Previous Article | Next Article 
Journal of Virology, December 2001, p. 11534-11543, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11534-11543.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Unique Monoclonal Antibody Recognizing the Third Extracellular
Loop of CXCR4 Induces Lymphocyte Agglutination and Enhances Human
Immunodeficiency Virus Type 1-Mediated Syncytium Formation and
Productive Infection
Reiko
Tanaka,1
Atsushi
Yoshida,1
Tsutomu
Murakami,1
Eishi
Baba,1
Julliane
Lichtenfeld,1
Takeru
Omori,1
Tohru
Kimura,2
Naomi
Tsurutani,3
Nobutaka
Fujii,4
Zi-Xuan
Wang,5
Stephen C.
Peiper,5
Naoki
Yamamoto,3 and
Yuetsu
Tanaka1,*
Department of Infectious Disease and Immunology,
Okinawa-Asia Research Center of Medical Science, Faculty of Medicine,
University of the Ryukyus, Okinawa,1
Department of Molecular Cell Biology, Research Institute for
Microbial Diseases, Osaka University,
Osaka,2 Department of Molecular
Virology, Bio-Response, Graduate School, Tokyo Medical and
Dental University, Tokyo,3 and
Graduate School of Pharmaceutical Science, Kyoto University,
Kyoto,4 Japan, and Henry Vogt Cancer
Research Institute, University of Louisville, Louisville,
Kentucky5
Received 8 May 2001/Accepted 28 August 2001
 |
ABSTRACT |
To increase insight into the structural basis of CXCR4 utilization
in human immunodeficiency virus type 1 (HIV-1) infection, a new
generation of three monoclonal antibodies (MAbs) was developed in WKA
rats. The A80 MAb, which binds an epitope in the third extracellular
loop (ECL3) of CXCR4, has unique biologic properties that provide novel
insights into CXCR4 function. This agent enhanced syncytium formation
in activated human peripheral blood mononuclear cells (PBMC) infected
with X4 or R5 and CEM cells infected with X4 HIV-1 strains. Exposure to
A80 increased the productive infection of activated CD4+ T
cells and CEM cells with R5 and X4 viruses, respectively. This antibody
uniquely induced agglutination of PBMC and CEM cells but did not
activate calcium mobilization. Agglutination induced by A80 was
inhibited by stromal cell-derived factor 1, T22, and phorbol
12-myristate 13-acetate but was not significantly altered by
pretreatment of cells with pertussis toxin, wortmannin, or MAbs to
LFA-1, ICAM-1, ICAM-2, and ICAM-3. The binding of the A145 and A120
MAbs was mapped to the N-terminal extracellular domain and a
conformational epitope involving ECL1 and ECL2, respectively. Both of
these MAbs inhibited HIV-1 infection and lacked the novel properties of
A80. These results suggest a new role for CXCR4 in homologous
lymphocyte adhesion that is ligand independent and in HIV-1 infection.
| |
INTRODUCTION |
Human immunodeficiency virus type 1 (HIV-1) infects target cells through sequential binding of the gp120
subunit of envelope glycoprotein with cellular receptors. Binding to
the primary receptor, CD4 (26, 47, 50, 51), induces a
gp120 conformation that is permissive for interaction with a
coreceptor, which is required for envelope-mediated fusion (3, 7,
21, 28, 30, 32, 35). CCR5 is the front line coreceptor for
commonly transmitted forms of HIV-1 and CXCR4 serves this role for
T-cell-tropic (T-tropic) strains that evolve late in the course of
infection (22, 24, 28, 29, 60, 70). CCR5 and CXCR4 belong
to the chemokine receptor family, which transmit signals through
heterotrimeric G proteins (3, 8, 7, 35). T-tropic HIV-1,
designated X4 strains based on the functional relationship with CXCR4,
has been suggested to be more virulent than R5 or
macrophage-tropic strains (7, 9, 23), possibly due
to the wider spectrum of target cells that express CXCR4
(13).
The exclusive ligand of CXCR4 is stromal cell-derived factor 1 (SDF-1),
a member of the family of chemo-attractant cytokines (54,
56). This chemokine has been demonstrated to play a critical role during embryologic development in the homing of hepatic
hematopoietic precursors to bone marrow, the arborization of small
blood vessels, the formation of the cerebellum, and B-cell
lymphopoiesis (54, 71). SDF-1 regulates homing and
directed the migration of lymphocytes and modulates the expression of
cell surface adhesion molecules (18, 66). SDF-1 can
interfere with infection by X4 strains of HIV-1 by receptor blockade
and downmodulation from the cell surface (54, 56, 68).
Activation of CXCR4 by SDF-1 or gp120 may induce cell activation and
apoptosis of neurons and CD4+ cells (10,
12, 27, 39, 42, 55, 69).
The structural basis for the interaction of CXCR4 with SDF-1 and HIV-1
envelope glycoproteins has not yet been elucidated. Structure-function
studies with chimeras, point mutants, or domain-specific monoclonal
antibodies (MAbs) indicate that these functions involve multiple
domains of the receptor and are not coincident (14, 16, 19, 20,
31, 33, 35, 41). Whereas the membrane-proximal region of the
N-terminal (NT) extracellular domain and the third extracellular loop
(ECL3) appear to be critical for SDF-1 binding and signaling, regions
contiguous to the second ECL have been implicated in coreceptor
activity (14, 15, 16, 31). Studies with CXCR4 mutants that
are not coupled to G proteins have revealed that coreceptor activity is
independent of signal transduction (31, 52). In contrast,
it has been shown that signaling through CCR5 is required for fusion of
R5 viruses with primary CD4+ T lymphocytes
(2), although signal transduction is not necessary for
infection of cell lines (4, 5, 34, 38).
Cell fusion with syncytium formation represents an important cytopathic
effect of HIV-1 infection that may be a critical mechanism for
depletion of CD4+ T lymphocytes (49, 50,
51, 62, 67). Syncytium formation results from the interaction of
the gp120 subunit of envelope glycoprotein expressed on infected cells
with CD4 and a coreceptor, typically CXCR4, on the surface of target
cells (3, 11, 28, 32, 35, 50, 51, 62, 67). The involvement
of cytoadhesion molecules in syncytium formation has been demonstrated
by inhibition with MAbs to LFA-1 and ICAM-1 (17, 37, 40,
65) and the observation that LFA-1-deficient
CD4+ T lymphocytes exhibit decreased syncytium
formation (57). Moreover, this process can be enhanced by
the modulation of LFA-1 conformation using the NKI-IL-16 MAb
(6).
In the physiologic response to SDF-1 signaling through CXCR4, rolling
of T lymphocytes and tight adhesion to endothelial cells is dependent
upon LFA-1 activation (18, 25, 45). Similarly, SDF-1
activates integrins (VLA-4 and VLA-5) in CD34+
cells (57, 66). These findings link CXCR4 signaling to
integrin activation in physiologic responses and implicate this
mechanism in HIV-1 infection as well. Here we demonstrate that an MAb
to the ECL3 of CXCR4, A80, has the unique properties of inducing cell
agglutination and enhancing syncytium formation by HIV-1, providing
additional evidence for the association between CXCR4 signaling and
cell adhesion. This unique activity of the A80 MAb provides important
insights into the mechanism for CXCR4 function in physiologic responses
and HIV-1 envelope-mediated membrane fusion.
| |
MATERIALS AND METHODS |
Reagents.
RPMI 1640 medium (Sigma Chemical Company, St.
