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B Factors1
The Picower Institute for Medical Research, Manhasset, NY 11030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B binding site recognized by the
transcription factors p50 (NF-B1), p65 (RelA) and c-rel.
Transcription factor binding to "classical" NF-B sites in the
HIV-1, the human IL-2, and lymphotoxin promoters, recognized by p50 and
p65 following CD3+28-mediated costimulation, was unaffected by Tat101
as was binding to the AP-1 motif in the IL-8 promoter. These
experiments identify the CD28RE in the IL-8 promoter as a
c-rel recognition site and a Tat101-responsive element. The
effect of Tat101 on CD28REs in the IL-8 promoter and the subsequent
up-regulation of IL-8 secretion is likely to contribute to the immune
dysregulation observed during HIV-1 infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed that the positive effect of Tat on IL-2 production occurred only in synergy with CD28-mediated signals, was dependent on the expression of Tat101, and was mediated through the CD28-responsive element (CD28RE)3 in the IL-2 promoter (4). CD28 provides the crucial second signal necessary for full T cell activation and differentiation (reviewed in Refs. 14 and 15). The first signal is delivered through the TCR via the CD3 complex in a MHC-restricted fashion. Signaling through CD28 regulates cytokine expression both at the transcriptional level, through induction of transcription factor binding to the CD28RE (16, 17) and AP-1 (18, 19, 20), and at the post-transcriptional level by inhibiting degradation of specific mRNAs (21).
To determine whether Tat101 expression could modulate the expression of other cytokines besides IL-2 in a CD28-dependent manner, we have examined the regulation of IL-8. IL-8 is a C-X-C chemokine and a potent chemotactic factor for neutrophil granulocytes and T lymphocytes (reviewed in 22 . Increased levels of circulating IL-8 are detected in HIV-1-infected individuals (23). Although not considered a T cell-derived cytokine, recent experiments have indicated that IL-8 is produced by primary human T cells and T cell lines in a CD28-dependent fashion (24).
During HIV-1 infection, IL-8 might play an important role in the recruitment of CD4-positive T cells to the lymph nodes, the site of continuous viral replication (25, 26). IL-8 binds to two distinct seven-transmembrane receptors called CXCR1 and CXCR2 (27, 28), which are constitutively expressed on the surface of neutrophils and of 5 to 25% of PBL (29). Binding of IL-8 leads to the activation of integrin receptors on the surface of target cells and ultimately promotes firm adhesion to the endothelium (reviewed in 22 . In contrast to the chemokine receptors CCR3, CCR5, and CXCR4, CXCR1 and CXCR2 are not considered HIV-1 coreceptors and no binding of IL-8 to CXCR4 has been detected (30, 31, 32, 33).
IL-8 production is mainly regulated at the transcriptional level,
although in the HL-60 monocytic cell line, a stabilizing effect on
IL-8-specific transcripts was observed after treatment with LPS or PMA
(34). Two clusters of transcription factor binding sites have been
identified in the promoter of the IL-8 gene (35). The sequence from
nucleotide (nt) -94 to nt -71 contains binding sites for AP-1, C/EBP
(NF-IL-6), and NF-B and confers to the IL-8 promoter responsiveness
to IL-1, TNF, and PMA treatment (36). The sequence of the negative
strand of the NF-B site in this region exhibits striking homology to
the CD28RE in the IL-2 promoter and has recently been linked to the
CD28 responsiveness of IL-8 production in T cells (24).
