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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1021-1027
Human Immunodeficiency Virus Type 1 Protease Inhibitor Modulates
Activation of Peripheral Blood CD4+ T Cells and Decreases
Their Susceptibility to Apoptosis In Vitro and In Vivo
By
Elaine M. Sloand,
Princy N. Kumar,
Sonnie Kim,
Aniruddho Chaudhuri,
Frank F. Weichold, and
Neal S. Young
From the National Heart, Lung and Blood Institute, National
Institutes of Health, Bethesda, MD; the Division of Infectious
Diseases, Georgetown University, Washington, DC; and the Institute of
Human Virology, University of Maryland, Baltimore, MD.
 |
ABSTRACT |
CD4+ T cells from patients with human immunodeficiency
virus (HIV) infection undergo apoptosis at an increased rate, which leads to their depletion during disease progression. Both the Fas-Receptor (Fas-R) and interleukin-1 (IL-1)-converting enzyme (ICE; caspase 1) appear to play a role in the mechanism of apoptosis of
CD4+ lymphocytes. Although Fas-R is upregulated on both
CD4+ and CD8+ cells in HIV-infected
patients, results from our laboratory and others indicate that, in
patients with advanced disease, CD4+ cells preferentially
express ICE. Protease inhibitors have successfully halted the
progression of HIV disease and increased CD4+ T counts.
In this study, we examined the effect of protease inhibitors on Fas-R
(CD95), ICE (caspase 1) expression, apoptosis, and cell death in
CD4+ T cells of (1) HIV-infected patients who were
receiving protease inhibitors, and (2) normal and patient
CD4+ T cells cultured with a protease inhibitor in vitro.
Fifteen patients with advanced HIV disease on treatment showed
dramatically decreased CD4+ T-cell ICE expression,
diminished apoptosis, and increased numbers of CD4+ cells
within 6 weeks of institution of protease inhibitor therapy, and before
down-modulation of Fas-R (CD95) expression was evident. To determine
the role of HIV infection, we studied the effect of ritonavir, a
protease inhibitor, on normal and patient cells in vitro. Stimulated
and unstimulated normal CD4+ T cells, cultured with
protease inhibitor, demonstrated markedly decreased apoptosis and ICE
expression (P = .01). While Fas-R expression was not
significantly altered during short-term culture by such treatment,
Fas-Ligand (Fas-L) membrane expression of phytohemagglutinin (PHA)-stimulated blood lymphocytes was decreased by protease inhibitor. In the presence of ritonavir, CD4+ T cells from
HIV-infected patients showed similar changes in ICE intracellular
levels without alteration of Fas expression. In conclusion, protease
inhibitors appear to decrease CD4+ T-cell ICE expression
and apoptosis before they affect Fas-R expression in HIV-infected
patients. This action was independent of HIV infection, as similar
effects were seen in CD4+ T cells from normal controls.
Some of the benefit of protease inhibitors may be related to
modification of programmed cell death, which increases
CD4+ T-cell number. Whether this is due to directly to
the changes effected in the caspase system remains to be determined.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INFECTION WITH THE human immunodeficiency
virus type 1 (HIV-1) is associated with apoptosis and subsequent
depletion of CD4+ cells, a process partially mediated by
the Fas-Receptor (Fas-R; CD95) and by interleukin-1
(IL-1)-converting enzyme (ICE; caspase 1).1-3 Apoptosis
of CD4+ cells is responsible for depletion of
CD4+ cells, independent of virally mediated
cytolysis.4-6 Spontaneous and activation-induced programmed
cell death leads to depletion of uninfected CD4+ cells in
patients with HIV infection.2,4,6 Fas-mediated apoptosis
may be responsible for a portion of CD4+ cell death. Fas-R
(CD95) expression is increased on both CD4+ and
CD8+ cells in HIV-infected individuals.7,8
Apoptosis is mediated by cross-linking of the Fas-R by Fas-Ligand
(Fas-L). Upregulation of the Fas-R, as well as Fas-L, is associated
with HIV infection and may be induced by HIV-Tat and glycoprotein
(gp)120.9,10 TH1 cells have a differential susceptibility
to gp120-mediated apoptosis.11 However, not all apoptosis
in HIV-infected individuals is mediated by Fas; for example, apoptosis
induced by activation of the CD4+ cells by CD3 antibody is
independent of Fas-R expression. Nef, a protein of HIV that binds to
and induces apoptosis of uninfected CD4+ cells, also acts
independently of Fas-R expression.12
ICE is integral to both Fas-R-independent and -dependent apoptosis,
as ICE inhibitors block apoptosis in most CD4+
cells.1-3 While Fas-R expression is upregulated in both
CD4+ and CD8+ cells of HIV-infected
patients,7,8 ICE protein is preferentially expressed in
CD4+ T cells in this group, but not in
normals.1 Uninfected CD4+ T cells express ICE
mRNA, but they do not express active ICE protein.1
Activation-induced cell death of CD4+ cells in HIV-infected
individuals is Fas-independent and can be blocked by inhibitors of
ICE.1-3 Because ICE protein is expressed in
CD4+ T cells of HIV-infected patients and ICE inhibitors
block apoptosis and cell death of these cells in culture, agents that
modulate ICE protein expression and activity might potentially modify
CD4+ T-cell depletion.
