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Departments of Immunology and Medicinal Chemistry, Berlex BioSciences, Richmond, CA 94804;
Laboratory of Immunology, National Institute on Aging, Baltimore, MD 21224;
Department of Surgery, Duke University Medical Center, Center for AIDS Research, Durham, NC 27710; and Departments of
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Medicine and
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Medicine and Pathology and the Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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and SDF-1ß with high
affinity. The CXCR4 Ab 12G5 inhibited both SDF-1 binding and
HIV-1LAI-mediated fusion of CEM. Scatchard analysis
revealed the presence of approximately 150,000 SDF-1-binding sites
per cell with a Kd between 5 and 10 nM.
Cross-competition experiments using unlabeled SDF-1 and SDF-1ß
revealed that both chemokines are equally capable of displacing their
radiolabeled counterparts. Internalization studies with
[125]I-SDF-1 revealed that Jurkat cells internalized greater than
90% of the ligand by 2 h at 37°C. SDF-1 was also chemotactic
for Jurkat cells and caused an increase in the rate of extracellular
acidification that was half-maximal at 18 nM SDF-1 and could be
inhibited by pretreatment with the SDF-1 proteins, pertussis toxin, or
the Ab 12G5. Finally, SDF-1 also caused an increase in the cytosolic
Ca2+ concentration in Sup-T1 cells that was abolished by
preincubating the cells with pertussis toxin or PMA and inhibited by
the Ab 12G5. This molecular characterization of CXCR4 receptors should
prove useful in clarifying receptor interaction with SDF-1 proteins and
with HIV-1 glycoprotein, with the ultimate aim of targeting the viral
interaction for therapeutic intervention. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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In common with other chemoattractant receptors, CXCR4 belongs to a family of seven transmembrane spanning proteins, the vast majority of which are receptors that couple to, and signal via, heterotrimeric guanine nucleotide-binding proteins (G-proteins) (11). Although many G-protein-coupled receptors have been identified and their biology is well understood, relatively little is known about the chemokine HIV-1 coreceptor, CXCR4. Until very recently CXCR4 was an orphan receptor, and only in the last 9 mo has stromal-derived factor (SDF-1)3 been identified as its natural ligand (12). SDF-1 binds to CXCR4 and stimulates cellular migration and actin polymerization in a dose-dependent manner (13, 14). SDF-1 has also been shown to block the invasion of T cell line-tropic strains of HIV-1, which utilize CXCR4 as a coreceptor (12, 15), but not M-tropic HIV-1 strains, which utilize CCR5 as coreceptors for invasion (9, 10). In the early stages of viral infection, CCR5 appears to be the major coreceptor for viral fusion; however, recent studies by Connor et al. (16) show that primary viral isolates obtained from patients later in infection switch to utilize CXCR4 as a major fusion partner in vivo and underscore the importance of understanding the molecular dynamics of CXCR4 interactions with their chemokine and viral counterparts.
To fully understand the role of CXCR4 as well as other chemokine
coreceptors in the pathogenesis of HIV-1 infection, these receptors
need to be fully characterized at the biochemical, kinetic, and
molecular levels. In this study we have begun to delineate the
molecular properties of the CXCR4 receptor. We have identified
CXCR4-binding sites on several human T cell lines and shown that they
bind with high affinity to the ligands SDF-1 and SDF-1ß.
Furthermore, when added to T cells expressing CXCR4, both SDF-1 ligands
proved equally capable of mediating biologic responses via a
G-protein-dependent mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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125I-SDF-1 and [125]I-SDF-1ß (sp. act. 2200 Ci/mmol) were
obtained from New England Nuclear, Boston, MA. Unlabeled chemokines
were from Peprotech, Rocky Hill, NJ. Reagents for electrophoresis were
from Novex (San Diego, CA) and FMC Bioproducts (Rockland, ME). All
other reagent grade chemicals were from Sigma, St. Louis, MO. The CXCR4
mAb 12G5 was as described previously (17). Ab 80709, a murine IgG2a Ab
directed against the bunyavirus G1 glycoprotein, was a gift from Dr.
