Interleukin-8-mediated Heterologous Receptor Internalization Provides Resistance to HIV-1 Infectivity

ROLE OF SIGNAL STRENGTH AND RECEPTOR DESENSITIZATION*

Ricardo M. RichardsonDagger §, Kenzo Tokunaga||, Robin Marjoram**, Tetsutaro Sata||, and Ralph Snyderman**

From the Dagger  Department of Biochemistry, Meharry Medical College, Nashville, Tennessee 37208, the § Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, the || Department of Pathology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan, and the ** Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, November 18, 2002, and in revised form, February 7, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human immunodeficiency virus type 1 (HIV-1) entry into CD4+ cells requires the chemokine receptors CCR5 or CXCR4 as co-fusion receptors. We have previously demonstrated that chemokine receptors are capable of cross-regulating the functions of each other and, thus, affecting cellular responsiveness at the site of infection. To investigate the effects of chemokine receptor cross-regulation in HIV-1 infection, monocytes and MAGIC5 and rat basophilic leukemia (RBL-2H3) cell lines co-expressing the interleukin-8 (IL-8 or CXCL8) receptor CXCR1 and either CCR5 (ACCR5) or CXCR4 (ACXCR4) were generated. IL-8 activation of CXCR1, but not the IL-8 receptor CXCR2, cross-phosphorylated CCR5 and CXCR4 and cross-desensitized their responsiveness to RANTES (regulated on activation normal T cell expressed and secreted) (CCL5) and stromal derived factor (SDF-1 or CXCL12), respectively. CXCR1 activation internalized CCR5 but not CXCR4 despite cross-phosphorylation of both. IL-8 pretreatment also inhibited CCR5- but not CXCR4-mediated virus entry into MAGIC5 cells. A tail-deleted mutant of CXCR1, Delta CXCR1, produced greater signals upon activation (Ca2+ mobilization and phosphoinositide hydrolysis) and cross-internalized CXCR4, inhibiting HIV-1 entry. The protein kinase C inhibitor staurosporine prevented phosphorylation and internalization of the receptors by CXCR1 activation. Taken together, these results indicate that chemokine receptor-mediated HIV-1 cell infection is blocked by receptor internalization but not desensitization alone. Thus, activation of chemokine receptors unrelated to CCR5 and CXCR4 may play a cross-regulatory role in the infection and propagation of HIV-1. Since Delta CXCR1, but not CXCR1, cross-internalized and cross-inhibited HIV-1 infection to CXCR4, the data indicate the importance of the signal strength of a receptor and, as a consequence, protein kinase C activation in the suppression of HIV-1 infection by cross-receptor-mediated internalization.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemokines are a diverse gene family of chemotactic cytokines that induce leukocyte accumulation and activation at sites of inflammation (1-3). They also mediate tumor cell trafficking and metastasis and participate in many acute and chronic inflammatory diseases (4, 5). Chemokine functions are mediated via cell surface G-protein-coupled receptors that couple predominantly to Gi (1-3, 18, 35). Chemokine receptors, most notably CCR5 and CXCR4, also serve as co-receptors for human immunodeficiency virus type 1 (HIV-1)1 entry into CD4+ cells (6, 7). To date, the relationship between the activation of these receptors and their role in HIV-1 infection is not well understood.

Like many members of the G-protein-coupled receptor family, CCR5 and CXCR4 become desensitized upon agonist exposure, resulting in a loss of cellular responsiveness to agonist followed by a decrease in the number of cell surface receptors (8-13). Phosphorylations of the carboxyl terminus of the receptors are responsible for the desensitization and down-regulation (8-13). We have previously shown that chemokine receptors cross-regulate the functions of each other (14, 35). The interleukin-8 (IL-8 or CXCL8) receptor CXCR1 cross-phosphorylated and cross-desensitized CCR1-mediated cellular responses to RANTES (CCL5) (14). The formyl peptide chemoattractant receptor also cross-desensitized CCR5-mediated cellular responses to RANTES in monocytes and diminished the ability of RANTES to mediate HIV-1 entry and infection (15, 16).

