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The oxygen consumption and 02 production that
occur during the respiratory burst are accounted forentirely by this reaction. H202 is produced by the
reaction of O� with itself, a dismutation in which one
molecule of 02 is oxidized by the other20:
Glucose oxidation through the HMP shunt accelerates
because of an increase in the rate of production of
NADP�, the availability ofwhich limits the activity of
the HMP shunt.2’ NADP� production increases dur-ing the respiratory burst through the actions of (a) the
02-forming enzyme and (b) a glutathione-dependent
system that uses NADPH to detoxify H202 by reduc-
ing it to water22’23:
2 GSH + H2O2 p GSSG + 2 H20glutathioneperoxidase
GSSG+NADPH . -‘2GSH+NADPglutathione
reductase
glucose-6-P dehydrogenase
Each glucose that is metabolized via the HMP shuntreduces two molecules of NADP to NADPH, thus
replenishing the supply of reducing agent necessary for
the continued operation of the respiratory burst:
Glucose-6-P + NADP
6-Phosphogluconate
+ NADPH
-p Ribulose-5-P
+ CO2 + NADPH
From the Blood Research Laboratory and the Department of
Medicine. Tufts-New England Medical Center. Boston.
Supported in part by US Public Health Service grant No.
AJ-11827 and by grant No. I349from the Councilfor Tobacco
Research-USA. Inc.
Submitted March 26, 1984; accepted July 10, 1984.
Address reprint requests to Dr Bernard M. Babior. Blood
Research Laboratory, Department of Medicine. Tufts-New
England Medical Center. Boston. MA 02/1/.in I 984 by Grune & Stratton. Inc.
0006-4971/84/6405--tX103/$03.0O/0
Blood, Vol 64, No 5 (November). 1984: pp 959-966 959
REVIEW
Oxidants From Phagocytes: Agents of Defense and Destruction
By Bernard M. Babior
0 F THE SYSTEMS that defend the host against
invading microorganisms, the professional pha-
gocytes (neutrophils, eosinophils, and mononuclear
phagocytes) act in the most primitive fashion. Unlike
cytotoxic lymphocytes and the complement system,which destroy their targets with a drop of poison,”2 the
professional phagocytes kill like Attila the Hun,deploying a battery of weapons that lay waste to boththe targets and the nearby landscape with the subtlety
of an artillery barrage. Among the most powerful ofthese weapons is a group of oxidizing agents that are
produced by the phagocytes when they encounter
invading microorganisms or other appropriate stimuli.These oxidants are reactive enough to destroy most
biologic molecules and are responsible for much of thedamage inflicted by phagocytes on both microorga-
nisms and surrounding tissues at sites of infection or
inflammation. In this article, I briefly review thenature of these oxidants, their mode of production, andtheir biologic effects, both good and bad.
THE REACTIVE OXIDANTS
Reactive oxidants are produced from oxygen
through a special metabolic pathway that, as far as isknown, is unique to phagocytes. The consumption ofoxygen through this pathway is initiated by the expo-sure of the cells to any one of a large number of stimuli,
among the most effective of which are several that are
likely to be present at sites of inflammation: opsonizedmicroorganisms,3 the complement fragment C5a,4’5
leukotriene B4 (produced by stimulated phagocytes),6’7
and N-formylated oligopeptides of bacterial origin4’8that are actively secreted or are released by lysis of
dead organisms. Activation of the pathway occurs
within a few seconds after stimulation9’1#{176}and is charac-
terized by an abrupt increase in oxygen uptaketogether with the onset of production of a series ofcompounds formed from this oxygen: superoxide (Ok),
hydrogen peroxide (H2O2), and a number of additional
oxygen-containing compounds, all of which are highly
reactive. In addition, there is a large increase in the
oxidation of glucose via the hexosemonophosphate
(HMP) shunt. These changes in oxidative metabolism
are collectively known as the “respiratory burst,” a
name derived from the sudden increase in oxygenuptake that is one of its invariable features.’
The biochemical basis for the respiratory burst is the
activation of an enzyme, dormant in resting cells, that
catalyzes the one-electron reduction of oxygen to 02 at
the expense of NADPH�9:
202 + NADPH-�2O2 + NADP� + H�.
