Desensitization of Chemokine Receptor CCR5 in Dendritic Cells at the Early Stage of Differentiation by Activation of Formyl Peptide Receptors

Clinical Immunology Vol. 99, No. 3, June, pp. 365–372, 2001 doi:10.1006/clim.2001.5021, available online at http://www.idealibrary.com on

Desensitization of Chemokine Receptor CCR5 in Dendritic Cells at the Early Stage of Differentiation by Activation of Formyl Peptide Receptors 1 Yingying Le,* Michele A. Wetzel,† Weiping Shen,* Wanghua Gong,‡ Thomas J. Rogers,† Earl E. Henderson,† and Ji Ming Wang* ,2 *Laboratory of Molecular Immunoregulation, Division of Basic Sciences, and ‡Intramural Research Support Program, SAIC Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702; and †Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Chemokine receptors are subjected to heterologous desensitization by activation of formyl peptide receptors. We investigated the cross-talk between formyl peptide receptors and the chemokine receptor CCR5 in human monocyte-differentiated immature dendritic cells (iDC). Monocytes cultured with GM-CSF and IL-4 for 4 days exhibit markers characteristic of iDC and maintain the expression of both formyl peptide receptors FPR and FPRL1, as well as CCR5. Pretreatment of iDC with W peptide (WKYMVm), a potent agonist for FPR and FPRL1 but with preference for FPRL1, resulted in down-regulation of CCR5 from the cell surface and reduced cell response to the CCR5 ligands through a PKC-dependent pathway. Furthermore, W peptide induced a PKC-dependent phosphorylation of CCR5 and inhibited infection of iDC by R5 HIV-1. Our results indicate that the expression and functions of CCR5 in iDC can be attenuated by W peptide, which activates formyl peptide receptors, and suggest an approach to the design of novel anti-HIV-1 agents. © 2001 Academic Press Key Words: chemokine receptor; receptor desensitization; N-formyl peptide; N-formyl peptide receptors; HIV-1. INTRODUCTION

Dendritic cells (DCs) are bone-marrow-derived leukocytes specializing in antigen capture, processing, and presentation to T lymphocytes and thus are indis1 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The U.S. Government’s right to retain a nonexclusive royalty-free license in and to any copyright covering this paper, for governmental purposes, is acknowledged. 2 To whom correspondence should be addressed at LMI, DBS, NCI–Frederick, Building 560, Room 31-40, Frederick, MD 21702. Fax: 301-846-7042. E-mail: [email protected].

pensable in initiating primary immune responses (1– 4). DC precursors originating in the bone marrow enter the blood and subsequently migrate into lymphoid and nonlymphoid tissues, where they develop into immature DCs, such as Langerhans cells or mucosal DCs, with high efficiency in antigen uptake and process, yet not in T cell stimulation. Upon the encounter of an antigen, DCs migrate from the site of residence to the T cell areas of regional lymph nodes via the afferent lymphatics. These migratory cells also undergo maturation from a “processing” to a “presenting” stage, characterized by the loss of antigen uptake capacity but an increased capacity to stimulate T cells (1– 4). In vitro studies have shown that DCs express CD4 and the chemokine receptors including CCR5 and can be infected by HIV-1 (5). HIV-1 is also efficiently transmitted from DCs to CD4 ⫹ T cells during T cell activation (6, 7). An ex vivo model revealed that HIV-1-infected human Langerhans cells transmit infection to lymphoid tissue (8). Furthermore, it is believed that DCs originating from the epithelial surface (i.e., Langerhans cells) play a major role in establishing HIV-1 infection in lymph node T cell populations (9, 10), where rigorous viral replication occurs (11, 12). Due to their central role in HIV-1 infection, DCs are considered one of the major targets for therapeutic interventions of AIDS. Human immune cells, including DCs, migrate and can be activated by a variety of exogenous and tissuederived chemotactic factors (13). A large number of chemoattractants, such as the bacterial formylated peptides and chemokines, bind and activate seven transmembrane (STM), G-protein-coupled receptors (14). The C-termini of these receptors contain serine and threonine residues which, upon phosphorylation, are involved in signaling and receptor desensitization. Agonist-induced phosphorylation of chemokine receptors, such as CCR5, can result in homologous desensitization and internalization of the receptors (15, 16).

