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*
Experimental Retrovirology Section, Center for Cancer Research and
HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and
Department of Internal Medicine II, Kumamoto University School of Medicine, Kumamoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we examined large-scale genome-wide effects of SDF-1 on CD4+ T cells using the cDNA microarray technique and demonstrate that SDF-1 promotes CD4+ T cell survival through the phosphatidylinositol 3-kinase (PI3K)- and mitogen-activated protein kinase (MAPK) signal transduction cascades without affecting cell cycle progression and that SDF-1 also exerts priming function to set CD4+ T cells to respond to immunologic stimuli.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An inhibitor of MAPK-extracellular signal-regulated kinases
(MEK) activity, PD098059, and a PI3K-specific inhibitor, LY294002, were
purchased from Biomol (Plymouth Meeting, PA) and New England Biolabs
Beverly, MA), respectively. Recombinant human IL-2, IL-4, IL-6, IL-15,
and SDF-1 (SDF-1
was used throughout this study) were purchased from
R&D Systems (Minneapolis, MN). The following mAbs were used for the
stimulation of T cells: anti-CD3 (UCTH1; Beckman Coulter,
Fullerton, CA) and anti-CD28 (LewTM-28; BD Biosciences,
Mountain View, CA). The following mAbs were purchased from BD
PharMingen (San Diego, CA) and used for staining surface Ags:
anti-CD3-FITC, anti-CD4-FITC and -PE, anti-CD8-FITC,
anti-CD14-FITC, anti-CD16-FITC, anti-CD20-FITC,
anti-HLA-DR-FITC, anti-CD25-FITC, anti-CD69-FITC, and
anti-CXCR4-PE (12G5). mAbs against human CD8, CD20, CD25, and
HLA-DR were produced, purified, and used for the purification of dense
resting CD4+ (drCD4+, see
below) T cells. Anti-human CD16 (LNK16) Ab was purchased from Serotec
(Oxford, U.K.). For Western blotting, Abs against various proteins were
purchased from New England Biolabs. Expression vectors for ribosomal S6
kinase (Rsk) proteins (pMT2-HA-RSK2-WT, pMT-HA-RSK2-KN, and a control
vector) were kind gifts from Dr. M. E. Greenberg (Children’s
Hospital, Harvard Medical School, Cambridge, MA). For transfection of
Jurkat cells, a green fluorescent protein expression vector (Clontech
Laboratories, Palo Alto, CA) and GenePORTER transfection reagents (Gene
Therapy Systems, San Diego, CA) were used.
Chemotaxis assays
Chemotactic activity of SDF-1 was determined using microchemotaxis plates (NeuroProbe, Gaithersburg, MD) as previously described (11). Two pore size (5- and 10-µm) filters were used for drCD4+ T and Jurkat cells, respectively.
Cell culture and cDNA microarray experiments
The Jurkat (clone E6-1; ATCC TIB-152) cell line was used
throughout this study. Cells (
7 x
105/ml) were maintained in RPMI 1640 containing 1
mM L-glutamine supplemented with 10% (v/v)
heat-inactivated FCS (BioWhittaker, Walkersville, MD) at 37°C in a
5% CO2 containing humidified air. To
study the effects of SDF-1 on CD4+ T cells as
cultured in the absence of FCS, cells were first maintained in RPMI
1640 containing 0.5% FCS for 36 h, thoroughly washed with
FCS-free RPMI 1640, divided into two flasks, and incubated further in
FCS-free medium for 6 h to deactivate the PI3K and MAPK signal
transduction cascades to an undetectable level. Cells in one flask were
stimulated with SDF-1 and harvested at 45 min, 3 h, 6 h, and
12 h for isolation of mRNA. mRNA was also isolated from
SDF-1-unexposed cells in another flask and served as a reference for
each time point. Preparation of mRNA and the labeling of the probe and
hybridization conditions are described in detail at
http://nciarray.nci.nih.gov/reference/index.shtml. In brief, total
mRNA was isolated using RNeasy kit (Qiagen, Chatsworth, CA) and 25 µg
of mRNA was used for generating fluorescence-labeled cDNA probes using
Cy3- and Cy5-dUTP (Amersham, Arlington Heights, IL), and the resulting
cDNA was subjected to hybridization on microarray slides. Methods used
for hybridization, fluorescein scanning, and image analysis are
described in detail at
http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/img_analysis.html. All
measurements were stored in a computer database for analyses and
interpretations. The microarray slides used contained 2304 elements
including 2140 human cDNAs which represented 1847 different genes (the
complete list of the genes can be seen at
http://nciarray.nci.nih.gov/cgi-bin/gipo) and were obtained from the
Advanced Technology Center, National Cancer Institute.
