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*
Max Delbrück Center for Molecular Medicine, Berlin-Buch;
GSF-National Research Center for Environment and Health, Institute of Immunology, Munich;
GSF-National Research Center for Environment and Health, Institute of Molecular Virology, Neuherberg, Oberschleissheim; and
§
Department of Hematology and Oncology, Städtisches Krankenhaus München-Schwabing, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, the natural
ligand for CXCR4, induces down-regulation of up to 60% of
surface-expressed receptors in a pertussis toxin-insensitive manner.
Interestingly, receptor cross-linking caused by incubation of cells
with anti-CXCR4 mAb triggers receptor trafficking, in that the
receptor is rapidly internalized and recycled to the cell surface.
Therefore, receptor internalization and recycling may regulate the
functional interaction of the receptor with envelope proteins during an
initial step of HIV-1 infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and macrophage inflammatory protein-1ß suppress HIV-1
infection, particularly of macrophage-tropic
(M-tropic)2 isolates
(2), indicating that chemokines or chemokine receptors might play a
role in HIV-1 infection. Recently, Feng and co-workers demonstrated
that the chemokine receptor CXCR4 (also termed fusin, LESTR, and
HUMSTR) is used by lymphotropic strains of HIV-1 as a cofactor for
viral entry (3), and others succeeded in identifying the CC chemokine
receptors CCR5 and CCR3 as cofactors necessary for infection with
M-tropic isolates (4, 5, 6, 7, 8). These findings shed light on previous
observations that expression of CD4 in nonhuman cell lines is required
for effective virus binding via envelope glycoproteins (Env) to the
cell surfaces but not sufficient for productive virus infection (9, 10). Although little is known about the interaction of HIV-1 and
chemokine receptors, it has been suggested that the V3 loop of gp120
may participate in this process. Exchange of only a few amino acids of
this loop, which is not engaged in CD4 interaction, alters the ability
of the virus to infect macrophages or transformed T cells (11, 12).
Therefore, available evidence suggests a direct interaction of gp120
with CXCR4 or with CCR5, which may be strong and efficient only in the
precence of CD4 (13, 14, 15). Chemokine receptors belong to the family of G protein-coupled receptors that are characterized by seven transmembrane-spanning domains and are coupled to heterotrimeric GTP binding proteins (reviewed in 16 . Sequence analysis of leukocyte chemoattractant receptors reveals a subgroup of primarily lymphocyte-expressed chemokine receptors, now consisting of BLR1, BLR2/EBI1, and CXCR4 (16). The ligand for BLR1 is unknown, and both stroma-derived factor-1 (SDF-1) and ECL were only recently identified as ligands for CXCR4 (17, 18) and BLR2/EBI1 (19), respectively. Therefore, little is known about the physiologic function of these receptors. Using gene-targeted mice lacking the chemokine receptor BLR1, we recently succeeded in identifying BLR1 as the first chemokine receptor involved in B cell migration and microenvironmental homing of recirculating B cells to the spleen and Peyer’s patches (20). To further characterize the role of this chemokine receptor subfamily in lymphocyte migration and viral pathogenesis, we generated mAbs against CXCR4. We demonstrate that CXCR4 is expressed on various leukocyte subpopulations, including monocytes, but not on T memory cells. Interestingly, the cellular localization of this molecule is not restricted to the surface, as large intracellular stores of the receptor can be identified in some cell types. Upon activation by PMA, these storage vesicles are translocated to the cell surface within 30 s, leading to a twofold increase in the concentration of surface-exposed receptor. Subsequently, all surface CXCR4 is internalized rapidly (2–4 min). The anti-CXCR4 mAb isolated by our group are able to inhibit HIV-1-mediated syncytia formation. Moreover, they were found to induce receptor internalization. While it is still controversial whether HIV uptake into coated vesicles is required for a productive infection, it is interesting that the HIV-1 virion docks to a receptor prone to endocytosis under receptor cross-linking conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA was isolated from human REH cells, and reverse transcriptase-PCR was performed to amplify a fragment coding for the complete cDNA of human CXCR4. A 1/20th volume of the reverse transcriptase reaction served as a template for PCR amplification, which was performed using vent polymerase (Biolabs, Schwalbach, Germany) and 50 pmol of each primer (5' primer, 5'-CAT GCC ATG GAG GGG ATC AGT ATA TAC AC-3'; 3' primer, 5'-CCG GGC CCG CTG GAG TGA AAA CTT GAA GAC-3') under the following conditions: 35 cycles of 94°C for 60 s, 55°C for 60 s, and 72°C for 90 s. After size fractionation, fragments were isolated and cloned into pGem4zMYCc vector, then transferred into the expression vector Rc/CMV, which allows the expression of a myc-tagged-CXCR4 fusion protein in accordance to the procedure described previously for human BLR1 (21, 22). A BstEII/BamHI fragment was cut from pGem4zCXCR4-MYCc containing the first N-terminal 190 bp of the cDNA of CXCR4. The fragment was cloned in the pGex-1 vector (Pharmacia, Piscataway, NJ). Using this p3x-NCXCR4 plasmid coding for a GST fusion protein containing the first 63 amino-terminal amino acids of CXCR4 (GST-NCXCR4), Escherichia coli were transformed and induced with isopropylthiogalactoside according to standard procedures. Cells were harvested and subjected to French press lysis, and the GST fusion protein was purified using glutathione-agarose and affinity chromatography. Lou/C rats were immunized with 100 µg of GST-NCXCR4 in CFA and were boosted once with the same amount of Ag. Rat splenocytes were fused with X63Ag8.653 cells as described previously (23), and 7 days later hybridomas were tested for the production of specific Abs by differential ELISA using GST-NCXCR4 and 10 irrelevant GST fusion proteins as controls, including those for the N-termini of BLR1 and BLR2/CCR7. The specificity of the mAb obtained was further tested using murine 3T3 cells transfected with expression plasmids coding for CXCR4, CXCR1, BLR1, and BLR2/CCR7 in accordance to the procedure described previously for human BLR1 (22). Only mAb binding to GST-NCXCR4- and CXCR4-transfected 3T3 cells, but not those binding to the other GST fusion proteins or to 3T3 cells transfected with the other plasmids, were considered to be specific for CXCR4.
