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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4202-4209
Constitutive Activation of STATs Upon In Vivo Human
Immunodeficiency Virus Infection
By
Chiara Bovolenta,
Laura Camorali,
Alessandro L. Lorini,
Silvia Ghezzi,
Elisa Vicenzi,
Adriano Lazzarin, and
Guido Poli
From the AIDS Immunophatogenesis Unit, DIBIT, and the Division of
Infectious Diseases, Centro San Luigi, San Raffaele Scientific
Institute, Milan, Italy.
 |
ABSTRACT |
Infection by the human immunodeficiency virus (HIV) either
upregulates or downregulates the expression of several cytokines and
interferons (IFNs) that use the Janus kinase/signal transducer and
activator of transcription (JAK/STAT) pathway for signal transduction. However, very little is known on the state of activation of the JAK/STAT pathway after HIV infection either in vivo or in vitro. In
this regard, we report here that a constitutive activation of a
C-terminal truncated STAT5 (STAT5 ) and of STAT1 occurs in the
majority (~75%) of individuals with progressive HIV disease. We have
further demonstrated that, among peripheral blood mononuclear cells
(PBMCs), STAT5 is activated preferentially in CD4+ T
cells. In contrast to a published report, expression of STATs from
PBMCs of infected individuals was comparable with that of seronegative
donors. In addition, in vitro infection of mitogen-activated PBMCs with
a panel of laboratory-adapted and primary HIV strains characterized by
differential usage of chemokine coreceptors did not affect STAT protein
levels. However, enhanced activation of STAT was observed after in
vitro infection of resting PBMCs and nonadherent PBMCs by different
viral strains. Thus, constitutive STAT activation in CD4+
T lymphocytes represents a novel finding of interest also as a
potential new marker of immunological reconstitution of HIV-infected individuals.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN IMMUNODEFICIENCY virus (HIV)
infection leads to a progressive deterioration of the human immune
system. In this regard, one of the early clinical manifestations of the
acquired immunodeficiency syndrome (AIDS) is the loss of both memory
and naive CD4+ T cells coinciding with a state of chronic
T-cell activation that favors apoptosis or anergy of both
CD4+ and CD8+ T-cell subsets.1
Consequently, these cells lose the capacity of controlling HIV
infection.2 Among other mechanisms, both cytokines and
interferons (IFNs) play a pivotal role in regulating proliferation,
differentiation, and activation of the immune cells through the
triggering of multiple intracellular signaling pathways.3,4 Among these, the far most studied and better-characterized is the Janus
kinase/signal transducer and activator of transcription (JAK/STAT)
pathway,4,5 a very rapid membrane-to-nucleus signaling system, based on the activation of the cytosolic latent transcription factors STATs by JAK-dependent tyrosine phosphorylation. STATs ultimately dimerize and translocate to the nucleus and activate cytokine-inducible gene transcription.5
Numerous studies indicate that several cytokines may have a direct or
indirect effect on HIV-infected cells by exerting either inductive,
suppressive, or bifunctional effects on virus
replication.1,6 In this regard, we have recently provided
evidence indicating a clear role of the IFN - and IFN -mediated
JAK/STAT pathways in inhibiting HIV replication in a human monocytic
system.7 Conversely, HIV replication or expression of viral
genes causes a profound dysregulation of the cytokine network, which
may play an important role in disease progression by impairing anti-HIV immune responses.1,6 Quite surprisingly, very little is
known on whether HIV infection in vitro or in vivo can result in the perturbation of the JAK/STAT pathway.8 In support of this
hypothesis, stimulation of glial cells by HIV gp120 envelope (Env)
glycoprotein has been shown to induce STAT1 and JAK2
activation.9 More recently, gp120 Env binding to CD4 has
been reported to result in the inhibition of interleukin-2 receptor
(IL-2R) expression and proliferation in T lymphocytes, effects that
have been correlated with the inhibition of both expression and
activation of JAK3.10 Finally, Pericle et al11
have reported a selective reduction of STAT5b expression in
phytohemagglutinin (PHA) blasts infected in vitro by the
HIV-1BZ167 dual-tropic isolate, but not by the
HIV-1BaL M-tropic strain, along with a reduced expression
of STAT5a, STAT5b, and STAT1 in T cells purified from HIV-infected
individuals. Unfortunately, no information on the state of activation
of STAT in HIV-infected individuals was provided in this
study.11
The aim of our study was to investigate the possible effect of HIV
infection on STAT activation and expression both in HIV-infected individuals and after in vitro infection of resting and activated peripheral blood mononuclear cells (PBMCs) by a panel of different HIV
strains differing for chemokine coreceptor usage. Our study provides
compelling evidence for a constitutive in vivo activation, mainly of a
truncated isoform of STAT5 and STAT1.