RNase protection assays
RNase protection assays were conducted using RiboQuant MultiProbe RNase Protection Assay kits (BD PharMingen) according to the manufacture’s instructions. Total mRNA was isolated from SDF-1 (0.38 µg/ml)-exposed cells cultured in 10% FCS-containing medium for 3, 6, 12, 24, and 36 h and the expression of 79 selected genes encoding cytokines, chemokines, apoptosis-related genes, and angiogenesis factors (hCK-1, hCK 2, hCK-3, hCR-1, hCR-5, hCR-6, hAPO-2c, hAPO-3c, hAPO-4, hSTRESS-1, hAngio-1) was examined.
Isolation of drCD4+ T cells
drCD4+ T cells were isolated from PBMC obtained from healthy donors as previously described by Sun et al. (12). In brief, PBMC were isolated over Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) and subjected to nylon wool fiber column (Polysciences, Warrington, PA) separation to deplete B cells and macrophages. The nonadherent cells were overlaid on a discontinuous Percoll (Pharmacia Biotech, Uppsala, Sweden) gradient and the dense T cells lodged in the interface between 60 and 65% Percoll were collected. CD4+ T cells were purified through negative selection using a magnetic cell separation system (Dynal MPC-1 column; Dynal Biotech, Great Neck, NY ) with a mixture of mAbs specific for CD8, CD16, CD20, CD25, and HLA-DR. Unstained cells were harvested with a magnetic column. The purity and the cell surface expression of various molecules were determined using a calibrated FACScan flow cytometer and were analyzed by CellQuest software (BD Biosciences). The purity of the drCD4+ T cells thus harvested routinely consisted of >97% CD4+ cells, <1.2% CD25+ cells, and <1.1% HLA DR+ cells.
Apoptosis and T cell proliferation assays
The percentages of apoptotic cells were determined using Annexin VFITC and propidium iodide (PI; BD PharMingen) costaining according to the manufacturer’s instructions and flow cytometry. Jurkat cells (104) or drCD4+ T cells (105) were cultured in the presence of various Abs or cytokines in 96-well flat-bottom microtiter culture plates. After 48 or 72 h in culture, each cell preparation was pulsed for 16 h with [3H]thymidine (1 µCi for drCD4+ T cells and 0.5 µCi for Jurkat cells). To determine whether SDF-1-exposed drCD4+ T cells progressed in the cell cycle, they were exposed to SDF-1 (0.38 µg/ml) for 72 h and their 5-bromo-2'-deoxyuridine (BrdU) incorporation and Ki-67 expression were determined using a BrdU Flow kit (BD PharMingen) and Ki-67 Ab kit (BD PharMingen), respectively.
Western blot analysis
Whole cell lysates derived from Jurkat, MAGI, and drCD4+ T cells were examined using 4–20% gradient polyacrylamide gels (Pharmacia Biotech). For Western blotting, 1/3000 and 1/10000 dilutions were used for the first Abs and a secondary HRP-conjugated anti-rabbit IgG (New England Biolabs), respectively. Proteins were visualized using a SuperSignal West Pico Chemiluminescent kit (Pierce, Rockford, IL) and Biomax-MR film (Kodak, Rochester, NY).
In vitro kinase assay
Jurkat cells were cultured with FCS (0.5%) containing RPMI 1640-based culture medium for 36 h, then in FCS-free medium for 6 h, and exposed to SDF-1 (0.38 µg/ml) for 10 min. Some cells were exposed to PD098059 (50 µM) or LY294002 (20 µM). Akt1 and Rsk2 were immunoprecipitated by Abs that are specific for Akt1 (New England Biolabs) and Rsk2 (Upstate Biotechnology, Lake Placid, NY, respectively). The immunoprecipitated kinases were subjected to in vitro kinase reaction with 200 µM ATP along with 1 µg of the substrate recombinant Bcl-2 antagonistic of cell death (BAD; Upstate Biotechnology). In vitro kinase assays were performed using a kit purchased from New England Biolabs according to the manufacturer’s instructions. Immunoblotting was performed using Abs that were specific for BAD phosphorylated at Ser112 or Ser136 and an Ab that was reactive with BAD regardless of phosphorylation, as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the profile of gene expression in CD4+ T cells upon SDF-1 exposure, we first determined the SDF-1 concentration that induced a maximal migration response. When Jurkat and drCD4+ T cells were exposed to serially diluted SDF-1 (0, 0.12, 0.38, 1.1, 3.3, and 10 µg/ml), the maximum migration occurred in the presence of 0.38 µg/ml SDF-1. We treated Jurkat cells with SDF-1 (0.38 µg/ml) in the presence and absence of FCS, since the PI3K- and MAPK-dependent signal pathways, known to be activated by SDF-1, are also activated by FCS (13, 14, 15, 16). Total mRNA was isolated from Jurkat cells at several time points following SDF-1 exposure and fluorescence-labeled cDNA probes were prepared using Cy3- and Cy5-dUTP as described in Materials and Methods. The Cy3- and Cy5-labeled cDNA probes were hybridized onto a cDNA microarray slide containing 2140 cDNA clones. Following thorough washing, the array was scanned and the fluorescence ratios were determined.
