Intracellular Localization and Constitutive Endocytosis of CXCR4 in Human CD34+ Hematopoietic Progenitor Cells
http://www.100md.com
《干细胞学杂志》
a INSERM U362 and
b Service Commun d’Imagerie, Institut Gustave Roussy,Villejuif, France
Key Words. CXCR4 receptor ? SDF-1 ? CD34+ cells ? Endocytosis ? Cell migration
Correspondence: Fawzia Louache, Ph.D., INSERM U362, Institut Gustave Roussy, PR1, 39 Rue Camille Desmoulins, 94805 Villejuif, France. Telephone: 33-1-42-11-42-33; Fax: 33-1-42-11-52-40; e-mail: fawl@igr.fr
ABSTRACT
The CXC chemokine ligand 12/stromal cell–derived factor-1 (SDF-1)/pre–B cell growth-stimulating factor was isolated from bone marrow (BM) stromal cells and first characterized as a pre–B cell growth-stimulating factor . CXCR4 is the primary physiologic receptor for SDF-1 and functions also as an entry coreceptor for strains of HIV-1 .
SDF-1 and CXCR4 knockout mice display abnormal B-cell development, impaired colonization of BM by hematopoietic progenitors, defects in blood vessel formation and the gastrointestinal tract, abnormal cardiac ventricular septum formation and cerebellar development, and embryonic lethality . In adult, CXCR4 receptors are involved in numerous biological processes, ranging from cell migration to proliferation and survival , indicating that this receptor plays a central role in cell biology. In the hematopoietic system, several data suggest that SDF-1 and CXCR4 play a critical role in the homing of hematopoietic progenitor cells (HPCs), with SDF-1 acting as a major chemoattractant for HPCs, severe combined immunodeficiency (SCID)–repopulating cells, and leukemic cells . Blocking CXCR4 with anti-CXCR4 antibodies on SCID–repopulating cells prevented their engraftment into nonobese diabetic (NOD)/SCID mice . Moreover, stimulation of mobilized peripheral CD34+ blood cells with a low concentration of CXCR4 antibody or SDF-1 resulted in an enhanced potential to engraft into NOD/SCID mice. Additionally, a critical role for CXCR4 in mobilization—the egress of HPC from BM to the peripheral blood—was recently suggested . Despite increasing evidence for a prominent role of SDF-1/CXCR4 in the regulation of several aspects of HPC biology, very little is known about the regulation of CXCR4 expression on the membrane of these cells.
Like many other G protein–coupled receptors, CXCR4 may undergo intracellular sequestration upon SDF-1 binding or after protein kinase C (PKC) activation. A constitutive internalization of CXCR4 was suggested in studies using green fluorescent protein (GFP)–tagged receptors, and a colocalization with internalized transferrin was demonstrated in several cell lines . Moreover, in T and B cells , cutaneous Langerhans cells , and various cell lines , CXCR4 exhibits an intracellular localization, suggesting that CXCR4 internalization may occur as both ligand-dependent and -independent processes . However, the degree of intracellular expression and spontaneous endocytosis was significantly different between cell types, suggesting the existence of a cell-specific regulation of CXCR4 trafficking .
To understand additionally the mechanisms regulating CXCR4 expression on hematopoietic progenitors, we investigated the cellular distribution and the subcellular localization of CXCR4 in freshly isolated BM and mobilized peripheral blood (MPB) and CD34+ cells. Independently of the source of CD34+ cells, we identified a large intracellular pool of CXCR4 receptors that was mainly localized in early and recycling endosome compartments but not in lysosomes. Using antibody feeding experiments to evaluate CXCR4 trafficking, we provide evidences for a rapid ligand-independent endocytosis. Thus, CXCR4 internalization and recycling in CD34+ cells may provide a dynamic regulation of HPC responses by altering the threshold for SDF-1 signaling, leading to a modulation in their migration and mobilization potential.
MATERIALS AND METHODS
CXCR4 Is Primarily an Intracellular Protein in CXCR4-Transduced Cell Lines and CD34+ Cells
In initial experiments, we used UT7 cells stably transduced with a GFP-tagged version of CXCR4 to visualize the cellular distribution of the receptor. As assessed by confocal microscopy, fluorescence was present on both the cell surface and intracellular compartments (Fig. 1). When UT7-CXCR4-GFP cells were stained with MAB172 after perme-abilization (Fig. 1A), GFP (Fig. 1B) and CXCR4 staining completely overlapped (Fig. 1C), confirming the specificity of MAB172 to intracellular CXCR4 molecules. A similar distribution of CXCR4 was seen in CXCR4-GFP–transduced K562 and DAMI cell lines (data not shown). To exclude the possibility that GFP tagging was causing a mis-location of CXCR4, an untagged CXCR4 version was expressed in UT7 cells. The overall staining pattern at the membrane (Fig. 1D) and in the cytoplasm (Fig. 1E) was similar to that observed in UT7-CXCR4-GFP cells, indicating that the GFP tagging did not interfere with CXCR4 localization. These observations are in line with previous studies that have reported an intracellular localization of GFP-tagged CXCR4 .
