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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1000-1010
Human Immunodeficiency Virus Type 1 Nef Protein Sensitizes
CD4+ T Lymphoid Cells to Apoptosis via Functional
Upregulation of the CD95/CD95 Ligand Pathway
By
Giorgio Zauli,
Davide Gibellini,
Paola Secchiero,
Hélène Dutartre,
Daniel Olive,
Silvano Capitani, and
Yves Collette
From the Human Anatomy Section, Department of Morphology and
Embryology, University of Ferrara, Ferrara; Institute of Microbiology,
University of Bologna, Bologna, Italy; Institute of Human Virology,
University of Maryland at Baltimore, Baltimore, MD; and Unité 119 Istitut National de la Santé et de la Recherche Medicale
(INSERM), Université dela Mediterrainée, Marseille, France.
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ABSTRACT |
Many viruses have evolved genes encoding proteins that regulate cell
death by apoptosis. The human immunodeficiency virus type 1 (HIV-1) Nef
protein alters T-cell development and signaling and is required for
optimal viral replication and pathogenicity in vivo. To analyze the
interference of Nef with cell survival, we used both regulated and
constitutively expressed nef alleles in stably transfected
T-cell lines. Nef-expressing cells were sensitized to cell death by
apoptosis, which was specifically exacerbated by an anti-CD95 IgM
monoclonal antibody (MoAb). Flow cytometric analysis showed that the
surface expression of both CD95 and CD95 ligand (CD95L) was upregulated
by endogenous Nef expression. Nef-mediated apoptosis was almost
completely suppressed by the addition in culture of an anti-CD95 Fab'
IgG MoAb, which specifically blocks CD95/CD95L interactions. Lastly,
mutation of a proline motif in the core region of the nef gene,
which disrupts its ability to interact with cellular kinases and
reduces HIV-1 replication in vitro, completely abrogated the
Nef-mediated induction of apoptosis as well as its ability to
upregulate surface CD95 and CD95L. These findings may provide molecular
insight into the role of endogenous Nef in the T-cell depletion
observed in vivo, particularly HIV-specific cytotoxic
CD8+ T cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
APOPTOTIC CELL DEATH has been proposed as
one of the key mechanisms involved in T-cell depletion during the
course of human immunodeficiency virus type 1 (HIV-1)
disease.1-3 It has also been shown that CD95 (Fas/Apo-1)
antigen stimulation induces marked apoptosis of T lymphocytes in
HIV-1-infected carriers,4,5 and the lack of chronic immune
activation in HIV-infected chimpanzees correlates with the resistance
of T cells to CD95-induced apoptosis.6
In vivo studies have clearly demonstrated that an intact nef
gene is critical to attain high virus loads and the development of an
acquired immune deficiency syndrome (AIDS)-like illness in rhesus
monkeys infected with simian immunodeficiency virus (SIV).7-10 A similar requirement for the maintenance of a
high virus load has been demonstrated for HIV-1 using the severe
combined immunodeficiency (SCID-Hu mouse) model,11 and
deletions of nef sequences were consistently identified in some
long-term nonprogressors with HIV-1 infection.12 Of note,
the Nef proteins of SIV and HIV are functionally
interchangeable.13 Enhanced virus replication and
infectivity are associated with nef expression upon propagation of HIV-1 in peripheral blood mononuclear cell (PBMC) cultures in
vitro.14 An increased inoculum of nef-deleted virus
can overcome the decreased infectivity of the viral progeny but still
does not induce illness in infected animals, suggesting a role for nef in HIV-1 pathogenicity.15 Indeed, mice
expressing a lymphoid-targeted nef transgene develop severe
T-cell depletion with altered T-cell function.16
Besides the enhancement of virus replication, in vitro studies also
indicate that the regulatory HIV-1 Nef protein plays a key role in AIDS
pathogenesis. One function of Nef is to mediate downregulation of cell
surface CD4, the major HIV-1 receptor.17 Nef also induces
dramatic effects on T-cell activation,18-21 leading to a
specific Th1 cytokine impairment, possibly via binding to cellular
kinases, including serine/threonine and tyrosine
kinases.21-27 Moreover, different investigators have
reported difficulties in establishing cell lines constitutively
expressing Nef protein. In fact, it has been shown that Nef may be
either cytotoxic or cytostatic when expressed in transfected cell
lines.28-30 In this context, we were able to establish
stably transfected clonal T-cell lines that can be propagated in
culture and allow for the controlled expression of an HIV-1-derived
nef allele.21 Taken together, these observations
imply that Nef has a role in perturbing T-cell activation pathways,
which are presumed to influence viral replication in the host and
possibly cause a dysfunction of cells in the immune system.
