of March 30, 2018.This information is current as

AntibodiesNaturalMechanism To Modulate CCR5 with

ERK1-Based Pathway as a New Selective

Agostino Riva, Maria Teresa Sciortino and Lucia LopalcoAssunta Venuti, Claudia Pastori, Gabriel Siracusano,

ol.1500708http://www.jimmunol.org/content/early/2015/08/30/jimmun

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The Journal of Immunology

ERK1-Based Pathway as a New Selective Mechanism ToModulate CCR5 with Natural Antibodies

Assunta Venuti,* Claudia Pastori,* Gabriel Siracusano,† Agostino Riva,‡

Maria Teresa Sciortino,† and Lucia Lopalco*

Natural human Abs, recognizing an epitope within the first extramembrane loop of CCR5 (the main HIV coreceptor), induce a long-

lasting internalization (48 h) of the protein, whereas all known CCR5modulating molecules show a short-term kinetics (60–90 min).

Despite extensive studies on the regulation of CCR5 signaling cascades, which are the effect of concomitant CCR5 internalization

by exogenous stimuli such as Abs, downstream signaling continues to be poorly understood. In this article, we report a hitherto

unrecognized mechanism of CCR5 modulation mediated by G protein–dependent ERK1 activity. We further demonstrate that

ERK1 is localized mainly in the cytoplasmic compartment and that it interacts directly with the CCR5 protein, thus provoking

possible CCR5 degradation with a subsequent de novo synthesis, and that re-expression of CCR5 on the cell membrane required

several days. In contrast, the RANTES treatment induces a recovery of the receptor on the cell membrane in short-term kinetics

without the involvement of de novo protein synthesis. The said new pathway could be relevant not only to better understand the

molecular basis of all pathologic conditions in which CCR5 is involved but also to generate new tools to block viral infections, such

as the use of recombinant Abs. The Journal of Immunology, 2015, 195: 000–000.

Gprotein–coupled receptors (GPCRs) represent the largestfamily of proteins (∼800) in the human genome. Be-cause of their central involvement in almost every aspect

of human physiology, they also represent the largest target fortherapeutic intervention (1). CCR5 is among the major drug tar-gets, because this receptor not only plays a critical role in leu-kocyte trafficking during inflammatory processes but also servesas a major HIV coreceptor for virus entry and cell-to-cell spread-ing (2). CCR5 internalization by chemokines starts with severalsequential events. Receptors associate with G proteins, whichin turn activate signaling processes, for example, changes inCa2+, whereby GPCR kinases induce receptor phosphorylation(3). This in turn leads to the association of b-arrestin 1/2 with thereceptor and to desensitization via uncoupling of the receptor andG protein (4). The internalization and recycling of the receptorinvolve short-term kinetics and are complete within a few hours(4, 5). Abs to CCR5, whether natural or elicited by virus exposure,induce CCR5 internalization with a mechanism differing from thatreported for other CCR5-specific molecules; Abs to CCR5 induce

full and long-lasting CCR5 downregulation, and hence very long-term kinetics, which cause HIV-blocking properties that mayprovide both systemic and local protection from the virus (6).CCR5 internalization, mediated by CCR5-specific Abs, may notrequire the involvement of b-arrestins to induce ligand-activatedreceptor into clathrin-coated pits, as reported for other GPCRs(7). However, the signaling mechanisms following CCR5 activa-tion are not completely understood, and the association betweenb-arrestin 1/2 and CCR5 receptor trafficking and signaling couldimply the existence of a new pathway. One potential nexus forCCR5 signaling and the multifunctional scaffold protein arrestinsconsists of MAPK families (8). It is noteworthy that G protein–dependent ERK activation stimulates the translocation of activatedERKs from the cytoplasm to the nucleus, but interaction withb-arrestins often restricts activated ERKs to the cytoplasm (8).Because arrestins preferentially bind to some, but not all, GPCRsin a ligand-dependent manner, we envision that arrestins exerttheir role in positive Ab-mediated long-lasting CCR5 downregu-lation by coordinating ERK activation. Such a regulatory mecha-nism and the distinct subcellular compartmentalization are essentialfor modulating MAPK signaling in various cellular functions (9,10). Data from the literature show that HIV viruses using CCR5 forentry purposes activate JNK and p38MAPKs efficiently (11), butthe role of MAPK ERK in the HIV-1 life cycle is not completelyunderstood. The present study aims to comprehensively investigatethe signaling pathways promoted by natural anti-CCR5 Abs thatregulate the receptor internalization ERK-mediated mechanismsand CCR5 recycling.

Materials and MethodsCell lines

The SupT1 cell line (NIBSC, CFAR, UK) was maintained in RPMI 1640(Lonza, Belgium) supplemented with 10% FCS (Lonza), 2 mM L-gluta-mine, 100 U/ml penicillin and 100 U/ml streptomycin. R5-SupT1-L23 andM10 cell lines are CCR5-expressing cell lines referred to as SupT1-R5clones L23 (low expression of CCR5) and M10 (medium expression ofCCR5). The clones were obtained by engineered SupT1 cells and kindlyprovided by H. Garg (Department of Biomedical Science, Texas Tech

*Division of Immunology, Transplantation and Infectious Diseases, San RaffaeleScientific Institute, 20127 Milan, Italy; †Department of Biological and EnvironmentalSciences, University of Messina, 98166 Messina, Italy; and ‡Third Division of In-fectious Diseases, Luigi Sacco Hospital, University of Milan, 20157 Milan, Italy

Received for publication March 26, 2015. Accepted for publication July 28, 2015.

This work was supported by the Italian Ministry of Health (Grant 40H15) to L.L. andby the Italian Ministry of University and Research, Research Projects of NationalInterest 2010–2011 (Grant 2010PHT9NF_005) to M.T.S.

A.V. performed and analyzed all data; C.P. conducted the preliminary experiments;G.S. performed immunofluorescence assays; A.R. recruited and analyzed all subjectsincluded in the study; and M.T.S. and L.L. designed experiments and wrote the paper.

Address correspondence and reprint requests to Dr. Lucia Lopalco or Dr. MariaTeresa Sciortino, San Raffaele Scientific Institute, Centro San Luigi, via StamiraD’Ancona 20, 20127 Milan, Italy (L.L.) or Department of Biological and Environ-mental Sciences, University of Messina, 98166 Messina, Italy (M.T.S.). E-mailaddresses: lopalco.lucia@hsr.it (L.L.) or mtsciortino@unime.it (M.T.S.)

Abbreviations used in this article: CHX, cycloheximide; dn, dominant negative; dnERK1,dnERK1 mutant; GPCR, G protein–coupled receptor; si, short interfering; t, time.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500708

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University, Health Sciences Center, Lubbock, TX). L23 and M10 cloneswere propagated in complete medium as described above and supple-mented with 3 mg/ml blasticidin (Calbiochem, Germany). Cell lines werecultured at 37˚C in a 5% CO2 incubator.

Reagents

Anti–CKR-5 (D6), anti–p-CKR-5(E11/19), anti–b-arrestin1/2 (21-B1),anti–a-adaptin 1/2 (C-8), anti-GAPDH mAbs, and anti-ERK1 (C-16), anti-ERK2 (C-14), anti–p-ERK1/2 (Thr202/Tyr204) polyclonal Abs were pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-histone H3polyclonal Abs were obtained from Cell Signaling Technology (Beverly,MA). PE Mouse anti-Human CD195 (2D7), PE-Cy5 Mouse anti-HumanCD38, and each Mouse IgG Isotype Control were purchased from BDPharmingen (San Diego, CA). Secondary HRP-conjugated goat anti-mouseIgG and goat anti-rabbit IgG were purchased from Millipore; secondaryFITC-conjugated goat anti-mouse IgG was from ICN Biomedicals (Aurora,OH). Secondary HRP-conjugated anti-mouse IgGVeriBlot and anti-rabbitIgGVeriBlot for immunoprecipitation were from Abcam (U.K.). The poolof serum samples from five LTNP positive for anti-CCR5 natural Abs(CCR5 Ab pos) was as follows: LTNP no. 21, LTNP no. 20, LTNP no. 11,LTNP no. 4, and LTNP no. 22, all of which had been characterized pre-viously (12). The pool of serum samples used as negative controls wastaken from LTNP negative for anti-CCR5 Abs (CCR5 Ab neg), as previ-ously described (12).

Ethics statement

The institutional review board Comitato Etico della Fondazione SanRaffeale del Monte Tabor, Milan, Italy, approved the investigations, Pro-tocol no. 95/DG. All subjects provided written informed consent.