Figure 1. Intracellular expression of CXCR4 in CXCR4-transduced UT7 cells. UT7 and UT7 cells overexpressing a fusion CXCR4-GFP protein (UT7-CXCR4-GFP) or a wild-type protein (UT7-CXCR4) were fixed, permeabilized, and stained with the anti-CXCR4 MAB172 monoclonal antibody. MAB172 binding was revealed with tetramethylrhodamine isothiocyanate anti-immunoglobulin G2b. (A): CXCR4 distribution in UT7-CXCR4-GFP cells stained with MAB172. (B): Distribution of GFP-tagged CXCR4 receptors. (C): Merged images showing significant overlapping. Membrane (D) and intracellular (E) expression of CXCR4 in UT7 cells overex-pressing wild-type receptors. Membrane (F) and intracellular (G) background staining with MAB172 was determined on untransduced UT7 cells. Bar = 10 μM. Abbreviation: GFP, green fluorescent protein.
We next performed FACS analysis and immunostaining to determine the location of CXCR4 expression in freshly isolated MPB and BM CD34+ cells. When cells were stained with either MAB172 or PE-12G5 before fixation and perme-abilization, a low but significant staining was detected at the cell surface, with, in most cases, an intensity slightly higher in BM than MPB CD34+ cells (Table 1, Figs. 2A, 2B). In contrast, after permeabilization, most of the freshly isolated MPB (Fig. 2A) and BM (Fig. 2B) CD34+ cells exhibited an intense staining throughout the cytoplasm. No staining was observed with isotype control antibody (not shown). These data indicate that CXCR4 is primarily found in a cytoplasmic compartment in both BM and MPB CD34+ cells.
Table 1. CXCR4 expression on freshly isolated MPB CD34+ cells and BM CD34+ cells
Figure 2. Membrane and intracellular expression of CXCR4 on MPB CD34+ and BM CD34+ cells. Freshly isolated CD34+ cells were stained with anti-CXCR4 MAB172 and fluorescein isothiocyanate–conjugated anti-CD34 monoclonal antibodies before (-fixation) or after (+fixation) fixation and permeabilization. Membrane and intra-cellular expression of CXCR4 and CD34 on MPB CD34+ cells (A) and BM CD34+ cells (B). Background staining with anti-CD34 monoclonal antibodies was determined on CD34– cells (C). Abbreviation: BM, bone marrow; MPB; mobilized peripheral blood.
Intracellular Compartment Containing CXCR4 Overlapped with Endocytic Compartment
To investigate more precisely CXCR4 intracellular location, we performed an immunofluorescence labeling with tetramethyrhodamine-conjugated Tf, because it is well documented that Tf internalizes with its receptor and constitutively recycles with the receptor from early endosomes to the recycling compartment before reappearing on the cell surface . After exposure to labeled Tf, UT7-CXCR4-GFP cells were incubated at 37°C for different lengths of time to allow internalization. At the end of each time point, cells were fixed and analyzed by confocal microscopy for Tf and CXCR4-GFP expression. Tf was first detected at the cell surface and then intracellularly into small vesicular structures concentrated in the perinuclear region (Fig. 3). As shown in merged images (Fig. 3), CXCR4-GFP colocalized with Tf, particularly in the perinuclear vesicular structures. These results indicate that CXCR4 is associated with the endocytic compartment and suggest that a spontaneous CXCR4 endocytosis occurs in UT7 cells.
Figure 3. Intracellular CXCR4 colocalized with transferrin, an endosome marker. UT7-CXCR4-GFP cells were labeled with tetramethylrhodamine-conjugated transferrin at 4°C, washed, and incubated at 37°C for 1–30 minutes to allow transferrin internalization. Cells were fixed and analyzed by confocal microscopy. Red fluorescence shows the endocytosis of transferrin. Green fluorescence shows the distribution of GFP-tagged CXCR4. The yellow color in merged images indicates significant colocalization. Abbreviation: GFP, green fluorescent protein.
To identify the subcellular compartment in which CXCR4 was localized in freshly isolated MPB CD34+ cells, we tried to use tetramethyrhodamine-conjugated Tf as above. However, our attempts have been unsuccessful so far, most likely because the level of Tf receptors expressed in these cells is low. We therefore used indirect immunofluorescence microscopy to dually label CD34+ cells with a CXCR4 MoAb and various antibodies directed against markers of the secretory and endocytotic pathways, including clathrin, EEA1, late endosome/lysosomal Lamp-1, and CD63. As shown in Figure 4A, a large proportion of CXCR4 molecules were colocalized with EEA1. Similar results were obtained with the clathrin marker (Fig. 4A). In contrast, most CXCR4 molecules did not show colocalization with Lamp-1 and CD63 (Fig. 4B), indicating that most CXCR4 molecules were localized in the endosomal compartment in human CD34+ cells.
Figure 4. Intracellular compartment containing CXCR4 overlapped with endocytic compartment in CD34+ cells. MPB CD34+ cells were fixed, permeabilized, and dually labeled with anti-CXCR4 MAB172, revealed with a phycoerythrin anti-Ig G2b (red fluorescence) and a panel of antibodies directed to EEA1 or clathrin (A); Lamp-1 or CD63 (B); or Rab4, Rab5, and Rab11 (green fluorescence) (C). Cells were analyzed by confocal microscopy; the yellow color in merged images (right columns) indicates significant colocaliza-tion. Background fluorescence was determined by staining with the conjugated secondary antibodies only for polyclonal antibodies and by using isotype controls followed by conjugated antibodies for monoclonal antibodies. Bar = 5 μm.Abbreviations: EEA1, early endosomal antigen 1; FITC, fluorescein isothiocyanate; Ig, immunoglobulin; MPB, mobilized peripheral blood.
