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Journal of Virology, October 2000, p. 9214-9221, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Induction of the Chemokines Interleukin-8 and IP-10
by Human Immunodeficiency Virus Type 1 Tat in Astrocytes
O.
Kutsch,1
J.-W.
Oh,1
A.
Nath,2 and
E. N.
Benveniste1,*
Department of Cell Biology, The University of
Alabama at Birmingham, Birmingham, Alabama,1
and Department of Neurology, University of Kentucky, Lexington,
Kentucky2
Received 9 May 2000/Accepted 14 July 2000
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ABSTRACT |
A finding commonly observed in human immunodeficiency virus type 1 (HIV-1)-infected patients is invasion of the brain by activated T cells
and infected macrophages, eventually leading to the development of
neurological disorders and HIV-1-associated dementia. The recruitment of T cells and macrophages into the brain is likely the result of
chemokine expression. Indeed, earlier studies revealed that levels of
different chemokines were increased in the cerebrospinal fluid of
HIV-1-infected patients whereas possible triggers and cellular sources
for chemokine expression in the brain remain widely undefined. As
previous studies indicated that HIV-1 Tat, the retroviral
transactivator, is capable of inducing a variety of cellular genes, we
investigated its capacity to induce production of chemokines in
astrocytes. Herein, we demonstrate that HIV-1 Tat72aa is a
potent inducer of MCP-1, interleukin-8 (IL-8), and IP-10 expression in
astrocytes. Levels of induced IP-10 protein were sufficiently high to
induce chemotaxis of peripheral blood lymphocytes. In addition,
Tat72aa induced IL-8 expression in astrocytes. IL-8
mRNA induction was seen less then 1 h after Tat72aa
stimulation, and levels remained elevated for up to 24 h, leading
to IL-8 protein production. Tat72aa-mediated MCP-1 and IL-8
mRNA induction was susceptible to inhibition by the MEK1/2 inhibitor
UO126 but was only modestly decreased by the inclusion of the p38
mitogen-activated protein kinase (MAPK) inhibitor SB202190. In
contrast, Tat-mediated IP-10 mRNA induction was suppressed by SB202190
but not by the MEK1/2 inhibitor UO126. These findings indicate that
MAPKs play a major role in Tat72aa-mediated chemokine
induction in astrocytes.
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INTRODUCTION |
Human immunodeficiency virus type 1 (HIV-1) infection causes neurological disorders in up to 90% of
infected patients, either by opportunistic infections of the brain
(i.e., toxoplasmosis) or by development of HIV-1-associated dementia
(HAD). HAD, a subcortical dementia characterized by cognitive deficits,
as well as motor and behavioral impairment, occurs in up to 30% of all
patients infected with HIV-1 (for reviews, see references
21 and 50). HAD develops
independently of opportunistic infections and is caused by direct
infection of cells in the central nervous system (CNS) by HIV-1. The
main sources of infected cells in the brain are infiltrating
macrophages and resident microglia cells (15, 32). Besides
the presence of infected cells in the brain, some characteristic
findings in HAD patients include astrogliosis, microgliosis, the
presence of activated T cells and macrophages in the brain, and the
loss of specific neuronal populations (for reviews, see references
17, 21, and 50).
A prerequisite for the recruitment of T cells and macrophages to the
brain is the secretion of chemokines, small proteins of 5 to 12 kDa.
Thus far, four major subfamilies of chemokines have been characterized
(1, 5, 29, 30). Of those, the CXC and CC chemokines are
distinguished according to the position of the first two cysteines from
four conserved cysteines linked by disulfide bonds. Those two cysteine
residues are adjacent in CC chemokines, whereas they are separated by
one amino acid in CXC chemokines. Gamma interferon (IFN-
)-inducible
protein 10 (IP-10) belongs to the family of CXC chemokines, along with
monokine induced by IFN- (Mig) and stromal cell-derived factor
(SDF-1). IP-10 was first discovered as an IFN--induced gene product
found to be expressed in delayed-type hypersensitivity reactions of the
skin (26). In the human system, IP-10 is reported to attract NK cells, monocytes, and T lymphocytes (63) although some of the reported results are controversial (41, 52). IL-8, the prototypic CXC chemokine, was initially described to be a
monocyte-derived factor known to attract neutrophils (2).
Subsequently, T cells, neutrophils, fibroblasts, endothelial cells, and
epithelial cells were also identified as sources for IL-8 production.
