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Phagocyte-derived reactive oxygen species as suppressors of inflammatory disease.

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Vol. 58, No. 10, October 2008, pp 2931–2935
DOI 10.1002/art.23941
© 2008, American College of Rheumatology
Phagocyte-Derived Reactive Oxygen Species as Suppressors of Inflammatory
Lena Björkman, Claes Dahlgren, Anna Karlsson, Kelly L. Brown, and Johan Bylund
important not only in terms of the quantity of radical
produced, but also in terms of where the production is
Neutrophils are phagocytic leukocytes that are
central for host defense and for rapid eradication of
infecting pathogens. These cells are armed with a variety
of potent antimicrobial systems, including the NADPH
oxidase that is capable of generating vast amounts of
reactive oxygen species (ROS) during the so-called
respiratory burst. In addition to playing a vital role in
microbial killing, ROS have long been considered important culprits of inflammatory tissue damage. The
finding by Ferguson et al, which is reported in this issue
of Arthritis & Rheumatism (1), that neutrophils from
patients with the autoinflammatory SAPHO syndrome
(synovitis, acne, pustulosis, hyperostosis, and osteitis)
have a specific defect in intracellular ROS production, is
as interesting as it is intriguing; it indicates that decreased ROS production may play a role in the development of this inflammatory disorder. The finding is
consistent with several recent reports describing how
absent or compromised phagocytic ROS production
confers a state of hyperinflammation instead of resulting
in a milder inflammatory response, as would have been
expected based on existing dogma. We may thus have
arrived at a point of reconsideration regarding the role
of ROS in inflammatory disease; the previous “bad
guys” accused of harming innocent bystanders may in
some instances be the “good guys” capable of dampening inflammatory responses and in this way limiting the
extent of tissue damage. Ultimately, it may be vitally
Phagocyte-derived ROS in inflammation
One major function of the respiratory burst became obvious when the defect leading to the syndrome
called fatal granulomatous disease of childhood (now
known as chronic granulomatous disease [CGD]) was
identified in the 1950s (for review, see ref. 2). This rare
disease stems from defects in the genes encoding subunits of phagocyte NADPH oxidase (phox) (Figure 1),
resulting in phagocytes incapable of ROS production.
The disease was originally named to depict the syndrome
of recurrent life-threatening infections often combined
with the development of sterile granuloma in hollow
organs, and only later was this condition demonstrated
to be associated with a lack of ROS production during
phagocytosis. Since then, there has been a broad, rigid
consensus that the ROS formed by phagocytes during
the respiratory burst are required to eradicate pathogenic microbes but may also cause “collateral damage”
to surrounding cells and tissues. An overproduction of
ROS would, according to this hypothesis, contribute to
the tissue-destructing activities associated with various
inflammatory disease states.
This idea has been hard to reconcile with the fact
that CGD is often associated with hyperinflammatory
syndromes that are remarkably similar to idiopathic
inflammatory diseases (2,3). Despite these overt inflammatory phenotypes in CGD, a potential link between
radicals and inflammation has been disregarded in favor
of the assumption that the inflammatory disorders in
CGD result from persistent underlying infections by
nonculturable microorganisms rather than from the
immunodeficiency itself. The possibility that low-grade
infections drive the inflammatory disease becomes less
compelling with increasing evidence that ROS have
roles in metabolism, cell death, apoptosis, induction of
Supported by the Swedish Medical Research Council, the
King Gustaf V Memorial Foundation, the Fredrik and Ingrid Thuring
Foundation, the Wenner-Gren Foundations, the Swedish Society for
Medicine, and the Swedish State under the LUA/ALF agreement.
Lena Björkman, MD, Claes Dahlgren, PhD, Anna Karlsson,
PhD, Kelly L. Brown, PhD, Johan Bylund, PhD: University of Gothenburg, Gothenburg, Sweden.
Address correspondence and reprint requests to Johan Bylund, PhD, Phagocyte Research Laboratory, Department of Rheumatology and Inflammation Research, University of Gothenburg, Guldhedsgatan 10, S-413 46 Gothenburg, Sweden. E-mail: johan.
Submitted for publication June 3, 2008; accepted in revised
form June 27, 2008.