The reported up-regulation of IL-8 during HIV-1 infection and the homology between the CD28RE in the IL-8 and the IL-2 promoters prompted us to test the effect of Tat on IL-8 production. In the present report, we show that Jurkat T cell lines stably expressing either Tat101 or Tat72 secreted markedly increased IL-8 levels following stimulation with anti-CD3 and CD28 mAbs. This increased IL-8 secretion was mediated through the CD28RE in the IL-8 promoter in the Tat101 cells, but not in the Tat72 cells. These results confirm the function of Tat101 as a regulator of host immune responses and underline its general property as a modulator of DNA binding activity to CD28REs in cytokine promoters.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia, Uppsala, Sweden). PBLs were obtained after three cycles of adherence-mediated depletion of macrophages. T cell-enriched cultures were obtained from PBMCs through negative selection with the use of T cell enrichment columns (R&D Systems, Minneapolis, MN). The purity of T cell fractions was confirmed by three-channel flow-cytometry analysis using chromophore-conjugated anti-CD3, anti-CD45, and anti-CD14 mAbs or appropriate isotype controls (Becton Dickinson, Mountain View, CA). The cell line WE17/10 was obtained from the AIDS Research and Reference Reagent Program-National Institute of Allergy and Infectious Diseases (Bethesda, MD) and maintained as recommended (37). The T cell lymphoma cell line Jurkat (clone E6-1) was obtained from the American Type Culture Collection (Rockville, MD) and served as the parental cell line for several clonal cell lines stably expressing HIV-1 Tat proteins (Tat72 and Tat101) or the empty vector cassette, described in Reference 4. The clones used in this study were frozen rapidly after identification and aliquots were thawed every 4 wk to maintain the identity of the clones. During that interval, all phenomena described here (Tat activity, IL-8 hyper-responsiveness) were stable and reproducible. All clones were negative for Mycoplasma (MycoTest; Life Technologies, Gaithersburg, MD). Cell lines were grown in complete RPMI 1640 medium (Life Technologies) supplemented with 600 µg/ml of geneticin (Life Technologies) and 10% FCS (HyClone, Logan, UT).
T cell activation
Exponentially growing Jurkat clones or primary cell cultures were stimulated with activating anti-CD3 mAbs (clone identification 454.3.21; a gift from N. Chiorazzi, North Shore University Hospital, Manhasset, NY), which had been precoated overnight at 4°C on tissue culture wells at a concentration of 3 µg/ml in 35 mM bicarbonate/15 mM carbonate buffer (pH 9.6) in the presence or absence of costimulatory anti-CD28 mAbs (clone identification 28.2; a gift from D. Olive, Marseilles, France) or a control isotype-matched mouse mAb (PharMingen, San Diego, CA). Supernatants were harvested 24 h after stimulation, and IL-8 concentrations in the supernatants were determined by ELISA (R&D Systems).
Transient transfection assay
The luciferase reporter (Beth Israel Hospital, Boston, MA)
plasmids pIL-8luc and pIL-8lucCD28REmut were a gift from K. LeClair and
are described in Reference 24. pIL-8luc contains a 318-bp fragment from
the human IL-8 promoter (nt -273 to nt +45) ligated into the
pGL2-Basic vector (Promega, Madison, WI). In pIL-8lucCD28REmut, nt -79
was mutated (G
A) (24). Supercoiled DNA from pIL-8luc,
pIL-8lucCD28REmut (3 µg DNA/5 x 106 cells)
were transfected in exponentially growing Jurkat clones
expressing either Tat72 or Tat101 using standard DEAE-dextran
techniques followed by an incubation with 0.1 mM chloroquine (38).
Twenty-four hours post-transfection, 106 viable cells,
determined by trypan blue exclusion, were induced with either
anti-CD3 mAbs alone or in combination with soluble anti-CD28
mAbs (anti-CD3+28). Cells were harvested 18 h later, washed
twice with PBS (pH 7.1), and resuspended in 25 µl of 25 mM Tris-HCl
(pH 7.8), 2 mM DTT, 2 mM EDTA, 10% glycerol, and 1% Triton X-100
(Promega). Luciferase activity was determined by the ATP-dependent
conversion of beetle luciferin substrate (Promega) to oxiluciferin, and
light production was measured as relative light units with the use of a
TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). The total protein
concentration of each extract was measured using the
detergent-compatible protein assay (Bio-Rad, Richmond, CA) with Igs as
standard. Values as relative light units were normalized to protein
concentrations and expressed in fold increase over unstimulated
controls. For transient cotransfections, pIL-8luc and pIL-8lucCD28REmut
(3 µg DNA/5 x 106 cells) were cotransfected with
the tat-expressing plasmids pRepTat72, pRepTat101 (4), or the empty
vector cassette pRep9 (Invitrogen, San Diego, CA) (1 µg DNA/5 x
106 cells) in the parental Jurkat cell line, followed by
activation treatment as described above.