IL-8 attracts T cells, neutrophils, basophils, and endothelial cells
(for review, see reference 3). In addition, IL-8
mediates shear flow-resistant adhesion of monocytes (20).
In recent publications, the role of chemokines in the development of
HAD and the correlation between their presence in the cerebrospinal
fluid (CSF) and the degree of neurological disorder observed in
HIV-1-infected patients have been described. Fontana and coworkers
reported that IP-10 is the only chemokine to be present in the CSF of
all HIV-1-infected patients tested and is absent in uninfected control
individuals (33). Other groups demonstrated that expression
of MCP-1 or RANTES in the CSF correlated with HIV-1 infection (7,
11, 13, 28), with some of those studies having conflicting
results. In addition, the chemokines MIP-1
and MIP-1
were
detected in the brains of HIV-1 patients using in situ PCR
(58). For all of the above chemokines except IP-10, the
cellular sources were identified as microglia and/or astrocytes. The
cellular source of IP-10 in the brains of HAD patients has not been determined.
HIV-1 Tat, the retroviral transactivator, is essential for viral
replication and directs viral gene expression by binding to the TAR
element within the long terminal repeat of the integrated viral genome.
HIV-1 Tat exists as different splice variants, of which
Tat72aa (one-exon Tat) and Tat86aa or
Tat101aa (two-exon Tat) are the most prominent
(31). Tat can be released from HIV-1-infected cells
(16) and then interact with nearby cells. Expression of Tat
in cells by transient or stable transfection, as well as stimulation of
cells with extracellular Tat, has been demonstrated to have different
effects, including inhibition of antigen-induced T-cell responsiveness
(62), apoptosis (39), and induction of several cytokines or chemokines, such as tumor necrosis factor alpha (TNF-), interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein 1 (MCP-1) (10, 25, 36, 40, 49, 55, 67). Putative receptors for
Tat that would mediate the effects of extracellularly applied Tat
include integrins and the CD26 molecule (22, 65). In
addition, Tat has been reported to be capable of penetrating the cell
membrane without interacting with any receptor (59).
In this study, we investigated the potential of HIV-1
Tat72aa to stimulate chemokine expression in human
astrocytes and demonstrated the involvement of the mitogen-activated
protein kinase (MAPK) signaling pathway in Tat-mediated induction of
MCP-1, IP-10, and IL-8 in human astrocytes.
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MATERIALS AND METHODS |
Reagents.
The p38 MAPK inhibitor SB202190 and the MEK1/2
inhibitor UO126, as well as the respective controls SB202474 and UO124
were obtained from Calbiochem (San Diego, Calif.) and dissolved in dimethyl sulfoxide to achieve a stock concentration of 20 mM. HIV-1
Tat72aa was produced as described earlier (14,
46) and was >98% pure. For some experiments, Tat protein was
heat inactivated by incubation at 85°C for 30 min. Ficoll-Paque for
the preparation of peripheral blood lymphocytes (PBL) was obtained from
Pharmacia (Uppsala, Sweden). Neutralizing anti-TNF- antibody and
recombinant human IL-2 and IP-10 were obtained from R&D Systems
(Minneapolis, Minn.).
Cell culture.
Human CRT-MG astroglioma cells were maintained
in RPMI medium with 2 mM L-glutamine, 100 U of penicillin
per ml, 100 µg of streptomycin per ml, and 10% heat-inactivated
fetal bovine serum (FBS) as previously described (47). Human
primary adult astrocytes were obtained from biopsy material of patients
undergoing surgery to treat intractable epilepsy as described earlier
(4). These cells are 95% positive for glial fibrillary
acidic protein expression. For passage of CRT-MG cells and human
primary astrocytes, medium was removed and cells were disloged by
trypsinization (0.05% trypsin).
PBL were isolated from healthy donors by Ficoll-Paque density gradient
centrifugation, followed by plastic panning to remove macrophages, and
were cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated FBS, 2 mM L-glutamine, 100 U of penicillin per ml, and 100 µg of streptomycin per ml. PBL were stimulated with
phytohemagglutinin (5 µg/ml; Boehringer GmbH, Mannheim, Germany) and
100 U of human IL-2 per ml for 10 days.
RNA isolation and RPA.