Purification of human CD4+ T lymphocytes

Human PBMCs from three healthy donors were isolated with Ficoll-Hypaque(Pharmacia, Uppsala, Sweden). CD4+ cells were purified from restingPBMCs by immune adsorption to anti-CD4 magnetic beads (Miltenyi Bio-tec, Italy). Purified CD4+ T lymphocytes were stimulated for 16 h in RPMI1640 supplemented with 10% FCS (Lonza), 2 mM L-glutamine, 100 U/mlpenicillin, and 100 U/ml streptomycin in the presence of recombinant IL-2(100 U/ml; Amersham, Buckinghamshire, U. K.); these lymphocytes werethen cultured at 37˚C in a 5% CO2 incubator. All donors were wild-type forCCR5, none carrying the D32 mutation.

CCR5 expression and internalization assay

R5-SupT1-L23 cells or CD4+ T lymphocytes were exposed to CCR5 Ab pos(1/30), and to 2 mg/ml RANTES (R&D Systems, Minneapolis, MN) aspositive control, at 37˚C for 30 min. Serum dilution and RANTES con-centration have been chosen based on previous titration experiments; inparticular, serum samples have been tested at 1:10, 1:30, and 1:50, and 1:30was the first nontoxic dilution compared with serum samples negative foranti-CCR5 Abs. RANTES was tested at 5, 2, and 0.5 mg/ml, and 2 mg/mlwas the minimum concentration able to bind CCR5 and induce the highestpercentage of receptor internalization (data not shown). The cells were thenwashed and incubated for an additional 120 min. To obtain a completedownregulation of the receptor, the cells were incubated with CCR5 Ab posat 37˚C for 48 h (12). CCR5 Ab neg was used as negative control (12). Whenindicated, the cells were pretreated for 1 h with hypertonic sucrose medium(0.45 M) or filipin (2.5 mg/ml) to inhibit CCR5 internalization, with U0126(a specific inhibitor of ERK pathway) (5 mg/ml) and with 2.5 mg/ml cy-cloheximide (CHX) (Sigma-Aldrich) to inhibit protein synthesis. As U0126has been reconstituted in DMSO, the same percentage of DMSO diluted inRPMI 1640 was used as negative control (data not shown). After 48 h ofCHX treatment, R5-SupT1-L23 and CD4+ T lymphocytes were washed andincubated in complete medium for an additional 24 h and 72 h, respectively.When indicated, after 48 h of Ab incubation, R5-SupT1-L23 cells and CD4+

T lymphocytes were washed and CCR5 cell surface re-expression wasevaluated by flow cytometry every day for 10 and 15 consecutive days, re-spectively. As the CCR5 expression was not homogeneous among the dif-ferent donors, CD4+ T lymphocytes from 3 of 10 different donors showingthe highest CCR5 expression have been selected and used for all experi-ments. FITC-conjugated anti-CD38 was tested on R5-SupT1-L23 and acti-vated CD4+ T lymphocytes.

Immunofluorescence was performed to detect cytoplasmic CCR5. Thesamples were collected, layered on slides treated with polylysine, and fixedin 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature,and permeabilized with 0.1% Triton X-100 in PBS for 15 min. Cells werethen incubated with anti–CKR-5 (D6) primary Ab for 45 min at 4˚C,followed by incubation with FITC-conjugated secondary Ab. Samples

were analyzed on a Leica DMRE fluorescence microscope. In some cases,flow cytometry was performed, as well, to detect cell surface expression ofCCR5. Treated cells were collected, washed, and incubated with thespecific Ab against CCR5, anti-CD195, for 45 min at 4˚C, and the analysiswas performed with FACSCalibur or multicolor LSR II (BD). For eachanalysis, 10,000 events were acquired and gated on CCR5 expression andside scatter properties. Samples were first run using an isotype control forcolor compensation.

Western blotting

Protein lysates were collected in radioimmunoprecipitation assay lysisbuffer (50 mM Tris-HCl, pH 7.5; 10 mM MgCl2; 150 mM NaCl; 0.5%sodium deoxycholate; 1% Nonidet P-40) supplemented with cOmpleteProtease Inhibitor mixture (Roche). Samples were resolved by SDS-PAGEand transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA).Membranes were blocked in 5% nonfat milk and incubated overnight at4˚C with the appropriate primary Ab. The membranes were then probedwith HRP-tagged secondary Abs at room temperature for 1 h. Immuno-reactive proteins were visualized by means of the ECL method (Euro-Clone). Comparative analysis of the bands was performed by quantitativedensitometry and concomitant use of the Tina software (version 2.10;Raytest, Straubenhardt, Germany). The normalization was done per eachband based on the density of GAPDH or histone H3 signals per each line.

Construction of plasmid dnERK1 and nucleofection

ERK1 CDS (NM_002746) was cloned into the expression vector pcDNA3.1(Invitrogen, Carlsbad, CA) with the following primers: forward 59-ATG-AATTCGACATGGATTACAAGGATGACGACGATAAGATGGCGGCGG-CGGCGGCTCAGGGG-39; reverse 59-TTGCGGCCGCTCACTTATCGT-CGTCATCCTTGTAATCCATGGGGGCCACCAGCACTCCGGGCTG-39.The sequences of FLAG epitope were also introduced. To produce a mu-tation in the ATP binding site (72 aa) and phosphorylation sites (202 and204 aa), two rounds of site direct mutagenesis were carried out accordingto the manufacturer’s instructions. The first round used the followingprimers: forward 59-cgcaagactcgAgtggcccgcaagaagCGgatcatcagccccttc-39;reverse 59-gaaggggctgatcCGcttgatggccacTcgagtcttgcg-39.

The double mutation, on positions 202 and 204 of the sequences, wasperformed with the aid of the following primers: forward 59-ggcttcc-tgGcggagtTtgtggctacgcgctggtaTcgggcccc-39; reverse 59-ggggcccgataccag-cgcgtagccacaaactccgccaggaagcc-39.

The mutated DNA obtained was used for the nucleofection experiments.A total of 2 mg dnERK1-DNA was used to nucleofect 2 3 106 R5-

SupT1-L23 cells by means of an AmaxaNucleofector 4D (Lonza) in ac-cordance with the manufacturer’s instructions. Subsequently, cells, 4 3105 per well, were seeded onto 24 multiwell plates for 24 h and treatedaccording to the experimental procedure for CCR5 internalization assay.

Cytoplasmic and nuclear protein extraction

The extracts were prepared as previously described (13). Briefly, 3 3 106

cells were collected, washed in PBS 13, and resuspended in hypotonicbuffer A (10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; 0.5 mMDTT; 0.2 mM PMFS). The cell suspensions were then incubated on iceand homogenized by 15 passages through a 25-gauge needle. Cytoplasmfractions were collected by centrifugation at 12,000 rpm for 1 min at 4˚C.Nuclei were washed in buffer A, centrifuged, and dissolved in hypertonicbuffer B (20 mM HEPES, pH 7.9; 25% glycerol; 0.42 M NaCl; 1.5 mMMgCl2; 0.2 mM EDTA; 0.5 mM DTT). The nuclear extracts were collectedby centrifugation at 12,000 rpm for 2 min at 4˚C. Equal amounts of cy-toplasmic or nuclear fractions were subjected to electrophoresis in 10% de-naturing polyacrylamide gels to resolve ERK1/2 and p-ERK1/2. GAPDHand histone H3 were used for housekeeping.

Immunoprecipitation

The cells were treated in accordance with the experimental procedure forCCR5 internalization assay. After 150 min, cells were washed twice in PBSand lysed with cold lysis buffer (20 mM Tris-HCl, pH 8; 1 mM EDTA;200mMNaCl; 1%Nonidet P-40; 2 mMDTT; 0.1mMNa3VO4; 10 mMNaF;0.1 mg/ml protease inhibitors). The supernatants were collected and preclearedwith 50% protein-A slurry for 16 h. Immunoprecipitations were performedwith 5 ml of the indicated Abs preadsorbed on Protein A-Sepharose beads(Amersham Pharmacia Biotech AB) for 2 h at 4˚C (14). After overnightincubation with the extracts, complexate-beads were resolved by SDS-PAGEand transferred to nitrocellulose membranes (Bio-Rad). Immunoblotting wasperformed with the indicated Abs, and immunoreactivity was revealed bymeans of secondary Abs specific for immunoprecipitation (Abcam) and ofPlatinum HD2 (Uvitec, Cambridge, U.K.).

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Real-time RT-PCR

Total RNAwas isolated from the CHX experiment with a QIAGEN RNeasyMini Kit (QIAGEN, Valencia, CA). Subsequently, 150 ng RNAwas reversetranscribed to cDNA by means of SuperScript III First-Strand SynthesisSystem (Invitrogen) in accordance with the manufacturer’s instructions.Gene transcript levels were analyzed with SYBR-Green-PCR Master Mixon an AB 7900HT real-time system (Applied Biosystems, Carlsbad, CA).The thermal profiles were 50˚C for 2 min; 95˚C for 10 min; 40 cycles at95˚C for 15 s; 60˚C for 1 min. GAPDH amplification was used to nor-malize the RNA content of the corresponding transcripts analyzed.