The transcriptional response upon SDF-1 exposure occurred within 3
h (Fig. 1
, A and
B). The initial response was much greater in cells exposed
to SDF-1 in the absence of FCS than in those treated similarly but in
the presence of FCS. In 6 h and beyond, the pattern of gene
expression was comparable in the two cell preparations, but the ratios
were greater in the cells cultured in the absence of FCS than in those
cultured in the presence of FCS (Fig. 1, A and
B). To corroborate these gene expression data and to confirm
its reproducibility, we chose 79 genes (see Materials and
Methods) encoding cytokines, chemokines, apoptosis, and
angiogenesis factors and examined their gene expression profiles in the
SDF-1-exposed cells using the MultiProbe RNase protection assay. The
levels of expression of these selected genes were comparable to the
ratio changes observed in the microarray assays (data not shown).
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, A and
C). Such genes included detoxification (members of
cytochrome P450 subfamilies)-, DNA repair (XRCC5, XRCC3, ERCC1, RAD54L,
MSH6, TDG)-, and antiapoptosis (defender against apoptotic death-1
(DAD-1) PPEF-1, PPEF-2, Nak1)-related genes (Fig. 2). We presumed that these genes were
associated with the promotion of cell survival. Genes associated with
TCR signaling (CD3
, Fyn, SLP-76, HS1, Nck1), inflammatory cytokine
receptors and related proteins, IFN receptors, and downstream molecules
(IFN regulatory factor 6 and IFN regulatory factor 3) were also
identified, leading us to postulate that the expression of these genes
represented immunologic response in CD4+ T cells
upon SDF-1 exposure. Moreover, the genes encoding cell adhesion
molecules (members of integrins, CD36, CD33, protocadherin 42), kinases
involved in the reorganization of actin cytoskeleton (ROCK1, MDK),
actin-binding proteins (HS1, GAS2, PKD1, filamin A, CDC10), chemokine
receptors (STRL33.1), and the genes involved in the remodeling of
extracellular matrix and metastasis (MMP-11) were identified (Fig. 2).
In addition, RNase protection assays using the RiboQuant MultiProbe
RNase Protection kit (hCR-5) revealed that the expression level of CCR8
which was not included in the microarray increased 20.8-fold in 36
h in Jurkat cells after SDF-1 exposure (data not shown). Thus, 6% of
the 101 genes were related to DNA repair enzymes, 5% detoxification
enzymes, 15% signal transduction molecules, and 18% cytokinesis/cell
morphology/cell adhesion molecules.
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In an attempt to examine whether the genes expressed seen in the
microarray technique were also phenotypically expressed, we conducted
functional assays for apoptosis and T cell signal transduction using
highly purified drCD4+ T cells as well as
CD4+ Jurkat cells. The
drCD4+ T cells are largely in the
G0 phase of the cell cycle and do not produce
detectable amounts of cytokines, making SDF-1-specific effects easily
identified. It was found that the removal of FCS from the culture of
drCD4+ T cells and Jurkat cells induced
apoptosis, but when cultured with SDF-1, the percentage of apoptotic
drCD4+ T cells was greatly decreased in the
absence of FCS to the level observed in the presence of FCS (Fig. 3, Aa and B). The
SDF-1-induced suppression of apoptosis was also observed in Jurkat
cells, although the suppression was not complete (Fig. 3C).
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-irradiation-induced apoptosis in
drCD4+ T and Jurkat cells was suppressed by
the addition of SDF-1 in culture (Fig. 3D, a and
b). These results strongly suggest that SDF-1 blocks
apoptosis in CD4+ T cells.