HIV-1 protease inhibitors cause substantial increases in
CD4+ cell counts, halt disease progression, and decrease
HIV viremia.13,14 The central mechanism of action of these
protease inhibitors is to block HIV protease activity, which is
necessary for the maturation of infectious virions. Previously, we
reported that a small group of HIV-infected patients who were receiving
protease inhibitors showed significantly reduced ICE expression in
CD4+T cells, although they had advanced
disease.1 Nucleoside analogs decrease Fas-L mRNA expression
and Fas-R expression in HIV-1-infected patients,15 and one
interpretation of this observation was that the increased
CD4+ cell count and the favorable clinical response
resulted from modulation of apoptosis in this lymphocyte population.
In the current study, we systematically examined the effects of HIV
protease inhibitors on CD4+ T-cell apoptosis, cell death,
and ICE expression in CD4+ T lymphocytes obtained from
HIV-infected patients who had previously failed to respond to treatment
with nucleoside analogs. We then determined if these effects were
dependent on the presence of HIV by examining the in vitro actions of
ritonavir, a protease inhibitor, on lymphocytes of both normal and
HIV-infected persons. We also tested the influence of protease
inhibitors on normal CD4+ cell expression of Fas-R and
Fas-L to further assess the mechanism by which they alter
susceptibility to apoptosis.
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MATERIALS AND METHODS |
Patient selection.
Peripheral blood (PB) samples were obtained from patients with AIDS and
normal volunteers who provided informed consent according to protocols
approved by the Institutional Review Boards of the National Heart,
Lung, and Blood Institute (Bethesda, MD) and the Georgetown University
Medical Center (Washington, DC). HIV-infected patients, who were not
currently taking protease inhibitors, but for whom the clinical
decision was already made by their primary care physician to place them
on protease inhibitors, were chosen from the patient population of the
HIV Clinic at Georgetown University Hospital. Patients received a
combination of protease inhibitors and were examined weekly to biweekly
intervals. Venous blood was obtained for studies immediately before
initiation of treatment with protease inhibitors and then at 3 to 6 weeks and 4 to 6 months.
Cell separation and culture.
PB mononuclear cells (PBMC) were separated using density gradient
centrifugation with lymphocyte separation media (Organon, Durham, NC).
Thereafter, cells were washed twice with phosphate-buffered saline
(PBS) and resuspended in RPMI 1640 supplemented with fetal calf serum
(FCS; both Life Technologies, Gaithersburg, MD). Cultures were
performed at a cell density of 0.5 × 106 cells/mL.
When appropriate, natural lymphocyte-derived IL-2 (Boehringer Mannheim,
Indianapolis, IN) or phytohemagglutinin (PHA; Boehringer) was used for
stimulation at concentrations of 10 U/mL or 5 µg/mL, respectively.
Anti-Fas monoclonal antibody (MoAb) CH11 (Kamyia, San Francisco, CA),
which mimics the Fas-L by cross-linking the Fas-R, was used in some cultures.
Flow cytometry.
For the measurement of Fas expression on CD4+ and
CD8+ PBMC by flow cytometry, a whole-blood test was used.
Briefly, 100 µL of cells were incubated with 20 µL phycoerythrin
(PE)-conjugated anti-CD4 or -CD8 MoAb (Becton Dickinson, Mountain View,
CA) combined with 20 µL of fluorescein isothiocyanate
(FITC)-conjugated anti-CD95 MoAb (UB2; PharMingen, San Diego, CA) or
anti-Fas-L (Immunotech, Miami, FL). After 15 minutes of
incubation, cells were washed and fixed in 0.2% paraformaldehyde.
Samples were analyzed using an Epics ELITE flow cytometer (Coulter,
Miami, FL).
Intracellular staining.