Commercial primers were Hs_MAPK3_1_SG (ERK1), Hs_MAPK1_1_SG(ERK2), Hs_MAPK14_1_SG (p38) QuantiTect Primer Assay (QIAGEN).Primer sequences were as follows: 59-ACCAGATCTCAAAAAGAAGGTCT-39 (CCR5-forward); 59-CATGATGGTGAAGATAAGCCTCA-39 (CCR5-reverse); 59-TGGAACACAACCACCCACAA-39 (CXCR4-forward); 59-CC-CTGCCCTCCTGCTGACTA-39 (CXCR4-reverse); 59-CCATGGAGAAGG-CTGGGG-39 (GAPDH-forward); 59-CAAAGTTGTCATGGATGACC-39(GAPDH-reverse). Relative expression was calculated with the DDCtstandardization method.

Short interfering RNA nucleofection

CCR5 GeneSolution short interfering (si) RNA was purchased fromQIAGEN. Briefly, 300 nM siCCR5 or NSsiRNAwas used to nucleofect 23106 cells. The cells were then seeded onto 24 multiwell plates for 5 h andtreated in accordance with the experimental procedure for CCR5 inter-nalization assay.

Statistical analysis

The two-tailed Student t test was used, and the data were analyzed byPrism version 5.0a (GraphPad Software, La Jolla, CA). Figs. 1–4 show thefollowing p values: *p # 0.05, **p # 0.01, ***p # 0.001.

ResultsAnti-CCR5 Abs induce CCR5 internalization with clathrin

Previous works published by our and other groups (15–18) provedthe existence of anti-CCR5 Abs, which induced long-lastinginternalization of CCR5 in the sera of a subset of LTNP andHIV-exposed uninfected subjects (12, 18–22). The mechanismsunderlying this unconventional and atypical long-lasting down-regulation of CCR5 required further investigation. Accordingly,in-depth analysis of the early events subsequent to the incubationof CCR5-specific Abs (CCR5 Ab pos) was performed with theT lymphoblastoid R5-SupT1-L23 cell line, which has cell sur-face levels very similar to those expressed in human CD4+ Tlymphocytes (23). Where indicated, the majority of the key ex-periments were confirmed in human CD4+ T lymphocytes, whichrepresent the natural target of HIV. As both T cells, R5-SupT1-L23 cells and human CD4+ T lymphocytes, have been used tocharacterize CCR5 modulation, the activation status of cells hasbeen evaluated by flow cytometry, and the mean fluorescenceintensity obtained with anti-CD38 Ab was 54.5 and 34.5 for R5-SupT1-L23 cells and activated CD4+ T lymphocytes, respectively.We used a pool of five LTNP sera, each containing CCR5 Abs aspreviously described and characterized in a paper by our group(12). Briefly, serum anti-CCR5 Abs recognized the cyclic ECL1peptide (YAAAQWDFGNTMCQ, aa 89–102), which is not in-volved in HIV binding. Purified Abs on ECL1 peptide were ableto induce long-lasting CCR5 internalization, both in CD4+ Tlymphocytes and in CCR5-transfected cell lines, and blocked in-fection from CCR5-dependent strains of HIV (12). We also useda pool of serum samples from five LTNP not containing anti-CCR5 Abs, as previously described (CCR5 Ab neg) (12).Procedurally, our first task was to examine whether CCR5 in-

ternalization by anti-CCR5 Abs is associated with clathrin- orcaveolae-mediated endocytosis. To this end, cells were pretreatedwith 0.45 M sucrose, a specific inhibitor of clathrin-coated pitspathway, and filipin (2.5 mg/ml), an inhibitor of caveolae. Thecells were then incubated for 30 min with either CCR5 Ab pos or

CCR5 Ab neg. We used a CCR5 ligand, the chemokine RANTES(2 mg/ml), as a positive control (24). After 30 min, the cells werecollected, washed, and separated into two groups. The first groupserved to compare the short incubation time (30 min) needed toobtain a specific and classical CCR5 internalization by naturalCCR5 ligands, such as RANTES (4, 5, 25–27). The second groupwas then incubated for a further 120 min (i.e., incubation for thisgroup totaled 150 min); this time was determined on the basis thatCCR5 internalization by CCR5 Ab pos becomes evident only after150 min. The first group, using CD4+ T lymphocytes, showeda marked CCR5 internalization with RANTES (75%) but withneither CCR5 Ab pos (3%) nor CCR5 Ab neg (8%). The secondgroup, as shown in Fig. 1A, demonstrated that at 150 min cellstreated with CCR5 Ab pos revealed a low CCR5 expression per-centage (53%) in comparison with CCR5 Ab neg (87.5%) anduntreated cells (88.1%).To reveal CCR5 localization at a single-cell level, we used static

fluorescence analysis. After treatment with the CCR5 modulatingmolecules described above, both R5-SupT1-L23 cells (Fig. 1B,1C) and CD4+ T lymphocytes (Fig. 1D, 1E) were collected at30 min and 150 min, permeabilized, and stained with a specific Abto human-CCR5; this Ab binds external regions of CCR5 (aa 66–250) (D6). At 30 min, the percentage of CCR5-positive cellsand the number of specific CCR5 puncta per cell treated withRANTES were statistically significant (p # 0.001 in R5-SupT1-L23 cells and p # 0.01 in CD4+ T lymphocytes for both evalu-ations) (Fig. 1B–E) in comparison with the analogous percentagein untreated cells. In contrast, the percentage of CCR5-positive cellsand the number of specific CCR5 puncta per cell treated with CCR5Ab pos increased less than those treated with RANTES, but theincrease was still statistically significant (p # 0.01 and p # 0.05,respectively, in both R5-SupT1-L23 cells and CD4+ T lymphocytes)in comparison with CCR5 Ab neg treatment (Fig. 1B–E). As ex-pected, both filipin and sucrose treatment inhibited RANTES-basedinternalization (p # 0.01) in R5-SupT1-L23 cells (Fig. 1B, 1C),as previously described by Mueller (4). In addition, CD4+ Tlymphocytes showed similar results, but with a statistically signif-icant lower percentage of punctuate cells, p # 0.05 with filipin andsucrose treatment (Fig. 1D), and p # 0.01 and p # 0.05 for thenumber of CCR5 puncta per cells for both treatments (Fig. 1E). Noeffect was evident after treatment with CCR5 Ab pos (Fig. 1B–E).Conversely, at 150 min the percentage of cytoplasmic CCR5 punctastructures had substantially increased (p # 0.001 in both R5-SupT1-L23 cells and CD4+ T lymphocytes) after treatment with anti-CCR5Ab pos, but not with RANTES. When incubated with anti-CCR5Ab pos, sucrose treatment inhibited the percentage of CCR5-positive cells in cytoplasm (p # 0.001) as well as the number ofpositive specific CCR5 puncta per cell (p # 0.001) in both R5-SupT1-L23 cells and CD4+ T lymphocytes (Fig. 1B, 1C for R5-SupT1-L23 cells and Fig. 1D, 1E for CD4+ T lymphocytes).Moreover, a less evident but statistically significant reduction

was observed in the presence of filipin with CCR5 Ab pos as well(p # 0.05 and p # 0.01 for the respective percentages of cyto-plasmic CCR5 punctate structures and of CCR5 puncta per cell inR5-SupT1-L23 cells, and p # 0.05 in CD4+ T lymphocytes forboth evaluations). The contribution of filipin, at an early stage ofCCR5 internalization (150 min), represents a general interferenceof such inhibitor in the clathrin-mediated pathway, as previouslydescribed by Signoret (27). At a later stage (48 h), the effect offilipin on CCR5 internalization was not evident, as demonstratedby Pastori (12).These observations confirm that anti-CCR5 Ab pos stimulation

prevalently triggers the translocation of CCR5 molecules intoclathrin-positive domains of the plasma membrane (12) at 150 min.

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FIGURE 1. CCR5 internalization through a clathrin-dependent pathway. (A) Evaluation of CCR5 expression by specific mAb to CCR5 (CD195) on the

cell surface of CD4+ T lymphocytes, in the presence of CCR5 Ab neg or CCR5 Ab pos, or in untreated cells. As the percentage (Figure legend continues)

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A single-cell section from a representative experiment is shown inFig. 1F.

