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Department of Cell Biology, University of Alabama, Birmingham, AL 35294
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein-10 and IL-8 and the CC chemokines monocyte
chemoattractant protein-1 and RANTES in response to stimuli such as
TNF-
, IL-1
, and IFN-. In this study, we investigated chemokine
receptor expression and function on human astroglioma cells.
Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was
observed upon treatment with the cytokines TNF- and IL-1. The
peak of CXCR4 expression in response to TNF- and IL-1 was 8 and
4 h, respectively. CXCR4 protein expression was also enhanced upon
treatment with TNF- and IL-1 (2- to 3-fold). To study the
functional relevance of CXCR4 expression, stable astroglioma
transfectants expressing high levels of CXCR4 were generated.
Stimulation of cells with the ligand for CXCR4, stromal cell-derived
factor-1 (SDF-1), resulted in an elevation in intracellular
Ca2+ concentration and activation of the mitogen-activated
protein kinase cascade, specifically, extracellular signal-regulated
kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest,
SDF-1 treatment induced expression of the chemokines monocyte
chemoattractant protein-1, IL-8, and IFN--inducible protein-10.
SDF-1-induced chemokine expression was abrogated upon inclusion of
U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK
signaling cascade is involved in this response. Collectively, these
data suggest that CXCR4-mediated signaling pathways in astroglioma
cells may be another mechanism for these cells to express chemokines
involved in angiogenesis and inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein, 10 kDa
(IP-10)3;
growth-related oncogene (GRO-), , and ; monokine-induced by
IFN-; and stromal cell-derived factor (SDF)-1 and . CXC
chemokines have been classically described as neutrophil
chemoattractants, and IP-10 and SDF-1 are involved in T cell
activation. The NH2 terminus of the majority of
CXC chemokines contains three amino acid residues (Glu-Leu-Arg: the ERL
motif), which precede the first cysteine residue of the primary
structure of these chemokines. Members with this ERL motif
(ERL+) are potent inducers of angiogenesis (for
review, see Ref. 4). The CC chemokine family is
characterized by the first two cysteine residues being adjacent to each
other, and include macrophage-inflammatory protein (MIP)-1,
MIP-1, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3,
RANTES, eotaxin, and thymus- and activation-regulated chemokine. CC
chemokines serve as chemoattractants for monocytes/macrophages,
activated T cells, B cells, eosinophils, basophils, and dendritic cells
(for review, see Ref. 3). Two other chemokine families
have recently been described. Lymphotactin, a chemoattractant for T
cells, lacks two of the four cysteine residues, and is the only member
of the C subfamily (5). Fractalkine (also known as
neurotactin) exists in two forms, either as a membrane-bound or soluble
glycoprotein in which the first two cysteine residues are separated by
three amino acids (CX3C) (6). This
chemokine functions as a chemoattractant for T cells and monocytes and
is highly expressed in brain (6, 7).
Chemokines are expressed locally in response to inflammatory stimuli
and act to recruit leukocytes via their chemoattractant properties and
ability to induce integrin activation. Other important physiological
functions have been ascribed to chemokines, including growth-regulatory
properties, T cell activation, and Th cell polarization (3, 8, 9). In addition, chemokine subfamilies have angiogenic and
angiostatic functions. The ERL+ CXC chemokines
IL-8 and GRO-, , and are angiogenic factors, while the
ERL- CXC chemokines IP-10 and monokine induced
by IFN- have angiostatic activity (for review, see Ref.
4). Aberrant expression of various chemokines has been
implicated in contributing to the pathogenesis of neurologic diseases
such as multiple sclerosis and HIV-1-associated dementia (for review,
see Ref. 10, 11, 12), as well as in animal models of CNS
disease, including experimental allergic encephalomyelitis, mechanical
injury/trauma, ischemia, virus-induced demyelination, and SIV-induced
encephalitis (13, 14, 15, 16). Astrocytes, the major glial cell
type in the CNS, have been shown to be a predominant source of
chemokine production within the diseased brain (17, 18, 19). A
growing literature also indicates that astrocytes are activated in
vitro to produce chemokines such as RANTES, IL-8, IP-10, MCP-1,
MIP-1, and MIP-1 (20, 21, 22, 23). Thus, astrocytes serve as
an important source of chemokines within the CNS.