02 + 02 + 2 H� -� H202 + 02.
Net: NADPH + H2O2 -‘ NADP + 2 H2O.
6-Phosphogluconate + NADP
6-phosphogluconate dehydrogenase
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960 BERNARD M. BABIOR
The reactive oxidants that are actually responsible
for oxygen-mediated damage by phagocytes are gener-
ated from the O� and H2O2 produced in the respiratory
burst. The latter, themselves relatively innocuous, are
converted by a complex series of secondary reactions to
two classes of highly reactive oxidizing agents: oxidiz-
ing radicals and the oxidized halogens. The oxidizing
radicals include the hydroxyl radical (OH’), the pro-
duction of which during the respiratory burst has been
strongly suggested by electron spin resonance spec-
trometry,24’25 and other radicals, as yet unidentified.
OH’ is thought to be produced at least partly by the
iron- or copper-catalyzed reaction between O� and
H2O2 (the Haber-Weiss reaction)26:
02 + H2O2-�OH’ + OH- + 02.
How other radicals are produced is not yet known,
though there is some evidence that O� participates in
their formation.
The production of the oxidized halogens begins with
the enzyme-catalyzed oxidation of the ubiquitous C1
ion by the H2O2 generated during the respiratory burst.
This oxidation is catalyzed by myeloperoxidase,27’28 a
heme enzyme, and yields as its product the very
reactive oxidizing agent, hypochiorous acid2931:
Cl + H2O2-� HOC1 + H20.
Much of this HOC1 is immediately consumed in
attacks on various biologic molecules, as will be
described subsequently. Some, however, may react
with O� to form another microbicidal species whose
identity is presently unknown.32 In addition, a substan-
tial portion3336 appears to react with low molecular
weight amines to yield chloramines:
R-NH2 + HOCI -� R-NHC1 + H2O.
Some of these chloramines, particularly those formed
from polar amines such as taurine (H2NCH2-
CH2SO3 ), are long-lived and relatively harmless3�36;
it may be that the formation of polar chloramines is a
way to detoxify HOd, a possibility of interest in
connection with the very high concentrations of taurine
found in neutrophils. In contrast, lipophilic chlora-
mines, such as those formed from ammonia and
spermidine [H2N(CH2)3NH(CH2)4NH2] , are very
toxic, dissolving in biologic membranes and wreaking
havoc on their components.34
Myeloperoxidase is not present in all phagocytes. It
is found in the azurophil granules of neutrophils and in
the lysosomes of juvenile mononuclear phagocytes
(monocytes and newly activated macrophages), but is
absent from eosinophils (which have a peroxidase of
their own that is incapable of catalyzing the oxidation
of Cl ) and from mature macrophages.37�#{176} Thus, it
would appear that oxidized halogens are employed as
destructive agents only by neutrophils and young
mononuclear phagocytes. There is some evidence, how-
ever, that mononuclear phagocytes lacking endogenous
myeloperoxidase can acquire the enzyme by ingesting
debris from neutrophils that have died in battle and
can use this secondhand weapon for their own pur-
poses.4’
THE O2-FORMING ENZYME
The key element in oxidant production by phago-
cytes is the Ok-forming oxidase. This enzyme is under
active investigation in many laboratories, and there is
(not surprisingly) some disagreement as to its proper-
ties. Virtually all agree that it is a plasma membrane-
bound flavoprotein that uses NADPH (Km ‘� 0.05
mmol/L) as the physiologic reducing agent, though in
the test tube it is also able to generate 02 at the
expense of NADH (Km 1 mmol/L))5”8’42�8 It is also
generally agreed that a b-type cytochrome found
exclusively in phagocytes is related in some manner to
the oxidase,4953 though the relationship usually postu-
lated-namely, that the cytochrome is part of a chain
of electron carriers that ferry electrons from NADPH
to oxygen-has yet to be reconciled with certain
experimental observations, and in my view remains
unproven.54 Beyond these facts, relatively little is
known about the enzyme, though several laboratories
are attempting to purify it, and additional answers
should be forthcoming once purification is accom-
plished.
