Free Radical Biology & Medicine, Vol. 13, pp. 651-657, 1992 0891-5849/92 $5.00 + .

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Printed in the USA. All rights reserved. Copyright © 1992 Pergamon Press Ltd.

Review Article
REACTIVE OXYGEN INTERMEDIATES AND HUMAN
IMMUNODEFICIENCY VIRUS (HIV) INFECTION

FREDRIK M U L L E R
Kaptein W. Wilhelmsen og flues Institute of Bacteriology, University of Oslo,
The National Hospital, Rikshospitalet, Oslo, Norway

(Received 27 January 1992; Revised 24 June 1992; Accepted 30 June 1992)

A b s t r a c t - - H I V infection affects various parts of the immune system, including the CD4 + lymphocytes and mononuclear
phagocytes, and causes a progressive immunodeficiency. This renders the patient susceptible to various opportunistic infections
and neoplasms. Reactive oxygen intermediates (ROI) are important for the intracellular killing of microorganisms by mononu-
clear phagocytes and neutrophils. Although data are discrepant, several studies suggest that the generation of ROI is impaired in
mononuclear phagocytes, and possibly also in neutrophils, from HIV-infected individuals. This may lead to deficient killing of
intracellular microorganisms predisposing the HIV-infected patient to certain opportunistic infections. Recently, in vitro stud-
ies have shown that ROI activate the intracellular transcription factor nuclear factor KB (NF-KB) which stimulates HIV replica-
tion. Intracellular antioxidant systems, such as the glutathione system, seem to be of importance for the regulation of ROI levels
and thus probably for HIV replication in vitro. However, the role of ROl in regulation of HIV replication in vivo is unknown at
present. The role of RO1 in HIV infection is thus difficult to assess, both at the cellular and clinical level. Reduced intracellular
concentrations of ROI may lead to impaired phagocyte microbicidal functions, thus predisposing HIV-infected patients to
various opportunistic infections. On the other hand, increased ROI levels may be associated with a stimulation of HIV replica-
tion leading to clinical deterioration.

Keywords--HIV, AIDS, Monocyte, Macrophage, Oxidative burst, Free radical, NF-rB, Glutathione

INTRODUCTION ing to more efficient microbial killing by cationic pro-

Reactive oxygen intermediates (ROI) play an im-
portant role in the killing of many microorganisms by
mononuclear phagocytes and neutrophils. 1 During
the oxidative burst (OB), the cells consume oxygen
and generate various ROI by means of the re-
duced nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase system/Several mechanisms may
contribute to the killing of microorganisms caused by
the ROI. Direct toxic effects, formation of free halo-
gens and hypohalous acids mediated by myeloperoxi-
dase, and alkalinization of the phagolysosomes lead-
teins are all probably of importance.
Another association between ROI and microbial
defense has been suggested recently. ROI seem to be
involved in the activation of gene expression by in-
duction of the transcription factor nuclear factor KB
(NF-KB), 3 leading to the translation ofimmunoregula-
tory proteins.
In this review, the possible role of ROI in the im-
munopathogenesis of HIV infection is discussed.
HIV AND THE IMMUNE SYSTEM

The course of human immunodeficiency virus

Address correspondence to: Fredrik Miiller, Institute of Bacteriol-
ogy, Rikshospitalet, Pilestredet 32, N-0027 Oslo l, Norway.
Fredrik Mtiller received his MD in 1983, and his PhD in 1992.
He was a research fellow from 1986 to 1990 at the Section of Virol-
ogy, Institute of Bacteriology and Laboratory for Immunohisto-
chemistry and Immunopathology, the National Hospital (Rikshos-
pitalet). His research interests are immunological aspects of HIV
disease, especially the monocyte/macrophage system and secretory
immunity.
(HIV) infection is characterized by a progressive im-
munodeficiency which renders the infected subject
susceptible to various opportunistic infections and
certain neoplastic diseases. HIV has the capability of
infecting certain cells of the immune system, includ-
ing CD4 ÷ lymphocytes and mononuclear phago-
cytes. 4
The immunopathogenesis of HIV infection is