Incubation of human primary adult
astrocytes and CRT-MG cells with Tat72aa for RNase
protection assays (RPA) was performed in 75-ml flasks containing 2 ml
of RPMI medium, 2 mM L-glutamine, 100 U of penicillin per
ml, and 100 µg of streptomycin per ml, either in the absence or in
the presence of 10% FBS. Stimulation in the absence or presence of
serum revealed the same results (data not shown). After addition of
Tat72aa protein, cells were incubated for the indicated
times on a rocker at 37°C. Adherent cells were then rinsed once with
ice-cold phosphate-buffered saline and dislodged by brief exposure to
trypsin-EDTA (0.05% trypsin, 0.02% EDTA; Gibco BRL). Cells were
washed twice with ice-cold phosphate-buffered saline, pelleted, and
frozen for subsequent RNA extraction. Cell pellets were lysed, and RNA
was extracted with guanidinium isothiocyanate and phenol and
precipitated with ethanol as described previously (60). A
linearized human chemokine multiprobe set (hCK-5; catalog no. 45035P;
Pharmingen, San Diego, Calif.) was transcribed with T7 RNA polymerase,
resulting in 10 antisense RNA probes of different lengths for
lymphotactin, RANTES, IP-10, MIP-1, MIP-1, MCP-1, IL-8, I-309,
and L32. As an internal control standard, a glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) probe is provided. RPA were performed as
previously described (47). Ten to 15 µg of total RNA was
hybridized with dUTP-labeled hCK-5 riboprobes and separated on a
denaturing (8 M urea) 5% polyacrylamide gel. For quantification of
protected RNA fragments, the gels were analyzed using a PhosphorImager
and ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.). Values
for each chemokine mRNA were normalized to GAPDH mRNA expression for
each experimental condition as previously described (47).
ELISA.
To determine secretion of chemokines by Tat-activated
primary astrocytes and astroglioma cells, enzyme-linked immunosorbent assays (ELISAs) were performed. Primary astrocytes or CRT-MG cells (3 × 105) were plated in six-well plates, incubated
for 24 h, and then stimulated with Tat72aa for 24 h. All experiments were done in the absence of serum. Expression of
MCP-1 and IL-8 in the supernatants was quantitated using a
dual-antibody solid-phase ELISA (Biosource International, Camarillo,
Calif.) in accordance with the manufacturer's instructions, as
previously described (47). The dual-antibody solid-phase
ELISA used for IP-10 was based on anti-human IP-10 antibodies (catalog
no. 266-IP and BAF266; R&D Systems), and the instructions of the
manufacturer were followed. Each supernatant sample was analyzed in duplicate.
Migration assays.
Migration assays were performed using
24-well Transwell plates (Costar). One milliliter of conditioned medium
from unstimulated astrocytes or astrocytes stimulated for 24 h
with HIV-1 Tat72aa was added to the lower chamber.
IL-2-activated PBL (106) in 200 µl of RPMI medium were
placed in the upper chamber. Chambers were separated by a
3-µm-pore-size polycarbonate membrane. The chambers were incubated
for 4 h at 37°C in 5% CO2, and then the Transwell
inserts were removed and 0.5 × 105 fluorescein
isothiocyanate-conjugated Calibrite-Beads (Becton Dickinson) were added
as a standard. Cells were then transferred to tubes, and ratios of
Calibrite-Beads to unstained, migrated cells were used to calculate the
total number of migrated cells per well. To investigate the role of
IP-10 in cell migration, 10 µg of neutralizing anti-IP-10 antibody
(catalog no. BAF266; R&D Systems) per ml or isotype-matched control
antibody (10 µg/ml) was added 30 min prior to the onset of the
migration assay.
Statistical analysis.
Levels of significance for comparisons
between samples were determined using Student's t-test distribution.
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RESULTS |
Dose-dependent regulation of chemokine mRNA expression by HIV-1
Tat72aa in primary human astrocytes and human astroglioma
cells.
Primary human adult astrocytes and the human astroglioma
cell line CRT-MG were tested for the ability to respond to
extracellular HIV-1 Tat72aa stimulation by the induction of
chemokine mRNA expression. Cells were stimulated for 6 h with
various amounts of HIV-1 Tat72aa, and then multiprobe RPA
was used to analyze chemokine expression. MCP-1 and IL-8 mRNAs were
constitutively expressed at low levels in primary astrocytes, whereas
there was no basal expression of any of the other chemokines (Fig.