CCR5 endocytosis and cytosolic retention by ERK1/2

To investigate the pathway of CCR5 internalization upon CCR5Ab pos stimulation, we assessed whether such internalization wasassociated with the regulation of core components of the GPCRsignal pathway. Western blotting analysis was performed to verifywhether b-arrestin 1/2, a-adaptin 1/2, CCR5 itself, and ERK1/2proteins were involved in CCR5 trafficking by means of CCR5 Abpos stimulation. R5-SupT1-L23 cells were incubated with CCR5Ab pos, CCR5 Ab neg, and RANTES for 30 min and 150 min, asspecified above. Fig. 2A shows that 30 min of stimulation withCCR5 Ab pos triggered early phosphorylation of CCR5 (p #0.01), as previously reported with other Abs to CCR5 (28). Incomparison with CCR5 Ab neg–treated cells, activated pCCR5correlated with the accumulation of b-arrestin 1/2 and a-adaptin1/2, p # 0.001 for both proteins. Although this effect was lowerin the samples treated with RANTES, and a-adaptin 1/2 andb-arrestin 1/2 did not accumulate significantly, the difference forpCCR5 activation was statistically significant (p # 0.01; seeFig. 2A). The ERK1/2 response did not reflect the activation ofCCR5 by RANTES, as the accumulation both of ERK1/2 and ofpERK1/2 was similar to that of untreated cells (lower graphs inFig. 2A). The cells incubated with CCR5 Ab pos showed greateraccumulation of ERK1/2 and pERK1/2 than did 1) cells incubatedeither with RANTES or with CCR5 Ab neg (statistically signifi-cant, p # 0.001) and 2) untreated cells, as shown in the lowergraphs of Fig. 2A. In contrast, at 150 min of CCR5 Ab pos in-cubation, accumulation of ERK1/2 and pERK1/2 was statisticallysignificant (p # 0.001 for both of them). These results correlatewith CCR5 accumulation and reflect the ability of CCR5, uponCCR5 Ab pos stimulation, to recruit ERK protein directly withoutextraneous intervention. Very low activation, not statistically sig-nificant, of ERK1/2 or pERK1/2 by CCR5 Ab neg, is shown inFig. 2A. At 150 min, no significant change in the accumulationof a-adaptin 1/2 was evident, irrespective of test conditions; incontrast, a moderate, but statistically significant, accumulation(p # 0.05) of b-arrestin 1/2 was observed, as shown in Fig. 2A.In addition, at 150 min of CCR5 Ab pos incubation, the accu-mulation of CCR5 within the cytoplasm was both evident andstatistically significant (p # 0.01). Because ERK1/2 protein, asrecruited by the b-arrestin 1/2 scaffold, is largely excluded fromthe nucleus and confined to cytoplasmic locations (e.g., endocyticvesicles) (29–34), our results raise the question of whether CCR5Ab pos–based ERK1/2 activation is the result of ERK1/2 proteinmigration to the nucleus. To address this question, we incubatedR5-SupT1-L23 cells with CCR5 Ab pos, CCR5 Ab neg, andRANTES for a total of 150 min; both cytosolic and nuclearfractions were then analyzed for pERK1/2 activity. In the cytosol,the stimulation of CCR5 Ab pos induced an increase in totalERK1/2 and a rapid increase in ERK1/2 phosphorylation com-pared with the nonincrease observed for CCR5 Ab neg (p # 0.001for both ERK1/2 and pERK1/2); in contrast, RANTES stimulationshowed a statistically significant reduction in total ERK1/2 form

(p # 0.05), as shown in Fig. 2B. Nuclear pERK1/2 accumulation,as observed with CCR5 Ab pos incubation, was significantlylower than that observed in the cytoplasmic fraction (Fig. 2B).Similar results emerged from the use of CD4+ T lymphocytesfrom three different healthy donors, although the statistical sig-nificance was lower than that obtained with the cell line (p # 0.001and p # 0.05, respectively). A representative assay is illustrated inFig. 2C. The double bands shown in Fig. 2B correspond to pERK1and pERK2 (molecular mass, 42 kDa and 44 kDa, respectively).The single band found in CD4+ T lymphocytes (Fig. 2C) corre-sponds to pERK1 (molecular mass, 42 kDa) and reflects the loweractivation status if compared with the T cell line; thus, the doublebands are lost in primary cells owing to the low level of phos-phorylation. Therefore, CCR5 Ab pos stimulation sustains ERK1/2activation throughout the b-arrestin–dependent pathway and leadsto ERK1/2 sequestration within the cytosolic fraction. On the basisof these results, we hypothesize that CCR5 activation under specificand unique stimuli is likely to influence ERK-activation dynamics.Overall, our data confirm that the reduction of surface CCR5 ex-pression level corresponds to internalization and accumulation ofthe receptor in the cytoplasm.

Inhibition of the ERK1 pathway affects CCR5 accumulation

To provide a more in-depth analysis of the role of ERK1/2, weinvestigated whether inhibition of ERK1 acts as a restriction fac-tor for CCR5 internalization. To this end, we chemically inhibitedERK1/2 phosphorylation by incubating R5-SupT1-L23 cells withU0126 (5 mM), an inhibitor of MAPK phosphorylation. Cellswere pretreated with U0126 for 1 h and incubated with CCR5 Abpos, CCR5 Ab neg, or RANTES for 150 min, at the end of whichWestern blotting analysis was performed. The results demon-strated that the inhibition of ERK1/2 mediated by U0126 hadgreatly reduced cytosolic CCR5 accumulation and phosphoryla-tion, and that b-arrestin 1/2 was similarly reduced under CCR5 Abpos stimulation; in contrast, neither CCR5 Ab neg incubationnor untreated cells produced comparable results (p # 0.001 forall tested conditions) (Fig. 3A). Similar results were obtained inCD4+ T lymphocytes from three different healthy donors, al-though the statistical significance was lower than that obtainedwith R5-SupT1-L23 (p # 0.001 versus p # 0.01 respectively).A representative assay is shown in Fig. 3B.To complement these findings, we further analyzed the in-

volvement of ERK1 in CCR5 outcomes and in CCR5 Ab pos–mediated recycling. To this end, we transiently nucleofected R5-SupT1-L23 cells with the dominant negative (dn) ERK1 mutant(dnERK1), which was mutated in lysine 72, threonine 202, andtyrosine 204 and was unable to translocate to the nucleus (35, 36).At 24 h post nucleofection, the cells were incubated with sera(either CCR5 Ab pos or CCR5 Ab neg) for 150 min and analyzedby Western blot. The functional loss in ERK1 activity induced bydnERK1 validates the data obtained with chemical treatmentand suggests the involvement of ERK1 rather than of ERK2. Theabsence of functional ERK1 protein in dnERK1-nucleofected cellsstrongly reduced both cytosolic CCR5 and b-arrestin 1/2 accu-mulation in cells stimulated with CCR5 Ab pos (p # 0.001 for all

of internalization varied between 25 and 55%, depending on the different donor, flow cytometry analysis of CCR5 expression from one of three different

donors showing the highest percentage of receptor internalization is shown. (B–E) Panels show the percentage of cells with the punctate form of CCR5 in

R5-SupT1-L23 cells (B and C), or CD4+ T lymphocytes (D and E) that were pretreated with sucrose (0.45 M) and filipin (2.5 mg/ml) for 1 h and then

stimulated with RANTES, CCR5 Ab neg and CCR5 Ab pos for 30 min. Cells were harvested at 30 min and 150 min. (B and D) The percentage of CCR5-

positive punctate cells and (C and E) the number of CCR5 puncta per cell are reported. (B)–(E) show bar graphs representing mean 6 SD of three in-

dependent experiments. Student t test was performed, and p values are shown. (F) Representative immunofluorescence images of CCR5-positive cells in

R5-SupT1-L23. *p # 0.05, **p # 0.01, ***p # 0.001.

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tested conditions) (Fig. 3C). In addition, cell surface expression ofthe CCR5 receptor in dnERK1-nucleofected cells, as determinedby FACS analysis, showed that CCR5 Ab pos stimulation reac-tivates long-lasting downregulation of CCR5 (2.66% of CCR5expression versus 12.9% and 13.2%, respectively, of untreatedcells and cells treated with CCR5 Ab neg). Fig. 3D shows theresults obtained with R5-SupT1-L23. Moreover, fluorescencemicroscopy analysis on R5-SupT1-L23 underscored that both thepercentage of cells showing CCR5-specific puncta in the cyto-plasm and the number of CCR5 puncta per cell had accumulatedin the cytoplasm only when ERK1 was functional (whether at150 min or at 48 h); for all tested conditions, statistical signifi-cance was high (p # 0.001) (Fig. 3E, 3F).