Louis, Mo.) supplemented with 10% fetal calf serum (FCS) (Sigma), 100 U of penicillin per ml, and 100 µg of streptomycin per ml
(hereinafter called RPMI medium) was used for cultivation of all of the
cells used. Anti-human CD3 MAb (OKT-3) was purchased from the American
Type Culture Collection (Rockville, Md.). Anti-human CD28 MAb was
purchased from R&D (Minneapolis, Minn.). Magnetic beads conjugated with anti-CD8, and unconjugated tosylactivated beads (M-450) were purchased from Dynal; the latter were conjugated with anti-CD3 and anti-CD28 MAb
(anti-CD3/CD28 beads) according to the manufacturer's recommendation. The fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled MAbs anti-human CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD28, CD29, CD50, CD54, CD69, and CD102 and FITC-labeled streptavidin were
purchased from Beckman-Coulter. The mouse anti-human CXCR4 MAbs
purchased were 12G5 (FAB170), FAB171, FAB172, and FAB173 (R&D). A rat
immunoglobulin G1 (IgG1) MAb against hepatitis C virus (HCV), Mo-8
(44), was used as a rat isotype-matched negative control.
Goat anti-rat IgG (heavy and light chains) labeled with either FITC or
peroxidase (POD) was purchased from American Corlex. Streptavidin-POD
was purchased from Dako. PE-labeled MAb anti-HIV-1 p24 was purchased
from Beckman Coulter. MAbs against adhesion molecules used for blocking
of adhesion were anti-LFA-1 alpha chain (CD11a) (clone TP1/32; Upstate
Biotechnology, Inc.), anti-LFA-1 beta chain (CD18, clone MHM23; Dako),
anti-ICAM-1 (CD54, clone 84H10; Beckman-Coulter), anti-ICAM-2 (CD102,
clone B-T1; Beckman-Coulter), anti-ICAM-3 (CD50, clone HP2/19;
Beckman-Coulter), and anti-VLA beta chain (CD29, clone BB4;
Beckman-Coulter). The CXCR4 ligands used were recombinant SDF-1 (R&D)
and T22 (53). Pertussis toxin (PTX) (Seikagakukogyo,
Tokyo, Japan) and wortmannin (WMN) (Wako Pure Chemicals, Kyoto, Japan)
were used to block SDF-1 signal pathway. Phorbol 12-myrisate 13-acetate
(PMA) was purchased from Sigma. WMN and PMA were dissolved at 100 µg/ml in dimethyl sulfoxide and diluted to a final concentration of
0.1 µg/ml in culture medium.
Cells.
Cell lines used included human T-cell lines (CEM,
Molt-4, Jurkat, Hut78, and MT-2), human B-cell lines (BJAB and Raji),
human myeloid cell line (THP-1), a human erythroblastoid cell line
(K-562), a human glioblastoma cell line (U-87 MG), a monkey kidney cell line (Cos-1), a WKA rat kidney cell line (W7KSV) and a rat T-cell line
(W7TM-1) (63), and a mouse myeloma cell line (SP2/0).
Fresh peripheral blood mononuclear cells (PBMC) were isolated from
heparinized blood of healthy donors by Ficoll-Hypaque density gradient
centrifugation. PBMC or PBMC depleted of CD8+ T
cells by an immunomagnetic method with anti-CD8 antibody-conjugated magnetic beads were cultured at 2 × 106
cells/ml in RPMI medium containing 50 U of human recombinant interleukin-2 (rIL-2; Shionogi Pharmaceutical, Osaka, Japan)/ml in the
presence of the anti-CD3 (OKT3 from the American Type Culture Collection)/CD28 beads at a cell/bead ratio of 1:1 in a 12-well plate
(Falcon). On days 3 and 6, the cells were collected, diluted 1:4
with fresh RPMI medium with 50 U of rIL-2/ml, and restimulated with the
anti-CD3/CD28 beads at a cell/bead ratio of 1:1.
Generation of rat MAbs.
The human CXCR4 expression plasmid
was kindly provided by K. Matsushima (University of Tokyo). The
CXCR4-expressing cells generated were A62 cells of Cos-1 origin, and
the 0.5a cells were of W7TM-1 origin (63). The human CCR5
gene was isolated by PCR performed directly on a human genomic DNA, and
its sequence was confirmed before transfer into XhoI and
NotI sites in the expression vector, BCMGSNeo
(46). The primers used for PCR amplification were as
follows: sense, 5'-CTCGAGAACAAGATGGATTATCAA-3'; and
antisense, 5'-GCGGCCGCGAGTCCGTGTCACAAGCCCACA-3'. The
CCR5-expressing cells generated were A41 cells of Cos-1 origin, and the
W516 cells were of of W7KSV origin. These transfectants were selected
and maintained in the presence of 0.5 mg of G418 (Gibco)/ml. Expression
of the transgenes was confirmed by a Northern blot assay and a flow
cytometric analysis with commercial antibodies against human CXCR4 and
human CCR5. Lines of MAb-producing hybridomas were generated as
described previously (64). Briefly, WKA rat spleen cells
immunized intraperitoneally with 0.5a or W516 cells at
107 cells/animal every 2 weeks for 2 to 4 months
were fused with SP2/0 cells by using polyethylene glycol 4000 and were
selected in hypoxanthine-aminopterin-thymidine medium. Specific
antibody screenings were performed by an indirect immunofluorescence
assay on A40 and A62 cells. Hybridomas were cloned by limiting
dilution, and MAbs were produced in culture medium or in ascitic fluid
of BALB/c nude mice. IgG fractions were obtained from ascitic fluid by
a gel filtration chromatography with Superdex G200 (Amersham Pharmacia). The immunoglobulin isotype and subclass of a new MAb were
determined by using an enzyme-linked immunosorbent assay (ELISA) kit (Zymed).
Immunofluorescence.
Sample cells (1 × 105 to 5 × 105) were
incubated in a volume of 100 µl of phosphate-buffered saline (PBS)
containing 2% FCS and 0.1% sodium azide (referred to as
fluorescence-activated cell sorting [FACS] buffer) and 100 µg of
normal human IgG/ml for 15 min on ice. The cells were reacted with 0.1 ml of hybridoma culture supernatants, 5 µg of purified antibody, or
appropriately diluted FITC-labeled antibodies/ml for 30 min on ice, and
then they were washed twice with FACS buffer. For the detection
of unlabeled rat IgG, these cells were further incubated with 100 µl
of 1:100 diluted goat anti-rat IgG labeled with FITC containing 100 µg of normal goat IgG/ml for 30 min on ice. After being washed, the cells were fixed with 1% paraformaldehyde in FACS buffer for 5 min at
room temperature and then analyzed on a flow cytometer (FACSCalibur) by
using the CellQuest software (Becton Dickinson). The area of positivity
was determined by using an isotype-matched mouse MAb (Beckman-Coulter)
or a rat IgG1 MAb (Mo-8).
Immunoprecipitation and immunoblot.
Cells (2 × 107cells) were lysed in 1 ml of lysis buffer
containing either 1% Brij 97 (Sigma) or 0.5% NP-40 in 20 mM Tris-HCl (pH 8.2)-0.15 M NaCl-5 mM iodoacetamide-1 mM phenylmethylsulfonyl fluoride. For immunoprecipitation, a volume of 100 µl of cell lysates
was incubated with 1 µg of MAb on ice for 30 min and precipitated with 10 µl of protein G-Sepharose (Amersham Pharmacia) at 4°C overnight. Washed precipitates were treated with the sample buffer at
37°C for 30 min in nonreducing conditions and separated by sodium
dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-10%
PAGE). The separated proteins were transferred to polyvinylidene difluoride membrane (Pierce). The membranes were reacted with rat MAbs,
followed by reaction with POD-labeled goat anti-rat IgG. The binding of
MAb was visualized by using the ECL substrate (Pharmacia), followed by
analysis with Fluor-S MAX MultiImager (Bio-Rad).