| |
MATERIALS AND METHODS |
Patients.
Sixteen HIV-seropositive individuals observed at the "Centro San
Luigi" Hospital (Milan, Italy) were studied after informed consent
had been obtained. All HIV-infected patients had CD4 counts greater
than 200/µL. Five patients were naive for antiretroviral therapy,
whereas 11 patients had been treated with different antiretroviral regimens at the time of the analysis. The main clinical parameters of
these individuals are reported in Table 1.
Cell separation, cell culture, and PBMC subsets separation.
Peripheral venous blood was collected from HIV+ patients
and from 6 healthy seronegative donors on both heparin and EDTA. PBMCs were prepared by centrifugation on a Ficoll-Hypaque density gradient (Pharmacia Biotech, Uppsala, Sweden), as previously
described.12 Aliquots of 1 × 106 cells
were washed twice in RPMI 1640 medium, and pellets were obtained by
centrifugation at 13,000g for 10 minutes and then frozen at
 80°C for further analyses.
Monocytes (M ) were separated from freshly isolated PBMCs of normal
uninfected donors by adherence onto plastic for 1 hour at 37°C in
RPMI 1640 supplemented with 10% human AB serum (BioWhittaker, Verviers, Belgium). Nonadherent PBMCs (NA-PBMCs) were removed from the
flask and, after 2 washes with phosphate-buffered saline (PBS), seeded
at 1 × 106 cells/mL in RPMI 1640 supplemented with 2 mmol/L glutamine, penicillin (100 U/mL), streptomycin (100 µg/mL;
BioWhittaker), and 10% fetal calf serum (FCS; HyClone Europe, Ltd,
Cramlington, UK). Adherent M were washed twice with warm medium and
further cultivated in RPMI 1640 supplemented with 10% FCS and 10%
human AB serum.
PHA blasts were prepared from PBMCs of normal donors resuspended at the
density of 1 × 106 cells/mL in RPMI 1640 supplemented
with 10% FCS and stimulated by PHA (5 µg/mL; Sigma Chemical Corp, St
Louis, MO) for 3 days. Cells were then washed 3 times with PBS and
seeded in RPMI 1640 medium supplemented with 10% FCS and recombinant
IL-2 (20 U/mL; Roche Diagnostics, SpA, Monza, Italy) at the density of
1 × 106 cells/mL.
In some experiments, CD4+ and CD8+ cells were
depleted from PBMCs of HIV-infected patients by using immunomagnetic
beads coated with anti-CD4 and anti-CD8 monoclonal antibody (MoAb;
Dynatech, Dynal, Oslo, Norway) following the manufacturer's
instructions. Cell phenotype was determined by cytofluorimetry after
immunostaining with anti-CD3/CD4 and anti-CD3/CD8 MoAb (Coulter Corp,
Hialeah, FL) by a FACScan (Becton Dickinson, San Jose, CA) and analyzed using CellQuest software (Becton Dickinson).
Quantification of viral RNAs in plasma.
Plasma viremia was measured by the Amplicor Monitor kit (Roche
Molecular Systems, Branchburg, NJ) following the manufacturer's instructions.
