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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 350-356
Activation of the Human Immunodeficiency Virus-1 Long Terminal
Repeat by Respiratory Burst Oxidants of Neutrophils
By
Seymour J. Klebanoff and
Catherine M. Headley
From the Department of Medicine, University of Washington, Seattle.
 |
ABSTRACT |
The human immunodeficiency virus type 1 (HIV-1) long terminal repeat
(LTR) introduced in association with the luciferase reporter gene into
Jurkat T cells was strongly activated by a combination of human
neutrophils and phorbol myristate acetate (PMA). Activation was not
observed when normal neutrophils were replaced by neutrophils which
lack a respiratory burst, ie, from a patient with chronic granulomatous
disease (CGD), was strongly inhibited by catalase, was potentiated by
vanadate, was stimulated by relatively low concentrations of azide, and
was inhibited by selective inhibitors of protein kinase C (PKC). The
PMA affected activation in three ways: (1) by directly activating the
LTR in Jurkat LTRluc; (2) by inducing a respiratory burst
in neutrophils with the formation of H2O2; and
(3) by increasing the sensitivity of Jurkat LTRluc to the
activating effect of H2O2. When PMA was
replaced by opsonized zymosan as the neutrophil stimulus, activation of
the LTR was low unless azide was added. Activation in the presence of
azide was not seen when CGD neutrophils were used or when catalase was added, suggesting that azide acts by inhibiting the degradation of
H2O2. These findings indicate that activation
of the HIV-1 LTR in Jurkat T cells can be induced by
H2O2 released by neutrophils, particularly when
PKC is concomitantly activated.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PHAGOCYTES (neutrophils, eosinophils,
monocytes/macrophages) respond to stimulation with a burst of oxygen
consumption and much, if not all, of the extra oxygen consumed is
converted initially to the superoxide anion and then to
H2O2. Among the potent inducers of a
respiratory burst in phagocytes is phorbol myristate acetate
(PMA), which is a direct stimulus of protein kinase C
(PKC), and opsonized zymosan, which acts as a phagocytic stimulus.
Myeloperoxidase (MPO) (released from the cytoplasmic granules of
neutrophils and monocytes) and eosinophil peroxidase (released from
eosinophil granules) can react with the H2O2
formed to oxidize a halide to form potent oxidants, which are toxic to ingested microorganisms and adjacent extracellular targets.
We have previously reported that neutrophils,1
monocytes,2 and eosinophils,3 when
appropriately stimulated, are viricidal to cell-free human
immunodeficiency virus type 1 (HIV-1), and we have provided evidence
for the involvement of the
peroxidase-H2O2-halide antimicrobial system in
this toxicity. Thus, the viricidal effect was inhibited by catalase,
but not by heated catalase or superoxide dismutase (SOD) implicating
H2O2 formed by the stimulus-induced respiratory
burst. This conclusion was supported by the absence of a viricidal
effect when neutrophils which lack a respiratory burst, ie, from
patients with chronic granulomatous disease (CGD), were used, unless a
source of H2O2 was added. Azide, a potent inhibitor of peroxidase, also was inhibitory, which is compatible with
the involvement of the phagocyte peroxidase. This was supported by the
finding that stimulated neutrophils and monocytes which lack MPO, ie,
from patients with hereditary MPO deficiency, were not viricidal to
HIV-1 unless MPO was added.
Biologic oxidants formed by phagocytes may also influence HIV-1 in
other ways. The long terminal repeat (LTR) of HIV-1 contains promoter
and enhancer sequences required for the initiation of gene
transcription. H2O2 can activate the HIV-1 LTR
introduced by transfection into HeLa,4
Jurkat,5,6 and THP-16 cells and can increase
HIV-1 replication in the chronically infected human monomyelocytic cell
line U-14,6 and the chronically infected T-lymphocyte cell
line Jurkat.5 This effect of H2O2 may be due, at least in part, to activation of Nuclear Factor  B
(NF-B), which can recognize NF-kB binding sites in the HIV-1 LTR and
thus facilitate gene transcription.5 Other mechanisms, however, cannot be excluded. The activation by
H2O2 of the HIV-1 LTR in THP-1 or Jurkat cells
and of viral replication in U-1 cells is greatly potentiated by
vanadate.6 Vanadate reacts with
H2O2 to form peroxides of vanadate with
heightened biologic activity.