While HIV-1 infection requires the CD4 receptor, the role of a chemokine receptor as the fusion cofactor depends on the target cell (17). Both macrophages and T lymphocytes express CCR5 and CXCR4 (18). Macrophages, however, utilize CCR5 for HIV-1 entry (M-tropism), whereas CD4+ T lymphocytes use CXCR4 (T-tropism) (18). In addition to CCR5 and CXCR4, macrophages and CD4+ T lymphocytes express other chemokine receptors including the IL-8 receptors CXCR1 and CXCR2 (1-8 × 106 receptors/cell) (1, 18-24). In the present study we sought to determine the role of cross-regulation by IL-8 receptors in CCR5- and CXCR4-mediated cellular activation and HIV-1 infection. For this purpose, monocytes and MAGIC5 and RBL-2H3 cells stably expressing different combination of receptors were used to study the mechanisms of cross-regulation among IL-8, CCR5, and CXCR4. The results demonstrate that IL-8 led to the cross-phosphorylation and cross-desensitization of both CCR5 and CXCR4. However, IL-8 down-regulated and inhibited HIV-1 infection to CCR5 but not CXCR4. Since CCR5 is a target for the entry of primary viruses in monocytes these results suggest a selective role for IL-8 in limiting HIV-1 infection through this receptor.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [32P]Orthophosphate (8500-9120 Ci/mmol), 125I-IL-8, and 125I-RANTES were purchased from PerkinElmer Life Sciences. IL-8 (monocyte-derived), melanoma growth-stimulating activity (MGSA or CXCL1), RANTES, MIP-1beta (CCL4), and SDF-1 were purchased from Peprotech. Geneticin (G418) and all tissue culture reagents were purchased from Invitrogen. Monoclonal 12CA5 antibody, protein G-agarose, and protease inhibitors were purchased from Roche Applied Science. Anti-human IL-8RA (CXCR1) and IL-8RB (CXCR2) antibodies were purchased from Pharmingen. Indo-1 acetoxymethyl ester and pluronic acid were purchased from Molecular Probes. Phorbol 12-myristate 13-acetate (PMA) and M2-FLAG antibody were purchased from Sigma. FuGENE 6 was purchased from Roche Applied Science. The enzyme-linked immunosorbent assay was obtained from PerkinElmer Life Sciences. All other reagents are from commercial sources.

Isolation of Monocytes-- Monocytes were isolated from heparinized human blood on a multiple density gradient and enriched for mononuclear cells as described previously (25, 26).

Construction of Epitope-tagged CXCR1, CXCR4, and CCR5-- Nucleotides encoding the nine-amino acid (YPYDVPDYA) hemagglutinin (HA) (CXCR1 and Delta CXCR1) or the octapeptide (DYKDDDDK) FLAG (CCR5 and CXCR4) epitope sequences were inserted between the amino-terminal initiator methionine and the second amino acid of each cDNA by polymerase chain reaction as described previously (9, 10, 27). The resulting PCR products were cloned into the eukaryotic expression vector pcDNA3, and the receptors were sequenced to confirm the intended mutations and lack of secondary mutations.

Cell Culture and Transfection-- RBL-2H3 cells were maintained as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 15% heat-inactivated fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 mg/ml) (27). RBL-2H3 cells (1 × 107 cells) were transfected by electroporation with 20 µg of pcDNA3 containing the receptor cDNAs, and geneticin-resistant cells were cloned into a single cell by fluorescence-activated cell sorter analysis. Levels of protein expression were monitored by fluorescence-activated cell sorter analysis and Western blotting using 12CA5 (HA)- and M2 (FLAG)-specific antibodies.

Radioligand Binding Assays and Receptor Internalization-- RBL-2H3 cells were subcultured overnight in 24-well plates (0.5 × 106 cells/well) in growth medium. Cells were then rinsed with Dulbecco's modified Eagles medium supplemented with 20 mM HEPES, pH 7.4, and 10 mg/ml bovine serum albumin and incubated on ice for 2-4 h in the same medium (250 µl) containing the radiolabeled ligand (0.1 nM). Reactions were stopped with 1 ml of ice-cold phosphate-buffered saline containing 10 mg/ml bovine serum albumin and washed three times with the same buffer. Then cells were solubilized with radioimmune precipitation assay buffer (200 µl) dried under vacuum, and bound radioactivity was counted. Nonspecific radioactivity bound was determined in the presence of a 500 nM concentration of the unlabeled ligand (14, 27).

GTPase Activity-- Cells were treated with appropriate concentrations of stimulants, and membranes were prepared as described previously (9, 14). GTPase activity using 10-20 µg of membrane preparations were carried out as described previously (14, 27).

Phosphoinositide Hydrolysis and Calcium Measurement-- RBL-2H3 cells were subcultured overnight in 96-well culture plates (50,000 cells/well) in inositol-free medium supplemented with 10% dialyzed fetal bovine serum and 1 µCi/ml [3H]inositol. The generation of inositol phosphates was determined as reported previously (9, 14). For calcium mobilization, RBL cells (5 × 106) or monocytes (107) were washed with HEPES-buffered saline and loaded with 1 µM Indo-1 acetoxymethyl ester for 30 min at room temperature. Then the cells were washed and resuspended in 1.5 ml of buffer. Intracellular calcium increase in the presence or absence of ligands was measured as described previously (27).

Phosphorylation of Receptors-- Phosphorylation of receptors was performed as described previously (27). RBL cells (5 × 106) expressing the receptors were incubated with [32P]orthophosphate (150 µCi/dish) for 90 min. Then labeled cells were stimulated with the indicated ligands for 5 min at 37 °C. Cells were then washed and solubilized in 1 ml of radioimmune precipitation assay buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS. Cells lysates were immunoprecipitated with specific antibodies against the epitope tags, analyzed by SDS electrophoresis, and visualized by autoradiography.