HIV infection and reverse transcriptase (RT) assay.
PHA blasts from normal uninfected donors were acutely infected in vitro
with 6 HIV-1 strains, characterized by different cellular tropism and
chemokine coreceptor use,13 at a multiplicity of infection
(moi) of 0.1. Three laboratory-adapted (HIV-1IIIB/LAI [T-tropic, X4], HIV-1BaL [M-tropic, R5], and
HIV-189.6 [multi-tropic, X4/R5/R2/R3]) and 3 primary
isolates (DU [dual-tropic X4/R5], SA [dual-tropic X4/R3], and BC
[multi-tropic X4/R5/R2]) were independently adsorbed to the cells for
1 hour at 37°C. Cells were then resuspended in complete medium and
seeded (1 × 106 cells/well) in duplicate
wells in 48-well polystyrene culture plates (Falcon, Becton Dickinson
Labware, Lincoln Park, NJ). Culture supernatants were harvested every 4 days and stored at 80°C until tested for
Mg2+-dependent RT-activity assay, as previously
described.14 Primary HIV isolates were established by
cocultivation of HIV-infected patients' PBMCs with PHA blasts of
uninfected individuals. Chemokine coreceptor usage was tested on U87
astrocytic cells stably transfected with human CD4 alone or together
with one of the following human chemokine receptors (as
reported15): CXCR4, CCR2B, CCR3, and CCR5 (kindly provided
by D. Littman, Skirball Institute, New York, NY). Resting PBMCs,
NA-PBMCs, and M were exposed in vitro to 0.1 moi of
HIV-1IIIB/LAI and HIV-1BaL strains.
Abs.
Affinity-purified rabbit polyclonal Abs raised against a C-terminal
(residues 711-727; sc-835) or an N-terminal (residues 5-24; sc-836)
epitope of STAT5 and the affinity-purified rabbit polyclonal Abs raised
against the C-terminal domain of JAK3 (sc-513) were purchased from
Santa Cruz Biotechnology Inc (Santa Cruz, CA); the pan-STAT5-reactive
MoAb raised in mouse against an internal epitope (residues 451-649) was
purchased from Transduction Laboratories (Lexington, KY); rabbit
polyclonal Abs raised against C-terminal epitopes of STAT5a and
STAT5b16 were a generous gift from R.A. Kirken (Laboratory
of Molecular Immunoregulation, National Cancer Institute, Frederick
Cancer Research and Development Center, Frederick, MD); anti-STAT1
rabbit polyclonal Ab raised against amino acids 741-750 was a generous
gift from K. Ozato (Laboratory of Molecular Growth Regulation, National
Institute of Child Health and Human Development, National Institutes of
Health, Bethesda, MD); and rabbit polyclonal Ab against human actin
(A2066) was purchased from Sigma.
Whole-cell extracts (WCEs) and electrophoretic mobility shift assay
(EMSA).
WCEs were prepared by repeated cycles of freezing and thawing. Fresh or
frozen cell pellets were resuspended in a high salt buffer (buffer C,
20 mmol/L HEPES, pH 7.9, 400 mmol/L NaCl, 1.5 mmol/L
MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L
dithiothreitol [DTT], 10% [vol/vol] glycerol)
supplemented with a cocktail of protease inhibitors that
included leupeptin (10 µg/mL), pepstatin A (10 µg/mL), aprotinin
(33 µg/mL), E-64 (10 µg/mL), AEBSF (1 mmol/L), diisopropyl
fluorophosphate (DFP; 3 mmol/L), and the phosphatases
inhibitors sodium vanadate (Na3VO4; 1 mmol/L),
sodium fluoride (NaF; 50 mmol/L), and 0.5% Nonidet P-40. Cell
disruption was achieved by 3 freeze-and-thaw cycles in liquid nitrogen.
Insoluble material was removed by centrifugation at 12,000g for
15 minutes at 4°C, and the resulting supernatants were aliquoted
and stored at 80°C before use. Protein concentration was
evaluated by a protein assay kit based on the Bradford method (Bio-Rad,
Hercules, CA).
