Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4 - PubMed (original) (raw)

. 1997 Nov 3;139(3):651-64.

doi: 10.1083/jcb.139.3.651.

J Oldridge, A Pelchen-Matthews, P J Klasse, T Tran, L F Brass, M M Rosenkilde, T W Schwartz, W Holmes, W Dallas, M A Luther, T N Wells, J A Hoxie, M Marsh

Affiliations

Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4

N Signoret et al. J Cell Biol. 1997.

Abstract

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficiency. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechanism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surface expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 undergoes slow constitutive internalization (1.0% of the cell surface pool/min). Addition of phorbol esters increased this endocytosis rate >6-fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 min. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time approximately 5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-terminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation through different cellular mechanisms. SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligand induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Together these studies indicate that endocytosis can regulate the cell-surface expression of CXCR4 and that SDF-1-mediated down regulation of cell-surface coreceptor expression contributes to chemokine-mediated inhibition of HIV infection.

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Figures

Figure 3

Figure 3

Endocytosis of CXCR4 in BC7 and Mv-1-Lu cells. BC7 cells in suspension (A) or confluent cultures of Mv-1-Lu-CD4/ CXCR4 cells (B) were labeled at 0 to 4°C with 125I-12G5, washed, and warmed to allow endocytosis of the ligand. The amount of internalized antibody was determined by acid washing as described in Materials and Methods. The plots show the acid-resistant activity as a proportion of the total cell-associated counts for cells warmed in the absence (○) or presence of PMA (•). In A the total cell-associated activity is shown for the course of the experiment (▵). B (▵ and ▴) shows the endocytosis kinetics of CXCR4ΔCyt in the absence and presence of PMA, respectively. All data points show means and standard deviations for triplicate samples of representative experiments.

Figure 1

Figure 1

Binding properties of 12G5 on CXCR4-expressing cells. (A) Concentration dependence of 12G5 binding. CHO cells expressing CXCR4-HA (○) or CCR4-HA (▴) and RD cells (•) were incubated with increasing concentrations of 125I-12G5 (up to 10 nM) for 5 h at 4°C. Aliquots of the unbound label (free) were taken for counting and the cells washed and harvested. The protein per well was determined and used to calculate the amount of antibody bound per 106 cells. The binding recorded on CCR4-HA cells was taken as background and was deducted from the other cell lines to generate the binding data used in the Scatchard analysis illustrated in B. (B) Scatchard analysis of 12G5 binding. The bound and free 12G5 activities derived from the experiment illustrated in A were used for Scatchard-type analysis of 12G5 binding to native CXCR4 expressed on RD cells (•) and CXCR4-HA expressed on CHO cells (○).

Figure 2

Figure 2

Phorbol ester-induced down modulation of CXCR4. (A) Immunofluorescence analysis of CXCR4 down modulation on SupT1 cells. Cells were incubated in medium with (A, C and D) or without (A, A and B) 100 ng/ml PMA for 60 min at 37°C. The cells were then fixed and stained with 12G5 either intact (A, A and C) or after permeabilization (A, B and D) with saponin. (B) SupT1 and BC7 cells were incubated in medium with (▪) or without (formula image ) 100 ng/ml PMA for 120 min at 37°C. Subsequently the cells were fixed and stained first with 12G5 and then with a FITC-conjugated anti–mouse reagent. The stained cells were analysed by FACScan® and the mean fluorescence intensity determined for each sample. (C) The dose dependence for PMA- induced down modulation of CXCR4 was determined on SupT1 cells. Cells were incubated in medium containing the indicated concentration of PMA for 60 min at 37°C. The cells were then fixed, stained as described for B, and analyzed by FACScan®. Down modulation was calculated from the mean fluorescence intensity for each sample and compared to cells stained without primary antibody. Bar, 25 μm.

Figure 2

Figure 2

Phorbol ester-induced down modulation of CXCR4. (A) Immunofluorescence analysis of CXCR4 down modulation on SupT1 cells. Cells were incubated in medium with (A, C and D) or without (A, A and B) 100 ng/ml PMA for 60 min at 37°C. The cells were then fixed and stained with 12G5 either intact (A, A and C) or after permeabilization (A, B and D) with saponin. (B) SupT1 and BC7 cells were incubated in medium with (▪) or without (formula image ) 100 ng/ml PMA for 120 min at 37°C. Subsequently the cells were fixed and stained first with 12G5 and then with a FITC-conjugated anti–mouse reagent. The stained cells were analysed by FACScan® and the mean fluorescence intensity determined for each sample. (C) The dose dependence for PMA- induced down modulation of CXCR4 was determined on SupT1 cells. Cells were incubated in medium containing the indicated concentration of PMA for 60 min at 37°C. The cells were then fixed, stained as described for B, and analyzed by FACScan®. Down modulation was calculated from the mean fluorescence intensity for each sample and compared to cells stained without primary antibody. Bar, 25 μm.