CXC chemokines bind to seven-transmembrane domain receptors (CXCR1 to CXCR5) that are coupled to heterotrimeric G proteins (for reviews, see Refs. 3 and 24). Signaling by CXC chemokines leads to an increase in intracellular calcium, tyrosine phosphorylation, and activation of mitogen-activated protein kinases (MAPKs) (25, 26, 27, 28). Little is known about the role of CXC chemokines in the CNS, although several CXC chemokine receptors are expressed in the brain; among these is CXCR4 (29, 30, 31, 32, 33). CXCR4 is involved in the directional migration of immune cells in response to its exclusive ligand SDF-1. Furthermore, CXCR4 functions as a coreceptor with CD4 in the binding and fusion of HIV-1, specifically interacting with the viral protein gp120 (34, 35, 36). CXCR4 is expressed in the CNS by neurons, astrocytes, endothelial cells, and microglia (27, 30, 32, 37, 38, 39, 40, 41, 42). CXCR4 is overexpressed in astroglioma tumors, and SDF-1 and CXCR4 expression is colocalized when both are expressed (43, 44, 45). Furthermore, SDF-1 and CXCR4 expression increases with increasing tumor grade (45). Mice with a targeted mutation in CXCR4 or SDF-1 have a defect in migration of cerebellar granule cells, indicating that CXCR4 signaling can mediate migration of neuronal progenitors (46, 47). SDF-1 binding to CXCR4 leads to a number of functional effects on cells of the CNS, including calcium mobilization and microglia migration (37, 38, 39, 40, 41).
In this study, we have examined the expression of CXCR4 by human
astroglioma cells in response to a variety of stimuli, including LPS,
TNF-, IL-1, IFN-, IL-6, IL-4, and IL-10. Our results
demonstrate that TNF- or IL-1 treatment of astroglioma cells
induces CXCR4 expression at both the mRNA and protein level, leading to
increased responsiveness to SDF-1. To assess the functional
importance of CXCR4 expression, astroglioma transfectants were
generated that expressed high levels of CXCR4. Stimulation of these
cells with SDF-1 led to intracellular Ca2+
concentration ([Ca2+]i)
mobilization and selective activation of the extracellular
signal-regulated kinase 2 (ERK2) MAPK pathway. Importantly, SDF-1
treatment also resulted in the induction of MCP-1, IL-8, and IP-10
chemokine gene expression in CXCR4 stable transfectants. CXCR4-mediated
signaling in astroglioma cells provides another pathway for these cells
to express chemokines involved in angiogenesis and inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CRT-J cells, a subclone of the CRT-MG human astroglioma cell line, were maintained in RPMI media with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FBS (HI-FBS), as described previously (48). U87-MG human astroglioma cells were maintained in HAM’s/F-12 DMEM medium with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% HI-FBS, as previously described (23). For passage, monolayers were rinsed with PBS, and then dislodged by trypsinization (0.25% trypsin, 0.02% EDTA).
Reagents
Human recombinant TNF- and IL-1 were purchased from
Genzyme (Cambridge, MA), and human recombinant IL-4, IL-10, and
SDF-1 were purchased from R&D Systems (Minneapolis, MN). Hybrid-IL-6
(H-IL-6) was the generous gift of Dr. S. Rose-John (University of
Mainz, Mainz, Germany). Human rIFN- was the generous gift of Biogen
(Cambridge, MA). LPS was from Sigma (St. Louis, MO). Pc.fusin plasmid
containing human CXCR4 cDNA was generously provided by the National
Institutes of Health AIDS Research and Reference Reagent Program
(Rockville, MD) (49). Mouse anti-human CXCR4 mAb and
anti-human SDF-1-neutralizing Ab were purchased from R&D
Systems. Mouse IgG2a and goat anti-mouse IgG2a-FITC were purchased
from Southern Biotechnology Associates (Birmingham, AL). Rabbit
anti-ERK2, anti-p38 MAPK, and anti-c-Jun N-terminal kinase
(JNK)/stress-activated protein kinase Ab were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The MAP/ERK kinase 1 (MEK1) inhibitor
U0126 was from Promega (Madison, WI). Pertussis toxin (PTX) and cholera
toxin (CTX) were purchased from Sigma. Myelin basic protein (MBP) and
GST c-jun for the in vitro kinase assays were from Sigma and
Stratagene (La Jolla, CA), respectively. HRP-conjugated secondary
anti-rabbit IgG Ab and ECL kits were purchased from
Amersham-Pharmacia Biotech (Little Chalfont, U.K.).
