| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Laboratory of AIDS Immunology, the Research Center for Immunodeficiency Virus, and
Department of Subcellular Biogenesis, Institute for Virus Research, Kyoto University, Kyoto; and
Shionogi Institute for Medical Science, Osaka, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and MIP-1ß, was identified as a cofactor for M-tropic HIV-1
infection (11, 12, 13, 14, 15), which gave a reasonable explanation for the
precedent observation that CD8+ T cell-derived factors,
mostly CC chemokines, could suppress infection of M-tropic HIV-1
strains (16). Two other members of this family, CCR-3 and CCR-2b, have
also been reported to support infection of some dual tropic HIV-1
strains (14, 15). Subsequently, a ligand for fusin was identified as
SDF-1, a bone marrow stromal cell-derived factor (17, 18) that had been
molecularly cloned and was known to stimulate the growth of pre-B cells
in mouse (19, 20). SDF-1, a CXC chemokine, has been shown to inhibit
entry of T-tropic HIV-1. Hence, redesignation of fusin as CXCR-4 has
been proposed. CCR-5 and CXCR-4 have been considered to be the major coreceptors for M-tropic and T-tropic HIV-1, respectively, while the number of HIV-1 coreceptor species has been increasing as several novel chemokine receptor-like molecules have been found to support HIV infection (21, 22, 23). CCR-5 plays an important role in sexual transmission of primary M-tropic viruses (24), which is supported by the recent genetic studies with Caucasian cohorts indicating that individuals homozygous for a CCR-5 deletion mutant are resistant to HIV-1 infection (25, 26, 27). On the other hand, T-tropic and syncytium-inducing viruses that use CXCR-4 emerge in many individuals during the late clinical stage of infection. The appearance of T-tropic HIV-1 correlates with the rapid decline of CD4+ T cells and the development of an immunodeficiency state (28, 29). The cell tropism of HIV-1 is though to be largely determined at the level of virus entry and mapped to the V3 region of the gp120 envelope protein (30, 31, 32, 33, 34). Recently, CXCR-4 as well as CCR-5 have been demonstrated to make a trimeric complex together with CD4 and gp120 (35, 36, 37). The V3 loop of M-tropic virus has been reported to be essential for interaction with CCR-5. Although these data suggest that gp120 of T-tropic or M-tropic virus directly interacts with the relevant coreceptor to initiate the process of viral entry, the precise mechanism of cell tropism remains to be elucidated partly because cell surface expression of these coreceptors on target cells has not been thoroughly described.
Apart from the unexpected link to HIV-1, SDF-1 has several unique properties distinct from those of other chemokines. First, it is a potent chemoattractant for resting lymphocytes (38) and CD34+ hemopoietic progenitor cells (39). Second, it is produced by bone marrow stromal cells constitutively (19, 20), whereas many other chemokines are in principle released by leukocytes upon proinflammatory stimulation. Third, it has several essential functions in development, because SDF-1 knockout mice have been reported to die perinatally, having hypoplasia of B cells in both liver and bone marrow, hypoplasia of myeloid cells in bone marrow, and a defect of the cardiac interventricular septum (40). These facts denote that the signals transmitted through CXCR-4, the receptor for SDF-1, cause unique and crucial events in various aspects, especially in the hemopoietic system. Thus, it is important to define the distribution and function of CXCR-4 in different cell types and biochemically characterize CXCR-4 as a membrane molecule in more detail.