Construction and Preparation of Viral Plasmids-- Infectious proviral clones expressing the luciferase gene in place of nef, pNL-Luc-ADA (CCR5-tropic) and pNL-Luc-HXB (CXCR4-tropic), were generated as described previously (28). The HIV-1-based expression vectors pNL-CXCR1 and pNL-Delta CXCR1 were generated from pNL-Luc/Rev- (28)2 by replacing the NotI-XhoI fragment encoding luciferase with a PCR-amplified NotI-XhoI fragment encoding either CXCR1 or Delta CXCR1 (CXCR1 minus amino acids 335-349 of the carboxyl terminus). HIV-1 virus stocks and HIV-1-based lentivirus vectors were prepared in 293T cells as described previously (30-32). Briefly, 293T cells were transfected with 2 µg of the proviral expression plasmids carrying a luciferase reporter gene, pNL-Luc-HXB or pNL-Luc-ADA, by using FuGENE 6. For HIV-1-based lentivirus, 293T cells were cotransfected with 0.5 µg of the Rev expression vector pcRev (32) and 0.5 µg of the vesicular stomatitis virus glycoprotein expression vector pHIT/G (33) with 1 µg of a lentivirus vector plasmid using FuGENE 6. The culture medium was replaced 16 h later, and the culture supernatants were harvested 40 h after transfection and filtered through 0.45-µm-pore-size filters, and virus yield was measured by enzyme-linked immunosorbent assay. Virus stocks were stored at -80 °C until needed.

Overexpression of CXCR1 and Delta CXCR1 on Human Cells and Luciferase Reporter Virus Assays-- MAGI (HeLa-CD4-LTR-beta -Gal) and MAGIC5 (MAGI stably expressing CCR5) (31) cells were maintained as monolayer in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum as described above. Cells (5 × 105) were transduced overnight with 10 ng of p24 antigen of vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vector virus encoding either CXCR1, Delta CXCR1, or a control vector, pNL-con. The cells were then washed with phosphate-buffered saline and cultured in fresh medium for an additional 24 h. CXCR1 and Delta CXCR1 expression was monitored by fluorescence-activated cell sorter analysis. Then the transduced cells were treated with 100 nM IL-8 for 30 min and incubated with 20 ng of p24 antigen of a luciferase reporter virus, NL-Luc-HXB or NL-Luc-ADA, for an additional 60 min. The cells were washed extensively with phosphate-buffered saline and cultured in fresh medium. After 48 h, the cells were lysed in 200 µl of lysis buffer, and luciferase activities were determined with a MicroLumatPlus LB96V microplate luminometer.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cross-desensitization of CCR5- and CXCR4-mediated Ca2+ Mobilization in Human Monocytes-- To study the cross-desensitization of CCR5 and CXCR4, intracellular Ca2+ mobilization in monocytes was elicited by RANTES and SDF-1 and used as a measure of CCR5 and CXCR4 activation, respectively. As shown in Fig. 1A, IL-8, which activates both CXCR1 and CXCR2, cross-desensitized Ca2+ responses to RANTES and SDF-1. MGSA, which only activates CXCR2, had no effect on RANTES or SDF-1. RANTES and SDF-1 pretreatment attenuated responses to both MGSA and IL-8 (Fig. 1A). RANTES, SDF-1, IL-8, and MGSA homologously desensitized (~90%) responses to a second dose of the same ligand (Fig. 1B).


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Fig. 1.   IL-8- and MGSA-mediated cross-desensitization of CCR5 and CXCR4-mediated intracellular calcium mobilization in human monocytes. Human monocytes (1 × 107) were loaded with the calcium indicator Indo-1 and exposed to a first EC100 dose (10 nM) of IL-8, MGSA, RANTES, or SDF-1. Cells were rechallenged 3 min later with a second dose of the same (B) or a different ligand (A) as indicated. Traces are representative of three experiments.

Cross-inhibition of CCR5- and CXCR4-mediated HIV-1 Infection by IL-8-- We determined whether IL-8 inhibited HIV-1 entry to CCR5 and CXCR4. MAGIC5 cells were infected with a lentiviral CXCR1 expression vector virus. Cells were pretreated with or without IL-8 (100 nM) and infected with NL-Luc-ADA (R5-tropic) or NL-Luc-HXB (X4-tropic), and luciferase activity was measured. As shown in Fig. 2, IL-8 pretreatment inhibited by ~50% CCR5-mediated R5-tropic virus infection but had no effect in X4-tropic infection to CXCR4.


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Fig. 2.   HIV-1 infection of MAGIC5 cells. MAGIC5 cells were transduced overnight with 10 ng of p24 antigen of vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vector virus encoding CXCR1. The cells were treated with 100 nM IL-8 for 30 min and incubated with 20 ng of p24 antigen of a luciferase reporter virus, NL-Luc-HXB (R5-tropic) or NL-Luc-ADA (X4-tropic), for 60 min. Luciferase activities in cell lysates were determined 48 h postinfection. The data are represented as percent infection efficiency, which is the total relative light units measured in control or untreated cells. Data are the means ± S.E. of three different experiments.