RNA isolation, riboprobes, and RNase protection assay (RPA)
Total cellular RNA was isolated from cell monolayers that were incubated for various time periods with the different cytokines/chemokines. Briefly, cells were lysed directly in the culture dish. RNA was extracted with guanidinium isothiocyanate and phenol, and precipitated with ethanol, as previously described (23).
Linearized human chemokine/chemokine receptor multiprobe sets
(hCK-5, catalog 45035P and hCR-6, catalog 45376P; PharMingen, San
Diego, CA) were in vitro transcribed with T7 RNA polymerase, resulting
in 10 antisense RNA probes. The probes generated from the kits are as
follows: for the hCK5 set, lymphotactin, RANTES, IP-10, MIP-1,
MIP-1, MCP-1, IL-8, I-309, L32, and GAPDH. For the hCR6 set, CXCR1,
CXCR2, CXCR3, CXCR4, BLR-1, BLR-2, V28, L32, and GAPDH.
RPA was conducted with a RPA kit according to the manufacturer’s instructions (PharMingen), as previously described (23). Briefly, 20 µg of total cellular RNA was hybridized with hCK5 riboprobes (3.1 x 105 cpm) or hCR6 riboprobes (2.1 x 105 cpm) and treated with RNase A/T1 (1/200 dilution in 200 µl of RNase digestion buffer) at 30°C for 1 h, then RNA was precipitated and analyzed by 5% denaturing (8 M urea) PAGE. The gels were exposed to x-ray film, and quantification of the protected RNA fragments was performed by scanning with the PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Values for each chemokine/chemokine receptor mRNA were normalized to GAPDH mRNA levels for each experimental condition.
Analysis of CXCR4 receptor expression by immunofluorescence flow cytometry
Human CRT-J and U87-MG astroglioma cells (2 x
105/well) were plated in six-well (35
mm2) plates (Costar, Cambridge, MA) and grown to 
80%
confluency. The cells were incubated with medium alone, TNF-,
IL-1, or a combination of both cytokines for 72 h. Cells were
trypsinized, suspended in PBS containing 1% BSA and 0.05% azide,
blocked with normal rabbit serum (1 µg/ml) for 15 min at room
temperature, and stained with mouse anti-human CXCR4 (1/10
dilution) mAb for 1 h on ice, washed twice, and then were stained
with FITC-conjugated goat anti-mouse Ab (1/50 dilution). After
washing three times, cells were fixed in 1% paraformaldehyde and
analyzed on the FACStar (Becton Dickinson, Mountain View, CA) for CXCR4
expression. Negative controls were incubated with an isotype-matched
(mouse IgG2a) control mAb. Ten thousand cells were analyzed for each
sample.
Stable transfection and FACS analysis
Human CXCR4 cDNA was subcloned into the BamHI sites
of the pcDNA3 expression vector. CRT-J and U87-MG (1 x
107 cells) were transfected by either
lipofectamine or electroporation with pcDNA3 plasmid containing CXCR4
cDNA and the geneticin (G-418)-resistant marker (20 µg). The CXCR4
transfectants were grown in medium containing 0.5 mg/ml G-418 (Life
Technologies, Rockville, MD). Mock cDNA, which has only the pcDNA3
plasmid, was used as a negative control. Cell surface expression of
CXCR4 on the transfectants was confirmed by FACS analysis, as described
above. The CRT-J13 and U87-T37 transfectants showed the best response
to SDF-1, as assessed by an increase in
[Ca2+]i (data not shown),
and were selected from more than 60 clones grown through the limiting
dilution method for further analysis.