Virus-specific cellular immune responses were also
studied. They were barely detectable, if at all, in
macaques infected with pathogenic SIV, whereas they were strongly
induced in animals infected with the nef-deleted
virus.31,32 In this last group of animals, increased Fas
expression and apoptotic cell death were noted on T-lymphocyte
populations.32 Furthermore, in vitro infection of macaque
PBMCs was associated with increased Fas ligand (FasL) expression in a
nef-dependent manner. It was thus proposed that expression of
FasL may protect infected cells from cytotoxic T lymphocyte (CTL)
attack, killing viral-specific CTLs in the process.
Whether nef itself can induce FasL expression and apoptosis
thus remains to be established. Here, we investigated the effect of
endogenous Nef on the degree of apoptosis and CD95/CD95 ligand (CD95L)
expression and function in a set of stably transfected T-cell
lines.21
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MATERIALS AND METHODS |
Cell lines.
JH6.2 and JBru.2 are CD4+ lymphoblastoid T stably
transfected clonal cell lines that have been previously
described.21 JBru.3 and JBru.mut.8 clonal cell
lines were obtained similarly in another round of stable transfections.
JBru.mut.8 was obtained by stable transfection of the Nef Bru
P72A-P75A mutant (Dutartre et al, manuscript
submitted for publication). Nef Bru
P72A-P75A was generated by substitution of Pro
residues 72 and 75 with Ala residues and yielded a construct allowing
for the expression of a stable Nef protein (data not shown). Cells were
cultured in RPMI 1640 (GIBCO, Grand Island, NY) plus 10% fetal calf
serum ([FCS] GIBCO) at an optimal cell density of 0.3 × 106 to 1 × 106/mL. In most experiments,
exponentially growing JH6.2, JBru.2, JBru.3, and JBru.mut.8
cell clones were cultured in the presence of low serum levels
(RPMI + 0.1% FCS) for up to 24 hours, and cell aliquots were
harvested at various time points to measure the percentage of apoptosis
and the expression of CD95, CD95L, and Nef proteins by flow cytometry.
Jurkat cell clones were either untreated or treated for 4 hours with 10 5 mol/L forskolin (FK), a potent protein
kinase A activator, which increases Nef expression due to the presence
of CREB consensus sequences in the cytomegalovirus
promoter33 driving the expression of Nef
cDNA.21 In some experiments, JH6.2, JBru.2, and JBru.3 cells were cultured in the presence of 50 ng/mL anti-CD95 IgM monoclonal antibody (MoAb) (Immunotech, Marseille Cedex, France), which
triggers apoptosis by interacting with surface CD95, or 100 ng/mL
anti-Apo-1 (CD95) Fab' MoAb (kindly provided by Dr Peter Kramer,
Heidelberg, Germany), which specifically blocks CD95/CD95L interactions. In other experiments, cells were treated with 100 ng/mL
anti-Apo-1 (CD95) Fab' MoAb for 1 hour and supplemented with 50 ng/mL
anti-CD95 IgM for an additional 23 hours.
Western blot analysis.
Samples derived from 2 × 106 cells, containing
approximately 100 µg protein, were migrated in 10% acrylamide gels
and blotted onto nitrocellulose filters. Blotted filters were blocked
for 30 minutes in a 3% suspension of dried skimmed milk in
phosphate-buffered saline (PBS) and incubated overnight at 4°C with a
1:200 dilution of anti-Nef MoAb (Transgene, Strasbourg, France) or
1:1,500 dilution of anti-tubulin MoAb (Sigma, St Louis, MO). The
filters were washed and further incubated for 1 hour at room
temperature with a 1:1,500 dilution of peroxidase-conjugated anti-mouse
IgG (Sigma) in 1% bovine serum albumin. Specific reactions were
revealed with the ECL Western blotting detection reagent (Amersham
Corp, Arlington Heights, IL).
Flow cytometric analysis of surface CD95 and CD95L and intracellular
Nef.
The detection of CD95 and CD95L surface expression was
performed on aliquots of 3 × 105 cells using unconjugated
anti-CD95 IgM MoAb (dilution 1:100; Immunotech) or anti-CD95L rabbit
polyclonal antibody (dilution 1:40; Santa Cruz Biotechnology, Santa
Cruz, CA), respectively, at 4°C for 30 minutes. After two washings
with PBS, the cells were stained with a polyclonal goat anti-mouse IgG
covalently linked to fluorescein isothiocyanate (GAM-FITC, dilution
1:100; Becton Dickinson, San Jose, CA) or a polyclonal goat anti-rabbit IgG covalently linked to FITC (GAR-FITC, dilution 1:100;
Becton Dickinson), respectively, at 4°C for 30 minutes. Nonspecific
fluorescence was assessed using irrelevant isotype-matched controls
(IgM for anti-CD95 MoAb or normal rabbit IgG for anti-CD95L) followed
by GAM-FITC or GAR-FITC.