Figure 1. Phagocyte NADPH oxidase and its activation. NADPH oxidase consists of 2 membrane-bound subunits (cytochrome b) present in the
plasma membrane and in the membranes of the specific granules, as well as at least 3 cytosolic components. Activation of NADPH oxidase occurs
by translocation of the cytosolic components to the cytochrome b. An electron from cytoplasmic NADPH is ferried across the membrane, resulting
in the formation of superoxide. In response to, for example, stimulation with phorbol myristate acetate, activation occurs both at the plasma
membrane, resulting in extracellular release of reactive oxygen species (ROS), and at an intracellular location. The exact nature of this intracellular
location is not yet established, but in order to give rise to an intracellular chemiluminescence (CL) reaction, the ROS need access to myeloperoxidase
(MPO) that is stored in azurophil granules. Colocalization of ROS and MPO is probably a result of a fusion event involving both specific and
azurophil granules. Details on the chemiluminescence techniques used for the detection of ROS generated at different sites are provided in ref. 13.
HRP ⫽ horseradish peroxidase; SOD ⫽ superoxide dismutase.
host defense genes, oxidative signaling, and the regulation of inflammation (4). Nevertheless, it has been
difficult to conclusively determine—and demonstrate—
whether ROS-deficient cells drive the development of
inflammatory diseases or whether repeated infections
that are slow to resolve in CGD patients are the catalyst
for the inflammatory disease. The study by Ferguson et
al, showing markedly decreased production of ROS
specifically in intracellular compartments (discussed below), seems to favor the former possibility (that ROSdeficient cells drive inflammatory disease), since the
patients with SAPHO syndrome had no history of
recurrent infections (1).
Inherent antiinflammatory roles of ROS are consistent with several in vivo studies. For example, rodents
with a polymorphism in the Ncf1 gene (encoding the
cytosolic oxidase component p47phox) (Figure 1) leading
to phagocytes with decreased capacity to produce ROS
are more susceptible to the development of autoimmune
inflammatory diseases (e.g., arthritis) (for review, see
ref. 5). The proposed mechanism for protection of
wild-type animals is that ROS released from phagocytes
decrease the levels of surface thiol groups on neighboring lymphocytes and thereby inhibit the activity of
arthritogenic T cells (5). Another mechanism was recently suggested to explain exaggerated inflammatory
responses of CGD (p47phox-knockout) mice, where superoxide was shown to be crucial for normal tryptophan
metabolism. A dysfunctional tryptophan conversion in
the absence of ROS was shown to result in unrestrained
activity of interleukin-17–producing T cells together
with decreased activity of regulatory T cells (6).
Such complex immunologic mechanisms may also
partly explain why CGD (gp91phox-knockout) mice are
susceptible to severe, T cell–dependent (collageninduced) arthritis (7) and persistent, severe, sterile hyperinflammation and tissue necrosis (8). We and others
have reported that isolated leukocytes from CGD pa-
tients (and mice) produce more proinflammatory mediators (chemokines and cytokines) than ROS-competent
cells in vitro (9,10). These cells could be viewed as
intrinsically hyperinflammatory, and the effect would
thus be independent of the production of extracellular
ROS as a means of decreasing T cell activity. Thus, there
is in vivo as well as in vitro evidence that ROS can limit
inflammatory responses in several ways. Several distinct
mechanisms probably account for this, both where the
ROS function as signaling molecules between neighboring immune cells and where they act directly on gene
expression and/or metabolism of a given cell.
It is known, although often overlooked, that the
oxidant–antioxidant balance influences immune cell
function (for review, see ref. 4). It has been noted that
signal transduction pathways and gene transcription are
sensitive to cellular redox states. Zinc-finger coordination, for example, is a redox-sensitive cellular event.
Since many transcription factors and RNA-(de)stabilizing
proteins contain zinc-finger domains, DNA binding,
transcription, and messenger RNA stabilization are subject to change with alterations in the cellular redox
states. Indeed, several important transcription factors
are known to be redox sensitive (11). Redox states can
also alter protein oxidation. In particular, this has been
shown to alter the activity of redox-sensitive phosphotyrosine kinases. Exactly how ROS (generated in fused
granules or elsewhere; see below) would interact with, or
even gain access to, the various signaling components in
the cytoplasm/nucleus remains to be determined. Hence,
redox-sensitive signal transduction is an area that warrants future investigation. Given the scarcity of individuals with a deficiency in ROS production, it is reasonable to use chemical inhibitors of NADPH oxidase to
produce ROS-deficient cells. However, it should be noted
that these inhibitors often have nonspecific (NADPH
oxidase–independent) side effects and are inaccurate
models for cells that are naturally deficient in radicals
(10). The use of chemical NADPH oxidase inhibitors
with nonspecific side effects is likely to explain many of
the contradictory results regarding the role(s) of ROS in
cellular processes (12).
