From the Departments of Neurology, Microbiology, and
Immunology, University of Kentucky, Lexington, Kentucky 40536-0284, the ¶ Laboratory of Molecular Medicine and Neuroscience, NINDS,
National Institutes of Health, Bethesda, Maryland 20892, and the
Department of Medical Microbiology, University of Manitoba,
Winnipeg, Manitoba R3E 0W3, Canada
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ABSTRACT |
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The pathological correlates of dementia due to
human immunodeficiency virus (HIV) infection are glial cell activation
and cytokine dysregulation. These findings occur in the setting of small numbers of productively infected cells within the brain. We
determined whether exposure of susceptible cells to Tat protein of HIV
could result in the production of select proinflammatory cytokines. In
a dose-responsive manner, Tat induced interleukin (IL)-1 The pathogenesis of dementia associated with
HIV1 infection involves the
complex interactions of viral products and cytokines, which eventually
result in neuronal dysfunction and cell loss. Several studies have
shown that viral proteins such as Tat and gp120 can induce cytokine
dysregulation in macrophages and glial cells as well as cause
neurotoxicity (1). However, it remains uncertain as to why infection of
the brain is limited in comparison with the severity of the clinical
presentation. In fact, macrophage infiltration, glial cell activation
and not viral load in the brain seem to correlate best with the
severity of dementia (2, 3). In keeping with these observations
cytokine levels in brain and cerebrospinal fluid are elevated in
patients with HIV dementia (2, 4).
The Tat protein of HIV is of particular interest, since it is released
extracellularly from unruptured, HIV-infected lymphoid and microglial
cells (5, 6). Tat exits from cells via a leaderless secretory pathway
in the absence of permeability changes (7). This protein thus has the
opportunity to interact with other uninfected cells. Further Tat can be
detected in mononuclear cells within the brain of patients with HIV
encephalitis (8) and in the sera of HIV-infected individuals (9).
Tat-mRNA levels are also elevated in the brains of patients with
HIV dementia (10, 11).
It has been shown previously that the Tat protein of HIV can induce
macrophage infiltration in the brain (12) likely via production of
monocyte chemoattractant protein-1 by astrocytes (13). Tat also causes
glial cell activation for several days post intracerebroventricular
inoculation (12). Furthermore, a Tat-derived peptide when injected into
the brain causes cytokine dysregulation (14) similar to that observed
in patients with HIV infection (10, 15). In this study, we examine the
possibility that a transient exposure of cells to Tat may be sufficient
to induce a sustained production of cytokines and we determine the role
of extracellular calcium and autoregulation in cytokine production.
Cell Culture--
Peripheral blood monocytes were isolated by a
Percoll gradient technique, and human fetal astrocyte cultures were
prepared by differential adhesion as described previously (16). The
human astrocytoma cell line U373 and monocytic cell line THP-1 were obtained from American Type Culture Collection (Rockville, MD). Human
astrocytes and U373 cells were maintained in minimal essential medium
with heat-inactivated 10% (v/v) fetal bovine serum and 1 mM sodium pyruvate. Monocytes were cultured in RPMI with
10% fetal bovine serum. All cell types were supplemented with 100 µg
of streptomycin/ml and 0.25 µg amphotericin/ml. THP-1 cells were
cultured in RPMI medium with 10% fetal bovine serum and 5.5 µM HIV-1 Tat Treatment--
Highly purified (>95%) recombinant
Tat protein was prepared from the tat gene encoding the
first 72 amino acids as outlined previously (17). The functional
properties of this protein were confirmed using a transactivation assay
in HL3T1 cells containing the HIV-1 long terminal repeat,
chloramphenicol acetyltransferase construct (17). For experiments
designed to determine the effect of a transient exposure to Tat, cells
were treated with 100 ng/ml Tat for either 5, 30, or 60 min, following
which the cells were washed five times and then reincubated in culture
media without Tat protein. In each case, culture supernatants were
collected at 30, 90, and 180 min following reincubation without Tat and analyzed for the presence of IL-1
To establish dose profiles for each cell type, Tat was used at 0, 10, 100, and 1000 ng/ml concentrations for 4 h with THP-1 cells for
IL-1 Quantitative Imunoassays for Cytokines--
Cell culture
supernatants were analyzed for IL-1 RNA Extraction, RT-PCR, and Southern Blot Analysis--
First
strand cDNA was prepared from total cellular RNA as per the
manufacturer's protocol (Amersham Pharmacia Biotech). PCR was
conducted using published primers for IL-1 Role of Extracellular Calcium and NF To determine the effect of a transient exposure of Tat on cytokine
production, we treated monocytes and astrocytes with concentrations of
Tat (100 ng/ml) previously shown to induce cytokine production in these
cells (18, 20-22). We found that a 5-min exposure to Tat was
sufficient to induce maximal amounts of proinflammatory cytokines
IL-1 production
in monocytic cells, while astrocytic cells showed an increase in
mRNA for IL-1
, but had a translation block for IL-1
protein
production. Conversely, IL-6 protein and mRNA productions were
strongly induced in astrocytic cells and minimally in monocytic cells.