To evaluate intracellular Nef expression, JH6.2, JBru.2, JBru.3, and
JBru.mut.8 cell lines were fixed in PBS-2% paraformaldehyde for 20 minutes at room temperature, washed twice with PBS containing 10 mmol/L glycine, and permeabilized in PBS-Triton X 1% for
5 minutes at 4°C. After two washings with PBS, the cells were
resuspended in PBS plus 10% normal goat serum for 10 minutes at room
temperature, before addition of anti-Nef IgG1 MoAb (Transgene; dilution
1:100) for 30 minutes at 4°C. After two washings with PBS, GAM-FITC
(dilution 1:100) was added to the cells and incubated for 30 minutes at 4°C. The negative controls consisted of an isotype-matched unreactive MoAb (anti-p66 IgG1 human cytomegalovirus, dilution 1:50; Du Pont Co,
Wilmington, DE) followed by identical second-layer labeling as before.
Surface or intracellular fluorescence was analyzed by a FACScan flow
cytometer (Becton Dickinson). Samples were assayed in duplicate (at
least 10,000 events recorded), and the mean fluorescence intensity
(MFI) was calculated for each sample by point-to-point subtraction of
positive counts on the negative controls using Lysis II software
(Becton Dickinson). MFI values are expressed in arbitrary units (AU).
Determination of apoptosis.
Apoptosis was evaluated as previously described34 by
combining two independent methods: (1) propidium iodide (PI) staining followed by flow cytometry and (2) the TdT-mediated d-UTP-biotin nick
end labeling (TUNEL) technique. For the first procedure, cells were
harvested and fixed in 70% ethanol for at least 1 hour at 4°C. They
were then treated with 0.5 µg RNase/mL (Type I-A; Sigma) and
resuspended in PBS containing 50 µg/mL PI. Analysis was performed by
FACScan with the FL2 detector in logarithmic mode, using Lysis II
software (Becton Dickinson). The threshold of PI fluorescence was
triggered on the Fl2 signal, where a clear-cut
distinction between cell debris and apoptotic cells was virtually
always present. As previously demonstrated by electron microscopic analysis,35 cells sorted from the hypodiploid peak were
virtually all apoptotic and cell debris was extremely rare.
The TUNEL technique was modified to stain the cells with 1 µg/mL
FITC-avidin to monitor DNA fragmentation in situ. Fluorescent nuclei
were scored using an MCR-1000 confocal microscope (Bio-Rad Microscience, Hemel, Hempstead, UK) equipped with a krypton/argon ion
laser emitting at 488 nm. The signal was achieved through an
Epidetector filter (passing band, 522/35 nm) (Bio-Rad
Microscience), analyzed by CoMOS software (Bio-Rad
Microscience), and printed on Ektachrome 64T Kodak film (Eastman Kodak,
Rochester, NY) by a Focus Imagerecorder Plus (Focus
Graphics Inc, Foster City, CA).
Statistical analysis.
The data are expressed as the mean ± SD for three or more experiments
performed in duplicate. Statistical analysis was performed using the
two-tailed Student's t-test.
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RESULTS |
HIV-1 Nef induces apoptosis of Jurkat cells that is counterregulated by
growth factors.
In this study, we aimed to characterize the mechanisms
underlying in vitro cytopathic effects mediated by endogenous
Nef.28-30 For this purpose, we used the HIV-1
nef-transfected JBru.2 and JBru.3 CD4+
lymphoblastoid T-cell clones and compared them with the control JH6.2
transfected with the backbone empty vector.21 Nef
expression is constitutively high in JBru.3 cells, whereas it is almost
undetectable in JBru.2 cells under basal conditions (Fig
1). We have previously shown21
that nef expression sharply increases in JBru.2 cells upon
treatment with FK, phorbol esters, or Ca2+ ionophores, due
to the presence of cAMP and NF-kB responsive elements in the human
immediate early cytomegalovirus promoter regions driving nef
expression in these cells.33 As in preliminary experiments,
FK alone did not modify the percentage of apoptosis in JH6.2 cells, at
variance with phorbol esters and Ca2+ ionophores, and this
agonist was chosen to induce Nef expression in JBru.2. The very weak or
absent Nef expression in JBru.2 readily increased (P < .01)
after stimulation of the cells for 5 hours with 105
mol/L FK (Fig 1A and B). On the other hand, the constitutively expressed Nef protein was only minimally (P > .1) enhanced
by the addition of 105 mol/L FK in JBru.3 (data not
shown). Of note, we have previously reported that nef
expression levels in stimulated JBru.2 cells are comparable to levels
detected in peripheral blood mononuclear cell cultures infected in
vitro with HIV-1 NL43 isolate,36 hence indicating that
physiologic levels of nef expression were achieved in our
experimental system.