651
652 F. MULLER
characterized by a depletion of CD4 + lymphocytes
with reduced "help" to other i m m u n e cells. 4 In addi-
tion, many functional responses associated with
CD4 + lymphocytes are reduced, 4 including reduced
production of regulatory cytokines such as interferon-
7 (IFN-7), 5'6 and interleukin-2 (IL-2). 7'8 The B lym-
phocyte system in HIV infection is characterized by
in vivo activation of the cells with raised immunoglob-
ulin concentrations in serum leading to hypergam-
maglobulinaemia, while antigen and mitogen re-
sponses are generally impaired. 4
HIV-infected macrophages produce low levels of
virus over a long period of time. 4 This persistent infec-
tion in macrophages suggests that this cell type may
serve as an important reservoir for virus in vivo and
possibly as a vehicle in the dissemination of the virus
to target organs. Macrophages appear to be important
target cells for HIV in the central nervous system, 9'1°
and this has been associated with the dementia fre-
quently seen in patients with AIDS. t~ Several reports
have described increased production of various cyto-
kines, partly derived from monocytes and macro-
phages, such as IL-6 (Ref. 12), t u m o r necrosis factor-a
(TNF-a), ~3 and transforming growth factor-/3 (TGF-
~).14 Several other monocyte/macrophage functional
defects have also been found, as discussed later.
HIV AND THE OXIDATIVE BURST OF PHAGOCYTES
Monon uclear phagocytes
The data concerning OB responses and microbici-
dal functions of mononuclear phagocytes from HIV-
infected subjects are discrepant. Several studies have
shown that the stimulated OB responses in m o n o n u -
clear phagocytes from HIV-infected subjects are re-
duced compared with HIV-seronegative controls, ~5-18
and the reduction was of borderline statistical signifi-
cance in one additional report) 9 We have previously
shown that the reduced monocyte OB responses were
present already in the asymptomatic state of the dis-
ease and were not considerably more aggravated with
the progression to AIDS. 17 Furthermore, treatment of
mononuclear phagocytes from subjects with asymp-
tomatic HIV infection with IFN-c~ or IFN-~ partially
reconstituted the OB responses in association with zy-
mosan phagocytosis. 2° Roux-Lombard et al. 21demon-
strated normal monocyte OB responses in patients
with a relatively early stage of HIV infection, while
the responses were reduced in three AIDS patients.
On the other hand, several investigators have not
been able to find any differences in OB responses
from HIV-infected subjects and controls. 22,23 When
patients with AIDS were treated with IFN-3,, their
monocytes showed increased HzO2 releasing capacity
during in vitro culture. 24 However, in another study 25
of AIDS patients treated with IFN-3,, depressed mono-
cyte OB responses were found during therapy com-
pared with pretreatment values.
The discrepant results concerning OB responses in
mononuclear phagocytes from HIV-infected patients
may have several causes. First, data from various stud-
ies may not be comparable due to differences in pa-
tient populations. Even AIDS patients constitute a
heterogeneous group. 26 A thorough characterization
of the patients is thus important in order to compare
the data from different studies. Second, differences in
experimental design, including methods for the quan-
tification of OB responses and the OB stimulants
used, might contribute to the discrepancies.
The reduced phagocyte OB responses in HIV in-
fection may have several causes that are probably op-
erative simultaneously. One possibility is a direct ef-
fect due to HIV infection o f m o n o n u c l e a r phagocytes,
as it has been shown that HIV infection in vitro modu-
lates monocyte OB responses. 27 Also, HIV-derived
proteins or other cellular products could affect the OB
generation. In line with this, a synthetic peptide ho-
mologous to a sequence of the HIV envelope protein
suppressed the OB of monocytes in vitro. 28 Further-
more, impaired growth and maturation of bone
marrow precursor cells 29'3° could lead to functionally
deficient monocytes. Several lines of evidence suggest
that this might be of importance. First, it has been
demonstrated that purified bone marrow precursor
cells are susceptible to HIV infection in vitro 31 and in