Measurements of [Ca2+]i
Parental U87-MG astroglioma cells and U87-T37 stable
transfectants (1 x 106 cells/ml) were
resuspended in HBSS supplemented with 1% FBS and 1.25 mM
CaCl2, and then incubated with 2 µM fura 2-AM
(Molecular Probes, Eugene, OR) for 30 min at 37°C. Subsequently,
excess fura 2-AM was removed by washing the cells twice, and the cells
were resuspended in 2 ml of HBSS containing 1% FBS and 1.25 mM
CaCl2 to a final concentration of 1 x
106 cells/ml.
[Ca2+]i was measured
using a spectrofluorometer (PTI System QM-1) with alternating
excitation at 340 and 380 nm, and fluorescence emission at 510 nm.
After a baseline level of
[Ca2+]i was established,
the cells were treated with various concentrations of SDF-1 or
bradykinin (BK; 2 µg/ml) as a positive control. When a stable peak of
[Ca2+]i in response to
SDF-1 was achieved, the cells were permeabilized with 0.1 mM
digitonin. EGTA was then added to a final concentration of 5 mM. The
digitonin and EGTA measurements were used to calibrate
[Ca2+]i vs fura 2-AM
fluorescence in each sample using a calibration equation originally
described by Grynkiewicz et al. (50).
Measurement of chemokine production
CRT-J13 and U87-T37 cells (3 x
105/well) were incubated with medium alone or
various concentrations of SDF-1 for 24 h, then supernatants
were collected, centrifuged, and stored at -70°C until use. MCP-1
and IL-8 in culture supernatants were quantitated using a dual-Ab
solid-phase ELISA (Biosource International, Camarillo, CA), as
previously described (23). The minimal detection limit for
the MCP-1 ELISA is 20 pg/ml, and for IL-8 is 10 pg/ml.
In vitro kinase assay
Soluble lysates (100–300 µg) were used to phosphorylate MBP
or GST c-jun, as previously described (51).
Lysates were incubated with 1 µg of anti-p38, anti-ERK2, or
anti-JNK/stress-activated protein kinase MAPK Ab overnight at
4°C, followed by an additional 1-h incubation with protein A/G gel
beads (Pierce, Rockford, IL). The immunocomplexes were washed, then
incubated for 20 min at 30°C in 20 µl of kinase buffer containing
20 µM ATP, 5 µCi [-32P]ATP, and 10 µg
MBP as the substrate for ERK and p38, and 2 µg of GST
c-jun for JNK. Substrate phosphorylation was stopped by
boiling in sample buffer, followed by 12% SDS-PAGE and
autoradiography. To assess for changes in the activation of each
protein kinase, levels of phosphorylated proteins were normalized
relative to the total MAPKs detected in each sample. The density of the
bands was quantified by a UMAXScan desktop scanner (Fremont, CA) using
National Institutes of Health Image software.
Statistical analysis
Levels of significance for comparisons between samples were determined using Student’s t test distribution.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-1
We were interested in determining whether CXCR4 receptor
expression on astroglioma cells could be modulated by cytokines known
to be present in the diseased CNS. To initiate this study, two human
astroglioma cell lines, CRT-J and U87-MG, were treated with the
proinflammatory cytokines TNF-, IL-1, and IFN-; the
antiinflammatory cytokines IL-4 and IL-10; the hybrid molecule of the
soluble IL-6R and IL-6 called H-IL-6 (52); or LPS for
10 h. The cells were harvested and CXCR4 mRNA expression was
analyzed by multiprobe RPA. As shown in Fig. 1
A, low constitutive CXCR4
mRNA expression was detected (lane 1), and expression
increased 2-fold upon TNF- or IL-1 treatment
(lanes 3 and 4). However, CXCR4 mRNA
expression was not influenced by LPS or the other cytokines (IFN-,
H-IL-6, IL-4, and IL-10) (lanes 2 and
5–8). The kinetics of TNF--enhanced CXCR4 mRNA
expression were next examined. As shown in Fig. 1, C and
D, CXCR4 mRNA expression is enhanced at 2 h, peaks
8 h after TNF- treatment (5-fold), and remains elevated
12–24 h after exposure to TNF-. Comparable results were seen in
U87-MG cells (data not shown). Astroglioma cells did not constitutively
express mRNA for any of the other CXC receptors (CXCR1, CXCR2, CXCR3),
nor was expression induced upon cytokine or LPS treatment (data not
shown).
