64
Nitric oxide, nitric oxide derivatives and reactive oxygen
intermediates are toxic molecules of the immune system which
contribute to the control of microbial pathogens and tumors.
There is recent evidence for additional functions of these
oxygen metabolites in innate and adaptive immunity; these
functions include the modulation of the cytokine response of
lymphocytes and the regulation of immune cell apoptosis, as
well as immunodeviating effects. Components of several signal
transduction pathways have been identified as intracellular
targets for reactive nitrogen and oxygen intermediates.
Addresses
*†Institute of Clinical Microbiology, Immunology and Hygiene,
University of Erlangen, Wasserturmstrasse 3, D-91054 Erlangen,
Germany
‡Department of Molecular and Cell Biology, 485 Life Sciences
Addition, University of California, Berkeley, CA 94720-3200, USA
Correspondence: Christian Bogdan;
e-mail: christian.bogdan@mikrobio.med.uni-erlangen.de
Current Opinion in Immunology 2000, 12:64–76
0952-7915/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
IFN interferon
LPS lipopolysaccharide
MPO myeloperoxidase
NK natural killer
NO nitric oxide
NOS NO synthase
RNI reactive nitrogen intermediate
ROI reactive oxygen intermediate
SOD superoxide dismutase
TGF-ββ transforming growth factor β
TNF-αα tumour necrosis factor α
Introduction
The main function of the immune system is the defense of
the host organism against infectious agents and malignant
tumors. Macrophages, neutrophils and other phagocytic
cells are key components of the antimicrobial and tumoricidal
immune responses, which is due to the fact that these
cells are capable of generating large amounts of highly
toxic molecules — reactive oxygen intermediates (ROIs)
and reactive nitrogen intermediates (RNIs) (Figure 1).
The production of ROIs is initiated by NADPH oxidase,
which becomes activated upon translocation of several
cytosolic proteins (i.e. gp40phox, gp47phox, gp67phox and the
Rho-family GTPase, Rac2) to the membrane-bound complex
carrying cytochrome C (gp91phox–gp22phox–Rap1a).
The activation of NADPH oxidase can be elicited by
microbial products (e.g. lipopolysaccharide [LPS; also
known as endotoxin] and lipoproteins), by IFN-γ, by IL-8
or by IgG-binding to Fc-receptors. The primary product of
the reaction catalyzed by the NADPH oxidase is superoxide
(O2
–), which can be converted to H2O2 by superoxide
dismutase (SOD), to hydroxyl radicals (.OH) and hydroxyl
anions (OH–) by the iron-catalyzed Haber-Weiss reaction
or, after dismutation to H2O2, to hypochlorous acid (HOCl)
and chloramines by myeloperoxidase (MPO) (reviewed in
[1]; Figure 1). Although each of these oxygen metabolites
exerts antimicrobial activity against extracellular bacteria, it
is still not clear to what extent phagocytes other than neutrophils
(notably macrophages) utilize ROIs for the control
of intracellular microbes in vivo. It is important to bear in
mind that an ROI (O2
–) is also generated by the mitochondrial
electron transport chain [2] and, under conditions of
L-arginine depletion, by nitric oxide (NO) synthases
(NOSs) (see [3] and references therein). Therefore, the
production of ROIs is by no means restricted to phagocytic
effector cells and there are more functions of ROIs in addition
to antimicrobial activities.
All NOSs convert the amino acid L-arginine and molecular
oxygen to L-citrulline and NO (·NO radicals or subsequent
intermediates), and require NADPH, FAD, FMN, tetrathydrobiopterin
(BH4) and a thiol donor as cosubstrates and
cofactors. Currently, there are three major NOS isoforms
known that are named after the prototypic cell type from
which the respective isoform was first isolated as well as after
the main characteristic of their regulation. The neuronal
NOS (also known as ncNOS or NOS1) and the endothelial
NOS (ecNOS or NOS3) are constitutively expressed; they
exist as preformed monomers in the cell, which dimerize and
gain activity upon Ca2+ influx and binding of calmodulin.
The macrophage NOS (macNOS, iNOS or NOS2), in contrast,
is an inducible isoform that is absent in strictly resting
cells, is strongly induced by cytokines and other immunological
stimuli and is regulated on transcriptional and
post-transcriptional levels involving a number of signal transduction
pathways and molecules: Jak1/Stat1α/IRF-1;
IκB/NF-κB; mitogen-activated protein kinases; protein
kinase C; phosphatidylinositol-3 kinase; protein tyrosine
phosphatases; and protein phosphatases 1 and 2A (reviewed
in [4,5•]). The various NOS isoforms are also subject to regulation
by the availability of L-arginine and BH4, which are
required for the formation of active NOS dimers. Virtually
every cell of the immune system has been described to
express one or several isoforms of NOS and most probably
every type of mammalian cell is capable of generating NO.
NOS activity (NOS1, NOS2) is found in the cytosol
(attached to cytoskeletal proteins) but can also be targeted to
plasmalemmal caveolae (NOS3) or localized in vesicles
(NOS2) or in mitochondria (reviewed in [6,7]). The primary
NO species that is synthesized by the NOSs has been a matter
of debate because it is very much dependent on the
Reactive oxygen and reactive nitrogen intermediates in innate
and specific immunity
Christian Bogdan*, Martin Röllinghoff† and Andreas Diefenbach‡
imc107.qxd 02/15/2000 02:00 Page 64
Reactive oxygen and reactive nitrogen intermediates in immunity Bogdan, Röllinghoff and Diefenbach 65
composition of the intra- and extra-cellular micromilieu. In
the strict absence of O2
–, the ·NO radical will be formed
whereas in the presence of ROIs, peroxynitrite (ONOO–)
and other RNIs might prevail (see [8] and references therein).