Reverse transcriptase (RT)-PCR analysis
Cells were treated with anti-CD3 or anti-CD3+28 mAbs for
the indicated time, pelleted, and resuspended in Trizol (Life
Technologies). Total cell RNA was extracted according to the
manufacturer’s instructions followed by DNAse I digestion for 30 min
(Boehringer Mannheim, Indianapolis, IN). The integrity of RNA was
verified on an 0.8% agarose gel, and RT reaction was performed on 0.5
µg of total RNA (Advantage RT for PCR Kit; Clontech, Cambridge,
U.K.). To test for DNA contamination in RNA samples, a negative control
was included in which this reaction was performed without reverse
transcriptase enzyme. Five microliters of diluted RT product was
amplified with Amplitaq (Roche Molecular System) in the presence of 1.5
mM MgCl2. Intron-spanning IL-8 and ß-actin-specific
primers were obtained from Clontech (RT-PCR Amplimer Sets). PCR
reactions were performed on a GeneAmpPCR System 9600 (Perkin-Elmer,
Norwalk, CT) using 30 cycles for the amplification of IL-8-specific
transcripts (TM = 60°C) and 25 cycles for ß-actin
(TM = 60°C). Two microliters from the PCR reaction were
subjected to electrophoresis on a 1.5% agarose gel, transferred to a
nylon membrane (Hybond; Amersham, Buckinghamshire, U.K.), and
hybridized with a [
-32P]ATP (>5000 Ci/mmol; Amersham)
5'end-labeled single stranded IL-8-specific oligonucleotide probe
(Clontech) at 42°C overnight. Blots were washed in 2x SSPE/0.1% SDS
at 55°C for 30 min and exposed on Kodak film for 1 h.
Intensities of bands were analyzed with the use of an InstantImager
(Packard, Meriden, CT).
Gel retardation assay
Double stranded oligonucleotides corresponding to binding sites
for transcription factors were synthesized (Genset, La Jolla, CA) and
purified on denaturing polyacrylamide gels and Sep-Pak cartridges
(Waters Associates, Milford, MA) (40). Double stranded oligonucleotides
were 5' end-labeled with [-32P]ATP (>5000
Ci/mmol; Amersham), purified after isolation from polyacrylamide gel,
and used as probes. Nuclear extracts were prepared from nuclei using a
rapid protocol (39). Binding reactions were performed as described in
References 4 and 40. Binding reactions for
NF-BHIV,LT,IL-2 included 0.1 mg/ml of
DNase-free BSA (Pharmacia). Poly(dI-dC) (Pharmacia) was included as
nonspecific competitor DNA at the following concentrations: 5
µg/reaction (NF-BHIV,LT,IL-2), 1 µg/reaction
(CD28REIL-8), 0.2 µg/reaction (CD28REIL-2), 1
µg/reaction (AP-1IL-8). Fifteen thousand cpm of
probe (10 to 40 fmol) was then added to the mixture with or without a
molar excess of an unlabeled specific DNA competitor, and the mixture
was incubated for 20 min at room temperature. Samples were subjected to
electrophoresis at room temperature on 6% polyacrylamide gels at 150 V
for 2 to 3 h in 1x TGE buffer (25 mM Tris-acetate, pH 8.3, 190 mM
glycine, 1 mM EDTA). Gels were dried and autoradiographed for 24 to
48 h at -70°C. For competition assays, increasing
concentrations (3-, 9-, 27-, 81-, and 243-fold molar excess) of the
homologous or heterologous unlabeled competitor oligonucleotides were
added simultaneously with the radiolabeled probe to the binding
reaction and assayed as usual. Abs against p50, p65, c-rel,
and p52 used in supershift assays were a gift from U. Siebenlist
(National Institutes of Health, Bethesda, MD). Anti-RelB Ab was
obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Abs were added
at a final concentration of 1 µg/rxn to the binding reaction at the
end of the binding reaction for an additional 30-min incubation at room
temperature before electrophoresis. The sequences of the coding strand
of the double stranded oligonucleotides used in this study are listed
below:
CD28REIL-2 5'-GATCAGAAATTCCAAA-3'
CD28REIL-8 5'-TCGTGGAATTTCCTCTGAC-3'
mCD28REIL-8 5'-TCGTGAAATTTCCTCTGAC-3'
NF-BIL-2 5'-AAAGAGGGATTTCACCTAAAT-3'
NF-BLT 5'-TCGACCCTGGGGGCTTCCCCGGGC-3'
NF-BHIV
5'-TACAAGGGACTTTCCGCTGGGGACTTTCCAGGG-3'
AP-1IL-8 5'-GTGATGACTCAGGT-3'
OAPIL-2 5'-TTTGAAAATATGTGTAATATGTAAAACATTTTG-3'.