Two endosome populations, early and recycling endosomes, are involved in receptor recycling . To characterize further the localization of CXCR4 with respect to these compartments, we performed colocalization studies using antibodies directed toward members of the Rab family of small GTPases. Double labeling with antibodies against Rab5, present in clathrin-coated vesicles, and CXCR4 antibodies showed good, although incomplete, colocalization(Fig. 4C). Similarly, in most of the cells, CXCR4 colocalized with Rab4, a marker of membranes emerging from Rab5-positive membranes. When the distribution of CXCR4 was examined with respect to recycling endosomes characterized by the presence of the Rab11 protein, a fairly good colocalization of the two proteins was seen (Fig. 4C). These results suggest that CXCR4 in CD34+ cells is associated with early and recycling endosomes but is not distributed in the lysosomal compartment.
Cycling of CXCR4 between Cell Surface and Intracellular Stores
The above data suggested that CXCR4 may cycle between the cell surface and the endocytic compartment. To determine whether surface CXCR4 undergoes ligand-independent endocytosis, we performed antibody feeding experiments. As a first step before using MAB172 as a probe for CXCR4 endocytosis, we characterized the effects of the antibody on UT7-CXCR4-GFP cells. First, we examined whether MAB172 had the ability to induce CXCR4 internalization. Cells were incubated with a saturating concentration of MAB172 at 4°C, washed, and warmed at 37°C to allow receptor internalization. Cells were then returned to 4°C and labeled again with MAB172, followed with PE-conjugated goat anti-mouse IgG2b. Under these conditions, the PE fluorescence reflects the cell-surface expression of CXCR4. Figure 5A shows that binding with MAB172 did not change the membrane expression of CXCR4. Moreover, no change in cell-surface binding level of the conformation-dependent 12G5 antibody was detected (Fig. 5B), indicating that MAB172 did not alter CXCR4 membrane expression and did not modify significantly its conformation. We also studied whether MAB172 had the capacity to interfere with CXCR4 signaling by measuring the dose-dependent migration response to SDF-1. MAB172-treated UT7-CXCR4-GFP cells exhibited comparable migration responses to SDF-1 as cells treated with the control antibody isotype or untreated cells (Fig. 5C). Other experiments (data not shown) showed that MAB172 did not interfere with SDF-1 binding and signaling. These data allow us to conclude that MAB172 did not affect significantly the localization or the function of CXCR4 and, thus, can be used as a trafficking tracer for CXCR4.
Figure 5. MAB 172 did not affect the localization or the function of CXCR4. UT7-CXCR4-GFP cells were labeled with a saturating amount (5 μg/ml) of the anti-CXCR4 MAB172 MoAb at 4°C, washed, and incubated at 37°C for 30 minutes to allow CXCR4 endocytosis. Cells were then treated with an acid buffer to stripe down the antibody molecules bound on the membrane. Cells were reincubated with MAB172 (A) or 12G5 (B) at 4°C, followed with PE-anti-IgG2b or PE-anti-IgG2a, respectively. Histograms of fluorescence show similar membrane expression of CXCR4 before incubation (bold line) and after 45 minutes of incubation at 37°C (thin line). Solid histograms are controls IgG2b (A) and IgG2a (B). (C): Chemotactic assay to SDF-1. UT7-CXCR4-GFP cells were labeled with MAB172 or IgG2b and subjected to an in vitro transwell migration assay in response to different concentrations of SDF-1. Data are expressed as the percentage of migration (in y axis). Results are from one representative experiment performed in triplicate. Each experiment was repeated three times with similar results. Abbreviations: GFP, green fluorescent protein; Ig, immunoglobulin; PE, phycoerythrin; SDF-1, stromal cell–derived factor-1.
The kinetic of CXCR4 internalization was quantified in both UT7-CXCR4-GFP and MPB CD34+ cells by flow cytometry. Cells were incubated with saturating amounts of unlabeled MAB172 at 4°C, washed, and incubated at 37°C for different periods of time. At the end of each time point, cells were labeled at 4°C with a PE-conjugated anti-mouse IgG2b to stain the MAB172 molecules remaining at the cell surface. As measured by FACS analysis, the intensity of membrane fluorescence decreased rapidly in both UT7-CXCR4-GFP (Fig. 6A) and CD34+ cells (Fig. 6B), with a reduction reaching approximately 40% within 5 minutes (Table 2). No diminution of fluorescence was observed using UT7 cells expressing a C-terminal truncated form of CXCR4 that cannot undergo endocytosis (data not shown), excluding the possibility that the time-dependent decrease in fluorescence after incubation at 37°C was related to a simple detachment of MAB172 from the cells. The reduction in membrane fluorescence is associated with a parallel augmentation of intracellular fluorescence (Figs. 6H, 6I). Interestingly, when MAB172-treated UT7-CXCR4-GFP (Fig. 6C) or CD34+ cells (Fig. 6D) were stained with APC-12G5 to assess cell-surface expression, very little change in CXCR4 membrane expression was detected, suggesting a redistribution of intra-cellular CXCR4 receptors to the cell surface. Examination by confocal microscopy of cells incubated at 37°C with MAB172 and treated with acid buffer to remove cell surface–associated antibodies revealed MAB172 staining (Fig. 6E) and numerous fluorescence-tagged vesicles inside the cells (Fig. 6F), and merged images (Fig. 6G) showed colocalization of GFP-tagged CXCR4 and internalized CXCR4 stained with the antibody. Kinetics of CXCR4 internalization were not affected by incubation of UT7-CXCR4 and CD34+ cells with neutralizing antibodies to SDF-1 (data not shown). These results show that CXCR4 receptors cycle continuously to and from the cell surface in a ligand-independent manner.