Intracellular staining for ICE expression was performed using the
PharMingen Intracellular Staining Kit.16 PBMC were stained with CD4 MoAb, fixed, and permeabilized and stained with anti-ICE antibody and FITC-labeled conjugated antirabbit antibody. Permeabilized isotypic controls were stained with secondary antibodies.
Apoptosis assays.
Annexin assay PBMC were prepared as described earlier, washed with PBS,
and stained with annexin and propidium iodide (Tat assay) as previously
described.17 Samples were analyzed using flow cytometry. An
in situ thymidine deoxyneuclease (TdT) assay (Apotag; Oncor,
Gaithersburg, MD) also was used to quantitate the number of cells that
underwent apoptosis. Cells were harvested, fixed with 4% neutral
buffered formalin, and centrifuged. Endogenous peroxidase was quenched
with 0.5% hydrogen peroxide and the cells were permeabilized using
company-supplied equilibration buffer. The 3'-OH ends of degraded
DNA were reacted with TdT and digoxygenin-labeled adenosine
triphosphate (ATP) for 30 minutes. After washing with PBS, cells were
reacted with antidigoxygenin MoAb conjugated to peroxidase, washed, and
exposed to 3,3' diaminobenzidine tetrahydrochloride (DAB; Pierce,
Rockford, IL) and the percent fluorescence quantitated by flow cytometry.
p24 enzyme-linked immunosorbent assay.
Viral replication was determined by p24 production. Tissue culture
supernatants were harvested and stored at  70°C until
analysis. p24 concentrations in tissue culture supernatants were
determined in duplicate using a commercially available enzyme-linked
immunosorbent assay (ELISA; Coulter) according to the manufacturer's
recommendations. To count for cell death, p24 production was expressed
per 1 × 106 viable cells.
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RESULTS |
Effect of ritonavir on viability, apoptosis, and ICE expression in
HIV-infected patients placed on protease inhibitors.
As an extension of our previous observations that HIV-infected patients
who received protease inhibitors demonstrated decreased ICE expression
in CD4+ T cells, we studied a group of patients before and
following institution of protease inhibitor therapy. Fifteen patients
with HIV disease, who had failed to respond to treatment with
nucleoside analogs, were studied before and following initiation of
treatment with a HIV protease inhibitor combination (Table
1). Patients had no active bacterial
infections, although 2 had stable cytomegalovirus (CMV) disease, for
which they were receiving ganciclovir therapy. The average increase in
CD4+ cell count during the period of evaluation was 20 cells/mL. When lymphocytes were stained with CD4+-PE, with
annexin and propidium iodide (PI), or with ICE antibody, CD4+ cells from patients after a mean of 6 weeks of
treatment demonstrated significantly decreased apoptosis, cell death,
and ICE expression (Fig 1). Fas-R
expression was not significantly changed after a mean of 6 weeks of
treatment. Patients who received protease inhibitors for longer periods
of time (3 to 4 months), with significant clinical responses and
undetectable viral titers, demonstrated normal Fas-R and ICE expression
(data now shown). In this group, apoptosis of CD4+ cells
was still increased compared with normals (Fig
2 and Table 2),
which suggests that excessive apoptosis and CD4+ T-cell
depletion continued to occur, despite optimal therapy, and that
pathways that lead to apoptosis, which do not involve ICE and FAS,
might be involved. TdT assay also demonstrated apoptosis (data not
shown). Staining for other effectors of apoptosis, tumor necrosis
factor receptor (TNF-R), bcl-2, and p53, was similar in normals
and this group of HIV-infected patients (data not shown).
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| Fig 1.
Combination treatment with protease inhibitors decreases
ICE expression and apoptosis in CD4 cells from HIV-infected patients.
Fifteen patients who failed nucleoside analog treatment were placed on
antiviral therapy that included a protease inhibitor. Blood was
analyzed before and 6 to 8 weeks after treatment. Cells were analyzed
for ICE and Fas-R expression, and apoptotic cells assessed by annexin V
binding. There were significant decreases in ICE expression and
apoptosis following treatment (P < .01), while there was no
significant change in Fas-R expression. CD4 counts increased a mean of
20 cells/mL.
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| Fig 2.
HIV-infected persons with undetectable viral loads still
demonstrate excessive apoptosis when compared with normal controls. Ten
HIV patients receiving combination antiviral therapy with a protease
inhibitor combination demonstrated normal Fas-R and ICE expression,
but excessive apoptosis when compared with 6 normal controls.
Scattergrams of apoptotic cells are seen above form-selected
HIV-infected patients and normals.
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Table 2.