Considering that CXCR4 is abundantly expressed in resting
CD4+ T cells, it is likely that SDF-1 contributes
to the maintenance and protection of unstimulated
CD4+ T cells. By contrast, high-affinity
receptors for a number of cytokines such as IL-2 and IL-4 appear only
upon immunologic stimulation of T cells, and such cytokines
elicit antiapoptotic activity, cell cycle progression, and
differentiation in CD4+ T cells. Thus, the
antiapoptotic effects of SDF-1 seem to differ from the antiapoptotic
activities of cytokines.
Next, we examined whether drCD4+ T cells were
immunologically activated upon SDF-1 exposure. There was no expression
of activation markers CD25 or CD69 in drCD4+ T
cells or Jurkat cells upon SDF-1 exposure, whereas those markers were
readily detected following stimulation with anti-CD3 Ab (Fig. 3E). We also failed to detect alterations in replication
rates or cell sizes in Jurkat cells following SDF-1 exposure (data not
shown). Moreover, we asked whether drCD4+ T cells
progressed to the G1 phase using a
[3H]thymidine incorporation assay (Fig. 3F), flow cytometry for BrdU incorporation, DNA content
determination (Fig. 3Ga), and the assay for expression of
Ki-67, a protein seen in all stages except the G0
phase of the cell cycle (Fig. 3Gb). We found no evidence for
the progression of drCD4+ T cells to the
G1 phase or beyond. Thus, we concluded that SDF-1
promotes survival of drCD4+ T cells without
forwarding them in the cell cycle.
Cell survival is promoted by SDF-1 through MEK- and PI3K-dependent pathways
Cell survival factors such as brain-derived neurotrophic factor
(BDNF) and insulin-like growth factor 1 activate the PI3K- and/or
MAPK-mediated signaling pathways and transduce cell survival signals
(17, 18, 19). Recently, Vicente-Manzanares et al. and others
(13, 14, 15, 16) reported that SDF-1 activates the MAPK-MEK- and
PI3K-serine/threonine kinase Akt (protein kinase B) pathways to induce
chemotaxis in CD4+ T cells. We therefore examined
whether the activation of these two pathways preceded the promoted
survival in SDF-1-exposed CD4+ T cells. To this
end, we used PD098059, an agent that blocks MEK activity (20, 21), and LY294002, a PI3K-specific inhibitor (22).
Both inhibitors almost completely suppressed the SDF-1-induced survival
in drCD4+ T cells and Jurkat cells (Fig. 3, Ab and C), suggesting that the activation of the
MAPK-MEK and PI3K signaling pathways is required for the SDF-1-induced
promoted survival of CD4+ T cells. For
biochemical confirmation of the involvement of the MAPK-MEK and PI3K
signaling pathways, we asked whether the signal transduction molecules
(MEK, extracellular signal-regulated kinase (ERK), Rsks (p90), and a
transcription factor CREB) in the MAPK signaling pathway were
phosphorylated in Jurkat cells upon SDF-1 exposure. When examined using
the immunoblotting technique, all such molecules were found to be
phosphorylated (Fig. 4A). When
Jurkat cells were cultured with PD098059, however, a substantial
suppression in the phosphorylation of ERK, Rsks, and CREB was observed
(Fig. 4A).
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B). To determine whether CREB phosphorylation follows
the phosphorylation of Rsks, we used transiently Rsk2-KN-overexpressing
CXCR4+ fibroblasts (MAGI cells)
(25). With respect to the use of MAGI cells, the
efficiency of transfection of dominant-negative protein Rsk2-KN in
Jurkat was very poor (only 8% of the cells became positive for
Rsk2-KN) and it was not possible to reliably perform the immunoblotting
assays. Therefore, we chose CXCR4-expressing MAGI cells, which we found
was a more transfection-efficient (70% of the cells became
positive) cell line, enabling us to study the effect of SDF-1. In this
respect, we had learned that SDF-1 exposure induced the phosphorylation
of Rsks and CREB in both Jurkat and MAGI cells, and that the MEK
inhibitor PD098059 inhibited the SDF-1-induced phosphorylation of Rsks
and CREB in both cell lines as shown in Fig. 4, A and
D. These data suggest that SDF-1 exposure induces the
phosphorylation of Rsks and CREB through the MEK-dependent pathway(s)
in both Jurkat and MAGI cells. When the Rsk2-KN-overexpressing
CXCR4+ MAGI cells were exposed to SDF-1, no
phosphorylation of CREB was seen (Fig. 4D). In this respect,
it has been shown that ERK serves as a substrate for MEK and the
activated ERK subsequently induces Rsk phosphorylation (17, 23, 24). As shown above, it appears that the SDF-1-induced
activation of CREB occurred through the MEK-ERK-Rsk pathway(s) (Fig. 4, A and D). Taken together, it is likely that the
SDF-1-induced cell survival promotion seen in this study occurs through
the MEK-ERK-Rsk-CREB cascade.