Co-expression, Characterization, and Cross-desensitization of CXCR1, CCR5, and CXCR4 in RBL-2H3 Cells-- To study the mechanism of cross-regulation among CXCR1, CCR5, and CXCR4, single transfectant RBL cells expressing FLAG-tagged CCR5 (CCR5-RBL) or CXCR4 (CXCR4-RBL) were first generated and characterized. The Kd values for RANTES binding to the CCR5 (4.3 ± 0.5 nM) and for SDF-1 binding to CXCR4 (5.7 ± 1 nM) were similar to those previously reported (9, 10). CCR5 induced a comparable peak of intracellular Ca2+ mobilization in response to both RANTES (Fig. 3A) and MIP-1beta (data not shown) but not IL-8, MGSA, or SDF-1 (Fig. 3A). CXCR4 was also specific for SDF-1-mediated Ca2+ mobilization (Fig. 3B). Cells expressing CXCR1 were specific for IL-8 (data not shown).


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Fig. 3.   Functional characterization of CCR5 and CXCR4 expressed in RBL-2H3 cells. RBL cells (5 × 106) stably expressing CCR5 (A) or CXCR4 (B) were loaded with Indo-1 and tested for either IL-8, MGSA, RANTES, or SDF-1 (10 nM)-stimulated Ca2+ mobilization. Representative tracings of five experiments are shown.

In cells co-expressing CXCR1 and CCR5 (ACCR5-RBL) IL-8 pretreatment cross-desensitized intracellular Ca2+ mobilization to both RANTES (58%) and MIP-1beta (56%) (Table I). IL-8 also inhibited SDF-1 (35%)-mediated Ca2+ response in cells co-expressing CXCR1 and CXCR4 (ACXCR4-RBL). Inhibition of responses to CCR5 was greater than that of CXCR4 (56-58 versus 35%, respectively). IL-8-mediated Ca2+ mobilization was also attenuated by pretreatment of the cells with RANTES, MIP-1beta , or SDF-1 (Table I). IL-8, RANTES, MIP-1beta , and SDF-1 also homologously desensitized (~90%) the response to a second dose of the same ligand (Table I). Pretreatment of ACCR5 (Fig. 4A) or ACXCR4 (Fig. 4B) cells with IL-8 (100 nM) also cross-desensitized RANTES (~55%)- and SDF-1 (~40%)-induced GTPase activity in membranes (Fig. 4). Both RANTES and SDF-1 cross-inhibited IL-8-mediated GTPase activity by ~35 and ~45%, respectively. PMA (100 nM) heterologously desensitized the GTPase response to IL-8 (~35%), RANTES (~60%), and SDF-1 (~50%).


                              
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Table I
Cross-desensitization among CCR5, CXCR4, and CXCR1 in transfected RBL cells
RBL cells stably co-expressing CXCR1 and CCR5 (ACCR5) or CXCR4 (ACXCR4) were Indo-1-loaded, and RANTES, MIP-1beta , SDF-1, or IL-8 (10 nM)-mediated Ca2+ mobilization was measured. Cells were rechallenged 3 min later with a second dose (10 nM) of the indicated ligand, and peak intracellular Ca2+ mobilization was determined. Data are the means ± S.E. of three different experiments.


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Fig. 4.   Cross-desensitization of CCR5- and CXCR4-mediated GTPase activity. Double transfected RBL-2H3 cells expressing CXCR1 and either CCR5 (ACCR5) (A) or CXCR4 (ACXCR4) (B) were treated with IL-8 (100 nM), RANTES (100 nM), SDF-1 (100 nM), or PMA (100 nM) for 5 min. Membranes were prepared and assayed for agonist-stimulated GTP hydrolysis. The data are presented as percentage of control, which is the net maximal stimulation, obtained with untreated cells. Data shown are representative of one of three experiments performed in triplicate.

Cross-internalization of CCR5 and CXCR4-- ACCR5 and ACXCR4 were treated with IL-8, RANTES, SDF-1, or PMA (100 nM), and receptor clearance from the cell surface was assessed by specific ligand binding. CXCR1 (Fig. 5, A and C), CCR5 (Fig. 5B), and CXCR4 (Fig. 5D) were homologously internalized by exposure of the cells to IL-8, RANTES, and SDF-1, respectively. IL-8 pretreatment cross-internalized CCR5 (Fig. 5B) but not CXCR4 (Fig. 5D). PMA pretreatment caused internalization of both CCR5 and CXCR4 (Fig. 5, B and D). RANTES, SDF-1, and PMA had no effect in CXCR1 internalization (Fig. 5, A and C).


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Fig. 5.   Cross-internalization of CCR5 and CXCR4. ACCR5 (A and B) and ACXCR4 (C and D) RBL cells (0.5 × 106 cells/well) were treated with a 100 nM concentration of either IL-8, RANTES, SDF-1, or PMA at different times. Cells were then washed and assayed for 125I-IL-8 (A and C), 125I-RANTES (B), or 125I-SDF-1 (D) binding. The values are presented as percentage of total, which is defined as the total amount of 125I-ligand bound to control (untreated) cells. The experiment was repeated four times with similar results.