IL-1
and IL-6 production were independent of tumor necrosis
factor-
production. An exposure to Tat for a few minutes was
sufficient for sustained releases of cytokines for several hours. This
prolonged cytokine production is likely maintained by a positive feed
back loop of Tat-induced nuclear factor
B activation and cytokine
production that is independent of extracellular calcium. Thus a
transient exposure may be sufficient to initiate a cascade of events
resulting in cerebral dysfunction and a "hit and run" approach may
be in effect. Hence cross-sectional measurement of viral load in the
brain may not be a useful indicator of the role of viral products in
the neuropathogenesis of HIV dementia.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. The cells were cultured to
approximately 95% confluence in 24-well plates (
1 × 105 cells/ml).
, IL-6, and TNF-
. For
experiments designed to determine the role of TNF-
in IL-1 and IL-6
production, the monocytes and astrocytes were preincubated with
antisera to TNF-
(0.5 µg/ml; R&D Systems) for 30 min followed by
incubation with 100 ng/ml Tat for either 5, 30, or 60 min. The cells
were then washed and reincubated with culture media containing antisera to TNF-
. Culture supernatants were then analyzed after another 90 and 180 min for the presence of IL-1, IL-6, or TNF-
. Cells from two
different donors were analyzed in triplicates. Data from a
representative experiment are shown.
and IL-6 mRNA detection in THP-1 cells and for 6 h
with U373 cells. IL-1
and IL-6 proteins were analyzed in culture
supernatants, 16 h post-Tat treatment. Since IL-1
could not be
detected in cell culture supernatants of U373 cells, cell extracts of
U373 cells were also measured at 1, 3, 6, 12, 24, and 48 h
following treatment with 1 µg/ml Tat for IL-1
by ELISA. Cells were
stimulated with lipopolysaccharide from Escherichia coli
type 055:B5 (Sigma) 1.0 µg/ml as positive controls. Negative controls
included mock (PBS)-treated cells and cells that were treated with
solutions from which Tat had been immunoadsorbed as described
previously (18).
, IL-6, or TNF-
. Additionally,
cell extracts of U373 cells were analyzed for IL-1
. In each case,
ELISA kits were used from R&D systems, and the procedure was followed
as per the manufacturer's instructions. Briefly, 200 µl of standard
or sample was added to each well, which had been precoated with a
murine monoclonal antibody (2 µg/ml) against the appropriate
cytokine. Following a 2-h incubation with the test sample and three
washes, 200 µl of a rabbit polyclonal antibody (diluted 1:1000)
directed against the appropriate cytokine was added. One hour later,
the plates were washed, color developed, and analyzed using a
microtiter plate reader. A standard curve was generated on each
microtiter plate, which was used for quantitating the amount of
cytokine in each sample. The sensitivity of detection for IL-1
was 4 pg/ml, while that of IL-6 and TNF-
was 5 pg/ml.
, IL-6, and
-actin (19).
-Actin primers served as internal controls in each reaction. PCR products were resolved in a 1.5% agarose gel and transferred to a
nylon membrane and probed with [32P]ATP end-labeled
oligonucleotide probes. IL-
, IL-6, and
-actin oligonucleotide
probes were designed based on products amplified using the above
primers (IL-1
, 5'-CTG CAC GCT CCG GGA CTC ACA CCA)-3'; IL-6, 5'AAT
CGG GTA CAT CCT CGA CGG CAT CT-3';
-actin, 5'-GAG ACC TTC AAC ACC
CCA GCC ATG T-3').
B Activation--
U373 or
THP-1 cells were washed in PBS, treated with EGTA (0.5 mM)
or BAPTA (200 µM) for 10 min, followed by incubation with 100 ng/ml Tat in a calcium-free buffer for 1 h. Alternatively, the
cells were pretreated with 100 µM TLCK (Sigma) for 30 min followed by treatment with 100 ng/ml Tat for 6 h in
serum-containing medium. mRNA expression in each case was analyzed
by RT-PCR and Southern blot analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IL-6, or TNF-
in the monocytes (Fig.