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| Fig 1.
Analysis of intracellular Nef protein expression in
untreated or FK-treated Nef transfectants by (A) Western blot and (B)
flow cytometric analyses. (A) JH6.2 (lane 1), J.Bru.2 cultured in the
absence (lane 2) or presence (lane 3) of FK, and J.Bru.3 (lane 4) were
subjected to Western blot analysis for Nef and tubulin. (B) Nef protein
levels were determined by indirect immunofluorescence revealed by flow
cytometry. Unshadowed histograms represent cells treated with anti-Nef
MoAb + GAM-FITC, and shadowed histograms are negative controls (cells
treated with anti-p66 of HCMV + GAM-FITC). X-axis, relative Nef
expression detected by fluorescence intensity (logarithmic scale);
y-axis, relative number of cells. A representative of four separate
experiments is shown. The MFI (mean ± SD) of the four experiments
expressed in AU was 3 ± 1 (JH6.2), 21 ± 3.5 (JBru.2), 254 ± 38 (JBru.2 + FK), and 322 ± 55 (JBru.3).
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To evaluate the presence of apoptosis, we used two independent
techniques: quantitative analysis by flow cytometry to measure the
percentage of subdiploid DNA after PI staining (Fig 2A and B) and
indirect immunofluorescence after application of the TUNEL technique
revealed by confocal microscopy to monitor DNA fragmentation in situ
(Fig 2C). Due to the use of strict criteria to exclude cell debris from
the population of apoptotic cells after PI staining,35 the
quantitative data obtained with both techniques were very similar.
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| Fig 2.
Evaluation of apoptosis by (A and B) flow cytometry after
PI staining and (C) indirect immunofluorescence after TUNEL in
untreated or FK-treated Nef transfectants cultured for up to 24 hours
in 0.1% FCS. (A) Representative experiment shows the presence of
apoptosis quantitatively evaluated by flow cytometry after PI staining
in 24-hour serum-starved JH6.2, J.Bru2, and J.Bru.3 cells untreated
(left) or treated with 105 mol/L FK (right). M1,
apoptotic cells; x-axis, PI fluorescence (log scale); y-axis, relative
number of cells. (B) Apoptosis was quantitatively evaluated by PI
staining followed by flow cytometry at different culture times. Data
are the mean ± SD of four-seven separate experiments in duplicate. (C) Representative of three separate
experiments in which apoptosis was evaluated by TUNEL in confocal
microscopy after 24 hours of serum starvation. Apoptotic cells were
identified for the presence of intense fluorescent nuclei.
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In the presence of low serum concentrations (0.1% FCS), only cell
lines displaying clearly detectable Nef expression (FK-treated JBru.2
and untreated and FK-treated JBru.3; Fig 1) showed a progressive increase of apoptosis (Fig 2B). In particular, the percentage of
apoptosis was significantly (P < .05) higher in
Nef-expressing cells versus control JH6.2 and untreated JBru.2 cells
from 9 hours of serum starvation onward.
HIV-1 nef-expressing cells are sensitized to CD95-induced apoptosis
and express high levels of CD95.
Fas is an important intermediate in T-cell apoptosis, and peripheral
blood mononuclear cells from HIV-infected patients demonstrate enhanced
CD95 expression that is correlated with an enhanced susceptibility to
the induction of apoptosis with anti-CD95 antibodies.37 For these reasons, we investigated CD95-mediated apoptosis in
nef-transfected cells. JH6.2, JBru.2, and JBru.3 were cultured
under low growth factor concentrations and in the presence of anti-CD95
IgM MoAb, which induces apoptosis in cells expressing functional CD95
(Fig 3A). The percentage of apoptotic cells
showed a progressive increase in all cell lines following the addition
of anti-CD95 IgM in culture. However, the kinetics of apoptosis was
much faster and reached significantly (P < .05) higher
levels in JBru.3 and FK-treated JBru.2 cell clones versus untreated
JBru.2 and JH6.2.
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| Fig 3.
