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Macrophage Signaling
and Respiratory Burst
Karen E. Iles
Henry Jay Forman
Abstract
Key Words
Signal transduction
NADPH oxidase
Superoxide
Nuclear factor-B
Mitogen-activated protein
kinase
Extracellular-regulated kinase
Activator protein-1
Inflammation
2002
Humana Press Inc.
0257277X/02/
26/13:95105/$12.75
Department of Environmental
Health Sciences, School of Public
Health, and Center for Free
Radical Biology, University
of Alabama at Birmingham
95
Introduction
Alveolar macrophages are the major resident phagocytes in the lung, and as such are
the first line of defense against bacteria and
particles in the lung. Part of the killing mechanism employed by phagocytes depends on
the generation of the superoxide anion in a
metabolic process known as the respiratory
burst (1). This process depends on the assembly and activation of a multisubunit complex
known as the NADPH oxidase. Assembly of
the oxidase is dependent upon the phosphorylation of critical subunits and the translocation of cytosolic components to the plasma
membrane (2,3). The assembly and activation
of the NADPH oxidase have been shown to be
preceded by increases in intracel lular calcium,
activation of IP3-mediated signaling and protein kinase C (PKC) activation (4). Superoxide generation by the NADPH oxidase is
rapidly converted to H2O2 spontaneously and
enzymatically by superoxide dismutase. The
bactericidal properties of the reactive oxygen
species (ROS) are well established. However,
compared to neutrophils, the size of the respiratory burst (i.e., the amount of superoxide
and H 2 O 2 produced) is much reduced in
macrophages.
Information transfer within and between
cells is critical to defending the lung against
infection and other insults. The appropriate
response to stimuli requires the consistent, regulated flow of information from the cell surface, through intracellular signaling pathways,
to the nucleus where the original stimuli is
transduced into changes in gene expression
and the generation of new products (i.e.,
inflammatory cytokines) in response to the initial stimuli. This transfer of information is collectively known as signal transduction. This
process may entail complex pathways that
include second messengers, kinases, and phosphatases. H2O2 has clearly been established
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Macrophage Signaling
and Respiratory Burst
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range of new products classified as eicosanoids, including leukotrienes and prostaglandins. It is not known if Ca2+-mediated
activation of the PLA2-signaling pathway activates the respiratory burst in alveolar macrophages. However, since calcium is required
for the receptor-mediated stimulation of the
NADPH oxidase, this does provide a plausible pathway that connects changes in [Ca2+]i
to activation of the respiratory burst.
In rat alveolar macrophages, mild oxidative
stress has an inverse dose-dependent effect on
the activation of the respiratory burst. Low
concentrations of H2O2 or t-BOOH (<50 M )
act as a priming agent, enhancing the respiratory burst triggered by subsequent stimulation
(20). By contrast, higher concentrations of
H2O2 (>50 M ) inhibit the respiratory burst.
Although both high and low concentrations of
peroxide increased [Ca2+]i, the temporal and
concentration-dependent effects of ROOH on
[Ca2+]i correlated with subsequent enhancement or inhibition of O 2 production (20).
This dual response was also observed in
NR8383 cells, a rat alveolar macrophage cell
line, using an in vitro assay system in which
a single population of cells was exposed to a
gradient of H2O2 (21). On subsequent stimulation with known activators of the respiratory
burst such as adenosine 5-diphosphate (ADP),
phorbol myristate acetate (PMA), and
zymosan, inhibition of the respiratory burst
was observed in cells exposed to the highest
concentration of H2O2, whereas the strongest
activation of the burst was observed in cells
exposed to an intermediate level of H2O2 (21).
The relationship between increases in [Ca2+]i
and the modulation of the respiratory burst was
further investigated by buffering [Ca2+]i. Interestingly, both the enhancement and the inhibition of the respiratory burst by low and
high concentrations of t-BOOH were attenuated by buffering [Ca2+]i (22). The effect of
buffering [Ca 2+ ] i on the assembly of the
98
NADPH oxidase was also investigated. Multiple phosphorylation events and the translocation of p47phox from the cytoplasm to the
plasma membrane are essential to the activation of the respiratory burst in phagocytes (23).
t-BOOH alone or t-BOOH priming followed
by PMA stimulation did not affect the phosphorylation or translocation of p47phox. By contrast, PMA-induced translocation of p47phox
was increased by priming with 25 M t-BOOH
and decreased by pretreatment with 100 M
t-BOOH (24), consistent with the effect of these
concentrations of t-BOOH on activating or
inhibiting the respiratory burst. Buffering
[Ca2+]i abolished the ability of PMA, primed
with low-dose t-BOOH, to activate the translocation of p47phox (24) and to activate the respiratory burst (22). Thus, it appears that changes
in [Ca2+]i are fundamental to this process.