Physical association between ERK1/2 and CCR5

To characterize the direct relationship between ERK1/2 proteins andCCR5 receptors, we determined whether the cytosolic accumulationof ERK was physically associated with CCR5. To this end, we usedR5-SupT1-M10 to immunoprecipitate both ERK1/2 and CCR5. R5-SupT1-M10 cells displayed a higher level of CCR5 expression thandid R5-SupT1-L23 cells (23). R5-SupT1-M10 or mock cells (SupT1not transduced with CCR5) were treated with CCR5 Ab pos for150 min. Cells were then lysed and incubated either with ERK1/2-preadsorbed beads or with CCR5-preadsorbed beads, as describedin Materials and Methods. Western blotting analysis performedafter overnight incubation demonstrated that ERK1/2 protein hadinteracted physically with the CCR5 receptor in both untreated andtreated cells. Fig. 3G shows a specific induction of CCR5 accu-mulation by CCR5 Ab pos stimulation. No reactivity was evidenteither in cells treated with CCR5 Ab neg or in untreated mockSupT1 cells (data not shown). This finding suggests that ERK1/2represents the CCR5 binding partner and that it possibly controlsreceptor recruitment upon CCR5 Ab pos stimulation.

siRNA-CCR5 reduces the cytosolic accumulation of thereceptor

We further examined the molecular mechanisms explaining thiscrosstalking by using specific siRNA to silence CCR5 expressionin the R5-SupT1-L23 cell line. Nucleofection was performed withsiRNA specific to CCR5 and NS-siRNA as a negative control; thecells were then treated with CCR5 Ab pos, CCR5 Ab neg, orRANTES, and collected at 150 min and 48 h. The 48-h incubationperiod was chosen on the basis of published data showing thatpreincubation of CD4+ T cells with CCR5 Ab pos induces completelong-lasting CCR5 downregulation. In contrast to all CCR5 ligands,which require only a few hours to achieve the downregulation,CCR5 Ab pos requires 48 h to obtain a complete CCR5 internali-zation (12, 20, 37, 38). The presence of CCR5-specific puncta wasanalyzed by fluorescence microscopy. Fig. 4A, 4B shows that bothat 150 min and at 48 h the depletion of CCR5 by siRNA had sig-nificantly reduced the long-lasting cytosolic accumulation of CCR5in terms both of the number per cell of CCR5-specific puncta and ofthe percentage of cells that show CCR5 puncta in the cytoplasmupon anti-CCR5 Ab pos stimulation (p# 0.001 at both 150 min and

48 h); no statistically significant reduction in cytosolic accumulationwas obtained from cells treated with NS-siRNA (control siRNA)(Fig. 4A, 4B).

CHX treatment influences the restoration of CCR5

To further investigate the mechanism, we blocked protein synthesisto evaluate the respective rates of recycling and CCR5 receptorsynthesis; cells were treated accordingly (or not) with the trans-lational inhibitor CHX and stimulated (or not) with CCR5 Ab posand their controls. The cells were collected at time(t) 0 (150 min),t1 (48 h), t2 (48 h, wash, further 24 h), and t3 (48 h, wash, further72 h), as described in Fig. 4C. At 150 min (t0), the cells treatedwith CHX displayed a regular cytoplasmic accumulation of CCR5by CCR5 Ab pos, although a reduction in CCR5 accumulationbecame evident in the number of CCR5 puncta per cell (p # 0.01)(t0 in Fig. 4C, 4D). Prolonged CHX treatment (48 h, t1), withits consequent ablation of translation, substantially reduced thenumber of CCR5-specific puncta per cell as well as the relevantpercentage of CCR5 punctate cells in comparison with the per-centage of CCR5 cells in untreated CHX cells (p # 0.001 for bothevaluations) (t1 in Fig. 4C, 4D). Of interest, at t2, in a parallelexperiment, when all stimuli were washed out and replaced withfresh complete medium for an additional 24 h (t2 in Fig. 4C, 4D),all samples displayed a recovery of the CCR5 receptor in terms ofpercentage of CCR5 punctate cells and the number of puncta percell in comparison with the CHX-treated cells at t1 (p # 0.001 forboth evaluations). These results were confirmed by flow cytometryanalysis. Fig. 4E shows a restoration of CCR5 expression on cellsurfaces at t2 in all samples that had previously been treated withCHX. To support these data, we used real-time PCR analysis toascertain whether the restoration of protein synthesis at t2 meanta de novo synthesis of CCR5 mRNA at a cytoplasmic level byCCR5 Ab pos (p # 0.001) (Fig. 4E). When CD4+ T lymphocyteswere analyzed, statistically significant CCR5 gene expression wasshown only at t3 with p # 0.01 (Fig. 4F).To verify the specificity of real-time PCR, four additional genes,

including ERK1, ERK2, p38, and CXCR4, were analyzed in bothR5-SupT1-L23 (Fig. 4G) and CD4+ T lymphocytes (Fig. 4H).A statistically significant gene expression was shown with ERK1only in both R5-SupT1-L23 (p # 0.01) and CD4+ T lymphocytes(p # 0.05), thus excluding the involvement of ERK2, p38, andCXCR4. The analysis of transcripts by real-time PCR has beenperformed for each time point, but statistically significant differ-ences in gene expression were shown at t2 or t3 only (see Fig. 4G,4H). Under physiologic conditions (wash after 48 h of Ab incu-bation), the restoration of CCR5 surface expression in R5-SupT1-L23 became evident 8 d after washing (Fig. 4I). Taken together,these data suggest the de novo synthesis of ERK1-mediated CCR5by CCR5 Ab pos, rather than a recycling of the receptor, as shownin the mechanistic model (Fig. 5).

DiscussionWe demonstrate that natural CCR5-specific Abs, found during HIVexposure or infection in humans, induce statistically significant

FIGURE 2. ECL1-CCR5–specific Abs (CCR5 Ab pos) recruit ERK1/2. (A) R5-SupT1-L23 cells were incubated with CCR5 Ab pos and appropriate

controls (CCR5 Ab neg and RANTES) for 30 min; untreated cells were also used as a negative control. Each sample was harvested at 30 min and 150 min.

Total cell lysates were analyzed by immunoblot for the detection of phosphorylated and nonphosphorylated forms of CCR5 and ERK1/2, b-arrestin 1/2, and

adaptin 1/2. (B and C) Cytoplasmic and nuclear extracts derived from R5-SupT1-L23 cells (B) and CD4+ T lymphocytes (C), untreated or stimulated with

RANTES, CCR5 Ab neg, and CCR5 Ab pos, were analyzed by immunoblot for localization of ERK1/2. GAPDH and histone H3 were used as respective

loading controls for the cytoplasmic fraction and for the nuclear fraction. Band density was determined with the T.I.N.A. program, and was expressed as

fold change over the appropriate housekeeping genes. Bar graphs represented mean 6 SD of three independent experiments. Student t test was performed,

and p values are shown. *p # 0.05, **p # 0.01, ***p # 0.001.

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FIGURE 3. Contribution of ERK1/2 protein to CCR5 regulation. (A and B) R5-SupT1-L23 cells and CD4+ T lymphocytes were incubated with the

U0126 inhibitor for 1 h and stimulated with CCR5 Ab pos and the appropriate controls for 30 min. Cells were collected at 150 min and lysed; total extracts

were separated by electrophoresis. The membranes were stained with Abs to phospho- or non-phospho-CCR5, and b-arrestin 1/2. (C) R5-SupT1-L23 cells

were nucleofected with plasmid dnERK1 and the relative control (pcDNA 3.1) for 24 h, then stimulated with CCR5 Ab pos and the appropriate controls for

30 min, and collected at 150 min. Total extracts were separated by electrophoresis and stained with Abs to phospho- or non-phospho-CCR5, and b-arrestin

1/2. GAPDH was used as a loading control. Bar graphs represented mean6 SD of three independent experiments. Student t test (Figure legend continues)

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receptor internalization through a clathrin-dependent pathway, asshown in Fig. 1. By so doing, we confirm previously publisheddata obtained in different experimental conditions (12). The char-acterization of five LTNP serum samples described in the cur-rent text derives from a previous study. During the follow-up tothe previous study, some of the LTNPs lost anti-CCR5 Abs di-rected to ECL1 and experienced a statistically significant increasein viremia, and thus became progressors in need of antiviraltherapy. Strikingly, subjects who retained anti-CCR5 Abs main-tained a stable condition without any treatment. This findingsuggests that the loss of anti-CCR5 Abs is associated with pro-gression toward the disease. In addition, anti-CCR5 Abs do notinduce any apparent alterations in immune function, as demon-strated by the continued health of subjects who retained anti-CCR5 Abs.The autoantibodies produced during the course of viral infec-

tions have been attributed to virus-induced self-alterations thatbecome autoimmunogenic (39). Therefore, the infrequent anti-CCR5 response might be due to a particular antigenic stimula-tion or to the particular reactivity of rare individuals. Lehner et al.(40) have shown that xenogenic immunization of macaques withSIV grown in human cells induces protective immunity and thegeneration of Abs reactive with simian CCR5 that are capable ofneutralizing SIV in in vitro assays. Allogenic immunization, ratherthan xenogenic, might have produced the same result in our LTNPindividuals. The first report, describing the presence of naturalAbs directed to CCR5, established that CCR5 can act as an allo-antigen in CCR5-D32 homozygous individuals, thus supporting ourhypothesis (15).To our knowledge, this is the first report that describes a new