Figure 2

Figure 2

Phorbol ester-induced down modulation of CXCR4. (A) Immunofluorescence analysis of CXCR4 down modulation on SupT1 cells. Cells were incubated in medium with (A, C and D) or without (A, A and B) 100 ng/ml PMA for 60 min at 37°C. The cells were then fixed and stained with 12G5 either intact (A, A and C) or after permeabilization (A, B and D) with saponin. (B) SupT1 and BC7 cells were incubated in medium with (▪) or without (formula image ) 100 ng/ml PMA for 120 min at 37°C. Subsequently the cells were fixed and stained first with 12G5 and then with a FITC-conjugated anti–mouse reagent. The stained cells were analysed by FACScan® and the mean fluorescence intensity determined for each sample. (C) The dose dependence for PMA- induced down modulation of CXCR4 was determined on SupT1 cells. Cells were incubated in medium containing the indicated concentration of PMA for 60 min at 37°C. The cells were then fixed, stained as described for B, and analyzed by FACScan®. Down modulation was calculated from the mean fluorescence intensity for each sample and compared to cells stained without primary antibody. Bar, 25 μm.

Figure 8

Figure 8

SDF-1- and PDB-induced down modulation of CXCR4 on SupT1 cells. (A) SupT1 cells were incubated in medium (□), medium containing 100 ng/ml PDB (•), or medium containing 500 nM SDF-1 (⋄) for up to 60 min at 37°C. At the indicated time points, aliquots of cells were placed on ice, washed with cold binding medium, fixed, and incubated with 0.5 nM 125I-12G5 for 2 h at room temperature. The values indicate the means and standard deviations for triplicate samples from a representative experiment. (B) SupT1 cells were incubated in twofold dilutions of SDF-1 for 30 min at 37°C. The cells were then cooled to 4°C and either fixed and labeled as in A with 125I-12G5 for 2 h (formula image ), or briefly incubated in low pH medium before fixation and labeling (formula image ). (C) SupT1 cells were incubated with (▪) or without (formula image ) 125 nM SDF-1 for 60 min at 37°C. The cells were then cooled to 4°C, fixed, and labeled as in A with 125I-12G5 or 0.3 nM 125I-Q4120 to detect cell surface CXCR4 and CD4, respectively.

Figure 8

Figure 8

SDF-1- and PDB-induced down modulation of CXCR4 on SupT1 cells. (A) SupT1 cells were incubated in medium (□), medium containing 100 ng/ml PDB (•), or medium containing 500 nM SDF-1 (⋄) for up to 60 min at 37°C. At the indicated time points, aliquots of cells were placed on ice, washed with cold binding medium, fixed, and incubated with 0.5 nM 125I-12G5 for 2 h at room temperature. The values indicate the means and standard deviations for triplicate samples from a representative experiment. (B) SupT1 cells were incubated in twofold dilutions of SDF-1 for 30 min at 37°C. The cells were then cooled to 4°C and either fixed and labeled as in A with 125I-12G5 for 2 h (formula image ), or briefly incubated in low pH medium before fixation and labeling (formula image ). (C) SupT1 cells were incubated with (▪) or without (formula image ) 125 nM SDF-1 for 60 min at 37°C. The cells were then cooled to 4°C, fixed, and labeled as in A with 125I-12G5 or 0.3 nM 125I-Q4120 to detect cell surface CXCR4 and CD4, respectively.

Figure 8

Figure 8

SDF-1- and PDB-induced down modulation of CXCR4 on SupT1 cells. (A) SupT1 cells were incubated in medium (□), medium containing 100 ng/ml PDB (•), or medium containing 500 nM SDF-1 (⋄) for up to 60 min at 37°C. At the indicated time points, aliquots of cells were placed on ice, washed with cold binding medium, fixed, and incubated with 0.5 nM 125I-12G5 for 2 h at room temperature. The values indicate the means and standard deviations for triplicate samples from a representative experiment. (B) SupT1 cells were incubated in twofold dilutions of SDF-1 for 30 min at 37°C. The cells were then cooled to 4°C and either fixed and labeled as in A with 125I-12G5 for 2 h (formula image ), or briefly incubated in low pH medium before fixation and labeling (formula image ). (C) SupT1 cells were incubated with (▪) or without (formula image ) 125 nM SDF-1 for 60 min at 37°C. The cells were then cooled to 4°C, fixed, and labeled as in A with 125I-12G5 or 0.3 nM 125I-Q4120 to detect cell surface CXCR4 and CD4, respectively.

Figure 4

Figure 4

EM immunolocalization of CXCR4 on the surface of SupT1 and BC7 cells. SupT1 (A and B) and BC7 (C and D) cells were labeled at 4°C with 12G5 followed by protein A-gold (PAG10) and either fixed directly (A) or warmed to 37°C for 2 min in medium containing PMA (100 ng/ml; B and C). Alternatively, cells were fixed in 2% paraformaldehyde/0.2% glutaraldehyde before labeling with 12G5 and PAG10 (D). Bars, 50 nm.