CD4+ T Cells From Normal and Patient
(selected because of undetectable viral titers) Samples Stained for PI
and Annexin
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Ritonavir decreases ICE expression, apoptosis, and cell death in
cultured normal CD4+ cells deprived of IL-2 or stimulated
with Fas agonist.
While adequate control of viral replication appeared to be the most
likely explanation of the beneficial effects and drug therapy on
apoptosis, we could not exclude a direct effect of protease inhibitors
on apoptotic pathways that were independent of the virus. To address
this hypothesis, we performed in vitro experiments using both patient
and normal uninfected PB lymphocytes (PBLs). When PBLs
from normal controls (n = 10) deprived of IL-2 so as to induce
programmed cell death were cultured with ritonavir for 72 hours, they
demonstrated significantly deceased apoptosis and cell death when
compared with cultures in the presence of protease inhibitor (P < .01; Fig 3). Similar results were
obtained using the TdT assay (Fig 4). Fas-R
expression was not significantly altered by drug exposure. High
ritonavir concentrations (>20 nmol/L) resulted in increased cell
death. Similar, but less pronounced changes were seen in
CD8+ cells (data not shown). CPP32 expression was
unaffected by ritonavir (data not shown).
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| Fig 3.
Ritonavir decreases ICE expression and apoptosis in
normal CD4+ cells deprived of IL-2. PBMC from normal
donors were cultured without IL-2. After 72 hours, cells were analyzed
by flow cytometry; ICE expression and apoptosis were significantly
decreased in cells cocultivated with ritonavir (P < .01).
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| Fig 4.
Ritonavir decreases apoptosis in normal
CD4+ T cells deprived of IL-2 using the TdT
assay. PBMC from normal donors prepared and cultured as described in
Fig 3 demonstrate decreased apoptosis as measured by the TdT assay
after exposure to ritonavir. The number of apoptotic cells was
determined using 2-color flow cytometry. Cells were gated to include
only CD4+ cells (stained with FITC-labeled
CD4-PE MoAb) are seen in the histogram above.
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In a second set of experiments, when normal CD4+ cells were
activated with IL-2 and PHA and then exposed to a Fas agonist (MoAb CH11). These cells also showed increased viability (Fig
5), decreased apoptosis (Fig
6), and decreased ICE expression (Fig
7) in the presence of ritonavir (P < .01). All effects were dose-dependent, and most pronounced after
stimulation with Fas agonist. Similar findings were obtained when
CD4+ cells from HIV+ patients (n = 4) were
examined (data not shown).
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| Fig 5.
Ritonavir increases cell viability of CD4+
cells treated with Fas agonist (CH11). PBMC from normal donors were
cultured with Fas agonist with and without ritonavir (5 nmol/L).
Viability is expressed as percentage of untreated (ie, without Fas
agonist) control. Cell viability was determined by PI staining.
Examples of scattergrams are shown.
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| Fig 6.
Ritonavir decreases apoptosis in CD4+cells
cultured with Fas agonist. Normal PBMC were cultured with Fas agonist,
with and without ritonavir (5 nmol/L). Cells were stained with CD4-PE
and FITC-annexin V and analyzed by flow cytometry. There was a
significant (P < .05) decrease in annexin V binding in cells
treated with ritonavir.
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| Fig 7.
Ritonavir decreases ICE expression in CD4+
cells cultured with Fas agonist. Normal PBMC were cultured with and
without ritonavir. Cells were surface-stained with CD4-PE,
permeabilized, and stained with ICE-FITC. CD4+ cells
expressing ICE are shown. There was a significant, dose-dependent
decrease in ICE expression (P < .01).
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We also tested whether a nucleoside analog had a similar effect on
CD4+ T cells from normal controls (n = 2) when cells were
cultured under similar conditions. There was no difference in
apoptosis, cell death, or ICE expression after 72 hours in culture with
IL-2 and Fas agonist (data not shown).
To assess whether protease inhibitors exerted their effect through
Fas-L, we cocultivated normal lymphocytes (n = 3) with ritonavir and
examined expression of Fas-L. When PBLs were cultured with IL-2 and
PHA, ritonavir-treated CD4+ cells showed decreased
expression of Fas-L, which was dose-dependent and independent of Fas-R
expression (Fig 8).
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| Fig 8.
Ritonavir decreases Fas-L expression, but does not alter
Fas-R expression in CD4+ cells stimulated with PHA.
Samples of normal PBMC were cultured with PHA for 72 hours at 37°C
at different concentrations of ritonavir. There was a significant
dose-dependent decrease in Fas-L expression with ritonavir, although
there was no change in Fas-R expression.