Reaction of ·NO with SH-groups of free amino acids, of
peptides or of proteins will give rise to S-nitrosothiols; this is
not only a mechanism for scavenging NO but also serves to
transport NO and is the molecular basis for many of the regulatory
functions of NO described below (see [9] and
references therein; see also Figure 1).
The original concept that the small quantities of NO generated
in a pulsatile fashion by constitutive NOSs mainly fulfil
regulatory functions required for normal homeostatic function
of the vasculature and the central nervous system, whereas
the high amounts of NO produced by NOS2 primarily exert
antimicrobial and cytotoxic effects in the immune system, has
recently seen several important modifications. In this brief
update covering the advances of the past year, we will highlight
new aspects of the interaction between infectious agents
and ROIs/RNIs, outline some immunoregulatory activities of
ROIs and NOS2-derived NO and discuss the evidence for a
role of ncNOS/ecNOS-derived NO in the immune system.
Regulation of NO production
Cytokines
The most characteristic feature of NOS2 is its prominent
regulation by activating cytokines (e.g. IFN-γ and TNF-α)
Figure 1
Enzyme NOS2 NADPH oxidaseMPO
(Co-)substrates H2O2, Cl– L-arginine, O2, NADPH O2, NADPH
Key products HOCl Citrulline
.NO
O2
–
.OH
1O2
NO2
–
S-nitroso-
thiols
ONOO– H2O2
Fe2+
(Fenton)
Fe3+/Fe2+
(Haber-Weiss)
OH–
.OH
OH–
SOD
.OH
NO2Cl
.NO2
.Cl
Examples of effects Oxidation
Chlorination
Tyrosine nitration
S-nitros(yl)ation
Disruption of FeSor
ZnS-clusters
Tyrosine
nitration
Oxidation
Examples of microbial
resistance genes
ahpC
metL
hmp
noxR1, noxR3
Urease? sodA
katG
gorA
ahpC
(a)
(c)
(d)
(e)
Current Opinion in Immunology
(b)
Pathways for the generation of ROIs and RNIs in antimicrobial effector
cells. Phagocytes generate ROIs and RNIs by MPO, NOS2 or
NADPH oxidase. There are several known interactions between these
antimicrobial effector pathways: (a) the utilization of NOS2-derived
nitrite (NO2
–) in the MPO pathway, leading to the production of nitryl
chloride (NO2Cl) and nitrogen dioxide (NO2); (b) the generation of
peroxynitrite (ONOO–) from nitric oxide (·NO) and superoxide (O2
–);
and (c) the consumption in the MPO pathway of peroxide (H202) that
(d) is generated from O2
– by the action of SOD. (e) Ferrous iron
(Fe2+) and H2O2 will yield OH–, ·OH and ferric iron (Fe3+) (Fenton
reaction). In the presence of O2
–, Fe3+ will be reduced to Fe2+, which
then again allows for the conversion of H2O2 to ·OH (Haber-Weiss
reaction). Microbes are equipped with a variety of defense
mechanisms (resistance genes) that help them to survive the oxidative
and nitrosative stress (see text). The expression of NOS2 is not
restricted to phagocytes but is also found in other cells (e.g.
fibroblasts, endothelial and epithelial cells, keratinocytes, and certain B
and T cell lines). Some of the biological effects of NOS2-derived NO
or of NADPH-oxidase-derived ROIs are likely to be also achieved by
NO derived from constitutive NO synthases (NOS1, NOS3) or by
ROIs generated in the mitochondrial respiratory chain (see text for
examples).
imc107.qxd 02/15/2000 02:00 Page 65
66 Innate immunity
Table 1
The course of infections in transgenic mice deficient for oxygen-dependent antimicrobial effector mechanisms.