Perhaps the most interesting of these answers could
be an understanding of the mechanism by which the
enzyme is activated upon stimulation of the cell. A
great deal of information has been gathered concern-
ing the activation event: it has been shown, for exam-
ple, that activation is a reversible,55 energy-requiring
process,56 that it requires the continuous presence of
the stimulus,57 and that it is reversibly blocked by
crosslinking agents.58 Evidence for synergism between
multiple activating agents applied to the same popula-
tion of cells (a “priming effect”) has also been
obtained.59 In addition, recent studies have provided
considerable information about ion fluxes,�#{176}� mem-
brane potential changes,65�7 alternations in protein
phosphorylation68’69 and membrane lipids,7#{176}72 and
degranulation-mediated transfers of hypothetical oxi-
dase components between subcellular compart-
ments53’73 that occur when phagocytes are exposed to
oxidase-activating stimuli. A connection between these
latter phenomena and the activation of the oxidase,
however, has not been established with certainty, and,
in some cases (eg, ion fluxes,” degranulation55) is
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OXIDANTS FROM PHAGOCYTES 961
rather questionable, in my opinion. In any event, it has
not yet been possible to put this information together to
describe the activation of the oxidase in well-defined
molecular terms.
What is really needed to achieve an understanding
of oxidase activation at a molecular level is a cell-free
oxidase-activating system that can be taken apart and
analyzed component by component using biochemical
techniques. In a major advance that has capped years
of work on this problem, activation of the oxidase in a
cell-free system has at last been realized, first in
disrupted macrophages,74 and more recently in neutro-
phil homogenates (J.T. Curnutte, Department of Pedi-
atrics, University of Michigan School of Medicine,
personal communication, December 1983; McPhail et
al75). Activation was accomplished by the addition of
arachidonic acid to a concentrated homogenate from
resting phagocytes. Components from both the soluble
and particulate fractions of the homogenate were
required, and O� production by the activated homog-
enates took place at rates close to those observed with
cell-free systems prepared from activated phagocytes.
The development of this system has clearly lifted the
lid from a corner of the mysterious black box that
contains the transducing apparatus rcsponsible for
converting an exogenous stimulus into a cellular
response. Further study should soon reveal much of
interest about the operation of this enigmatic appara-
tus.
CONGENITAL FAILURE OF OXIDANT PRODUCTION:
CHRONIC GRANULOMATOUS DISEASE
An inherited abnormality in the O2-forming oxidase
or its activating system gives rise to the condition
known as chronic granulomatous disease. In this disor-
der (actually, a group of related disorders), phagocytes
are unable to express a respiratory burst because of the
defect in oxidase activity.76 As a result, their oxidant-
dependent microbial systems are inoperative, leading
to a pronounced impairment in their ability to kill
certain microorganisms.
Recurrent infections caused by this defect in micro-
bial killing is the hallmark of chronic granulomatous
disease.77 Formerly, these infections led typically to
early death from sepsis after a life punctuated by
repeated hospitalizations for the treatment of severe
bacterial pneumonias and deep tissue abscesses. The
routine use of prophylactic antibiotics has changed the
course of chronic granulomatous disease, and now the
problems are principally related to the complications
of imperfectly suppressed chronic infections, including
pulmonary fibrosis, bronchiectasis, and strictures of
the gastrointestinal or urinary tract, with death ensu-
ing from these complications or from an acute infec-
tion by some resistant microorganism (eg, Aspergil-
lus).
Recent investigations have been undertaken to try to
define the biochemical defect in chronic granuloma-
tous disease. These investigations have suggested that
some cases of chronic granulomatous disease result
from failure to activate the oxidase,65’66’7’ while others
are caused by an oxidase whose affinity for NADPH is
fivefold to tenfold lower than normal.788’ Implicit in
these findings is a classification of chronic granuloma-
tous disease into cases that are related to a defect in
activation and cases that are caused by a defect in the
enzyme itself. In another classification, chronic granu-
lomatous disease patients may be divided into those
whose phagocytes lack the b-type cytochrome or pos-
sess a cytochrome that shows abnormal properties,
those whose phagocytes lack a flavoprotein, and those
whose phagocytes are missing both the cytochrome
and a flavoprotein8184; at least one of those whose
phagocytes lack the cytochrome has a low-affinity
oxidase as well.8’ Finally, the disease may be classified
by mode of transmission, being transmitted in some
families as an X-linked disorder and in other families
as an autosomal recessive condition.85’86 The relation-
ships among these classifications are rather poorly
defined at present. The overall picture is of a single
clinical entity caused by a highly diverse group of
biochemical abnormalities whose exact delineation will
probably have to await a better understanding of the
normal oxidase and its activating system.