Francisco Gonzalez-Scarano (University of Pennsylvania). Both Abs were
purified from ascites before use.
Cell culture
The human T lymphocyte cell lines Jurkat, HUT78, SupT1, and CEM were obtained from the American Type Culture Collection, Rockville, MD, and were maintained in RPMI 1640 medium containing 10% FBS and 50 µg per ml of penicillin and streptomycin. For binding assays, the cells were harvested and washed twice with PBS. Cell viability was assessed by trypan blue exclusion, and cell number was determined by counting the cells in a Coulter Electronics Cell Counter, Hialeah, FL.
Chemokine-binding studies
For binding assays, cells (5 x 105 cells per ml) were incubated in PBS with 125I-labeled ligands (0.2 nM) and varying concentrations of unlabeled ligands at 4°C for 1 h. The incubation was terminated by removing aliquots from the cell suspension and separating cells from buffer by centrifugation through a silicone/paraffin oil mixture as described previously (18). Nonspecific binding was determined in the presence of 1 µM unlabeled ligand. The binding data were curve fit with the computer program IGOR (Wavemetrics, Lake Oswego, OR) to determine the affinity (Kd), number of sites, and nonspecific binding.
Receptor internalization
The distribution of 125I-labeled SDF-1 between the cell surface and the cell interior was determined by measuring the total amount of specific SDF-1 binding and then subtracting from this the amount of labeled ligand associated with the cell interior to yield an estimate of surface-bound ligand. Cell-associated radioactivity was determined by acid extraction of cells, which removed cell surface-bound material (19).
Measurement of extracellular acidification with the microphysiometer
Jurkat cells were washed once with a low-buffering media (1 mM
of sodium phosphate, supplemented with 1 mg/ml of BSA) and resuspended
in the same media at a density of 6 to 12 x 106
cells/0.1 ml. A cell suspension of 150 µl was mixed with 50 µl of
agarose cell entrapment medium (Molecular Devices, Palo Alto, CA), and
7 µl of the cell-agarose mixture was spotted into the center of a
disposable polycarbonate cell capsule (Molecular Devices) (2, 20). The
cell numbers in each well were about 300 to 600,000. To measure the
rate of acidification, the assembled cell capsules with the
agarose-entrapped cells were loaded into the chambers of the
microphysiometer (Molecular Devices). The chambers were perfused with
the low-buffering media at a rate of 100 µl/min. For each cycle of
operation, the cells were perfused with the media for 80 s, and
then the flow was interrupted for 40 s, during which the rate of
acidification of the media was measured and recorded. The flow was
resumed and the next cycle begun. The chamber temperature was 30°C.
After stabilization, the cells were perfused with the indicated
concentrations of SDF-1 or -ß for about 16 min, during which time
the rate of acidification was measured. For the pertussis toxin
studies, the cells were perfused with 5 nM of pertussis toxin for 90
min before perfusing with SDF-1.
Chemotaxis
Jurkat cell migration was examined using a 48-well microchemotaxis assay as previously described (21). Briefly, various concentrations of chemokine were placed in the lower wells of a 48-well microchemotaxis chamber. Jurkat cells (2 to 5 x 106 cells/ml) were then placed in the upper compartment of the chamber. The upper and lower wells of the chamber were separated by a 5-µm polycarbonate filter coated with laminin (Sigma Chemical Co.), which seems to be optimal for human T cell migration in vitro. The chambers were incubated for 4 h at 37°C (a time period over which chemokine equilibrium between the upper and lower chambers is optimally achieved) after which the filters were scraped, washed, fixed with methanol, and stained with Diff-Quik (Sigma, St. Louis, MO). Cell migration was measured by counting the number of cells attached to the lower surface of the filter in three high-power fields, and each concentration of chemokine was tested in either triplicate or sets of six wells. The results were expressed as the number of migrating cells per three high-power fields (±SEM).
