CCL2 Increases X4-tropic HIV-1 Entry into Resting CD4+ T Cells*

During human immunodeficiency virus type 1 (HIV-1) infection, there is a strong positive correlation between CCL2 levels and HIV viral load. To determine whether CCL2 alters HIV-1 infection of resting CD4+ T cells, we infected purified resting CD4+ T cells after incubation with CCL2. We show that CCL2 up-regulates CXCR4 on resting CD4+ T cells in a CCR2-dependent mechanism, and that this augmentation of CXCR4 expression by CCL2 increases the ability of these cells to be chemoattracted to CXCR4 using gp120 and renders them more permissive to X4-tropic HIV-1 infection. Thus, CCL2 has the capacity to render a large population of lymphocytes more susceptible to HIV-1 late in the course of infection.

During human immunodeficiency virus type 1 (HIV-1) infection, there is a strong positive correlation between CCL2 levels and HIV viral load. To determine whether CCL2 alters HIV-1 infection of resting CD4 ؉ T cells, we infected purified resting CD4 ؉ T cells after incubation with CCL2. We show that CCL2 up-regulates CXCR4 on resting CD4 ؉ T cells in a CCR2-dependent mechanism, and that this augmentation of CXCR4 expression by CCL2 increases the ability of these cells to be chemoattracted to CXCR4 using gp120 and renders them more permissive to X4-tropic HIV-1 infection. Thus, CCL2 has the capacity to render a large population of lymphocytes more susceptible to HIV-1 late in the course of infection.
Human immunodeficiency virus type 1 (HIV-1) 2 establishes a persistent long lived and latent infection of resting CD4 ϩ T cells that eludes immune surveillance and current drug therapy, and it is the major barrier to the treatment of HIV. Chemokine (C-C motif) ligand 2 (CCL2; also known as MCP-1 (monocyte chemoattractant protein 1)) is a pro-inflammatory chemokine induced during many inflammatory and autoimmune diseases and during bacterial and viral infections. During HIV-1 infection, there is a strong positive correlation between CCL2 levels and HIV viral load with higher expression of CCL2 mRNA and elevated serum CCL2 concentrations being found in viremic (viral load Ͼ100,000 RNA copies/ml plasma) compared with aviremic (viral load Ͻ50 copies/ml) HIV-1-infected patients (1). CCL2 is produced from infected macrophages (2), and extracellular secreted HIV-1 proteins such as Tat and gp120 can induce the production of CCL2 from uninfected bystander cells either directly or indirectly by up-regulating tumor necrosis factor production (3)(4)(5). The cognate receptor for CCL2 is chemokine (C-C motif) receptor 2 (CCR2). CCL2 chemoattracts CCR2 ϩ cells such as monocytes (6), activated and memory T cells (7), and natural killer cells (8) to the site of inflammation and infection, rendering them more susceptible to HIV-1 infection. This provides an ideal environment for HIV-1 to replicate in freshly recruited target cells that leads to the appearance of high viral load. As with other C-C chemokines, CCL2 plays a role in maintaining immune homeostasis. However, unlike the other C-C chemokines, CCL2 regulates T helper (Th) cell differentiation by polarizing Th0 cells toward a Th2 phenotype as opposed to a Th1 phenotype (9). Conversely, CCL2, along with CCL3, CCL4, and CCL5, is able to increase the replication of X4-tropic strains in activated CD4 ϩ T cells (10).
CXCR4 is a seven-transmembrane receptor coupled to a pertussis toxin (PTX)-sensitive heterotrimeric G i protein, which modulates the levels of intracellular cAMP by inhibiting the activity of adenylate cyclase. It also links to phosphoinositide 3-kinase, which is intrinsically linked to cell motility by promoting reorganization of the actin cytoskeleton (20). CXCR4 also activates Src kinase, which, in conjunction with other proteintyrosine kinases of the Syk, Tec, and focal adhesion kinase families, promotes the activation of mitogen-activated protein kinases (MAPK) and Ras-related GTPases that change the transcriptional profile of the cell and promote actin remodeling (20). IL-4 specifically enhances cell surface expression of CXCR4 on resting CD4 ϩ T cells (21), whereas stimulation of resting CD4 ϩ T cells with phytohemagglutinin (PHA), anti-CD3, or anti-CD28 antibodies leads to down-regulation. Th2 cells express variable levels of CXCR4; however, CXCR4 is undetectable on polarized Th1 cells (21).