We report here on the activation of the HIV-1 LTR in a
T-lymphocyte-derived cell line by intact neutrophils stimulated either by PMA or opsonized zymosan.
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MATERIALS AND METHODS |
Special reagents.
PMA, sodium orthovanadate, and H2O2 (30%
wt/wt) were obtained from Sigma Chemical Co (St Louis, MO). The
H2O2 was assayed by its absorption at 230 nm,
using an extinction coefficient of 81 mol/L 1
cm1. Human recombinant tumor necrosis factor-
(TNF-) was generously provided by Genentech (South San Francisco,
CA), and recombinant human interleukin-2 (IL-2) by Hoffman-La Roche
(Nutley, NJ). Bovine liver catalase (CTS, 57,622 U/mg) was obtained
from Worthington Biochem Corp (Freehold, NJ), and human erythrocyte
catalase (50,000 U/mg) was obtained from Calbiochem (La Jolla, CA). The
bovine liver catalase was dialyzed against water overnight and the
catalase preparations were heated at 100°C for 20 minutes where
indicated. Endotoxin contamination of the catalase preparations was
determined using a Limulus Amebocyte Lysate kit (BioWhittaker, Inc,
Walkersville, MD). Endotoxin was removed from the bovine liver catalase
preparation by passage through a column containing polymyxin B
immobilized on agarose (Detoxi-gel; Pierce, Rockford, IL). Superoxide
dismutase (SOD, bovine erythrocytes, 5,000 U/mg) was obtained from
Boehringer Mannheim Biochemicals (Indianapolis, IN).
Bisindolylmaleimide 1 (BIM) and Gö 6976 were obtained from
Calbiochem.
Cells.
IG5 (Jurkat LTRluc), a Jurkat T-cell-derived cell line
containing a stably integrated HIV LTR-luciferase construct was
obtained through the Research and Reference Reagent Program, Division
of Acquired Immune Deficiency Syndrome (AIDS), National Institute of
Allergy and Infectious Diseases (NIAID), National Institutes of Health
(NIH) and maintained in RPMI-1640 containing 10 mmol/L Hepes buffer, 2 mmol/L L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin sulfate, and 10% heat inactivated fetal bovine serum (RPMI-FBS) (GIBCO, Grand Island, NY). On the day of the experiment, the
cells were suspended in RPMI without antibiotics or FBS (RPMI).
Neutrophils were isolated from venous blood collected from normal human
volunteers and a CGD patient as previously described.7 The
preparation contained greater than 97% neutrophils of which greater
than 96% were viable as measured by trypan blue exclusion. The
neutrophils were suspended in RPMI and used immediately.
Activation of the HIV-1 LTR.
Into 12 × 75-mm polypropylene tubes (Falcon 2063; Becton
Dickinson Labware, Lincoln Park, NJ) were added 2 × 106 Jurkat LTRluc, the components indicated in
the legends to the figures and table, and RPMI to a final volume of 2.0 mL. The tubes were incubated at 37°C in a CO2 incubator
(5% CO2, 95% air) for 6 hours and the luciferase activity
was determined using a luciferase assay kit (Promega Biotec, Madison,
WI) and a Monolight 1500 luminometer (Analytical Luminescence
Laboratories, San Diego, CA). Photons were counted over a 30-second
period and designated as relative light units (RLU).
Chemiluminescence.
The components indicated in the legend to the figure were placed in 12 × 75 mm polypropylene tubes (Falcon 2063), which were incubated
at 37°C in a CO2 incubator (5% CO2, 95%
air). At intervals, chemiluminescence was measured over a 10-second
period using the Monolight 1500 luminometer and the results expressed
as RLU.
Statistical analysis.
The results are presented as mean ± standard error of mean (SEM)
and the Mann Whitney U rank-sum test (unpaired, two-tailed) was used to
analyze differences for significance unless otherwise indicated (NS,
not significant P > .05).