HIV-1-mediated cell-cell fusion assay
Fusion assays were performed as previously described (22) except CEM cells were used as the uninfected fusion partner. Briefly, uninfected CEM cells (7 x 104) were incubated with CEM cells (1 x 104) chronically infected with HIV-1LAI in 96-well half-area flat-bottom plates (Costar, Cambridge, MA) in a final volume of 100 µl of culture medium. Abs and SDF-1 preparations at various concentrations were added in 10 µl of culture medium at initial setup and were incubated with the cell mixtures at 37°C for 24 h. Multinucleated syncytia were enumerated by microscopic examination of the entire contents of each well.
Cytosolic Ca2+ measurements in Sup-T1 cells
SupT1 cells were loaded with 5 µM of fura-2 AM (Molecular Probes, Eugene, OR) for 1 h at 37°C in RPMI 1640 (Life Technologies, Grand Island, NY) containing 10% serum, penicillin, and streptomycin. Cells were pelleted, washed with serum-free RPMI 1640 lacking phenol red, and resuspended in the same medium at a density of 2 x 106 cells/ml. In all, 1.5 ml of this suspension or 3 x 106 cells were used for each experiment. Changes in the cytosolic free Ca2+ concentration were measured with an SLM/Aminco model AB2 fluorescence spectrophotometer. Where indicated, cells were incubated overnight with 200 ng/ml of pertussis toxin (Sigma). PMA (Sigma) was dissolved in ethanol to a stock concentration of 50 µM.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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is the natural
ligand for the C-X-C chemokine receptor CXCR4. Although SDF-1 has been
shown to stimulate T cell migration and actin polymerization in a
dose-dependent manner (13) and can block the invasion of T cell
line-tropic strains of HIV-1, which utilize CXCR4 as a coreceptor (12, 15), very little information is available regarding the binding of this
ligand or of the related chemokine SDF-1ß to its receptor. Here we
have sought to determine the expression of CXCR4 in a number of human T
cell lines and to further characterize the interaction of the receptor
with its ligand(s).
The two forms of SDF-1, and ß, differ in primary sequence at the
carboxyl-terminal end. SDF-1ß has a four-amino acid extension, RFKM,
compared with SDF-1 (23). In addition, human SDF-1 is almost
identical to the murine homologue, differing in only one amino acid
residue (24). This strong conservation of the primary structure of
SDF-1 is uncommon for a chemokine and suggests that the SDF-1 family of
proteins has important physiologic functions. Initial binding
experiments with Jurkat cells using radiolabeled SDF-1 and -ß
revealed the presence of specific binding sites that were equally
displaced by the addition of either unlabeled SDF-1, suggesting the
presence of a common receptor (Fig. 1
).
Furthermore, the binding of both ligands was inhibited by the addition
of the anti-CXCR4 Ab, 12G5 (Fig. 1), supporting the finding that
SDF-1 proteins are binding to CXCR4 receptors.
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and -ß binding (Fig. 2). The
competition curves were almost superimposable for both of these ligands
and, as indicated in the insets to Figure 2, Scatchard analysis of the
competition curves yielded linear plots with almost identical
Kds. The plots are linear, consistent with a
single class of binding sites with a Kd of 7.5
nM ± 2.6 and 163,521 ± 35,875 binding sites per cell for
SDF-1 and a Kd of 13.7 nM ± 4.2 and
224,841 ± 42,361 binding sites per cell for SDF-1ß. The only
previously reported binding studies with radiolabeled SDF-1 was to
human hNT neurons, and the affinity for SDF-1 was reported to be 54
nM (14).