ERK1-based pathway that triggers the CCR5-negative phenotypemediated by CCR5 Ab pos in T cells, including a T cell line (R5-SupT1-L23 cells), and in human CD4+ T lymphocytes, and wecannot exclude that this mechanism is specific for T cells only.Indeed, at a mucosal site, the role of CCR5 seems to be differentfrom that observed in CD4+ T lymphocytes, as HIV binds CCR5intracellularly in the endosome (41, 42) and HIV transcytosisinhibition with anti-CCR5 Ab pos performed in epithelial cellsrequires 1 h of Ab incubation to block HIV infection and not 48 h,as for T lymphocytes (22); thus we hypothesize that at a mucosalsite, CCR5 Ab pos bind the receptor and reach the CCR5 intra-cellularly, thereby precluding interaction with HIV and thentranscytosis (22), rather than inducing long-lasting internalization.In this work we used RANTES/CCL4 ligand as a control of thecommon internalization pathway of CCR5 previously described inT cells (26, 43, 44).The involvement of clathrin-coated pits under stimulation by Abs

to CCR5 leads to cytoplasmic CCR5 accumulation and consequentactivation of ERK1/2. We believe that activating plasma membraneCCR5 receptor with CCR5 Ab pos internalizes the receptor viaclathrin-mediated macropinocytosis into recycling endosomes. Theendosomes provide a platform for compartment-specific molecularinteractions leading to a correct ERK1 signaling and promote propercell response. On the basis of this thinking, we cannot exclude thatthe failure of ERK1 phosphorylation and recruitment in experiments

in which CCR5 signal transduction cascades, triggered by CCR5 Abpos, could depend on other factors that affect functional activity ofthe receptor, such as a lipid modification in carboxyl terminus in-duced by RANTES, as previously described by Kraft (45). Arrestin-mediated activation seems to be involved in such processes as thoseof clathrin adaptor proteins, as reported earlier by other groups (9,25, 46) (Fig. 2). The specificity of the said pathway is demonstratedin this study by a dnERK1, which demonstrated high statisticalsignificance (p # 0.001) in its reduction of CCR5, pCCR5, andb-arrestin 1/2 in the presence of ECL1-CCR5–specific Abs. In ourexperiments, we obtained an unequivocal correlation between theabsence of functional ERK1 protein and the reduction of both cy-tosolic CCR5 and b-arrestin 1/2 accumulation under stimulation byCCR5 Ab pos at 150 min (Fig. 3A–C). Although the activation ofERK1/2 by several GPCRs can occur through b-arrestin 1/2, bypromoting cytoplasmic retention of phosphorylated ERK1/2 (9),in our model, the relationship between receptor phosphorylationand b-arrestin 1/2 binding could be a complex mechanism,requiring further studies to definitively understand the role ofb-arrestin 1/2.Based on Western blotting data and on real-time PCR, these

findings demonstrate that ERK1, not ERK2, plays a key role in denovo synthesis of CCR5 by human Abs (Figs. 3, 4).Data obtained by chemical inhibition of protein synthesis and

by CCR5 silencing seem to indicate an involvement of de novoCCR5 synthesis (p # 0.001 after either 150 min or 48 h of Abstimulation for both siRNA and CHX) (Fig. 4). Although we didnot find direct evidence of CCR5 degradation, the restoration ofprotein synthesis at t2 (for R5-SupT1-L23 cells) and t3 (for CD4+

T lymphocytes) in CHX experiments clearly showed a new syn-thesis of CCR5 mRNA at a cytoplasmic level after CCR5 Ab posstimulation (Fig. 4F), followed by the re-expression of CCR5 onthe membrane of R5-SupT1-L23 cells (Fig. 4E). On the basis ofthis evidence, we can assume that the prolonged sequestrationof CCR5 into the cytoplasm can lead to the final destiny, whichis degradation of the receptor. Furthermore, under physiologicalconditions the restoration of CCR5 surface expression in R5-SupT1-L23 was clearly seen 8 d after washing (Fig. 4I). WhenCCR5 expression was analyzed on the membrane of CD4+ Tlymphocytes for 15 consecutive days after washing, it remainednegative (data not shown). Further time points have not beenanalyzed owing to the high level of cell deaths. Taken together,these data suggest a de novo CCR5 synthesis rather than a recy-cling of the protein.Finally, data on a physical interaction between CCR5 and ERK1

suggest a new scenario for MAPK ERK1 as mediated by CCR5interaction. Our model (see Fig. 5) shows a new and distinctspatio-temporal profile of ERK1 localization that is mediated bythe CCR5/ERK1 complex; such a complex hosts a hitherto un-known mechanism of CCR5 regulation mediated by ERK1 asa sequestration. This sequestration can lead to the accumulationand consequent possible degradation of the receptor within thecytoplasm. Other groups have demonstrated that when CCR5 in-ternalization is mediated by RANTES (where both caveolae- andclathrin-dependent internalization pathways are used), the level of

was performed, and p values are shown. (D) CCR5 expression on the cell surface was evaluated by flow cytometry as applied to untreated cells or cells

incubated with CCR5 Ab neg and CCR5 Ab pos for 48 h. A representative experiment is shown. (E) The percentage of cells with the punctate form of

CCR5 and (F) the number of CCR5 puncta per cell in untreated and treated cells with Abs (CCR5 pos and CCR5 neg) are reported in nucleofected R5-

SupT1-L23. (E) and (F) show bar graphs representing the mean 6SD of three independent experiments. Student t test was performed, and p values are

shown. (G) The interaction between ERK1/2 and CCR5 was evaluated by immunoprecipitation. R5-SupT1-M10 cells were treated or not with CCR5 Ab

pos. After incubation, the cells were lysed and both CCR5 and ERK1/2 immunoprecipitates (IP) were evaluated by Western blotting analysis. Untreated

cells were used as negative controls. One representative of three independent experiments is shown. **p # 0.01, ***p # 0.001.

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FIGURE 4. Role of siRNA specific to CCR5 in the internalization of the receptor, CCR5 protein modulation, and its gene expression pathway under

CHX treatment. (A and B) Data obtained by fluorescence microscopy on R5-SupT1-L23 cells nucleofected with siRNA-CCR5 are reported. After 5 h of

nucleofection, cells were incubated for 150 min and 48 h with CCR5 Ab neg and CCR5 Ab pos. NSsiRNA and untreated cells (Figure legend continues)

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receptor on the cell surface recovered to nearly 100% after 120-minincubation without the involvement of de novo protein synthesis (4,26, 27). In this article, we reveal the mechanism of action of Absthat induce an ERK1-based CCR5-negative phenotype, which inturn is responsible for long-term kinetics in the internalization(48 h), the possible degradation, and hence the de novo synthesisof CCR5.This mechanism in turn raises the question of whether it is ex-

clusively associated with ECL1 or whether other extramembranedomains are involved and, if so, which ones. It has been demon-strated that the differing properties of each CCR5 extramembraneregion, as revealed by the use of mAbs to the N terminus and to

the second loop of CCR5, differentially modulate and influencereceptor activity (47).A recent study performed in mice systematically addressed

several aspects of anti-CCR5 immunization, including the use ofall the extramembrane domains of CCR5, to define the optimalschedule to elicit strong and long-lasting immune responses. ECL1and ECL2 gave rise to stronger responses than did the N terminus;they achieved almost total CCR5 downregulation, and they blockedHIV infection (38). These findings possibly suggest that the CCR5receptor–induced ERK1 signaling via crosstalk with b-arrestin1/2does not exclusively depend on the ECL1 domain but on otherspecific stimuli, as well.

were used as negative controls. Panels report the percentage of cells with the punctate form of CCR5 and the number of CCR5 puncta per cell. (C) A time

course analysis was performed to evaluate CCR5 expression in the presence or not of CHX. Cells were harvested at t0 (corresponding to 150 min of

incubation with CCR5-modulating factors), t1 (corresponding to 48 h of incubation with factors), t2 (corresponding to 48 h of incubation with factors,

washing, and a further 24 h of cell incubation in medium without stimuli), and t3 (corresponding to 48 h of incubation with factors, washing, and a further

72 h of cell incubation in medium without stimuli). (C) The percentage of R5-SupT1-L23 cells with punctate form of CCR5 and (D) the number of CCR5

puncta per cell are reported. (E) CCR5 re-expression on cell surface was evaluated by flow cytometry at t2 in R5-SupT1-L23 cells previously treated with

CHX. (F) CCR5-normalized mRNA levels were analyzed with real-time quantitative PCR at t2 and t3 time points in R5-SupT1-L23 cells and CD4+ T

lymphocytes untreated or incubated with CCR5 Ab neg and CCR5 Ab pos in the presence or not of CHX, as described above. (G and H) Normalized mRNA

levels in additional genes, including ERK1, ERK2, p38, and CXCR4, were analyzed with real-time quantitative PCR at t2 in R5-SupT1-L23 (G) and t3 in

CD4+ T lymphocytes (H). Cells were untreated or incubated with CCR5 Ab neg and CCR5 Ab pos in the presence or not of CHX, as described above. (I)

CCR5 re-expression on cell surface was evaluated by flow cytometry at 8 d after washing in R5-SupT1-L23. All panels, except (E) and (I), show bar graphs

representing mean 6 SD of three independent experiments; Student t test was performed and p values are shown. In (E) and (I), one representative of three

independent experiments is shown. *p # 0.05, **p # 0.01, ***p # 0.001.