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Results |
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We first examined the IL-8 production of human T cells following
cross-linking of CD3 and costimulation through CD28. Purified human T
cells, isolated from PBMC (>90% CD3/CD45-double positive,
CD14-negative) from three independent donors were induced with
immobilized anti-CD3 mAb either in the presence of soluble
anti-CD28 mAb or soluble isotype control. IL-8 secretion was
clearly detected 48 h after stimulation with anti-CD3 alone or
in combination with anti-CD28 mAb (Fig. 1
A). No IL-8 secretion
was observed in the absence of stimulation. Costimulation with
anti-CD28 mAb amplified the IL-8 response stimulated through CD3 on
average two- to threefold, confirming observations obtained with the
IL-8 promoter in transient transfection assays (24). The IL-8 response
of anti-CD3- or anti-CD3+28-treated T cell cultures increased
slowly over time to reach a maximum 4 days after induction (Fig. 1A).
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B). Since IL-2 has been reported to up-regulate IL-8
secretion in monocytes/macrophages (41), we performed the experiment in
the presence (+IL-2) or absence (-IL-2) of exogenous IL-2 (50 U/ml).
Addition of exogenous IL-2 only slightly increased the IL-8 secretion
both in anti-CD3- and anti-CD3+28-treated cultures (Fig. 1B, left side of
panel). Since WE17/10 does not secrete significant
amounts of IL-2 in response to T cell activation, this lack of response
to IL-2 suggests that the IL-8 response obtained in activated T cells
is driven primarily by T cell activation signals. Tat superinduces IL-8 secretion in response to CD3 and CD28 stimulation
To examine the effect of Tat expression on IL-8 secretion, we used
15 clonal cell lines derived from Jurkat following stable transfection
with expression vectors for either Tat72, Tat101, or the control empty
vector cassette (five clones in each group). IL-8 secretion was
measured in culture supernatants 24 h after treatment with
anti-CD3 and anti-CD28 mAb. Expression of either Tat72 or
Tat101 resulted in the superinduction of IL-8 secretion in comparison
to control clones in response to either anti-CD3 alone or
anti-CD3+28 mAb (Fig. 2). After
treatment with anti-CD3+28 mAb, a 20-fold increase in IL-8
secretion was measured in cells expressing Tat101 (Fig. 2, black bars)
and an 8-fold increase was noted in Tat72 cells (Fig. 2, hatched bars).
The amount of IL-8 secreted following treatment with anti-CD3+28
Abs correlated positively (r2 = 0.6) with the amount
of Tat101 expressed in individual clones. No such correlation was
observed in Tat72 clones.
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). No response or a low response
was observed in control clones and Tat72 clones. This suggested that
the activation requirements for IL-8 and IL-2 secretion differ in T
cells and that Tat101, in addition to superinducing CD28-mediated
signals, could also substitute for weak or missing costimulatory
signals to elicit a maximal IL-8 response. Tat101, but not Tat72, enhances IL-8 transcription
When IL-8-specific mRNA levels were examined with the use of
RT-PCR followed by Southern blotting with an IL-8-specific radiolabeled
oligonucleotide, a marked increase of IL-8-specific mRNAs was
noted in Tat101 and Tat72 cells. This increase in IL-8 mRNA was
detectable as early as 3 h following induction and occurred in
response to both anti-CD3 Ab alone or anti-CD3+28 mAb (Fig. 3A). To determine the
mechanism of the Tat72 and Tat101 effects on IL-8 mRNA, i.e.,
transcriptional vs post-transcriptional, we conducted transient
transfection assays using an IL-8 promoter construct driving the
luciferase gene (pIL-8luc). Transfection of this construct in Jurkat
cell lines expressing Tat101 demonstrated a strong increase in promoter
activity (Fig. 3B, black bars). In contrast, no effect of
Tat72 was detected on the IL-8 luciferase construct, suggesting that
the Tat72 effect on IL-8 mRNA does not occur at the transcriptional
level (Fig. 3B, hatched bars).