Cross-phosphorylation of CCR5 and CXCR4-- To assess the role of receptor phosphorylation in cross-internalization ACCR5 and ACXCR4 cells were labeled with 32P and treated with IL-8 (100 nM), RANTES (100 nM), SDF-1 (100 nM), or PMA (100 nM). Cells were lysed, immunoprecipitated with the M2-FLAG (CCR5 and CXCR4)- or HA (CXCR1)-specific antibodies, and analyzed by SDS electrophoresis and autoradiography. The identities of the phosphorylated bands for the respective receptors (CXCR1, ~70 kDa; CCR5, ~40 kDa; and CXCR4, ~45 kDa) have been previously demonstrated (9, 10, 27). As shown in Fig. 6, CCR5 (A, lane 2) and CXCR4 (B, lane 2) were homologously phosphorylated by RANTES and SDF-1, respectively. CCR5 (A, lane 4) and CXCR4 (B, lane 4) were also cross-phosphorylated by IL-8. CXCR1 was homologously phosphorylated by IL-8 (A and B, lanes 7) and cross-phosphorylated by both RANTES and SDF-1 (A and B, lanes 6). PMA induced phosphorylation of CCR5 (A, lane 3), CXCR4 (B, lane 3), and CXCR1 (A and B, lanes 8).


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Fig. 6.   Cross-phosphorylation of CCR5 and CXCR4. 32P-Labeled ACCR5 (A) and ACXCR4 (B) RBL cells (5 × 106/60-mm plate) were incubated for 5 min with or without stimulants as shown. Cells were lysed, immunoprecipitated first with an anti-FLAG (CCR5 and CXCR4) and second with anti-HA (CXCR1) antibodies specific for the M2 and HA epitope tags, respectively, expressed at the amino terminus of the receptors, and then analyzed by SDS-PAGE and autoradiography. The results are from a representative experiment that was repeated three times.

Role of Protein Kinase C in CCR5 Cross-internalization-- Pretreatment of ACCR5 with the PKC inhibitor staurosporine (100 nM) partially inhibited RANTES-mediated CCR5 internalization (Fig. 7A) and phosphorylation (Fig. 7B). Cross-internalization and cross-phosphorylation by IL-8 as well as heterologous internalization and phosphorylation by PMA were totally inhibited by staurosporine (Fig. 7, A and B).


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Fig. 7.   Effect of staurosporine on CCR5 cross-internalization and cross-phosphorylation. A, ACCR5 cells (0.5 × 106 cells/well) were incubated with and without staurosporine and treated with a 100 nM concentration of either IL-8, RANTES, or PMA for 60 min. Cells were then washed and assayed 125I-RANTES binding as indicated in the legend of Fig. 5. The values are presented as percentage of total, which is defined as the total amount of 125I-RANTES bound to control (untreated) cells. The experiment was repeated twice with similar results. B, 32P-labeled ACCR5 cells were incubated with and without staurosporine for 5 min and then stimulated with a 100 nM concentration of either RANTES (lanes 3 and 4), IL-8 (lanes 5 and 6), or PMA (lanes 7 and 8). Cells were lysed, immunoprecipitated with anti-FLAG antibody, electrophoresed into a 10% SDS-polyacrylamide gel, and autoradiographed. Two other experiments yielded similar results. Cont, control; Stau, staurosporine.

Delta CXCR1-mediated Cross-internalization and Cross-inhibition of CXCR4-- The role of IL-8 in CXCR4 cross-internalization was further assessed by co-expressing a carboxyl terminus-deficient mutant of CXCR1, Delta CXCR1, along with CXCR4 (Delta ACXCR4). The Kd and Bmax of Delta CXCR1 (2.1 ± 1.10 nM and 6898 ± 523 receptors/cell, respectively) were similar to those of CXCR1 (1.7 ± 0.33 nM and 7013 ± 311 receptors/cell, respectively). Delta CXCR1 mediated greater phosphoinositide hydrolysis (Fig. 8A), G-protein activation, secretion of beta -hexosaminidase, and sustained Ca2+ response relative CXCR1.2 IL-8 pretreatment of Delta ACXCR4 but not ACXCR1 cells resulted in cross-internalization of CXCR4 (Fig. 8B). In contrast to CXCR1, Delta CXCR1 activation also cross-inhibited CXCR4-mediated virus entry into MAGI cells (Fig. 8C).


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Fig. 8.   Delta CXCR1-mediated phosphoinositide hydrolysis, cross-internalization, and cross-inhibition of CXCR4. A, for the generation of [3H]inositol phosphates, RBL cells (50,000 cells/well) co-expressing CXCR4 and either CXCR1 (ACXCR4) or Delta CXCR1 (Delta ACXCR4) were cultured overnight in the presence of [3H]inositol (1 µCi/ml). Cells were preincubated (10 min at 37 °C) with HEPES-buffered saline containing 10 mM LiCl in a total volume of 200 µl and stimulated with 100 nM IL-8 or SDF-1 for 10 min. Supernatant was used to determine the release of [3H]inositol phosphates. Data are represented as -fold stimulation over basal. The experiment was repeated three times with similar results. B, ACXCR4 and Delta ACXCR4 RBL cells (0.5 × 106 cells/well) were treated with a 100 nM concentration of IL-8 at different times and assayed for 125I-SDF-1 binding as described in the legend of Fig. 5. C, MAGI cells were transduced overnight with 10 ng of vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vector encoding either CXCR1 or Delta CXCR1. Cells were treated with IL-8 (100 nM) and infected with X4-tropic virus, and luciferase activities in cell lysates were determined as described in the legend of Fig. 2.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemokines and chemokine receptors are redundant in that many chemokines activate more than one chemokine receptor and many chemokine receptors are activated by multiple chemokines (8, 34). To date, the structural basis and biological significance of these redundancies remain unclear. Initial studies in our laboratory, however, provided evidence that chemokine receptors are capable of cross-regulating the functions of each other, thus limiting cellular responsiveness to chemokines. The IL-8 receptor CXCR1 was shown to cross-desensitize responses to the CC receptor CCR1 at two levels: receptor/G-protein uncoupling via receptor cross-phosphorylation and inhibition of phospholipase Cbeta activity via phosphorylation of the enzyme, which diminishes its activation by G-protein (14, 35). The data herein describe another level of cross-regulation among chemokines that may have important consequences in their action: chemokine-mediated receptor cross-internalization. Chemokine-mediated receptor cross-internalization appears to be selective and distinct from receptor desensitization. This contention is based on the following observations. First, IL-8 activation of CXCR1, but not CXCR2, cross-internalized CCR5 (Fig. 5 and data not shown). Second, while CXCR1 cross-phosphorylated and cross-desensitized Ca2+ mobilization and GTPase activity to both CCR5 and CXCR4, receptor internalization occurred only with CCR5.