FIGURE 5. A mechanistic model of CCR5 regulation by CCR5 Ab pos. Upon binding of CCR5 Ab pos, the CCR5 receptor is phosphorylated and

b-arrestin 1/2 can initiate desensitization, thus contributing to the internalization of CCR5 by clathrin-coated pits. Activation of b-arrestin 1/2 leads to

receptor recruitment and subsequent internalization. The CCR5-bound b-arrestin 1/2 assembles a signaling complex, which recruits (Gq-coupled) CCR5

receptor–induced ERK1. The CCR5–b-arrestin 1/2–ERK1 complex sequesters induced ERK1 into cytosol, thus preventing nuclear translocation. In ad-

dition, CCR5 Ab pos engagement leads to the activation and consequent sequestration of ERK1 into the cytoplasm. The interaction between CCR5 and

ERK1, mediated by CCR5 Ab pos stimulation, ultimately leads to the accumulation and consequent possible degradation of the receptor within the cy-

toplasm. As a consequence, CCR5 is de novo synthesized and reappears on the cell surface with long-lasting kinetics. The right section of the figure

represents the classical endocytosis of CCR5 induced by natural ligands such as RANTES.

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Taken together, these findings can implement previous data ob-tained by our group, whereby murine Abs at the first loop of CCR5induced CCR5 internalization in vivo following very slow kinetics,similar to that observed in humans. Of note is that murine CCR5expressionwas gradually recovered 4wk subsequent to immunization(20). Although the current study does not provide a direct analysis ofmurine CCR5 internalization and recycling by Abs directed againstECL1-CCR5, we can hypothesize that regulation of CCR5 receptor–induced ERK1 signaling via crosstalk with b-arrestin 1/2 could bethe mechanism responsible for the in vivo findings in murine models.Although CCR5-specific Abs act through an immunologically ratherthan a genetically mediated mechanism, as instanced by D32 mu-tation and reviewed by Venuti (48), this finding is potentially per-tinent to the design of appropriate therapeutic candidates that mayprevent HIV infection for several days after specific treatment.

AcknowledgmentsWe thank Tonia Lorraine Russouw for the English revision of the whole

manuscript.

DisclosuresThe authors have no financial conflicts of interest.

References1. Lin, S. H., and O. Civelli. 2004. Orphan G protein-coupled receptors: targets for

new therapeutic interventions. Ann. Med. 36: 204–214.2. Lederman, M. M., A. Penn-Nicholson, M. Cho, and D. Mosier. 2006. Biology of

CCR5 and its role in HIV infection and treatment. JAMA 296: 815–826.3. Pitcher, J. A., N. J. Freedman, and R. J. Lefkowitz. 1998. G protein-coupled

receptor kinases. Annu. Rev. Biochem. 67: 653–692.4. Mueller, A., E. Kelly, and P. G. Strange. 2002. Pathways for internalization and

recycling of the chemokine receptor CCR5. Blood 99: 785–791.5. Signoret, N., A. Pelchen-Matthews, M. Mack, A. E. Proudfoot, and M. Marsh.

2000. Endocytosis and recycling of the HIV coreceptor CCR5. J. Cell Biol. 151:1281–1294.

6. Lopalco, L. 2011. Natural anti-CCR5 antibodies in HIV-infection and -exposure.J. Transl. Med. 9(Suppl 1): S4 doi:10.1186/1479-5876-9-S1-S4.

7. Fraile-Ramos, A., T. A. Kohout, M. Waldhoer, and M. Marsh. 2003. Endocytosisof the viral chemokine receptor US28 does not require beta-arrestins but is de-pendent on the clathrin-mediated pathway. Traffic 4: 243–253.

8. Lefkowitz, R. J., and S. K. Shenoy. 2005. Transduction of receptor signals bybeta-arrestins. Science 308: 512–517.

9. DeFea, K. A. 2011. Beta-arrestins as regulators of signal termination andtransduction: how do they determine what to scaffold? Cell. Signal. 23: 621–629doi:10.1016/j.cellsig.2010.10.004.

10. Eishingdrelo, H., and S. Kongsamut. 2013. Minireview: Targeting GPCR acti-vated ERK pathways for drug discovery. Curr. Chem. Genomics Transl. Med. 7:9–15. doi: 10.2174/2213988501307010009. eCollection 2013.

11. Popik, W., and P. M. Pitha. 1998. Early activation of mitogen-activated proteinkinase kinase, extracellular signal-regulated kinase, p38 mitogen-activated pro-tein kinase, and c-Jun N-terminal kinase in response to binding of simian im-munodeficiency virus to Jurkat T cells expressing CCR5 receptor. Virology 252:210–217.

12. Pastori, C., B. Weiser, C. Barassi, C. Uberti-Foppa, S. Ghezzi, R. Longhi,G. Calori, H. Burger, K. Kemal, G. Poli, et al. 2006. Long-lasting CCR5 in-ternalization by antibodies in a subset of long-term nonprogressors: a possibleprotective effect against disease progression. Blood 107: 4825–4833.

13. Sciortino, M. T., M. A. Medici, F. Marino-Merlo, D. Zaccaria, M. Giuffre-Cuculletto, A. Venuti, S. Grelli, and A. Mastino. 2008. Involvement of HVEMreceptor in activation of nuclear factor kappaB by herpes simplex virus 1 gly-coprotein D. Cell. Microbiol. 10: 2297–2311 doi:10.1111/j.1462-5822.2008.01212.x.

14. Sciortino, M. T., M. Suzuki, B. Taddeo, and B. Roizman. 2001. RNAs extractedfrom herpes simplex virus 1 virions: apparent selectivity of viral but not cellularRNAs packaged in virions. J. Virol. 75: 8105–8116.

15. Ditzel, H. J., M. M. Rosenkilde, P. Garred, M. Wang, K. Koefoed, C. Pedersen,D. R. Burton, and T. W. Schwartz. 1998. The CCR5 receptor acts as an allo-antigen in CCR5Delta32 homozygous individuals: identification of chemo-kineand HIV-1-blocking human antibodies. Proc. Natl. Acad. Sci. USA 95:5241–5245.

16. Osbourn, J. K., J. C. Earnshaw, K. S. Johnson, M. Parmentier, V. Timmermans,and J. McCafferty. 1998. Directed selection of MIP-1 alpha neutralizing CCR5antibodies from a phage display human antibody library. Nat. Biotechnol. 16:778–781.

17. Bouhlal, H., H. Hocini, C. Quillent-Gregoire, V. Donkova, S. Rose, A. Amara,R. Longhi, N. Haeffner-Cavaillon, A. Beretta, S. V. Kaveri, and M. D. Kazatchkine.2001. Antibodies to C-C chemokine receptor 5 in normal human IgG block in-

fection of macrophages and lymphocytes with primary R5-tropic strains of HIV-1.J. Immunol. 166: 7606–7611.

18. Grene, E., L. A. Pinto, A. L. Landay, H. A. Kessler, S. A. Anderson, M. J. Dolan,and G. M. Shearer. 2001. Anti-CCR5 antibodies in sera of HIV-positive indi-viduals. Hum. Immunol. 62: 143–145.

19. Lopalco, L., C. Barassi, C. Pastori, R. Longhi, S. E. Burastero, G. Tambussi,F. Mazzotta, A. Lazzarin, M. Clerici, and A. G. Siccardi. 2000. CCR5-reactiveantibodies in seronegative partners of HIV-seropositive individuals down-modulate surface CCR5 in vivo and neutralize the infectivity of R5 strains ofHIV-1 in vitro. J. Immunol. 164: 3426–3433.