vivo. 32 Second, HIV has a strong inhibitory effect on
the growth capacity of such cells, at least partly me-
diated by the HIV surface glycoprotein gp 120 (Ref.
33). Also, growth of bone marrow precursor cells
from HIV-infected subjects was impaired by T N F - a
and TGF-¢/. 34 These cytokines have been shown to be
secreted at increased levels in HIV-infected sub-
jects. 13,J4
The microbicidal activity of mononuclear phago-
cytes depends on the generation of ROI through the
OB. Reduced phagocyte microbicidal functions pre-
disposes the individual to various infections. 1 Con-
flicting results have been achieved concerning the mi-
crobicidal capacity of monocytes and macrophages
from individuals with HIV infection. Normal killing
of Candida albicans, Cryptococcus neoJormans,
Aspergillus Jumigatus, and Staphylococcus aureus by
monocytes or macrophages from subjects with HIV
infection has been demonstrated. 22'35'36 However,
others have found that the killing of Toxoplasma gon-
dii, various Candida spp., and Staphylococcus aureus
is impaired. 6'21'37'38
 ROI and HIV infection
Neutrophils
The results concerning the OB responses ofneutro-
phils from HIV-infected subjects are also discrepant.
Several reports conclude that neutrophils from HIV-
infected subjects show normal or even enhanced OB
responses. 19'39'4° On the other hand, Srnnerborg and
Jarstrand 4~ found reduced OB responses in both rest-
ing and stimulated neutrophils from AIDS patients.
Several studies have shown reduced microbicidal
effects of neutrophils from HIV-infected subjects. 4°,42
On the contrary, Estevez et al. 37 found normal killing
ofCandida spp. by neutrophils from HIV-infected pa-
tients. Lazzarin et al. 43 found reduced killing of Can-
dida albicans in drug addicts with AIDS but not in
homosexual men with AIDS and suggested that the
defective killing was related to drug abuse rather than
to HIV infection.
The observed defects might be due to impaired
growth and maturation of bone marrow precursor
cells as discussed in relation to mononuclear phago-
cytes. In line with this, the bactericidal defect of neu-
trophils from HIV-infected children was partially
corrected by granulocyte-macrophage colony-stimu-
lating factor (GM-CSF),4° which is a growth factor for
bone marrow precursor cells. It has even been sug-
gested that neutrophil functions may be modulated
by T lymphocytes.44 If so, the reduced CD4 + lympho-
cyte functions in HIV infection might contribute to
the observed neutrophil defects.
The virus
The HIV proviral genome contains an env- (enve-
lope) gene coding for the envelope glycoproteins, a
gag- (group specific antigen) gene coding for struc-
tural proteins forming the viral core, and a pol- (poly-
merase) gene coding for reverse transcriptase and
other enzymes.45 Furthermore, at least six other genes
coding for regulatory proteins have been described. 45
The HIV genes are bounded by long terminal repeats
(LTR) with important sequences for the regulation of
virus expression. 45
HIV infection of susceptible cells may lead to la-
tency, where no or very little viral RNA and protein
are synthesized.45 The level of viral transcription de-
pends on the overall state of cellular activation,45
which in turn is influenced by a variety of factors in-
cluding antigens, cytokines such as TNF-a, 46 and
trans-activators produced by other viruses. 47 Many of
these activating agents induce the expression of host
transcription factors, notably NF-KB, which normally
regulates the expression of various genes involved in
cellular activation.48 However, NF-KB has also been
653
shown to bind to and activate a KB enhancer element
in the HIV proviral LTR, leading to increased viral
gene expression. 49 During this early phase ofgene ex-
pression, the splicing pattern of viral mRNA results in
the preferential translation of regulatory proteins, in-
cluding Tat (transactivation of transcription). Tat is
essential for HIV replication 5° and transactivates the
expression of all viral genes. 5~Another regulatory pro-
tein, Rev (regulator of expression of virion proteins),
leads to a preferential translation of structural and
enzymatic proteins, 45 leading to the production of
progeny virus.
REACTIVE OXYGEN INTERMEDIATES AND
INDUCTION OF NF-KB
The transcription factor NF-KB is present in