Figure 6. Cycling of CXCR4 between the cell surface and intracellular stores in UT7-CXCR4-GFP and MPB CD34+ cells. UT7-CXCR4-GFP cells (A, C) and CD34+ cells (B, D) were labeled with a saturating amount, 5 μg/ml, of unlabeled MAB172 at 4°C, washed, and incubated at 37°C for different times. At the end of each time point, half of the cells were labeled at 4°C with PE-anti-IgG2b to detect MAB172 remains on the cell surface (A, B). Half of the cells were treated with an acidic buffer to strip down the MAB172 on membrane and reincubated with APC-12G5 (C, D). (A, B): Time-dependent internalization of CXCR4 on UT-7-CXCR4-GFP cells (A) and MPB CD34+ cells (B). (C, D): Stable surface expression of CXCR4 on UT7-CXCR4-GFP (C) and MPB CD34+ (D) cells. Solid histograms, control IgG2b; bold line, 0 minutes; thin line, 1 minute; dotted line, 5 minutes; dashed line, 45 minutes. (E–G): Detection of internalized CXCR4 by confocal microscopy. UT7-CXCR4-GFP cells were stained with MAB172, incubated for 30 minutes at 37°C, treated with acid to strip down the remaining MAB172 combined on the cell membrane, fixed, permeabilized, and incubated with tetramethylrhodamine isothiocyanate–conjugated anti-mouse IgG2b. The yellow color in merged image indicated significant colocalization of MAB172 and GFP staining. (H, I): Time-dependent endocytosis of CXCR4 on UT7-CXCR4-GFP cells (bar = 10 μm) (H) and CD34+ cells (bar = 5 μm) (I). CXCR4 membrane expression (0 minutes) and internalized antibody for different times (1, 5, 15, and 45 minutes) are shown. Background staining was determined by labeling the cells with an IgG2b isotype antibody. A representative experiment out of three performed is shown. Abbreviation: GFP, green fluorescent protein; IgG2b, immunoglobulin G2b; PE, phycoerythrin.
Table 2. Constitutive internalization of CXCR4 on UT7-CXCR4-GFP cells and CD34+ cells
Ligand-Independent CXCR4 Internalization Is Clathrin Mediated
Hypertonic sucrose medium blocks the assembly of coated pits and prevents endocytosis of receptors that use clathrin for internalization. In addition, it has been established that SDF-1 and phorbol myristate acetate–induced CXCR4 endocytosis occurs via coated pits and is blocked by hyper- tonic sucrose . To determine whether the ligand-independent endocytosis of CXCR4 also occurs via coated pits, MPB CD34+ cells were incubated at 37°C in medium lacking or containing high sucrose (0.45 M) for different lengths of time. Upon incubation for 3–15 minutes in sucrose-rich medium, an increase in CXCR4 membrane expression was observed (Fig. 7A), whereas no significant variation was detected in cells maintained in control medium (Fig. 7B). On the other hand, expression of CD45 and CD34 was not affected by sucrose treatment (data not shown). These results are consistent with the notion that CXCR4 undergoes a constitutive internalization through coated pits.
Figure 7. Spontaneous internalization of CXCR4 is clathrin mediated. MPB CD34+ cells were incubated with (A) or without (B) sucrose (0.45 M) for 0 minutes (bold line), 3 minutes (thin line), or 15 minutes (dotted line). Membrane CXCR4 was detected by phycoerythrin-12G5 and analyzed by flow cytometry. A representative experiment out of three performed is shown.Abbreviation: MPB, mobilized peripheral blood.
DISCUSSION
We are grateful to Novartis for recombinant human granulocyte-macrophage colony-stimulating factor. We are grateful to the surgeons for providing bone marrow samples and to Dr. C. Bocaccio at Institut Gustave Roussy (Villejuif, France) for mobilized peripheral blood samples. We thank Pascal Roux (Institut Pasteur, Paris) for confocal microscopy images, and Frederic Larbret and Yann Lecluse (IFR54,Villejuif, France) for cell-sorting experiments. We are also grateful to Dr. Francoise Wendling, Inserm U362, Villejuif, France, for discussion and critical reading of the manuscript. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Institut Gustave Roussy, and the Association pour la Recherche contre le Cancer (grant 4309 to F.L.). Y.Z., A.F., and D.B. are supported by a fellowship from the Ministère de la Recherche. M.B. is supported by la Ligue Nationale contre le Cancer (comité Yvelines). J.F.G. was a recipient of Comité de Recherche Clinique (Institut Gustave Roussy). Yanyan Zhang and Adlen Foudi, contributed equally to this study.