(A) Effect of addition of anti-CD95 IgM on the percentage
of apoptosis in untreated or FK-treated Nef transfectants cultured in
RPMI + 0.1% FCS for up to 24 hours. Apoptosis was quantified by PI
staining and flow cytometry at different culture times after serum
starvation. Data are the mean ± SD of four separate experiments in
duplicate. (B) Surface expression of CD95 in untreated or FK-treated
Nef transfectants cultured in RPMI 0.1% FCS for 24 hours. Unshadowed
histograms represent cells treated with anti-CD95 + GAM-FITC, and
shadowed histograms are negative controls (cells treated with anti-p66
of HCMV + GAM-FITC). X-axis, relative CD95 expression detected by
fluorescence intensity (log scale); y-axis, relative number of cells. A
representative of four separate experiments is shown. The MFI of the
four experiments (AU) was 189 ± 28 (JH6.2), 277 ± 51 (JBru.2), 325 ± 64 (JBru.2 + FK), and 394 ± 76 (JBru.3).
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To explain this increased susceptibility to apoptosis, we next
investigated the expression of CD95 (Fig 3B) on the cell surface of
JH6.2, JBru.2, and JBru.3. CD95 expression was clearly detectable in
all Jurkat cell clones, but it reached significantly higher (P < .01) MFI values in JBru.3 and FK-treated JBru.2 versus
untreated J.Bru.2 or JH6.2. These results suggested that the
nef-mediated susceptibility of transfected cells to
CD95-triggered apoptosis could relate to enhanced membrane CD95 expression.
HIV-1 Nef induces CD95-dependent apoptosis through induction of CD95L
membrane expression.
CD95 triggers apoptosis upon oligomerization by CD95L.38 We
thus determined CD95L expression at the surface of JH6.2, JBru.2, and
JBru.3 cells (Fig 4). In cells cultured in the presence
of low serum (RPMI + 0.1% FCS) for 24 hours, membrane-bound CD95L was clearly detectable. However, the expression was significantly (P < .01) higher in JBru.3 and FK-treated JBru.2 versus
untreated JBru.2 or control JH6.2 cells (Fig 4). These findings
suggested that an upregulated expression of both CD95 and CD95L may be
involved in the nef-dependent apoptosis observed in
nef-expressing Jurkat cells.
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| Fig 4.
Surface expression of CD95L in untreated or FK-treated
Nef transfectants cultured in RPMI + 0.1% FCS for 24 hours.
Unshadowed histograms represent cells treated with anti-CD95L + GAM-FITC, and shadowed histograms are negative controls (cells treated
with anti-p66 of HCMV + GAM-FITC). X-axis, relative CD95L
expression detected by fluorescence intensity (log scale); y-axis,
relative number of cells. A representative of four separate experiments
is shown. The MFI of the four experiments (AU) was 11 ± 4 (JH6.2), 63 ± 11 (JBru.2), 175 ± 46 (JBru.2 + FK), and 248 ± 70 (JBru.3).
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Because significant amounts of CD95L can be shed from the cell surface
and released in the culture medium,39 we next investigated the role of nef-induced CD95L in nef-dependent
apoptosis. Cells were cultured in the presence of an anti-CD95 Fab'
IgG, which blocks CD95/CD95L interactions and prevents apoptosis
induced by this pathway (Fig 5A). When cells were cultured in the
presence of an anti-CD95 Fab' IgG, the degree of apoptosis remained
very low over time, never exceeding 6% in all of the Jurkat
transfectants cultured for up to 24 hours under low growth factor
concentrations. Although nef-expressing cells showed slightly
higher levels of apoptosis, no statistically significant
(P > .1) differences were observed among the various Jurkat
cell clones treated with anti-CD95 Fab' IgG (Fig 5A).
Moreover, when cells were pretreated for 1 hour with anti-CD95 Fab' IgG
and then with anti-CD95 IgM for an additional 23 hours, the fraction of
apoptosis never exceeded 10% (Fig 5B), a much lower percentage
(P < .01) than previously observed in cultures supplemented
with anti-CD95 IgM alone (Fig 3A). Thus, the anti-CD95 Fab' IgG was
very efficient in blocking the CD95-mediated induction of apoptosis.
Taken together, these data demonstrated that the CD95/CD95L pathway in
nef-expressing cells was not only upregulated but also
functional, and fully accounted for the Nef-dependent apoptosis in
lymphoid T-cell lines reported herein.
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| Fig 5.