various cell types, including lymphocytes, monocytes,
and macrophages/2 NF-KB activates genes that are
involved in immune responses, leading to the transla-
tion of several cytokine proteins and major histocom-
patibility complex (MHC) antigens. 52 NF-KB is
usually associated with the inhibitor protein IKB in
cytoplasm as an inactive, non-DNA-binding com-
plex. Dissociation of IKB from the complex leads to
translocation of NF-KB to the nucleus, binding to
DNA, and induction of gene transcription. 53 Induc-
tion of NF-KB is mediated by diverse agents such as
viral transactivator proteins, phorboi myristate ace-
tate (PMA), lectins, TNF-a, and interleukin-I (IL-
l). 52 As discussed earlier, induction of NF-KB has
been shown to activate HIV replication in infected T
cells 49 and monocytes. 54
N-acetyl-L-cysteine (NAC) is a direct ROI scaven-
ger and restores intracellular glutathione (GSH) lev-
els. 55'56 Furthermore, NAC effectively inhibits TNF-
a- and PMA-induced activation of the HIV LTR
leading to reduced HIV replication in infected
cells. 57'5sThis effect of NAC is due to a specific block-
ing of the induction of NF-KB.59,6°The NAC effect on
NF-KB in addition to its known antioxidant effects led
Schreck et al. 3 to investigate the role of ROI in the
induction of NF-KB. They found that HIV replication
was induced in the human Jurkat T cell line by the
addition of 30 #M H/O2 to the culture medium. How-
ever, the authors suggested that the intracellular H202
concentration was much lower. The effect on HIV
replication was mediated by induction of NF-KB, al-
though H202 on its own was not able to activate puri-
fied NF-KB. Recent studies suggest that production of
the hydroxyl radical, "OH, derived from H20 2 is re-
quired for activation of NF-KB. In contrast, superox-
ide anion, O~-, did not lead to such activation. 61 Possi-
bly, the generated "OH could mediate oxidative dam-
654
age oflrB causing dissociation of the NF-KB/IKBcom-
plex. Oxidation of a single cystein-residue in the jun
and los proteins has been shown to modulate their
binding to DNA. 62 Alternatively, proteolytic degrada-
tion of IKB might also lead to NF-KB activation. In line
with this, ROI are able to activate latent metallopro-
teinases, as reviewed by Weiss. 63 Also, activation of
protein kinase C (e.g., by PMA) leads to phosphoryla-
tion of IKB causing activation of NF-KB.64
The effect of H202 on HIV replication was pre-
vented by NAC. NAC and other thiol compounds
blocked the induction of NF-KB by agents such as
PMA, lectins, TNF-,, and IL-l. Schreck et al. 3 sug-
gested that the various agents inducing NF-KB might
act through a common pathway involving the synthe-
sis of ROI.
The significance of these findings in relation to
HIV replication in vivo is unknown. Studies of HIV-
infected subjects have shown decreased levels of acid-
soluble thiols in plasma and reduced GSH concentra-
tions in peripheral blood mononuclear cells, plasma,
and lung epithelial lining fluid.65-67 When rhesus ma-
caques were infected with the simian immunodefi-
ciency virus, an animal model closely related to HIV
infection in humans, the plasma level of acid-soluble
thiols dropped 1 week after infection.68 Furthermore,
significantly reduced GSH levels in CD4 ÷ and CD8 ÷
lymphocytes from HIV-infected patients have been
demonstrated, possibly due to a selective loss of cells
with high intracellular GSH levels. 69'7° On the other
hand, GSH levels were not significantly reduced in
monocytes from HIV-infected patients. 69 This is of
interest in relation to the reduced ROI levels in these
cells, as discussed earlier. However, even if the mono-
cyte/macrophage GSH levels are normal, other mech-
anisms must be operative to cause reduced ROI gener-
ation in these cells.
In vitro studies in the mouse have shown that mac-
rophages regulate the intracellular GSH concentra-
tion in lymphocytes.7' In light of the macrophage dys-
functions in patients with HIV infection as discussed
earlier, this may be a possible mechanism for the re-
duced GSH level in lymphocytes from these patients.
Based on the inhibitory effect of NAC and other
thiols on HIV replication in vitro and the reduced
levels of thiols and GSH in vivo, several authors have
proposed clinical trials with NAC or other antioxi-