For EMSA, WCEs were incubated with different
[-32P]ATP-end-labeled double-stranded oligonucleotides
corresponding to either the prolactin-responsive element (PRE) located
within the promoter of the  -casein promoter,17 the
IFN-responsive region (GRR) located within the promoter of the
FcRI gene, or the high-affinity synthetic derivative of the
c-sis-inducible element (SIE), hSIE-m-67,18 and
the DNA-protein complexes were resolved as previously
described.18 In selected experiments, the radiolabeled
double-stranded oligonucleotide corresponding to the Ying Yan (YY1)
binding site located within the Moloney murine leukemia virus promoter
(UCR)19 was added to the binding mixture together with the
PRE probe to verify that similar amounts of proteins were used in each sample.
Immunoblot analyses.
Cellular proteins were denatured by the addition of an equal volume of
sample buffer 2× (50 mmol/L Tris-base, pH 6.8, 4% sodium dodecyl
sulfate [SDS], 10% 2--mercaptoethanol, and 20% glycerol) and
heated for 5 minutes at 100°C before electrophoretic separation on
7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent
transfer to nitrocellulose membrane Hybond enhanced chemiluminescence
system (ECL; Amersham, Little Chalfont, UK) by electroblotting.
Membranes were blocked in 7.5% bovine serum albumin (BSA), 20 mmol/L
Tris, pH 7.6, 137 mmol/L NaCl, and 0.2% Tween 20 for 1 hour at room
temperature and further incubated (overnight at 4°C) with the
desired primary Ab. Anti-STAT5 C- or N-terminal Abs were diluted
1:1,000, whereas anti-STAT5 MoAb and anti-actin Abs were diluted 1:250
and 1:500, respectively, as recommended by the manufacturers. Ab
binding was visualized by using the appropriate horseradish
peroxidase-conjugated secondary Ab (antimouse or antirabbit Abs,
diluted 1:5,000 or 1:15,000, respectively). The signal was shown by the
ECL (Amersham) according to the manufacturer's instructions.
| |
RESULTS |
Constitutive activation of STATs in HIV+ patients.
We analyzed WCEs obtained from unstimulated PBMCs of either
HIV+ or HIV individuals by EMSA
using a STAT-specific radiolabeled probe corresponding to the PRE, in
combination with a YY1-specific radiolabeled probe corresponding to the
UCR element. Because YY1 is a ubiquitously expressed nuclear
transcription factor, the UCR probe was included to verify that equal
amounts of proteins were present in the extracts of each sample
(Fig 1). We examined a total
of 14 HIV+ patients with progressive disease, 9 of whom
were under antiretroviral therapy and 5 who were naive subjects. In
addition, PBMCs were prepared from 6 healthy seronegative donors. Eight
of 9 (89%) and 4 of 5 (80%) HIV+ individuals, treated and
untreated with antiretrovirals, respectively, but none of the healthy
controls, as expected, showed activation of STATs, as shown in Fig 1.
No significant difference in YY1 binding was observed, indicating that
equal amounts of proteins were loaded in all samples. Similar results
were obtained using oligonucleotides corresponding to binding elements
with selectivity for different STATs, such as the GRR and the hSIE-m67
(data not shown). In this regard, PRE binds preferentially STAT5 but
also STAT1, whereas GRR preferentially recognizes STAT1 and STAT5 and, finally, hSIE/m-67 exclusively recognizes STAT1 and STAT3. Thus, a
constitutive STAT-DNA binding activity was observed in vivo in most
HIV-infected individuals.
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| Fig 1.
Constitutive activation of STAT in HIV+
patients. EMSA using WCEs (8 µg), obtained from PBMCs isolated from 6 healthy HIV and 14 HIV+ individuals, and a
mixture of PRE and UCR radiolabeled oligonucleotides. Constitutive
YY1-DNA binding serves as a control.