Cell agglutination assay.
Anti-CD3/CD28 bead-activated PBMC,
CEM cells, and BJAB cells were washed once and resuspended in RPMI
medium at 2 × 106 cells/ml, separated into
aliquots in 48-well flat-bottom plates (0.5 ml/well), and then
preincubated with 1 µg of PTX/ml, 100 µM WMN, 1 µg of
SDF-1
/ml, 5 µM T22, or 20 µg of MAbs (including anti-CD11a,
CD18, CD50, CD54, CD102, and CD29)/ml for 30 or 60 min at 37°C,
followed by incubation with 2 µg of A80 IgG/ml. After overnight
incubation, cells were observed for agglutination under an inverted
microscope at magnification of ×40 or ×100. PMA was used to activate
LFA-1 on BJAB cells and to downmodulate CXCR4 on CEM cells at 0.1 µg/ml.
HIV-1 infection.
The HIV-1 strains used were molecular
clones of NL4-3 (1) and JR-CSF (48). These
HIV-1 stocks produced in Cos-1 cells were obtained from Y. Koyanagi
(Tohoku University). Infectious titers of each virus stock were
determined by endpoint dilution assay with activated PBMC and are
expressed as 50% tissue culture infective doses
(TCID50). Either ten million PBMC activated for 6 days with anti-CD3/CD28 beads and depleted of
CD8+ T cells or CEM cells were washed once with
RPMI medium and incubated with 1,000 TCID50 of
HIV-1 in a 0.2-ml volume for 3 h at 37°C. The infected PBMC and
CEM cells were subsequently washed twice with fresh RPMI medium and
cultured at 2 × 105 cells/ml for 5 and 8 days, respectively, in 48-well culture plates (0.5 ml/well) in the
presence or absence of 10 µg of MAbs/ml. CEM cell cultures were split
1:2 with fresh medium on days 4 and 6. Culture supernatants were
examined for production of cell-free HIV-1 p24 by using an ELISA kit
(Zepto Metrix Corporation). The number of syncytia per culture well was
microscopically determined by using a hemocytometer at a magnification
of ×100. Data were analyzed by using the Student's t test.
Epitope mapping.
Peptides with amino acid sequences of four
of the extra cellular regions of the human CXCR4
(35)
i.e., amino acids 1 to 39 (MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKI), amino acids 97 to 110 (DAVANWYFGNFLCK), amino acids 176 to 201 (NVSEADDRYICDRFYPNDLWVVVFQFQ), and amino acids 262 to 282 (DSFILLEIIKQGCEFENTVHK)and those of the human CCR5
(59)i.e., amino acids 1 to 31 (MDYQVSSPIYDINYYTSEPCQKINVKQIAAR), amino acids 89 to 102 (YAAAQWDFGNTMCQ), amino acids 168 to 197 (RSQKEGLHYTCSSHFPYSQYQFWKNFQTLK),
and amino acids 258 to 279 (NTFQEFFGLNNCSSSNRLDQAM)were synthesized by the step-wise solid-phase procedure of
Na9-fluorenylmethoxycarbonyl chemistry on an automated peptide
synthesizer (PSSM-8; Shimadzu, Kyoto, Japan). All peptides used were
>90% pure as judged by a high-pressure liquid chromatographic
analysis (data not shown). These peptides were immobilized onto 96-well
flat-bottom ELISA plates (Nunc) by incubation at 100 µl/well of a
10-µg/ml concentration of peptide in PBS at 4°C overnight. The
immobilized peptides were then tested for reactivity with an antibody
by an ELISA as described previously (64). Epitopes
recognized by anti-CXCR4 MAbs were also determined by using chimeric
receptors. The chimeric receptors composed of CXCR4 and CXCR2 were
constructed by the PCR-ligation-PCR approach as described previously
(52). Cos-1 cells transfected with each receptor plasmid
by means of electroporation by using the Gene Pulser II (Bio-Rad).
After culture for 48 h, the cells were harvested and examined for
reactivity with MAbs by an indirect immunofluorescence assay.
Ca2+ mobilization assay.
CEM cells at
107 cells/ml were loaded with 4 µM
fluo-3-acetylozymethyl ester (Fluo-3; Molecular Probes, Eugene, Oreg.),
a Ca2+ indicator, for 30 min at 37°C. After
either stimulation with 0.5 µg of SDF-1/ml or treatment with each
MAb at 10, 50, and 100 µg/ml, the intracellular
Ca2+ concentration was then measured by use of
FACSCalibur with analysis software (Flow Jo; Tree Star, Inc.).
| |
RESULTS |
Generation of novel MAbs to CXCR4: A145 is active in
immunoblotting.
Three MAbs to human CXCR4 were generated by
immunization of WKA rats with a syngeneic transfectant cell line
programmed to express human CXCR4. The specificity of these IgG1 MAbs,
designated A80, A120, and A145, was established by indirect
immunofluorescence analysis of CXCR4 transfectants by using anti-CCR5
MAbs as control myeloma proteins. These MAbs lacked reactivity with
control cells and CCR5 transfectants. Analysis of a panel of cell lines
demonstrated that all known to express CXCR4, including CEM (Fig.
1), Molt4, Jurkat, Hut78, K562, THP-1,
BJAB, and Raji cells (data not shown), stained positively with A80,
A120, and A145. The three MAbs did not react with U-87 MG cells, which
lack CXCR4 expression, or with CCR1, CCR2B, CCR3, or CCR5 HOS-CD4
transfectants (data not shown).
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 1.
Reactivity of MAbs generated from WKA rats. Cos-1 cells
expressing human CXCR4 (Cos/X4) and CCR5 (Cos/R5) and CEM cells were
reacted with 5 µg of MAbs/ml, followed by incubation with goat
anti-rat IgG-FITC. The binding of MAb was determined by flow cytometry.
The shaded area indicates cells stained with negative control rat IgG1
MAb (Mo-8) specific for HCV. T227 and T312 were control rat IgG MAbs to
anti-human CCR5. Representative results from four independent
experiments are shown.
|
|
The reactivity of these MAbs with CXCR4 was further analyzed by
immunoprecipitation, followed by immunoblotting. Analysis
of CEM cell
lysates by immunoblotting revealed that A145, but
not A80 or A120,
detected a 50-kDa protein corresponding to CXCR4
(data not shown).
Figure
2 shows that A145 had the unique
ability
among the three MAbs to specifically immunoprecipitate CXCR4
from
detergent lysates (Brij 97 or NP-40) of Cos-1 cells transfected
with CXCR4 but not CCR5.
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 2.
Immunoprecipitation and immunoblot of CXCR4 by MAb.
Cos-1 cells expressing human CXCR4 (Cos/X4) and CCR5 (Cos/R5) were
lysed with either NP-40 (NP) or Brij 97 (Brij), and the lysates were
reacted with anti-CXCR4 or anti-CCR5 MAbs. Immunocomplexes collected by
protein G-Sepharose were separated by SDS-PAGE, blotted on
polyvinylidene difluoride sheets, and reacted with either anti-CXCR4
A145 or anti-CCR5 T312, followed by reaction with POD-labeled goat
anti-rat IgG. Arrows a and b indicate 50-kDa CXCR4 and 40-kDa CCR5
molecules, respectively. Representative results from three independent
experiments are shown.
|
|
Effects of anti-CXCR4 MAbs on HIV-1 infection: enhancement by
A80.