Defective Infectious agent Route of infection; form of disease Effect on References
molecule disease severity
NOS2 Coxsackie virus B3 i.p.; myocarditis ↑ [112]*
Ectromelia virus i.v.; mouse pox ↑ [113]†
(liver, spleen, lung)
Herpes simplex virus type 1 s.c.; cutaneous infection, ↑ [114]*
dorsal root ganglia (reactivation)
Influenza virus A i.n.; pneumonitis ↓ [32]†
Chlamydia trachomatis (MoPn strain) i.vg.; vaginal infection – [115,116]†
i.vg.; mouse pneumonia (spleen infection) ↑ [117]†
Chlamydia pneumoniae i.n.; pneumonitis ↑ [118]†
Helicobacter pylori p.o.; chronic gastritis – [58]†
Listeria monocytogenes i.v. or i.p.; listeriosis ↑ [119]†
(EGD 3B or ATCC 10403 strains) (liver, spleen) [30••]†
Mycobacterium tuberculosis i.v.; tuberculosis (lung, liver, spleen) ↑ [120]†
Mycobacterium avium i.v.; systemic infection (lung, spleen, liver) — or ↓ [121,122]†
Mycoplasma pulmonis (UAB CT strain) i.n.; pneumonia ↑ [40]†
Salmonella typhimurium (14028 strain) i.p.; systemic infection ↑ [30••]†
Salmonella typhimurium (recBC mutant)‡ i.p.; systemic infection – [30••]†
Shigella flexneri i.n.; bronchopulmonary infection – [123]†
Staphylococcus aureus (TSST-1+ LS-1 strain) i.v.; septic arthritis ↑ [124]*
Leishmania major s.c.; cutaneous leishmaniasis ↑ (non-healing) [13•]†,[33]*
↑ (healing) [34•]*
Leishmania donovani i.v.; visceral leishmaniasis ↑ [125•]†
Plasmodium berghei ANKA i.v.; murine cerebral malaria – [126]†
Plasmodium berghei XAT i.v.; blood-stage malaria – [127]†
Trypanosoma cruzi (Tulahuen strain) i.p.; experimental Chagas´ disease ↑ [128]†
(spleen, liver, heart)
Trypanosoma brucei i.p.; experimental sleeping sickness ↓ or – [129]*;[130]†
Toxoplasma gondii i.p. or p.o.; cerebral toxoplasmosis ↑ [131]†
p.o.; inflammation of small intestine and liver ↓ [132]†
Schistosoma mansoni p.c.; immunized NOS2+/+ or NOS2–/– (↑)§ [133]†
mice challenged with live cercariae [134]*
gp47phox Commensal Staphylococci Spontaneous development of lethal deep ↑ [135]
and fungi infections (CGD mouse model)
Mycobacterium avium Mycobacteriosis (spleen and lung) – [73]
Listeria monocytogenes Listeriosis (liver, spleen) ↑ [136]
gp91phox Commensal bacteria CGD mouse model ↑ [137]
Listeria monocytogenes i.p.; listeriosis (liver, spleen) ↑ (day 2) [30••,138]
– (day 3 or 6)
Salmonella typhimurium (14028 strain) i.p.; systemic infection ↑ [30••,50]
Salmonella typhimurium (recBC mutant)‡ i.p.; systemic infection ↑ [30••]
Leishmania donovani i.v.; visceral leishmaniasis ↑ (but healing) [125•]
Aspergillus fumigatus i.t.; pulmonary aspergillosis ↑ (lethal) [74]
Rac2 Aspergillus fumigatus i.v.; disseminated aspergillosis (kidney, brain) ↑ [139]
NOS2 plus Listeria monocytogenes i.p.; listeriosis (liver, spleen) – (50%) or ↑ (50%) [30••]†
gp91phox (ATCC 10403 strain)
Salmonella typhimurium (14028 strain) i.p.; systemic infection ↑ [30••]†
Salmonella typhimurium (recBC mutant)‡ i.p.; systemic infection ↑ [30••]†
MPO Staphylococcus aureus i.p.; peritonitis – [43]
Candida albicans i.t., pneumonia; ↑ [43]
i.p., disseminated candidiasis ↑
*In these studies NOS2-mutant mice were used, in which the intended
deletion of exon 1 to 5 of the NOS2 gene had not occurred, and an
alternative mRNA transcript of NOS2 was expressed [33] that later turned
out to express reduced, but clearly functional, levels of NOS2 [34•]. †In
these studies NOS2-deficient mice were used, in which the proximal 585
basepairs of the NOS2 promotor and exon 1 to 4 of the NOS2 gene were
deleted and no NOS2 mRNA, protein or activity was detectable. ‡recBC
mutants of S. typhimurium have a strongly reduced ability for DNA repair
after oxidative damage. §The protective effect of vaccination is reduced in
NOS2–/–, compared with NOS2+/+ mice. CGD, chronic granulomatous
disease; i.n., intranasal; i.p., intraperitoneal; i.t., intratracheal; i.v.,
intravenous; i.vg., intravaginal; p.c., percutaneous; p.o., per os.
imc107.qxd 02/15/2000 02:00 Page 66
Reactive oxygen and reactive nitrogen intermediates in immunity Bogdan, Röllinghoff and Diefenbach 67
or inhibitory cytokines (e.g. IL-4, IL-10, IL-13 and transforming
growth factor β [TGF-β]) [4,5•]. These are
produced by alternative populations of Th cells (Th1 or
Th2), which therefore strongly affect the degree of NOS2
expression [10•]. The regulation of NOS2 can occur on
multiple levels: NOS2 gene transcription, mRNA stability,
mRNA translation and protein stability; the availability of
substrates, cofactors and endogenous substrate analogues
acting as NOS inhibitors; and the feedback effects of the
end product (NO). More than 30 cytokines or cytokine-like
factors have been described to date that increase or inhibit
the expression of NOS2 activity in cells participating in the
immune response: macrophages; microglia; Kupffer cells;
neutrophils; eosinophils; mast cells; dendritic cells; natural
killer (NK) cells; certain T cell and B cell lines; keratinocytes;
fibroblasts; endothelial and epithelial cells; and
vascular smooth muscle cells. Some cytokines —such as
IFN-α/β, IL-4 or IL-10 — can exert bimodal effects on the
production of NO by macrophages (reviewed in [5•]). IFNα/β,
which on their own are unable to induce NOS2 except
in human monocytes [11,12], synergize with LPS or
Leishmania major for the induction of NOS2 under costimulation
conditions but have an inhibitory effect when
added to rodent macrophages prior to the second stimulus
(IFN-γ, LPS or L. major) ([13•,14•]; C Bogdan, unpublished
data). In the latter situation, IFN-α/β have been shown to
inhibit the activation of NF-κB [14•] or Stat1α (C Bogdan,
unpublished data), two transcription factors that are critical
for the induction of NOS.