In the present study, three independent mAbs against human CXCR-4 have been established to detect its expression at the single cell level and dissect the postbinding events of T-tropic HIV infection. By using these mAbs, we analyzed cell surface expression of CXCR-4 on cell lines and subsets of peripheral blood leukocytes. In parallel, we examined the effects of these mAbs on env-mediated fusion and infection of T-tropic and M-tropic viruses. Here we show that cell surface expression of CXCR-4 is not ubiquitous but cell type specific in hemopoietic cells and that three anti-CXCR-4 mAbs suppressed env-mediated fusion and infection of a T-tropic virus with different potencies but had no effect on those of an M-tropic virus.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Jurkat, HPB-ALL, HSB-2, SupT1, Molt-4, Raji, Daudi, HL-60, U937, KG-1, and HeLaS3 were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS and 30 µg/ml tobramycin. Kit225, a human IL-2-dependent T cell line (41), was cultured with 0.5 nM recombinant human IL-2 (provided by Shionogi Pharmaceutical Co., Osaka, Japan), and BAF-B03, a mouse pro-B cell line (42) (provided by Dr. S. Yonehara, Institute for Virus Research, Kyoto University, Kyoto, Japan), was cultured with 5 x 10-5 2-ME and 1% WEHI-3-conditioned medium in RPMI 1640 supplemented with 10% FCS and 30 µg/ml tobramycin. COS-7, NIH-3T3, GI (gliosarcoma) (43), U-251 (glioblastoma) (44), and IMR-32 (neuroblastoma) (45) were cultured in DMEM (Life Technologies) supplemented with 10% FCS and 30 µg/ml tobramycin. PBMC were isolated by Ficoll-Hypaque density gradient centrifugation from heparinized venous blood taken from healthy donors as previously described (41). Neutrophils were separated according to the standard method of Ficoll sedimentation followed by removal of mononuclear cells by density gradient centrifugation and hypotonic lysis of RBC (46).
Generation of a murine cell line stably transfected with human CXCR-4
Human CXCR-4 (LESTR) cDNA (10) was amplified by PCR from a cDNA library of ATL-43T (47), an ATL-derived human T cell line, using the LA-PCR kit (Takara Shuzo, Otsu, Japan) with an EcoRI site-tagged forward primer (5'-GGAATTCCAGTAGCCACCGCATCTGGAGA) and an XbaI site-tagged reverse primer (5'-GCTCTAGAGTTAGCTGGAGTGAAAACTTGA) covering an entire coding region. The 1.1-kb PCR product was isolated, digested with EcoRI and XbaI, and inserted into a mammalian expression vector, pMKITneo (provided by Dr. K. Maruyama, Tokyo Medical and Dental University, Tokyo, Japan). The cloned cDNA was confirmed to be CXCR-4 by nucleotide sequencing using an automated sequencer ABI Prism310 (Perkin-Elmer, Foster City, CA). The plasmid was linearized by digestion with CpoI and introduced into BAF-B03 by electroporation. After 2 days of culture, cells were plated in 96-well plates in medium containing 1.5 mg/ml geneticin (Life Technologies). Geneticin-resistant transfectant clones were screened for expression of CXCR-4 mRNA by reverse transcriptase-PCR using a forward primer (5'-GGAAATGGGCTCAGGGGACTAT) and a reverse primer (5'-GACCACCTTTTCAGCCAACAGC). One of the clones with the highest CXCR-4 mRNA expression was chosen and expanded for immunization.
Establishment of anti-CXCR-4 mAbs
Eight-week-old BALB/c female mice were inoculated at bilateral footpads with 1 x 107 CXCR-4-transfected BAF-B03 cells emulsified with CFA (Sigma Chemical Co., St. Louis, MO) on day 0. On days 7 and 14, 1 x 107 cells suspended in PBS were injected into the same footpads. On day 15, the regional lymph nodes were removed, minced, and passed through a steel mesh. Released cells were fused with PAI, a partner myeloma cell line, cultured in 96-well plates, and subjected to hypoxanthine-aminopterin-thymidine selection. After 8 to 12 days, culture supernatants were screened by flow cytometry based on difference in reactivity between CXCR-4 transfectant and parental BAF-B03 cells. The culture supernatants of the candidate wells were next examined for the reactivity with COS-7 cells transiently transfected by the DEAE-dextran method with CXCR-4 as well as other irrelevant membrane molecules as described previously (47). After recloning, culture supernatants containing mAbs were typed by a mouse mAb isotyping kit (Amersham Life Science, Arlington Heights, IL). Each mAb was purified from ascitic fluid using a mouse IgG purification kit (Amersham).