Antibodies
The following Abs were used in this study: anti-CD4-FITC, anti-CD8-PE, anti-CD14-PE, anti-CD19-FITC, and anti-CD62L-PE (all purchased from Becton Dickinson, Mountain View, CA). Anti-CD45RA-FITC, anti-CD45R0-PE, and mouse anti-rat F(ab')2-FITC were purchased from Dianova (Hamburg, Germany). The anti-BLR1 mAb RF8B2 (rat IgG2b) was produced at our laboratory and was characterized previously (24). Cy3 and Cy5 conjugates of Abs were prepared as recommended by the manufacturer (Amersham, Arlington Heights, IL).
Cell preparations
PBL were isolated from venous blood of healthy donors or of HIV+ individuals at various stages of the disease. Mononuclear cells were separated by centrifugation on Ficoll gradients (Biochrom, Berlin, Germany) for 30 min at 1200 x g. The interphase was harvested, and cells were washed twice in staining buffer (PBS, 4% FCS, 5 mM EDTA, and 0.1% NaN3, pH 7.4) at 4°C and kept on ice until used. In some experiments leukocytes were isolated by lysis of erythrocytes using the NH4Cl method. Normal tonsil tissues were obtained from tonsillectomies of children 4 to 14 yr of age. Tonsil tissues were minced and strained through a stainless steel mesh. To remove debris, cells were centrifuged over a Ficoll cushion as described above. Thymic tissue was obtained from children 3 to 5 yr of age undergoing cardiovascular surgery at the Deutsches Herzzentrum (Munich, Germany). Suspensions were made as described for tonsillar lymphocytes, omitting the Ficoll separation step.
Cell culture and Western blotting
B and T cell lines used were cultured in RPMI/10% FCS in a humidified incubator at 37°C 5% CO2. Western blotting was performed as described previously (21).
Immunohistology and flow cytometry
For immunohistochemical analysis of lymphoid tissue, tonsils were embedded in Tissue-Tek (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -80°C. Eight-micron cryostat sections on gelatin-covered slides were dried overnight and fixed with acetone. Slides were rehydrated and preincubated for 1 h in staining buffer (0.1 M Tris, pH 8.0, and 0.1% Tween-20) containing 5% heat-inactivated rat serum. Sections were incubated with conjugated primary Abs in a humidified chamber for 1 h at room temperature. After three washes in staining buffer, binding of biotinylated Abs was revealed with streptavidin-TRITC (tetramethyl rhodamine isothiocyanate) (The Jackson Laboratory, Bar Harbor, ME; 1/150) or streptavidin-Cy5 (Amersham; 1/150). After three final washes the slides were mounted in Moviol (Calbiochem, La Jolla, CA) and analyzed by confocal microscopy (Leica, Heidelberg, Germany). Digitized images were printed on a Sony video printer (UP-D8800; Sony, Berlin, Germany). Flow cytometric analysis of lymphoid cells was performed as described previously (24).
Endocytosis assay
PBL were adjusted to 2 x 106/ml in
RPMI/5% FCS, incubated at 37°C, and then stimulated with PMA (50
ng/ml) and ionomycin (800 ng/ml). After various incubation periods at
37°C, aliquots were added to 10-fold volumes of ice-cold FACS buffer
(PBS, 2% FCS, 10 mM EDTA, and 0.1% NaN3, pH 7.2) and
placed on ice. Cells were stained with saturating amounts of
FITC-labeled anti-CXCR4 and various PE-labeled mAb (CD4, CD8, CD14,
and CD19) at 4°C for 30 min and analyzed by flow cytometry.
Linearized mean fluorescence values of cell populations were calculated
using Winlist 3.0 software (Verity Software House, Inc., Topsham, ME)
and used as a parameter to monitor relative changes in the amount of
cell surface-expressed CXCR4 and CD4 (see below). To quantitate the
uptake of anti-CXCR4 mAb and BLR1 mAb, cells were incubated with
saturating amounts of fluorochrome (FITC or CY3)-conjugated mAb for 30
min on ice. Some aliquots were transferred to 37°C, while others were
kept on ice for various periods of time. Uptake of labeled Ab was
analyzed by flow cytometry or confocal microscopy. To determine the
amount of surface CXCR4 remaining on the cell surface after various
incubation periods with anti-CXCR4, cells were incubated with
unlabeled anti-CXCR4 mAb for 30 min on ice before some samples were
transferred to 37°C. After various incubation periods, aliquots were
removed, washed, and stained with FITC-labeled anti-species
conjugate. In some experiments cells were coincubated with FITC-labeled
transferrin (Sigma Chemical Co., St. Louis, MO) and anti-CXCR4-Cy3
mAb to check for colocalization of transferrin-containing endosomes and
anti-CXCR4-containing endosomes by confocal microscopy. To assess
the influence of pertussis toxin (PTX) on CXCR4 internalization, cells
were preincubated with various amounts of PTX for 2 or 18 h before
FITC-labeled anti-CXCR4 was added. To test whether SDF-1 induces
down-regulation of surface CXCR4, cells were incubated with recombinant
SDF-1 (Peprotech, London, U.K.) at 4 or 37°C. After 1 h,
cells were washed extensively and stained with anti-CXCR4 mAb (30
min on ice) to determine the amount of surface-expressed CXCR4.