Effect of addition of (A) anti-CD95 Fab' IgG alone or (B)
followed by anti-CD95 IgM on the percentage of apoptosis in untreated
or FK-treated Nef transfectants cultured in RPMI + 0.1% FCS for up
to 24 hours. Apoptosis was quantified by PI staining and flow cytometry
at different culture times after serum starvation. Data are the mean ± SD of three to four separate experiments in duplicate.
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Mutation in the core sequence of Nef abrogates its ability to induce
apoptosis and CD95/CD95L upregulation.
It has been previously shown that both HIV-1 and SIV Nef interact with
src-like tyrosine kinase(s), and with a member of the p21-activated kinase (PAK) family of
kinases.24 Moreover, it has been suggested that the
association of SIV Nef with these kinases is important for the
development of AIDS in rhesus macaques and may provide a novel target
for clinical intervention.24,40,41 Therefore, in this group
of experiments, the induction of apoptosis upon serum withdrawal was
evaluated in a Jurkat cell clone stably transfected with a
nef-expressing plasmid in which the nef core region has
been mutated by substitution of Pro residues 72 and 75 by Ala residues
(JBru.mut.8). This mutant is unable to activate src-like tyrosine kinase(s) or PAK (Dutartre et al, manuscript submitted for publication). Nef expression in
JBru.mut.8 was very low under basal conditions, whereas it
rapidly and significantly (P < .01) increased upon
treatment with FK, reaching levels similar to those observed in
FK-treated JBru.2 (Fig 6A and B). The
percentage of apoptosis in both untreated and FK-treated
JBru.mut.8 cultured in RPMI + 0.1% FCS was very low (Fig
7A). When anti-CD95 IgM was added to the
culture, a progressive increase of apoptosis was noted (Fig 7B),
reaching values similar to those previously observed in JH6.2 control
cells (Fig 3A). Moreover, the surface expression of both CD95 (Fig 7C)
and CD95L (Fig 7D) in JBru.mut.8 was significantly (P < .05) lower versus FK-treated JBru.2 or JBru.3 cells
(Figs 3 and 4), while the CD4 surface expression was similar to that of
JBru.2 and JBru.3 (data not shown). Thus, the Nef core domain responsible for the interaction with src-like tyrosine
kinase(s) and PAK is critical for the ability of Nef to induce
apoptosis and upregulate CD95/CD95L.
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| Fig 6.
Analysis of intracellular Nef protein expression in
untreated or FK-treated Nef transfectants by (A) Western blot and (B)
flow cytometric analyses. (A) JBru.mut.8 cells were cultured in
the absence (lane 1) or presence (lane 2) of FK and then subjected to
Western blot analysis for Nef and tubulin. (B) Nef protein levels were
determined by indirect immunofluorescence revealed by flow cytometry.
Unshadowed histograms represent cells treated with anti-Nef MoAb + GAM-FITC, and shadowed histograms are negative controls (cells treated
with anti-p66 of HCMV + GAM-FITC). X-axis, relative Nef expression
detected by fluorescence intensity (log scale); y-axis, relative number
of cells. A representative of four separate experiments is shown. The
MFI of the four experiments (AU) was 4 ± 1.5 (JBru.mut.8) and
151 ± 29 (JBru.mut.8 + FK).
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| Fig 7.
Evaluation of (A and B) apoptosis and surface expression
of (C) CD95 and (D) CD95L in untreated and FK-treated
JBru.mut.8. (A and B) Apoptosis was quantitatively evaluated by
PI staining followed by flow cytometry at different culture times after
serum starvation in the (A) absence or (B) presence of anti-CD95 IgM.
Data are the mean ± SD of three separate experiments in duplicate. (C
and D) Surface expression of CD95 and CD95L was detected by indirect
staining in cells cultured for 24 hours in RPMI + 0.1% FCS.
Unshadowed histograms represent cells treated with (C) anti-CD95 + GAM-FITC or (D) anti-CD95L + GAR-FITC, and shadowed histograms are
negative controls (cells treated with anti-p66 of HCMV + GAM-FITC
and cells treated with normal rabbit IgG + GAR-FITC). X-axis,
relative (C) CD95 or (D) CD95L expression detected by fluorescence
intensity (log scale); y-axis, relative number of cells. A
representative of four separate experiments is shown. The MFI of the
four experiments (AU) was 233 ± 48 (JBru.mut.8) and 251 ± 62 (JBru.mut.8 + FK) in (C) and 43 ± 9 (JBru.mut.8) and 49 ± 17 (JBru.mut.8 + FK) in
(D).
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| |
DISCUSSION |
The loss of functional immune cells is a hallmark of AIDS. Although the
magnitude of the viral burden increases with disease progression42 and much emphasis has been placed on the
direct cytopathic effect of a productive HIV-1 infection of
CD4+ T lymphocytes,43,44 a considerable loss of
uninfected or abortively infected bystander T lymphocytes occurs in
HIV-infected individuals.