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C). Again,
Tat101 expression resulted in increased IL-8 transcription and Tat72
had no effect in comparison to the control plasmid. These results
indicate that Tat101 and Tat72 affect IL-8 production via different
mechanisms and that, in contrast to Tat101, the effect of Tat72 on IL-8
production is not mediated at the transcriptional level. The observed
effect of Tat101 on IL-8 transcription in response to anti-CD3 mAbs
alone (Fig. 3, B and C) is in good
agreement with the increased IL-8 secretion observed under the same
conditions in Tat101 clones (Fig. 2). No difference in IL-8 promoter
activity was measured between Tat-expressing and control clones in
uninduced cultures (data not shown). Tat101 increases nuclear factor binding to the CD28RE in the IL-8 promoter
Our studies on the effect of Tat101 on factors bound to the CD28RE
in the IL-2 promoter have demonstrated that CD28REs and NF-B
recognition sites responded differently to Tat101 (4). This
differential responsiveness was unexpected since both sites bind
factors belonging to the NF-B family of transcription factors and
exhibit strong sequence homology (Fig. 4A). To determine
whether Tat101 can also influence the binding of factors to the IL-8
CD28RE, we performed gel shift experiments using nuclear extracts
purified from either control or Tat101-expressing clones.
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, B and C). No difference in binding
activity between Tat101 clones and control clones was noted when an
oligonucleotide corresponding to the NF-B motif in the lymphotoxin
(LT) promoter or the AP-1 site from the IL-8 promoter were used as
probes (Fig. 4, B and C). These
observations confirm results previously obtained with the IL-2 and
HIV-1 NF-B sites (4), underlining the selective effect of Tat101 on
CD28REs. Tat72 had no effect on nuclear factor binding to either the
IL-2 or IL-8 CD28RE or on several NF-B binding sites (data not
shown).
CD28REs in the IL-2 and IL-8 promoter are low-affinity NF-B
sites
To further study the mechanism of action of Tat101 on CD28REs, we
examined the transcription factors binding to this site in comparison
to the related IL-2 CD28RE and to "classical" NF-B sites, which
are unaffected by Tat101. Competition electrophoretic mobility shift
assays were performed using anti-CD3+28-treated Jurkat
nuclear extracts. As expected, the retarded complex obtained when the
IL-8 CD28RE was used as a probe (Fig. 5A, left
panel) was inhibited by competition with an excess of
oligonucleotides carrying either the HIV-1 NF-B, the LT NF-B, or
the IL-2 CD28RE motifs. The unrelated oligonucleotide IL-2 OAP did not
compete for binding activity to the IL-8 CD28RE (Fig. 5A). Comparison of competition assays between
different sites allowed their ranking in terms of affinity for the
complex binding to the CD28RE as NF-BHIV >
CD28REIL-8 > NF-BLT >
CD28REIL-2 (Fig. 5B).
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BHIV site as a probe (Fig. 5A,
right panel). Remarkably, both CD28REs
were very poor competitors of factors binding to this "classical"
NF-B site, which is in good agreement with their classification as
low-affinity NF-B sites (Fig. 5A).
