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The Journal of Immunology, 1998, 160: 180-188.
Copyright © 1998 by The American Association of Immunologists

Detection and Delineation of CXCR-4 (Fusin) as an Entry and Fusion Cofactor for T Cell-Tropic HIV-1 by Three Different Monoclonal Antibodies1

Toshiyuki Hori2,*, Hitoshi Sakaida*, Akihiko Sato{ddagger}, Toshihiro Nakajima{ddagger}, Hisatoshi Shida{dagger}, Osamu Yoshie{ddagger} and Takashi Uchiyama*

* Laboratory of AIDS Immunology, the Research Center for Immunodeficiency Virus, and {dagger} Department of Subcellular Biogenesis, Institute for Virus Research, Kyoto University, Kyoto; and {ddagger} Shionogi Institute for Medical Science, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A chemokine receptor, CXCR-4, has been identified as an entry cofactor for T cell line-tropic (T-tropic) HIV-1. To detect expression of CXCR-4 at the single cell level and dissect postbinding events of HIV-1 infection, we generated three mAbs against human CXCR-4. These mAbs inhibited SDF-1-induced intracellular Ca2+ mobilization, and one of the mAbs immunoprecipitated a specific 47-kDa component from CXCR-4+ cells. Flow cytometric analysis showed that most human cell lines examined expressed CXCR-4. A fraction of normal PBMC expressed CXCR-4, but neutrophils were negative. Two-color analysis revealed that the majority of T cells, virtually all B cells, and all monocytes expressed CXCR-4, while it was only weakly present on NK cells. Thus, expression of CXCR-4 is not ubiquitous but cell type specific in hemopoietic cells. The three mAbs were shown to suppress cell fusion mediated by envelope proteins of a T-tropic NL432 virus but not by those of an M-tropic JRCSF virus. Likewise, they suppressed infection of NL432 but not that of an M-tropic NL162 virus. In both cases it was noted that the suppressive activity varied considerably among the mAbs. These data confirmed that CXCR-4 is directly involved in env-mediated entry and fusion of T-tropic HIV-1 and suggest that the epitopes on CXCR-4 recognized by the three mAbs may have different roles in interaction with the envelope proteins of T-tropic HIV-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
For more than a decade, accumulating evidence had indicated that a cell surface cofactor(s) in addition to CD4 is required for entry and fusion of HIV-1 (1, 2, 3, 4). In 1996, this idea was finally substantiated by functional cDNA cloning of a cofactor for T cell-tropic (T-tropic)3 HIV-1 infection (5). The first described HIV-1 entry cofactor, named fusin, was found to be a seven-transmembrane domain glycoprotein identical with one of the orphan chemokine receptors whose cDNA had been reported (6, 7, 8, 9, 10). It was shown that fusin enabled cells in conjunction with CD4 to support entry and fusion of T-tropic but not of macrophage-tropic (M-tropic) HIV-1 strains. Before long, CCR-5, a receptor for RANTES, MIP-1{alpha}, 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and culture conditions

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{alpha}), 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 {alpha} 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 {alpha} subunit of ß-galactosidase, respectively. NIH-3T3 cells were infected with three vaccinia virus vectors containing human CD4, the {omega} 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of anti-CXCR-4 mAbs

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. 1Go). These mAbs also did not react with BAF-B03 stably transfected with CCR-5 (data not shown).



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  		FIGURE 1. Reactivity of the three mAbs with COS-7 cells transfected with CXCR-4. COS-7 cells were transfected with CXCR-4 cDNA or CD23 cDNA (control) inserted in a mammalian expression vector, pME18S, by the DEAE-dextran method. After 2 days, cells were detached with PBS containing 0.5 mM EDTA and 0.02% NaN3; stained with control mouse IgG1, IgG2b, IgG3, H107 (anti-CD23), IVR7, AI58, or THS123 by indirect immunofluorescence using FITC-conjugated F(ab')2 of goat anti-mouse IgG and IgM; and subjected to flow cytometric analysis.

 
Immunoprecipitation and SDS-PAGE analysis of CXCR-4

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. 2Go). 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.



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  		FIGURE 2. Immunoprecipitation by anti-CXCR-4 mAb. A, SupT1 and BAF-B03 transfected with human CXCR4 (BAF-CXCR-4) were cell surface biotinylated by NHS-LC-biotin and extracted in lysis buffer with 10% glycerol. After preclearing, cell lysates were incubated with THS123 mAb or isotype-matched irrelevant mouse IgG and protein A-Sepharose. The immunoprecipitates were electrophoresed through 7.5% polyacrylamide gel and visualized by Western blot using horseradish peroxidase-conjugated streptavidin and the ECL detection system. B, Immunoprecipitates from biotinylated SupT1 cells extracted in lysis buffer with 20% glycerol were electrophoresed through 10% polyacrylamide gel and visualized as described in A. The arrowhead indicates a specific band immunoprecipitated by anti-CXCR-4 mAb.

 
Effects of anti-CXCR-4 mAbs on intracellular Ca2+ mobilization

To determine whether the three mAbs had antagonistic activities against SDF-1, transient increases in intracellular Ca2+ were measured. As shown in Figure 3Go, 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.



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  		FIGURE 3. Effects of anti-CXCR-4 mAbs on SDF-1-induced Ca2+ mobilization. After loading of fura-2, cells were pretreated without (a) or with 50 µg/ml of control mouse IgG (b), IVR7 (c), AI58 (d), or THS123 (e) and then stimulated with 1 µg/ml SDF-1 (indicated by solid triangles). Increases in the cytosolic free Ca2+ concentration were measured by a fluorescence spectrophotometer.