The patients with SAPHO syndrome investigated
by Ferguson and coworkers (1) did not completely lack
intracellular ROS production, and it is worth noting that
small differences in the levels of ROS may have a
noticeable impact on cellular function and biologic
phenotype. This is illustrated by the fact that inflammatory manifestations of CGD are more commonly seen in
the X-linked form (gp91phox deficiency), in which patients’ phagocytes completely lack ROS production, than
in the autosomal-recessive form (most often p47phox
deficiency) (3), in which cells are able to produce
minute, but detectable, levels of ROS (13). In addition,
female carriers of X-linked CGD, whose phagocyte
populations represent a mosaic of normal and ROSdeficient cells, have a higher incidence of discoid lupus,
an autoimmune disease of the rheumatic family, compared with the general population (3). It should also be
mentioned that even in CGD patients with identical
genetic defects, the inflammatory disease is clinically
heterogeneous, thereby pointing to other factors, perhaps genetic (polymorphisms) or environmental, that
may be important in the progression and severity of
inflammatory conditions in both CGD and SAPHO
The link between reduced/absent ROS production and defective microbial killing by neutrophils has
been the main focus of research on phagocyte-derived
ROS for many years. More recent evidence, including
the report by Ferguson et al, challenges conventional
thinking on the role(s) of radicals. Today we are in the
middle of a paradigm shift, with increasing evidence
pointing to the fact that ROS limit the inflammatory
response, thus curbing the extent of inflammatory tissue
damage and the development of inflammatory disorders.
Like many elements of the immune and inflammatory
response, ROS appear to be a double-edged sword,
contributing directly to tissue damage, yet clearly capable of suppressing the development of inflammatory
diseases. Many of the suggested regulatory functions of
ROS involve modification of extracellular (or membranebound) proteins/peptides, which may be the easiest way
to visualize extracellular ROS released from phagocytes
as having an influence on cellular and biologic processes.
Interestingly, added scavengers, such as superoxide dismutase (SOD) (which dismutates superoxide to H2O2)
or catalase (which removes H2O2), which are known to
decrease the severity of inflammatory conditions such as
arthritis (for review, see ref. 14), are not cell permeable.
Thus, the roles of ROS in inflammation probably differ
depending not only on the levels of ROS produced, but
also on where the ROS are generated.
The report by Ferguson et al (1) correlates the
intracellular production of radicals with the incidence of
inflammatory disease. Intracellular ROS production has
long been synonymous with intraphagosomal production
(i.e., ROS being produced inside the phagosomal compartment after uptake of a prey). However, many phagocytes have the ability to generate ROS in the absence of
a particulate prey (i.e., in an intracellular compartment
distinct from the phagosome) (15). The details are
gradually becoming clearer regarding this activity and
the cellular compartment in which it occurs.
Production and detection of intracellular ROS
Phagocyte NADPH oxidase consists of 2 membranebound components (comprising the cytochrome b) and
at least 3 cytosolic components (Figure 1). In the resting
neutrophil, a minor fraction of cytochrome b is localized
in the plasma membrane, while the major part is found
in membranes of intracellular organelles, mainly the
specific granules (16). Upon cell activation, the cytosolic
proteins translocate to the membrane-bound cytochrome b, and a functional multicomponent electrontransfer system is formed that reduces molecular oxygen
to superoxide anion at the expense of cytosolic NADPH
(Figure 1). This activation can take place either at the
plasma membrane, leading to release of oxygen radicals
to the extracellular environment, or at intracellular
membranes, leading to ROS formation in intracellular
Neutrophil NADPH oxidase can be activated in
response to a number of physiologic and synthetic
agonists, and the nature of the cell-activating agent
determines where in the cell the NADPH oxidase is
activated (i.e., in the plasma membrane or in intracellular membranes). Signaling through chemoattractant
receptors, for example, mainly activates the plasma
membrane–localized NADPH oxidase that releases
ROS extracellularly, while engulfment of a particulate
prey primarily generates ROS within the phagosome. It
is, however, becoming increasingly clear that a substantial part of the oxidants produced by activated neutrophils are generated at an intracellular site distinct from
the phagosome; activation of CR3 on the neutrophil
surface is one example of physiologic stimulation that
can result in intracellular ROS production in the absence of phagosome formation (15). In cell-free systems,
NADPH oxidase assembly and activation can take place
on specific granule membranes, but the precise compartment in the intact cell where intracellular (nonphagosomal) activation takes place has yet to be defined.