Cell surface receptors
NOS2 is regulated by crosslinking of cell-surface receptors
such as CD23 (Fcγ receptor IIb) [5•] or CD8 (α- or β-chain)
[15]. Crosslinking of CD23 or CD8 leads to induction of
NOS2 in human monocytes or rat macrophages, respectively.
The adenosine receptor A2B, which is induced by IFN-γ,
in contrast mediates a feedback inhibitory effect on the
expression of NOS2 mRNA in mouse macrophages upon
binding of adenosine or adenosine analogues [16•].
Microbial products
Another group of NOS2 regulators are products of viruses,
bacteria, protozoa or fungi. LPS is the prototypic example; it
stimulates rodent macrophages to express NOS2, at least
partly via induction of endogenous IFN-α/β [17]. LPS usually
synergizes with IFN-γ for the induction of NOS2 but
can also have antagonistic effects, depending on the concentration
and sequence of the stimulation (reviewed in
[5•]). Similar to LPS, other microbial lipoproteins (e.g. the
19 kDa Mycobacterium tuberculosis lipoprotein or Borrelia
burgdorferi OspA [18•]) and bacterial exotoxins (e.g. pneumolysin
of Streptococcus pneumoniae or tracheal cytotoxin of
Bordetella pertussis) induced the expression of NOS2 activity
in macrophages and other cell types. In some cases their
Table 2
Examples of NO as a signalling molecule in immunity.
Cell population Stimulus Source of NO Phenotypic effect of NO Molecular effect Reference
and target of NO
Mouse macrophages Mitogen (ConA) Endogenous (NOS2) Reduced expression of NF-κB- Inhibition of NF-κB p50 [103•]
(ANA-1) dependent genes DNA binding by
(e.g. NOS2 and M-CSF) S-nitrosylation of p50
Mouse macrophages IFN-γ + LPS Endogenous (NOS2) Inhibition of IL-1β and Inhibition of caspase-1 [102•]
(RAW264.7) or NO donor (SNAP) IL-18 release* and caspase-1-mediated
cleavage of pro-IL-1β
and pro-IL-18
Primary mouse T Mitogen (ConA) Mouse alveolar or Inhibition of T cell proliferation Inhibition of Jak3 and [99]
lymphocytes peritoneal Stat5 tyrosine
macrophages or phosphorylation
NO donor (SNAP)
Human Burkitt’s Anti-Fas Endogenous (NOS2) or n.a. Inhibition of caspase-3 by [69••]
lymphoma (10C9) or NO donor (SNAP) S-nitrosylation; anti-Fas
T cell leukemia lines leads to caspase-3
(Jurkat, CEM) denitrosylation
Mouse pro-B cell-line IL-3 NO gas or NO donor Inhibition of proliferation Inhibition of Jak2 kinase [98]
(Ba/F3) (>1 mM DETA-NO) activity
Mouse NK cells (cell IL-12 Endogenous (NOS2) or Prerequisite for IL-12- Cofactor for Tyk2 kinase [31••]
line or IL-2-expanded) NO donor (1–100 µM induced IFN-γ production* activity
primary NK cells) SNAP or DETA-NO)
Murine endothelial TNF-α NO donor Enhanced (25–250 µM) or Inhibition of IκB-kinase-α [100•]
cell line (TC10) (glycerol-trinitrate) diminished (>500 µM)
DNA-binding of NF-κB
*Study also presents evidence for a similar effect of NO in vivo. DETA-NO, diethylenetriamine nitric oxide; M-CSF, macrophage-colony-stimulating
factor; n.a., not analysed; SNAP, S-nitroso-N-acetylpenicillamine.
imc107.qxd 02/15/2000 02:00 Page 67
68 Innate immunity
action was dependent on the presence of LPS or of IFN-γ
[19,20•] and was strongly associated with deleterious rather
than host-protective effects [20•].
Several infectious pathogens (e.g. Leishmania spp. or
Candida albicans) were reported to inhibit the expression of
NOS2 in macrophages [21•,22]; this might contribute to
the very limited expression of NOS2 early during infection
[13•] and facilitate the initial survival of the parasites in the
host. In the case of L. major, the suppression appears to be
mediated by glycoinositolphospholipids of the parasites
[5•]. For L. donovani, it was reported that the parasites
might activate cellular phosphotyrosine phosphatases and
thereby impair the signalling cascade that otherwise leads
to the expression of NOS2 [21•].
Other regulatory factors
A recent study showed that the human heat shock protein
(hsp)60 has some capacity to stimulate the J774 mouse
macrophage line for the production of NO but the
(patho)physiologic relevance of this finding is presently
unclear despite the known release of intracellular hsp60
during cell necrosis [23•]. In addition to the macromolecular
regulators of NOS2, several small inorganic molecules
have been described to affect the expression and/or activity
of NOS2. Depletion of non-ferritin-bound iron (Fe2+)
[24], a decrease of oxygen tension [25] or low environmental
pH [26] have all been found to enhance NOS2
gene transcription. This might accelerate the resolution of
infections with intracellular pathogens but could also promote
tissue damage during chronic inflammatory
processes. Finally, the expression of NOS2 is also positively
or negatively affected by a number of drugs
(e.g. glucocorticoids, aspirin, taxol, tetracyclines or antifungal
imidazoles) that are used for the treatment of
autoimmune, infectious or malignant diseases [5•,27–29].
To date it is unclear to what extent the induction or suppression
of NOS2 promotes or counteracts the therapeutic
value of these compounds in humans.