The induction of apoptosis following viral infection of a cell is
generally viewed as an attempt by the host cell to limit virus
replication. In fact, many viruses have their own antiapoptosis genes
or upregulate antiapoptotic cellular genes, which can block the
premature death of infected cells and thereby facilitate a persistent
infection or prolong the survival of lytically infected cells to
maximize the production of viral progeny.45 On the other
hand, it has also been shown that apoptotic death may favor HIV-1
replication.46 Thus, like other animal viruses, HIV-1 may
have developed strategies to modulate the apoptotic cell death of its
target cells.
The regulatory HIV-1 Nef protein represents an interesting candidate
for HIV-1-mediated induction of apoptosis. In fact, (1) it induces
severe T-cell depletion in transgenic mice,16 (2) it is
required for HIV pathogenicity in SCID-Hu mice independently of its
impact on viral replication,15 (3) it is required for CTL
killing in SIV-infected macaques31 and possibly also in humans,47 (4) it is abundantly expressed during acute HIV-1 infection,48 (5) it plays a relevant role in AIDS
pathogenesis,7-11 (6) it shows cytotoxic/cytostatic
activity in transfected cell lines,30 and (7) in its
extracellular form, it binds to the cell surface and induces apoptosis
of a variety of murine and human blood cells.49-51
In this study, we have established that endogenous expression of the
HIV-1 Nef protein in cycling mature CD4+ T cells can
regulate cell survival at two different levels. Firstly, it induces
functional CD95L expression, which in turn triggers CD95-mediated
T-cell death. This pathway correlates with the ability of Nef to
interact with cellular kinases and T-cell signaling in vitro and
relates to the role of nef in the evasion by infected cells of
CTL responses in SIV-infected macaques.31 Secondly, Nef
increases CD95-mediated T-cell death. Remarkably, Nef induced both
effects in a dose-dependent fashion, as JBru.2 was unable to induce
apoptosis unless stimulated to produce high levels of intracellular Nef
by FK.
Okada et al49-51 have convincingly shown that extracellular
Nef protein induces apoptosis in both lymphoid and myeloid cells of
human and mouse origin through a CD95-independent pathway. The apparent
discrepancy with our findings may be reconciled by taking into account
that we explored the effect of intracellular Nef and Okada et al
studied the effects of nonmyristylated recombinant Nef protein. It is
possible that intracellular and extracellular Nef protein trigger
apoptosis in their target cells through complementary (CD95-dependent
and -independent, respectively) mechanisms, which can meet upstream to
IL-1 converting enzyme (ICE).51
Several cell surface receptors including TNF-R1 and CD95 trigger
apoptosis upon contact with their counterreceptors or ligands, TNF and
CD95L for TNF-R1 and CD95, respectively.52 In particular, it has been demonstrated that the interaction between CD95 and CD95L
plays an important role in the homeostatic regulation of the normal
immune response. The expression of CD95 is diffuse, being found on a
variety of extra-lymphoid tissues, whereas the expression of CD95L is
much more tightly controlled, being restricted to activated
lymphocytes53 and selected sites of immune privilege such
as Sertoli cells, stromal cells of the anterior chamber of the eye, and
neurons.54
The ability of Nef to upregulate CD95 and induce CD95L is twofold. It
is particularly remarkable since CD95 expression per se does not lead
to cell death, as ligation of CD95 by CD95L is required to trigger
apoptosis. In addition, CD95 was also found to transduce activation
signals in normal human T lymphocytes.53 Here, using
anti-CD95 IgM antibodies, we found that Nef increased the sensitivity
to CD95-mediated T-cell death. This may have resulted from the
upregulation of CD95 membrane expression. Alternatively, Nef may
operate by increasing CD95 apoptotic signaling.51 The increased CD95 expression and sensitivity to CD95-mediated cell death
thus parallel those found in ex vivo T lymphocytes from HIV-infected
patients.1-3
Furthermore, we demonstrated that nef can induce CD95L
expression. Our data establish that the induction of CD95L membrane expression is instrumental in nef-induced cell death, since it was blocked by CD95 Fab' antibodies, and the proline-mutated Nef protein failed to upregulate CD95L and to induce apoptosis. In this
respect, it has been previously shown that Nef perturbs eye lens
development in transgenic mice.55 It is likely that
nef-expressing cells bearing CD95L at the cell surface induce
CD95-based cytotoxicity of CD95-expressing cells. Our results may thus
provide a molecular basis for the severe nef-induced T-cell
depletion found in nef-transgenic mice.16 However,
in situ labeling of lymph nodes from HIV-infected children and