Inhibition of HIV-1/RF-induced cytotoxicity by anti-CXCR4 mAb
The c8166 cells (2 x 104) were incubated with 100 µl of serially diluted mAb in RPMI 1640/10% FCS. After 2 h at 37°C, cells were infected by addition of 100 µl of virus stock (HIV-1/RF; multiplicity of infection = 0.05) and subsequently cultivated for 3 to 5 days at 37°C. Virus-induced cytotoxic syncytia formation was quantified by measuring metabolization of MTT dye as described previously (25). Culture medium in hexplicates (SD = <12%) and rat isotype mAb in serial dilutions served as controls. The OD values were compared with the mean OD value of the culture medium controls (virus without mAb), which is given as 100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rats were immunized with a GST fusion protein containing the first
63 amino-terminal amino acids of CXC4 (GST-NCXCR4), and hybridoma
supernatants were tested for the presence of CXCR4-specific mAb by
differential ELISA using either GST-NCXCR4 or various irrelevant GST
fusion proteins (see Materials and Methods). This
procedure enabled us to identify 44 clones binding to GST-NCXCR4 but
not to the control GST fusion proteins (data not shown). The
specificity of the hybridomas obtained was further tested by using
murine 3T3 cells transiently transfected with a myc-tagged
expression plasmid encoding for the entire sequence of CXCR4 or other
chemokine receptors. Using these cells and flow cytometry, three mAb
(1D9, 2B11, and 3D2) were found to bind to CXCR4-MYC transfected 3T3
cells, but not to BLR1-MYC transfected 3T3 cells (Fig. 1
A) or to 3T3 cells
transfected with plasmids coding for the chemokine receptors CXCR1 or
BLR2/CCR7 (data not shown). These Abs were applied in protein
immunoblot analysis and were found to identify a major protein band
with an apparent molecular mass of 45 kDa, which is close to the
predicted molecular mass derived from the amino acid sequence (40 kDa;
see Fig. 1B). All mAb reacted with the same
specificity, and all additional experiments were performed using
anti-CXCR4 2B11.
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Using anti-CXCR4 mAb and three-color flow cytometry we found
CXCR4 to be expressed in a variety of lymphoid cells. On peripheral
blood leukocytes, expression of CXCR4 was observed on all PMN (Fig. 2A), on about 75% of
monocytes (Fig. 2B), and on about 90% of B cells
(Fig. 2C). The expression pattern of CXCR4 on T cells
was analyzed in more detail. Seventy percent of CD4+
lymphocytes and 40% of CD8+ T cells were associated with
this receptor. Using mAb against L-selectin (CD62L) or CD45RA,
CXCR4-positive T cells were primarily identified as naive,
Ag-inexperienced T cells (reviewed in 26 , as 90% of the
CD4+CXCR4+ cells and about 75% of the
CD8+CXCR4+ cells coexpressed L-selectin (Fig. 2, D and E) or CD45RA (data not shown). In
contrast, only 20 to 40% of CD45RO+ T memory cells were
associated with this chemokine receptor (data not shown). As CXCR4 was
expressed strongly on naive T cells, we also looked for CXCR4
expression at earlier stages of T cell development. In total, CXCR4 is
expressed on the majority (>90%) of T cells isolated from juvenile
thymi. Interestingly, a similar expression pattern was observed for
both immature (CD4+CD8+) and mature
(CD4+CD8- or
CD4-CD8+) T cells, and no difference in CXCR4
expression during these stages of T cell development was observed (Fig. 3, A–C). In contrast
to high CXCR4 expression levels on T cells isolated from peripheral
blood or thymus, CXCR4 was barely detectable on T cells isolated from
three (of four) different tonsils (data not shown and compare Fig. 4A, T zone). In one case,
however, tonsillar T cells were found to express CXCR4 at levels
similar to those expressed by peripheral blood T cells (data not
shown). In contrast, almost all CD19+ B cells (>80%)
isolated from tonsils expressed CXCR4 at a high rate. Interestingly,
counterstaining with anti-CD10 or anti-CD38 mAb, which both
stain defined germinal center B cells, marked a subpopulation of
germinal center B cells not expressing CXCR4 (Fig. 3, Dand E).