Monoclonal Abs and flow cytometry
FITC-conjugated mAbs, Leu 4 (anti-CD3), Leu 3
(anti-CD4), Leu 2 (anti-CD8), Leu 12 (anti-CD19), Leu
11a (anti-CD16), Leu M3 (anti-CD14), and control mouse IgGs
were purchased from Becton Dickinson (San Jose, CA). H107
(anti-CD23) mAb (48) was provided by Dr. J. Yodoi (Institute for
Virus Research, Kyoto University, Kyoto, Japan). OKT4 (anti-CD4)
mAb was purified from ascitic fluid in our laboratory. Anti-CXCR-4 mAbs
and isotype-matched control mouse IgGs (Sigma) were biotinylated with
EZ-Link Biotin-BMCC (Pierce Chemical Co., Rockford, IL) according to
the manufacturer’s instructions. Direct or indirect immunofluorescence
staining was performed as described previously (49). Two-color analysis
was conducted as follows. Cells were first incubated with 1 mg/ml human
IgG on ice for 30 min to block nonspecific binding and then with
biotinylated anti-CXCR-4 mAb and FITC-labeled mAb on ice for 30
min. After washing, cells were incubated with PE-conjugated
streptavidin (Becton Dickinson) on ice for 30 min, washed, and
subjected to flow cytometric analysis using a FACScan (Becton Dickinson
Imunocytometry Systems, San Jose, CA). Reactivity was determined by
comparison to control stainings with biotinylated or FITC-conjugated
irrelevant mouse IgG.
Cell surface biotinylation and immunoprecipitation
Cell surface biotinylation and immunoprecipitation were performed as previously described (50). In brief, cells were washed with PBS and incubated in PBS containing 0.5 mg/ml NHS-LC-biotin (Pierce) at room temperature for 15 min. Free succinimide groups were then blocked by the addition of 5 ml of nonsupplemented RPMI 1640 medium at room temperature for 5 min. After washing, cells were lysed in 0.5 ml of lysis buffer (1% Triton X-100, 137 mM NaCl, 10 or 20% glycerol, and 20 mM Tris-HCl, pH 8.0) supplemented with 1 mM PMSF and a mixture of protease inhibitors (5 µg/ml antipain, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 7.5 µg/ml bestatin, and 5 µg/ml trypsin inhibitor) on ice for 20 min. Cell lysates were centrifuged at 15,000 rpm for 20 min, and the supernatants were collected and precleared by incubation with protein A-Sepharose FF (Pharmacia Biotech AB, Uppsala, Sweden) at 4°C for 1 h. Precleared lysates were incubated with mAbs and protein A-Sepharose at 4°C for 4 h with gentle mixing by a rotator. Then Sepharose beads were washed with lysis buffer five times. The immunoprecipitates were eluted in SDS sample buffer, electrophoresed through 7.5 or 10% acrylamide gel, and visualized by Western blot using horseradish peroxidase-conjugated streptavidin (Amersham) and an enhanced chemiluminescence (ECL) detection system (Amersham).
Measurement of Ca2+ mobilization
Intracellular Ca2+ mobilization was measured as previously described (17). In brief, 1 x 107 SupT1 cells were loaded with 5 µM fura-2/AM fluorescence dye (Molecular Probes, Eugene, OR) in loading buffer (136 mM NaCl, 48 mM KCl, 1 mM CaCl2, 1 mg/ml glucose, and 20 mM HEPES, pH 7.4) at 37°C for 20 min. The loaded cells were centrifuged and resuspended in fresh loading buffer, incubated with or without 50 µg/ml mAbs for 30 min, and then applied to a fluorescence spectrophotometer F2000 (Hitachi, Tokyo, Japan). Human SDF-1ß (R&D Systems, Minneapolis, MN) was added to a final concentration of 1 µg/ml, and increases in intracellular Ca2+ were measured.