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RESULTS |
Figure 1 demonstrates the activation of the
HIV-1 LTR in Jurkat T cells by normal neutrophils + PMA. Neutrophils
alone had no effect on the HIV-1 LTR at concentrations ranging from 3 × 104 to 107/mL. The Jurkat cell
concentration was 106/mL so that the ratio of
neutrophils:Jurkat cells ranged from 0.03:1 to 10:1. The LTR in Jurkat
cells was activated by PMA alone (background RLU 20,218 ± 2,725;
PMA 373,473 ± 65,262, n = 9, P < .001).
However, when neutrophils and PMA were combined, activation of the LTR
was significantly increased at a neutrophil concentration of 3 × 105/mL, reached a maximum at 106/mL
(neutrophil:Jurkat ratio 1:1) and then decreased to a level significantly below that of PMA alone when the neutrophil concentration was increased to 107/mL. Activation of the LTR in the
presence of PMA was significantly greater than that in the absence of
PMA (P < .05) at all the neutrophil concentrations used
except 107/mL. When normal neutrophils were replaced by
neutrophils from a patient with CGD, the stimulatory effect in the
presence of PMA was lost. The decrease in activity at high neutrophil
concentration, however, was still evident.
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| Fig 1.
Activation of the HIV-1 LTR in Jurkat T cells by
neutrophils and PMA. The reaction mixture contained 2 × 106 Jurkat LTRluc and, where indicated, 100 ng/mL PMA and either normal or CGD neutrophils at the concentrations
indicated, in RPMI at a final volume of 2.0 mL. The effect of PMA alone
is indicated by the ( ) (or  ) at zero neutrophils/mL. The results
with normal neutrophils are the mean ± SEM of 4 to 11 values and the
results with CGD neutrophils are the mean ± SEM of 2 to 4 values. The
asterisk indicates a significant difference from PMA alone (P < .05).
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A PMA dose-response curve is shown in Fig
2. A significant effect of PMA alone was observed at concentrations
down to 1 ng/mL, whereas when PMA and 106 neutrophils/mL
were combined, activation of the LTR was significantly greater than
that produced by PMA alone at PMA concentrations down to 3 ng/mL. The
decrease in activity at high neutrophil concentrations (107/mL) was observed at all of the PMA concentrations
used.
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| Fig 2.
PMA dose response curve. The reaction mixture contained
2 × 106 Jurkat LTRluc, either
106 or 107 neutrophils/mL and PMA at the
concentrations indicated in RPMI at a final volume of 2.0 mL. The
results are the mean ± SEM of four experiments. The y-axis was broken
to allow for an increased scale in the lower range to illustrate the
inhibitory effect of high neutrophil concentrations.
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Catalase had no effect on the LTR when added alone or with either
neutrophils (106 or 107/mL; data not shown) or
PMA (Fig 3). However, when catalase was added to cells exposed to a combination of neutrophils
(106/mL) and PMA, a significant inhibition was observed,
with the activation of the LTR decreasing to the level observed with
PMA alone. This inhibitory effect of catalase was lost on its heat inactivation. At the high neutrophil concentration (107/mL)
where activation of the LTR by neutrophils + PMA was lower than that of
PMA alone (Fig 1), the addition of catalase had no effect (Fig 3). SOD
at 1 µg/mL had no effect on the activation of the LTR under any of
our experimental conditions.
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| Fig 3.
Effect of catalase, heated catalase, or SOD on the
activation of the LTR by neutrophils and PMA. The reaction mixture
contained 2 × 106 Jurkat LTRluc
and, where indicated, 100 ng/mL PMA, 106 or 107
neutrophils/mL, 8.2 µg/mL catalase, 8.2 µg/mL heated catalase, or 1 µg/mL SOD in RPMI at a final volume of 2.0 mL. The results are the
mean + SEM of three to six experiments, with the asterisk indicating
a significant difference from the absence of catalase, heated catalase
and SOD (P < .05).