1A, lane 2). In response to Tat72aa (1 nM), primary human astrocytes showed induction
of mRNA for MCP-1, IL-8, and IP-10 (Fig. 1A, lane 3). Increased
expression of MCP-1, IL-8, and IP-10 mRNAs was observed when 10 nM
Tat72aa was used, while a higher concentration of
Tat72aa (100 nM) did not further enhance expression (Fig.
1A, lanes 4 and 5, and 1B, C, and D). Depending on the donor or the
passage number of the primary astrocytes, the degree of chemokine mRNA
induction after Tat72aa treatment was variable (Table
1). Nevertheless, a consistent pattern of
chemokine mRNA induction in response to Tat72aa stimulation was observed. CRT-MG astroglioma cells reacted in a fashion comparable to that of primary astrocytes. CRT-MG cells constitutively expressed small amounts of MCP-1 and IL-8 mRNAs (Fig.
2A, lane 2). A Tat72aa concentration of 0.5 nM stimulated MCP-1, IL-8, and IP-10 mRNA expression (Fig. 2A, lane 4). In a dose-dependent manner,
Tat72aa increased the expression of MCP-1, IL-8, and IP-10
mRNAs (Fig. 2A, lanes 5 to 7), reaching saturation between 10 and 50 nM
(Fig. 2B, C, and D). RANTES mRNA was found to be induced only at high concentrations of Tat72aa in CRT-MG cells (Fig. 2A, lanes 6 and 7). The specificity of the Tat72aa effect was confirmed
by immune precipitation of Tat72aa prior to stimulation of
the cells, resulting in >70% abrogation of
Tat72aa-induced chemokine mRNA expression (data not
shown).
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FIG. 1.
Induction of IP-10, MCP-1, and IL-8 mRNAs in primary
human astrocytes by HIV-1 Tat72aa. (A) Human primary
astrocytes were incubated with medium or Tat72aa (1 to 100 nM) for 6 h, and total RNA was isolated and analyzed for induction
of chemokine mRNA using RPA. Probe alone is shown in lane 1. Quantitative analysis of chemokine mRNA induction for IP-10 (B), for
MCP-1 (C), and for IL-8 (D) is shown. Expression of different chemokine
mRNAs was normalized to the respective expression of GAPDH mRNA, and
fold induction was calculated in comparison to cells cultured in medium
alone. These results are representative of three independent
experiments.
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FIG. 2.
Induction of IP-10, MCP-1, and IL-8 mRNAs in the human
astroglioma cell line CRT-MG by HIV-1 Tat72aa. (A) CRT-MG
human astroglioma cells were incubated with medium or HIV-1
Tat72aa (0.1 to 50 nM) for 6 h, and total RNA was
isolated and analyzed for induction of chemokine mRNA using RPA. Probe
alone is shown in lane 1. Quantitative analysis of chemokine mRNA
induction for IP-10 (B), for MCP-1 (C), and for IL-8 (D) is shown.
Expression of different chemokine mRNAs was normalized to the
respective expression of GAPDH mRNA, and fold induction was calculated
in comparison to cells cultured in medium alone. Bars represent the
mean ± the standard deviation of the experiment shown in panel A
and two additional experiments. Statistical analysis was performed
comparing chemokine mRNA induction between
Tat72aa-stimulated CRT-MG cells and untreated controls
(*, P < 0.05).
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Kinetics of chemokine mRNA induction by HIV-1 Tat72aa
in human astroglioma cells.
Kinetic analysis of
Tat72aa induction of chemokines revealed differences in the
pattern of induction time and stability. CRT-MG cells were incubated
with Tat72aa (50 nM) for various amounts of time (1 to
24 h), and then mRNA expression was detected by RPA. MCP-1 and
IL-8 mRNAs were induced within the first hour after Tat72aa
stimulation (Fig. 3A, lane 3). MCP-1 mRNA
peaked at 6 h but was still elevated after 24 h (Fig. 3C).
IL-8 mRNA exhibited a biphasic pattern of expression; levels first
peaked after 2 h, declined slightly, and then reached a second
peak at 12 h (Fig. 3D). Levels of IL-8 mRNA were still increased
24 h after stimulation (Fig. 3D). IP-10 induction was delayed;
IP-10 mRNA was first detectable 2 h after Tat72aa
stimulation, peaked at 12 h, and returned to basal levels 24 h after stimulation (Fig. 3B).
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FIG. 3.