To test our hypothesis that the chemotactic and polarizing nature of CCL2 drives the recruitment of target cells to the site of HIV infection favoring viral infection and eventually a higher viral load in infected individuals, we investigated the modula-tory effects of CCL2 on CXCR4 expression on resting CD4 ϩ T cells and tested the effect of CCL2-induced CXCR4 expression on gp120-induced chemotaxis and HIV-1 infection.
CD4 ϩ T Cells-Human peripheral blood mononuclear cells (PBMC) were isolated from the heparinized blood of healthy HIV-1 seronegative donors by density centrifugation over Ficoll-Hypaque (Amersham Biosciences). CD4 ϩ T cells were isolated from PBMC by negative selection using the CD4 ϩ T cell isolation kit from Miltenyi Biotec (Auburn, CA) in accordance with the manufacturer's instructions. The final cultures of CD4 ϩ T cells were always Ͼ95% pure, as determined by twocolor flow cytometry analysis using anti-CD4-PerCp and anti-CD3-FITC. After purification, cells were resuspended in AIM-V medium with human serum albumin (AIM-V; Invitrogen) at 1 ϫ 10 6 CD4 ϩ T cells/ml for 24 h to allow for maximal unstimulated expression of CXCR4 before use.
Viruses-The following viruses were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health: HIV-1 Lai was from Dr. Jean-Marie Bechet and Dr. Luc Montagnier (22,23), and HIV-1 93In905 was from Dr. Robert Bollinger and the UNAIDS Network for HIV Isolation and Characterization, and the DAIDS, NIAID (24). Both were propagated on PHA/IL-2-activated PBMC, treated with RNase-free DNase I (Invitrogen), and purified using Vivaspin 20 columns with a 300,000 molecular weight cutoff (Sartorius Stedim Biotech, Edgewood, NY). The 50% tissue culture infective dose (TCID 50 ) was determined by serial dilution and calculated using the Spearman-Kärber method as described elsewhere (25). The HIV-1 p24 antigen concentration in culture supernatants was determined using the Alliance HIV-1 p24 antigen enzyme-linked immunosorbent assay kit (PerkinElmer Life Sciences).

Flow Cytometry
All flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences). All cytogram analysis was performed using CellQuest Prosoftware (BD Biosciences).
Receptor internalization was measured by retention of anti-CXCR4 antibody binding following elimination of cell surfacebound antibody via acid wash as described previously (27). Receptor internalization was quantified as the fraction of total anti-CXCR4 fluorescence intensity (pH 7 wash) retained following acid wash (pH 4).

Western Blot Analysis
CD4 ϩ T cells were treated with CCL2 for 4 h, washed three times with ice-cold DPBS, and lysed in CelLytic M (Sigma). Cell debris was removed by centrifugation (15,000 ϫ g, 15 min). 40 g per sample was then heated at 70°C for 10 min in NuPAGE lithium dodecyl sulfate sample buffer containing reducing agent and separated by 3-8% Tris acetate-polyacrylamide gel (Invitrogen) and transferred to nitrocellulose membranes (Fisher). Membranes were blocked by 1 h of incubation at room temperature with DPBS supplemented with 0.1% (v/v) polysorbate 20 (Sigma) and 5% (w/v) dried nonfat milk (Genesee Scientific, San Diego) and then probed with polyclonal antibodies against plectin followed by detection using the WesternBreeze chemiluminescence kit (Invitrogen). Membranes were subsequently stripped and reprobed with anti-␤-actin antibody to confirm equal loading. The relative density of the protein bands was analyzed using the freely available ImageJ (National Institutes of Health).