dants that replenish GSH levels. 72-74NAC is presently
used as an antidote for paracetamol (acetaminophen)
overdose 75and in the treatment of various respiratory
diseases, 76 and it can be given orally and is well toler-
ated. The use of NAC or related anti-oxidants is a
novel and interesting approach to anti-HIV therapy.
However, NAC might also reduce the chemotaxis and
F. MULLER
generation of ROI by phagocytes,77 possibly predis-
posing patients with HIV infection to various opportu-
nistic infections. Lymphocyte functions could proba-
bly also be impaired by antioxidants, as ROI consti-
tute positive signals for lymphocyte activation. 78'79
Furthermore, in vitro studies of other antioxidants
such as ascorbic acid 8°'8~ and vitamin E 82 have shown
that they can inhibit HIV replication. The efficacy in
vivo of these compounds is presently unknown.
The immunomodulating drug dithiocarb sodium
(diethyldithiocarbamate, Imuthiol) is a free radical
scavenger, 83 and several clinical trials have been
carried out in patients with HIV infection.84 In a re-
cent study, a derivative of dithiocarbamate inhibited
the activation of NF-KB with approximately 200-fold
higher potency than NAC. Dithiocarbamates have
metal chelating activities in addition to the antioxi-
dant properties, and both mechanisms might be of
importance for the inhibition of NF-KB activation. 6~
However, recent data concerning mono-therapy with
dithiocarb sodium in HIV-infected patients suggest
that this drug is without clinical benefit. 85
CONCLUSIONS
Several reports suggest an impaired generation of
ROI during the oxidative burst by phagocytes from
HIV-infected subjects. Probably several mechanisms
are operative, such as direct infection ofmononuclear
phagocytes, abnormalities of phagocyte function due
to defective growth and maturation of bone marrow
precursor cells, and reduced cytokine "help" from the
CD4 + lymphocytes.
In vitro studies have shown that ROI activate NF-
KB leading to increased HIV replication. The intracel-
lular regulation of antioxidants such as the glutathi-
one system might be of importance for the effect of
ROI on NF-KB. However, the effect of ROI on HIV
replication in vivo is unknown at present.
The role of ROI in HIV infection is difficult to
assess, both at the cellular and clinical level. Reduced
intracellular concentrations of ROI may lead to im-
paired phagocyte microbicidal functions, thus predis-
posing HIV-infected patients to various opportunistic
infections. On the other hand, increased ROI levels
may be associated with a stimulation of HIV replica-
tion leading to clinical deterioration.
The use of antioxidants such as NAC may have
interesting possibilities in anti-HIV therapy and mer-
its clinical testing. However, suppression of physiolog-
ical ROI levels might lead to impaired lymphocyte
activation as well as reduced antimicrobial activity of
phagocytes.
 ROI and HIV infection
Acknowledgements - - ! would like to thank Dr. Stig S. Froland, Dr.
Pill Aukrust, and Dr. Halvor Rollag for helpful discussions during
preparation of the manuscript.
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ABBREVIATIONS
AIDS--acquired immunodeficiency syndrome
e n v - g e n e - - e n v e l o p e gene ( c o d i n g for the surface gly-
coproteins of HIV)
g a g - g e n e - - g r o u p a n t i g e n gene ( c o d i n g for H I V struc-
tural proteins)
GM-CSF--granulocyte-macrophage colony stimulat-
ing factor
gp 1 2 0 - - g l y c o p r o t e i n 120 ( H I V surface protein re-
s p o n s i b l e for b i n d i n g to C D 4 )
GSH--glutathione
H I V - - h u m a n i m m u n o d e f i c i e n c y virus
IFN--interferon
IL- - - i n t e r l e u k i n -
L T R - - l o n g t e r m i n a l r e p e a t (part o f the H I V p r o v i r a l
genome)
MHC--major histocompatibility complex
NAC--N-acetyl-L-cysteine
N F - K B - - n u c l e a r factor k a p p a B (cellular t r a n s c r i p t i o n
factor)
OB--oxidative burst
P M A - - p h o r b o l m y r i s t a t e acetate
p o l - g e n e - - p o l y m e r a s e gene (gene c o d i n g for H I V re-
verse t r a n s c r i p t a s e a n d o t h e r e n z y m e s )
ROI--reactive oxygen intermediates
T G F - f l - - t r a n s f o r m i n g g r o w t h factor b e t a
TNF-,--tumor necrosis factor a l p h a