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or IL-1 treatment on
astroglioma cells was examined by flow cytometry (Fig. 2). CRT-J and U87-MG cells were treated
with TNF-, IL-1, or a combination of both cytokines for 72
h, then CXCR4 surface expression was analyzed. In unstimulated CRT-J
and U87-MG cells, 9.6% and 23.8%, respectively, were CXCR4 positive.
Upon TNF- treatment, 41.2% of CRT-J and 60.6% of U87-MG cells
became CXCR4 positive. IL-1 stimulation also enhanced CXCR4
expression (28.8% for CRT-J and 41.6% for U87-MG cells). The combined
treatment of CRT-J cells with TNF- plus IL-1 for 72 h had an
additive effect on CXCR4 induction (61.9% positive for CXCR4). In
contrast, in the U87-MG cells, TNF- and IL-1 did not exhibit an
additive effect. CXCR4 protein expression was also detected by
immunofluorescence analysis (data not shown).
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(50 ng/ml) treatment. CRT-J cells were treated
with TNF- for 72 h, then SDF-1 was added. SDF-1 treatment
increased the [Ca2+]i
concentration in TNF--treated CRT-J cells, while no change was
observed upon SDF-1 treatment of unstimulated cells (Fig. 3). These results indicate that cytokine
treatment of astroglioma cells induces CXCR4 expression at both the
mRNA and protein level, leading to responsiveness to SDF-1.
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-induced elevation in [Ca2+]i
We next wished to discern the functional consequences of CXCR4
expression on astrogliomas. However, the use of TNF-/IL-1 to
up-regulate CXCR4 expression complicates this analysis due to the
varied effects of TNF-/IL-1 themselves on astroglioma function.
Therefore, stable transfectants overexpressing CXCR4 were generated.
After transfection of CRT-J and U87-MG parental cells with a CXCR4
expression plasmid, two transfectants, CRT-J13 and U87-T37, were chosen
based on high CXCR4 expression, as determined by flow cytometry (see
Fig. 4). Ca2+
mobilization following ligand stimulation is a characteristic of most
chemokine receptors. The effect of SDF-1 on intracellular
Ca2+ modification was determined in U87-T37 and
CRT-J13 cells. SDF-1 increased the
[Ca2+]i concentration in
U87-T37 cells, whereas no increase was detected in U87-MG parental
cells or U87-mock-transfected cells (Fig. 5A). CRT-J13 cells also showed
a similar pattern as that of U87-T37 cells (data not shown). As a
positive control for Ca2+ influx, cells were
treated with BK. The effect of SDF-1 was dose dependent, with a
clear increase in [Ca2+]i
observed at a SDF-1 concentration of 5 ng/ml, and optimal effects
noted at 50 ng/ml (Fig. 5B). To establish the specificity of
this effect, neutralizing Ab to SDF-1 was used, which abolished the
SDF-1-mediated Ca2+ response (Fig. 5C). Anti-human TGF-1 Ab had no effect in this system
(Fig. 5C), indicating that the increase in
[Ca2+]i was induced by
SDF-1 through CXCR4.
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i proteins, and
SDF-1-induced responses can be inhibited by PTX (27, 28, 37, 53). Thus, we tested whether PTX treatment would inhibit
SDF-1-induced Ca2+ mobilization in U87-T37
cells. As shown in Fig. 5
D, PTX pretreatment blocked
SDF-1-induced Ca2+ mobilization, while CTX had
no effect. These results indicate that Gi
proteins couple to the CXCR4 receptor in astroglioma cells to control
intracellular Ca2+ homeostasis.