Real Time-PCR
CXCR4 mRNA expression was measured by real time PCR. Target cells were harvested and total cellular RNA prepared with the RNeasy mini kit using the optional DNase step in accordance with the manufacturer's directions (Qiagen, Valencia, CA). CXCR4 mRNA expression in relation to RNA polymerase II (RPII) expression (internal standard) was determined using the LightCycler System and the FastStart RNA Master SYBR Green I kit (Roche Applied Science) according to the manufacturer's instructions. Primers used were as follows: CXCR4 sense 5Ј-CAGCGGTTACCATGGAGGG-3Ј and antisense 5Ј-TTCCTCGGTGTAGTTATCTGAAGTGT-3Ј; RPII sense 5Ј-GCACCACGTCCAATGACAT-3Ј and antisense 5Ј-GTGCGGCTGCTTCCATAA-3Ј. Amplification was performed for 45 cycles, with the following cycle parameters: 5 s of denaturation at 95°C, 12 s of primer annealing at 52°C, and 15 s of fragment elongation at 72°C.
The quantity of HIV-1 long terminal repeat (LTR) DNA in relation to RPII (internal standard) was determined using the LightCycler System and the FastStart DNA Master SYBR Green I kit (Roche Applied Science). PCRs were carried out in a 20-l mixture composed of 3 mM MgCl 2 , 0.5 M of each primer, 5 l of sample, and 1-fold LightCycler FastStart DNA Master SYBR Green I. The primers for HIV-1 amplify a 128-bp fragment in the LTR R/U5 region, which was designed to detect early steps in reverse transcription: LTR sense 5Ј-CTCTCTGGCTAAC-TAGG-3Ј and antisense 5Ј-ACTGACTAAAAGGGTCT-3Ј. The reaction mixture was initially incubated at 95°C for 10 min to denature the DNA. Amplification was performed for 45 cycles, with the following cycle parameters: 10 s of denaturation at 95°C, 10 s of primer annealing at 62°C, and 15 s of fragment elongation at 72°C.
Quantification and melting curve were analyzed with Light-Cycler analysis software, RelQuant (Roche Applied Science). All results are expressed as the ratio between the copy number of the target gene and the copy number of RPII and normalized so that CXCR4 mRNA expression and HIV-1 LTR in unconditioned cells equals 1.00.

Chemotaxis
The cell migration of CD4 ϩ T cells toward a gp120 gradient after 4 h of conditioning with CCL2 in AIM-V was evaluated using 24-well Transwell migration chambers with 5-m pore size polyvinylpyrrolidone-free polycarbonate filters (Corning Life Sciences, Lowell, MA) as described previously (5, 28) using gp120 in the basal chamber and 5 ϫ 10 6 cells/ml in the apical chamber. Data are expressed as the percentage of migrated cells and are representative of three independent experiments carried out in triplicate.

HIV Infection Assay
After 4 h of conditioning with CCL2, CD4 ϩ T cells were infected with X4-tropic HIV-1 Lai or the R5-tropic HIV-1 93In905 at a multiplicity of infection of 0.01 for 3 h and then washed three times with DPBS. Cells were then cultured in RPMI 1640 medium supplemented with 15% (v/v) fetal bovine serum, 2 mM glutamine, and antibiotics (all Invitrogen). After 14 h, cells were either harvested and total DNA prepared with the QIAamp DNA mini kit in accordance with the manufacturer's directions (Qiagen, Valencia, CA) or, alternatively, media were supplemented with 20 units/ml IL-2, and cells were cultured for a further 96 h. Supernatants were then collected and assayed using the Alliance HIV-1 p24 antigen enzyme-linked immunosorbent assay kit (PerkinElmer Life Sciences).

Statistics
The results are expressed as the means Ϯ S.E. of three or more experiments performed in triplicate. All p values correspond to two-tailed t tests.

RESULTS
CXCR4 Antibody-binding Sites Are Increased on CCL2treated Resting CD4 CD4 ϩ T Cells-We first examined whether CCL2 could activate or regulate surface expression of receptors on CD4 ϩ T cells that had been freshly isolated from healthy donors and cultured for 24 h as described under "Experimental Procedures." CD4 ϩ T cells were exposed to CCL2 for 4 h, harvested, stained, and analyzed by flow cytometry for the surface expression of CD4, CD3, CD25, CD45RO, CD25, CD69, HLA-DR, CXCR4, and CCR5. We observed a CCL2-specific dose-dependent increase in the surface expression of CXCR4 (Fig. 1A). Flow cytometry demonstrated that stimulation with CCL2 did not alter the expression of any activation markers (HLA-DR, CD25, or CD69) and had no effect on the surface expression of CD4, CD3, CCR5, or CD45RO (data not shown). Furthermore, although natural variations in CXCR4 surface expression exist between individuals, we observed a uniform up-regulation across all donors tested.