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Chemokine receptors may also be subjected to “heterologous desensitization” when the cells are activated by agonists selective for unrelated STM receptors. For example, activation of formyl peptide receptor (FPR), the receptor for the bacterial chemotactic peptide fMLF, effectively desensitizes a number of chemoattractant receptors, including the receptors for the chemokine IL-8 (17). Both homologous and heterologous desensitization results in impairment of various biological functions of chemokine receptors in response to further stimulation by their cognate ligands. Since human dendritic cells express a variety of chemoattractant receptors, the “cross-talk” among those receptors may be important for fine-tuning of cellular responses in the presence of multiple stimulants. We therefore investigated the expression of fMLF receptors during the differentiation of peripheral blood monocytes into DCs and the desensitization of CCR5 by the agonists for fMLF receptors. In this study, we show that DCs at the early stages of differentiation express functional FPR and FPRL1 (formyl peptide receptor-like 1). In addition, WKYMVm, a synthetic peptide which is derived from a random peptide library and is a highly potent agonist for fMLF receptors with preference for FPRL1 (18, 19), attenuates the function of CCR5 in immature DCs in association with a PKC-dependent serine phosphorylation of CCR5. Furthermore, the function of CCR5 as an HIV-1 coreceptor is impaired by the activation of formyl peptide receptors. MATERIALS AND METHODS

Reagents WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met, designated W peptide) was synthesized and purified by the Department of Biochemistry, Colorado State University (Fort Collins, CO), according to the published sequence (18, 19). The protein kinase C inhibitor staurosporine was purchased from Sigma (St. Louis, MO). Recombinant human (rh) GM-CSF, IL-4, SDF-1␣, RANTES, MIP-1␣, MIP-1␤, MCP-1, and MCP-3 were purchased from Pero Tech, Inc. (Rocky Hill, NJ). Anti-CD1a, HLA-DR, CD86, and CD83 antibodies, and FITC-conjugated anti-CCR5 monoclonal antibody were purchased from PharMingen (San Diego, CA). Generation of Immature DCs Human peripheral blood monocytes were isolated by Percoll gradient centrifugation as described previously (18). Monocytes were further purified (⬎98%) by incubation in RPMI 1640 containing 10% FBS, 2 mM glutamine, 25 mM Hepes, 100 U/ml penicillin, and 100 ␮g/ml streptomycin for 2 h at 37°C in a plastic flask. The adherent cells were incubated with rhGM-CSF (50

ng/ml) and rhIL-4 (1000 U/ml) at 37°C in humidified air with 5% CO 2. The cultures were refreshed with the same cytokines every other day. The detached cells were harvested at different times for receptor expression and phenotype analysis using FACS (Coulter Epics). The cells expressing markers for early immature DCs (iDCs) were used for receptor expression and function experiments. FACS Analysis iDCs were first washed three times with FACS buffer (PBS, 1% FBS, 0.02% NaN 3, pH 7.4) and then stained with various antibodies at 4°C for 1 h. Subsequently, the cells were stained with FITC-conjugated goat anti-mouse IgG (Sigma) for 30 min at 4°C, fixed with 1% paraformaldehyde in PBS, and analyzed with a flow cytometer (Coulter Epics). RNA Isolation and RT-PCR Total RNA was extracted from iDCs with the RNeasy Mini Kit (Qiagen). A total of 0.5 ␮g of RNA was used for RT-PCR with the High Fidelity ProSTAR HF SingleTube RT-PCR System (Stratagene). Forty cycles were used for the amplification for FPR and FPRL1 cDNA. Twenty-two cycles were used for the amplification for GAPDH sequences. The primers for FPRL1 were 5⬘CTGCTGGTGCTGCTGGCAAG-3⬘ and 5⬘-AATATCCCTGACCCCATCCTCA-3⬘, to yield a 1.1-Kb product. The primers for FPR were 5⬘-CTCCAGTTGGACTAGCCACA-3⬘ and 5⬘-CCATCACC CAGGGCC CAATG-3⬘ to yield a 500-base-pair product. PCR products were visualized on 1% agarose gel with ethidium bromide staining. Chemotaxis Assays The migration of iDCs was assessed using a 48-well microchemotaxis chamber (Neuro Probe, Cabin John, MD) and polycarbonate filter (5-␮m pore size, Neuro Probe) as previously described (18). The results were expressed as a chemotaxis index (CI) which represents the fold increase in the number of migrated cells in three high-power fields in response to chemoattractants over the spontaneous cell migration in response to control medium. Each experiment was performed in triplicate. The statistical significance of the increase in cell migration was determined by unpaired Student’s t test. Calcium Mobilization iDCs (2 ⫻ 10 7 cells/ml) were incubated with 5 ␮M Fura-2 AM (Molecular Probes, Eugene, OR) in loading