Activation of Signaling
by Respiratory Burst
The role of ROS produced by the respiratory burst on bacterial killing and tissue
damage is well established. Extensive literature also exists on the pathology inflicted by
high doses of exogenous agonists. In the alveolar macrophage, this may result in the production of lipid peroxides and other damaging
molecules that can alter cell function. For
example, ROS production by macrophages can
induce oxidation of low-density lipoproteins
(LDLs), which are taken up more readily than
normal LDLs. Oxidized LDL has recently been
shown to interfere with normal signaling in
macrophages (25).
By contrast, relatively few studies have been
conducted on the products of the NADPH oxidase per se, specifically on H2O2 as a second
messenger in the signaling pathways of alveolar macrophages. H2O2 is not a radical. It is
highly diffusible and relatively unreactive
unless it is in the presence of a reduced tran-
sition metal; the hydroxyl radical can be produced via Fenton chemistry. Any OH produced is sufficiently reactive that it will oxidize
whatever is closest to it. As such, it cannot
function as a second messenger because it lacks
specificity. Conversely, because H2O2 is relatively unreactive, it can act with specificity,
and function effectively as a second messenger. H2O2 can also be elevated transiently and
regulated enzymatically, additional characteristics required of second messengers. Not
long ago, ROS were considered either as
mimics of signaling or more often as disruptive of signaling and other metabolic processes.
Today there is ample evidence that ROS function as signaling molecules (2630). Antioxidant enzymes such as superoxide dismutase
(SOD) and catalase, and the glutathione
(GSH/GSSG) and thioredoxin (Trx) systems
prevent the toxicity of O 2 and H2O2 and restore
the redox status of the cell. It is now known
that several key intracellular signaling pathways may be activated by the products of the
respiratory burst, in particular, H2O2. A new
concept is emerging that antioxidant enzymes,
by restoring the resting state concentrations
of ROS such as H2O2, control signal termination and, therefore, are essential to regulated
intracellular signaling.
Activation of NF-B Signaling Pathway
via Respiratory Burst
The NF-B signaling pathway was one of
the first major pathways shown to be activated
by H2O2 produced by the respiratory burst (5).
NF-B controls the inducible expression of a
variety of genes, particularly those involved
in inflammatory and other immune responses
(31,32). In resting cells, NF-B is predominantly found in the cytoplasm and is typically
a heterodimer composed of p50 and p65
(Rel A) subunits, where it is complexed with
IB, an inhibitory protein that prevents the
migration of NF-B into the nucleus (33).
Macrophage Signaling
and Respiratory Burst
When cells are stimulated, NF-B is translocated to the nucleus, where it binds to a B
transcription element and, along with the binding of other transcription factors and the RNA
polymerase II complex, initiates the transcription of NF-B-inducible genes. In the
classic pathway of NF-B activation, activation of the cell triggers the phosphorylation of
IB by IB-specific kinases (IKKs) (3436).
The IKKs are multicomponent complexes that
contain IKK, IKK, along with several other
proteins (36). Once IB is phosphorylated, it
is targeted for ubiquitination and degradation
(37). This facilitates the release of IB from
the NF-B complex, which is then translocated to the nucleus to initiate transcription of
B-dependent genes. Recently, NF-B activation independent of IB phosphorylation
has also been reported (38). In a given cell
type, IB-dependent and IB-independent
activation of NF-B may be inducible.
The NF-B/Rel family was the prototype
of the redox-sensitive transcription factor.
A model was proposed based on studies with
antioxidants that NF-B was redox sensitive,
and that regardless of the stimulus or agonist
it was activated owing to the production of
ROS (39). Later studies revealed that ROSmediated induction of NF-B is not a universal phenomenon (40). NF-B is activated by
many diverse compounds and stresses including proinflammatory cytokines such as TNF-
and IL-1, T and B cell mitogens, intact viruses,
viral proteins, double-stranded RNA, bacteria, lipopolysaccharide, PMA, ADP, and H2O2
(5,41 43). Several of these compounds have
also been shown to activate the respiratory
burst in alveolar macrophages and to increase
the production of ROS (5).
Early studies in nonmacrophage cell lines
first established that exogenous H2O2 can activate NF-B binding. This was first shown in
Jurkat T cells (39). As well, Schmidt et al. (44)
reported that TNF- and okadaic acidinduced
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and Respiratory Burst
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