Assay of cell fusion mediated by HIV-1 envelope proteins
HIV-1 env-mediated fusion was assayed by the method
using subunit complementation of ß-galactosidase and will be
described in detail elsewhere (K. Igarashi, S. Minoguchi, M. Murata,
and H. Shida, in preparation). In brief, HeLaS3 cells were infected
with two vaccinia virus vectors containing env of HIV-1
NL432 (T-tropic) (51) or JRCSF (M-tropic) (52) and the subunit of
ß-galactosidase, respectively. NIH-3T3 cells were infected with three
vaccinia virus vectors containing human CD4, the 
subunit of
ß-galactosidase, and T7 RNA polymerase, respectively, which were
subsequently transfected with CXCR-4 or CCR-5 inserted in the
downstream of T7 promotor of pBluescript using Lipofectamine (Life
Technologies). After 16 h of incubation at 37°C, cells were
washed with PBS containing 0.5 mM CaCl2 and resuspended in
HBSS, pH 7.65, containing 3 mM CaCl2. NIH-3T3 cells were
incubated with or without 500 µg/ml mAbs for 1 h and then mixed
with HeLaS3 cells. For comparison, OKT-4 (anti-CD4 mAb), which has
modest inhibitory effects on HIV infection (53), was used. Cells were
plated in 24-well plates (Biocoat Cellware, rat tail collagen, type I,
Becton Dickinson), centrifuged at 1300 rpm for 5 min, and incubated at
37°C overnight. Then cells were lysed in 200 µl of chlorophenol
red-ß-D-galactopyranoside solution (8 mM chlorophenol
red-ß-D-galactopyranoside, 45 mM 2-ME, 1 mM
MgCl2, 0.1 M HEPES (pH 8.0), and 0.1 mg/ml DNase) and
incubated at 37°C for 30 min. Reactions were stopped by adding SDS
solution (final concentration, 1%), and the OD590 nm of
each sample was measured.
Detection and measurement of HIV-1 infection
The wild-type NL432 strain of HIV-1 was produced by transfection of the human colon carcinoma cell line SW480 with pNL432 infectious clone (51). M8166 cells were then infected with the virus, and the culture supernatants were collected after the appearance of cytopathic effects, filtrated, and stored frozen in aliquots at -80°C. These culture supernatants (reverse transcriptase activity = 4,000 cpm/µl) were used as T-tropic HIV-1 virus. The NL162 strain of HIV-1 was produced from a hybrid clone, pNL162, in which the region including env (EcoRI/BamHI) of the backbone pNL432 was replaced by that of SF162 (54). The culture supernatants containing NL162 virus (reverse transcriptase activity = 2000 cpm/µl) were prepared as described above and used as M-tropic virus. HIV-1 infection was measured by tat-induced activation of ß-galactosidase as follows. HeLa cells that had been stably transfected with CD4 and HIV-1 long terminal repeat (LTR)-ß-galactosidase were first incubated with or without mAbs in flat-bottom 48-well plates at 37°C for 1 h. Cells were then mixed with NL432 or NL162 viruses in a total volume of 200 µl and cultured at 37°C in a humid atmosphere with 5% CO2. After 4 days, culture supernatants were discarded, and cells were lysed in 200 µl of reporter lysis buffer (Promega, Madison, WI) by freezing and thawing. Twenty microliters of soluble fraction from each sample was assayed with a luminometer using a ß-Galassay kit (Clontech, Palo Alto, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
BALB/c mice were immunized with a stable human CXCR-4 transfectant
of a mouse BAF-B03 pro-B cell line. Culture supernatants of hybridomas
growing in 96-well plates were screened by flow cytometry for
differences in reactivity with CXCR-4 transfectants and parental cells.
Three mAbs, named IVR7, AI58, and THS123, were found to react with the
transfectants but not with the parental cells. These mAbs were
typed as IgG1, IgG2b, and IgG3, respectively, and were demonstrated to
react specifically with COS-7 cells transiently transfected with CXCR-4
but not those transfected with CD23, indicating that they specifically
recognize CXCR-4 expressed on the cell surface (Fig. 1
). These mAbs also did not react with
BAF-B03 stably transfected with CCR-5 (data not shown).
|
Biochemical analysis of the Ag molecule recognized by the mAbs was
first tried by SDS-PAGE and immunoblotting. However, despite extensive
trials under various conditions, Western blotting by none of the three
mAbs was successful. Therefore, we next conducted immunoprecipitation
of the Ag molecule by cell surface biotinylation followed by incubation
of the cell lysates with mAbs and protein A-Sepharose. The
immunoprecipitates were visualized by Western transfer and the ECL
detection system. It was difficult to decrease nonspecific bands
without losing most of the immunoprecipitates. Changing lysis buffers
and washing conditions resulted in little improvement. Nevertheless, a
cellular component with an apparent molecular mass of 47 kDa was
immunoprecipitated by THS123 but not by isotype-matched control mouse
IgG from human SupT1 as well as from BAF-B03 transfected with human
CXCR-4 (Fig. 2). The molecular mass of
this specific band was consistent with the size of fusin (CXCR-4)
determined previously by immunoblotting with rabbit antisera against
the synthetic peptide of its NH2-terminus (5). To
date, attempts to immunoprecipitate with IVR7 or IA58 have not been
successful.