WKYMVm DESENSITIZES CCR5 AND CXCR4 IN DENDRITIC CELLS

medium (DMEM, 10% FBS, 2 mM glutamine) for 30 min at room temperature. Ca 2⫹ mobilization induced by stimulants was measured with a fluorescence spectrometer (LS-50B, Perkin–Elmer, Beaconsfield, England) as previously described (18). The ratio of fluorescence at 340 and 380 nm was calculated using a FL-WinLab program (Perkin–Elmer). CCR5 Phosphorylation CCR5 phosphorylation was examined as described previously (20). Briefly, iDCs stimulated with chemoattractants were lysed and immunoprecipitated with an anti-phosphoserine antibody (Zymed Laboratory, Inc., South San Francisco, CA). The immunocomplexes were absorbed with Protein–A Sepharose beads, electrophoresed on 10% SDS–PAGE precast gel (Novex) under nonreducing conditions, and transferred to Immobilon P membranes (Millipore, Bedford, MA). The membranes were incubated with a polyclonal anti-CCR5 antibody (Millenium Biotechnology, Ramona, CA) followed by incubation with a horseradish-peroxidaseconjugated goat anti-rabbit IgG (1:20,000) (Pierce, Rockford, IL). The phosphorylated receptor was detected with Super Signal Chemiluminescent Substrate Stable Peroxide solution (Pierce) and Biomax-MR film (Eastman Kodak Company, Rochester, NY).

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important for their migration, trafficking, and activation during immunological responses. We obtained iDCs by incubating human blood monocytes with GMCSF and IL-4 and examined the expression and function of the receptors that can be activated by the bacterial chemotactic peptide fMLF. By using RT-PCR, we found that the high-affinity fMLF receptor FPR and its low-affinity variant FPRL1 were expressed by day 4 when monocytes were cultured in the presence of GMCSF and IL-4 (Fig. 1A). At this stage, most cells expressed markers typical of iDCs (HLA-DR ⫹, CD1a ⫹, and CD86 ⫹), but less CD83, a marker for mature DCs (Fig. 1B). By day 7, the iDCs expressed a reduced level of fMLF receptor genes, which were no longer detectable by day 9 (Fig. 1A). In agreement with the pattern of gene expression, the iDCs at day 4 responded to FPR- or FPRL1-specific ligands by cell migration and Ca 2⫹ mobilization (data not shown). These cells also showed potent chemotactic and Ca 2⫹ mobilization responses to a synthetic hexapeptide WKYMVm (W peptide) (Figs. 1C and 1D), which activates both FPR and FPRL1, with higher efficacy for FPRL1. iDCs at day 7 also responded, albeit at a lower level compared with that at day 4, to W peptide in migration and Ca 2⫹ flux assays (data not shown). These results indicate that at early stages of differentiation iDCs express functional FPR and FPRL1. Down-Regulation of CCR5 Expression and Function in iDCs by W Peptide