SDF-1-mediated activation of the MAPK signaling cascade
We next determined whether SDF-1 stimulation could induce the
activation of ERK1/2, p38, and/or JNK MAPK in the CXCR4 stable
transfectants. CRT-J13 or U87-T37 cells were stimulated with SDF-1
for various periods of time (0–60 min), cells were lysed, and
subjected to immunoprecipitation with anti-ERK2, anti-p38, or
anti-JNK Ab, and then in vitro kinase assays were performed, as
previously described (51). MBP was used as the substrate
for ERK2 and p38, and GST c-jun as the substrate for JNK.
Fig. 6A illustrates that
phosphorylation of MBP by ERK2 kinase was seen after 1 min of
stimulation with SDF-1 (lane 2), reached a maximum
level after 30 min (lane 5), and was sustained for 60
min (lane 7). SDF-1 did not activate p38 or JNK
MAPK. Pretreatment of cells with PTX, but not CTX, inhibited
SDF-1-induced ERK2 activation (Fig. 6B, lanes
3 and 4), indicating that this signaling was mediated
by Gi proteins.
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induction of chemokine expression in astroglioma cells
Recent studies have suggested that MAPKs play an important role in
the signal cascades that lead to the induction of various inflammatory
molecules, including chemokines (54). As we demonstrated
that SDF-1 stimulation of astroglioma cells led to the activation of
ERK2 MAPK, we wished to further determine whether SDF-1 stimulation
could induce chemokine expression in these cells. CRT-J13 cells were
stimulated with SDF-1 for various time periods (0–12 h), then
chemokine mRNA expression was assessed using multiprobe RPA. As shown
in Fig. 7A, mRNA induction of
IP-10 and IL-8, two CXC chemokines, was detected as early as 1 and
2 h (lane 2 for IL-8, and lane 3 for
IP-10); reached a maximum at 4 h (lane 4 for
IL-8) and 8 h (lane 5 for IP-10); and was
sustained until 12 h (lane 6). mRNA encoding
MCP-1, a CC chemokine, was constitutively expressed (lane
1), and upon stimulation with SDF-1, MCP-1 mRNA expression was
enhanced, reaching maximum levels at 8 h (lane
5). Quantification of the data is shown in Fig. 7, B–D. Similar results were observed in U87-T37 cells (data
not shown). The astroglioma cells did not constitutively express mRNA
for the other chemokines that can be detected by this multiprobe RPA
kit (lymphotactin, RANTES, MIP-1, MIP-1), and SDF-1 treatment
did not induce expression of these chemokines (data not shown).
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induction of
chemokine expression
To test the involvement of MAPK activation in SDF-1-induced
chemokine expression, we examined the effect of U0126, an inhibitor of
MEK1/2, a MAPK that phosphorylates ERK1/2 (55). The cells
were pretreated for 20 min with the indicated amounts of U0126 and
stimulated with SDF-1 for an additional 4 h, and chemokine mRNA
expression was analyzed using multiprobe RPA. SDF-1-induced IP-10,
MCP-1, and IL-8 mRNA expression was inhibited by U0126 in a
dose-dependent manner, with inhibition observed using 0.005–5 µM of
U0126 (Fig. 8, lanes 4–8).
Optimal inhibition was detected using 5 µM of U0126
(lane 8). The diluent DMSO had a negligible effect on
chemokine expression (lane 3).
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for 24 h, then
supernatants were analyzed for IL-8 and MCP-1 protein expression by
ELISA. The chemokines were induced at the protein level, and
pretreatment of the cells with U0126 inhibited IL-8 and MCP-1 protein
secretion (Fig. 9). Similar results were
obtained for IP-10 expression (data not shown). These results
collectively indicate that the ERK signaling pathway regulates
SDF-1-induced IP-10, IL-8, and MCP-1 gene expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1 were the only cytokines capable
of up-regulating CXCR4 expression, as shown by RPA and flow cytometry.
It has also been reported that IL-1 can up-regulate CXCR4 expression
on primary human fetal astrocytes (12, 56), indicating
that this finding is relevant for both primary astrocyte cultures as
well as astroglioma cell lines. Cytokine regulation of CXCR4 expression
appears to be cell-type specific; TNF- also up-regulates CXCR4
expression on PBMCs and eosinophils, while for endothelial cells,
TNF- inhibits expression in a time-dependent manner (53, 57, 58, 59). The cytokine IL-4 strongly up-regulates CXCR4
expression on CD4+ T cells, specifically Th2
cells (57, 60), and inhibits expression on eosinophils
(53). Our results indicate that IL-4 has no effect on
CXCR4 expression on astroglioma cells, although these cells express
functional IL-4R (61). Expression of IL-1 and TNF- is
elevated in astroglioma tumors (for review, see Ref. 62).