Kinetic analysis of chemokine mRNA expression in the
human astroglioma cell line CRT-MG after stimulation with HIV-1
Tat72aa. (A) CRT-MG cells were incubated with medium (lane
2) or with 50 nM Tat for the times indicated (1 to 24 h; lanes 3 to 8). Free probe is shown in lane 1. Total RNA was isolated and
analyzed for chemokine expression by RPA. Quantitative analysis of
chemokine mRNA expression is shown for IP-10 (B), MCP-1 (C), and IL-8
(D). Expression of different chemokine mRNAs was normalized to the
respective expression of GAPDH mRNA, and fold induction was calculated
in comparison to cells cultured in medium alone. Bars represent the
mean ± the standard deviation of five independent experiments.
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As Tat has previously been described to stimulate TNF-
expression
(
10), and TNF-
, in turn, can induce expression of IL-8,
MCP-1, and IP-10 in astrocytes (
47), we wanted to exclude a
possible influence of TNF-
produced by an autocrine mechanism.
Therefore, CRT-MG cells were stimulated with 50 nM Tat
72aa
in
the absence or presence of neutralizing anti-human TNF-
antibody
(5 µg/ml). Under these conditions, Tat
72aa-mediated
induction
of IL-8, MCP-1, or IP-10 mRNA was not affected (data not
shown),
suggesting that endogenous production of TNF-
is not
responsible
for Tat
72aa-induced chemokine
expression.
Secretion of chemokine protein after stimulation of primary
astrocytes and astroglioma cells with HIV-1 Tat72aa.
To test whether Tat72aa stimulation of astrocytes would
lead not only to the induction of chemokine mRNA but also to the
synthesis and secretion of the encoded chemokine proteins, primary
human adult astrocytes and CRT-MG cells were stimulated with
Tat72aa and secretion of IL-8, IP-10, and MCP-1 was
detected after 24 h using ELISA. All three chemokines were
efficiently synthesized and secreted into the culture supernatants
(Fig. 4).
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FIG. 4.
Expression of chemokine protein by HIV-1
Tat72aa-stimulated astrocytes. Primary human astrocytes (A,
B, and C) and CRT-MG cells (D, E, and F) were stimulated with 50 nM
Tat72aa for 24 h. Supernatants were collected and
analyzed for the expression of IP-10 (A and D), MCP-1 (B and E), and
IL-8 (C and F) by ELISA. Bars represent the mean ± the standard
deviation of three independent experiments.
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Capacity of supernatants from Tat72aa-stimulated
astrocytes to induce migration of PBL.
To assess the functionality
of the secreted chemokines, we performed migration assays with
Transwell plates using PBL which consisted primarily of T cells (90%
of the PBL used in the migration assays expressed the T-cell receptor,
as assessed by fluorescence-activated cell sorter analysis). Within the
4-h time frame of the experiment, minimal migration of PBL was observed
in response to supernatants from unstimulated astrocytes. In response
to supernatants from Tat72aa-stimulated CRT-MG cells, PBL
exhibited greatly increased migratory activity (Fig.
5). Interestingly, this activity was completely abrogated upon the addition of neutralizing anti-IP-10 antibody to the supernatants of astrocytes stimulated with
Tat72aa, whereas a control antibody had no effect on
migration. Supernatants from CRT-MG cells stimulated with
heat-inactivated Tat did not cause migration. Migration also was not
detected when Tat72aa (50 nM) was used directly as a
chemoattractant. In contrast, IP-10 (10 ng/ml) induced migration of PBL
to a degree similar to that of supernatants from
Tat72aa-stimulated astrocytes (Fig. 5). Comparable results
were obtained by using supernatants from Tat72aa-stimulated primary astrocytes (data not shown). These findings suggest that IP-10
in the supernatants from Tat72aa-stimulated astrocytes is the major attractant for T cells.
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FIG. 5.