A number of techniques are used to determine
cellular ROS production in/from neutrophils (and other
cells) (13). The chemiluminescence technique used by
Ferguson et al (1) to specifically detect intracellular
respiratory burst activity uses a membrane-permeable
detector molecule, luminol, which is excited by ROS and
emits light when returning to its ground state. To
specifically detect intracellular ROS, luminol is combined with SOD and catalase, both of which are unable
to cross biologic membranes and will therefore consume
the extracellular radicals (13). The intracellular luminolamplified chemiluminescence reaction in neutrophils is
highly dependent on the participation of myeloperoxidase (MPO) that is stored in another granule type, the
azurophil granules. Theoretically, ROS generated in
specific granules could reach MPO in the azurophil
granules by diffusion. However, it is more reasonable to
assume that the ROS reach the MPO-containing compartment through fusion between the 2 granule types
involved, a process that would bring the 2 entities
required for the chemiluminescence reaction (superoxide anion and MPO) into the same compartment (15)
(Figure 1).
The fusion hypothesis gains support from the
facts that a similar process occurs during eosinophil
activation (17) and that the intracellular chemiluminescence reaction can be selectively inhibited without alterations in either intracellular NADPH oxidase activity (as
measured by an alternative, peroxidase-independent
technique) or MPO activity (Karlsson A: unpublished
observations). Ferguson et al report normal MPO activity as well as normal amounts and sizes of 4 NADPH
components in the phagocytes from their subjects (1),
and it is tempting to speculate that the decreased
intracellular ROS production could be due to dysfunctional intracellular fusion processes.
Ferguson et al launch the hypothesis that the
SAPHO syndrome is a genetic disorder resulting in
aberrant intracellular ROS production while the extracellular production is intact (i.e., that the 2 pools of
NADPH oxidase are affected differently) (1). The intracellular signals that induce NADPH oxidase activation
are far from defined. However, the differences between
the patterns of extracellular and intracellular production
of ROS induced by different stimuli indicate a diversity
in the regulating pathways to the 2 pools of NADPH
oxidase, and studies of dependencies for intracellular
calcium fluxes between the 2 pools support such a
scenario (18). Further, the use of signal transduction
inhibitors has revealed that activation of the intracellular
NADPH oxidase pool by phorbol myristate acetate (a
stimulus used by Ferguson et al) is dependent on
phosphatidylinositol 3-kinase (PI 3-kinase) activation,
while activation of the plasma membrane–localized pool
is not, and that different protein kinase C isozymes
(upstream of PI 3-kinase) appear to take part in the 2
signal transduction pathways (15). Consequently, a molecular explanation for the SAPHO syndrome may hypothetically be sought among the signal transduction
molecules responsible for activation of NADPH oxidase
in intracellular membranes or, as suggested above, among
those leading to intracellular granule fusion.
The neutrophil respiratory burst has long been
considered to be of main importance for the generation
of ROS inside the phagosome, where it promotes the
killing of microbes, or for the release of extracellular
ROS mainly thought to contribute to tissue damage.
However, intracellular ROS production often occurs in
the absence of phagosome formation through a process
that most likely involves fusion events of at least 2 types
of distinct granules. In addition, ROS may act as essential signaling molecules with the capacity to affect a
variety of cellular processes of importance for immune
regulation. The scientific community is gradually opening up to the somewhat counterintuitive idea that ROS
may actually curb inflammatory responses. The article
by Ferguson et al (1) could be an important contribution
to our understanding of the exaggerated inflammatory
responses following diminished or absent ROS production. We hope that future research will determine exactly how (and where!) oxygen radicals dampen inflammatory processes, but clearly, the old view of ROS
merely as antimicrobials and mediators of tissue damage
is about to change.
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