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b Service Commun d’Imagerie, Institut Gustave Roussy,Villejuif, France
Key Words. CXCR4 receptor ? SDF-1 ? CD34+ cells ? Endocytosis ? Cell migration
Correspondence: Fawzia Louache, Ph.D., INSERM U362, Institut Gustave Roussy, PR1, 39 Rue Camille Desmoulins, 94805 Villejuif, France. Telephone: 33-1-42-11-42-33; Fax: 33-1-42-11-52-40; e-mail: fawl@igr.fr
ABSTRACT
The CXC chemokine ligand 12/stromal cell–derived factor-1 (SDF-1)/pre–B cell growth-stimulating factor was isolated from bone marrow (BM) stromal cells and first characterized as a pre–B cell growth-stimulating factor . CXCR4 is the primary physiologic receptor for SDF-1 and functions also as an entry coreceptor for strains of HIV-1 .
SDF-1 and CXCR4 knockout mice display abnormal B-cell development, impaired colonization of BM by hematopoietic progenitors, defects in blood vessel formation and the gastrointestinal tract, abnormal cardiac ventricular septum formation and cerebellar development, and embryonic lethality . In adult, CXCR4 receptors are involved in numerous biological processes, ranging from cell migration to proliferation and survival , indicating that this receptor plays a central role in cell biology. In the hematopoietic system, several data suggest that SDF-1 and CXCR4 play a critical role in the homing of hematopoietic progenitor cells (HPCs), with SDF-1 acting as a major chemoattractant for HPCs, severe combined immunodeficiency (SCID)–repopulating cells, and leukemic cells . Blocking CXCR4 with anti-CXCR4 antibodies on SCID–repopulating cells prevented their engraftment into nonobese diabetic (NOD)/SCID mice . Moreover, stimulation of mobilized peripheral CD34+ blood cells with a low concentration of CXCR4 antibody or SDF-1 resulted in an enhanced potential to engraft into NOD/SCID mice. Additionally, a critical role for CXCR4 in mobilization—the egress of HPC from BM to the peripheral blood—was recently suggested . Despite increasing evidence for a prominent role of SDF-1/CXCR4 in the regulation of several aspects of HPC biology, very little is known about the regulation of CXCR4 expression on the membrane of these cells.
Like many other G protein–coupled receptors, CXCR4 may undergo intracellular sequestration upon SDF-1 binding or after protein kinase C (PKC) activation. A constitutive internalization of CXCR4 was suggested in studies using green fluorescent protein (GFP)–tagged receptors, and a colocalization with internalized transferrin was demonstrated in several cell lines . Moreover, in T and B cells , cutaneous Langerhans cells , and various cell lines , CXCR4 exhibits an intracellular localization, suggesting that CXCR4 internalization may occur as both ligand-dependent and -independent processes . However, the degree of intracellular expression and spontaneous endocytosis was significantly different between cell types, suggesting the existence of a cell-specific regulation of CXCR4 trafficking .
To understand additionally the mechanisms regulating CXCR4 expression on hematopoietic progenitors, we investigated the cellular distribution and the subcellular localization of CXCR4 in freshly isolated BM and mobilized peripheral blood (MPB) and CD34+ cells. Independently of the source of CD34+ cells, we identified a large intracellular pool of CXCR4 receptors that was mainly localized in early and recycling endosome compartments but not in lysosomes. Using antibody feeding experiments to evaluate CXCR4 trafficking, we provide evidences for a rapid ligand-independent endocytosis. Thus, CXCR4 internalization and recycling in CD34+ cells may provide a dynamic regulation of HPC responses by altering the threshold for SDF-1 signaling, leading to a modulation in their migration and mobilization potential.
MATERIALS AND METHODS
CXCR4 Is Primarily an Intracellular Protein in CXCR4-Transduced Cell Lines and CD34+ Cells
In initial experiments, we used UT7 cells stably transduced with a GFP-tagged version of CXCR4 to visualize the cellular distribution of the receptor. As assessed by confocal microscopy, fluorescence was present on both the cell surface and intracellular compartments (Fig. 1). When UT7-CXCR4-GFP cells were stained with MAB172 after perme-abilization (Fig. 1A), GFP (Fig. 1B) and CXCR4 staining completely overlapped (Fig. 1C), confirming the specificity of MAB172 to intracellular CXCR4 molecules. A similar distribution of CXCR4 was seen in CXCR4-GFP–transduced K562 and DAMI cell lines (data not shown). To exclude the possibility that GFP tagging was causing a mis-location of CXCR4, an untagged CXCR4 version was expressed in UT7 cells. The overall staining pattern at the membrane (Fig. 1D) and in the cytoplasm (Fig. 1E) was similar to that observed in UT7-CXCR4-GFP cells, indicating that the GFP tagging did not interfere with CXCR4 localization. These observations are in line with previous studies that have reported an intracellular localization of GFP-tagged CXCR4 .