Antimicrobial activity of RNIs and ROIs
In vivo models
The antimicrobial activity of ROIs and RNIs has been
most convincingly demonstrated by the use of transgenic
mice deficient for one or several antimicrobial effector
pathways. These studies have been informative in several
respects. First, they showed that a broad spectrum of infectious
agents (ranging from viruses to helminths) is directly
or indirectly controlled by RNIs and/or ROIs in vivo;
importantly, these studies did not rely on ROI or RNI
inhibitors or scavengers with questionable activity or specificity
(Table 1). Second, Salmonella typhimurium or
Listeria monocytogenes infection of macrophages from
mice lacking both NADPH oxidase and NOS2 provided
evidence for an additional mechanism that impedes the
replication of these bacteria [30••]. Third, in some models
NOS2-derived NO was produced and functional
already during the very early (innate) phase of infection;
this demonstrates that the expression of NOS2 is not
restricted to the adaptive phase of the immune response,
when IFN-γ-producing Th cells prevail [13•,31••]. Fourth,
the analysis of NOS2–/– mice revealed that NOS2-derived
NO is dispensable for the control of several pathogens
(Table 1) or might be even counterprotective [5•,32]. Some
of these results, however, will require re-evaluation
because they were obtained with a strain of NOS2-mutant
mice [33] in which the NOS2 gene was not deleted and an
alternative NOS2 transcript was expressed. Although these
mice were originally reported to lack NOS2 activity and to
develop progressive leishmaniasis upon infection with
L. major [33], a recent analysis by the same group demonstrated
residual NOS2 activity in these mice that was
functional and sufficient to heal an infection with L. major
[34•]. These data disprove the recently proposed existence
of an antimicrobial mechanism that even in the absence of
NOS2 is sufficient to control L. major in vivo [35].
Mechanisms of antimicrobial activity
In the past year several studies have provided important
new insights into the mechanisms of antimicrobial function
of RNIs and ROIs (for previous reviews, see [36,37]).
Saura et al. [38••] identified (in Coxsackie-virus-infected
epithelial cells) the viral cysteine protease 3C as the target
of macrophage-derived NO, which inactivates the protease
via S-nitrosylation of the cysteine residue in the
active site and thereby interrupts the viral life cycle; this
study provided strong evidence for a direct antiviral effect
of NO. Although NO donors could substitute for
cytokine-activated macrophages in this system, this does
not imply that the ·NO radical is always the active molecular
species. In macrophages cocultured with the
extracellular protozoon Trypanosoma musculi, the NOS2dependent
killing of the parasites was strictly dependent
on the presence of albumin; S-nitrosylated albumin, but
not free NO, exerted the antiparasitic effect [39]. In other
systems, peroxinitrite (ONOO–) rather than NO or O2
–
turned out to account for the killing of the infectious
pathogen by macrophages [40]. Although NO and O2
– are
usually derived from host cells, significant amounts of O2
–
can also be produced by certain bacteria (e.g. Helicobacter
pylori); such O2
–, upon reaction with host-cell-derived NO
and formation of peroxinitrite (see Figure 1), blocks
microbial respiration [41].
Another form of agonistic interaction between RNIs and
ROIs was recently reported by Andonegui et al. [37], who
found that long-term treatment (8–18 hours) of human
neutrophils with NO donors leads to an increased production
of O2
– and H2O2. However, previous studies have
reached the opposite conclusion — they showed inhibition
of NADPH oxidase activity after short-term exposure
(2 minutes) to NO (see [42] and references therein).
Although the actual level to which the MPO/H2O2/Cl– pathway
contributes to the defense against microbial pathogens
in vivo has been controversial throughout the years [1],
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Reactive oxygen and reactive nitrogen intermediates in immunity Bogdan, Röllinghoff and Diefenbach 69
recent in vitro and in vivo studies have refueled ideas supporting
its biological significance [43,44]. Experiments with
MPO–/– mice (Table 1) confirmed earlier clinical experience
with MPO-deficient patients, some of whom developed
major systemic (fungal) infections — in particular with
C. albicans (see [45] and references therein). Furthermore,
activated human neutrophils were shown to use MPO for
conversion of nitrite (NO2
–) into the oxidants nitryl chloride
(NO2Cl) and nitrogen dioxide (.NO2) that can cause tyrosine
nitration and chlorination of target molecules [46••]
(Figure 1). NO2
–-dependent tyrosine nitration reactions also
occur in human eosinophils from MPO-deficient individuals,
which possibly explains why most of these patients are
not at an increased risk of infection [47]. In vivo, the nitrite
might be supplied by activated macrophages in the vicinity
of the neutrophils or eosinophils, or might originate from the
primary granules of neutrophils, in which MPO was found to
colocalize with NOS2 [48]. The convergence of the NOS2
and MPO pathways might represent a novel mechanism of
the antimicrobial machinery of granulocytes.
Resistance to ROIs and RNIs
Phenotypic resistance to ROIs has been observed in a
number of bacteria (e.g. koagulase-negative staphylococci,
Escherichia coli, Salmonella typhimurium and M. tuberculosis)
and involves the expression of O2
–- and H2O2-scavenging
enzymes such as SOD, catalase and glutathione reductase.
In M. tuberculosis, expression of even small amounts of
catalase activity strongly protected the bacilli against
oxidative killing by H2O2 [49]. In S. typhimurium, periplasmic
copper/zinc-SOD detoxified O2
– and thereby most
probably impeded the formation of ONOO–, thus protecting
the bacteria against O2
– and NO [50]. In E. coli,
exposure to O2
– activates a distinct set of antioxidant
genes that are under the control of the SoxRS regulon
(e.g. genes for manganese-containing SOD [the gene is
called sodA], oxidative DNA repair enzyme endonuclease
IV [nfo] and glucose-6-phosphate dehydrogenase [zwf]).