Since the PI3K-Akt-induced phosphorylation and inactivation of Forkhead
family transcription factors are reportedly involved in cell survival
(26, 27), we also asked whether Akt and FKHR (a member of
the Forkhead family) were involved in SDF-1-induced signaling pathways.
Upon SDF-1 stimulation, both Akt and FKHR were found phosphorylated and
the PI3K-specific inhibitor, LY294002, inhibited the phosphorylation of
these two molecules (Fig. 4A). These data strongly suggest
that FKHR inactivation (phosphorylation) was mediated through the
PI3K-Akt pathway.
We also asked whether SDF-1 stimuli were transduced through pathways
other than the MAPK and PI3K signaling pathways and examined the
activation of three signal transduction molecules (p38 MAPK, c-Jun
N2-terminal kinase 1/2, and I
B) associated
with other signal transduction pathways, but no phosphorylation was
detected in any of these three molecules (data not shown). Furthermore,
we examined whether the genes associated with DNA repair,
detoxification, and apoptosis, which we identified to be up-regulated
upon SDF-1 exposure in the microarray assays, were linked to the
MAPK-MEK and PI3K signaling pathways using PD098059 or LY294002,
respectively, in Jurkat cells (Fig. 2). These two inhibitors blocked
the up-regulation of those genes, suggesting that the two pathways are
involved in the SDF-1-induced transcription.
SDF-1 exposure induces BAD phosphorylation
The Bcl-2 family proteins consist of molecules that enhance cell
survival as well as those that promote apoptosis (28, 29).
Recently, one such Bcl-2 family protein, BAD, which is bound to Bcl-2
and/or Bcl-xL and exerts a proapoptotic function,
has been shown to be phosphorylated at Ser112 and
Ser136 by Rsks and Akt and to be involved in cell
survival (17, 18, 19). We therefore examined whether the
phosphorylation of BAD occurred in SDF-1-exposed
CD4+ Jurkat cells using the immunoblotting assay
with Abs that recognized phosphorylated BAD. It was revealed that the
MEK inhibitor PD098059 decreased BAD phosphorylation at
Ser112 and the PI3K inhibitor LY294002 the
phosphorylation at Ser136 (Fig. 4C),
suggesting that SDF-1 causes the phosphorylation of BAD at
Ser112 or Ser136 via the
MEK and PI3K pathways, respectively. We next overexpressed both Rsk2
and BAD in CXCR4+ MAGI cells and examined whether
BAD phosphorylation was enhanced upon SDF-1 exposure. With the
overexpression of wild-type Rsk2 (Rsk2-WT), BAD phosphorylation at
Ser112 was clearly enhanced (Fig. 4D),
whereas when the dominant-negative Rsk2-KN was overexpressed such
phosphorylation was suppressed. Moreover, to determine whether BAD is a
substrate for Rsks and/or Akt, we employed an in vitro kinase assay
using the cell lysates of SDF-1-exposed Jurkat cells. It was then shown
that Rsk2, which was immunoprecipitated from the lysates of
SDF-1-exposed cells, clearly phosphorylated BAD protein at
Ser112 (Fig. 4E). When Jurkat cells
were incubated concomitantly with SDF-1 and PD098059, Rsk2-induced
phosphorylation of BAD at Ser112 was reduced,
whereas Rsk2-induced BAD phosphorylation was not suppressed by LY294002
treatment. We also determined the in vitro kinase activity of Akt1,
which was immunoprecipitated from the cell lysates of SDF-1-exposed
Jurkat cells. Immunoprecipitated Akt1 phosphorylated BAD at
Ser136; however, treatment of the cells with
LY294002, not with PD098059, substantially reduced Akt1 kinase activity
to phosphorylate BAD at Ser136. Therefore,
immunoprecipitated Rsk2 and Akt1, phosphorylated upon SDF-1 exposure,
caused the phosphorylation of recombinant BAD protein, indicating that
BAD protein served as a substrate for both kinases.