Figure 1. Intracellular expression of CXCR4 in CXCR4-transduced UT7 cells. UT7 and UT7 cells overexpressing a fusion CXCR4-GFP protein (UT7-CXCR4-GFP) or a wild-type protein (UT7-CXCR4) were fixed, permeabilized, and stained with the anti-CXCR4 MAB172 monoclonal antibody. MAB172 binding was revealed with tetramethylrhodamine isothiocyanate anti-immunoglobulin G2b. (A): CXCR4 distribution in UT7-CXCR4-GFP cells stained with MAB172. (B): Distribution of GFP-tagged CXCR4 receptors. (C): Merged images showing significant overlapping. Membrane (D) and intracellular (E) expression of CXCR4 in UT7 cells overex-pressing wild-type receptors. Membrane (F) and intracellular (G) background staining with MAB172 was determined on untransduced UT7 cells. Bar = 10 μM. Abbreviation: GFP, green fluorescent protein.
We next performed FACS analysis and immunostaining to determine the location of CXCR4 expression in freshly isolated MPB and BM CD34+ cells. When cells were stained with either MAB172 or PE-12G5 before fixation and perme-abilization, a low but significant staining was detected at the cell surface, with, in most cases, an intensity slightly higher in BM than MPB CD34+ cells (Table 1, Figs. 2A, 2B). In contrast, after permeabilization, most of the freshly isolated MPB (Fig. 2A) and BM (Fig. 2B) CD34+ cells exhibited an intense staining throughout the cytoplasm. No staining was observed with isotype control antibody (not shown). These data indicate that CXCR4 is primarily found in a cytoplasmic compartment in both BM and MPB CD34+ cells.
Table 1. CXCR4 expression on freshly isolated MPB CD34+ cells and BM CD34+ cells
Figure 2. Membrane and intracellular expression of CXCR4 on MPB CD34+ and BM CD34+ cells. Freshly isolated CD34+ cells were stained with anti-CXCR4 MAB172 and fluorescein isothiocyanate–conjugated anti-CD34 monoclonal antibodies before (-fixation) or after (+fixation) fixation and permeabilization. Membrane and intra-cellular expression of CXCR4 and CD34 on MPB CD34+ cells (A) and BM CD34+ cells (B). Background staining with anti-CD34 monoclonal antibodies was determined on CD34– cells (C). Abbreviation: BM, bone marrow; MPB; mobilized peripheral blood.
Intracellular Compartment Containing CXCR4 Overlapped with Endocytic Compartment
To investigate more precisely CXCR4 intracellular location, we performed an immunofluorescence labeling with tetramethyrhodamine-conjugated Tf, because it is well documented that Tf internalizes with its receptor and constitutively recycles with the receptor from early endosomes to the recycling compartment before reappearing on the cell surface . After exposure to labeled Tf, UT7-CXCR4-GFP cells were incubated at 37°C for different lengths of time to allow internalization. At the end of each time point, cells were fixed and analyzed by confocal microscopy for Tf and CXCR4-GFP expression. Tf was first detected at the cell surface and then intracellularly into small vesicular structures concentrated in the perinuclear region (Fig. 3). As shown in merged images (Fig. 3), CXCR4-GFP colocalized with Tf, particularly in the perinuclear vesicular structures. These results indicate that CXCR4 is associated with the endocytic compartment and suggest that a spontaneous CXCR4 endocytosis occurs in UT7 cells.
Figure 3. Intracellular CXCR4 colocalized with transferrin, an endosome marker. UT7-CXCR4-GFP cells were labeled with tetramethylrhodamine-conjugated transferrin at 4°C, washed, and incubated at 37°C for 1–30 minutes to allow transferrin internalization. Cells were fixed and analyzed by confocal microscopy. Red fluorescence shows the endocytosis of transferrin. Green fluorescence shows the distribution of GFP-tagged CXCR4. The yellow color in merged images indicates significant colocalization. Abbreviation: GFP, green fluorescent protein.
To identify the subcellular compartment in which CXCR4 was localized in freshly isolated MPB CD34+ cells, we tried to use tetramethyrhodamine-conjugated Tf as above. However, our attempts have been unsuccessful so far, most likely because the level of Tf receptors expressed in these cells is low. We therefore used indirect immunofluorescence microscopy to dually label CD34+ cells with a CXCR4 MoAb and various antibodies directed against markers of the secretory and endocytotic pathways, including clathrin, EEA1, late endosome/lysosomal Lamp-1, and CD63. As shown in Figure 4A, a large proportion of CXCR4 molecules were colocalized with EEA1. Similar results were obtained with the clathrin marker (Fig. 4A). In contrast, most CXCR4 molecules did not show colocalization with Lamp-1 and CD63 (Fig. 4B), indicating that most CXCR4 molecules were localized in the endosomal compartment in human CD34+ cells.
Figure 4. Intracellular compartment containing CXCR4 overlapped with endocytic compartment in CD34+ cells. MPB CD34+ cells were fixed, permeabilized, and dually labeled with anti-CXCR4 MAB172, revealed with a phycoerythrin anti-Ig G2b (red fluorescence) and a panel of antibodies directed to EEA1 or clathrin (A); Lamp-1 or CD63 (B); or Rab4, Rab5, and Rab11 (green fluorescence) (C). Cells were analyzed by confocal microscopy; the yellow color in merged images (right columns) indicates significant colocaliza-tion. Background fluorescence was determined by staining with the conjugated secondary antibodies only for polyclonal antibodies and by using isotype controls followed by conjugated antibodies for monoclonal antibodies. Bar = 5 μm.Abbreviations: EEA1, early endosomal antigen 1; FITC, fluorescein isothiocyanate; Ig, immunoglobulin; MPB, mobilized peripheral blood.