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binding sites in both cell lines with a
Kd of 7.1 nM ± 3.5 and 120,968 ±
38,946 binding sites per cell for CEM cells and a
Kd of 18 nM ± 5.1 and 101,733 ±
28,209 binding sites per cell for HUT78 cells. The detection of
high-affinity CXCR4 receptors on CEM cells is consistent with the
observed susceptibility of this cell line to infection by T cell-tropic
strains of HIV-1 (25), which use CXCR4 as a coreceptor for fusion (6).
That CXCR4 functions as a coreceptor on CEM cells is indicated by our
finding that both SDF-1 and SDF-1ß inhibit
HIV-1LAI-mediated cell-cell fusion of CEM cells equally
well with an apparent IC50 of 125 nM (S.F. Lacey, C.B.
McDanal, R. Horuk, and M. Greenberg, unpublished observations). We have
also have been able to demonstrate CXCR4 receptor expression in CEM
cells by flow cytometry using the CXCR4-specific Ab, 12G5 (data not
shown). Similar studies by two separate groups have demonstrated cell
surface expression of CXCR4 receptors on Jurkat cells (26, 27).
Interestingly, the 12G5 Ab was able to inhibit
HIV-1LAI-mediated cell-cell fusion of CEM cells with an
apparent IC50 of 39 nM (Fig. 3). McKnight et al. (26) have
demonstrated that the ability of the 12G5 Ab to inhibit HIV-1-mediated
cell-cell fusion varies as a function of both the cell type and the
strain of HIV-1 employed, hypothesizing that either the processing or
presentation of CXCR4 may differ among cell types. Although 12G5 failed
to inhibit HIV-1LAI on several CD4+
CXCR4+ T cell lines (26), our results indicate that
HIV-1LAI-mediated fusion of some cell lines may be
susceptible to inhibition by this Ab. It is possible that the way in
which the CXCR4 receptor is displayed on the cell surface of different
cell types could alter its interaction with the 12G5 Ab (its lipid
environment could be very different depending on the cell in which it
is expressed), and this may have consequences for other CXCR4-dependent
processes, such as HIV-1 infectivity. In addition to inhibiting
HIV-1-mediated fusion of CEM cells, the 12G5 Ab inhibited the binding
of the CXCR4 ligand SDF-1 to these cell with an IC50 of
5 nM (data not shown).
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and measured CXCR4
internalization. At any given time, the amount of [125]I-SDF bound is
the sum of the amount bound to receptors on the cell surface and the
amount included in internalized receptor:ligand complexes. To determine
what proportion of cell-associated radioactivity is intracellular and
what portion is bound to cell surface receptors at 37°C, Jurkat cells
were incubated with radioligand at 4°C and 37°C and subjected to
the acid stripping procedure described in the experimental procedures.
With this approach, non acid-extractable material is taken as a measure
of internalized radioactivity and extractable material is considered to
be surface-bound [125]I-SDF-1.
At 37°C, the rate of internalization of SDF-1 by CXCR4 was rapid with
almost 90% of the radiolabeled SDF-1 being internalized by 2 h
(Fig. 4). The ability of the acid wash to
efficiently remove cell surface counts was assessed in control
experiments, since it is known that some receptors are resistant to
this procedure (28). Acid wash experiments with Jurkat cells at 4°C,
a temperature at which radioligand binding is almost exclusively on the
cell surface, reduced total specific binding by about 95%,
demonstrating the efficiency of this procedure.
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The biologic activity of CXCR4 receptors on Jurkat cells was initially
assessed by stimulating the cells with SDF-1 and measuring their
increase in extracellular acidification rate. As shown in Figure 5A, SDF-1 induced a rapid
increase in the extracellular acidification rate, reaching a maximum
after about 4 min and returning to close to baseline levels within 10
to 16 min. These kinetics are quite similar to those reported for
chemokines in human monocytes and in transfected cell lines (29, 30).
Pretreatment of Jurkat cells with pertussis toxin totally inhibited the
ability of SDF-1 to induce changes in the metabolic activity of the
cells (Fig. 5A), demonstrating that the CXCR4
receptor in Jurkat cells is coupled to G-proteins of the Gi
class, which are known to be sensitive to pertussis toxin
(31).