HIV-1 Infection The monocyte-derived immature iDCs were treated with W peptide at the designated concentrations, and after 1 h, the cells were infected with HIV-1 SF162 at an MOI of 0.1 for 2 h. The cells were then washed, the GM-CSF and IL-4 were replaced, and the cells were incubated in medium for 4 days. HIV-1 p24 levels were determined by enzyme-linked immunosorbent assays using capture and detection antibodies in a sandwich ELISA (ELISA kit obtained from the AIDS Vaccine Program, SAIC Frederick, NCI–Frederick, Frederick, MD). These treatments did not change the cell number or viability. RESULTS

Expression and Function of Formyl Peptide Receptors in iDCs It has been reported that DCs can be differentiated in vitro from peripheral blood monocytes in the presence of GM-CSF and IL-4 or IL-13 (21–23). Kinetic studies showed that monocytes treated with IL-4 and GM-CSF acquired the phenotype of immature DCs at day 4 and exhibited significant stimulating activity on allogeneic T lymphocytes (24). DCs express a number of chemoattractant receptors that are thought to be

We next examined the expression and function of CCR5 in iDCs and its regulation by activation of the cells with W peptide. FACS analyses using a specific monoclonal anti-CCR5 antibody showed that a substantial proportion of iDCs express CCR5 on the cell surface (Fig. 2B). The chemokines RANTES, MIP-1␣, and MIP-1␤ which use CCR5 as a functional receptor significantly down-regulated the cell surface expression of CCR5 (Fig. 2C and data not show). The expression of CCR5 on iDCs was also markedly decreased by prior treatment of the cells with W peptide and fMLF (37°C, 60 min) (Figs. 2D and 2E). In order to elucidate the mechanistic basis for CCR5 down-regulation, we used staurosporine, a reported inhibitor of protein kinase C (PKC). Pretreatment of iDCs with staurosporine did not affect the effect of chemokine ligand on down-regulation of CCR5 (data not shown). However, cells treated with staurosporine maintained a high level of CCR5 expression even after W peptide or fMLF treatment (Figs. 2F and 2G). These results suggest that the down-regulation of CCR5 on iDCs by formyl peptide receptor agonists is dependent on PKC activation. To determine whether the down-regulation of cell surface CCR5 expression by iDCs was correlated with

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FIG. 1. Expression of functional FPR and FPRL1 by monocyte-derived dendritic cells. (A) Expression of FPRL1 and FPR transcripts in monocyte-derived iDCs was determined by RT-PCR. (B) Phenotype of iDCs at day 4 of culture in the presence of GM-CSF and IL-4. (C) W-peptide-induced migration of iDCs at day 4 differentiation with cytokines. Chemotaxis index represents the fold increase of cell migration induced by W peptide over the spontaneous cell migration in response to control medium. *P ⬍ 0.01 compared with migration in the absence of stimulants. (D) Calcium mobilization induced by W peptide in iDCs. iDCs at day 4 differentiation were stained with 5 ␮M Fura-2 AM and stimulated with different concentrations of W peptide. The ratio of fluorescence at 340 and 380 nm was monitored with a fluorescence spectrometer and calculated using the FL-WinLab program.

reduced function, we tested the cell response to chemokine ligands after treatment with W peptide. As shown in Fig. 3A, pretreatment of iDCs with W peptide at 37°C for 1 h completely inhibited cell migration in response to MIP-1␤. The CXCR4 ligand SDF-1␣-induced chemotaxis of iDCs was also significantly reduced by pretreatment of the cells with W peptide, while little effect was observed with the cell response to MCP-3, a chemokine that does not activate CCR5. The capacity of iDCs treated with W peptide to respond to CCR5 ligands was also examined by calcium mobilization experiments. iDCs preincubated with W peptide (10 ⫺8 M) at 37°C for 60 min showed a significantly reduced Ca 2⫹ flux in response to RANTES and SDF-1␣, but not to MCP-1 (Figs. 3B–3D). The PKC inhibitor staurosporine reduced the inhibitory effect of W peptide on iDC responses to RANTES and SDF-1␣ as measured by chemotaxis and Ca 2⫹ flux (Fig. 3). These