1, A-C) and IL-6 in the
astrocytes (Fig. 1D). IL-1
or TNF-
could not be
detected in the astrocyte culture supernatants at all time points
tested. To determine whether the IL-1
or IL-6 production in these
cells was regulated via TNF-
, we analyzed the ability of TNF
antisera to inhibit IL-1
or IL-6 production. IL-1
and IL-6
induction by Tat was independent of TNF-
production (Fig. 1,
A, B, and D).
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Fig. 1.
Tat-induced IL-1 and IL-6 are independent of
TNF- production. Peripheral blood
monocytes (A-C) and human fetal astrocytes (D)
were treated with Tat (100 ng/ml) for either 5, 30, or 60 min, and
cytokine levels were measured in the culture supernatants 3 h
later. A 5-min exposure was sufficient to induce maximal amounts of
cytokine production. Sets of cultures were also pretreated with
antisera to TNF-
for 30 min followed by Tat exposure. The antiserum
was maintained in the medium for the duration of the experiment. IL-1
or IL-6 production by monocytes (A, B) or IL-6
production by astrocytes (D) was not inhibited by antisera
to TNF-
.
Even though we used highly purified cultures of monocytes and
astrocytes, we could not exclude the possibility of small amounts of
other contaminating cell types in these cultures. Hence we used a human
monocytoid (THP-1) and astrocytic (U373) cells for further experiments.
We have previously characterized Tat-induced TNF- in these cells
(18). Hence, to determine the effect of Tat on the production of
cytokines IL-1
and IL-6, we treated THP-1 and U373 cells with Tat
and measured IL-1
and IL-6 in the culture supernatants. Increasing
levels of IL-1
were produced by THP-1 cells in a
dose-dependent manner. Significant increases were noted
with concentrations of 100 ng/ml Tat (Fig.
2A).
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To determine whether the induction of IL-1 occurred at the level of
transcription or translation, we estimated IL-1
mRNA levels in
THP-1 cells by RT-PCR. Tat induced IL-1
mRNA expression in a
dose-dependent manner. The RT-PCR was much more sensitive and was able to detect IL-1
mRNA induction with 10 ng/ml Tat (Fig. 2D).
The U373 cells did not produce detectable levels of IL-1 in the
culture supernatants, even with 1 µg/ml Tat. Since stimuli such as
IL-1
and IL-2 result in production of cell associated IL-1
(23,
24), we tested cell extracts for the presence of IL-1
but were still
unable to detect any IL-1
protein (data not shown). To determine
whether the block in IL-1
production was at the level of
transcription or translation, we measured mRNA levels in Tat
treated U373 cells. IL-1
mRNA levels in U373 cells were
comparable with that of THP-1 cells (Fig. 2, D and E). Hence there was a translation block in the U373 cells.
Significant amounts of IL-6 were produced in the culture supernatants of THP-1 cells and the U373 cells (Fig. 2, B and C). Comparatively, however, the U373 cells produced nearly 20-fold more IL-6 than the THP-1 cells at both 100 ng/ml and 1 µg/ml concentrations of Tat. IL-6 mRNA production paralleled the production of IL-6 protein in U373 cells (Fig. 2, C and F) and THP-1 cells (data not shown).
We next conducted experiments to determine whether a transient exposure
to Tat would be sufficient to induce cytokine production in U373 and
THP-1 cells. A 5-min incubation was insufficient for inducing TNF-
or IL-1
in THP-1 cells, but was sufficient to induce IL-6 production
in U373 cells at 180 min post Tat exposure (Fig.
3). Following a 30- or 60-min exposure to
Tat, all three cytokines could be induced. A 60-min exposure led to
higher levels and earlier release of cytokines (Fig. 3).
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We used highly purified Tat protein in all our experiments and have
previously shown the specificity of Tat action (18, 25). However, to
further determine whether there were any contaminating substances that
may result in IL-1 or IL-6 production, we immunoadsorbed Tat and
used the remaining solution for treating the cells. The cell extracts
were analyzed for IL-1
and IL-6 mRNA levels, since this was a
much more sensitive technique for detection of Tat effects as compared
with cytokine detection in culture supernatants. A complete block in
IL-1
and IL-6 mRNA production was noted as shown in the U373
cells (Fig. 4), demonstrating that our
Tat preparations are devoid of other bioactive substances.
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Previous studies have shown that Tat induces NFB activation (26) and
that Tat-induced TNF-
production is NF
B-dependent (18). Hence, we pretreated the cells with TLCK, an inhibitor of NF
B
activation followed by incubation with Tat. A complete block in IL-1
and IL-6 production was noted in the U373 cells (Fig.
5, A and B) and the
THP-1 cells (data not shown). We examined the role of extracellular
divalent cations including calcium in Tat-induced cytokine production.
Neither EGTA nor BAPTA was able to block cytokine production. Fig.