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The catalase used in Fig 3 was from bovine liver. It contained 58 ng
endotoxin per mg protein as measured by the Limulus Amebocyte Lysate
assay. Passage of the catalase preparation through a polymyxin B column
resulted in the removal of greater than 99% of the endotoxin. The
resultant endotoxin-poor catalase preparation inhibited the activation
of the HIV-1 LTR by neutrophils + PMA to a degree comparable to that of
the original catalase preparation, as did erythrocyte catalase, which
contained 4.0 ng endotoxin per mg protein.
Vanadate greatly potentiates the activation of the HIV-1 LTR in Jurkat
cells by H2O2.6 Under the
conditions used in Fig 4, vanadate at 3 × 105 mol/L had no effect on the activation of
the LTR by a combination of neutrophils (106/mL) and PMA.
However, when the neutrophil concentration was decreased to
105/mL, activation by neutrophils + PMA was decreased and
now a significant stimulation by vanadate was observed.
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| Fig 4.
Effect of vanadate on the activation of the LTR by
neutrophils and PMA. The reaction mixture contained 2 × 106 Jurkat LTRluc and, where indicated, 3 × 105 mol/L vanadate, 100 ng/mL PMA, and neutrophils at
the levels indicated, in RPMI at a final volume of 2.0 mL. The results
are the mean + SEM of four to five experiments, and the asterisk
indicates a significant difference from the absence of vanadate
(P < .05).
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Azide is an inhibitor of hemeproteins, such as catalase and MPO, which
degrade H2O2. Under the conditions used in
Fig 5A,activation of the LTR by neutrophils + PMA was significantly increased by azide at 3 × 106 mol/L, whereas, when the azide concentration was
increased to 104 to 103 mol/L,
activation of the LTR was inhibited. Azide at concentrations ranging
from 3 × 106 mol/L to 103
mol/L had no effect on the activation of the LTR in Jurkat cells induced by PMA alone (Fig 5A). The stimulation by azide at 3 × 106 mol/L was not seen when normal neutrophils were
replaced by CGD neutrophils.
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| Fig 5.
Effect of azide on the activation of the LTR by
neutrophils and either PMA or opsonized zymosan. The reaction mixture
contained 2 × 106 Jurkat LTRluc, either 100 ng/mL PMA (A) or 1 mg/mL opsonized zymosan (OZ) (B), azide at the
concentrations indicated and, where indicated, 106 normal
or CGD neutrophils/mL in RPMI at a final volume of 2.0 mL. The results
are the mean ± SEM of three to six experiments, with the asterisk
indicating a significant difference from the absence of azide
(P < .05).
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These findings suggested that the H2O2 formed
by the respiratory burst of PMA-stimulated neutrophils can activate the
HIV-1 LTR in Jurkat T cells. It was, therefore, surprising that another potent stimulus of the respiratory burst of neutrophils, opsonized zymosan, when combined with neutrophils, had only a small effect on the
LTR (Table 1). The combination of
neutrophils and opsonized zymosan was significantly different from
background by paired (P < .01), but not by unpaired,
analysis. Azide at concentrations ranging from 3 × 106 to 3 × 104 mol/L
significantly increased the activation of the LTR by normal neutrophils + opsonized zymosan, with an optimum effect observed at 3 × 105 mol/L azide (Fig 5B). Azide had no effect on the
activation of the LTR either alone (data not shown), with normal
neutrophils alone (data not shown), with opsonized zymosan alone (Fig
5B), or with CGD neutrophils + opsonized zymosan (Fig 5B). The
activation of the LTR by opsonized zymosan-stimulated normal
neutrophils in the presence of azide was strongly inhibited (P < .001), but not abolished (P < .001) by catalase, whereas
heated catalase was without effect (Table 1), implicating
H2O2.