We tested the hypothesis that CCL2 triggers the up-regulation of surface CXCR4 expression through a CCR2-dependent mechanism by incubating the cells with RS102895, a potent and specific spiropiperidine class inhibitor for CCR2b. This resulted in the complete inhibition of surface CXCR4 up-regulation (Fig. 1B). To determine whether the CCL2-induced upregulation of surface CXCR4 was because of de novo protein synthesis or merely the relocation of pre-existing intracellular receptor to the cell surface, CD4 ϩ T cells were incubated with 2 nM CCL2, and the expression of both surface and total (intracellular and surface) CXCR4 was quantified by flow cytometry (Fig. 1C), and CXCR4 mRNA was quantified by real time PCR (Fig. 1D). Although cell surface expression of CXCR4 increases significantly (1.92-fold increase, p Ͻ 0.0007), there was no significant increase of either total CXCR4 (mean 100% versus 97.96%, p ϭ 0.5735; Fig. 1C) or CXCR4 mRNA (fold increase 1 versus 0.89, p ϭ 0.4662; Fig. 1D). NOVEMBER 7, 2008 • VOLUME 283 • NUMBER 45

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CCL2 Reduces CXCR4 Internalization-To determine whether altered receptor trafficking might play a role in CCL2 up-regulation of CXCR4 cell surface expression, receptor internalization rates were quantified by flow cytometric analysis of internalized anti-CXCR4 antibody following removal of surface-bound antibody by acid washing. Residual antibody binding reflects internalized receptors not exposed to extracellular low pH (29). Low pH washing efficiently removed cell surfacebound antibody as demonstrated by the abrogation of the fluorescent signal of both anti-CD4-PerCp and anti-CXCR4-PE (data not shown). After 1 h of incubation at 37°C, acid-resistant anti-CXCR4 antibody binding averaged 7% of total CXCR4 fluorescence intensity (Fig. 2A). Parallel incubation in the presence of 2 nM CCL2 decreased acid-resistant anti-CXCR4 Ab binding by 42%, to an average of 3.2% of total fluorescence intensity at 60 min (p ϭ 0.03). Thus, CCL2 appears to up-regulate cell surface expression of CXCR4 in part by reducing receptor internalization rates rather than by increasing the size of the total CXCR4 receptor pool.
In the absence of ligand binding, the precise mechanisms that regulate CXCR4 signaling and intracellular trafficking are not well understood. However, recent studies have suggested a role for plectin, a cytolinker protein that plays an important role as a scaffolding platform for proteins involved in cellular signaling, and in CXCR4 signaling and trafficking (30). Therefore, to determine whether CCL2 has an effect on plectin expression, plectin levels were quantified by Western blot (Fig. 2B). We found that CCL2 significantly increased the expression of plectin in CD4 ϩ T cells (mean fold increase 1 versus 1.6, p ϭ 0.01; Fig. 2C). These data suggest that plectin may play a role in CCL2-dependent up-regulation of surface CXCR4 expression.
CCL2 Increases CXCR4 Signaling, Leading to an Increase in X4 gp120-induced Actin Polymerization and Chemotaxis-Using a Transwell assay, we tested the hypothesis that CCL2-induced surface CXCR4 up-regulation would enhance CD4 ϩ T cell chemotaxis toward gp120. Initially, we compared the chemotactic effect of 200 nM X4 gp120 MN , the reported K d value of X4-tropic gp120 for CXCR4 (31), on unconditioned resting CD4 ϩ T cells with known CXCR4 expression levels. We observed that CD4 ϩ T cells with greater CXCR4 surface expression had a higher chemotactic index than those with lower surface expression, but the difference was not significant (data not shown). We then compared the percentage of unconditioned resting CD4 ϩ T cells that migrated toward gp120 to those with prior conditioning with 1 nM CCL2 for 4 h. CD4 ϩ T cell migration toward 200 nM X4 gp120 MN was enhanced 43% by prior exposure to 1 nM CCL2 (p Ͻ 0.0001), whereas migration toward R5 gp120 SF162 remained unaffected (Fig. 3). Preincubation of CD4 ϩ T cells with 20 M RS102895 before CCL2 exposure reduced migration toward X4 gp120 MN to 34%, the same as for non-CCL2 treated X4 gp120 MN -specific migration. All chemotactic activity toward gp120 MN was abrogated by pretreating the cells with 1 M AMD3100 or 1 g/ml PTX, indicating a requirement for CXCR4 and G␣ i proteins in X4 gp120 MN -induced migration. All chemotactic responses to the R5 gp120 SF162 were at significantly inferior levels to that of gp120 MN (p Յ 0.0001) and were unaffected by treatment with either AMD3100 or PTX. To establish whether anti-CD3 stimulation would inhibit the migration of CD4 ϩ T cells toward X4 gp120 MN , the response of CD4 ϩ T cells pretreated with 10 g/ml anti-CD3 antibody was evaluated. The response was reduced to 2% of the migration of cells in response to X4 gp120 MN without prior anti-CD3 treatment (p Ͻ 0.0001).