HIV-1 entry into CD4+ cells requires the presence of chemokine receptors such as CCR5 and CXCR4 as co-fusion proteins. Several studies have indicated that signaling by these receptors is not required for virus fusion and infection (28, 36-38). Activation of CXCR1, however, diminished the ability of CCR5 to mediate virus entry (Fig. 2). This suggests that while chemokine-mediated activation of CCR5 and CXCR4 may not be required, activation of signaling through other chemokine receptors may, through cross-internalization, prevent the target receptors to serve as co-factors for HIV-1 infection. Despite receptor cross-desensitization, CXCR4-mediated virus entry was resistant to cross-inhibition by CXCR1. This may be due to its relative resistance to cross-internalization since the stronger signaling Delta CXCR1 or PMA, which induced cross-internalization as well as cross-desensitization of CXCR4, also inhibited HIV-1 infection through CXCR4. Activation of the formyl peptide receptor also cross-phosphorylated and cross-inhibited HIV-1 infection to CCR5 (16), but these chemoattractants are less commonly present at sites of inflammation than the chemokines. Chemokine production can be induced by many stimuli including cytokines, lipopolysaccharides, and viral products (39). Modulation of chemokine receptor internalization may therefore be a useful target for therapeutic intervention against HIV-1 infection.

An interesting finding in these studies is the importance of signal strength in cross-desensitization, cross-internalization, and inhibition of HIV-1 infectivity. Upon activation by IL-8, CXCR2 internalizes rapidly (~90% after 2-5 min) and, as a consequence, does not mediate cross-regulatory signals (14, 29, 40). CXCR1, which is more resistant to internalization (~50% after 20-40 min), mediated cross-phosphorylation and cross-desensitization of both CXCR4 and CCR5 but cross-internalized and inhibited HIV-1 entry to CCR5 but not CXCR4 (Figs. 2, 4, and 5 and Table I) (14, 32, 33). Delta CXCR1, however, which is far more resistant to internalization (~10% after 60 min) and mediated greater cellular responses (i.e. phosphoinositide hydrolysis, exocytosis, and Ca2+ mobilization), cross-internalized CXCR4 and inhibited T4-tropic virus entry (Fig. 8).2 This indicates a hierarchy in receptor-mediated cross-regulation that is directly correlated with the receptor resistance to desensitization, internalization, and, as a consequence, signaling time. Supporting that contention is that in monocytes isolated from mice deficient in beta -arrestin-2 in which CXCR2 internalization is delayed (~25% after 5 min) MGSA cross-desensitized Ca2+ mobilization to RANTES by ~50% relative to control or wild type mice (~90% after 5 min).3

CXCR1-mediated cross-desensitization and cross-internalization of CCR5 and CXCR4 as well as desensitization and internalization by PMA were inhibited by the PKC inhibitor staurosporine (Fig. 7 and data not shown). These results indicate that PKC may play a key regulatory role in the modulation of HIV-1 infection.

The resistance of CXCR4 to cross-internalization by CXCR1 may be explained in two ways. First, it could be that cross-regulation of CXCR4 is mediated via a PKC isoform different from that of CCR5, which requires greater second messenger production for its activation. Indeed Delta CXCR1, which mediated greater phosphoinositide hydrolysis and Ca2+ mobilization, cross-internalized CXCR4. Second, previous studies in our laboratory have shown that PMA-induced CXCR4 internalization occurs via a mechanism distinct from receptor phosphorylation (9). It is likely that phosphorylation of (an)other component(s) distal from the receptor/G-protein coupling is necessary for the PKC-mediated internalization and may require a higher level of second messenger production. Orsini et al. (11) have shown that a dileucine motif of the carboxyl terminus of the receptor that binds the adaptor protein-2 was necessary for the phosphorylation-independent internalization of the receptor. Whether or not adaptor protein-2 plays a role in the immunomodulation of HIV-1 infection by CXCR4 remains to be explored. Thus far our preliminary studies have shown that, upon activation, both CXCR4 and CCR5 bind adaptor protein-2 (data not shown).