5C shows the effect on IL-1
production in U373 cells. A
similar lack of response was seen for IL-6 production in U373 cells and
IL-1
and IL-6 production in THP-1 cells (data not shown).
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DISCUSSION |
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Our studies show that cytokine expression in monocytes and
astrocytes are differentially regulated by Tat. While monocytes could
be induced to produce all three cytokines, i.e. IL-1,
IL-6, and TNF-
, astrocytes produced only IL-6. The levels of IL-6
produced by astrocytic cells were nearly 20-fold greater than those
produced by monocytic cells. The astrocytic cells did not produce
measurable amounts of IL-1
, even though mRNA for IL-1
could
be induced. Similarly, we have shown previously that Tat could induce
only small amounts of TNF-
in the astrocytic cells (18).
Furthermore, we were unable to inhibit the production of IL-6 with
antisera to TNF-
in astrocytes or monocytes. This is an interesting
observation, since IL-1
and TNF-
have been shown to induce IL-6
gene expression in astrocytes (27). This suggests that the effect of
Tat on IL-6 production is specific.
Within the brain, IL-1 is primarily produced by activated microglia
(brain macrophages) (28). HIV-infected macrophages also release high
levels of IL-1
(29). Our studies show that Tat protein may, at least
in part, contribute to the elevation of IL-1
levels in the brain of
patients with HIV dementia. Tat-induced IL-1
is independent of
TNF-
production. The increase in IL-1
may induce astrocytosis
(30), promote HIV-1 replication (31), and induce other cytokines such
as TNF-
(28), resulting in further brain injury.
Tat-treated astrocytic cells not only produced large amounts of IL-6, but the elevated levels were present for prolonged periods. Several studies have shown that astrocytes are an important source of IL-6 (32). IL-6 has prominent effects on the brain, which include activation of the hypothalamic pituitary-adrenal axis, decreased appetite, and neuronal growth (33). Furthermore, IL-6 has been implicated in neuronal degeneration. Transgenic mice with IL-6 develop severe neurologic disease accompanied with neurodegeneration and astrocytosis (34). IL-6 has also been implicated in pathogenesis of neuronal injury in Alzheimer's disease (35).
Tat has been shown to induce changes in intracellular calcium in neurons through an influx of extracellular calcium (36). We hence examined the role of extracellular calcium in Tat-induced cytokine production. Removal of extracellular calcium had no effect on cytokine production. These observations are interesting since a recent study showed that calcium channel antagonists do not significantly alter the course of HIV dementia (37).
Previous studies have shown NFB activation in the brains of patients
with HIV infection (38). Further Tat induces NF
B activation in glial
cells (26). We now show that Tat-induced cytokine production is likely
NF
B-dependent. Interestingly, the same cytokines have
also been shown to activate NF
B itself (39). Thus cytokine
production once initiated by Tat could result in a positive feedback
loop between NF
B and cytokine production. This process may therefore
lead to an amplification of cytokine production without requiring the
continued presence of Tat.
Importantly, in this study we show that exposure to Tat for even a few
minutes is sufficient to induce cytokine production in monocytes and
astrocytes for prolonged periods of time. These findings are consistent
with our previous observations that, following a single
intraventricular injection of Tat in rats, progressive glial activation
and macrophage infiltration could be seen for several days, even though
Tat itself could not be detected in the brain after a few hours (12).
Furthermore, an exposure of Tat in the order of only milliseconds is
sufficient to induce prolonged depolarization in neurons (25, 40).
Together, these studies suggest that a transient exposure to HIV-Tat
protein results in a cascade of events leading to glial cell activation
and neuronal degeneration. We thus propose that a "hit and run"
phenomenon may be operative in neuropathogenesis of HIV infection,
which may also explain why cross-sectional measurements of viral load in the brain at autopsy do not always correlate with neuronal degeneration and dementia.
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ACKNOWLEDGEMENTS |
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We thank Tanis Benidictson and Carol Anderson for technical assistance and Jonathan Geiger and Melina Jones for helpful comments.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Neurology, University of Kentucky, Kentucky Clinic, Rm. L-445, Lexington, KY 40536-0284. Tel.: 606-323-6702; Fax: 606-323-5943; E-mail: anath{at}pop.uky.edu.
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ABBREVIATIONS |
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The abbreviations used are:
HIV, human
immunodeficiency virus;
ELISA, enzyme-linked immunosorbent assay;
RT, reverse transcriptase;
PCR, polymerase chain reaction;
IL, interleukin;
TLCK, N--tosyl-L-lysine chloromethyl
ketone;
TNF-
, tumor necrosis factor-
;
PBS, phosphate-buffered
saline;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
NF
B, nuclear factor
B.
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