The considerably greater responsiveness of the LTR to neutrophils
stimulated by PMA as compared with neutrophils stimulated by opsonized
zymosan, despite both acting as strong stimuli of the neutrophil
respiratory burst appears to be due, at least in part, to a synergism
between H2O2 and PMA in the activation of the
LTR. Under the conditions used in Fig 6,
H2O2 alone at the optimum concentration of
104 mol/L increased luciferase activity from a
background of 23,379 RLU to 110,956 RLU and PMA alone at 100 ng/mL
increased luciferase activity to 403,823 RLU. When
H2O2 and PMA were combined, luciferase activity
was 3,096,165 RLU, which was significantly greater than the effect of
H2O2 (P < .005) or PMA (P < .005) alone or than the additive effect of H2O2
and PMA (P < .005). Thus, under these conditions,
H2O2 and PMA act together to produce an effect,
which is considerably greater than the sum of the effects of each
alone. Synergism with H2O2 was not observed
when PMA was replaced by TNF- or IL-2 at 100 U/mL (Fig 6). IL-2
alone had no effect on the LTR in Jurkat cells under our experimental
conditions.
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| Fig 6.
Synergistic effect of H2O2 and
PMA on activation of the LTR. The reaction mixture contained 2 × 106 Jurkat LTRluc and, where indicated, 100 ng/mL PMA, 100 U/mL TNF-, 100 U/mL IL-2, and
H2O2 at the concentrations indicated in RPMI at
a final volume of 2.0 mL. The effect of PMA, TNF-, or IL-2 alone is
indicated by the () at zero H2O2
concentration. The results are the mean ± SEM of four to six
experiments. The asterisk indicates a significantly greater effect of a
combination of PMA (or TNF- or IL-2) and
H2O2 than the additive effect of each alone
(P < .05).
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Because PMA activates PKC, the synergism between PMA and
H2O2 may reflect a priming effect of PKC
activation on the activation of the LTR by
H2O2. This prompted a study of the effect of
the PKC selective inhibitors BIM and Gö 6976 on the activation of the HIV-1 LTR in Jurkat T cells. BIM strongly inhibited the activation of the LTR by PMA, H2O2,
H2O2 + PMA, and neutrophils + PMA, with an
inhibitory effect observed at a concentration of 107
mol/L, and in some instances, lower (Fig
7). Gö 6976 was less effective with an inhibition observed at
106 mol/L in all instances. BIM and Gö 6976 at
concentrations ranging from 3 × 109 to
106 mol/L were without effect on the activation of
the LTR by TNF-.
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| Fig 7.
Effect of the PKC inhibitors BIM and Gö 6976 on
activation of the LTR. The reaction mixture contained 2 × 106 Jurkat LTRluc and where indicated 100 ng/mL
PMA, 104 mol/L H2O2,
106 neutrophils/mL, 100 U of TNF-/mL, and BIM and
Gö 6976 at the concentrations indicated in RPMI at a final volume
of 2.0 mL. The results are the mean + (Gö 6976) or  (BIM)
SEM of four experiments with the asterisk indicating a significant
difference from the absence of the PKC inhibitors (P < .05).
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The effect of BIM on the respiratory burst of neutrophils stimulated
with either PMA or opsonized zymosan as measured by either luminol or
lucigenin-enhanced chemiluminescence is shown in
Fig 8. Luminol-enhanced chemiluminescence
by neutrophils is predominantly dependent on peroxidase-catalyzed
reactions, whereas lucigenin-enhanced chemiluminescence is largely
peroxidase-independent and related to superoxide anion
release.8 Opsonized zymosan was more effective than PMA as
a stimulus of luminol-enhanced chemiluminescence by neutrophils,
whereas, when lucigenin-enhanced chemiluminescence was measured, PMA
was more effective than opsonized zymosan as the stimulus. This may
reflect a greater MPO release by the phagocytic stimulus opsonized
zymosan. With both luminol and lucigenin, BIM almost completely
inhibited PMA-induced chemiluminescence, whereas opsonized
zymosan-induced chemiluminescence was decreased approximately 50%.
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| Fig 8.
Effect of the PKC inhibitor BIM on the respiratory burst
of neutrophils induced by PMA and opsonized zymosan. The reaction
mixture contained 106 neutrophils/mL and where indicated
105 mol/L luminol, 104 mol/L lucigenin,
100 ng/mL PMA, 1 mg/mL opsonized zymosan, and 106 mol/L
BIM in a standard salt solution (4 × 103 mol/L sodium
phosphate buffer pH 7.4, 0.128 mol/L NaCl, 1.2 × 102
mol/L KCl, 103 mol/L CaCl2, 2 × 103 mol/L MgCl2, 2 × 103
mol/L glucose) at a final volume of 0.5 mL. The results are the mean + SEM of four experiments.