We further asked whether the phosphorylation of Rsks, Akt, and BAD
occurred via the MEK and PI3K pathways in drCD4+
T cells exposed to SDF-1. In immunoblotting assays, we found that
PD098059 but not LY294002 decreased the phosphorylation of Rsk and
Ser112 of BAD while LY294002 but not PD098059
suppressed the phosphorylation of Akt (Fig. 4F). These
findings suggest that the activation of MEK-ERK-Rsk and PI3K-Akt causes
BAD phosphorylation, leading to the promoted survival of SDF-1-exposed
CD4+ T cells.
Bonni et al. (17) reported that BDNF-mediated nerve cell
protection was associated with the phosphorylation of CREB and an
enhanced expression of Bcl-2. We therefore asked whether the expression
level of the Bcl-2 family mRNA was altered upon SDF-1 exposure. As
assessed by the RNase protection assay, no changes in any of detectable
Bcl-2 family mRNA were observed in CD4+ Jurkat T
cells (Fig. 4G).
SDF-1 primes CD4+ T cells for TCR-CD3-mediated stimulation
Finally, we examined whether anti-CD3-induced proliferation of drCD4+ T cells was affected in the presence of SDF-1. When drCD4+ T cells were stimulated with plate-bound anti-CD3 at 10, 20, and 40 µg/ml, CXCR4 down-regulation occurred in 52 ± 2, 62 ± 2, and 75 ± 3% of those cells, respectively, in agreement with reports by Jourdan et al. and others (30, 31, 32). Our kinetic studies of CXCR4 expression showed that the anti-CD3-induced CXCR4 down-regulation started as early as 6 h in culture and reached a maximum level at around 16 h. Once the anti-CD3-induced down-regulation of CXCR4 occurred, it generally took >7 days to fully regain CXCR4 on the cellular surface (data not shown).
Interestingly, when drCD4+ T cells were
pretreated with SDF-1 and then exposed to anti-CD3, a significant
level of proliferation was observed (Fig. 5A). When
drCD4+ T cells were concomitantly stimulated with
anti-CD3 and SDF-1, an enhanced cell proliferation was also
observed. However, the anti-CD3 response was less than that of the
cells pre-exposed to SDF-1 (Fig. 5A). The percentage of
CD69+ cells in SDF-1-pre-exposed,
anti-CD3-exposed drCD4+ T cells was greater
than the percentage in concomitantly SDF-1/anti-CD3-exposed
drCD4+ T cells (82 vs 58%) as assessed in
16 h following the exposure, whereas the viability was comparable
in the two drCD4+ T cell populations,
corroborating the data illustrated in Fig. 5A that the
SDF-1-pre-exposed drCD4+ T cells underwent a
greater magnitude of activation. The observation that once exposed to
SDF-1, the presence of SDF-1 in culture was not required for the
enhanced activation and proliferation of drCD4+ T
cells suggests that SDF-1 primed CD4+ T cells for
stimuli incoming through the TCR-CD3 complexes rather than promoted
their survival.
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B). The combination of anti-CD3 and anti-CD28,
however, enhanced the proliferation of Jurkat cells when they were
exposed to SDF-1 (Fig. 5C). These data suggest that the
mechanism of the cell proliferation enhancement with IL-2 or IL-15
along with SDF-1 is different from that of anti-CD3-mediated cell
proliferation (Fig. 5, A and B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokine (C-X-C)
family of cytokines and is a ligand for the chemokine receptor CXCR4,
which is also known as a coreceptor for HIV-1 and is expressed largely
in various B cell and CD4+ T cell subsets
(1). In addition to its role as a chemoattractant for T
cells, B cells, monocytes, and neutrophils, SDF-1 is known to be
essential for viability of the embryo and to play a critical role in
bone marrow hemopoiesis, cardiogenesis, and B lymphopoiesis
(4, 5, 6).
Well-defined studies by Nagasawa et al. and others (4, 5, 6)
have shown that mice genetically deficient in SDF-1 or CXCR4 suffer
profound defects in the hemopoietic and nervous systems. However, in
such SDF-1-deficient mice, no alterations were identified in T cell
development or in T cell trafficking to lymphoid organs (6, 7), leading to an assumption that SDF-1 may not have significant
effects on T cells. It is possible, however, that the specific role of
SDF-1 on T cells could not be examined since the embryo died so early
in its development. Recently, Nanki and Lipsky (8)
reported that SDF-1 serves as a costimulating factor with anti-CD3
for CD4+ T cell activation, proliferation, and
production of IL-2, IFN-, IL-4, and IL-10, although SDF-1 alone
reportedly does not induce CD4+ T cell activation
or cytokine production. In this study, we used the complementary DNA
microarray and examined whether certain genes were induced in
CD4+ T cells when they were exposed to SDF-1.