CD28REs, and not NF-B sites, bind c-rel in response
to CD28 costimulation
To further characterize the factor(s) recognizing each site, we
used specific antisera against different NF-B family members in
supershift assays. While NF-B sites in the IL-2 and HIV-1 promoters
were recognized by the "classical" heterodimer composed of p50
(NF-B1) and p65 (RelA), we observed that antisera against p50, p65,
and c-rel supershifted the complex binding to the IL-8
CD28RE and IL-2 CD28RE (Fig. 5C). These experiments
therefore define the CD28REs in the IL-2 and IL-8 promoter as
low-affinity NF-B sites that are recognized by p50, p65, and
c-rel. The same factors were found to occupy these sites
irrespective of whether nuclear extracts derived from Tat101 or control
cell lines were used, demonstrating that the mechanism of Tat action
does not lie in the recruitment of specific DNA binding subunits to the
CD28RE (data not shown).
An intact CD28RE is critical for Tat101 action on the IL-8 promoter
To confirm the role of the CD28RE in the Tat101-mediated increase
in IL-8 transcription, we repeated transient transfection experiments
in Tat-expressing and control Jurkat clones using an IL-8 promoter
construct harboring a mutated CD28RE. Substitution of a single cytidine
residue in the IL-8 CD28RE motif to a thymidine residue abolished DNA
binding activity to the IL-8 CD28RE, as previously reported (24) (Fig. 6, A and B).
Comparison of luciferase activity following transient transfections of
the wild-type pIL-8luc construct or pIL-8lucCD28REmut in Tat72 (Fig. 6C, hatched bars), Tat101 (Fig. 6C, black
bars), or control clones (Fig. 6C, white bars) demonstrated
that an intact CD28RE in the IL-8 promoter is critical for Tat101 to
increase IL-8 transcription. It also confirmed that the Tat101 effect
on IL-8 secretion and transcription observed after stimulation with
anti-CD3 Abs alone was equally dependent on the superinduction of
factors binding to the CD28RE.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B binding sites that bind
c-rel in addition to the classical heterodimer p50
(NF-B1) and p65 (RelA).
These results confirm the unique biologic property of the
full-length Tat101 protein as an HIV-1-encoded immune activator.
Through enhanced transcription factor binding to the CD28REs in the
IL-2 and IL-8 promoters, Tat101 modifies the expression of both
cytokines in response to CD28-mediated costimulation. Since CD28REs
have been identified in the promoters of several other cytokines, such
as granulocyte macrophage-CSF, IFN-ß and -, and IL-3 (16, 24, 42),
it is likely that Tat101 also modulates the expression of these genes,
thereby contributing to the chronic immune hyperactivation observed in
HIV-1-infected individuals. Our results indicate that in uninfected T
cell cultures, IL-8 appears as a "late" cytokine following
induction with anti-CD3 and anti-CD28 mAbs. In
Tat101-transfected T cell lines, however, IL-8 production was markedly
enhanced 24 h after stimulation. This increase in IL-8 secretion
in response to Tat represents a likely source for the IL-8
overproduction observed in HIV-1-infected individuals. In support of
this model, we detected elevated levels of IL-8-specific transcripts in
PBLs isolated from HIV-1-infected individuals compared with healthy
donors (data not shown). Others have reported that PBLs and
lymphocyte-enriched cultures from HIV-1-infected individuals secreted
elevated levels of IL-8, both spontaneously and upon activation with
phytohemagglutinin (43).
However, macrophages are also a known source for IL-8, and HIV-1-mediated dysregulation of macrophages could also result in increased IL-8 secretion by these cells. Controversial findings have been reported with regard to the effect of HIV-1 infection on IL-8 production in monocytes/macrophages (44, 45, 46, 47). Macrophages do not express CD28 receptors on their surface, and it is not clear whether CD28REs are occupied by specific proteins in macrophages. However, soluble factors derived from HIV-1-infected T cells might influence IL-8 expression in an autocrine or paracrine fashion. The ability of Tat to exit the infected cell and enter the nucleus of a neighboring cell is well established (48). In addition, IL-2, IL-1, and TNF have been found to induce IL-8 production in monocyte/macrophages (reviewed in 49 , and IL-8 itself has been described to autostimulate its own production in CD4-positive T cells (50). We did not detect a marked effect of exogenous IL-2 on IL-8 production in an IL-2-responsive T cell line.