Similarly, H2O2 stimulates the thiol-containing transcriptional
activator OxyR which controls the expression of
genes for catalase/hydroperoxidase (katG), glutathion
reductase (gorA) and alkyl hydroperoxidase reductase subunit
C (ahpC) in E. coli and S. typhimurium. Interestingly,
both the SoxRS and the OxyR regulon are also responsive
to NO or S-nitrosothiols, suggesting a common defense
strategy against nitrosative and oxidative stress (see
[51,52••] and references therein; see also Figure 1).
Recently, several genes and proteins were identified that
confer protection against RNIs in E. coli, S. typhimurium
and/or M. tuberculosis as shown by gene overexpression or
by complementation of mutants. These include four
genes products in particular: first, flavohemoglobin (hmp),
which exhibits an NO dioxygenase activity (i.e. it detoxifies
NO to NO3
–) and is induced by NO via inactivation
of an iron-dependent repressor [53–55]; second, alkyl
hydroperoxidase reductase subunit C (ahpC), which is
regulated by OxyR but whose mechanism of protection
against RNIs is unknown [52••]; third, the bifunctional
enzyme aspartokinase II-homoserine dehydrogenase II
(metL), which is critical for the synthesis of homocysteine
— an antagonist of S-nitrosothiols in S. typhimurium
[56]; and, fourth, two novel genes (noxR1 and noxR3) that
were isolated from M. tuberculosis, protect against both
RNIs and ROIs by an as-yet unknown mechanism and
are not expressed in nonpathogenic or opportunistic
mycobacteria (see [57] and references therein). In
H. pylori, CO2/HCO3
– produced by the bacterial urease
might protect the microbe against peroxynitrite [58].
Thus, pathogenic bacteria are most probably equipped
with a whole array of mechanisms that mediate resistance
to oxidative and nitrosative effector molecules of the
host (Figure 1).
Although studied to a much smaller extent, antioxidant
mechanisms also exist in protozoa (reviewed in [59]) and
fungi (see [60] and references therein) and are similarly
critical for the survival of the parasites in the host.
Immunoregulation by RNIs and ROIs
The first immunoregulatory activity that could be assigned
to ROIs and RNIs was their ability to inhibit the proliferation
of lymphocytes. Together with prostaglandins, the
production of NO by NOS2 and of ROIs by the NADPH
oxidase are the key mechanisms by which ‘suppressor
macrophages’ impair the proliferative response of T lymphocytes
to antigens or mitogens. This ROI/RNI-mediated
inhibition of T cell proliferation at least partially accounts
for the immunosuppressed state seen in certain infectious
diseases, malignancies and graft-versus-host reactions but
might also serve to control inflammatory processes or to
delete autoreactive T cells and thereby fulfils host-protective
functions (reviewed in [5•]). Recently, a number of
molecularly defined mechanisms have emerged by which
ROIs and RNIs modulate the immune response.
Cytokine production
To date, (NOS2-derived) NO is known to affect the production
of more than 20 cytokines (including IL-1, IL-6, IL-8,
IL-10, IL-12, IFN-γ, TNF-α and TGF-β) by various
immune cells (e.g. macrophages, T lymphocytes, NK cells
and endothelial cells) as assessed by the use of NOS
inhibitors, NOS2–/– mice and/or NO donors (reviewed in
[5•]). Although in most cases the pathway that is subject to
regulation by NO remains to be identified, a number of signal-transduction
molecules are already known to be positively
or negatively regulated by NO (see below and Table 2). For
several cytokines, conflicting results have been reported as to
the effect of NO; in some cases this might reflect the use of
different cell populations. The production of IL-12 (p40 or
p70) by macrophages (for example) was facilitated, inhibited
or remained unaltered by NOS2-derived NO [13•,61–63]. In
rat and mouse NK cells, NOS2-derived NO was partially or
absolutely required for the production of IFN-γ in response to
IL-2 or IL-12 (respectively) whereas in human NK cells
expression of NOS2 inhibited the subsequent secretion of
imc107.qxd 02/15/2000 02:00 Page 69
70 Innate immunity
IFN-γ [13•,31••,64,65•]. NOS2-dependent production of
IFN-γ by NK cells has also been observed during the innate
phase of the immune response of mice infected with
L. major [13•,31••].
Whether NO also regulates the cytokine production of Th
cells has received considerable attention during the past
years because it might be relevant for the development of
a Th1 response compared with a Th2 response. The published
results have not been uniform. Controversy exists
with respect to three aspects: first, the level of expression
of NOS2 by T lymphocytes [66–68,69••]; second, the
effect of exogenous NO on the cytokine production by primary
T cells [34•,68,70]; and, third, with respect to a
(possibly selective) effect of exogenous NO on the
cytokine release by established Th1 (compared with Th2)
lines and clones [34•,66,68,71,72]. Differences in the cell
type (primary compared with cloned, mouse compared
with human, transformed compared with nontransformed),
the donors (e.g. agents releasing ·NO, NO+, ·NO plus O2
–
or ·NO plus cyanide) and their concentrations, and the
stimulation conditions (mitogen compared with antigenpresenting
cells [APCs] plus antigen) are parameters that
are likely to determine whether NO stimulates, inhibits or
does not alter T cell differentiation and function. This is
underlined by a recent study which demonstrates that low
doses (1–10 µM) of S-nitrosopenicillamin upregulated,
whereas higher concentrations (100 µM) suppressed, the
production of IFN-γ by primary T cells cultured with
APCs, antigen and IL-12. The cytokine response of established
T cell lines, in contrast, was largely inert in response
to the NO donor [34•]. As the actual concentrations of
NOS2-derived NO in situ are variable and not predictable,
it is impossible to make a definitive statement on the
effect of NO on Th1/Th2 development. Accordingly,
NOS2 deficiency in mice was found to be associated with
an enhanced, an unaltered or a diminished Th1 response
in different infectious disease models (reviewed in [5•]).