Migration of IL-2-activated PBL toward supernatants from
HIV-1 Tat72aa-stimulated astrocytes. PBL were stimulated
with phytohemagglutinin and cultivated in the presence of IL-2 (100 U/ml) for 5 days. Migration assays to measure the capacity of
supernatants from Tat72aa-stimulated CRT-MG cells to induce
chemotaxis were performed in 24-well Transwell chambers. Supernatants
from unstimulated control cultures (CS) and from Tat-stimulated
cultures (TS) were added to the lower chamber of Transwell plates, and
106 PBL were placed in the upper chamber. After 4 h,
migration was quantitated by flow cytometry using Calibrite beads as a
standard. (A) Histogram analysis of PBL migration induced by
supernatants from unstimulated CRT-MG cells (CS; black), supernatants
from Tat-stimulated CRT-MG cells (TS; green), supernatants from
Tat-stimulated astrocytes in the presence of neutralizing anti-IP-10
antibody (10 µg/ml; TS + -IP10; red), supernatants derived
from CRT-MG cells stimulated with heat-inactivated Tat72aa
(hTS; blue), and Tat72aa (50 nM), used as a
chemoattractant (Tat; yellow). (B) Quantitative analysis of
migrated cells under different conditions. In addition to the culture
conditions shown in panel A, the migratory responses of PBL to
supernatants from Tat72aa-stimulated CRT-MG cells treated
with isotype control antibody (TS + IgG) and to recombinant IP-10
(10 ng/ml), used as a chemoattractant (IP10), are shown. Bars represent
the mean ± the standard deviation of three independent
experiments.
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Inhibition of Tat72aa-induced chemokine expression by
MEK and p38 MAPK inhibitors.
We next examined the signal
transduction pathways activated upon Tat72aa that are
involved in chemokine induction, focusing on the involvement of MAPKs
in this response. MAPKs are activated by a variety of extracellular
stimuli and can be divided into three major signal transduction
cascades: the ERK1/2 MAPK pathway, involving mainly Ras, Raf, and
MEK1/2; the JNK/SAPK pathway; and the p38 pathway. The ERK1/2 pathway
is thought to be activated mainly by growth factors, whereas the
JNK/SAPK pathway is activated by heat shock or inflammatory cytokines,
as is the p38 pathway (for a review, see reference
12).
Using the highly specific MEK1/2 inhibitor UO126 and its negative
control UO124, IL-8 mRNA induction by Tat was almost completely
blocked
at a UO126 concentration of 1 µM while UO124 at 10 µM
had no
inhibitory effect (Fig.
6C). UO126 also
partially abrogated
MCP-1 mRNA induction by Tat
72aa (Fig.
6B), whereas IP-10 induction
remained unaffected (Fig.
6A). In
accordance with these findings,
we have determined that ERK1/2 is
phosphorylated 20 min after
stimulation with 50 nM Tat
72aa
(data not shown). Using the p38-specific
inhibitor SB202190 (negative
control, SB202474), we demonstrate
that increasing concentrations of
SB202190 strongly inhibit the
induction of IP-10 mRNA by
Tat
72aa (Fig.
6D), with modest inhibition
of MCP-1 (Fig.
6E) or IL-8 (Fig.
6F). SB202474 was without effect
on IP-10, MCP-1, and
IL-8 expression. These findings indicate
that the MAPK pathway is of
major importance in the signal transduction
pathway activated by
extracellular Tat
72aa.
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FIG. 6.
Inhibition of HIV-1 Tat72aa-mediated
induction of chemokine mRNA by the MEK1/2 inhibitor UO126 and the p38
inhibitor SB202190. CRT-MG cells were incubated with medium ( ) or
stimulated with 50 nM Tat (T) for 6 h. Cells were preincubated
with the MEK1/2 inhibitor UO126 (A to C) or the p38 inhibitor SB202190
(D to F) for 1 h at the concentrations indicated (0.1 to 10 µM)
before stimulation of the cells with 50 nM Tat. As a negative control
for UO126, the compound UO124, at a concentration of 10 µM, was used
(C; A to C). For inhibition experiments using SB202190, the control
compound SB202474, at a concentration of 10 µM, was used (C; D to F).
The histograms show the quantitative analysis of chemokine mRNA
expression for IP-10 (A and D), MCP-1 (B and E), and IL-8 (C and F).
Expression of different chemokine mRNAs was normalized to the
respective expression of GAPDH mRNA and corrected for the background,
and relative induction was calculated in comparison to the expression
of mRNA in cells stimulated with Tat alone (100%). Bars represent the
mean ± the standard deviation of four independent experiments.