|
To determine whether the three mAbs had antagonistic activities
against SDF-1, transient increases in intracellular
Ca2+ were measured. As shown in Figure 3, an addition of 1 µg/ml SDF-1
elicited rapid and transient Ca2+ mobilization in SupT1
cells in the absence of mAbs, which abrogated responsiveness to a
subsequent stimulation with the same ligand. Pretreatment with the
three mAbs inhibited SDF-1-induced Ca2+ mobilization with
different magnitudes compared with that of the control mouse IgG; IVR7
completely inhibited this response, while THS123 and AI58 exhibited
partial and slight inhibition, respectively. These data further confirm
the specificity of the three mAbs to CXCR-4 and suggest that they react
with different epitopes on CXCR-4.
|
Cell lines of various cell lineages were examined for expression
of CXCR-4 by indirect immunofluorescence staining and flow cytometry.
The specificity and staining patterns of the three mAbs were
essentially the same, except that AI58 stains cells slightly weaker
than IVR7 or THS123. Therefore, we used THS123 as a representative for
the following analyses of CXCR-4 expression. The summary of the results
is shown in Table I. As indicated from
Northern blot analysis data reported by others (9, 10), most
hemopoietic cell lines and some nonhemopoietic cell lines expressed
CXCR-4, although the levels of expression varied considerably. In
general, T cell lines and B cell lines expressed high levels of
CXCR-4. Two glial cell lines, GI and U-251, did not express detectable
levels of CXCR-4. It was unexpected that KG-1, a CD34+
myeloid cell line, expressed no cell surface CXCR-4, since normal
CD34+ hemopoietic progenitor cells have been reported to
respond well to SDF-1, the ligand of CXCR-4 (39). Next we examined the
expression of CXCR-4 on normal peripheral blood leukocytes. As shown in
Figure 4, >50% of fresh PBMC expressed
detectable levels of CXCR-4. The percentage of positive cells and
fluorescence intensity were somewhat variable among donors.
Furthermore, these values depended on the method of staining, since the
amount of CXCR-4 on individual cells varied widely, and there were
cells that expressed low levels of CXCR-4 close to the detection limit.
In contrast to mononuclear cells, we did not detect any cell surface
expression of CXCR-4 on neutrophils. This finding was reproducible even
in experiments using more sensitive PE-conjugated reagents (data not
shown). To examine expression of CXCR-4 on subsets of PBMC, two-color
flow cytometric analysis was conducted with a sensitive detection
system using biotinylated anti-CXCR-4 mAb and PE-conjugated
streptavidin. Analyses with five different donors gave similar results,
and data from a representative experiment are shown in Figure 5. The majority of, but not all, T cells
(CD3+) expressed CXCR-4, virtually all B cells
(CD19+) and all monocytes (CD14+) expressed
CXCR-4, while NK cells (CD16+) expressed very low levels
of, if any, CXCR-4. Both CD4+ and CD8+ subsets
of T cells contained subpopulations negative for CXCR-4. When PBMC were
activated with mitogens, expression of CXCR-4 was up-regulated in both
T cells and NK cells, but some T cells still remained negative for
CXCR-4 (data not shown).
|
|
|
We first examined the effects of anti-CXCR-4 mAbs on HIV-1
env-mediated fusion by a sensitive and specific assay system
in which the magnitude of fusion could be measured by subunit
complementation of ß-galactosidase upon fusion between effector
HeLaS3 cells expressing the envelope protein of HIV-1 and target
NIH-3T3 cells expressing CD4 together with CXCR-4 or CCR-5. As shown in
Figure 6, HeLaS3 cells that expressed the
envelope protein of NL432 (51), a T-tropic strain, efficiently fused
with NIH-3T3 cells that expressed CXCR-4, which was suppressed by the
anti-CXCR-4 mAbs. IVR7 and THS123 showed strong suppression
comparable to that by OKT4, while that of AI58 was modest. These mAb
had no effect on the fusion between HeLaS3 cells expressing the
envelope protein of JRCSF (52), an M-tropic strain, and NIH-3T3 cells
expressing CCR-5, while OKT4 was effective. These results confirmed the
specificity of our mAbs to CXCR-4 and support the idea that the cell
surface CXCR-4 is directly involved in interaction with the envelope
protein of T-tropic HIV-1 to initiate the process of fusion.