The effects of the three anti-CXCR4 MAbs on HIV-1 infection
were determined in experiments with R5 and X4 strains and activated PBMC and CEM cells as targets. Figure 3
shows that, unlike A120 and A145, A80 induced syncytium formation in
PBMC cultures infected with either X4 or R5 strains. A80 also
significantly enhanced productive infection of CD8-depleted PBMC with
R5 HIV-1 and of CEM cells with X4 HIV-1 (P < 0.05), as
demonstrated in Table 1. A80 also induced
syncytium formation by R5 and X4 strains in the PBMC and X4 strains in
CEM cells (Table 1). The novel properties of inducing syncytium
formation and enhancing infection are unique among MAbs to CXCR4,
including the commercial MAbs, 12G5, F171, F172, and F173.
View larger version (45K):
[in this window]
[in a new window]
|
FIG. 3.
Induction of large syncytia in activated PBMC infected
by HIV-1 in the presence of A80. Anti-CD3/CD28 bead-activated PBMC were
infected with X4 HIV-1NL4-3 or R5 HIV-1JR-CSF
at a multiplicity of infection of 0.005 for 3 h. After being
washed, the cells were cultured in the presence of various MAbs at 10 µg/ml for 5 days. The control culture contained Mo-8. Syncytia were
observed under an inverted microscope at an original magnification of
×100. Representative results from five independent experiments are
shown.
|
|
Induction of cell agglutination by A80.
It was reasoned that
the induction of syncytium formation and enhancement of HIV-1 infection
by A80 may be the result of increased intercellular adhesion. The role
of cytoadhesion molecules and CXCR4 signaling in the mechanism for the
development of agglutination of cells by A80 in the absence of HIV-1
infection was determined. As shown in Fig.
4A and B, MAbs to LFA-1
(CD11a and CD18), ICAM-1 (CD54), ICAM-2 (CD102), and ICAM-3 (CD50) had
minimal effects on agglutination mediated by A80. In addition, a MAb to
the beta chain of VLA (CD29) lacked inhibitory activity (data not
shown), and it is doubtful that LFA-2 (CD2) plays a role because CEM
cells did not express this protein.
View larger version (178K):
[in this window]
[in a new window]
|
FIG. 4.
Effects of MAbs against various adhesion molecules,
CXCR4 ligand, CXCR4 antagonist, and SDF-1 signal-blocking reagents on
A80-induced cell agglutination. Activated PBMC (A), CEM cells (B), and
BJAB cells (C) were preincubated with 20 µg of anti-LFA-1 CD11a,
anti-LFA-1 CD18, anti-ICAM-1 (CD54), or anti-ICAM-2 (CD102) and
anti-ICAM-3 (CD50)/ml; 1 µg of SDF-1/ml; 5 µM T22; 0.1 µg of
PTX/ml; 0.1 µg of WMN/ml; or various mixtures thereof at
37°C. After 1 h, either A80 (final concentration, 2 µg/ml) (A and B) or PMA (0.1 µg/ml) (C) was added, and the mixtures
were incubated at 37°C overnight. Cell agglutination was observed
under an inverted microscope at an original magnification of ×40. The
"medium" panel was cultured in the absence of A80. Representative
results from three independent experiments are shown.
|
|
Blocking studies were performed to ensure that the MAbs to cytoadhesion
molecules inhibit adhesion of BJAB cells induced by
PMA. As shown in
Fig.
4C, agglutination after exposure to PMA
was completely blocked by
the MAbs to LFA-1 (CD11a and CD18) and
ICAM-1 (CD54) but not by the MAb
to ICAM-2. In contrast, A80 induced
agglutination of BJAB cells that
was not inhibited by the MAbs
to LFA-1, ICAM-1, or ICAM-2. These
results confirm that binding
of A80 to CXCR4 on the cell surface
induces agglutination via
a novel
mechanism.
Engagement of CXCR4 by SDF-1 or T22, a polypeptide CXCR4 antagonist,
did not result in intercellular agglutination, but exposure
to both
inhibited the ability of A80 to induce this effect (Fig.
4A and B).
Downmodulation of CXCR4 with PMA (
61) completely
abolished
the agglutination of CEM cells after A80 binding (data
not shown).
Figure
4A and B also show that inhibitors of G
i,
PTX, and
G-protein-coupled receptor signaling, WMN, did not influence
the
induction of agglutination by A80. These findings indicate
that CXCR4
signaling is not required for the A80-induced agglutination
but that
the role of CXCR4 is
critical.
A80 binds to ECL3 of CXCR4.
The epitopes recognized by A80,
A120, and A145 MAbs were mapped by enzyme-linked immunosorbent assay
(ELISA) with a panel of synthetic peptides spanning the four
extracellular domains of CXCR4. An analogous panel of peptides from
CCR5 served as controls. As shown in Table
2, A145 bound specifically to the peptide
corresponding to the NT domain of CXCR4 and A80 and A120 did not bind
to any of the peptides. To determine whether the latter two MAbs bound to epitopes that required expression of the native receptor on the cell
surface, they were tested for binding to an array of CXCR4/CXCR2
chimeras in transient-expression experiments. The binding of A80 to
chimeras corresponded to the presence of CXCR4-ECL3 (Table
3). In contrast, A120 showed optimal
binding to chimeras that contained ECL1 and ECL2 of CXCR4, a result
similar to that for MAb 12G5.
A80, A120, and A145 do not induce CXCR4 signaling.
Since the
binding of A80 to CXCR4 induced agglutination of CEM cells and PBMC,
experiments were performed to determine whether binding of any of the
three MAbs to CXCR4 induced a prototypic Gi signaling
response, i.e., mobilization of cytosolic calcium ions (Fig.
5). Whereas SDF-1 stimulated an influx of
free calcium ions in CEM cells labeled with Fluo-3, exposure to A80,
A120, or A145 did not induce calcium mobilization. Pretreatment of CEM cells with A120, but not A80 or A145, blocked the calcium response induced by SDF-1.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 5.
Ca2+ influx assay. CEM cells were
loaded with Fluo-3 and then treated with 0.5 µg of SDF-1/ml either
before or after treatment with 20 µg of MAb/ml. Ca2+
influx was monitored by flow cytometry. The arrows indicate the time
when SDF-1 or MAbs were added. Representative results from four
independent experiments are shown.
|
|
| |
DISCUSSION |
Here we report the characterization of a new set of three rat MAbs
to human CXCR4, one of which, A80, has unique biologic properties.
Binding of A80 to ECL3 of CXCR4 on the surface of target cells enhanced
productive infection by X4 and R5 strains of HIV-1 and augmented
syncytium formation. A80 also induced agglutination of PBMC and CEM
cells. The agglutination induced by A80 was blocked by SDF-1 and T22, a
specific ligand and an antagonist of CXCR4, respectively, but not by
inhibitors of CXCR4 signal transduction or MAbs to cytoadhesion
molecules. The A120 MAb recognized an epitope that involved ECL1 and
ECL2, and A145 bound to a peptide corresponding to the NT extracellular
domain. A145 MAb bound CXCR4 in immunoprecipitation and Western
blotting experiments, indicating that A145 bound an epitope that was
not dependent upon native conformation of this domain. A120 blocked
infection via CXCR4.
The mechanism for the enhancement of HIV-1 infection by A80 is novel.