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DISCUSSION |
A high percentage of HIV-1 patients develop neurological disorders
during the course of the disease, caused either by HAD or by
opportunistic infections. HAD is caused by direct effects of HIV-1
infection on the different cell types of the CNS. Pathological hallmarks of HAD are the recruitment of activated T cells and infected
macrophages into the CNS, astrogliosis, and apoptosis of defined
population of neurons (for a review, see reference 17). Opportunistic infections within the CNS of
HIV-1 patients (i.e., toxoplasmosis) occur due to systemic immune
suppression of HIV-1 patients. Leukocytes infected with different
parasites or virus are not eliminated due to the compromised systemic
immune response, thereby potentially serving as vehicles by which
infectious agents invade the brain. Chemokines are capable of
participating in both syndromes, either by the recruitment of immune
cells into the brain or by alteration of vital cellular functions.
RANTES, SDF-1, and IP-10 exhibit the ability to recruit T cells
(8, 9, 57, 63), while MCP-1 and MIP-1 are major attractants for macrophages (19, 68). SDF-1 also has the capacity to
induce apoptosis in neurons (23, 27). Therefore, it is of
major importance to elucidate sources and stimulators of chemokine
production in the CNS during the course of an HIV-1 infection as
control of chemokine expression may be beneficial for HAD patients.
HIV-1-infected cells release the viral Tat protein, either by secretion
or due to the cytopathic effect of the virus (16). Externally applied Tat72aa has been shown to be a
potent inducer of different cellular genes; in various cell types,
it is capable of stimulating expression of IL-6 (45,
56, 69), TNF (10, 55), IL-8 (25, 49), and
MCP-1 (13, 43, 67). In this study, we demonstrated that
primary human adult astrocytes and CRT-MG human astroglioma cells
are stimulated by extracellularly applied Tat72aa to
express elevated levels of the CC chemokine MCP-1, as well as of the
CXC chemokines IL-8 and IP-10. One nanomolar Tat72aa was
sufficient to induce significant levels of all three chemokine mRNAs in
both primary astrocytes and CRT-MG astroglioma cells. The kinetics of
mRNA induction after Tat72aa stimulation differed for all
three chemokines. MCP-1 and IL-8 mRNA induction was evident at 1 h, whereas IP-10 mRNA induction was not strongly detected until 4 h. MCP-1 mRNA induction peaked after 6 h, and MCP-1 mRNA was still
present at elevated levels after 24 h. IP-10 mRNA peaked at
12 h but returned to basal levels after 24 h. IL-8 mRNA
induction exhibited a first peak after 2 h and a second after 12 h. Due to the fast initial induction of the different chemokine mRNAs, these findings suggest that the initial mRNA induction is due
only to the extracellularly applied Tat72aa. As Tat has been reported to induce TNF- in astrocytes (10) and, in
turn, TNF- is capable of inducing chemokine expression
(47), neutralizing anti-TNF- antibody was added to the
Tat-stimulated cultures and found to have no effect on chemokine
induction. This supports the idea that Tat can directly stimulate
chemokine expression. Nevertheless, we cannot exclude the possibility
that secretion of other cytokines due to Tat72aa
stimulation activates chemokine expression in astrocytes in an
autocrine fashion. The biphasic pattern of IL-8 mRNA expression
suggests the involvement of other factors, possibly IL-1, at a later
stage of the response (47).
As induction of chemokines in astrocytes by different stimuli has been
demonstrated to be dependent on the MAPK signaling pathway
(38) and Tat has been demonstrated to be capable of activating the MAPK signaling pathway in glial cells (44),
we investigated the involvement of the MAPK signaling pathway in Tat-mediated chemokine induction. Finding that ERK1/2 became
phosphorylated after Tat72aa stimulation (data not
shown), UO126, a very potent and highly specific MEK1/2 inhibitor
(18), was utilized to demonstrate the involvement of the ERK
pathway. We found that UO126 partially inhibited
Tat72aa-mediated induction of MCP-1 and abrogated the induction of IL-8 but had no effect on IP-10 mRNA induction. In contrast, SB202190, a specific inhibitor of p38 MAPK activation (37), efficiently suppressed IP-10 mRNA induction by
Tat72aa, while expression of MCP-1 and IL-8 mRNAs was
affected less. These findings suggest that Tat activation of MCP-1
and IL-8 gene expression is only partially dependent on p38 MAPK
activation, as even high concentrations of SB202190 could not
completely suppress expression. Tat-mediated IL-8 gene regulation is
stringently controlled by the ERK1/2 pathway, while IP-10 regulation by
Tat72aa exclusively involves the p38 MAPK pathway.