Compared with the results on RNIs there is much less evidence
for a cytokine-regulatory function of ROIs in vivo. This
might be due to the fact that there is more redundancy in the
production of ROIs compared with RNIs. In mice lacking the
gp47phox protein that were infected with Mycobacterium avium
or Schistosoma mansoni eggs, there was no difference in the
development of the Th cell cytokine response when compared
with wild-type mice [73]. In mice deficient for the
gp91phox and challenged with sterile Aspergillus fumigatus
hyphal cell walls, the expression of IL-1β, TNF-α, the
chemokines KC and JE, and of TGF-β were significantly elevated
in the lungs compared with wild-type controls [74].
Apoptosis
Native NO or S-nitrosothiols can induce or block apoptosis
in many cell types. Proapoptotic effects (e.g. in
macrophages or T lymphocytes) were observed with high
(100–200 µM) concentrations of exogenous NO and were
associated with the accumulation of p53, which at least in
part is due to an NO-mediated inhibition of the degradation
of polyubiquitinated p53 by the 26S proteasome (see
[75•] and references therein). Peroxynitrite (ONOO–)
primed T lymphocytes to undergo apoptotic cell death
after subsequent activation by phorbol esters or anti-CD3.
The effect of ONOO– is likely to involve an inhibition of
protein tyrosine phosphorylation via nitration of tyrosine
residues [76]. Other mechanisms by which (endogenous)
NO might promote apoptotic events include the upregulation
of the cell surface receptor Fas (CD95) or its ligand
(FasL, CD95L) [77,78•]. NO, however, can also induce
apoptosis without measurable modulation of Fas/FasL
[79]. There is considerable evidence that NOS2-derived
NO contributes to the deletion of double-positive T lymphocytes
in the thymus and to the elimination of
autoreactive T cells (see [80,81•] and references therein).
In the immune system, an antiapoptotic function of endogenous
NO generated by NOS2 has been observed in B cell
lines, macrophages, eosinophils and T lymphocytes
(reviewed in [5•]). In mouse macrophages, nontoxic concentrations
of NO lead to the activation of the transcription
factors NF-κB and AP-1 and the subsequent expression of
cyclooxygenase-2 which protected the macrophages against
apoptosis [82]. In human eosinophils, NO inhibited Fasreceptor-induced
activation of Jun kinase and the
proteolysis of lamin B1 — a protein that is known to rapidly
decrease in apoptotic cells [83]. In resting human B and T
cell lines, the unprocessed (zymogen) form of caspase-3, one
of the downstream caspases executing apoptosis, was found
to be S-nitrosylated on its active-site cysteine. Upon activation
of the cells via crosslinking of Fas, the caspase-3
zymogen was cleaved into its active subunits and the activesite
thiol was denitrosylated [69••]. As NO inhibits the
activity of caspase-3 in cell-free systems (via S-nitrosylation)
as well as in intact cells (see [69••,84] and references therein),
these data suggest that S-nitrosylation helps to maintain
the proenzyme in an inactive form and that denitrosylation
of caspase-3 is an integral part of the Fas apoptotic pathway.
Similar observations have also been made with hepatocytes,
in which NO protected against TNF-α-induced activation
of caspase-3 and -8 and apoptotic cell death [85]. This might
explain the impaired liver regeneration after injury in mice
lacking NOS2 activity [86]. Despite this cell-protective
function of NO, it is important to bear in mind that NO,
while inhibiting apoptotic events, can still cause necrotic
cell death in the same cells [87]. The reversion of blocked
apoptosis into necrosis presumably requires high concentrations
of NO, is therefore most likely to occur during strong
cytokine responses and might account for the tissue damage
seen in chronic inflammatory processes.
Similar to RNIs, reactive oxygen species were reported to
induce or suppress apoptosis of various cells of the
immune system. Human NK cells were readily killed by
H2O2 produced by monocytes whereas human T cells
were two- to five-times more resistant to H2O2-induced
apoptosis [88]. In mouse macrophages, endogenously proimc107.qxd
02/15/2000 02:00 Page 70
Reactive oxygen and reactive nitrogen intermediates in immunity Bogdan, Röllinghoff and Diefenbach 71
duced O2
– was shown to contribute to resistance to NOmediated
apoptosis (presumably via scavenging of
NO) — indicating that the balance between intracellularly
produced ROIs and RNIs could determine macrophage
apoptosis/survival (see [89] and references therein). In
human neutrophils stimulated with a protein kinase C activator
(phorbol myristate acetate), NADPH oxidase activity
completely suppressed the rapid induction of caspase
activity but promoted the early externalization of phosphatidyl
serine (which serves as a recognition signal of
apoptotic cells by macrophages). Thus ROIs, similar to
endogenous NO (see above), might prevent caspases from
functioning but at the same time an oxidant-dependent
pathway is used to mediate exposure of phosphatidylserine,
cell death and subsequent neutrophil clearance. The
existence of two apoptosis pathways might explain why
neutrophils from NADPH-oxidase-deficient patients can
still undergo apoptosis spontaneously or in response to
Fas-triggering (see [90] and references therein).