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abolished the responsiveness of the cells to any further
stimulation with the same chemokine (Fig. 5
B). In
addition, following an initial response to 100 nM SDF-1ß no further
stimulation of Jurkat cells with 100 nM SDF-1 was observed (Fig. 5C). These cross-desensitization experiments further
confirmed that both of the SDF-1 proteins were able to signal through
the CXCR4 receptor in Jurkat cells. Finally, preincubation of
Jurkat cells with Abs to CXCR4 blocked their ability to signal in
response to the SDF-1 proteins in the microphysiometer (data not
shown). The same Abs have recently been shown to partially block the
ability of SDF-1 to increase intracellular calcium and induce
chemotaxis in human PBMCs (27).
Receptor-binding experiments established that the relative affinity of
the SDF-1 proteins for the CXCR4 receptor in Jurkat cells ranged from 7
to 14 nM (Fig. 2). To determine whether the potency of the SDF-1
proteins was in line with their binding affinities, we measured the
dose-response relationship of SDF-1 for CXCR4 by microphysiometry.
As shown in Figure 5D, increasing concentrations of SDF-1
were able to induce a dose-dependent increase in biologic activity in
Jurkat cells. The maximal effect was observed at ligand concentrations
of 100 nM while the half-maximal response, EC50, occurred
at a ligand concentration of 18 nM. This EC50 for SDF-1
is in line with its Kd for receptor binding
measured above and also with those reported previously (27).
Chemokines were originally defined and classified as potent leukocyte
chemoattractants mediating their effects through G-protein-coupled
receptors (36). Thus, another measure of the biologic activity of
SDF-1 on CXCR4 receptors on Jurkat cells would be to determine their
ability to induce the migration of Jurkat cells toward a directed
gradient of chemokine. Table I shows that
the C-X-C chemokines SDF-1, and to a lesser extent IL-8, but not
platelet factor 4 were able to induce the migration of Jurkat cells
toward the chemokine gradient. Furthermore, the activation of
Jurkat cells with anti-CD3 Abs greatly increases this directed
migration of cells. The effect of SDF-1 appears to be close to
maximal at a ligand concentration of 100 nM, mirroring the effect
observed by microphysiometry (Fig. 5D).
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, we
measured the ability of SDF-1 to elicit a transient rise in
intracellular Ca2+. To do this, we measured the change in
intracellular Ca2+ concentration in response to different
concentrations of SDF-1 by fluorometry using the indicator fura-2.
These studies were performed in SupT1 cells, which were demonstrated by
flow cytometry with Ab 12G5 to express CXCR4 (data not shown). As shown
in Figure 6, panel
A, 5 µg/ml of SDF-1 produced a transient rise in
intracellular Ca2+ of approximately 100 nM. This increase
was concentration-dependent across the range from 0.1 to 5 µg/ml and
was no greater at 20 µg/ml than at 5 µg/ml. Preincubating the SupT1
cells briefly with Ab 12G5 inhibited the response to SDF-1 by
approximately 67% (Fig. 6, panels E,
F, and G). An isotype-matched control Ab
directed against bunyavirus glycoprotein G1 had no effect (Fig. 6, panel F). This partial inhibition of
SDF-1-mediated responses agrees with the partial inhibition of
SDF-mediated chemotaxis observed in PBMCs and SDF-1-mediated
Ca2+ flux observed in Chinese hamster ovary cells stably
expressing CXCR4 (27).
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(Fig. 6, panel
B), suggesting that this response, like
acidification, is mediated by a pertussis toxin-sensitive member of the
Gi family of G proteins. Decreasing the extracellular
Ca2+ with EGTA partially inhibited the response to
SDF-1, showing that the rise in intracellular Ca2+ is
due primarily to the release of Ca2+ from intracellular
stores (data not shown). Preincubating the cells with the phorbol
ester, PMA, also blocked the rise in cytosolic Ca2+
triggered by SDF-1 (Fig. 6, panels C and
D). Although PMA, like SDF-1, has been shown to cause
internalization of CXCR44
(37), the rapidity with which exposure to PMA abolished the response to
SDF-1 in this case suggests that it is likely to cause receptor
desensitization as well.