The ability of TNF- and IL-1 to up-regulate CXCR4 expression on
astroglioma cells (this study) may be important for the growth of these
cells because Sehgal et al. (43) have shown that SDF-1
induces proliferation of astroglioma cells.
Engagement of CXCR4 by its ligand SDF-1 leads to a diverse array of
biological effects, including chemotaxis, adhesion molecule expression,
apoptosis, activation of ion channels, and angiogenesis (37, 45, 63, 64, 65, 66). We wished to discern the functional consequences of
CXCR4 expression on astroglioma cells; thus, stable CXCR4 astroglioma
transfectants were generated. The function of CXCR4 was first confirmed
by SDF-1 stimulation of
[Ca2+]i elevation.
Indeed, [Ca2+]i levels
were elevated in response to SDF-1, and abrogated by the inclusion
of anti-SDF-1 Ab, demonstrating the specificity of the response.
As mentioned previously, a wide array of intracellular signaling events
occurs upon SDF-1/CXCR4 interaction. These include tyrosine
phosphorylation of FAK, Pyk2, and paxillin (26);
activation of phosphatidylinositol 3-kinase (26, 38);
activation of MAPKs (26, 27, 63); NF-
B activation
(26); and activation of the JAK/STAT signaling pathway
(28). Regarding MAPK, we observed selective activation of
ERK2 MAPK by SDF-1 in the astroglioma cells, with no effects on p38
or JNK MAPK. Activation of MAPKs by SDF-1 also occurs in a
cell-type-specific manner; in rat cerebrocortical cultures, SDF-1
activates the p38 MAPK pathway (63).
Given that MAPK activation contributes to the induction of genes
involved in inflammation and angiogenesis, including chemokines, we
next examined whether SDF-1 stimulation led to the induction of
chemokine gene expression in astroglioma cells. Indeed, our results
demonstrated that SDF-1 enhanced expression of three chemokines,
IP-10, IL-8, and MCP-1, in the CXCR4 stable transfectants.
SDF-1-mediated expression of IP-10, IL-8, and MCP-1 was inhibited by
U0126, a very potent and highly selective MEK1/2 inhibitor, indicating
an involvement of the ERK MAPK pathway in this response. It appears
that the signaling pathways ultimately leading to chemokine expression
vary depending on the stimulus used. In this regard, we have recently
demonstrated that HIV-1 Tat is a potent inducer of MCP-1, IL-8, and
IP-10 expression in astrocytes (67). U0126 completely
blocked IL-8 induction by Tat, and partially abrogated MCP-1
expression; however, no inhibitory effect was observed on IP-10
expression. In contrast, the p38 MAPK-specific inhibitor SB202190
suppressed IP-10 induction by Tat (67). Thus, numerous
signaling pathways can be activated, ultimately leading to chemokine
expression.
In this study, we have identified SDF-1 as an important inducer of chemokine expression in astroglioma cells. Regarding potential CNS sources of SDF-1, astrocytes and monocyte-derived macrophages (MDM) constitutively express SDF-1 mRNA transcripts, with higher levels of expression in astrocytes (38). Interestingly, regulation of SDF-1 expression differs in these two cell types; in MDM, SDF-1 expression was inhibited after HIV-1 infection or LPS treatment of the cells, while in astrocytes, SDF-1 expression was enhanced upon LPS treatment or exposure of cells to conditioned media from HIV-1-infected MDM (38, 39). We have also observed enhanced SDF-1 expression in human astroglioma cells upon LPS treatment (data not shown). SDF-1 is detected at low levels in brains from normal controls, is overexpressed in astroglioma tumor tissue, and increases with increasing tumor grade (45). SDF-1 was localized to astroglioma cells, neurons, and phagocytic cells around vessels in the tumors (45). These findings indicate that astroglioma cells can serve as an autocrine source of SDF-1, and other CNS cells (neurons, microglia) also contribute to SDF-1 expression within the brain.