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A truncated STAT5 isoform and STAT1 are
constitutively activated in HIV+ individuals.
To determine which protein(s) of the STAT family was responsible for
the binding activity observed in infected individuals, supershift
experiments were performed by using specific Abs raised against several
STAT proteins from STAT1 to STAT6. Partial and complete supershifting
of the DNA binding complex was observed using the PRE and the GRR
oligonucleotides or the hSIE/m-67 oligonucleotide, respectively, by
anti-STAT1 Abs, which recognize the last C-terminal aa
(Fig 2A). To characterize the nature of the
residual DNA binding activity observed with the PRE oligo, we tested
Abs recognizing specifically the other STATs capable of binding the
STAT binding elements (from STAT3 to STAT6). In contrast to anti-STAT3,
anti-STAT4, and anti-STAT6 Abs (data not shown), the N-terminal, but
not the C-terminal anti-STAT5 Ab almost completely eliminated the
PRE/STAT binding complex (Fig 2B). These results indicate that both
full-length (fl)-STAT1 and a C-terminal truncated isoform of STAT5
(STAT5- ) are constitutively activated in HIV+
individuals.
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| Fig 2.
STAT1 and STAT5- are constitutively activated in
HIV+ patients. (A) Supershift analysis using the extract
of patient no. 4 of Fig 1, anti-STAT1 rabbit polyclonal Ab, and the
different oligonucleotides PRE, GRR, and hSIE/m-67. (B) Supershift
analysis using the PRE oligo and 4 different rabbit polyclonal Abs
raised against C- (C) or N-terminal (N) epitopes of STAT5a/b
proteins.
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STAT5a/b expression in HIV-infected patients.
Because supershift analysis on EMSA showed the presence of C-terminal
truncated STAT5 in the DNA-protein complex, in addition to fl-STAT1,
we further investigated the presence of truncated STAT5 isoforms in
both HIV+ and HIV individuals.
Unexpectedly, immunoblot analyses of WCEs obtained from 5 HIV+ patients and 2 uninfected donors using a mixture of
anti-STAT5a and anti-STAT5b Abs demonstrated that both STAT5a and
STAT5b fl proteins (96 and 94 kD, respectively) were expressed in both
controls and HIV+ patients (Fig
3). When the same filter was stripped and rehybridized with an
anti-STAT5 Ab recognizing an internal epitope of STAT5, a truncated
isoform of approximately 80 kD in size, in addition to the fl-STAT5a/b
proteins, was clearly detectable both in HIV-seronegative and
-seropositive individuals (Fig 3).
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| Fig 3.
STAT5 expression in HIV-infected patients. Immunoblotting
of WCEs (10 µg) of PBMCs isolated from 4 HIV-infected and 2 healthy,
HIV individuals using a mixture of anti-STAT5a and
anti-STAT5b rabbit polyclonal Abs in the upper panel and, after
stripping of the filter, using an anti-STAT5 mouse MoAb that recognizes
an internal epitope of STAT5 (lower panel).
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Thus, the immunoblot analysis confirmed the existence of a truncated
STAT5 isoform also in PBMCs of uninfected healthy individuals. These
results are in partial disagreement with a recent report by Pericle et
al11 indicating a downmodulated expression of STAT5a/b in T
lymphocytes of HIV-infected individuals.
STAT-DNA binding activity in PBMC subsets.
To assess whether STAT5 and STAT1 were differentially activated in
PBMC subsets of HIV-seropositive individuals, WCEs of CD4+-
and CD8+-depleted PBMCs were compared with WCEs of
unfractionated PBMCs obtained from 2 HIV-infected individuals (no. 15 and 16) by EMSA (Fig 4). In WCEs of both
patients, STAT5 and STAT1 migrated as 2 independent bands, with the
upper band corresponding to STAT5 and the lower band to STAT1, as
confirmed by supershift analysis (data not shown). It is noteworthy
that depletion of CD4+ cells, but not of CD8+,
resulted in the disappearance of the upper band (STAT5) compared with unfractionated PBMCs, indicating that activation of STAT5 occurred selectively in CD4+ cells. In contrast, STAT1-DNA
binding activity remained unchanged compared with the whole PBMCs after
depletion of either CD4+ or CD8+ cells (Fig 4).