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DISCUSSION |
Activation of the HIV-1 LTR occurs by a variety of mechanisms, of which
reaction of the product of the TAT gene with the TAR sequence in the
LTR appears to be the most important. Activation of the LTR by
H2O2 is an additional mechanism, raising the
possibility that biologic sources of H2O2 such
as stimulated phagocytes, may influence the replication of HIV-1. We
report here that neutrophils, when combined with the stimulus PMA,
strongly activate the HIV-1 LTR introduced by transfection into the
Jurkat T-lymphocyte cell line. This activation by neutrophils and PMA
is due to, at least, three processes: (1) the direct activation of
Jurkat LTRluc by PMA; (2) the activation of the LTR by
H2O2 formed by the PMA-induced respiratory
burst of neutrophils; and (3) synergism between
H2O2 and PMA in the activation of the LTR.
The direct activation of the LTR in Jurkat cells by PMA could account
for only 12% to 13% of the total luciferase synthesis induced by
neutrophils (106/mL) + PMA. Activation by PMA alone was
unaffected by catalase or by vanadate (see also Kazazi et
al6), which would argue against the involvement of
H2O2, at least H2O2
accessible to these agents. PMA is a potent activator of PKC. The
involvement of PKC in the activation of the LTR by PMA is suggested by
the inhibitory effect of the selective PKC inhibitors BIM and Gö
6976. BIM had an IC50 for PMA-induced LTR activation of
0.02 µmol/L and Gö 6976 was approximately 10-fold less
effective.
That H2O2 formed by the PMA-induced respiratory
burst of neutrophils is also responsible, in part, for the activation
of the LTR is supported by the inhibitory effect of catalase, the
stimulatory effect of vanadate at suboptimal neutrophil concentrations
and the loss of activity when normal neutrophils were replaced by neutrophils from a CGD patient. The effect of catalase is lost on its
heat-inactivation and is unrelated to endotoxin contamination. Catalase
reduces the activation of the LTR by neutrophils + PMA to the level
observed with PMA alone, as did the substitution of CGD for normal
neutrophils, suggesting that the total additional effect on the
addition of neutrophils to PMA is due to the
H2O2 formed.
Although both PMA and H2O2 can individually
activate the HIV-1 LTR in Jurkat cells,6 the effect of
their combination is considerably greater than the sum of each alone.
The combination of PMA and reagent H2O2
increases the activation of the LTR to the level observed with
neutrophils + PMA. This suggests that PMA acts not only directly and by
inducing H2O2 formation by the neutrophils, but
also by increasing the sensitivity of the LTR in Jurkat cells to
activation by H2O2. Some selectivity for PMA was observed, as H2O2 did not synergize with
either TNF- or IL-2 in the activation of the LTR. A synergism
between PMA and H2O2 in the activation of
NF-B has been reported,9 and this may form the basis for
the synergistic activation of the LTR by these agents. Activation of
the LTR by PMA in the presence of neutrophils or
H2O2 is inhibited by BIM and Gö 6976 suggesting the involvement of PKC in the synergism. PKC consists of a
number of isoforms, which have in common catalysis of the
phosphorylation of protein serine/threonine residues. PKC has been
implicated in the replication of HIV-1 at a number of sites including
the activation of the HIV-1 LTR10,11 where PKC may act in
part through its activation of NF-B.12-14 Activation of
the LTR by H2O2 alone also is inhibited by the
PKC inhibitors raising the possibility of an autostimulatory mechanism
by which activation of PKC by H2O2 amplifies
the subsequent response to H2O2. The PKC
inhibitors at concentrations up to 106 mol/L did not
inhibit the activation of the LTR by TNF-, indicating some
selectivity for PMA- and/or
H2O2-induced activation.