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A), some cells within the germinal center could be
identified that were not stained with anti-CXCR4 mAb. However,
extrafollicular cells could be identified that were stained brightly
with this mAb. These cells were in close proximity to the tonsillar
epithelium (Fig. 4B, arrowheads) and along tonsillar septae
(Fig. 4B, long arrows). They were classified as B cells
because they coexpress CD19 (data not shown). Further functional and
flow cytometric experiments must be performed to further delineate the
phenotype of these cells, but the data presented here are in accordance
with the localization and phenotype recently described for
subendothelial B cells (27, 28). Surprisingly, in those cells not only
was CXCR4 expressed on the cell surface as expected for members of this
family, but they accumulated in large intracellular compartments (Fig. 4C). Rapid up-regulation and subsequent complete internalization of surface CXCR4 upon activation with phorbol esters
As we had identified preformed cytoplasmic CXCR4 in some cell
types (compare Fig. 4C), we tested whether activation
influences the distribution between pools of cytoplasmic and
surface-expressed CXCR4. PBL were therefore stimulated with PMA and the
calcium ionophore ionomycin. The amount of surface CXCR4 on different
leukocyte subpopulations was determined by flow cytometry using
FITC-labeled anti-CXCR4 mAb and various PE-labeled mAb specific for
leukocyte subpopulations. As shown in Figure 5, activation of PBL with PMA/ionomycin
rapidly releases cytoplasmic CXCR4 to the cell surface. Thirty seconds
poststimulation, the mean fluorescent intensities of the lymphocyte,
monocyte, and neutrophil (not shown) subpopulations within the sample
increased approximately twofold compared with those at the zero time
point. This peak in expression was followed by a steep decline in CXCR4
surface concentration within <10 min. However, receptor
down-regulation occurred with different kinetics on different leukocyte
subpopulations. Four to six minutes after activation, surface CXCR4 was
completely undetectable on the lymphocyte surface. On monocytes,
down-regulation was significantly slower; after the same incubation
period, approximately identical levels of surface CXCR4 could be
detected as at the beginning of the experiment. Here, incubation
periods had to be extended to 15 min until surface CXCR4 was completely
down-regulated on these cells (Fig. 5B). As PMA is
also known to induce down-regulation of CD4, we compared the
down-regulation kinetics of CD4 and surface CXCR4 on peripheral blood
Th cells. In contrast to the pattern observed for CXCR4, no initial
increase in surface-bound CD4 could be observed, and even 20 min
poststimulation, >50% of the surface CD4 was still detectable. To
address the question of whether CXCR4 is internalized or shed from the
cell upon activation, we stained PBL with FITC-labeled anti-CXCR4
mAb. Cells were washed and activated with PMA/ionomycin as described
above. After different incubation periods aliquots were removed and
subjected to flow cytometry. During a 30-min period there was no change
in fluorescence activity, indicating that the receptor/mAb moiety
remains cell associated. This observation was confirmed by confocal
microscopy demonstrating that the receptor/mAb complex is transferred
from the cell surface (Fig. 5C) into intracellular
vesicles following activation of cells with PMA/ionomycin (Fig. 5D).
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induces receptor down-regulation
As receptor down-regulation was observed upon activation with PMA,
we asked whether SDF-1, the natural ligand for CXCR4 would induce
this effect as well. White blood cells (WBC) were therefore incubated
with different concentrations of this chemokine for 1 h at either
4 or 37°C before cells were washed, stained with anti-CXCR4 mAb,
and subjected to flow cytometry. The different forward/side scatter
properties of lymphocytes, monocytes, and neutrophils were used to set
electronic gates on these cell populations, and changes in the relative
amount of surface-expressed CXCR4 were determined using the mean
fluorescence intensity of fluorochrome-labeled anti-CXCR4 mAb. As
demonstrated in Figure 6A, we
did not observe any difference in surface-expressed CXCR4 on any
population regardless of the presence or the absence of SDF1 when
cells were incubated at 4°C (hatched bars in Fig. 6A). This observation indicates that binding of
SDF-1 to CXCR4 does not interfere with binding of mAb 2B11 to the
receptor. However, incubating cells with SDF-1 at 37°C induced
down-regulation of up to 60% of surface-expressed CXCR4 on all WBC
populations (Fig. 6A, filled bars). Interestingly, we
constantly observed a marked increase in CXCR4 on lymphocytes and
monocytes after a 1-h incubation period in RPMI/5% FCS at 37°C but
not at 4°C (compare hatched and closed bars in Fig. 6A at
0 ng/ml SDF-1), indicating that internal stores of the receptor are
translocated to the cell surface under this condition (see also below).
As both the SDF-1-induced increase in free intracellular
Ca2+ and cell migration are sensitive to PTX (29), we
tested whether inhibitory G proteins (Gi) are also
involved in receptor internalization. Interestingly, preincubation of
WBC with 100 ng/ml PTX for 2 h at 37°C could not prevent
ligand-induced internalization of CXCR4 on PMN, lymphocytes, or
monocytes (Fig. 6B, data shown for lymphocytes only).
Together with the observations made by others, these data demonstrate
that PTX-sensitive G proteins are involved in defined cellular
signaling pathways but not in receptor internalization, thus resembling
the situation described by others for the angiotensin I receptor
(30).
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We then asked whether anti-CXCR4 mAb could induce receptor
activation or internalization in a similar fashion. Therefore, various
T and B cell lines were incubated with FITC-labeled anti-CXCR4 mAb
for various periods at 4 or 37°C. Aliquots were removed, washed, and
subjected to flow cytometry. Within the incubation period observed, the
fluorescence intensity of CEM T cells continuously increased (Fig. 7A),
reaching a plateau at approximately 90 min. A similar effect was
observed for other T cell lines when incubated at 37°C (Fig. 7B, closed symbols). In contrast, in none of the cell lines
tested was increased fluorescence intensity observed when cells were
incubated at 4°C (Fig. 7B, open symbols). Neither CEM
cells nor ESIII cells (see below) possessed noticeable amounts of
internal CXCR4 as observed for primary lymphocytes. Thus, the release
of preformed cytoplasmic CXCR4 to the cell surface could not account
for the increased cell-bound fluorescent label. Likewise, de novo
synthesized and mAb-stained receptor did not add to the increased
fluorescence intensity, since the uptake kinetics did not change when
cells were treated with cycloheximide, which is known to prevent de
novo protein synthesis (data not shown). Therefore, we suggest that
incubation with anti-CXCR4 mAb may induce receptor recycling in a
way that internalized receptor becomes redistributed to the cell
surface, initiating a new round of Ab transport inside the cell. To
test this more vigorously, we first asked whether Ab-induced receptor
internalization is characteristic of other chemokine receptors as well.