|
We next investigated the effects of anti-CXCR-4 mAbs on
cell-free infection of HIV-1 using HeLa-CD4 stably transfected with
ß-galactosidase under the regulation of HIV-1 LTR. The extent of
virus infection was measured by ß-galactosidase activity induced by
HIV-1 tat after infection of T-tropic and M-tropic viruses. It is known
that a T-tropic virus can infect HeLa-CD4 through endogenous CXCR-4. In
the case of M-tropic virus infection, CCR-5 was transfected into cells
in advance to confer on otherwise insensitive cells susceptibility to
M-tropic virus infection. As shown in Figure 7, the three anti-CXCR-4 mAbs
suppressed infection of a T-tropic virus, NL432 (51). In particular,
IVR7 mAb exhibited >98% suppression at 500 µg/ml, while THS123 and
AI58 showed modest, but significant, suppression as well. In contrast,
infection of an M-tropic hybrid virus, NL162 (54), was not affected by
any of these mAbs. Both viruses were inhibited by OKT4 or dextran
sulfate. Figure 8 shows a dose-response
suppression of infection of NL432 by anti-CXCR-4 mAbs. IVR7,
THS123, and AI58 in this order showed strong to relatively weak
suppressive effects. At 20 µg/ml, IVR7 still suppressed T-tropic
HIV-1 infection considerably, while AI58 exerted no effect.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous studies have indicated that most hemopoietic cell lines and some nonhemopoietic cell lines express CXCR-4 (9, 10). Our data overall are consistent with this, and we detected expression of CXCR-4 on cell lines of various cell lineages. It was unexpected, however, that KG-1, a CD34+ hemopoietic cell line, expressed no cell surface CXCR-4. In normal peripheral blood leukocytes, CXCR-4 was expressed on mononuclear cells but not on neutrophils. The absence of cell surface CXCR-4 on neutrophils was confirmed by staining with sensitive PE-conjugated reagents. As far as we know, two independent studies demonstrated that neutrophils expressed significant amounts of CXCR-4 mRNA (9, 10), in sharp contrast to our results using flow cytometry. Nevertheless, our results are in accordance with those of a recent functional study in which it was shown that neutrophils did not respond to SDF-1 (38). It may be that translation or transfer to the cell membrane of the CXCR-4 protein is blocked in neutrophils. Another study of SDF-1 has recently indicated that CD34+ hemopoietic progenitor cells respond to SDF-1 with chemotaxis, suggesting that these cells express cell surface CXCR-4 (39). Accordingly, it seems likely that progenitors of neutrophils down-modulate the expression of cell surface CXCR-4 as they mature until they are finally detached from bone marrow. In this context, KG-1 may represent abnormal loss of cell surface CXCR-4 at the myeloblast stage that might result in detachment and peripheral circulation of leukemic cells. To clarify the mechanism of homing and release of hemopoietic progenitors, it is important to analyze the expression of cell surface CXCR-4 at various stages of maturation. Two-color analysis with normal PBMC indicated that the majority of, but not all, T cells, virtually all B cells, and all monocytes expressed CXCR-4, while it was only weakly present on NK cells. It was noted that there were subpopulations of T cells negative for CXCR-4 in both CD4+ and CD8+ subsets. Furthermore, we observed that the intensity of CXCR-4 expression on PBMC is variable among individuals. Therefore, it is theoretically possible that the susceptibility of an individual to T-tropic HIV-1 is affected by the proportion of CXCR-4+ cells and the average density of CXCR-4 in PBMC.