results provide further evidence that both CCR5 and CXCR4 in iDCs are subjected to desensitization by formyl peptide receptor agonists and this cross-desensitization is PKC dependent. CCR5 Phosphorylation Induced by W Peptide Since our previous studies showed that the heterologous desensitization of CCR5 following activation of FPR and FPRL1 in monocytes was associated with a PKC-mediated serine phosphorylation of CCR5 (20, 25), we examined the effect of W peptide on CCR5 phosphorylation in iDCs. An anti-phosphoserine antibody was used to immunoprecipitate cell lysates followed by immunoblotting with a polyclonal anti-CCR5 antibody to detect the phosphorylation state of CCR5. We observed that, as in human monocytes (20, 25), CCR5 in iDCs appeared as a molecular species at about

WKYMVm DESENSITIZES CCR5 AND CXCR4 IN DENDRITIC CELLS

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peptide, or the FPRL1 peptide ligand MMK-1 (26), for 1 h, followed by infection with the R5 HIV-1 strain SF162. Cells pretreated with these peptides all showed significantly reduced HIV replication as measured by p24 production. Surprisingly, the maximal inhibition of p24 production in iDCs by W peptide and MMK-1 occurred at concentrations much lower than those required to desensitize the functions of CCR as a chemoattractant receptor (Fig. 5). The reasons for a more sensitive attenuation of HIV-1 coreceptor function by

FIG. 2. Down-regulation of CCR5 from iDC surface by W peptide. iDCs incubated with different stimulants were stained with a FITC-conjugated anti-CCR5 monoclonal antibody (FITC-anti-CCR5) and analyzed by FACS. (A) Cells incubated with medium and stained with FITC-conjugated mouse IgG. (B) Cells incubated with medium and stained with FITC-anti-CCR5. (C–E) Cells treated with RANTES (1 ␮g/ml, 15 min, 37°C), W peptide (10 ⫺6 M, 60 min, 37°C), or fMLF (10 ⫺6 M, 60 min, 37°C), and then stained with FITC-antiCCR5. (F and G) Cells pretreated with staurosporine (stau, 2.5 ng/ml, 30 min, 37°C), followed by W peptide or fMLF (10 ⫺6 M, 60 min, 37°C), and then stained with FITC-anti-CCR5.

75 kDa under nonreducing conditions and exhibited a low level of serine phosphorylation which could be rapidly increased by stimulation of the cells with CCR5 chemokine agonists MIP-1␣, RANTES, and MIP-1␤ (Fig. 4A and data not shown). Stimulation of iDCs with W peptide also induced a significantly increased serine phosphorylation of CCR5 in a time- and dose-dependent manner (Figs. 4A and 4B). The maximal CCR5 phosphorylation induced by W peptide was observed at 30 min after stimulation (Fig. 4B). These results are in agreement with the notion that heterologous receptor desensitization requires a longer incubation time with the stimulants and an accumulation of second messengers that culminates in phosphorylation of an unrelated STM receptor. In fact, when iDCs were pretreated with the PKC inhibitor staurosporine, W peptide no longer significantly induced phosphorylation of CCR5, whereas staurosporine treatment did not affect MIP-1␤-induced CCR5 phosphorylation (Fig. 4C). Thus, the mechanism of agonist-induced homologous CCR5 phosphorylation differs from that induced by the formyl peptide receptor ligand W peptide. W Peptide Inhibits HIV-1 Infection of iDC Finally, we wish to determine the impact of FPR and FPRL1 activation on the capacity of CCR5 to act as an HIV-1 coreceptor in iDCs. iDCs were treated with W