BIOLOGIC EFFECTS OF THE RESPIRATORY BURSTOXIDANTS
The physiologic function of the respiratory burst
oxidants is the destruction of invading microorga-
nisms. The range of natural targets is very wide and
includes all kinds of bacteria, fungi (eg, Candida),
unicellular parasites (leishmania, toxoplasma), and
metazoa (larval stages of schistosomes). A small tar-
get, such as a bacterium, is usually attacked after it has
been ingested by the phagocyte, with oxidants being
delivered into the phagocytic vesicle containing the
internalized organism. A target such as a fungal hypha
or a schistosome larva, however, is too large to be
ingested by the phagocyte. In this case, the phagocyte
will work from the outside, spreading itself onto the
surface of the target and delivering oxidants into the
space between itself and the target wall. This space
appears to be sealed at the edges, so the oxidants
delivered into it are not dissipated into the surrounding
environment but are concentrated onto the small
region of the target that lies under the phagocyte. It is
apparent from these topographic considerations that
the plasma membrane is the ideal location for the
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962 BERNARD M. BABIOR
Of-forming oxidase, because from this location the
enzyme is able to deliver oxidants onto the target with
maximum efficiency.The phagocyte is also susceptible to damage by
these reactive oxidants, so attacks by at least some
kinds of phagocytes (for example, neutrophils) are
kamikaze assaults in which the attacker dies along
with the target. However, phagocytes are able to
defend themselves against their oxidants, at least to alimited extent. The antioxidant systems of the phago-
cytes include superoxide dismutase,87 which converts
Oi to oxygen and H2O2; catalase88 and the gluta-thione-dependent H2O2-detoxifying system referred to
above, both of which reduce H2O2 to water; taurine,89which may detoxify HOC1 (see above); and assorted
other antioxidants, such as a-tocopherol�#{176} and ascorbic
acid.9’ These may not preserve the life of the activated
phagocyte forever, but they permit it to survive long
enough to fire a lethal volley of oxidants at its target.Work has recently begun on the biochemical lesions
inflicted on living systems by the lethal oxidants of
phagocytes. HOC1, recognized for some time as a
highly potent microbicidal agent, has been shown to
oxidize -SH groups to disulfides and higher sulfuroxides,92’93 to convert thioethers to sulfoxides,94 and to
oxidize amino compounds to chloramines and dichlor-amines95 (see above). Halogenation of pyridine nucleo-
tides has been demonstrated,96 as has the destruction of
the biologic activity of nucleotides such as adenosine
triphosphate (ATP).97 In addition, HOC1 has been
shown to inactivate certain redox enzymes, including
heme-containing enzymes and iron-sulfur proteins, but
not flavoproteins.97 The inactivation of iron-sulfur pro-
teins is a particularly rapid reaction. The hydroxyl
radical (OH) as an isolated oxidant of biologic sys-
tems has not been studied nearly as thoroughly, butimportant information as to its effects can be inferred
from the extensive work that has been carried out withionizing radiation, most of the biologic actions ofwhich are mediated through OH. Particularly impor-
tant in this regard are studies that have been carried
out on the effects of ionizing radiation on DNA. Thesestudies have shown oxidative alterations of bases,
perhaps the most characteristic of which is the oxida-
tion of thymine to dihydrothymine glycol,98 as well as
the introduction of strand breaks in the DNA chain,some resulting from the action of excision repair
enzymes� and some caused by direct damage to thedeoxyribose moieties ofthe DNA chain.’#{176}#{176}Despite this
mass of information, the identity of the lesion(s)
immediately responsible for the death of an organism
attacked by these lethal oxidants has not yet beenestablished. My own guess is that the fatal lesion willultimately be shown to involve the membrane of the
microorganism under attack and will result in a loss of
permselectivity such that the organism is no longer
able to control its internal environment. If this shouldprove correct, tHen the mechanism of killing by oxi-dants will be similar to the mechanism of complement-
mediated killing, at least in the most general sense.