It is now widely accepted that cell tropism is determined by selection of the coreceptor at the level of cell entry. Recently, both CXCR-4 and CCR-5 have been demonstrated to make a trimeric complex with CD4 and gp120 (35, 36, 37). Particularly, in the case of gp120 of M-tropic virus, direct interaction of V3 loop with CCR-5 has been strongly implicated by experiments using chimeric gp120 variants and neutralizing mAb against V3 epitopes. Thus, direct interaction of gp120 with the relevant coreceptor is postulated to trigger a series of events leading to viral entry. However, the mechanism of cell tropism could not be thoroughly understood without knowing the cell surface expression of the coreceptors on target cells. To date, it has been conceived that cell type-specific expression of a certain type of coreceptor may explain the susceptibility to T-tropic or M-tropic virus. Expression of CCR-5 has been reported to be narrowly restricted to certain cell types of hemopoietic lineage, including monocytes (56, 57), which may be the reason why M-tropic virus only inefficiently infect T cells. However, the inefficient infection of monocytes by T-tropic virus cannot be explained in the same way, since we have shown that significant levels of cell surface CXCR-4 is expressed on peripheral blood monocytes. Our results are again consistent with those of the functional study showing that monocytes respond well to SDF-1 (38). These data suggest that the cell tropism or the susceptibility to a certain type of virus is not simply the consequence of cell type-specific expression of the relevant coreceptor. At least three possible explanations should be considered as follows. First, gp120 of T-tropic virus may require a high density of CD4, as expressed on T cells, to interact with CXCR-4, while a low density of CD4, as expressed on monocytes, is sufficient for gp120 of M-tropic virus to interact with CCR-5. Second, coexpression of cell surface CCR-5 may interfere with the interaction between gp120 of T-tropic virus and CXCR-4. Third, there may be a cell type-specific difference in the presentation of CXCR-4 that determines its availability as a coreceptor, as suggested by others (58).
The three mAbs were shown to suppress cell fusion mediated by the envelope protein of T-tropic virus and CXCR-4 but not that mediated by the envelope protein of M-tropic virus and CCR-5, which further confirmed the specificity of our mAbs to CXCR-4 and supported the hypothesis that interaction of the viral envelope protein with cell surface CXCR-4 is essential for HIV-1 env-mediated fusion. Similarly, these mAbs suppressed infection of T-tropic virus, but not that of M-tropic virus, measured by tat-induced ß-galactosidase activity. In both experiments using a T-tropic strain, NL432, it was noted that the suppressive activity varied considerably among the mAbs, suggesting that the epitopes on the CXCR-4 molecule recognized by the three mAbs may have different roles in interaction with the envelope protein of T-tropic HIV-1. It has been reported that 12G5, an anti-CXCR-4 mAb, could not always inhibit entry and fusion of T-tropic HIV-1 and that the inhibition was both cell type and virus strain dependent (58). Although we have not tested many other HIV-1 strains, preliminary experiments have indicated that at least one of our mAbs is able to strongly suppress the infection of Jurkat cells by HIV-1 IIIB. Our future studies will examine the interaction between the HIV-1 envelope protein and CXCR-4 by comparing the suppressive effects of the three mAbs on infection of different strains of HIV-1 as well as HIV-2 in different cell types. In conclusion, the newly developed three mAbs against CXCR-4 enable us to study the function of the SDF-1/CXCR-4 system in various types of cells and delineate the CXCR-4 molecule with regard to interaction with the HIV-1 envelope protein.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Toshiyuki Hori, Institute for Virus Research, Kyoto University, 53 Kawaracho, Shogoin, Sakyoku, Kyoto 606, Japan. E-mail address:
3 Abbreviations used in this paper: T-tropic, T cell-tropic; M-tropic, macrophage-tropic; MIP, macrophage-inflammatory protein; PE, phycoerythrin; LTR, long terminal repeat.
Received for publication March 11, 1997. Accepted for publication September 11, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, MIP-1ß receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955.[Abstract]
, and MIP-1ß as the major HIV-suppressive factors produced by CD8+ T cells. Science 270:1811.