In summary, these data demonstrate that the IL-8 chemokine can inhibit HIV-1 infection via CCR5 through activation of CXCR1 but not CXCR2. Inhibition of HIV-1 infection is not blocked by receptor desensitization alone but requires receptor internalization. CXCR4 is susceptible to CXCR1-mediated cross-desensitization but is resistant to cross-internalization. Delta CXCR1 and PMA, which mediated greater cellular responses, cross-internalized and cross-inhibited CXCR4-mediated virus entry. This suggests that signaling through other chemokine receptors with stronger signal strengths may regulate the ability of CXCR4 as well as CCR5 to function as co-fusion proteins with CD4+. This observation presents new targets for therapeutic intervention against the infection and propagation of HIV-1.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants AI-38910 (to R. M. R.) and DE-03738 (to R. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Meharry Medical College, 1005 Dr. D. B. Todd, Jr. Blvd., Nashville, TN 37208. Tel.: 615-327-6749; Fax: 615-327-6442; E-mail: mrrichardson@mmc.edu.

Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M211745200

2 Richardson, R. M., Marjoram, R. J., Barak, L. S., and Snyderman, R. (2003) J. Immunol. 70, 2904-2911.

3 R. M. Richardson and R. Marjoram, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus type 1; IL-8, interleukin-8; CXCR1, IL-8 receptor A; CXCR2, IL-8 receptor B; PMA, phorbol 12-myristate 13-acetate; RANTES, regulated on activation normal T cell expressed and secreted; SDF, stromal derived factor; RBL, rat basophilic leukemia; MGSA, melanoma growth-stimulating activity; HA, hemagglutinin; PKC, protein kinase C.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Baggiolini, M., Dewald, B., and Moser, B. (1997) Annu. Rev. Immunol. 15, 675-705[CrossRef][Medline] [Order article via Infotrieve]
2. Zlotnik, A., Morales, J., and Hedrick, J. A. (1999) Crit. Rev. Immunol. 19, 1-4[Medline] [Order article via Infotrieve]
3. Cyster, J. G. (1999) Science 286, 2098-2102[Abstract/Free Full Text]
4. Gerard, D., and Rollins, B. J. (2001) Nat. Immunol. 2, 108-115[CrossRef][Medline] [Order article via Infotrieve]
5. Muller, A., Homey, B., Soto, H., Ge, N., Cartron, D., Buchanan, M. E., McClanahan, T., Murphy, E., Yuan, W., Wagner, S. N., Barrera, J. L., Mohar, A., Verastegui, E., and Zlotnik, A. (2001) Nature 410, 50-56[CrossRef][Medline] [Order article via Infotrieve]
6. Dimitrov, D. S., Xiao, X., Chabot, D. J., and Broder, C. C. (1998) J. Membr. Biol. 166, 75-90[CrossRef][Medline] [Order article via Infotrieve]
7. Locati, M., and Murphy, P. M. (1999) Annu. Rev. Med. 50, 425-440[CrossRef][Medline] [Order article via Infotrieve]
8. Richardson, R. M., Snyderman, R., and Haribabu, B. (2001) in Chemokine Receptors and AIDS (O'Brien, T., ed) , pp. 31-50, Marcel Dekker Inc., New York
9. Haribabu, B., Richardson, R. M., Fisher, I., Sozzani, S., Peiper, S. C., Horuk, R., Ali, H., and Snyderman, R. (1997) J. Biol. Chem. 272, 28726-28731[Abstract/Free Full Text]
10. Oppermann, M., Mack, M., Proudfoot, A., and Olbrich, H. (1999) J. Biol. Chem. 274, 8875-8885[Abstract/Free Full Text]
11. Orsini, M. J., Parent, J.-L., Mundell, S. J., and Benovic, J. L. (1999) J. Biol. Chem. 274, 31076-31086[Abstract/Free Full Text]
12. Kraft, K., Olbrich, H., Majoul, I., Mack, M., Proudfoot, A., and Oppermann, M. (2001) J. Biol. Chem. 276, 34408-34418[Abstract/Free Full Text]
13. Hüttenrauch, F., Nitzki, A., Lin, F.-T., Höning, S., and Oppermann, M. (2002) J. Biol. Chem. 277, 30769-30777[Abstract/Free Full Text]
14. Richardson, R. M., Pridgen, B. C., Haribabu, B., and Snyderman, R. (2000) J. Biol. Chem. 275, 9201-9208[Abstract/Free Full Text]
15. Deng, X., Ueda, H., Su, S. B., Gong, W., Dunlop, N. M., Gao, J., Murphy, P., and Wang, J. M. (1999) Blood 94, 1165-1173[Abstract/Free Full Text]