The strong activation of the LTR by neutrophils + PMA reached a maximum
at 106 neutrophils/mL and then decreased to levels below
that of PMA alone when the neutrophil concentration was increased to
107/mL. The mechanism of this decrease in activity at high
neutrophil concentration is unclear. The generation of reactive oxygen
species as measured by chemiluminescence was increased at high
neutrophil concentration raising the possibility of a toxic effect of
products of the respiratory burst on the Jurkat LTRluc
cells; however, the decrease in LTR activation was not prevented by
catalase suggesting that H2O2 was not a toxic
species under these conditions. Neutrophils heated at 65°C for 15 minutes lose their ability to respond to PMA with a respiratory burst,
but remain inhibitory of the LTR at high concentration (0.9 × 107 heated neutrophils + 106 normal
neutrophils/mL) (data not shown). It is possible that a product of
neutrophils unrelated to the respiratory burst is toxic to Jurkat cells
at high neutrophil concentrations.
When opsonized zymosan was used instead of PMA as the stimulus of the
neutrophil respiratory burst, activation of the LTR was considerably
decreased. A small, but significant, activation was observed, which was
less than 1% of that observed with neutrophils + PMA. Unlike PMA,
opsonized zymosan did not have a direct effect on the Jurkat
LTRluc nor did opsonized zymosan act synergistically with
H2O2 to activate the LTR (data not shown).
Azide is a potent inhibitor of the heme enzymes catalase and
myeloperoxidase, which degrade H2O2 and, thus,
the amount of H2O2 detected in the
extracellular fluid on stimulation of neutrophils by PMA or opsonized
zymosan is greatly increased by azide.15 The addition of
azide thus would be expected to increase the availability of
H2O2 for LTR activation. When Jurkat
LTRluc were exposed to neutrophils + opsonized zymosan in
the presence of azide, increased activation of the LTR was observed,
which was strongly inhibited by catalase, but not by heated catalase,
and was not seen when normal neutrophils were replaced by CGD
neutrophils. In contrast, addition of azide to neutrophils stimulated
by PMA had only a small stimulatory effect at low (3 × 106 mol/L) azide concentration, possibly due to the
strong activation in the absence of azide.
Our findings suggest that H2O2 generated by the
respiratory burst of neutrophils may influence the survival and
replication of HIV-1 in two opposing ways: (1) by reaction with
peroxidase and a halide to produce a viricidal effect1; and
(2) by activation of the HIV-1 LTR with consequent stimulation of gene
transcription and viral replication. The latter effect is favored by
the addition of azide (which promotes the accumulation of
H2O2 and its release into the extracellular
fluid) or vanadate (which forms a bioactive complex with
H2O2). Further, activation by
H2O2 is amplified by procedures, such as the
addition of PMA, which activates PKC. Activation of PKC occurs when
receptor-mediated hydrolysis of inositol phospholipids induced by a
variety of stimuli, including mitogens, growth factors, and cytokines
results in the release of diacylglycerol.16
H2O2, as well as other forms of oxidant stress,
can also activate PKC.17-21 Activation of PKC by these and
other mechanisms would be expected to increase the sensitivity of the
HIV-1 LTR to further activation by H2O2
generated by adjacent phagocytes, certain microorganisms such as
vaginal lactobacilli, as well as from other sources.
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ACKNOWLEDGMENT |
We thank Peggy Sue O'Brien for the preparation of the manuscript.
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FOOTNOTES |
Supported in part by Grant No. AI07763 from the National Institutes of
Health, Bethesda, MD.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Seymour J. Klebanoff, MD, PhD,
Department of Medicine, Box 357185, University of Washington, Seattle,
WA 98195-7185; e-mail: seym{at}u.washington.edu.
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Viricidal effect of polymorphonuclear leukocytes on HIV-1: Role of the myeloperoxidase system.
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Viricidal effect of stimulated human mononuclear phagocytes on human immunodeficiency virus type 1.
Proc Natl Acad Sci
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Klebanoff SJ, Coombs RW:
Virucidal effect of stimulated eosinophils on human immunodeficiency virus type 1.
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Abstract
Introduction