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| Fig 4.
STAT activation in PBMC subsets of 2 HIV-infected
patients. EMSA using WCEs (15 µg) obtained from PBMCs,
CD4+- and CD8+-depleted PBMCs, and a
mixture of PRE and UCR radiolabeled oligonucleotides.
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Expression of STAT5a and STAT5b in PHA blasts infected in vitro with
6 different HIV-1 strains.
We next assessed whether STAT5 downmodulation occurred during acute
infection in vitro, as reported in activated PBMCs infected with the
dual-tropic strain HIV1BZ167 strain.11 To this
end, 6 HIV-1 isolates, characterized by different cellular tropisms and
coreceptor usage, were used to infect PHA blasts obtained from normal
donors. Kinetics of infection were observed up to at least 20 days. As
shown in Fig 5, the viruses were divided in
2 groups according to the day of peak RT activities. All of the
infections were productive, as demonstrated by the comparable levels of
the peak RT activities (Fig 5). In parallel, we analyzed WCEs obtained
from the PBMCs at the peaks of infection by immunoblotting experiments
using specific anti-STAT5a and anti-STATb Abs. No evidence of
downregulation of either STAT5a or STAT5b protein expression was
observed after in vitro HIV infection with these different viruses. In
addition, we found no difference in the expression of STAT5- between
mock-infected and HIV-infected cells (data not shown), as previously
observed by comparing WCEs obtained from HIV+ and
HIV individuals by using an anti-STAT5 Ab raised
against an internal epitope of STAT5.
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| Fig 5.
Expression of STAT5a and STAT5b in PHA blasts infected in
vitro with 6 different HIV-1 viral strains. (Upper panel) Peak of RT
values of kinetics of infection, observed up to 20 days, of PBMCs
purified from a normal healthy donor and then infected in vitro with
comparable moi of either 3 laboratory-adapted HIV-1 strains (IIIB/LAI,
BaL, and 89.6) or 3 primary isolates (DU, SA, and BC). The peak of RT
values was at day 13 for the IIIB, BaL, DU, and SA strains and at day
20 for the 89.6 and BC strains. (Lower panel) Immunoblotting of WCEs
obtained at the indicated peaks of blast infection using first
anti-STAT5a and then, after stripping of the filter, anti-STAT5b rabbit
polyclonal Abs.
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HIV upregulates STAT activation in vitro.
To investigate whether HIV infection could lead to STAT activation, we
analyzed WCEs of resting cells exposed to HIV-1IIIB/LAI and
HIV-1BaL strains at 0.1 moi by EMSA
(Fig 6). Infection of PHA blasts, although
more efficient than that of resting cells, was not informative, because
mock-infected cells showed a very strong basal level of STAT activation
due to PHA and IL-2 stimulatory effects (data not shown). Therefore, we
analyzed resting PBMCs, NA-PBMCs, and M isolated from a healthy
normal donor cultured for 7 days in standard medium not containing
mitogens such as PHA or IL-2. Mock-infected PBMCs and NA-PBMCs showed a
basal level of STAT activation likely as a result of cytokine
expression during the 7 days of culture. In support of this hypothesis,
WCEs produced from mock-infected cells harvested after 1 and 4 days of
culture did not show evidence of STAT activation (data not shown).
However, in WCEs from either IIIB- or BaL-infected cells, STAT
activation was clearly enhanced over the basal level of uninfected WCEs
(Fig 6). It is noteworthy that WCEs prepared from either uninfected or
infected M did not show STAT activation. Thus, in vitro STAT activation seems to occur after infection of resting T cells, but not
of M.
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| Fig 6.