ESIII Burkitt’s lymphoma cells that express BLR1, BLR2, and CXCR4 were
incubated with specific mAbs. As observed for T cell lines, all
anti-CXCR4 mAb used generated continuously induced receptor
internalization over a period of 2 h before reaching a plateau
indicative of saturation (Fig. 7, C and E, data
shown for clone 2B11). In contrast, neither anti-BLR1 mAb nor
anti-BLR2 mAb induced receptor internalization at all (Fig. 7, D and E, data for BLR2 not shown). To address the
question of whether receptor internalization is a continuous process
independent of Ab-mediated cross-linking, we used FITC-labeled Fab of
anti-CXCR4 mAb in the experiment outlined in Figure 7E
with ESIII cells. Although these Fab bound CXCR4, they did not induce
internalization of CXCR4, suggesting that receptor cross-linking is
required for receptor uptake (data not shown). As mentioned before (see
also Fig. 7E), receptor uptake was saturated 2 h
after the start of incubation with mAb. It is conceivable that previous
Ab incubation cleared all CXCR4 molecules from the cell surface,
preventing ongoing Ab uptake. Therefore, ESIII cells were incubated
with a large excess of unlabeled anti-CXCR4 mAb. Samples were
withdrawn after various incubation periods, and levels of surface-bound
anti-CXCR4 mAb were determined by counterstaining with
FITC-conjugated anti-rat Ab. As demonstrated in Figure 7F, expression levels of sCXC4 did not depend on either the
incubation temperature (4 or 37°C) or the incubation time, indicating
that the number of surface CXCR4 remains constant during the process of
Ab internalization. As incubating cells in hypertonic medium has been
shown to prevent the formation of clathrin-coated pits, we incubated
CEM T cells in medium containing 0.6 M sucrose to determine whether
mAb-mediated receptor internalization and recycling use this pathway.
As shown in Figure 7G, incubating cells in hypertonic medium
completely inhibited receptor internalization, indicating that
clathrin-coated pits are involved in receptor internalization and
recycling. In summary the data presented provide evidence that CXCR4
cycles from the surface to the cell interior and back upon
cross-linking mediated by anti-CXCR4 mAb. To address the question
of whether inhibitory G proteins are involved in receptor
internalization, ESIII cells were incubated with pertussis toxin before
labeled Ab was added. As described above for SDF-1 (compare Fig. 6B), at no concentration tested did PTX exert any
influence on receptor internalization, indicating that this process is
also independent of Gi (Fig. 7H).
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A, most of the Ab bound to
the cell surface, indicating the absence of large quantities of
cytoplasmic CXCR4 in these cells. A similar staining pattern was
observed analyzing unfixed cells when incubated with anti-CXCR4 at
4°C for 1 h (Fig. 8B). As expected from flow
cytometry data, following incubation of cells for the same time at
37°C, large cytoplasmic vesicles stained by the Ab could be observed
(Fig. 8C). Next, ESIII cells were incubated with
Cy3-labeled anti-CXCR4 and FITC-labeled transferrin. Confocal
microscopy (Fig. 8D) demonstrated partially
overlapping endocytotic pathways for both transferrin and the
anti-CXCR4 complex, as some vesicles contained transferrin (green)
or anti-CXCR4 (red) only, whereas others contained both molecules
(yellow).
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As CXCR4 has been identified as a coreceptor for HIV-1, we used
HIV-1/RF to test CXCR4 mAb for their ability to prevent HIV-1
infection. In the presence of anti-CXCR4 mAb, c8166 cells were
infected with HIV-1/RF, and virus-induced syncytia formation was
quantitated indirectly analyzing metabolic activity of the cells. As
demonstrated in Figure 9A,
anti-CXCR4 mAb reduces HIV-1 cytotoxicity in a dose-dependent
manner, demonstrating that this Ab could inhibit virus infection. Since
receptor internalization was observed upon addition of anti-CXCR4
Ab (see Fig. 7), it was possible that inhibition of virus infection was
due to Ab-induced down-regulation of surface CXCR4. However, when
tested, slightly increased levels of sCXCR4 could be detected after
prolonged incubation periods (24 h) with the Ab (data not shown). As
CXCR4 is expressed on various lymphocyte subpopulations, we
investigated whether altered expression patterns of this receptor can
be observed in HIV-1+ individuals. Analyzing PBL from 30
patients at different stages of the disease, we found an overall
significantly reduced percentage of both CD4+ or
CD8+ cells coexpressing this HIV coreceptor (Fig. 9B).
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C). Interestingly, when incubating CEM cells with
gp120-coated latex beads, surface-expressed CXCR4 was primarily found
at those areas of the cell where latex beads had bound (Fig. 9D). As we did not observe binding of gp120 latex
beads to ESIII cells that do not express CD4 but high levels of CXCR4
(data not shown), these observations indicate that CD4 and CXCR4 are
closely associated at the cell surface, and that CD4 and CXCR4 are
cocapped upon binding of latex-bound gp120. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced
receptor internalization observed on lymphocytes, monocytes, and
neutrophils. As both monocytes and naive T cells express similar levels
of sCXCR4, it is unlikely that the number of surface receptors
expressed on monocytes or macrophages is too low to allow infection
with T-tropic strains. Interestingly, mutation analysis of CCR5
demonstrated that different M-tropic strains require different residues
of the first 10 N-terminal amino acids or the first extracellular loop
of the receptor for membrane fusion and infection (38). Although there
are currently no data available, it seems plausible that 1) due to
differential splicing or post-translational modifications, different
forms of CXCR4 are expressed on macrophages and T cells that cannot be
distinguished with any of the three mAb but can be distinguished by
different HIV-1 strains. 2) In addition to CXCR4 (and other chemokine
receptors identified to date), further cofactors are required to
determine cell tropism, i.e., to allow for productive infection.