In conclusion, we demonstrate here that a number of human T cell lines
express significant numbers of CXCR4-binding sites. Furthermore, we
show for the first time that these receptors bind both SDF-1 and
SDF-1ß with high affinity and that ligand binding is accompanied by a
rapid down-regulation of cell surface receptors. In addition, we show
that both ligands are able to mediate a biologic response via CXCR4
measured by microphysiometry. This activity can be blocked by
pretreatment with pertussis toxin, suggesting that SDF-1 signaling is
mediated through a G-protein-dependent receptor. Repeated exposure to
ligand also desensitizes the CXCR4 receptors to any further biologic
response. Finally, SDF-1 is also able to induce the directed cell
migration of Jurkat cells.
These studies are the first to extensively characterize the CXCR4 receptor. Since this receptor functions not only to mediate the biologic effects of SDF-1, but also serves as an HIV-1 coreceptor involved in promoting T cell viral fusion, it is important that we characterize its molecular properties as completely as possible. These studies should pave the way toward providing a more complete understanding of CXCR4 receptor/chemokine interactions and may ultimately be useful in elucidating the mechanism of viral gp120 binding to the CXCR4 receptor. With this information it may be possible to design specific antagonists of CXCR4 that are targeted to inhibit HIV infection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard Horuk, Berlex BioSciences, Department of Immunology, 15049 San Pablo Ave, Richmond, CA 94804. E-mail address:
3 Abbreviations used in this paper: SDF-1, stromal-derived factor.
4 N. Signoret, et al., Submitted for publication.
Received for publication July 11, 1997. Accepted for publication October 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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K. S.C. Weber, L. B. Klickstein, and C. Weber Specific Activation of Leukocyte beta 2 Integrins Lymphocyte Function-associated Antigen-1 and Mac-1 by Chemokines Mediated by Distinct Pathways via the alpha Subunit Cytoplasmic Domains Mol. Biol. Cell, April 1, 1999; 10(4): 861 - 873. [Abstract] [Full Text]
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R. K. Ganju, S. A. Brubaker, J. Meyer, P. Dutt, Y. Yang, S. Qin, W. Newman, and J. E. Groopman The alpha -Chemokine, Stromal Cell-derived Factor-1alpha , Binds to the Transmembrane G-protein-coupled CXCR-4 Receptor and Activates Multiple Signal Transduction Pathways J. Biol. Chem., September 4, 1998; 273(36): 23169 - 23175. [Abstract] [Full Text] [PDF]
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D. J. Chabot and C. C. Broder Substitutions in a Homologous Region of Extracellular Loop 2 of CXCR4 and CCR5 Alter Coreceptor Activities for HIV-1 Membrane Fusion and Virus Entry J. Biol. Chem., July 28, 2000; 275(31): 23774 - 23782. [Abstract] [Full Text] [PDF]
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J. Hirose, H. Kawashima, O. Yoshie, K. Tashiro, and M. Miyasaka Versican Interacts with Chemokines and Modulates Cellular Responses J. Biol. Chem., February 9, 2001; 276(7): 5228 - 5234. [Abstract] [Full Text] [PDF]
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L. L. Chen, A. Whitty, D. Scott, W.-C. Lee, M. Cornebise, S. P. Adams, R. C. Petter, R. R. Lobb, and R. B. Pepinsky Evidence That Ligand and Metal Ion Binding to Integrin alpha 4beta 1 Are Regulated through a Coupled Equilibrium J. Biol. Chem., September 21, 2001; 276(39): 36520 - 36529. [Abstract] [Full Text] [PDF]
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