We considered the possibility that the observed IL-8 up-regulation in Tat72 clones was partly dependent on an autocrine loop via the secondary effect of TNF/LT on IL-8 production. LT production has been previously described to be positively regulated by Tat (10, 51) and we have confirmed the spontaneous and activation-induced up-regulation of this cytokine in Tat72 and Tat101 cells (M. Ott and E. Verdin, unpublished observation).
Our data indicate that Tat72 superinduces IL-8 production via a post-transcriptional mechanism, as no effect of Tat72 on IL-8 promoter activity was observed in a transfection assay. We cannot, however, totally exclude that Tat72 functions at the transcriptional level in the IL-8 promoter since a Tat72-responsive element might be located upstream of the promoter region, which we tested in this study. Indeed, we focussed our study on the region -273 to +45 of the IL-8 promoter, since this region contains the binding sites necessary for IL-8 gene activation in response to IL-1, TNF, and PMA as previously reported (36). This region contains recognition sites for the CD28-responsive complex (nt -81 to -71), the AP-1 site described in this study (nt -126 to -120), as well as two recognition sites for the C/EBP-like nuclear factor, NF-IL-6 (nt -94 to -81 and nt -68 to -57). Therefore, we cannot rule out a role for upstream transcription factor binding sites, including an IFN regulatory factor 1 binding site (nt -425 to -420), a hepatocyte nuclear factor-1 binding site (nt -381 to -376), and a glucocorticoid-responsive element (nt -330 to -325) (35).
An intact CD28RE (nt -81 to -71) in the IL-8 promoter is critical for
the Tat101 effect, and binding activity to this site is markedly
enhanced in the presence of Tat101. Further support for this critical
role of the CD28RE in Tat action comes from our observation that a
single base pair mutation in this site abolished Tat101-mediated
superinduction of the IL-8 promoter. CD28REs are binding sites
recognized by rel-like transcription factors such as NF-B
family members (17, 42, 52, 53) and the nuclear factor of activated T
cells (NF-AT) (54, 55). Here, we demonstrate that CD28REs act as
low-affinity NF-B binding sites and are occupied by c-rel
in addition to the classical NF-B heterodimer p50 (NF-B1) and p65
(RelA). We have not determined whether the CD28-responsive complex is
composed of all three proteins (c-rel, p50, and p65) or
whether the supershifted p50 protein originates from a small fraction
of resting, nonactivated cells that harbor nuclear NF-B1/p50
homodimers in the absence of transcriptional activation. Cotransfection
experiments have indicated that NF-B p65 and c-rel
proteins are sufficient to transactivate transcription from the IL-8
promoter (53), and that p50 weakly transactivated IL-8 gene expression
in combination with the C/EBP-like nuclear factor NF-IL-6 (56, 57, 58, 59). On
the other hand, a cooperative interaction between NF-B and C/EBP
factors plays a role in modulating gene expression of the IL-6 and
HIV-1 promoters (59, 60). However, the role of p50 in the modulation of
transcription of the IL-8 promoter has not been established.
The molecular mechanism of Tat101 action on transcription factor binding to CD28REs is at present unclear. While Tat101 expression results in an increase in binding of the CD28-responsive complex to the CD28RE, we have not detected any qualitative differences in the factors participating in this complex in Tat101 clones or control clones (data not shown). Irrespective of whether Tat101 was present, p50, p65, and c-rel were found in the complex binding to the CD28RE. We also tested for a possible influence of Tat101 on NF-AT binding to this site, but could not detect any NF-AT binding activity to the IL-2 CD28RE in the presence or absence of Tat101 in our system (our unpublished observation). Recent reports have described the binding of the nuclear factor of mitogen-activated T cells (NF-MAT) and of the high-mobility group protein HMGI(Y) to the IL-2 CD28RE (42, 61). Each of these two factors could in theory be targeted by Tat101. Additional experiments will examine this possibility.