Cell polarization and motility are governed by actin cytoskeleton rearrangements. Actin turnover is also involved in HIV-1 entry, which requires disintegration and reconstitution of the membrane structure. Thus, by inducing F-actin polymerization, CXCR4 can regulate both X4 HIV-1 entry and chemotaxis. CD4 ϩ T cells conditioned with 1 nM CCL2 for 4 h were treated with 200 nM gp120 and probed with FITC-phalloidin for F-actin polymerization at various time points. F-actin polymerization was rapidly induced following the addition of 200 nM X4 gp120 MN . Kinetics in all instances was rapid with the peak response achieved within 30 s post-stimulation and was completely abrogated in the presence of AMD3100, indicating a dependence on signaling through CXCR4. CCL2 conditioning increased the gp120 MN -induced F-actin polymerization (p ϭ 0.022; Fig. 4). The R5-tropic gp120 SF162 also induced rapid polymerization of F-actin, although we observed no difference between the CCL2-conditioned and nonconditioned cells (1.32-fold increase versus 1.33-fold increase, p ϭ 0.89). The peak response achieved using gp120 SF162 in nonconditioned cells was significantly less compared with gp120 MN alone (2.48fold increase versus 1.32-fold increase, p ϭ 0.033; Fig. 4).

FIGURE 2. Effect of CCL2 on CXCR4 internalization and on plectin content.
A, CXCR4 internalization was quantified by flow cytometric assessment of anti-CXCR4 antibody binding following acid stripping of cell surface-bound antibody. Acid-resistant binding (internalized CXCR4) is expressed as a fraction of total antibody binding over 1 h of incubation at 37°C. CCL2 reduced CXCR4 internalization rates by 42% across four experiments (**, p ϭ 0.03). B, effect of CCL2 on plectin expression. Western blot was used to quantify plectin levels in resting CD4 ϩ T cells cultured for 4 h in the presence of 0 (U) or 2 nM CCL2 (C). Parallel determination of ␤-actin verified equivalent protein loading. C, densitometric analysis of the protein bands revealed that CCL2 conditioning of resting CD4 ϩ T cells significantly increased plectin content (*, p ϭ 0.01). Experiments were performed three times in triplicate. Chemokinesis was minimal when equal concentrations of gp120 were added to both the apical and basal chambers and has been subtracted from these histograms. X4 gp120 MN induced chemotaxis of CD4 ϩ T cells and was augmented by preconditioning with 1 nM CCL2. The augmentation was abrogated in the presence of RS102895 (p Ͻ 0.0001). Treatment with AMD3100, PTX, or anti-CD3 antibody inhibited X4 gp120 MN induced chemotaxis. R5 gp120 SF162 induced minimal chemotaxis and was unaffected by all inhibitor and stimulus treatments. *, p Ͻ 0.0001. CCL2 Enhances X4 gp120-induced Erk 1/2 Phosphorylation in Primary CD4 ϩ T Cells-Erk 1/2 has been implicated in regulating chemotaxis in some systems (32,33). Therefore, we analyzed whether gp120 could induce the phosphorylation of Erk 1/2 in resting unstimulated CD4 ϩ T cells. At both 40 and 80 nM, we observed no effect of either gp120 MN or gp120 SF162 (data not shown). However, at 200 nM, the reported K d value of X4-tropic gp120 for CXCR4 (31), X4 gp120 MN induced significant phosphorylation of Erk 1/2 MAPK (1.86-fold increase, p Ͻ 0.0001; Fig. 5). Furthermore, Erk 1/2 MAPK phosphorylation was greater in cells conditioned with 1 nM CCL2 than in unconditioned cells (1.86-fold versus 3.47-fold increase, p Ͻ 0.0001; Fig.  5). X4 gp120 MN -induced phosphorylation of Erk 1/2 MAPK was abrogated if cells were pretreated with either AMD3100, a specific CXCR4 antagonist, or PTX, which irreversibly inhibits the G i subunits of heterotrimeric G proteins by ADP-ribosylation, indicating the inhibition of the Ras/MAPK pathway and a requirement for CXCR4 in X4 gp120 MN -induced Erk 1/2 phosphorylation; conditioning with CCL2 had no effect on this result (Fig. 5). Conversely, the R5-tropic gp120 SF162 had no effect on Erk 1/2 MAPK phosphorylation at either its K d for CCR5, 6 nM (34; data not shown) or at 200 nM (Fig. 5).