STAT activation in resting PBMCs exposed in vitro to
HIV-1. EMSA using WCEs (8 µg) obtained from resting PBMCs, NA-PBMCs,
and M exposed for 7 days to HIV-1IIIB/LAI and
HIV-1BaL strains (0.1 moi) and a mixture of PRE and UCR
radiolabeled oligonucleotides. After a single round of purification,
CD4+-depleted PBMCs of patients no. 15 and 16 contained
3% and 1% CD4+ cells, respectively, whereas
CD8+-depleted PBMCs contained 0.3% and 1%
CD8+ cells, respectively.
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| |
DISCUSSION |
In the present study, we report for the first time that a constitutive
activation of STAT proteins occurs in the majority of HIV-infected
individuals (75%; Table 1), in contrast to healthy seronegative
controls. This activity was accounted for by activation of both
STAT1 and, mostly, of a truncated STAT5 isoform; the latter was
comparably expressed, as inactive protein, in PBMCs of both
HIV-infected and uninfected individuals. No evidence of diminished
STAT5 expression was observed either from infected individuals or after
in vitro infection of PHA blasts by a panel of HIV strains differing
for chemokine coreceptor usage. However, activation of STAT was
observed in resting PBMCs and NA-PBMCs infected in vitro with
HIV-1IIIB/LAI or HIV-1BaL.
In agreement with our findings, it has been previously shown that STATs
are constitutively activated after viral (HTLV-I) or oncogenic (v-Abl,
raf/myc, abl/myc, and v-src) transformation.4 In the case
of HIV-infected individuals, we have shown here that the STAT proteins
activated in vivo and in vitro were STAT1 and STAT5-. In vivo,
STAT1 and STAT5- activation unlikely represents the consequence
of HIV infection per se in that only a minority of PBMCs (1:100 to
1:1,000) are actually infected in vivo,20 although we
observed enhancement of STAT activation in resting T cells infected in
vitro after 7 days of culture. Given that STAT binding to the DNA
requires JAK-dependent phosphorylation of 1 or more tyrosine
residues,4,5 dysregulated expression of specific cytokines
or some other factors, resulting in a constitutive JAK activation, is
likely the key event underlying STAT activation in HIV-infected
individuals. Unfortunately, we could not further investigate the
enzymatic activity of JAKs in HIV-seropositive individuals by
immunoprecipitation as a consequence of the limited material allowing
us to perform exclusively immunoblotting analyses and because of the
lack of commercially available Abs specific for phosphorylated JAKs.
The nature of the possible cytokine(s) or factor(s) that might
constitutively trigger JAK activation and, consequently, activation of
STAT1 and STAT5- will require further studies. In this regard, STAT1 and STAT5 are activated by both IFN and IFN as well as by
several cytokines, such as IL-2, IL-3, IL-5, IL-7, IL-9, IL-15, and
granulocyte-macrophage colony-stimulating factor, several hormones, and
growth factors such as growth hormone, prolactin, erythropoietin, and
thrombo- poietin.4 In particular, IFN, IFN, and
IL-2 are critical cytokines for several functions of the immune
system,21-23 which are profoundly dysregulated by HIV infection.6 In par- ticular, Abs against IFN
were shown to revert the immunosuppressed state of HIV-infected PBMC
cultures.24 However, IFN-dependent STAT5 activation in
human T lymphocytes,25 in contrast to other cell types and
several cell lines,26-28 has not been demonstrated so far.