20. Lopalco, L., C. Barassi, C. Paolucci, D. Breda, D. Brunelli, M. Nguyen,J. Nouhin, T. T. Luong, L. X. Truong, M. Clerici, et al. 2005. Predictive value ofanti-cell and anti-human immunodeficiency virus (HIV) humoral responses inHIV-1-exposed seronegative cohorts of European and Asian origin. J. Gen. Virol.86: 339–348.

21. Barassi, C., A. Lazzarin, and L. Lopalco. 2004. CCR5-specific mucosal IgA insaliva and genital fluids of HIV-exposed seronegative subjects. Blood 104: 2205–2206.

22. Bomsel, M., C. Pastori, D. Tudor, C. Alberti, S. Garcia, D. Ferrari, A. Lazzarin,and L. Lopalco. 2007. Natural mucosal antibodies reactive with first extracellularloop of CCR5 inhibit HIV-1 transport across human epithelial cells. AIDS 21:13–22.

23. Joshi, A., A. M. Nyakeriga, R. Ravi, and H. Garg. 2011. HIV ENV glycoprotein-mediated bystander apoptosis depends on expression of the CCR5 co-receptor atthe cell surface and ENV fusogenic activity. J. Biol. Chem. 286: 36404–36413doi:10.1074/jbc.M111.281659.

24. Choi, W. T., and J. An. 2011. Biology and clinical relevance of chemokines andchemokine receptors CXCR4 and CCR5 in human diseases. Exp. Biol. Med.(Maywood) 236: 637–647 doi:10.1258/ebm.2011.010389.

25. Oppermann, M. 2004. Chemokine receptor CCR5: insights into structure,function, and regulation. Cell. Signal. 16: 1201–1210.

26. Venkatesan, S., J. J. Rose, R. Lodge, P. M. Murphy, and J. F. Foley. 2003.Distinct mechanisms of agonist-induced endocytosis for human chemokinereceptors CCR5 and CXCR4. Mol. Biol. Cell 14: 3305–3324.

27. Signoret, N., L. Hewlett, S. Wavre, A. Pelchen-Matthews, M. Oppermann, andM. Marsh. 2005. Agonist-induced endocytosis of CC chemokine receptor 5 isclathrin dependent. Mol. Biol. Cell 16: 902–917.

28. Huttenrauch, F., A. Nitzki, F. T. Lin, S. Honing, and M. Oppermann. 2002.b-Arrestin binding to CC chemokine receptor 5 requires multiple C-terminalreceptor phosphorylation sites and involves a conserved Asp-Arg-Tyr sequencemotif. J. Biol. Chem. 277: 30769–30777.

29. DeFea, K. A., J. Zalevsky, M. S. Thoma, O. Dery, R. D. Mullins, andN. W. Bunnett. 2000. beta-Arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.J. Cell Biol. 148: 1267–1281.

30. Luttrell, L. M., F. L. Roudabush, E. W. Choy, W. E. Miller, M. E. Field,K. L. Pierce, and R. J. Lefkowitz. 2001. Activation and targeting of extracellularsignal-regulated kinases by beta-arrestin scaffolds. Proc. Natl. Acad. Sci. U. S. A.98: 2449–2454.

31. Tohgo, A., K. L. Pierce, E. W. Choy, R. J. Lefkowitz, and L. M. Luttrell. 2002.beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activitybut inhibits ERK-mediated transcription following angiotensin AT1a receptorstimulation. J. Biol. Chem. 277: 9429–9436.

32. Tohgo, A., E. W. Choy, D. Gesty-Palmer, K. L. Pierce, S. Laporte,R. H. Oakley, M. G. Caron, R. J. Lefkowitz, and L. M. Luttrell. 2003. Thestability of the G protein-coupled receptor-beta-arrestin interaction determinesthe mechanism and functional consequence of ERK activation. J. Biol. Chem.278: 6258–6267.

33. Ebisuya, M., K. Kondoh, and E. Nishida. 2005. The duration, magnitude andcompartmentalization of ERK MAP kinase activity: mechanisms for providingsignaling specificity. J. Cell Sci. 118: 2997–3002.

34. Jafri, F., H. M. El-Shewy, M. H. Lee, M. Kelly, D. K. Luttrell, and L. M. Luttrell.2006. Constitutive ERK1/2 activation by a chimeric neurokinin 1 receptor-beta-arrestin1 fusion protein. Probing the composition and function of the G protein-coupled receptor “signalsome.” J. Biol. Chem. 281: 19346–19357.

35. Robbins, D. J., E. Zhen, H. Owaki, C. A. Vanderbilt, D. Ebert, T. D. Geppert, andM. H. Cobb. 1993. Regulation and properties of extracellular signal-regulatedprotein kinases 1 and 2 in vitro. J. Biol. Chem. 268: 5097–5106.

36. Izumi, Y., S. Kim, M. Namba, H. Yasumoto, H. Miyazaki, M. Hoshiga,Y. Kaneda, R. Morishita, Y. Zhan, and H. Iwao. 2001. Gene transfer of dominant-negative mutants of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase prevents neointimal formation in balloon-injured rat artery.Circ. Res. 88: 1120–1126.

37. Pastori, C., A. Clivio, L. Diomede, R. Consonni, G. M. De Mori, R. Longhi,G. Colombo, and L. Lopalco. 2008. Two amino acid substitutions within thefirst external loop of CCR5 induce human immunodeficiency virus-blockingantibodies in mice and chickens. J. Virol. 82: 4125–4134 doi:10.1128/JVI.02232-07.

38. Pastori, C., L. Diomede, A. Venuti, G. Fisher, J. Jarvik, M. Bomsel, F. Sanvito,and L. Lopalco. 2014. Induction of HIV-blocking anti-CCR5 IgA in Peyers’spatches without histopathological alterations. J. Virol. 88: 3623–3635 doi:10.1128/JVI.03663-13.

39. Bachmann, M. F., and R. M. Zinkernagel. 1997. Neutralizing antiviral B cellresponses. Annu. Rev. Immunol. 15: 235–270.

40. Lehner, T., Y. Wang, C. Doyle, L. Tao, L. A. Bergmeier, E. Mitchell, W. M.J. Bogers, J. Heeney, and C. G. Kelly. 1999. Induction of inhibitory antibodies to

12 NEW CCR5 MODULATION PATHWAY

by guest on March 30, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

the CCR5 chemokine receptor and their complementary role in preventing SIVinfection in macaques. Eur. J. Immunol. 29: 2427–2435.

41. Meng, G., X.Wei, X.Wu, M. T. Sellers, J. M. Decker, Z. Moldoveanu, J. M. Orenstein,M. F. Graham, J. C. Kappes, J. Mestecky, et al. 2002. Primary intestinal epithelial cellsselectively transfer R5 HIV-1 to CCR5+ cells. Nat. Med. 8: 150–156.

42. Bomsel, M., and V. David. 2002. Mucosal gatekeepers: selecting HIV viruses forearly infection. Nat. Med. 8: 114–116.

43. Yuan, J., H. Y. Ren, Y. J. Shi, and W. Liu. 2015. In vitro immunological effects ofblocking CCR5 on T cells. Inflammation 38: 902–910 doi:10.1007/s10753-014-0052-6.

44. Signoret, N., M. M. Rosenkilde, P. J. Klasse, T. W. Schwartz, M. H. Malim,J. A. Hoxie, and M. Marsh. 1998. Differential regulation of CXCR4 and CCR5endocytosis. J. Cell Sci. 111: 2819–2830.

45. Kraft, K., H. Olbrich, I. Majoul, M. Mack, A. Proudfoot, and M. Oppermann.2001. Characterization of sequence determinants within the carboxyl-terminaldomain of chemokine receptor CCR5 that regulate signaling and receptor in-ternalization. J. Biol. Chem. 276: 34408–34418.

46. Rodrigues, A. R., H. Almeida, and A. M. Gouveia. 2012. Melanocortin 5 receptorsignaling and internalization: role of MAPK/ERK pathway and b-arrestins 1/2.Mol. Cell. Endocrinol. 361: 69–79 doi:10.1016/j.mce.2012.03.011.

47. Blanpain, C., J. M. Vanderwinden, J. Cihak, V. Wittamer, E. Le Poul, H. Issafras,M. Stangassinger, G. Vassart, S. Marullo, D. Schlndorff, et al. 2002. Multipleactive states and oligomerization of CCR5 revealed by functional properties ofmonoclonal antibodies. Mol. Biol. Cell 13: 723–737.

48. Venuti, A., and L. Lopalco. 2014. Tackling HIV: genetic vs. immune CCR5targeting. J. AIDS Clin. Res. 5: 3442014 doi:10.4172/2155-6133.1000344.

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