Two endosome populations, early and recycling endosomes, are involved in receptor recycling . To characterize further the localization of CXCR4 with respect to these compartments, we performed colocalization studies using antibodies directed toward members of the Rab family of small GTPases. Double labeling with antibodies against Rab5, present in clathrin-coated vesicles, and CXCR4 antibodies showed good, although incomplete, colocalization(Fig. 4C). Similarly, in most of the cells, CXCR4 colocalized with Rab4, a marker of membranes emerging from Rab5-positive membranes. When the distribution of CXCR4 was examined with respect to recycling endosomes characterized by the presence of the Rab11 protein, a fairly good colocalization of the two proteins was seen (Fig. 4C). These results suggest that CXCR4 in CD34+ cells is associated with early and recycling endosomes but is not distributed in the lysosomal compartment.
Cycling of CXCR4 between Cell Surface and Intracellular Stores
The above data suggested that CXCR4 may cycle between the cell surface and the endocytic compartment. To determine whether surface CXCR4 undergoes ligand-independent endocytosis, we performed antibody feeding experiments. As a first step before using MAB172 as a probe for CXCR4 endocytosis, we characterized the effects of the antibody on UT7-CXCR4-GFP cells. First, we examined whether MAB172 had the ability to induce CXCR4 internalization. Cells were incubated with a saturating concentration of MAB172 at 4°C, washed, and warmed at 37°C to allow receptor internalization. Cells were then returned to 4°C and labeled again with MAB172, followed with PE-conjugated goat anti-mouse IgG2b. Under these conditions, the PE fluorescence reflects the cell-surface expression of CXCR4. Figure 5A shows that binding with MAB172 did not change the membrane expression of CXCR4. Moreover, no change in cell-surface binding level of the conformation-dependent 12G5 antibody was detected (Fig. 5B), indicating that MAB172 did not alter CXCR4 membrane expression and did not modify significantly its conformation. We also studied whether MAB172 had the capacity to interfere with CXCR4 signaling by measuring the dose-dependent migration response to SDF-1. MAB172-treated UT7-CXCR4-GFP cells exhibited comparable migration responses to SDF-1 as cells treated with the control antibody isotype or untreated cells (Fig. 5C). Other experiments (data not shown) showed that MAB172 did not interfere with SDF-1 binding and signaling. These data allow us to conclude that MAB172 did not affect significantly the localization or the function of CXCR4 and, thus, can be used as a trafficking tracer for CXCR4.
Figure 5. MAB 172 did not affect the localization or the function of CXCR4. UT7-CXCR4-GFP cells were labeled with a saturating amount (5 μg/ml) of the anti-CXCR4 MAB172 MoAb at 4°C, washed, and incubated at 37°C for 30 minutes to allow CXCR4 endocytosis. Cells were then treated with an acid buffer to stripe down the antibody molecules bound on the membrane. Cells were reincubated with MAB172 (A) or 12G5 (B) at 4°C, followed with PE-anti-IgG2b or PE-anti-IgG2a, respectively. Histograms of fluorescence show similar membrane expression of CXCR4 before incubation (bold line) and after 45 minutes of incubation at 37°C (thin line). Solid histograms are controls IgG2b (A) and IgG2a (B). (C): Chemotactic assay to SDF-1. UT7-CXCR4-GFP cells were labeled with MAB172 or IgG2b and subjected to an in vitro transwell migration assay in response to different concentrations of SDF-1. Data are expressed as the percentage of migration (in y axis). Results are from one representative experiment performed in triplicate. Each experiment was repeated three times with similar results. Abbreviations: GFP, green fluorescent protein; Ig, immunoglobulin; PE, phycoerythrin; SDF-1, stromal cell–derived factor-1.
The kinetic of CXCR4 internalization was quantified in both UT7-CXCR4-GFP and MPB CD34+ cells by flow cytometry. Cells were incubated with saturating amounts of unlabeled MAB172 at 4°C, washed, and incubated at 37°C for different periods of time. At the end of each time point, cells were labeled at 4°C with a PE-conjugated anti-mouse IgG2b to stain the MAB172 molecules remaining at the cell surface. As measured by FACS analysis, the intensity of membrane fluorescence decreased rapidly in both UT7-CXCR4-GFP (Fig. 6A) and CD34+ cells (Fig. 6B), with a reduction reaching approximately 40% within 5 minutes (Table 2). No diminution of fluorescence was observed using UT7 cells expressing a C-terminal truncated form of CXCR4 that cannot undergo endocytosis (data not shown), excluding the possibility that the time-dependent decrease in fluorescence after incubation at 37°C was related to a simple detachment of MAB172 from the cells. The reduction in membrane fluorescence is associated with a parallel augmentation of intracellular fluorescence (Figs. 6H, 6I). Interestingly, when MAB172-treated UT7-CXCR4-GFP (Fig. 6C) or CD34+ cells (Fig. 6D) were stained with APC-12G5 to assess cell-surface expression, very little change in CXCR4 membrane expression was detected, suggesting a redistribution of intra-cellular CXCR4 receptors to the cell surface. Examination by confocal microscopy of cells incubated at 37°C with MAB172 and treated with acid buffer to remove cell surface–associated antibodies revealed MAB172 staining (Fig. 6E) and numerous fluorescence-tagged vesicles inside the cells (Fig. 6F), and merged images (Fig. 6G) showed colocalization of GFP-tagged CXCR4 and internalized CXCR4 stained with the antibody. Kinetics of CXCR4 internalization were not affected by incubation of UT7-CXCR4 and CD34+ cells with neutralizing antibodies to SDF-1 (data not shown). These results show that CXCR4 receptors cycle continuously to and from the cell surface in a ligand-independent manner.