Following the exposure of CD4+ T cells with
SDF-1, significantly altered expression profiles of 269 genes were
seen (Fig. 1A). Among the genes that exhibited significant
ratio changes upon SDF-1 exposure, three conspicuous clusters, whose
ratios continued to increase throughout a 12-h culture following SDF-1
exposure, were identified (Fig. 1, A and C).
These genes were those associated with DNA repair, apoptosis,
detoxification, cell morphology, cell adhesion, and signal transduction
(Fig. 2). The number of drCD4+ T cells we can
obtain from a single donor is limited and their use for the microarray
assay could not be chosen since at least 4 x
108 drCD4+ T cells yielding
25 µg of total mRNA were required. Therefore, in the
present study we used Jurkat T cells for the microarray experiment for
the assay. However, we found that SDF-1 exposure caused the
phosphorylation of the proteins involved in the two major cascades,
MEK- and PI3K-dependent pathways, in both Jurkat T cells and
drCD4+ T cells (Fig. 4, A,
C, and F). Moreover, the phosphorylation of such
proteins and the expression of the genes induced upon SDF-1 exposure
were blocked by the MEK and/or PI3K inhibitors (Figs. 2 and 4),
suggesting that the signals from CXCR4 receptors are transduced to the
nucleus via the MEK and/or PI3K pathways. Considering that the
activation of the same cascades were seen in SDF-1-exposed Jurkat T
cells and drCD4+ T cells (Fig. 4, A
and C), it seems that the gene induction seen in
SDF-1-exposed Jurkat T cells is likely to occur in
drCD4+ T cells as well. Indeed, using
Multiple-Probe RNase Protection kits with SDF-1-treated
drCD4+ T cells, we found that SDF-1 exposure also
induced DAD-1 mRNA expression in drCD4+ T
cells (data not shown). Thus, it appears that certain antiapoptotic
genes are induced in normal CD4+ T cells by SDF-1
exposure.
Although no significant effects of SDF-1 on CD4+
T cells have been reported (6, 7), as described above, the
involvement of a large number of genes linking to
CD4+ T cell functions was seen upon SDF-1
exposure, which was rather unexpected. With this respect, Onai et al.
(33) recently reported an impaired T cell maturation in
the thymus of SDF-1-transduced and SDF-1-intrakine-transduced mice.
They hypothesized that SDF-1 is associated with the migration of T cell
progenitors to thymus and the ensuing intrathymic selection of T cells.
Our data show that SDF-1 also plays a significant role in T cell
functions. We found that 1) activation of MEK-ERK-Rsk and PI3K-Akt
causes BAD phosphorylation, leading to inactivation of its proapoptotic
function and that 2) the expression of cell survival-related genes was
associated with SDF-1-activated-MAPK-MEK and PI3K signaling (Figs. 2, 3, Ab and C, and 4), suggesting that
SDF-1-induced survival promotion occurred through at least two
mechanisms: posttranslational inactivation of the cell death machinery
and an increased transcription of cell survival-related genes. With
this respect, Kaul and Lipton (10) reported that SDF-1
induced apoptosis in neuronal cells through the p38 MAPK signaling
pathway. However, we detected no phosphorylation of p38 MAPK protein in
SDF-1-stimulated Jurkat T cells (Y. Suzuki, unpublished data).
It is possible that different signal transduction pathways used in
those different types of cells have brought about the different
outcomes in response to SDF-1.