This effect was not altered by inhibition of signaling by the
Gi protein pathway or the phosphatidylinositol 3-kinase cascade and was accompanied by the induction of syncytium formation. A80 alone did not affect G-protein-mediated signaling, and SDF-1 did
not induce agglutination; thus, the mechanism for stimulating this
intercellular adhesion must involve an alternative pathway. The
engagement of CXCR4 by SDF-1 or T22 blocks the effect of A80 on the
agglutination of target cells. Analysis of structural requirements for
the activation of signaling by SDF-1 has implicated ECL3 and adjacent
transmembrane domains of CXCR4 (31). Thus, it is possible that binding of this CXCR4 domain by A80 may induce conformational changes in the receptor that activate a novel signaling pathway. The
agglutination mediated by A80 binding to CXCR4 involves the development
of intercellular adhesion. The stimulation of T lymphocyte and
hematopoietic progenitor interactions with endothelial cells by SDF-1
has been shown to involve LFA-1 and VLA4/5, respectively (18, 45,
58). However, although a single MAb against a single epitope on
an adhesion molecule may be not sufficient to interfere with adhesion
function, cell adhesion blocking experiments with a set of MAbs against
LFA-1, ICAMs, and CD29 (a common chain of the 
1 integrin family)
suggested that A80-mediated cell agglutination is independent of LFA-1,
VLA4, and VLA5. Recent studies have demonstrated that the CX3C
chemokine (fractalkine or neurotactin
[fractalkine/neurotactin]) is capable of mediating firm
intercellular adhesion through CX3CR1 that is resistant to pertussis
toxin, exposure to EDTA or EGTA, and MAbs to 1 or 2 integrins
(36, 43). Both CXCR4 and CX3CR1 effects are independent of
receptor coupling to Gi signaling and the function of the
1 or 2 integrins, but the latter mechanism requires that the
chemokine module of fractalkine/neurotactin be attached to the mucin
stalk that tethers it to the cell surface (43). Our
preliminary studies showed that A80-mediated cell agglutination did not
occur in the presence of 1 mM EDTA or EGTA and at 4°C (data not
shown), suggesting that the cell agglutination is dependent on both
calcium and temperature. Future experiments will determine whether
monovalent fragments of A80 can induce syncytium formation and agglutination.
The set of new anti-CXCR4 MAbs will also serve as critical
domain-specific reagents to study the function of CXCR4. Whereas A120,
which recognizes an epitope involving ECL1 and ECL2 of CXCR4, blocked
infection with X4 HIV-1, A80 enhanced productive infection. Binding of
A145 to the NT extracellular domain resulted in low-level inhibition of
HIV-1 infection. ECL2 and adjacent segments of CXCR4 have been
implicated in its coreceptor activity with T-tropic envelope
glycoproteins, but chimeras containing the NT extracellular domain did
not support envelope-mediated fusion with dual- and T-tropic envelope
glycoproteins (14-16, 19, 20, 31). Similarly, A120, but
not A80 or A145, inhibited SDF-1 signaling through CXCR4. Binding and
signaling studies with CXCR4/CXCR2 chimeras have implicated the segment
between Cys28 and the interface with the first transmembrane helix of
CXCR4 in the interaction with SDF-1 (31). It is therefore likely that A145 binds with the portion of this domain that is N
terminal to Cys28, which contains more residues that are divergent between human and rodent CXCR4 homologs. The distinct biologic properties of these three MAbs establish the specificity of these reagents and illustrate the complexity of the structural basis of CXCR4
functions, both physiologic and pathological. The activities of the
MAbs to epitopes in the NT and ECL1/2 domains show excellent correlation with our understanding of CXCR4 function derived from molecular genetic approaches. This set of domain-specific immunologic reagents should be a critical tool for dissecting the structural basis
for CXCR4 functions, including novel activities in cytoadhesion.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan; the Health Sciences
of Organization for Drug ADR Relief, R&D Promotion, and Product Review
of Japan; the Ministry of Health, Labor, and Welfare of Japan; the
Japan Human Heath Sciences Foundation; and CREST of the Japan Science
and Technology Corporation (JST).
We are grateful to Y. Koyanagi for providing HIV-1 and to H. Sato and
Y. Takebe for unpublished data on HOS-CD4 cells.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Infectious Disease and Immunology, Okinawa-Asia Research Center of
Medical Science, Faculty of Medicine, University of the Ryukyus, Uehara 207, Nishihara, Okinawa 903-0215, Japan. Phone: 81-98-895-1202. Fax:
81-98-895-1437. E-mail:
yuetsu{at}ma.kcom.ne.jp.
| |
REFERENCES |
| 1.
|
Adachi, A.,
H. E. Gendelman,
S. Koenig,
T. Folks,
R. Willey,
A. Rabson, and M. A. Martin.
1986.
Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.
J. Virol.
59:284-289[Abstract/Free Full Text].
|
| 2.
|
Alfano, M.,
H. Schmidtmayerova,
C. A. Amella,
T. Pushkarsky, and M. Bukrinsky.
1999.
The B-oligomer of pertussis toxin deactivates CC chemokine receptor 5 and blocks entry of M-tropic HIV-1 strains.
J. Exp. Med.
190:597-606[Abstract/Free Full Text].
|
| 3.
|
Alkhatib, G.,
C. Combadiere,
C. C. Broder,
Y. Feng,
P. E. Kennedy,
P. M. Murphy, and E. A. Berger.
1996.
CC CKR5: a RANTES, MIP-1, MIP-1 receptor as a fusion cofactor for macrophage-tropic HIV-1.
Science
272:1955-1958[Abstract].
|
| 4.
|
Alkhatib, G.,
M. Locati,
P. E. Kennedy,
P. M. Murphy, and E. A. Berger.
1997.
HIV-1 coreceptor activity of CCR5 and its inhibition by chemokines: independence from G protein signaling and importance of coreceptor downmodulation.
Virology
234:340-348[CrossRef][Medline].
|
| 5.
|
Aramori, I.,
S. S. Ferguson,
P. D. Bieniasz,
J. Zhang,
B. Cullen, and M. G. Cullen.
1997.
Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor.
EMBO J.
16:4606-4616[CrossRef][Medline].
|
| 6.
|
Barbeau, B.,
J. F. Fortin,
N. Genois, and M. J. Tremblay.
1998.
Modulation of human immunodeficiency virus type 1-induced syncytium formation by the conformational state of LFA-1 determined by a new luciferase-based syncytium quantitative assay.
J. Virol.
72:7125-7136[Abstract/Free Full Text].
|
| 7.
|
Berger, E. A.,
P. M. Murphy, and J. M. Farber.
1999.
Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease.
Annu. Rev. Immunol.
17:657-700[CrossRef][Medline].
|
| 8.
|
Berger, E. A.,
R. W. Doms,
E. M. Fenyo,
B. T. Korber,
D. R. Littman,
J. P. Moore,
Q. J. Sattentau,
H. Schuitemaker,
J. Sodroski, and R. A. Weiss.
1998.
A new classification for HIV-1.
Nature
391:240[CrossRef][Medline].
|
| 9.
|
Berkowitz, R. D.,
S. Alexander,
C. Bare,
V. Linquist-Stepps,
M. Bogan,
M. E. Moreno,
L. Gibson,
E. D. Wieder,
J. Kosek,
C. A. Stoddart, and J. M. McCune.
1998.
CCR5- and CXCR4-utilizing strains of human immunodeficiency virus type 1 exhibit differential tropism and pathogenesis in vivo.
J. Virol.
72:10108-10117[Abstract/Free Full Text].
|
| 10.
|
Berndt, C.,
B. Mopps,
S. Angermuller,
P. Gierschik, and P. H. Krammer.
1998.
CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4+ T cells.
Proc. Natl. Acad. Sci. USA
95:12556-12561[Abstract/Free Full Text].
|
| 11.
|
Bieniasz, P. D.,
R. A. Fridell,
I. Aramori,
S. S. Ferguson,
M. G. Caron, and B. R. Cullen.
1997.
HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 co-receptor.
EMBO J.
16:2599-2609[CrossRef][Medline].
|
| 12.
|
Blanco, J.,
E. Jacotot,
C. Cabrera,
A. Cardona,
B. Clotet,
E. De Clercq, and J. A. Este.
1999.
The implication of the chemokine receptor CXCR4 in HIV-1 envelope protein-induced apoptosis is independent of the G protein-mediated signalling.
AIDS
13:909-917[CrossRef][Medline].
|
| 13.
|
Bleul, C. C.,
L. Wu,
J. A. Hoxie,
T. A. Springer, and C. R. Mackay.
1997.
The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes.
Proc. Natl. Acad. Sci. USA
94:1925-1930[Abstract/Free Full Text].
|
| 14.
|
Brelot, A.,
N. Heveker,
K. Adema,
M. J. Hosie,
B. Willett, and M. Alizon.
1999.
Effect of mutations in the second extracellular loop of CXCR4 on its utilization by human and feline immunodeficiency viruses.
J. Virol.
73:2576-2586[Abstract/Free Full Text].
|
| 15.
|
Brelot, A.,
N. Heveker,
M. Montes, and M. Alizon.
2000.
Identification of residues of CXCR4 critical for human immunodeficiency virus coreceptor and chemokine receptor activities.
J. Biol. Chem.
31:23736-23744.
|
| 16.
|
Brelot, A.,
N. Heveker,
O. Pleskoff,
N. Sol, and M. Alizon.
1997.
Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity.
J. Virol.
71:4744-4751[Abstract].
|
| 17.
|
Butini, L.,
A. R. De Fougerolles,
M. Vaccarezza,
C. Graziosi,
D. I. Cohen,
M. Montroni,
T. A. Springer,
G. Pantaleo, and A. S. Fauci.
1994.
Intercellular adhesion molecules (ICAM)-1, ICAM-2, and ICAM-3 function as counter-receptors for lymphocyte function-associated molecule 1 in human immunodeficiency virus-mediated syncytium formation.
Eur. J. Immunol.
24:2191-2195[Medline].
|
| 18.
|
Campbell, J. J.,
J. Hedrick,
A. Zlotnik,
M. A. Siani,
D. A. Thompson, and E. C. Butcher.
1998.
Chemokines and the arrest of lymphocytes rolling under flow conditions.
Science
279:381-384[Abstract/Free Full Text].
|
| 19.
|
Chabot, D. J.,
H. Chen,
D. S. Dimitrov, and C. C. Broder.
2000.
N-linked glycosylation of CXCR4 masks coreceptor function for CCR5-dependent human immunodeficiency virus type 1 isolates.
J. Virol.
74:4404-4413[Abstract/Free Full Text].
|
| 20.
|
Chabot, D. J.,
P. F. Zhang,
G. V. Quinnan, and C. C. Broder.
1999.
Mutagenesis of CXCR4 identifies important domains for human immunodeficiency virus type 1 X4 isolate envelope-mediated membrane fusion and virus entry and reveals cryptic coreceptor activity for R5 isolates.
J. Virol.
73:6598-6609[Abstract/Free Full Text].
|
| 21.
|
Choe, H.,
M. Farzan,
Y. Sun,
N. Sullivan,
B. Rollins,
P. D. Ponath,
L. Wu,
C. R. Mackay,
G. LaRosa,
W. Newman,
N. Gerard,
C. Gerard, and J. Sodroski.
1996.
The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.
Cell
85:1135-1148[CrossRef][Medline].
|
| 22.
|
Connor, R. I.,
K. E. Sheridan,
D. Ceradini,
S. Choe, and N. R. Landau.
1997.
Change in coreceptor use correlates with disease progression in HIV-1-infected individuals.
J. Exp. Med.
185:621-628[Abstract/Free Full Text].
|
| 23.
|
Connor, R. I., and D. D. Ho.
1994.
Human immunodeficiency virus type 1 variants with increased replicative capacity develop during the asymptomatic stage before disease progression.
J. Virol.
68:4400-4408[Abstract/Free Full Text].
|
| 24.
|
Connor, R. I., and D. D. Ho.
1994.
Transmission and pathogenesis of human immunodeficiency virus type 1.
AIDS Res. Hum. Retrovir.
10:321-323[Medline].
|
| 25.
|
Constantin, G.,
M. Majeed,
C. Giagulli,
L. Piccio,
J. V. Kim,
E. C. Butcher, and C. Laudanna.
2000.
Chemokines trigger immediate 2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow.
Immunity
13:759-769[CrossRef][Medline].
|
| 26.
|
Dalgleish, A. G.,
P. C. Beverley,
P. R. Clapham,
D. H. Crawford,
M. F. Greaves, and R. A. Weiss.
1984.
The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus.
Nature
312:763-767[CrossRef][Medline].
|
| 27.
|
Davis, C. B.,
I. Dikic,
D. Unutmaz,
C. M. Hill,
J. Arthos,
M. A. Siani,
D. A. Thompson,
J. Schlessinger, and D. R. Littman.
1997.
Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.
J. Exp. Med.
186:1793-1798[Abstract/Free Full Text].
|
| 28.
|
Deng, H.,
R. Liu,
W. Ellmeier,
S. Choe,
D. Unutmaz,
M. Burkhart,
P. Di Marzio,
S. Marmon,
R. E. Sutton,
C. M. Hill,
C. B. Davis,
S. C. Peiper,
T. J. Schall,
D. R. Littman, and N. R. Landau.
1996.
Identification of a major co-receptor for primary isolates of HIV-1.
Nature
38:1661-1666.
|
| 29.
|
Dimitrov, D. S.,
X. Xiao,
D. J. Chabot, and C. C. Broder.
1998.
HIV coreceptors.
J. Membr. Biol.
166:75-90[CrossRef][Medline].
|
| 30.
|
Doranz, B. J.,
J. Rucker,
Y. Yi,
R. J. Smyth,
M. Samson,
S. C. Peiper,
M. Parmentier,
R. G. Collman, and R. W. Doms.
1996.
A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors.
Cell
85:1149-1158[CrossRef][Medline].
|
| 31.
|
Doranz, B. J.,
M. J. Orsini,
J. D. Turner,
T. L. Hoffman,
J. F. Berson,
J. A. Hoxie,
S. C. Peiper,
L. F. Brass, and R. W. Doms.
1999.
Identification of CXCR4 domains that support coreceptor and chemokine receptor functions.
J. Virol.
73:2752-2761[Abstract/Free Full Text].
|
| 32.
|
Dragic, T.,
V. Litwin,
G. P. Allaway,
S. R. Martin,
Y. Huang,
K. A. Nagashima,
C. Cayanan,
P. J. Maddon,
R. A. Koup,
J. P. Moore, and W. A. Paxton.
1996.
HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.
Nature
381:667-673[CrossRef][Medline].
|
| 33.
|
Endres, M. J.,
P. R. Clapham,
M. Marsh,
M. Ahuja,
J. D. Turner,
A. McKnight,
J. F. Thomas,
B. Stoebenau-Haggarty,
S. Choe,
P. J. Vance,
T. N. Wells,
C. A. Power,
S. S. Sutterwala,
R. W. Doms,
N. R. Landau, and J. A. Hoxie.
1996.
CD4-independent infection by HIV-2 is mediated by fusin/CXCR4.
Cell
87:745-756[CrossRef][Medline].
|
| 34.
|
Farzan, M.,
H. Choe,
K. A. Martin,
Y. Sun,
M. Sidelko,
C. R. Mackay,
N. P. Gerard,
J. Sodroski, and C. Gerard.
1997.
