enzyme function. GS-NO, the
product of the reaction between NO and GSH, acts
as a storage molecule for NO and GSH, both of
which can be released to S-nitrosate or S-glutathio-
late target enzymes and other proteins w54x.
RNOS react preferentially with sulfydryl contain-
ing amino acids, and will therefore irreversibly inac-
tivate enzymes and other proteins with functionally
important surface accessible thiol groups, usually on
cysteine residues w8x. Many enzymes are targeted in
this way by NO w8,16x, particularly those important
in mitochondrial respiratory pathways that are re-
quired for ATP synthesis and energy generation
w55,56x. NO and peroxynitrite inhibit mitochondrial
respiration by distinct mechanisms. Low concentra-
tions of NO directly and reversibly inhibit cy-
tochrome oxidase in competition with oxygen. How-
ever, NO binding to cytochrome c oxidase leads to
generation of superoxide that then forms peroxyni-
trite. NO itself at higher concentrations nitrosates
protein thiols on heme containing cytochrome en-
zymes, leading to inhibition of other respiratory chain
pathways w55,56x. Peroxynitrite irreversibly damages
mitochondrial complexes I, II, IV and V, as well as
acetonitase, creatine kinase, mitochondrial DNA and
superoxide dismutase. Mitochondria themselves con-
tain a calcium-dependent NOS that may be a source
of localised NO w55x. NO inhibits glyceraldehyde-3-
phosphate dehydrogenase GAPDH. by S-nitrosa-
tion, thus inhibiting glycolysis and ATP generation
w56x. Inhibition of mitochondrial respiratory path-
ways by NO leading to depletion of energy resources
would have multiple consequences and in fact might
explain many of the reported inhibitory effects of
NO on immune cell function and growth.
Other than guanylyl cyclase that has already been
discussed, a number of proteins and enzymes impor-
tant in cell signalling may be targeted by NO, as
reviewed by Schindler and Bogdan w52x in this issue.
These include the I k BrNFk B and JAKrSTAT
pathways important in cytokine signalling in immune
and inflammatory cells. NO, through RNOS, may
enhance or inhibit a range of key signalling proteins
including the kinases mitogen-activated protein ki-
nase MAPK., Erk, Jun N-terminal kinase JNK.,
stress-activated protein kinase SAPK. and src fam-
ily kinases. RNOS modify key signalling enzymes
including the G protein p21ras , protein tyrosine ki-
nases such as Fyn, protein tyrosine phosphatases and
caspases. Although in most cases the structural target
for NO is not defined, NO is known to nitrosate
cysteine118 on the GTP-binding protein p21ras , caus-
ing a conformational change that enhances the
GTPase activity of the protein w57x. p21ras is an
important signalling molecule that controls cell pro-
liferation, differentiation and apoptosis by interacting
with several downstream signalling proteins such as
Raf-1, phosphatidyl-inositol 3-kinase and MAP ki-
nase, ultimately leading to activation of the transcrip-
tion factor NFk B w52x.
NOS-2-derived NO inhibits expression of many
cytokine genes, including IL-1, IL-6, IL-8, IFN-g ,
and TNF-a by various immune cell types w11,16x. In
addition to possible effects of NO on kinases and
upstream signalling molecules w52x, NO can S-nitro-
sate, through RNOS, cysteine-rich transcription fac-
tors w11,16x. For example, NO inhibits the DNA
binding activity of NFk B, AP-1 and c-Myb w11x.
Each of these contains a cysteine residue in or near
their DNA binding region, which probably represents
the target site for S-nitrosation. Also blocked by NO
are transcription factors such as SP1 and EGR-1,
which incorporate the Cys 2 His 2 zinc finger type
DNA-binding motif. SP1 and EGR-1 are expressed
in all cell types and are responsible for constitutive
rather than induced gene expression w58x. Kroncke
et
al. w11,59x provide evidence for direct nitrosation of
SP1 and EGR-1 by the NO donor S-nitrosocysteine,
as well as nitration of the same nuclear proteins by
exposure of intact cells to NO. NFAT, which is also
involved in IL-2 regulation but lacks a zinc finger
motif or cysteine residues in its DNA binding do-
main, is insensitive to NO w11,59x. SP1 can also act
as a suppressor and its inactivation therefore explains
the reported TNF-a-inducing effect of NO in some
systems w60x.
9. Conclusions
NO is generated, largely by the NOS-2 enzyme,
in many cell types involved in immunity and inflam-
mation. It exerts complex regulatory activity on the
function, growth and death of many immune and
inflammatory cell types both in vitro and in vivo.
 J.W. Colemanr International Immunopharmacology 1 (2001) 13971406
The signalling processes through which NO acts to
regulate these cells are extremely complex and are
only just beginning to be unraveled, but are largely
indirect Fig. 1. through generation of reactive nitro-
gen oxide species that chemically modify enzymes,
signalling proteins and transcription factors. There
remain considerable gaps in our knowledge regard-
ing the role of NO in vivo, particularly in humans.
As always in science, what we do know is overshad-
owed by what we do not know. Future major direc-
tions will focus on molecular mechanisms of action
of NO, its target molecules and cells, and its role in
infection and immunologically mediated diseases.
Acknowledgements
The authors work is supported by the Medical
Research Council and The Wellcome Trust.
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