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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1984 64: 959-966  

BM Babior Oxidants from phagocytes: agents of defense and destruction 

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