Supernatants from Tat72aa-stimulated astrocyte cultures
exhibited a strong capacity to induce migration of IL-2-stimulated PBL,
which was completely abrogated using neutralizing anti-IP-10 antibody.
This finding is very interesting in the context of a recent publication
in which Fontana and coworkers demonstrated that elevated IP-10 levels
correlate with the degree of HAD and IP-10 is the main factor in the
CSF to cause migration of T cells in HIV-1-infected patients
(33). Our findings suggest that astrocytes are a major
source of IP-10 in the brains of HIV-1-infected patients. In this
regard, the p38 MAPK signal transduction pathway would be an
interesting target for pharmaceutical drugs to control IP-10 expression
in the brains of HIV-1-infected patients. Other signaling pathways may
be involved in Tat-mediated chemokine induction. We found that
Tat72aa induction of all three chemokine mRNAs could be
abrogated by preincubation of the astrocytes with tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK), a potent inhibitor of NF-
B
activation (data not shown). Involvement of NF-B has been described
in the induction of IL-8 (49) and IP-10 (48) in other cell types using other stimuli and in astrocytes for the stimulation of TNF- (10). Thus, further investigation is
needed to definitely determine the involvement of NF-B in the
induction of the monitored chemokines by Tat72aa in
astrocytes and possible cross talk with the MAPK signaling pathway.
Recent publications suggest that other T-cell attractants, such as
RANTES, are increased in the CSF of HIV-1 patients (28), as
well as MIP-1 and MIP-1 (58), two potent macrophage
attractants. In our study, Tat72aa did not lead to an
increase in MIP-1 or MIP-1 expression in astrocytes and only a
minor increase in RANTES production could be seen. An earlier study in
our laboratory also demonstrated that MIP-1 and MIP-1 could
not be induced in astrocytes by cytokines such as TNF-, IFN-, and
IL-1 (47). Therefore, these chemokines may be produced by
cells in the CNS other than astrocytes, such as microglia and/or
macrophages (42).
The presence of elevated levels of IP-10 can be connected to the
migration of T cells into the CNS. IP-10 has been shown to be increased
not only in the brains of HIV-1-infected patients (33) and
macaques with SIV encephalitis (54) but also in a variety of
other diseases, all of which exhibit increased levels of activated T
cells in the brain, such as Theiler's virus-mediated demyelination
(24, 64), experimental allergic encephalomyelitis (51), or multiple sclerosis (61). A possible role
for IL-8 in HAD is less obvious. Thus far, IL-8 has been reported to
attract mainly neutrophils, which are absent in the brains of
HIV-1-infected patients. Nevertheless, there is evidence that IL-8 is
of major importance in the brain. IL-8 release into the CSF after brain injury is associated with blood-brain barrier dysfunction
(6) and nerve growth factor production (34). IL-8
is produced by astrocytes under acidosis (66), by blood
mononuclear cells after ischemic stroke (35), and in
neoplastic and infectious diseases of the human CNS (35).
Also, a recent publication suggests that IL-8 contributes to shear
flow-resistant adhesion of macrophages to vascular endothelial cells
(20). In this context, IL-8 secreted by astrocytes may
facilitate the extravasation of macrophages through the blood-brain
barrier. Of interest is the finding that depletion of IL-8 by addition
of neutralizing antibodies to astrocyte cultures renders astrocytes
susceptible to Fas-mediated apoptosis (53). Increased IL-8
expression in the brain could be a response to stress, promoting the
survival of astrocytes. A better understanding of chemokine expression
in the brain may therefore be of great interest not only for HAD
therapy but also as a strategy to prevent opportunistic infections in
the brain.
| |
ACKNOWLEDGMENTS |
This work was supported in part by National Institutes of Health
grants MH55795, NS36765, and NS29719 (to E.N.B.). O.K. is supported by
a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft, and
J.-W.O. is supported by a postdoctoral fellowship from the National
Multiple Sclerosis Society.
We thank Y. Gillespie (the University of Alabama at Birmingham) for the
cultures of primary adult astrocytes and Shaun Sparacio for assistance
in running the laboratory.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Cell Biology, 350 MCLM, The University of Alabama at Birmingham, 1918 University Blvd., Birmingham, AL 35294-0005. Phone: (205) 934-7667. Fax: (205) 975-6748. E-mail: tika{at}uab.edu.
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Journal of Virology, October 2000, p. 9214-9221, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