FIG. 3. Effect of W peptide on iDC response to chemokines. (A) Migration of iDCs treated with W peptide. iDCs preincubated with W peptide (10 ⫺6 M, 37°C, 1 h, hatched histogram,) were tested for migration in response to MIP-1 ␤, SDF-1␣, or MCP-3 (100 ng/ml). In parallel experiments, iDCs were preincubated with staurosporine (2.5 ng/ml, 30 min at 37°C), followed by W peptide (10 ⫺6 M, 37°C, 1 h) (shaded histogram), and then were measured for chemotaxis induced by chemokines. Results are expressed as the fold increase (chemotaxis index, CI) of cell migration in response to chemokines over the spontaneous cell migration in response to control medium. *P ⬍ 0.01 compared with the CI of cells preincubated with medium in response to chemokines (black histogram). (B–D) Ca 2⫹ mobilization in iDCs in response to chemokines. iDCs were preincubated with medium (medium) or W peptide (W pep, 10 ⫺6 M) for 60 min at 37°C and then were loaded with Fura-2 and measured for Ca 2⫹ mobilization in response to RANTES (A, 10 ng/ml), SDF-1␣ (〉, 100 ng/ml), or MCP-1 (C, 100 ng/ml). In parallel experiments, cells were first treated with staurosporine (2.5 ng/ml, 30 min at 37°C) followed by W peptide (10 ⫺6 M, 37°C, 60 min, Stau ⫹ W peptide) and then were measured for Ca 2⫹ flux in response to chemokines.

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FIG. 4. Phosphorylation of CCR5 in iDCs induced by W peptide. (A) Serine phosphorylation of CCR5 in iDCs treated with MIP-1␤ (100 nM, 37°C, 5 min) or different concentrations of W peptide (37°C, 60 min). (B) iDCs treated with W peptide (10 ⫺6 M) for different time points. (C) Effect of staurosporine (Stau., 2.5 ng/ml, 37°C, 30 min) on CCR5 phosphorylation induced by MIP-1␤ (100 nM, 5 min at 37°C) or W peptide (10 ⫺6 M, 60 min). The inset in C shows the detection of CCR5 in 40 ␮g of cell lysates by immunoblot prior to immunoprecipitation.

lower doses of formyl peptide receptor ligands are currently not known and are under investigation. DISCUSSION

Leukocyte recruitment at sites of inflammation and infection is an early host response to invading pathogens. A number of leukocyte chemotactic factors have been identified that specifically induce migration of leukocyte subpopulations. These chemotactic factors activate a class of STM G-protein-coupled receptors on the cell surface, including the receptors for the classical chemotactic factors such as bacterial fMLF, C5a, and LTB4, as well as newly defined members of the chemokine superfamily (14). The prototype receptor for formyl peptides designated FPR is mainly expressed by phagocytic leukocytes and can be activated by a diverse array of agonists, including the bacterial fMLF, and many synthetic small peptides with or without N-terminal modifications (14, 27, 28). Based on their ability to recognize bacterial chemotactic peptides, formyl peptide receptors have been proposed to play an important role in host defense against microbial invasion. This hypothesis is supported by the results obtained with mice depleted of the FPR gene that exhibited higher susceptibility to microbacterial infection, although these mice maintain an apparently normal phenotype (29). The recent identification of a novel and host-derived FPR agonist annexin I suggests additional biological functions of this receptor (30). FPRL1 is a variant of FPR and reacts with bacterial fMLF with low affinity. Recently, a number of highly