HIV-1 entry and macrophage inflammatory protein-1-mediated signaling are independent functions of the chemokine receptor CCR5.
J. Biol. Chem.
272:6854-6857[Abstract/Free Full Text].
|
| 35.
|
Feng, Y.,
C. C. Broder,
P. E. Kennedy, and E. A. Berger.
1996.
HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.
Science
272:872-877[Abstract].
|
| 36.
|
Fong, A. M.,
L. A. Robinson,
D. A. Steeber,
T. F. Tedder,
O. Yoshie,
T. Imai, and D. D. Patel.
1998.
Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow.
J. Exp. Med.
188:1413-1419[Abstract/Free Full Text].
|
| 37.
|
Fortin, J. F.,
B. Barbeau,
H. Hedman,
E. Lundgren, and M. J. Tremblay.
1999.
Role of the leukocyte function antigen-1 conformational state in the process of human immunodeficiency virus type 1-mediated syncytium formation and virus infection.
Virology
257:228-238[CrossRef][Medline].
|
| 38.
|
Gosling, J.,
F. S. Monteclaro,
R. E. Atchison,
H. Arai,
C. L. Tsou,
M. A. Goldsmith, and I. F. Charo.
1997.
Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity.
Proc. Natl. Acad. Sci. USA
94:5061-5066[Abstract/Free Full Text].
|
| 39.
|
Hesselgesser, J.,
D. Taub,
P. Baskar,
M. Greenberg,
J. Hoxie,
D. L. Kolson, and R. Horuk.
1998.
Neuronal apoptosis induced by HIV-1 gp120 and the chemokine SDF-1 is mediated by the chemokine receptor CXCR4.
Curr. Biol.
8:595-598[CrossRef][Medline].
|
| 40.
|
Hildreth, J. E., and R. J. Orentas.
1989.
Involvement of a leukocyte adhesion receptor (LFA-1) in HIV-induced syncytium formation.
Science
244:1075-1078[Abstract/Free Full Text].
|
| 41.
|
Hori, T.,
H. Sakaida,
A. Sato,
T. Nakajima,
H. Shida,
O. Yoshie, and T. Uchiyama.
1998.
Detection and delineation of CXCR-4 (fusin) as an entry and fusion cofactor for T-tropic [correction of T cell-tropic] HIV-1 by three different monoclonal antibodies.
J. Immunol.
160:180-188[Abstract/Free Full Text].
|
| 42.
|
Horuk, R.
1999.
Chemokine receptors and HIV-1: the fusion of two major research fields.
Immunol. Today
20:89-94[CrossRef][Medline].
|
| 43.
|
Imai, T.,
K. Hieshima,
C. Haskell,
M. Baba,
M. Nagira,
M. Nishimura,
M. Kakizaki,
S. Takagi,
H. Nomiyama,
T. J. Schall, and O. Yoshie.
1977.
Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.
Cell
91:521-530.
|
| 44.
|
Inudo, M.,
N. Kato, and Y. Tanaka.
1998.
New monoclonal antibodies against a recombinant second envelope protein of hepatitis C virus.
Microbiol. Immunol.
42:875-877[Medline].
|
| 45.
|
Kantele, J. M.,
S. Kurk, and M. A. Jutila.
2000.
Effects of continuous exposure to stromal cell-derived factor-1 on T cell rolling and tight adhesion to monolayers of activated endothelial cells.
J. Immunol.
164:5035-5040[Abstract/Free Full Text].
|
| 46.
|
Karasuyama, H.,
N. Tohyama, and T. Tada.
1989.
Autocrine growth and tumorigenicity of interleukin 2-dependent helper T cells transfected with IL-2 gene.
J. Exp. Med.
169:13-25[Abstract/Free Full Text].
|
| 47.
|
Klatzmann, D.,
E. Champagne,
S. Chamaret,
J. Gruest,
D. Guetard,
T. Hercend,
J. C. Gluckman, and L. Montagnier.
1984.
T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV.
Nature
312:767-768[CrossRef][Medline].
|
| 48.
|
Koyanagi, Y.,
S. Miles,
R. T. Mitsuyasu,
J. E. Merrill,
H. V. Vinters, and I. S. Y. Chen.
1987.
Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms.
Science
236:819-822[Abstract/Free Full Text].
|
| 49.
|
LaBonte, J. A.,
T. Patel,
W. Hofmann, and J. Sodroski.
2000.
Importance of membrane fusion mediated by human immunodeficiency virus envelope glycoproteins for lysis of primary CD4-positive T cells.
J. Virol.
74:10690-10698[Abstract/Free Full Text].
|
| 50.
|
Lifson, J. D.,
G. R. Reyes,
M. S. McGrath,
B. S. Stein, and E. G. Engleman.
1986.
AIDS retrovirus induced cytopathology: giant cell formation and involvement of CD4 antigen.
Science
232:1123-1127[Abstract/Free Full Text].
|
| 51.
|
Lifson, J. D.,
M. B. Feinberg,
G. R. Reyes,
L. Rabin,
B. Banapour,
S. Chakrabarti,
B. Moss,
F. Wong-Staal,
K. S. Steimer, and E. G. Engleman.
1986.
Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein.
Nature
323:725-728[CrossRef][Medline].
|
| 52.
|
Lu, Z.,
J. F. Berson,
Y. Chen,
J. D. Turner,
T. Zhang,
M. Sharron,
M. H. Jenks,
Z. Wang,
J. Kim,
J. Rucker,
J. A. Hoxie,
S. C. Peiper, and R. W. Doms.
1997.
Evolution of HIV-1 coreceptor usage through interactions with distinct CCR5 and CXCR4 domains.
Proc. Natl. Acad. Sci. USA
94:6426-6431[Abstract/Free Full Text].
|
| 53.
|
Murakami, T.,
T. Nakajima,
Y. Koyanagi,
K. Tachibana,
N. Fujii,
H. Tamamura,
N. Yoshida,
M. Waki,
A. Matsumoto,
O. Yoshie,
T. Kishimoto,
N. Yamamoto, and T. Nagasawa.
1997.
A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infection.
J. Exp. Med.
186:1389-1393[Abstract/Free Full Text].
|
| 54.
|
Nagasawa, T.,
T. Nakajima,
K. Tachibana,
H. Iizasa,
C. C. Bleul,
O. Yoshie,
K. Matsushima,
N. Yoshida,
T. A. Springer, and T. Kishimoto.
1996.
Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin.
Proc. Natl. Acad. Sci. USA
93:14726-14729[Abstract/Free Full Text].
|
| 55.
|
Nanki, T., and P. E. Lipsky.
2000.
Stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation.
J. Immunol.
164:5010-5014[Abstract/Free Full Text].
|
| 56.
|
Oberlin, E.,
A. Amara,
F. Bachelerie,
C. Bessia,
J. L. Virelizier,
F. Arenzana-Seisdedos,
O. Schwartz,
J. M. Heard,
I. Clark-Lewis,
D. F. Legler,
M. Loetscher,
M. Baggiolini, and B. Moser.
1996.
The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1.
Nature
382:833-835[CrossRef][Medline].
|
| 57.
|
Pantaleo, G.,
L. Butini,
C. Graziosi,
G. Poli,
S. M. Schnittman,
J. J. Greenhouse,
J. I. Gallin, and A. S. Fauci.
1991.
Human immunodeficiency virus (HIV) infection in CD4+ T lymphocytes genetically deficient in LFA-1: LFA-1 is required for HIV-mediated cell fusion but not for viral transmission.