Though the phagocyte seems to be designed to
restrict oxidant production to the smallest possible
region (there is evidence, for example, that the O�-
forming oxidase is activated only in that region ofmembrane that is in contact with the target),’#{176}’ some
of the reactive oxidants inevitably leak into the sur-
rounding tissues, where they have the capacity toinflict considerable damage. The organ where this
damage has been best documented is the lung, in which
phagocyte-generated oxidants have been implicated in
the pathogenesis of both acute and chronic disease.Acutely, these oxidants may be responsible for much of
the alveolar damage and pulmonary edema seen in the
adult respiratory distress syndrome (ARDS; shocklung). One explanation for the pathogenesis of shock
lung involves a train of events in which the release of
the complement-derived anaphyllatoxin C5a into the
blood stream causes neutrophils to aggregate and to
begin producing O� ; these Of-generating neutrophil
aggregates become trapped in the lungs, where the
liberated oxidants destroy the pulmonary capillary
endothelium, permitting plasma to exude into the
alveoli.’#{176}2’#{176}5This explanation is supported by animal
and organ perfusion models of shock lung in which the
pulmonary damage induced by the inciting agent is
prevented if the neutrophils in the model system are
unable to manufacture reactive oxidants or are elimi-
nated entirely.’#{176}�”#{176}7Furthermore, recent studies in
patients with ARDS have provided unequivocal cvi-
dence for the release of oxidants into the pulmonarytissues of affected individuals, but not normal con-
trols.’#{176}�On the other hand, the foregoing train ofevents cannot be the only route to ARDS, because thiscondition can occur in patients with neutropenias so
severe that pathogenetically significant neutrophil
aggregates probably cannot be formed.A role for phagocyte-generated oxidants has also
been proposed in the pathogenesis of chronic lungdisease. In this condition, neutrophils accumulate in
lungs subjected to chronic irritation, drawn there per-
haps by a chemotactic factor released by alveolar
macrophages that have been activated by the irri-
tant.’#{176}�The following sequence of events is then postu-
lated to take place. The neutrophils in the lungs
become activated to generate reactive oxidants and torelease the contents of their granules into the extracel-
lular environment. Among these contents is elastase, a
powerful protease that is able, among other things, to
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OXIDANTS FROM PHAGOCYTES 963
digest the elastic fibers in the walls of the alveoli.”0’t”
Ordinarily, the release of a small quantity of elastase
would do little harm, because it would be rapidly
inactivated by a,-antiproteinase, a circulating inhibi-
tor of elastase and other proteases that are released
from time to time into the tissues.”2 This inhibitor,
however, is destroyed by the neutrophil-derived oxi-
dant HOC1, which acts by converting a critical methio-
nine to methionine sulfoxide”3”4 (but see Campbell et
al”6). The elastase therefore remains active and grad-
ually destroys the lungs through its unopposed action
on the elastin and other proteins of the alveolar wall.
A potentially harmful effect of these oxidants of
perhaps even wider significance has recently been
discovered. This is their ability to induce mutations in
DNA. Originally described in bacteria (Ames tester
strains),”7 the production of mutations in the DNA of
mammalian cells (hamster fibroblasts) by oxidants
generated by phagocytes has now been 8 As
evidence increases that mutations are etiologically
important in carcinogenesis, it becomes at least plausi-
ble to postulate that mutations caused by oxidants of
phagocytic origin are responsible for the development
of malignancies in nude mice inoculated with cells
exposed to activated � and may perhaps
account for the cancers that are known to occur at sites
of chronic inflammation, which are continuously infil-
trated by activated phagocytes. Mutations are also
coming under increasing suspicion as a mechanism of
aging, implying that phagocytes, with their capacity to
produce mutagenic oxidants, may also participate in
the aging process. From the foregoing considerations,
it appears that phagocytes may be playing at least
some role in the disintegration, as well as in the
defense, of the host.
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BM Babior Oxidants from phagocytes: agents of defense and destruction
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