CCL2 Increases Entry of X4-tropic HIV-1 Lai but Not R5-tropic HIV-1 93In905 into Resting CD4 ϩ T Cells-The previous results suggest that CCL2 conditioning of resting CD4 ϩ T cells selectively affects X4-tropic HIV-1 gp120-CXCR4 interactions. Therefore, we tested the hypothesis that increased surface expression of CXCR4 will enhance X4 HIV-1 entry into resting CD4 ϩ T cells while having no effect on R5 HIV-1 entry. To test this hypothesis directly, we measured viral entry by using PCR. Highly purified resting CD4 ϩ T cells were conditioned with 1 nM CCL2 or vehicle control for 4 h before infection with HIV-1 (multiplicity of infection of 0.01) for 3 h. After an additional 14 h culture in fresh medium, samples were analyzed by real time PCR for the presence and quantity of strong-stop HIV-1 DNA (with LTR R/U5 primers), an early product of reverse transcription. CCL2 conditioning significantly enhanced X4-tropic HIV-1 IIIB infection 5-fold (p ϭ 0.039; Fig. 6A). Conversely, there was no enhancement of R5-tropic HIV-1 93In905 infection (p ϭ 0.36).
Virus replication was then induced using 20 units/ml IL-2 for a further 96 h, and p24 antigen was quantified at the end of this period. The increase in viral DNA found in CCL2-conditioned cells with the X4-tropic HIV-1 IIIB correlated with a significant 35-46% increase in p24 antigen concentration at 4 days postinfection (p Ͻ 0.05; Fig. 6B).
We then tested the hypothesis that Erk 1/2 phosphorylation is important to the infection of primary resting CD4 ϩ T cells with X4 HIV-1 by incubating the cells with U0126, a selective inhibitor of Erk 1/2, prior to infection. This resulted in the inhibition of viral replication at a point before the reverse transcription step (data not shown). Treatment of the cells with CCL2 had no effect on this inhibition.

DISCUSSION
In this study, we show that CCL2 up-regulates the expression of CXCR4 on the surface of resting CD4 ϩ T cells through a dose-and CCR2-dependent mechanism that does not require de novo protein synthesis. Furthermore, we show that surface CXCR4 levels on resting CD4 ϩ T cells are positively associated with both the efficiency of X4-tropic but not R5-tropic gp120 to chemoattract resting CD4 ϩ T cells and of X4 but not R5 HIV-1 entry into resting CD4 ϩ T cells. CXCR4 functional receptor protein is detectable in a diverse range of cells and, with its natural ligand CXCL12, is involved in the regulation of numerous biological processes, including fundamental roles in the development of numerous organs during embryonic development, proliferation, survival, and the retention of primitive hematopoietic progenitor cells in the bone marrow. The CXCR4/CXCL12 pair have been shown to be critically involved in inflammatory conditions, such as rheumatoid arthritis, and they are often highly overexpressed in cancers contributing to tumor metastasis, as well as CXCR4 acting as a coreceptor for X4-tropic HIV infection.