J. Exp. Med.
173:511-514[Abstract/Free Full Text].
|
| 58.
|
Peled, A.,
O. Kollet,
T. Ponomaryov,
I. Petit,
S. Franitza,
V. Grabovsky,
M. M. Slav,
A. Nagler,
O. Lider,
R. Alon,
Zipori, and T. Lapidot.
2000.
The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice.
Blood
95:3289-3296[Abstract/Free Full Text].
|
| 59.
|
Samson, M.,
O. Labbe,
C. Mollereau, and G. Parmentir.
1996.
Molecular cloning and functional expression of a new human CC-chemokine receptor gene.
Biochemistry
35:3362-3367[CrossRef][Medline].
|
| 60.
|
Schuitemaker, H.,
M. Koot,
N. A. Kootstra,
M. W. Dercksen,
R. E. de Goede,
R. P. van Steenwijk,
J. M. Lange,
J. K. Schattenkerk,
F. Miedema, and M. Tersmette.
1992.
Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus population.
J. Virol.
66:1354-1360[Abstract/Free Full Text].
|
| 61.
|
Signoret, N.,
J. Oldridge,
A. Pelchen-Matthews,
P. J. Klasse,
T. Tran,
L. F. Brass,
M. M. Rosenkilde,
T. W. Schwartz,
W. Holmes,
W. Dallas,
M. A. Luther,
T. N. Wells,
J. A. Hoxie, and M. Marsh.
1997.
Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4.
J. Cell Biol.
139:651-664[Abstract/Free Full Text].
|
| 62.
|
Sodroski, J.,
W. C. Goh,
C. Rosen,
C. Campbell, and W. A. Haseltine.
1986.
Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity.
Nature
322:470-474[CrossRef][Medline].
|
| 63.
|
Tanaka, Y.,
H. Tozawa,
Y. Koyanagi, and H. Shida.
1990.
Recognition of human T cell leukemia virus type I (HTLV-I) gag and pX gene products by MHC-restricted cytotoxic T lymphocytes induced in rats against syngeneic HTLV-I-infected cells.
J. Immunol.
144:4202-4211[Abstract].
|
| 64.
|
Tanaka, Y.,
L. Zeng,
H. Shiraki,
H. Shida, and H. Tozawa.
1991.
Identification of a neutralization epitope on the envelope gp46 antigen of human T cell leukemia virus type 1 and induction of neutralizing antibody by peptide immunization.
J. Immunol.
147:354-360[Abstract].
|
| 65.
|
Valentin, A.,
K. Lundin,
M. Patarroyo, and B. Asjo.
1990.
The leukocyte adhesion glycoprotein CD18 participates in HIV-1-induced syncytia formation in monocytoid and T cells.
J. Immunol.
144:934-937[Abstract].
|
| 66.
|
Wang, J. F.,
I. W. Park, and J. E. Groopman.
2000.
Stromal cell-derived factor-1 stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C.
Blood
95:2505-2513[Abstract/Free Full Text].
|
| 67.
|
Wyatt, R., and J. Sodroski.
1998.
The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens.
Science
280:1884-1888[Abstract/Free Full Text].
|
| 68.
|
Yang, O. O.,
S. L. Swanberg,
Z. Lu,
M. Dziejman,
J. McCoy,
A. D. Luster,
B. D. Walker, and S. H. Herrmann.
1999.
Enhanced inhibition of human immunodeficiency virus type 1 by Met-stromal-derived factor 1 correlates with down-modulation of CXCR4.
J. Virol.
73:4582-4589[Abstract/Free Full Text].
|
| 69.
|
Zheng, J.,
A. Ghorpade,
D. Niemann,
R. L. Cotter,
M. R. Thylin,
L. Epstein,
J. M. Swartz,
R. B. Shepard,
X. Liu,
A. Nukuna, and H. E. Gendelman.
1999.
Lymphotropic virions affect chemokine receptor-mediated neural signaling and apoptosis: implications for human immunodeficiency virus type 1-associated dementia.
J. Virol.
73:8256-8267[Abstract/Free Full Text].
|
| 70.
|
Zhu, T.,
H. Mo,
N. Wang,
D. S. Nam,
Y. Cao,
R. A. Koup, and D. D. Ho.
1993.
Genotypic and phenotypic characterization of HIV-1 patients with primary infection.
Science
261:1179-1181.
|
| 71.
|
Zou, Y. R.,
A. H. Kottmann,
M. Kuroda,
I. Taniuchi, and D. R. Littman.
1998.
Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development.
Nature
393:595-599[CrossRef][Medline].
|
Journal of Virology, December 2001, p. 11534-11543, Vol. 75, No. 23
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.23.11534-11543.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Murakami, T., Kumakura, S., Yamazaki, T., Tanaka, R., Hamatake, M., Okuma, K., Huang, W., Toma, J., Komano, J., Yanaka, M., Tanaka, Y., Yamamoto, N.
(2009). The Novel CXCR4 Antagonist KRH-3955 Is an Orally Bioavailable and Extremely Potent Inhibitor of Human Immunodeficiency Virus Type 1 Infection: Comparative Studies with AMD3100. Antimicrob. Agents Chemother.
53: 2940-2948
[Abstract]
[Full Text]
-
Zhang, L. F., Okuma, K., Tanaka, R., Kodama, A., Kondo, K., Ansari, A. A., Tanaka, Y.
(2008). Generation of Mature Dendritic Cells with Unique Phenotype and Function by In Vitro Short-Term Culture of Human Monocytes in the Presence of Interleukin-4 and Interferon-{beta}. Exp. Biol. Med.
233: 721-731
[Abstract]
[Full Text]
-
Kubo, Y., Yokoyama, M., Yoshii, H., Mitani, C., Tominaga, C., Tanaka, Y., Sato, H., Yamamoto, N.
(2007). Inhibitory role of CXCR4 glycan in CD4-independent X4-tropic human immunodeficiency virus type 1 infection and its abrogation in CD4-dependent infection. J. Gen. Virol.
88: 3139-3144
[Abstract]
[Full Text]
-
Nimura, F., Zhang, L. F., Okuma, K., Tanaka, R., Sunakawa, H., Yamamoto, N., Tanaka, Y.
(2006). Cross-linking cell surface chemokine receptors leads to isolation, activation, and differentiation of monocytes into potent dendritic cells.. Exp. Biol. Med.
231: 431-443
[Abstract]
[Full Text]
-
Navenot, J.-M., Wang, Z., Chopin, M., Fujii, N., Peiper, S. C.
(2005). Kisspeptin-10-Induced Signaling of GPR54 Negatively Regulates Chemotactic Responses Mediated by CXCR4: a Potential Mechanism for the Metastasis Suppressor Activity of Kisspeptins. Cancer Res.
65: 10450-10456
[Abstract]
[Full Text]
-
Ichiyama, K., Yokoyama-Kumakura, S., Tanaka, Y., Tanaka, R., Hirose, K., Bannai, K., Edamatsu, T., Yanaka, M., Niitani, Y., Miyano-Kurosaki, N., Takaku, H., Koyanagi, Y., Yamamoto, N.
(2003). A duodenally absorbable CXC chemokine receptor 4 antagonist, KRH-1636, exhibits a potent and selective anti-HIV-1 activity. Proc. Natl. Acad. Sci. USA
100: 4185-4190
[Abstract]
[Full Text]
-
Kollet, O., Petit, I., Kahn, J., Samira, S., Dar, A., Peled, A., Deutsch, V., Gunetti, M., Piacibello, W., Nagler, A., Lapidot, T.
(2002). Human CD34+CXCR4- sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation. Blood
100: 2778-2786
[Abstract]
[Full Text]
Top
Abstract
Introduction