R(+) human lymphocytes. J. Immunol. 137:1258.[Abstract]
, MIP-1ß, and RANTES. J. Leukocyte Biol. 60:147.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  A. Miles, E. Liaskou, B. Eksteen, P. F. Lalor, and D. H. Adams CCL25 and CCL28 promote {alpha}4{beta}7-integrin-dependent adhesion of lymphocytes to MAdCAM-1 under shear flow Am J Physiol Gastrointest Liver Physiol, May 1, 2008; 294(5): G1257 - G1267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ouellet, S. Mercier, I. Pelletier, S. Bounou, J. Roy, J. Hirabayashi, S. Sato, and M. J. Tremblay Galectin-1 Acts as a Soluble Host Factor That Promotes HIV-1 Infectivity through Stabilization of Virus Attachment to Host Cells J. Immunol., April 1, 2005; 174(7): 4120 - 4126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Tanaka, A. Yoshida, T. Murakami, E. Baba, J. Lichtenfeld, T. Omori, T. Kimura, N. Tsurutani, N. Fujii, Z.-X. Wang, et al. 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 J. Virol., December 1, 2001; 75(23): 11534 - 11543. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. K. Gupta, K. Pillarisetti, and N. Aiyar CXCR4 undergoes complex lineage and inducing agent-dependent dissociation of expression and functional responsiveness to SDF-1{alpha} during myeloid differentiation J. Leukoc. Biol., September 1, 2001; 70(3): 431 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Dragic An overview of the determinants of CCR5 and CXCR4 co-receptor function J. Gen. Virol., August 1, 2001; 82(8): 1807 - 1814. [Full Text] [PDF]
|
|
|
|
 |
|
 T. Koshiba, R. Hosotani, Y. Miyamoto, J. Ida, S. Tsuji, S. Nakajima, M. Kawaguchi, H. Kobayashi, R. Doi, T. Hori, et al. Expression of Stromal Cell-derived Factor 1 and CXCR4 Ligand Receptor System in Pancreatic Cancer: A Possible Role for Tumor Progression Clin. Cancer Res., September 1, 2000; 6(9): 3530 - 3535. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. Wünschmann and J. T. Stapleton Fluorescence-Based Quantitative Methods for Detecting Human Immunodeficiency Virus Type 1-Induced Syncytia J. Clin. Microbiol., August 1, 2000; 38(8): 3055 - 3060. [Abstract] [Full Text]
|
|
|
|
|
|
P. Jourdan, J.-P. Vendrell, M.-F. Huguet, M. Segondy, J. Bousquet, J. Pene, and H. Yssel Cytokines and Cell Surface Molecules Independently Induce CXCR4 Expression on CD4+ CCR7+ Human Memory T Cells J. Immunol., July 15, 2000; 165(2): 716 - 724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 C. Chelucci, I. Casella, M. Federico, U. Testa, G. Macioce, E. Pelosi, R. Guerriero, G. Mariani, A. Giampaolo, H.J. Hassan, et al. Lineage-Specific Expression of Human Immunodeficiency Virus (HIV) Receptor/Coreceptors in Differentiating Hematopoietic Precursors: Correlation With Susceptibility to T- and M-Tropic HIV and Chemokine-Mediated HIV Resistance Blood, September 1, 1999; 94(5): 1590 - 1600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
H. Sakaida, T. Hori, A. Yonezawa, A. Sato, Y. Isaka, O. Yoshie, T. Hattori, and T. Uchiyama T-Tropic Human Immunodeficiency Virus Type 1 (HIV-1)-Derived V3 Loop Peptides Directly Bind to CXCR-4 and Inhibit T-Tropic HIV-1 Infection J. Virol., December 1, 1998; 72(12): 9763 - 9770. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
T. Shioda, H. Kato, Y. Ohnishi, K. Tashiro, M. Ikegawa, E. E. Nakayama, H. Hu, A. Kato, Y. Sakai, H. Liu, et al. Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1alpha (SDF-1alpha ) and SDF-1beta are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage PNAS, May 26, 1998; 95(11): 6331 - 6336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Jourdan, C. Abbal, N. Nora, T. Hori, T. Uchiyama, J.-P. Vendrell, J. Bousquet, N. Taylor, J. Pene, and H. Yssel Cutting Edge: IL-4 Induces Functional Cell-Surface Expression of CXCR4 on Human T Cells J. Immunol., May 1, 1998; 160(9): 4153 - 4157. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