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DISCUSSION |
Protease inhibitors have favorably altered the quality of life of
HIV-infected patients. HIV protease inhibitors block cleavage of gag
and gag-pol protein precursors, aborting virus
infectivity.18
These drugs increase CD4+ T-cell counts, increase naive and
memory cells, enhance lymphoproliferative responses, and reduce plasma
concentrations of cytokines such as TNF- that promote apoptosis.19 Protease inhibitors are presumed to exert
their positive effect on CD4+ T-cell number and immune
function by inhibiting viral replication. However, clinical data
suggest that the beneficial consequences of protease inhibitors persist
even after viral resistance to the drug has developed,20
and these drugs may thus affect the immune system, independent of their
inhibition of retroviral replication.
The current work demonstrates that protease inhibitor therapy is
associated with decreased ICE (caspase 1) expression, apoptosis, and
cell death of CD4+ T cells. We further show that these
changes in ICE protein expression and in apoptotic cell death may not
solely be related to changes in viremia, viral products, or cytokines.
In vitro experiments, using CD4+ T cells from
normal uninfected controls, showed similar significant declines in ICE
expression, apoptosis, and cell death when lymphocytes exposed to Fas
agonist or cultured in the absence of IL-2. Protease inhibitors may
thus exert effects on apoptosis, independent of their effects on HIV,
by affecting directly or indirectly the generation of active ICE
protein in the apoptotic pathway.
Others have reported that effective treatment of HIV-infected patients
with antiviral therapy lowered Fas-L and Fas-R expression with
decreased apoptosis in the CD4+ T-cell
population.15 Our study demonstrates that, in the
HIV-infected patient, protease inhibitors block apoptosis and cell
death, while they inhibit active ICE formation in CD4+
cells. We confirm the observations of others that Fas-R expression declines after initiation of antiviral therapy, which is probably a
result of lower levels of viremia and fewer viral products to promote
Fas-R expression.13 However, findings due to changes in
Fas-R expression temporally followed changes in apoptosis and ICE
expression, and therefore decreases in Fas-R expression do not appear
alone sufficient to account for decreased apoptosis in the lymphocytes
observed with treatment. This observation is consistent with those of
others who showed that HIV kills CD4+ cells by a
Fas-independent mechanism.21
The pathophysiologic pathway responsible for inhibition of ICE
expression by protease inhibitors is unknown. Expression of active ICE
protein in mammalian cells is regulated posttranscriptionally by
control and cleavage of an aspartate residue from the inactive pro-ICE
protein. To determine if protease inhibitors could potentially bind to
the inactive ICE precursor in a manner analogous to their binding to
the HIV protease, we compared the sequences of HIV-1 protease and
caspase 1 (ICE), but no significant similarity was found between them
nor did HIV-1 protease sequence show similarity to either caspase 2 (ICH-1) or caspase 3 (CPP32). Further studies are needed to determine
if protease inhibitors actually bind to caspase 1 or to pro-caspase 1.
The number of apoptotic and dead CD4+ T cells remained
abnormally high in the protease inhibitor-treated patients, despite undetectable viral loads, even with apparently normal ICE and Fas-R
expression. This suggests that an additional ICE- and Fas-independent pathway is responsible for apoptosis and cell death in this population. HIV infection is associated with an increased rate of
activation-associated necrosis,22 although in this study,
apoptosis was excessive and appeared to account for most of the
lymphocyte death seen. Other caspases also are involved in apoptosis,
including CPP32 and FLICE (caspase 8), and more recent studies suggest
that these caspases may have more prominent roles in apoptosis than
caspase 1.23 TNF-related apoptosis-inducing ligand
(TRAIL)24 also has been shown to be responsible for
apoptosis in HIV infection and is independent of ICE.
One implication of our findings is that inhibitors of ICE or CD4
apoptosis, without antiviral activity, may have potential therapeutic
utility in the treatment of HIV infection, even in patients with low
viral loads. However, caution is indicated, as increased viral
replication in infected cells subjected to ICE blockade has been
reported in vitro,25 possibly related to the role of
apoptosis in controlling viral replication.26,27
| |
FOOTNOTES |
Submitted October 9, 1998; accepted April 1, 1999.
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 Elaine M. Sloand, MD, National Institutes
of Health, National Heart, Lung, and Blood Institute, 31 Center Dr, MSC
2490, Building 31, Room 4A11, Bethesda, MD 20892-2490.
| |
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Apoptosis, lymphocytotoxicity and the containment of viral infections.
Med Hypoth
18:399, 1985[Medline]
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