Figure 6. Cycling of CXCR4 between the cell surface and intracellular stores in UT7-CXCR4-GFP and MPB CD34+ cells. UT7-CXCR4-GFP cells (A, C) and CD34+ cells (B, D) were labeled with a saturating amount, 5 μg/ml, of unlabeled MAB172 at 4°C, washed, and incubated at 37°C for different times. At the end of each time point, half of the cells were labeled at 4°C with PE-anti-IgG2b to detect MAB172 remains on the cell surface (A, B). Half of the cells were treated with an acidic buffer to strip down the MAB172 on membrane and reincubated with APC-12G5 (C, D). (A, B): Time-dependent internalization of CXCR4 on UT-7-CXCR4-GFP cells (A) and MPB CD34+ cells (B). (C, D): Stable surface expression of CXCR4 on UT7-CXCR4-GFP (C) and MPB CD34+ (D) cells. Solid histograms, control IgG2b; bold line, 0 minutes; thin line, 1 minute; dotted line, 5 minutes; dashed line, 45 minutes. (E–G): Detection of internalized CXCR4 by confocal microscopy. UT7-CXCR4-GFP cells were stained with MAB172, incubated for 30 minutes at 37°C, treated with acid to strip down the remaining MAB172 combined on the cell membrane, fixed, permeabilized, and incubated with tetramethylrhodamine isothiocyanate–conjugated anti-mouse IgG2b. The yellow color in merged image indicated significant colocalization of MAB172 and GFP staining. (H, I): Time-dependent endocytosis of CXCR4 on UT7-CXCR4-GFP cells (bar = 10 μm) (H) and CD34+ cells (bar = 5 μm) (I). CXCR4 membrane expression (0 minutes) and internalized antibody for different times (1, 5, 15, and 45 minutes) are shown. Background staining was determined by labeling the cells with an IgG2b isotype antibody. A representative experiment out of three performed is shown. Abbreviation: GFP, green fluorescent protein; IgG2b, immunoglobulin G2b; PE, phycoerythrin.
Table 2. Constitutive internalization of CXCR4 on UT7-CXCR4-GFP cells and CD34+ cells
Ligand-Independent CXCR4 Internalization Is Clathrin Mediated
Hypertonic sucrose medium blocks the assembly of coated pits and prevents endocytosis of receptors that use clathrin for internalization. In addition, it has been established that SDF-1 and phorbol myristate acetate–induced CXCR4 endocytosis occurs via coated pits and is blocked by hyper- tonic sucrose . To determine whether the ligand-independent endocytosis of CXCR4 also occurs via coated pits, MPB CD34+ cells were incubated at 37°C in medium lacking or containing high sucrose (0.45 M) for different lengths of time. Upon incubation for 3–15 minutes in sucrose-rich medium, an increase in CXCR4 membrane expression was observed (Fig. 7A), whereas no significant variation was detected in cells maintained in control medium (Fig. 7B). On the other hand, expression of CD45 and CD34 was not affected by sucrose treatment (data not shown). These results are consistent with the notion that CXCR4 undergoes a constitutive internalization through coated pits.
Figure 7. Spontaneous internalization of CXCR4 is clathrin mediated. MPB CD34+ cells were incubated with (A) or without (B) sucrose (0.45 M) for 0 minutes (bold line), 3 minutes (thin line), or 15 minutes (dotted line). Membrane CXCR4 was detected by phycoerythrin-12G5 and analyzed by flow cytometry. A representative experiment out of three performed is shown.Abbreviation: MPB, mobilized peripheral blood.
DISCUSSION
We are grateful to Novartis for recombinant human granulocyte-macrophage colony-stimulating factor. We are grateful to the surgeons for providing bone marrow samples and to Dr. C. Bocaccio at Institut Gustave Roussy (Villejuif, France) for mobilized peripheral blood samples. We thank Pascal Roux (Institut Pasteur, Paris) for confocal microscopy images, and Frederic Larbret and Yann Lecluse (IFR54,Villejuif, France) for cell-sorting experiments. We are also grateful to Dr. Francoise Wendling, Inserm U362, Villejuif, France, for discussion and critical reading of the manuscript. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Institut Gustave Roussy, and the Association pour la Recherche contre le Cancer (grant 4309 to F.L.). Y.Z., A.F., and D.B. are supported by a fellowship from the Ministère de la Recherche. M.B. is supported by la Ligue Nationale contre le Cancer (comité Yvelines). J.F.G. was a recipient of Comité de Recherche Clinique (Institut Gustave Roussy). Yanyan Zhang and Adlen Foudi, contributed equally to this study.
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