The CCL2-mediated CXCR4 up-regulation was not accompanied by the up-regulation of other cell markers such as HLA-DR, CD69, CD25, CD45RO, CCR5, CD4, and CD3. Moreover, the inability of CCL2 to up-regulate the activation markers CD69, CD25, and HLA-DR suggests that the CCL2-induced up-regulation of CXCR4 is not dependent upon activation of The change in F-actin content is expressed as a relative fold change in the median fluorescence intensity, when the base-line fluorescence intensity before addition of ligand (time 0) is expressed as 100%. CCL2 preconditioning of resting CD4 ϩ T cells significantly increased the content of X4 gp120-induced F-actin (solid squares; *, p ϭ 0.022) but had no effect on R5 gp120-induced F-actin (solid circles; p ϭ 0.89). AMD3100 inhibited the increase in X4 gp120-induced F-actin content (solid triangles). All data presented are representative of three independent experiments. these cells. The efficient inhibitory effect of RS102895 on the CCL2-induced increase in the CXCR4 surface expression suggests that the CCL2-mediated up-regulation of CXCR4 is through a CCR2b-dependent mechanism. Furthermore, the inability of CCL2 to induce an increase in total CXCR4 receptor or an increase in CXCR4 mRNA, combined with the reduction in CXCR4 internalization rates and increase in plectin content, suggests that CCL2-mediated CXCR4 up-regulation is because of the relocation of preexisting receptor to the cell surface combined with a decrease in internalization rates that does not require de novo CXCR4 synthesis. CXCR4, like many G protein-coupled receptors, including the ␣ 1a -adrenoreceptor and the -opioid receptor, actively and continuously undergoes internalization in the absence of ligand, which fine-tunes the threshold at which cells sense ligand. The C-terminal cytoplasmic domain of CXCR4 is known to regulate its function and spatiotemporal expression (35) and is involved in the plectin complex formation (30). However, little is known about the mechanism underlying constitutive internalization and surface expression of CXCR4 compared with internalization mediated by its ligand CXCL12. The present data indicate that CCL2 up-regulates CXCR4 surface expression primarily by altering receptor trafficking through up-regulation of plectin. However, the precise mechanisms by which CXCR4 signaling and intracellular trafficking are regulated remain not fully understood.
We next determined whether CCL2 modulates the functional responses of CXCR4. X4 tropic gp120 MN , when used as a ligand, induced CXCR4 signaling leading to F-actin polymerization, Erk 1/2 phosphorylation, and a chemotactic response more efficiently in CCL2-conditioned resting CD4 ϩ T cells than in unconditioned cells. The response was CXCR4-and G␣ i protein-specific as pretreating the cells with either AMD3100 or PTX abrogated the response; thus X4 tropic gp120 MN functions as a full CXCR4 agonist. Treating CD4 ϩ T cells with anti-CD3 antibody induced a marginal down-regulation of CXCR4 surface expression (data not shown), mediated via protein kinase C activation that phosphorylates CXCR4, followed by receptor internalization (36). However, it is FIGURE 5. CCL2 augments X4 gp120-induced Erk 1/2 phosphorylation in primary resting CD4 ؉ T cells. Primary resting CD4 ϩ T cells were stimulated with gp120 for 2 min, fixed, permeabilized/ and stained with a fluorescently tagged antibody specific for phosphorylated Erk 1/2 and then analyzed by flow cytometry. A, Erk 1/2 phosphorylation induced by X4 tropic gp120 MN . Histograms shown are from a representative donor. Solid gray histogram in each image corresponds to cells not stimulated with 200 nM gp120 MN . Light gray line corresponds to unconditioned cells stimulated with 200 nM gp120 MN . Black line corresponds to pretreatment with either 1 nM CCL2, 1 M AMD3100, or 1 g/ml PTX. B, Erk 1/2 phosphorylation induced by R5-tropic gp120 SF162 . Histograms shown are from a representative donor. Solid gray histogram in each image corresponds to cells not stimulated with 200 nM gp120 SF162 . Light gray line corresponds to unconditioned cells stimulated with 200 nM gp120 SF162 . Black line in ϪCCL2 corresponds to pretreatment with 1 nM CCL2. C, bars represents the mean Ϯ S.E. of the relative change in fluorescence intensity indicative of Erk 1/2 phosphorylation after stimulation with X4 gp120 MN (black