Interleukin-8-mediated heterologous receptor internalization provides resistance to HIV-1 infectivity. Role of signal strength and receptor desensitization.

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, DeltaCXCR1, produced greater signals upon activation (Ca(2+) 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 DeltaCXCR1, 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.

Chemokines are a diverse gene family of chemotactic cytokines that induce leukocyte accumulation and activation at sites of inflammation (1)(2)(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 G i (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 ϩ 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 ϩ T lymphocytes use CXCR4 (T-tropism) (18). In addition to CCR5 and CXCR4, macrophages and CD4 ϩ T lymphocytes express other chemokine receptors including the IL-8 receptors CXCR1 and CXCR2 (1-8 ϫ 10 6 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. (8500 -9120 Ci/mmol), 125 I-IL-8,  and 125 I-RANTES were purchased from PerkinElmer Life Sciences. IL-8 (monocyte-derived), melanoma growth-stimulating activity (MGSA or CXCL1), RANTES, MIP-1␤ (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.

Materials-[ 32 P]Orthophosphate
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 ⌬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 ϫ 10 7 cells) were transfected by electroporation with 20 g 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 ϫ 10 6 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 l) 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 l) 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 g 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 Ci/ml [ 3 H]inositol. The generation of inositol phosphates was determined as reported previously (9,14). For calcium mobilization, RBL cells (5 ϫ 10 6 ) or monocytes (10 7 ) were washed with HEPES-buffered saline and loaded with 1 M 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 ϫ 10 6 ) expressing the receptors were incubated with [ 32 P]orthophosphate (150 Ci/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-⌬CXCR1 were generated from pNL-Luc/Rev Ϫ (28) 2 by replacing the NotI-XhoI fragment encoding luciferase with a PCRamplified NotI-XhoI fragment encoding either CXCR1 or ⌬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 g 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 g of the Rev expression vector pcRev (32) and 0.5 g of the vesicular stomatitis virus glycoprotein expression vector pHIT/G (33) with 1 g 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-m-pore-size filters, and virus yield was measured by enzyme-linked immunosorbent assay. Virus stocks were stored at Ϫ80°C until needed.
Overexpression of CXCR1 and ⌬CXCR1 on Human Cells and Luciferase Reporter Virus Assays-MAGI (HeLa-CD4-LTR-␤-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 ϫ 10 5 ) were transduced overnight with 10 ng of p24 antigen of vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vector virus encoding either CXCR1, ⌬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 ⌬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 l of lysis buffer, and luciferase activities were determined with a MicroLumatPlus LB96V microplate luminometer.

Cross-desensitization of CCR5-and CXCR4-mediated Ca 2ϩ
Mobilization in Human Monocytes-To study the cross-desensitization of CCR5 and CXCR4, intracellular Ca 2ϩ 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 Ca 2ϩ 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 (ϳ90%) responses to a second dose of the same ligand (Fig. 1B).
Cross-phosphorylation of CCR5 and CXCR4 -To assess the role of receptor phosphorylation in cross-internalization 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).
⌬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, ⌬CXCR1, along with CXCR4 (⌬ACXCR4). The K d and B max of ⌬CXCR1 (2.1 Ϯ 1.10 nM and 6898 Ϯ 523 receptors/cell, respectively) were similar to those of CXCR1 (1.7 Ϯ 0.33 nM and 7013 Ϯ 311 receptors/cell, respectively). ⌬CXCR1 mediated greater phosphoinositide hydrolysis (Fig. 8A), G-protein activation, secretion of ␤-hexosaminidase, and sustained Ca 2ϩ response relative CXCR1. 2 IL-8 pretreatment of ⌬ACXCR4 but not ACXCR1 cells resulted in crossinternalization of CXCR4 (Fig. 8B). In contrast to CXCR1, ⌬CXCR1 activation also cross-inhibited CXCR4-mediated virus entry into MAGI cells (Fig. 8C). DISCUSSION 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 chemo-  kines. The IL-8 receptor CXCR1 was shown to cross-desensitize responses to the CC receptor CCR1 at two levels: receptor/Gprotein uncoupling via receptor cross-phosphorylation and inhibition of phospholipase C␤ 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 FIG. 6. Cross-phosphorylation of CCR5 and CXCR4. 32 P-Labeled ACCR5 (A) and ACXCR4 (B) RBL cells (5 ϫ 10 6 /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. CCR5 ( Fig. 5 and data not shown). Second, while CXCR1 crossphosphorylated and cross-desensitized Ca 2ϩ mobilization and GTPase activity to both CCR5 and CXCR4, receptor internalization occurred only with CCR5.
HIV-1 entry into CD4 ϩ 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 ⌬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 (ϳ90% after 2-5 min) and, as a consequence, does not mediate cross-regulatory signals (14,29,40). CXCR1, which is more resistant to internalization (ϳ50% after 20 -40 min), mediated cross-phosphorylation and crossdesensitization 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). ⌬CXCR1, however, which is far more resistant to internalization (ϳ10% after 60 min) and mediated greater cellular responses (i.e. phosphoinositide hydrolysis, exocytosis, and Ca 2ϩ 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 ␤-arrestin-2 in which CXCR2 internalization is delayed (ϳ25% after 5 min) MGSA cross-desensitized Ca 2ϩ mobilization to RANTES by ϳ50% relative to control or wild type mice (ϳ90% 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 crossregulation of CXCR4 is mediated via a PKC isoform different from that of CCR5, which requires greater second messenger production for its activation. Indeed ⌬CXCR1, which mediated greater phosphoinositide hydrolysis and Ca 2ϩ 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. ⌬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 ϩ . This observation presents new targets for therapeutic intervention against the infection and propagation of HIV-1.