Secondary lymphatic organs are known as a major source of HIV
replication and virus spread. Various cell populations such as
macrophages, dendritic cells, and T cells have been identified as major
players in this process (39, 40, 41). Interestingly, we found that
expression of CXCR4 on T cells is primarily restricted to naive T
cells, as thymocytes and L-selectin+ or
CD45RA+ peripheral blood T cells were stained with the mAb.
This is of particular interest as on T cells, expression of most of the
known chemokine receptors is restricted to activated or memory
(CD45R0+) cell types (24, 42, 43). CXCR4 is therefore the
first chemokine receptor showing preferential expression on naive T
cells. Another remarkable feature regarding expression of this receptor
is the increase in surface expression following incubation of WBC in
culture medium. After a 1-h incubation period we usually observed a
twofold increase in the amount of surface CXCR4 on lymphocytes and
monocytes (see Fig. 6A). It seems possible that
storage temperature of blood samples before FACS analysis is a crucial
parameter when analyzing the expression pattern of this receptor.
We have shown that the mAb generated can inhibit HIV-1/RF-induced
cytotoxicity in c8166 cells. However, rather high amounts of mAb
(50–80 µg/ml) were needed to completely inhibit infection with this
strain. Recently, others demonstrated that their mAb against CXCR4 is
able to inhibit infection with some strains of HIV-2 at lower
concentration (10 µg/ml), but also reported that inhibition of HIV-1
infection with their Ab depends on the viral isolate and the target
cell used (35), indicating that viruses might contact different
epitopes of CXCR4. Cellular receptors for enveloped viruses are
characteristically removed from the cell surface to prevent
superinfection (44). Our data indicate that receptor down-regulation is
not a mechanism underlying the neutralizing effect of anti-CXCR4
mAb, as even after prolonged incubation periods (<24 h), this Ab does
not induce receptor clearance from the cell surface. It is therefore
more likely that the Ab block receptor epitopes necessary for infection
with individual strains of HIV-1. Although we frequently observed
reduced proportions of CXCR4+ T cells in HIV+
individuals, it seems unlikely that this finding directly reflects
virus-induced down-regulation of the chemokine receptor as both
CD4+ and CD8+ cells were equally affected (see
Fig. 7B).
CXCR4 is a member of the G protein-coupled receptor family that is
characterized by seven hydrophobic transmembrane-spanning domains. Some
members of this family are known to be internalized upon binding with
their natural ligand (45, 46). However, this report describes for the
first time the existence of lymphocytes that contain large
intracellular stores of a preformed chemokine receptor. One of the most
interesting features of this receptor was the rapid translocation upon
activation with PMA (Fig. 5). Similar phenomena have been described for
various molecules, including CD4 and CD40L (47, 48). Preformed CD40L
has been identified in a subpopulation of Th memory cells, and
activation with PMA resulted in a permanent translocation to the cell
surface, reaching the maximum after 15 min (47). In contrast,
activation of resting PBL with PMA immediately transferred preformed
CXCR4 to the cell surface, reaching a maximum within 30 s.
Furthermore, in contrast to the observation made for CD40L, CXCR4 did
not remain at the cell surface, but was completely internalized within
2 to 5 min. Interestingly, receptor internalization upon activation
with PMA has also been observed for CD4, the primary HIV receptor (48).
In contrast to the observation made for CXCR4, we never observed an
increase in surface CD4 upon stimulation, and receptor internalization
occurred considerably slower, as after 20 min >50% of the CD4
molecules were still found at the cell surface (Fig. 5).
Internalization of CXCR4 could also be induced upon incubation with any
of the three mAb specific for the receptor. Under these conditions,
receptor internalization occurred at a constant rate for the first 40
to 60 min and reached saturation after approximately 2 h. After
this incubation period, the amount of mAb found in the cell was about 3
times higher than mAb bound to the cell surface, demonstrating a high
frequency of receptor trafficking. Interestingly, although
internalization was observed at high frequency, the amount of surface
CXCR4 remained constant over that period. This finding together with
the observation that intracellular stores of CXCR4 were not detectable
in ESIII cells indicates that CXCR4 is internalized upon binding by mAb
and is then completely recycled to the cell surface once the Ab is
released to intracellular vesicles. In control experiments we could not
observe an increase in cell-associated Fab derived from parental 2B11
mAb with time. This indicates that cross-linking of CXCR4 by bivalent
mAb is required for uptake, and that unlike the situation observed for
CTLA-4 (49), continuous circulation of unstimulated CXCR4 between
intracellular vesicles and the cell surface is not responsible for Ab
uptake. As observed for the mAb, incubating cells with SDF-1 also
resulted in receptor internalization. In contrast, binding of SDF-1 to
CXCR4 resulted in an internalization of the receptor without
significant concomitant receptor recycling. Therefore, we assume that
CXCR4 enters different pathways for receptor internalization upon
stimulation with either ligand or mAb. It is likely, however, that the
described processes, CXCR4 internalization caused by PMA stimulation,
Ab cross-linking, or binding to SDF-1, trigger different secondary
signaling events. Several lines of evidence would support this idea: 1)
the different time kinetics of receptor internalization (compare
Figs. 5–7 and 2) complete receptor clearance from the cell surface upon PMA
treatment as opposed to unchanged steady state levels of
surface-exposed receptor upon Ab cross-linking and partially
down-regulated expression after SDF-1 binding. However, it remains
to be determined if activation events caused by either mechanism merge
into identical signaling pathways. Our observations that both SDF-1-
and mAb-triggered receptor trafficking are insensitive to PTX
treatment, whereas cell migration toward SDF-1 is sensitive to PTX
(29), would argue against this.