bars) or R5 gp120 SF162 (gray bars) from three independent donors carried out in triplicate. The change in Erk 1/2 phosphorylation is expressed as the relative fold change in the mean fluorescence intensity, with the base-line fluorescence intensity before the addition of gp120 expressed as 1.00. CCL2 preconditioning of resting CD4 ϩ T cells significantly increased X4 gp120-induced phosphorylation of Erk 1/2 (p Ͻ 0.0001) but had no effect on R5 gp120-induced events. AMD3100 and PTX inhibited Erk 1/2 phosphorylation. *, p Ͻ 0.0001. NOVEMBER 7, 2008 • VOLUME 283 • NUMBER 45 unlikely that this down-regulation accounted for the almost complete inhibition of gp120 MN -induced chemotaxis that we observed. Previous studies have shown that CXCR4 physically interacts with the TCR in response to signaling via CXCL12 and uses TCR signaling machinery from signal initiation to signal termination. CD3-mediated activation of protein kinase C leads to phospholipase C-␤-3 phosphorylation, which in turn inhibits the activation of G q ␣ (37). However, it is also possible that the signaling pathways of both the TCR and CXCR4 use the same G q ␣ subunit. Therefore, by stimulating the cells with anti-CD3 antibody prior to the gp120 chemotaxis assay, CXCR4 is transiently desensitized, rendering the cell insensitive to the agonistic effects of the gp120 MN -CXCR4 interaction.

CCL2 and CXCR4 Up-regulation
In addition to the surface up-regulation of CXCR4 by CCL2, the enhanced X4 tropic gp120 MN -induced chemotaxis signaling events that accompanied it suggested that CCL2 might increase the susceptibility of resting CD4 ϩ T cells to infection by X4 HIV-1. The best characterized latently infected cells are resting CD4 ϩ T cells that are formed while in an activated state prior to returning to a resting state (38). However, HIV-1 can be isolated from CD45RA ϩ naive CD4 ϩ T cells in HIV-1-infected organ cultures and from HIV-1-infected individuals (39,40). Moreover, resting CD4 ϩ T cells can be latently infected in vivo without the requirement for a secondary stimulus (41). Together, this suggests that an activation step is not required for either HIV-1 entry or for HIV-1 integration. In this study we show that pretreatment of resting CD4 ϩ T cells from peripheral blood with CCL2 increased the efficiency of X4 but not R5-tropic viral entry.
Genetic variations of CCL2 and CCR2 play a role in HIV-1 pathogenesis. In European-Americans, homozygosity for the CCL2-2518G allele, i.e. the GA/GA haplotype pair, is associated with accelerated progression to AIDS and death in infected adults (42). Moreover, in vitro studies show that PBMCs from individuals homozygous or heterozygous for CCL2-2518G produce larger amounts of CCL2 than PBMCs from individuals homozygous for CCL2-2518A (43) and that this correlated with in vivo sera studies (42). Interestingly, in an in vitro model of HIV-1 infection, blocking endogenous CCL2 production decreased viral replication (2). Furthermore, higher CCL2 levels correlate with higher HIV load (1) and enhanced leukocyte trafficking to tissues (42), possibly playing a role in the etiology of HIV-1-associated dementia. The CCR2-V64I allele also has a significant influence on the rate of HIV-1 disease progression in adults, with the presence of CCR2-V64I associated with a 2-3year delay in progression to AIDS and death, although it has no impact upon HIV-1 transmission where CCR5 using variants are dominant (44). Interestingly, although PBMCs isolated from CCR2-V64I heterologous donors have normal levels of both CCR5 and CCR2, and the calcium flux mediated by CCL2 through CCR2-V64I was unaffected, they have reduced surface expression of CXCR4 (45). It is possible that in late stage HIV disease, the increase in CCL2 expression and secretion up-regulates the surface expression of CXCR4 on resting CD4 ϩ T cells, increasing their permissiveness to X4 HIV-1 infection and contributing to the switch from R5 to X4 using virus and more rapid disease progression.
In summary, CCL2 up-regulates CXCR4 expression on resting CD4 ϩ T cells through a CCR2-dependent mechanism that augments the ability of these cells to be chemoattracted to X4 gp120 and renders them more permissive to X4-tropic HIV-1 entry. Thus, CCL2 has the capacity to render a large population of lymphocytes more susceptible to X4 HIV-1 late in the course of infection.