SIV-infected macaques has indicated that apoptosis occurs predominantly
in bystander cells and not in the productively infected cells
themselves.3 Our study suggests that Nef-expressing CD4+ T cells with upregulated CD95L expression are a good
candidate for the induction of apoptosis found in both CD4+
and CD8+ uninfected cells.1-3,31 Our
demonstration at the protein level of CD95L induction by Nef is also
consistent with the demonstration of CD95L induction in HIV-infected
macrophages and CD4+ T cells by RT-PCR.56 We
propose that the nef-dependent regulation of CD95L may
contribute in vivo to protect infected cells from CTL killing by
inducing the cell death of Fas-bearing CTLs upon contact with the
nef-expressing cells. Our current model is consistent with the
recent observation of a nef-dependent evasion from CTL responses in SIV-infected macaques.31 The investigators
suggested such a nef-dependent CD95L induction in PBMCs
infected in vitro with SIV, yet the direct implication of nef
could not be demonstrated. It is remarkable that the various described
functions of nef, including downregulation of Th1
cytokines,20 together with the herein-described function in
the regulation of the CD95/CD95L pathway all may contribute in vivo to
allow nef-expressing cells to escape from CTL responses and
hence to increase viral replication.
Studies to characterize intracellular targets of Nef have shown that
HIV-1 and SIV Nef associate with cellular serine/threonine kinases, as
well as src-like tyrosine kinases.21-23 The
serine/threonine kinase may represent a member of the PAK
family,24 while tyrosine kinases comprise Hck in
monocytes57 and Lck in T lymphocytes.21,23 A
proline motif in HIV-1 Nef was found to be required for binding these
various kinases to increase viral replication and infectivity and for
impairment of CD3 signaling, but not for downregulation of membrane
CD4.21,26,27 Here, we show that this motif was also
required for induction of CD95L and apoptosis by Nef, suggesting that
common cellular targets are implicated in these different pathways.
Consistently, on the one hand, CD95L is known to be expressed following
activation of the T-cell receptor. This expression is inhibited by cell
treatment with immunosuppressive drugs including tyrosine kinase
inhibitors58 and requires Lck in mature cycling T
cells.59,60 Recently, using a reporter gene construct
containing elements from the CD95L promoter, Latinis et
al61 demonstrated that T-cell receptor-stimulated
activation of the small G-protein Ras signaling pathway is also
required for optimal activation of CD95L. Hence, both pathways may
contribute to the transcriptional induction of CD95L, the PAK effectors
being regulated by Ras and also by a Lck-Vav signaling
pathway.62 Similarly, various viral infections are
associated with increased CD95 expression, which was found to be
inhibited by tyrosine kinase inhibitors.63 These results
indicate a role for tyrosine kinases in common activation pathways
initiated by different stimuli. On the other hand, CD95 ligation
induces apoptosis and the small G-protein signaling cascade independently of Lck in human T cells.64 The relative
contribution of these various signaling molecules in the Nef function
awaits site-directed mutagenesis analysis to identify the specific
regions of Nef implicated in Lck and/or PAK regulation.
Other HIV-1 gene products such as extracellular HIV-1 Tat and gp120
have been shown to increase the expression of CD95L on activated T
cells.65 Together with Nef, these proteins may act in
concert to induce CD95L and to increase CD95-based cytotoxicity. Because Nef is abundantly expressed early after
infection,47,48 it may operate in early infection to induce
CD95L, followed by Tat and then gp120 to further support it. Indeed,
the concentrations (nanomolar to micromolar) of gp120 and Tat required
to upregulate CD95L expression are very high and difficult to achieve
in vivo also at sites of high viral replication such as lymph nodes.
The present report provides the first description of a twofold
mechanism triggered by a viral protein to regulate cell death via the
CD95-CD95L pathway. It also suggests cellular pathways regulating the
expression of this cell death machinery.
| |
FOOTNOTES |
Submitted May 1, 1998; accepted September 30, 1998.
Supported by the AIDS Project of the Italian Ministry of Health, the
Association Nationale de Recherche sur le syndrome d'Immunodeficience Acquise, and the INSERM, and in part by a fellowship from the European
Community (ERB-CHRX CT94-0537 to Y.C.).
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 Giorgio Zauli, MD, PhD, Human Anatomy
Section, Department of Morphology and Embryology, University of
Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy; e-mail:
zlg{at}dsn.unife.it.
| |
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Top
Abstract
Introduction