Recent evidence suggests cell surface association between CXCR4 and CD4
upon binding with HIV gp120 (15). The interaction between gp120 and
CXCR4 is apparently weak, as exemplified by the lack of detectable
binding of gp120-coated latex beads to CXCR4 expressing ESIII cells
(see Results). Therefore, in most cases CD4 remains
the key determinant for initial attachment of virus to the cell
surface. It is remarkable that this seven transmembrane receptor whose
affinity toward the virus is low in most cases is involved in viral
entry. However, the CD4 gp120 interaction itself causes conformational
changes in the gp120 molecule (50, 51). This, in turn, may not only
strengthen the binding of gp120 toward CD4, but may also lead to the
presentation of binding epitopes for CXCR4. We showed that CXCR4 is
internalized readily and quickly upon activation by PMA, mAb
cross-linking, and binding to SDF-1. Assuming that both Ab
cross-linking and cross-linking caused by interaction with the virus
trigger similar events, it is tempting to speculate that HIV exploits
the property of this activated coreceptor to promote membrane
rearrangements and connections to the cytoskeleton of the cell. These
represent processes necessary to imitate receptor uptake, but may be
required as well to render a large patch of membrane competent for
fusion (a step in viral entry probably triggered by viral gp41).
Indeed, we found that CXCR4 cocaps with a gp120/CD4 complex (see Fig. 9D). As chemokine receptors have been repeatedly
postulated to activate adhesion molecules during cell migration (52),
the cross-linking of CXCR4 by attached virus may also activate adhesion
molecules on the cell surface via the CXCR4 signaling pathway. This
could lead to intimate and broad contact areas between membranes of the
virus and the cell.
Note added in proof.
During the reviewing process of the manuscript, Bleul et al. and Amara
et al. reported similar results for CXCR4 expression on lymphocyte
subpopulations and for SDF-1-induced CXCR4 internalization (53, 54).
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: M-tropic, macrophage-tropic; SDF-1, stroma-derived factor-1; GST, glutathione S-transferase; PE, phycoerythrin; PTX, pertussis toxin; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; WBC, white blood cells; T-tropic, T cell tropic; CD40L, CD40 ligand.
Received for publication January 21, 1997. Accepted for publication October 22, 1997.
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M. L. Colamussi, P. Secchiero, A. Gonelli, M. Marchisio, G. Zauli, and S. Capitani Stromal derived factor-1{alpha} (SDF-1{alpha}) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95L) pathway J. Leukoc. Biol., February 1, 2001; 69(2): 263 - 270. [Abstract] [Full Text]
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R. D. Chernock, R. P. Cherla, and R. K. Ganju SHP2 and cbl participate in {alpha}-chemokine receptor CXCR4-mediated signaling pathways Blood, February 1, 2001; 97(3): 608 - 615. [Abstract] [Full Text] [PDF]
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 P. Schaerli, K. Willimann, A. B. Lang, M. Lipp, P. Loetscher, and B. Moser Cxc Chemokine Receptor 5 Expression Defines Follicular Homing T Cells with B Cell Helper Function J. Exp. Med., December 4, 2000; 192(11): 1553 - 1562. [Abstract] [Full Text] [PDF]
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T. Nanki, K. Hayashida, H. S. El-Gabalawy, S. Suson, K. Shi, H. J. Girschick, S. Yavuz, and P. E. Lipsky Stromal Cell-Derived Factor-1-CXC Chemokine Receptor 4 Interactions Play a Central Role in CD4+ T Cell Accumulation in Rheumatoid Arthritis Synovium J. Immunol., December 1, 2000; 165(11): 6590 - 6598. [Abstract] [Full Text] [PDF]
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I. J. Crane, C. A. Wallace, S. McKillop-Smith, and J. V. Forrester CXCR4 Receptor Expression on Human Retinal Pigment Epithelial Cells from the Blood-Retina Barrier Leads to Chemokine Secretion and Migration in Response to Stromal Cell-Derived Factor 1{alpha} J. Immunol., October 15, 2000; 165(8): 4372 - 4378. [Abstract] [Full Text] [PDF]
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T.-J. Lin, T. B. Issekutz, and J. S. Marshall Human Mast Cells Transmigrate Through Human Umbilical Vein Endothelial Monolayers and Selectively Produce IL-8 in Response to Stromal Cell-Derived Factor-1{alpha} J. Immunol., July 1, 2000; 165(1): 211 - 220. [Abstract] [Full Text] [PDF]
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H. Nagase, M. Miyamasu, M. Yamaguchi, T. Fujisawa, K. Ohta, K. Yamamoto, Y. Morita, and K. Hirai Expression of CXCR4 in Eosinophils: Functional Analyses and Cytokine-Mediated Regulation J. Immunol., June 1, 2000; 164(11): 5935 - 5943. [Abstract] [Full Text] [PDF]
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T. Nanki and P. E. Lipsky Cutting Edge: Stromal Cell-Derived Factor-1 Is a Costimulator for CD4+ T Cell Activation J. Immunol., May 15, 2000; 164(10): 5010 - 5014. [Abstract] [Full Text] [PDF]
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P. Secchiero, D. Zella, S. Curreli, P. Mirandola, S. Capitani, R. C. Gallo, and G. Zauli Engagement of CD28 Modulates CXC Chemokine Receptor 4 Surface Expression in Both Resting and CD3-Stimulated CD4+ T Cells J. Immunol., April 15, 2000; 164(8): 4018 - 4024. [Abstract] [Full Text] [PDF]