High levels of IFN and/or its related molecule neopterin have been
reported in the plasma/serum of HIV-infected individuals,29,30 whereas its expression in the germinal
centers (GC) of the lymph nodes by infiltrating CD8+ T
lymphocytes is a peculiar feature of HIV infection.31
Conflicting results regard IL-2 expression in HIV-infected
individuals32 in that the defective IL-2 secretion, which
was first described as one of the hallmarks of AIDS,33 was
not observed in a per cell-based analysis.34 Furthermore,
an increased level of IL-2 in the cerebrospinal fluid35 and
in the GC of the lymph nodes of AIDS patients31 has also
been shown. IL-2 plays a critical role in the activation of
lymphocytes,21,22 whereas its role on STAT5 activation in
lymphocyte proliferation is still controversial.36-38
At present, it remains unclear why STAT5- is preferentially
activated in CD4+ cells of HIV-infected individuals,
whereas such restriction is not observed for STAT1 activation. As
discussed previously, direct infection and replication of HIV-1 in vivo
could only minimally account for this phenomenon. However,
amplification loops leading to cytokine dysregulation and/or anergy and
apoptosis of CD4+ T cells have been associated with
improper signaling caused by shed gp120 Env either alone or complexed
with Abs or complement components.1,6,39
We have also observed that STAT5- is expressed in PBMC-seronegative,
healthy individuals. In this regard, Lokuta et al40 observed that a smaller isoform of STAT5, identical in size to the
STAT5- described in the present study, was preferentially expressed
in immature macrophages among 3 murine macrophage cell lines at
different stages of maturation and in primary cells. In contrast, we
did not observe STAT5- and STAT1 activation in either unexposed or
HIV-1-exposed unstimulated M. Although the ultimate function of
this truncated STAT5 isoform(s) is unknown at present, several studies
have demonstrated that truncated STAT5 isoforms can exhibit a higher
DNA binding affinity compared with fl-STAT5.41-44
Furthermore, we44 and others41-43 have
previously demonstrated that activated C-terminal truncated STAT5 might
exert a dominant negative effect in a variety of experimental systems, including murine erythroid progenitor and early hematopoietic cells.
These findings suggest that activated STAT5- may possess similar
characteristics in HIV-infected patients' PBMCs, potentially playing a
role in the reduced lymphocyte proliferation and anergy observed in
these individuals.
STAT activation did not correlate with viral load, CD4+
T-cell counts, or being either naive or under antiretroviral therapy, likely indicating a long-lasting perturbation of cell activation not
reflective of the downregulation of in vivo HIV replication, as judged
by the levels of viremia (Table 1). Whether immune based therapies,
such as the combined used of IL-2 and antiretrovirals, can affect STAT
activation is currently under scrutiny in our institute.
Finally, we have also shown that both HIV+ patients and in
vitro-infected PHA blasts expressed normal levels of STAT5a and STAT5b,
in contrast to what was recently reported by Pericle et al.11 In this regard, we have examined a larger number of
HIV patients (14 v 5) than reported11; however, a
different technical approach (immunoblotting in our study v
immunoprecipitation followed by immunoblotting in that of Pericle et
al11) has been adopted in the analyses of STAT5 isoforms
expression. It is also conceivable that the discrepancy of in vivo
results of these 2 studies may depend on the fact that we examined
total unfractionated PBMCs and not purified T lymphocytes.
In vitro, although we could not test the highly virulent
HIV-1BZ167 strain described in the study of Pericle et
al,11 we have analyzed a panel of HIV isolates differing
for coreceptor usage, including multitropic viruses such as 89.6 and
BC, in the same experimental conditions reported. However, we failed to
obtain evidence supporting a downmodulation of STAT5b upon in vitro HIV infection.
In conclusion, the STAT-binding activity found in most HIV-infected
individuals may represent a new surrogate marker for monitoring both a
global activation of the cytokine network as well as potentially their
immunological reconstitution.
| |
FOOTNOTES |
Submitted January 4, 1999; accepted August 9, 1999.
Supported by grants of the IX National Project for research against
AIDS of the Istituto Superiore di Sanità (ISS). C.B. and S.G. are
fellows of the ANLAIDS and ISS, Italy, respectively.
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 Chiara Bovolenta, PhD, P2/P3 Laboratories,
San Raffaele Scientific Institute, via Olgettina n. 58, 20132 Milano,
Italy; e-mail: bovolenta.chiara{at}hsr.it.
| |
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ABSTRACT
INTRODUCTION