The nonresponsiveness of "classical" NF-B sites to Tat101 is
intriguing and indicates a selective effect of Tat101 either on
c-rel binding, a factor known to play a central role in T
cell activation (62), or on HMGI(Y), since both factors appear to be
binding exclusively to CD28REs and not to NF-B sites. The Tat101
effect is therefore distinct from the well-studied effect of the
transactivator Tax, encoded by the human T cell leukemia virus
(HTLV-I), which enhances binding activities to NF-B sites (63) as
well as CD28REs (64, 65, 66). NF-B proteins are regulated by the
selective nucleo/cytoplasmic partitioning mediated by the IB family
of inhibitory molecules (reviewed in Refs. 67 and 68). IB proteins
retain NF-B proteins in the cytoplasm by masking their nuclear
localization sequences. Upon cell activation with various stimuli,
IB is phosphorylated and rapidly degraded, allowing the NF-B
homo- or heterodimers to migrate into the nucleus. Rel proteins are
also known to interact with the cytoplasmic precursors p100 and p105,
which mature to NF-B p50 and p52 upon proteolytic removal of their
ankyrin-containing carboxyl-terminal segments. Tax expression activates
NF-B binding independently from any activation stimulus through the
degradation of the cytosolic inhibitor molecules IB
and IBß
and the accelerated proteolysis of p105 (reviewed in 3 .
In contrast to Tax, the Tat101 effect was dependent on CD28-mediated costimulation. We observed, however, that Tat101 could partly replace CD28-mediated signals to activate IL-8 transcription as demonstrated by increased IL-8 production, increased promoter activity, and increased transcription factor binding to the CD28RE in the IL-8 promoter following CD3 stimulation alone (data not shown). Tat101 did not stimulate IL-8 production in the absence of stimulatory signals in contrast to what has been reported for Tat-induced LT production (10). These experiments underline the pleiotropic effects of HIV-1 Tat proteins, which appear dependent on the unique architecture and signaling requirements of the different Tat-responsive promoters. We have previously observed that Tat101 could increase costimulatory signals for IL-2 transcription and binding to the CD28RE in the absence of CD28-mediated signals; however, no costimulatory effect of Tat101 on IL-2 secretion had been observed following treatment with anti-CD3 mAb alone (4). These experiments indicate that although Tat101 can partly replace the transcriptional effect of CD28-mediated signals, CD28-mediated costimulation provides extra signals that are critical for IL-2, but less for IL-8 secretion and cannot be substituted by Tat101. One possibility is that CD28 costimulation enhances the stability of IL-2-, but not IL-8-specific transcripts (21).
Our studies define a new immunomodulatory region in Tat101 that is located in the carboxyl-terminal 29 amino acids of the protein encoded by the second exon of the viral tat gene. This Tat domain enhances IL-8 expression by modulating factors binding to the CD28RE in the IL-8 promoter. Tat-mediated superinduction of IL-8 is likely to play an important physiologic role during HIV-1 infection given the potential of IL-8 to attract T cells, in addition to its known function as a neutrophil chemoattractant. During HIV-1 infection, IL-8 might play a unique role in the recruitment of target cells to the sites of viral replication in the lymph nodes. We could not detect any negative effect of IL-8 on HIV-1 replication using either macrophage- or T cell-tropic strains (data not shown). This finding is in agreement with the reported observation that IL-8 does not cross-react with the HIV-1 coreceptor CXCR4 (33). Increased migration of T cells to lymph nodes provides a constant supply of target cells necessary for continuous HIV-1 replication and is likely to contribute to lymphadenopathy and the progressive loss of lymph node architecture. Therefore, targeting Tat101 action on CD28REs should be considered for future therapeutic efforts to control the pleiotropic manifestations of immune activation during HIV-1 infection.
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Acknowledgments |
|---|
B family members, Drs. N.Chiorazzi and D. Olive
for mAbs against CD3 and CD28, respectively, and Dr. Christine Metz for
the purification of mAbs against CD3 and CD28. We thank members of the
Verdin laboratory and Dr. Narendra Chirmule for discussion and
suggestions. We thank Nuriza Yarlett for performing the
Mycoplasma testing of our cell lines. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric Verdin, The Gladstone Institute for Virology and Immunology, University of California, 365 Vermont St, San Francisco, CA 94103. E-mail address:
3 Abbreviations used in this paper: CD28RE, CD28-responsive element; nt, nucleotide; RT, reverse transcriptase; LT, lymphotoxin.
Received for publication August 27, 1997. Accepted for publication November 25, 1997.
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References |
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