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D. Daelemans, D. Schols, M. Witvrouw, C. Pannecouque, S. Hatse, S. van Dooren, F. Hamy, T. Klimkait, E. de Clercq, and A.-M. VanDamme A Second Target for the Peptoid Tat/Transactivation Response Element Inhibitor CGP64222: Inhibition of Human Immunodeficiency Virus Replication by Blocking CXC-Chemokine Receptor 4-Mediated Virus Entry Mol. Pharmacol., January 1, 2000; 57(1): 116 - 124. [Abstract] [Full Text]
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Y. Sotsios, G. C. Whittaker, J. Westwick, and S. G. Ward The CXC Chemokine Stromal Cell-Derived Factor Activates a Gi-Coupled Phosphoinositide 3-Kinase in T Lymphocytes J. Immunol., December 1, 1999; 163(11): 5954 - 5963. [Abstract] [Full Text] [PDF]
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J. A. Burger, M. Burger, and T. J. Kipps Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells Blood, December 1, 1999; 94(11): 3658 - 3667. [Abstract] [Full Text] [PDF]
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M. Honczarenko, R. S. Douglas, C. Mathias, B. Lee, M. Z. Ratajczak, and L. E. Silberstein SDF-1 Responsiveness Does Not Correlate With CXCR4 Expression Levels of Developing Human Bone Marrow B Cells Blood, November 1, 1999; 94(9): 2990 - 2998. [Abstract] [Full Text] [PDF]
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 C. Abbal, P. Jourdan, T. Hori, J. Bousquet, H. Yssel, and J. Pene TCR-mediated activation of allergen-specific CD45RO+ memory T lymphocytes results in down-regulation of cell-surface CXCR4 expression and a strongly reduced capacity to migrate in response to stromal cell-derived factor-1 Int. Immunol., September 1, 1999; 11(9): 1451 - 1462. [Abstract] [Full Text] [PDF]
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N. Zimmermann, J. J. Conkright, and M. E. Rothenberg CC Chemokine Receptor-3 Undergoes Prolonged Ligand-induced Internalization J. Biol. Chem., April 30, 1999; 274(18): 12611 - 12618. [Abstract] [Full Text] [PDF]
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D. Misse, M. Cerutti, N. Noraz, P. Jourdan, J. Favero, G. Devauchelle, H. Yssel, N. Taylor, and F. Veas A CD4-Independent Interaction of Human Immunodeficiency Virus-1 gp120 With CXCR4 Induces Their Cointernalization, Cell Signaling, and T-Cell Chemotaxis Blood, April 15, 1999; 93(8): 2454 - 2462. [Abstract] [Full Text] [PDF]
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 R. Salcedo, K. Wasserman, H. A. Young, M. C. Grimm, O. M. Z. Howard, M. R. Anver, H. K. Kleinman, W. J. Murphy, and J. J. Oppenheim Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Induce Expression of CXCR4 on Human Endothelial Cells : In Vivo Neovascularization Induced byStromal-Derived Factor-1{alpha} Am. J. Pathol., April 1, 1999; 154(4): 1125 - 1135. [Abstract] [Full Text] [PDF]
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S. W. Cole, B. D. Jamieson, and J. A. Zack cAMP Up-Regulates Cell Surface Expression of Lymphocyte CXCR4: Implications for Chemotaxis and HIV-1 Infection J. Immunol., February 1, 1999; 162(3): 1392 - 1400. [Abstract] [Full Text] [PDF]
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J. W. Peacock and F. R. Jirik TCR Activation Inhibits Chemotaxis Toward Stromal Cell-Derived Factor-1: Evidence for Reciprocal Regulation Between CXCR4 and the TCR J. Immunol., January 1, 1999; 162(1): 215 - 223. [Abstract] [Full Text] [PDF]
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P. Secchiero, D. Zella, O. Barabitskaja, M. S. Reitz, S. Capitani, R. C. Gallo, and G. Zauli Progressive and Persistent Downregulation of Surface CXCR4 in CD4+ T Cells Infected With Human Herpesvirus 7 Blood, December 15, 1998; 92(12): 4521 - 4528. [Abstract] [Full Text] [PDF]
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J. L. Riley, B. L. Levine, N. Craighead, T. Francomano, D. Kim, R. G. Carroll, and C. H. June Naive and Memory CD4 T Cells Differ in Their Susceptibilities to Human Immunodeficiency Virus Type 1 Infection following CD28 Costimulation: Implications for Transmission and Pathogenesis J. Virol., October 1, 1998; 72(10): 8273 - 8280. [Abstract] [Full Text] [PDF]
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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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 Q. Ma, D. Jones, P. R. Borghesani, R. A. Segal, T. Nagasawa, T. Kishimoto, R. T. Bronson, and T. A. Springer Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice PNAS, August 4, 1998; 95(16): 9448 - 9453. [Abstract] [Full Text] [PDF]
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 N Signoret, M. Rosenkilde, P. Klasse, T. Schwartz, M. Malim, J. Hoxie, and M Marsh Differential regulation of CXCR4 and CCR5 endocytosis J. Cell Sci., January 9, 1998; 111(18): 2819 - 2830. [Abstract] [PDF]
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P. Loetscher, A. Pellegrino, J.-H. Gong, I. Mattioli, M. Loetscher, G. Bardi, M. Baggiolini, and I. Clark-Lewis The Ligands of CXC Chemokine Receptor 3, I-TAC, Mig, and IP10, Are Natural Antagonists for CCR3 J. Biol. Chem., January 26, 2001; 276(5): 2986 - 2991. [Abstract] [Full Text] [PDF]
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