Another example for the existence of a caspase-independent
pathway of apoptosis, mediated by endogenous
(mitochondrial?) ROIs, has recently been reported for primary
T lymphocytes isolated from mice after treatment
with superantigens. In vitro, a large percentage of these
cells spontaneously developed signs of apoptosis (exposed
phosphatidylserine, DNA fragmentation, nuclear condensation
and loss of mitochondrial membrane potential) and
died (due to acquisition of membrane permeability) unless
they were cultured in the presence of a mimetic of superoxide
dismutase — Mn (III) tetrakis (5,10,15,20-benzoic
acid) porphyrin (MnTBAP). Caspase inhibitors, in contrast,
only prevented DNA degradation but not cell death.
The ROI-mediated apoptosis did not require Fas- or
TNF-receptor signalling [91••]. Thus, based on the primary
apoptotic signal, T cell apoptosis is triggered either by
cell surface death-receptor-dependent caspase activation
or by caspase-independent ROI-mediated mitochondrial
damage. Both pathways will finally result in loss of mitochondrial
membrane potential, DNA degradation and
intracellular disintegration.
Signalling
In vitro experiments with purified target proteins or intact
cells have identified RNIs or ROIs as regulators of a broad
spectrum of signalling pathways. Examples include ion
channels, G proteins (e.g. the proto-oncogene p21ras), protein
tyrosine kinases (e.g. Fyn and other members of the src
family of tyrosine kinases), protein tyrosine phosphatases,
Janus kinases (Jak1, Jak2, Jak3 and Tyk2), mitogen-activated
protein kinases (extracellular signal-regulated kinases,
Jun N-terminal kinase, stress-activated protein kinase), caspases
and various transcription factors (e.g. NF-κB, AP-1,
Sp1 and c-Jun) (reviewed in [5•,92–95]). Many of these
studies have relied on the use of exogenous sources of
RNIs or ROIs (frequently at concentrations which are not
compatible with life) and therefore did not allow claims of
a physiological role of RNIs or ROIs in this respect.
More recently, however, several reports convincingly
demonstrated a signalling role of small quantities of
exogenous or endogenously produced RNIs
[31••,69••,96,97•,98,99,100•,101,102•,103•] (see Table 2).
Molecular mechanisms by which NO inhibits or stimulates
the action of transcription factors and other
members of signal-transduction pathways include Snitrosylation,
S-glutathionylation, disruption of zinc
fingers and the formation of iron–nitrosyl complexes (in
the case of heme proteins) (reviewed in [5•]). As to the
role of ROIs, there is evidence that H2O2 (or an oxygen
species generated through Fenton chemistry [Figure 1])
facilitates the tyrosine-phosphorylation of Stat3 and Jak2
[94,95]. Endogenous or exogenous ROIs have been
implicated in the activation of NF-κB in a number of cell
types but chronic exposure to oxidative stress
(100 µM H2O2) inhibited the phosphorylation and degradation
of IκBα and thereby impeded NF-κB-dependent
transcriptional activity in T lymphocytes (see [104] and
references therein).
Constitutive NO synthases and the
immune system
The expression of the constitutive NO synthases is not
restricted to neurones or endothelial cells, as is suggested
by the commonly used acronyms ncNOS and ecNOS.
Ca2+-dependent NO synthase activity was found in rat thymocytes
[105] and NOS1 or NOS3 was present in human
monocytes/macrophages and in B and T lymphocytes (see
[69••,106] and references therein). NOS1 or NOS3 can also
assume typical immunological functions previously
assigned to NOS2, such as the induction of apoptotic cell
death [78•] and the control of viruses [107,108]. Similarly,
a recently described Ca2+-dependent NOS isoform of
plants was shown to exert an antibacterial effect against
Pseudomonas syringae that was inhibitable by cNOS
inhibitors [109].
Conclusions
Even in modern textbooks on immunology a frequently
conveyed message is that ROIs as products of neutrophils
primarily act as antimicrobial effector molecules in the
innate defense against extracellular bacteria whereas RNIs
are mainly generated by macrophages in the context of a
specific type 1 Th cell response, where they contribute to
the control of intracellular pathogens. This review intended
to illustrate that the functions assumed by ROIs and RNIs
in the immune system are far more varied. To date, there is
no doubt that both groups of inorganic intermediates are
integral parts of immunologically important signalling pathways,
regulate cytokine responses and cell survival and
contribute to tissue damage as seen in autoimmune diseases
or other forms of chronic inflammatory processes. There is
also no reason to assume that these effects are restricted to
animal models and do not occur in humans. In particular, to
deny the importance of the production of NO by human
monocytes, macrophages and other human cells or to dismiss
it as functionally irrelevant (on the grounds that the
imc107.qxd 02/15/2000 02:00 Page 71
72 Innate immunity
levels are often considerably lower compared with rodent
cells) is without scientific basis: human cells (including
blood monocytes) do express NOS2 activity — as has been
shown in many different diseases [110•,111•] — and even
small amounts of NO can exert immunoregulatory effects,
as reviewed above. One major task for the future, however,
will be to delineate the actual function(s) of the NO produced
by human cells in the various disease states.
Acknowledgements
This research was supported by grants from the Deutsche
Forschungsgemeinschaft (SFB 263 A5).
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