Figure 5

Figure 5

Immunofluorescence analysis of CXCR4 and CD4 internalization on SupT1 cells. Top figures (CD4-green/CXCR4-red): SupT1 cells were labeled with 12G5 and FITC-conjugated L120 (anti-CD4) at 4°C before warming to 37°C in the medium containing 100 ng/ml PMA. At the indicated times the cells were cooled, fixed, permeabilized, and stained with a biotin-conjugated isotype-specific anti–mouse reagent to detect 12G5, followed by streptavidin-Texas red. Lower figures (CXCR4-green/LAMP1-red): SupT1 cells were initially labeled with 12G5 alone. After fixation and permeabilization the cells were stained with anti-LAMP1 and Rhodamine-conjugated anti–rabbit antibodies and with a biotin-conjugated anti–mouse reagent and streptavidin-FITC to detect 12G5.

Figure 6

Figure 6

Recycling of internalized CXCR4. SupT1 cells were either left untreated or incubated in medium containing 100 ng/ml PDB for 60 min at 37°C. Aliquots of the treated cells were placed on ice or left in PDB for a further 90 min (+90). The remaining cells were washed three times with media to remove the PDB and incubated in fresh 37°C medium for the indicated time periods (formula image ). Subsequently, all cells were cooled to 4°C and incubated with 125I-12G5 to determine cell surface CXCR4 levels. In parallel, a duplicate set of cells was treated in the same way with 100 μg/ml cycloheximide present throughout (formula image ). The data show the means and standard deviations from triplicate samples.

Figure 7

Figure 7

12G5 binding to CXCR4 in the presence of SDF-1. (A) SupT1 cells were washed, fixed (3% PFA, 15 min), or cooled on ice and incubated in twofold dilutions of SDF-1 for 2 h at room temperature (formula image ) or 3 h at 4°C (formula image ), respectively. The cells incubated at 4°C were then fixed, and all the cells were labeled with 0.5 nM 125I-12G5 for 2 h at room temperature. Subsequently, the cells were washed and the amount of bound antibody determined. Antibody bound in the presence of SDF-1 is expressed as a percentage of antibody bound in the absence of ligand and represents the means and SD for triplicate samples. (B) SDF-1 inhibition of HIV-1 infection. Mv-1-Lu-CD4/CXCR4 (▪) and Mv-1-Lu-CD4/CXCR4ΔCyt (formula image ) were cultured in 96-well plates. The cells were incubated with twice the indicated concentration of SDF-1 (GlaxoWellcome) for 30 min before the addition of HIV-1IIIB. After 14 h the virus and chemokines were removed and the cells incubated for a further 2 d. Finally, the cells were fixed and stained for infected cell foci and each focus scored as a single infection event. The number of focus-forming units per 100 μl of virus innoculum is plotted on the y axis. The bars show the means of two wells; the error bars are one standard deviation.

Figure 9

Figure 9

The COOH-terminal cytoplasmic domain is required for SDF-1 down regulation of CXCR4. Mv-1-Lu-CD4/CXCR4 and Mv-1-Lu-CD4/ CXCR4ΔCyt were incubated in BM (□) or BM containing 500 nM SDF-1 (○) at 37°C. At the indicated times the cells were cooled on ice, washed with BM, and then incubated in BM adjusted to pH 2.0 (acid medium) for 10 min. The cells were then returned to BM (pH 7.4) at 4°C and cell surface CXCR4 determined using 0.5 nM 125I-12G5. Each point shows the mean and SD of triplicate samples from a representative experiment.

Figure 10

Figure 10

Effect of PKC inhibitors on SDF-1- and phorbol ester-induced down modulation of CXCR4 and CD4. SupT1 cells were washed twice by centrifugation and resuspended in 6 ml of BM, alone or with 0.5 μM staurosporin or 1 μM calphostin C, and incubated for 30 min at 37°C. For each condition 6 × 1 ml of cell suspension (∼3.25 × 106 cells/ ml) were diluted 1:1 with BM or BM containing 200 ng/ml PDB or 250 nM SDF-1 at 37°C for 30 min. The cells were then rapidly cooled on ice by dilution with 10 ml of cold PBS, centrifuged, and washed once in cold PBS. They were then fixed for 15 min in 3% PFA, washed, and quenched in 50 mM NH4Cl and labeled with 0.5 nM 125I-12G5 (for CXCR4) or 0.3 nM 125I-Q4120 (for CD4) for 2 h at room temperature. The cell-associated activity was determined as described in Materials and Methods. Binding medium alone (□), 100 ng/ml PDB (formula image ), and 125 nM SDF-1 (▪).

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