efficacious agonists of FPRL1 have been identified, including peptide domains derived from HIV-1 envelope proteins and at least five host-derived molecules, lipid metabolite lipoxin A4, serum amyloid A, ␤ amyloid peptide, prion protein fragment, mitochondrial peptide fragment, and cathelicidin fragment (28, 31–36). Compared with FPR, FPRL1 is expressed by a greater variety of cell types including T and B lymphocytes and certain cells of nonhematopoietic origin (28). In this study, we have shown that both FPR and FPRL1 are expressed and functional during the early stages of DC differentiation from peripheral blood monocytes. The implication of the loss of the expression of these receptors by more differentiated DCs is not clear. It is hypothesized that, in addition to chemokine receptors, such as CCR6, that mediate the cell response to hostderived agonists, the functional expression of FPR and FPRL1 by iDCs may also help localization and activation of these cells at the sites of infection which may favor the uptake and processing of bacterial or viral antigens by iDCs. During further differentiation, a new pattern of chemoattractant receptor expression favors migration of DCs to regional lymphatic tissues in response to highly specialized chemokines, where specific immune responses are initiated (37). As classical G-protein-coupled chemoattractant receptors, FPR and FPRL1 have been studied extensively for their property of signal transduction and cross-talk with other receptors. The binding of these receptors by agonists results in a cascade of G-proteinmediated signaling events leading to phagocytic cell adhesion, chemotaxis, release of oxygen intermediates, enhanced phagocytosis, and bacterial killing, as well as mitogen-activated protein kinase activation and gene transcription (14, 27, 28). Activation through FPR and FPRL1 can also lead to heterologous desensitization of a subsequent cell response to other G protein receptor ligands, such as the chemokines, presumably by pro-

FIG. 5. Susceptibility of peptide-treated iDCs to infection with HIV-1. iDCs were treated with the designated concentrations of W peptide (black histogram) or MMK-1 (hatched histogram) for 1 h, infected with HIV-1 SF162 for 2 h, washed to remove free virus, and cultured for an additional 96 h. Levels of HIV-1 p24 produced by these cells were determined by ELISA.

WKYMVm DESENSITIZES CCR5 AND CXCR4 IN DENDRITIC CELLS

tein-kinase-mediated receptor phosphorylation. Our present study provides novel evidence that CCR5 in iDCs is also a target of desensitization and phosphorylation induced by a synthetic peptide that activates both FPR and FPRL1. This rapid and progressively increased level of CCR5 phosphorylation is accompanied by down-regulation of the surface expression and function of CCR5 in iDCs, including its capacity to act as HIV-1 coreceptor. It should be pointed out that although the down-regulation of CCR5 on iDC surface by formyl peptide receptor activation was only partial, the important functions of this receptor for chemotaxis and HIV-1 infection are significantly attenuated. It is yet not clear whether W peptide acted at the level of HIV-1 viral entry via utilization of CCR5. Although further research is underway to clarity this issue, in our previous study, HIV-1 envelope mediated fusion was inhibited in macrophages after heterologous desensitization of CCR5 by activation of FPR (25). Thus, W peptide may interfere in HIV-1 infection of iDCs at the level of viral entry. Our results are in support of the observation that activation of human PBMC with the B oligomer, a subunit of the pertussis toxin, desensitizes CCR5 and inhibits HIV-1 fusion and infection (38). B oligomer did not affect the cell surface expression of CCR5, suggesting that interference with the signaling cascade at the level of CCR5 may compromise its capacity to act as an HIV-1 coreceptor. Therefore, in addition to strategies directed at virus or at host cells, desensitization of chemokine receptors may represent an additional approach to the development of anti-HIV-1 agents. W peptide has only six amino acids and therefore may not be antigenic in vivo. It contains a D amino acid at the C-terminus and may be more resistant to peptidase degradation and maintain a longer half-life in vivo. Furthermore, as a potent agonist for both FPR and FPRL1, it recruits leukocytes including iDCs and enhances phagocytosis and release of reactive mediators that support host defense. Thus, W peptide may be utilized as a basis for designing more efficient antiHIV-1 agents and immune adjuvants. ACKNOWLEDGMENTS The authors thank J. J. Oppenhiem for reviewing the manuscript, N. M. Dunlop for technical support, and C. Fogle and C. Nolan for secretarial assistance. This project has been funded in whole or in part with Federal Funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-56000, and by NIH Grants DA06650 and DA11130 (TJR), F31DA05894 and T32DA07237 (MAW), and DA12113 (EEH). REFERENCES 1. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., and Palucka, K., Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767– 811, 2000.

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Received December 13, 2000; accepted with revision February 21, 2001