We also observed that the anti-CD3-stimulated proliferation of
CD4+ T cells was greatly enhanced when the cells
had been pre-exposed to SDF-1 for 12 h and thoroughly washed to
remove SDF-1 from culture (Fig. 5A). We thought that SDF-1
induced signal transduction molecules and preset
CD4+ T cells to readily and effectively transduce
the incoming TCR/CD3 stimuli to the nucleus. Unexpectedly, the
magnitude of CD4+ T cell proliferation was
substantially reduced when the cells were concomitantly exposed to
anti-CD3 and SDF-1 (Fig. 5A). We presume that with the
anti-CD3 stimulation, the down-regulation of CXCR4 occurred and
SDF-1 binding to CD4+ T cells was substantially
prevented throughout the 3-day culture, although SDF-1 could bind to
CD4+ T cells to some extent at the beginning of
the culture. Indeed, a profound down-regulation of CXCR4 occurred as
early as 6 h after anti-CD3 stimulation and once the
anti-CD3-induced down-regulation of CXCR4 occurred, it generally
took >7 days to fully recover CXCR4 in drCD4+ T
cells. It should be noted that anti-CD3 stimulation does not induce
CXCR4 down-regulation in Jurkat CD4+ T cells and this
may explain why the magnitude of proliferation of Jurkat T cells
stimulated with anti-CD3 plus anti-CD28 was comparable
regardless of SDF-1 pretreatment or concomitant exposure (Fig. 5C). We thus conclude that SDF-1 exerts priming functions on
CD4+ T cells for stimuli incoming through the
TCR-CD3 complexes but does not serve as a costimulation factor to
anti-CD3 stimulation. There is a possibility that upon SDF-1
pre-exposure, a certain population of drCD4+ T
cells remained more robust than others and were ready to receive
activation stimuli because of the survival-promoting property of SDF-1.
Regarding the viability of drCD4+ T cells, the
experiment illustrated in Fig. 5A was conducted in the
presence of FCS; therefore, as expected virtually all
drCD4+ T cells (>90%) were viable in any of
those drCD4+ T cell populations in the initial
24 h of culture. Furthermore, the percentage of
CD69+ cells in SDF-1-pre-exposed and
anti-CD3-exposed drCD4+ T cells was greater
than the percentage in concomitantly SDF-1/anti-CD3-exposed
drCD4+ T cells, whereas the viability was
comparable in the two drCD4+ T cell populations,
corroborating the data illustrated in Fig. 5A that the
SDF-1-pre-exposed drCD4+ T cells underwent a
greater magnitude of activation. Thus, it seems likely that the priming
effect of SDF-1 rather than its survival-promoting property was
involved.
The data we generated in this study in SDF-1-exposed CD4+ T cells led us to postulate that SDF-1, beyond its known functions as a chemoattractant, plays a critical role in the CD4+ T cell-mediated immune response. SDF-1 is known to be produced in a variety of tissues including lymphoid organs, liver, lung, and mesenchymal cells surrounding endothelial cells (5). Thus, one can assume that CD4+ T cells constantly receive SDF-1 signal and gain their survival and are primed at a low, basic level. Indeed, a single i.v. infusion of a CXCR4 antagonist AMD3100 reportedly caused dose-related elevation of white blood cell counts (polymorphonuclear leukocytes, lymphocytes, and monocytes) within 2 h, suggesting that circulating blood cells constantly receive signal(s) from CXCR4 and lodge onto endothelia cells (34). However, when CXCR4 receptors were blocked by AMD3100, a "demargination" effect occurred in which white blood cells including lymphocytes were released from attachment to the endothelial cell surface into circulation. Thus, it appears that SDF-1 undertakes the housekeeping or maintenance role for lymphocytes. On the other hand, once inflammation occurs, various cells, attracted by chemoattractants, migrate into regions where immunologic and other related stimuli are imposed, and generate oxygen radicals, NO, and numerous tissue-destructive factors. In such an environment, SDF-1-mediated cell survival promotion, DNA damage repair, and priming function would become critical for the effective defense. It is also worth noting that with regard to the pathogenesis of HIV-1 infection, when HIV-1 or its envelope glycoprotein gp120 binds to CXCR4 receptors on CD4+ T cells and blocks the binding of SDF-1 to CXCR4 and/or down-regulates CXCR4 expression, a decrease or cancellation of the effect of SDF-1 to maintain and protect CD4+ T cells ensues, thus contributing to the premature death of CD4+ T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hiroaki Mitsuya, Experimental Retrovirology Section, Center for Cancer Research, National Cancer Institute, Building 10, Room 5A11, 9000 Rockville Pike, Bethesda, MD 20892. E-mail address: hmitsuya{at}helix.nih.gov
3 Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK-extracellular signal-regulated kinase; ERK, extracellular signal-regulated kinase; Rsk, ribosomal S6 kinase; BAD, Bcl-2 antagonistic of cell death; dr, dense resting; PI, propidium iodide; BrdU, 5-bromo-2'-deoxyuridine; BDNF, brain-derived neurotrophic factor; DAD-1, defender against apoptotic death-1.
Received for publication February 5, 2001. Accepted for publication July 16, 2001.
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