 
Expression of CXCR-4 on cell lines and subsets of peripheral blood leukocytes

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 IGo. 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 4Go, >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 5Go. 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).


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  		Table I. Reactivity of anti-CXCR-4 mAb with various cell lines and peripheral blood leukocyte subsetsa

 


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  		FIGURE 4. Expression of CXCR-4 on normal PBMC and neutrophils. Mononuclear cells and neutrophils were isolated from peripheral blood as described in Materials and Methods. Cells were first incubated with 1 mg/ml human IgG to block nonspecific binding, then stained with THS123 or isotype-matched irrelevant mouse IgG by indirect immunofluorescence using THS123 and FITC-conjugated F(ab')2 of goat anti-mouse IgG and IgM. Expression of CXCR-4 was analyzed by flow cytometry using a FACScan.

 


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  		FIGURE 5. Two-color analysis of CXCR-4 expression on subsets of PBMC. Normal PBMC were first incubated with 1 mg/ml human IgG and then stained with biotinylated THS123 and FITC-conjugated mAbs (anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD16, and anti-CD14). Binding of biotinylated mAb was detected by incubation with PE-conjugated streptavidin. Left and lower quadrants correspond to the cut-off range in control stainings with FITC-conjugated or biotinylated irrelevant mouse IgG.

 
Effects of anti-CXCR-4 mAbs on env-mediated fusion of HIV-1

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 {alpha} 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 6Go, 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.



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  		FIGURE 6. Effects of anti-CXCR-4 mAbs on env-mediated fusion. The env-mediated fusion was measured in the presence or the absence of anti-CXCR-4 mAbs by an assay system using {alpha} subunit complementation of ß-galactosidase upon fusion. HeLaS3 cells expressing the envelope protein of NL432, a T-tropic strain, were fused with NIH-3T3 cells expressing CD4 and CXCR-4 (left). HeLaS3 cells expressing the envelope protein of JRCSF, an M-tropic strain, were fused with NIH-3T3 cells expressing CD4 and CCR-5 and (right). All experiments were performed in duplicate. Irrelevant mouse IgG and OKT4 were included as negative and positive controls, respectively.

 
Effects of anti-CXCR-4 mAbs on infection of HIV-1

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 7Go, 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 8Go 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.



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  		FIGURE 7. Effects of anti-CXCR-4 mAbs on cell-free infection of HIV-1. Viruses of the T-tropic wild-type NL432 strain and the M-tropic hybrid NL162 strain of HIV-1 were produced as described in Materials and Methods. HeLa-CD4 cells that had been stably transfected with the ß-galactosidase gene under the control of HIV-1 LTR were incubated with 500 µg/ml anti-CXCR-4 mAbs for 1 h before incubation with NL432 virus or NL162 virus in flat-bottom 48-well plates. In the case of NL162, CCR-5 was transfected in advance to confer upon the cells susceptibility to the virus. After 4 days, ß-galactosidase activity in cell lysates was measured by a luminometer. All experiments were performed in triplicate, and relative ß-galactosidase activity was calculated as the percentage of the value of each culture to that of cells and virus. The suppressive activity of the mAbs was compared with that of control mouse IgG. OKT4 and dextran sulfate (20 µg/ml) were included as positive controls.

 


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  		FIGURE 8. Dose-response suppression of T-tropic HIV-1 infection by anti-CXCR-4 mAbs. HeLa-CD4 cells with the reporter gene of HIV-1 LTR-ß-galactosidase were incubated with serial dilutions of anti-CXCR-4 mAbs and then infected by T-tropic NL432 virus and cultured for 4 days. Infection of the virus was measured by ß-galactosidase activity in the same way as in the experiments shown in Figure 7Go. All experiments were performed in triplicate, and each point indicates the mean value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have established three mAbs against human CXCR-4 by immunizing mice with a mouse cell line stably transfected with human CXCR-4. The specificity of these mAbs was validated by their reactivities with murine cells or COS-7 cells transfected with CXCR-4, but not those transfected with other membrane molecules, including another chemokine receptor, CCR-5. One of the mAbs, THS123, immunoprecipitated a specific band with an apparent molecular mass of 47 kDa from CXCR-4+ cells, although the signal of this band was relatively weak, and it was difficult to decrease nonspecific bands. The molecular mass of this specific band was consistent with that of fusin (CXCR-4), as estimated by immunoblotting with rabbit antisera against the synthetic peptide of its NH2-terminus (5). Western blotting with none of these mAbs has been successful. When we were characterizing the three mAbs, a report was published in which one mAb against CXCR-4, named 12G5, was described. This mAb was reported to be unable to immunoblot or immunoprecipitate the Ag molecule, CXCR-4 (55). Thus, it is probable that mAbs recognizing the extracellular domains of the CXCR-4 molecule are in general inclined to react with conformational epitopes that are sensitive to detergents. Furthermore, the three mAbs were shown to inhibit intracellular Ca2+ mobilization induced by SDF-1, the ligand of CXCR-4. These mAbs had different potencies of inhibition, suggesting that they react with different epitopes on CXCR-4. It is noteworthy that one of the mAbs, IVR7, inhibited SDF-1-induced Ca2+ immobilization completely, suggesting that this mAb reacts with an epitope on CXCR-4 identical or very close to the ligand binding site.

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
 
We thank Dr. K. Maruyama (Tokyo Medical and Dental University) for providing the plasmid vectors, and Ms. K. Fukunaga for excellent technical assistance.


   Footnotes
 
1 This work was partly supported by grants-in-aid from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan. Back

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: Back

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. Back

Received for publication March 11, 1997. Accepted for publication September 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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