SDF-1/CXCR4 engagement on astroglioma cells leads to diverse biological
effects, ranging from cell proliferation, chemokine induction, and
angiogenesis. There are several possibilities for the participation of
SDF-1/CXCR4 in the angiogenic process. SDF-1 stimulation of human
endothelial cells induces the production of the angiogenic factor
vascuilar endothelial growth factor (68), while in this
study, we have shown that SDF-1 induces IL-8 production by astroglioma
cells. Both of these angiogenic factors are increased in brain tumors
(69, 70). At present, the contribution of IL-8 to CNS
angiogenesis is unclear. IL-8 stimulates angiogenesis in the rat and
rabbit cornea, suggesting the possibility that IL-8 may also
participate in glioma neovascularization (71).
Interestingly, the ERL- CXC chemokine IP-10 has
been reported to have angiostatic effects to angiogenic factors such as
IL-8 on endothelial cells (72). IP-10 is also induced upon
SDF-1 stimulation of astroglioma cells; however, little is known
about the functional relevance of IP-10 in astroglioma tumors. It will
be interesting to determine whether in fact IL-8 and IP-10 have
opposing effects on the angiogenic process.
The other chemokine induced upon SDF-1 stimulation of astroglioma
cells is MCP-1. MCP-1 is a CC chemokine that induces the migration of
monocytes, memory T cells, and NK cells. MCP-1 is expressed by human
astrogliomas in vivo and in vitro (73, 74). Infiltrating
macrophages are a common feature of human gliomas, and the extent of
macrophage infiltration correlates with the level of MCP-1 expression
(74). The functional significance of infiltrating
macrophages is not known; however, given the ability of macrophages to
produce a wide array of tumoricidal mediators, this may be a mechanism
to provoke an immune attack on gliomas.
The importance of CXCR4 in glioma proliferation is controversial. One
group has shown that blockade of CXCR4 (by CXCR4 Abs or antisense
CXCR4) inhibited the proliferation of human glioblastoma tumors
(43, 44), suggesting an important role for CXCR4 in cell
transformation. However, Rempel et al. (45) determined by
immunohistochemical analysis that SDF-1/CXCR4 expression, and that of
the proliferation marker MIB-1, were mutually exclusive. We are
currently conducting studies to assess the ability of SDF-1 to
induce proliferation of the CXCR4 astroglioma transfectants, which
should be informative with respect to the role of CXCR4 in astroglioma
proliferation.
Our results highlight another functional role for SDF-1/CXCR4, that
leading to chemokine expression. In this regard, two recent studies
have documented the ability of SDF-1 to activate chemokine expression;
SDF-1 up-regulated production of the CXC chemokines IL-8 and GRO- by
human colon epithelial cells (75) and MCP-1, IL-8, and
GRO- by human retinal pigment epithelial cells (76).
These findings suggest that SDF-1 activation of chemokine expression
may occur in a cell-type-specific manner. It is clear, then, that
chemokines have broader functional properties than initially
anticipated, and with respect to the CNS, are important for both
inflammatory and angiogenic events within this organ.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jae-Wook Oh, Department of Cell Biology, 308 McCallum Basic Health Sciences Building (MCLM), University of Alabama, 1918 University Boulevard, Birmingham, AL 35294-0005.
3 Abbreviations used in this paper: IP-10, IFN--inducible protein 10; BK, bradykinin; [Ca2+]i, intracellular Ca2+ concentration; CTX, cholera toxin; CXCR, CXC chemokine receptor; ERK, extracellular signal-regulated kinase; GRO, growth-related oncogene; H-IL-6, hybrid-IL-6; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MCP, monocyte chemoattractant protein; MDM, monocyte-derived macrophage; MEK, MAP/ERK kinase; MIP, macrophage-inflammatory protein; PTX, pertussis toxin; RPA, RNase protection assay; SDF, stromal cell-derived factor.
Received for publication August 15, 2000. Accepted for publication December 5, 2000.
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