16. Shen, W., Proost, P., Li, B., Gong, W., Le, Y., Sargeant, R., Murphy, P., Damme, J. V., and Wang, J. M. (2000) Biochem. Biophys. Res. Commun. 272, 276-283[CrossRef][Medline] [Order article via Infotrieve]
17. Berger, E. A., Murphy, P. M., and Farber, J. M. (1999) Annu. Rev. Immunol. 17, 657-700[CrossRef][Medline] [Order article via Infotrieve]
18. Murphy, P. M., Baggiolini, M., Charo, I. F., Hebert, C. A., Horuk, R., Matsushima, K., Miller, L. H., Oppenheim, J. J., and Power, C. A. (2000) Pharmacol. Rev. 52, 145-176[Abstract/Free Full Text]
19. Tani, K., Su, S. B., Utsonomiya, I., Oppenheim, J. J., and Wang, M. (1998) Eur. J. Immunol. 28, 502-507[CrossRef][Medline] [Order article via Infotrieve]
20. Xu, L., Kelvin, D. J., Ye, G. Q., Taub, D. D., Ben-Baruch, A., Oppenheim, J. J., and Wang, J. M. (1995) J. Leukoc. Biol. 57, 335-342[Abstract]
21. Bacon, K. B., Flores-Romo, L., Life, P. F., Taub, D. D., Premack, B. A., Arkinstall, S. J., Wells, T. N., Schall, T. J., and Power, C. A. (1995) J. Immunol. 154, 3654-3666[Abstract/Free Full Text]
22. Qin, S., LaRosa, G., Campbell, J. J., Smith-Heath, H., Kassam, N., Shi, X., Zeng, L., Butcher, E. C., and Mackay, C. R. (1996) Eur. J. Immunol. 26, 640-647[Medline] [Order article via Infotrieve]
23. Wang, J. M., Xu, L., Murphy, W. J., Taub, D. D., and Chertov, O. (1996) Methods Enzymol. 10, 135-144[CrossRef]
24. Utsunomiya, I., Tani, K., Oppenheim, J. J., and Wang, J. M. (1997) Eur. J. Immunol. 27, 1406-1412[Medline] [Order article via Infotrieve]
25. Tomhave, E. D., Richardson, R. M., Didsbury, J. R., Menard, L., Snyderman, R., and Ali, H. (1994) J. Immunol. 153, 3267-3275[Abstract/Free Full Text]
26. Xu, L. L., McVicar, D. W., Ben-Baruch, A., Kuhns, D. B., Johnston, J., Oppenheim, J. J., and Wang, J. M. (1995) Eur. J. Immunol. 25, 2612[Medline] [Order article via Infotrieve]
27. Richardson, R. M., DuBose, R. A., Ali, H., Tomhave, E. D., Haribabu, B., and Snyderman, R. (1995) Biochemistry 34, 14193-14201[Medline] [Order article via Infotrieve]
28. Tokunaga, K., Greenberg, M. L., Morse, M. A., Cumming, R. I., Lyerly, H. K., and Cullen, B. R. (2001) J. Virol. 75, 6776-6786[Abstract/Free Full Text]
29. Richardson, R. M., Pridgen, B. C., Haribabu, B., Ali, H., and Snyderman, R. (1998) J. Biol. Chem. 273, 23830-23836[Abstract/Free Full Text]
30. Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D. (1993) Proc. Natl. Acad. Sci. U. S. A.  90, 8392-8396[Abstract/Free Full Text]
31. Mochizuki, N., Otsuka, N., Matsuo, K., Shiino, T., Kojima, A., Kurata, T., Sakai, K., Yamamoto, N., Isomura, S., Dhole, T. N., Takebe, Y., Matsuda, M., and Tatsumi, M. (1999) AIDS Res. Hum. Retrovir. 15, 1321-1324[CrossRef][Medline] [Order article via Infotrieve]
32. Malim, M. H., Hauber, J., Fenrick, R., and Cullen, B. R. (1988) Nature 335, 181-183[CrossRef][Medline] [Order article via Infotrieve]
33. Fouchier, R. A. M., Meyer, B. E., Simon, J. H. M., Fischer, U., and Malim, M. H. (1997) EMBO J. 16, 4531-4539[Abstract/Free Full Text]
34. Montovani, A. (1999) Immunol. Today 20, 254-257[CrossRef][Medline] [Order article via Infotrieve]
35. Ali, H., Richardson, R. M., Haribabu, B., and Snyderman, R. (1999) J. Biol. Chem. 274, 6027-6030[Free Full Text]
36. Alkhatib, G., Locati, M., Kennedy, P. E., Murphy, P. M., and Berger, E. A. (1997) Virology 234, 340-348[CrossRef][Medline] [Order article via Infotrieve]
37. Atchison, R. E., Gosling, J., Monteclaro, F. S., Franci, C., Digilio, L., Charo, I. F., and Goldsmith, M. A. (1996) Science 274, 1924-1926[Abstract/Free Full Text]
38. Farzan, M., Choe, H., Martin, K. A., Sun, Y., Sidelko, M., Mackay, C. R., Gerald, N. P., Sodroski, J., and Gerald, C. (1997) J. Biol. Chem. 272, 6854-6857[Abstract/Free Full Text]
39. Wuyts, A., Proost, P., and Van Damme, J. (1998) in The Cytokine Handbook (Thompson, A. W., ed), 3rd Ed. , pp. 271-311, Academic Press, London
40. Chuntharapai, A., and Kim, K. J. (1995) J. Immunol. 155, 2587-2594[Abstract]


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