Published online before print November 2, 2004
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,
* Department of Medicine, Karolinska Systems Biomedicine Center, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden;
Department of Clinical and Experimental Medicine, University of Bologna, Italy; and
Abteilung Virologie, Universitätsklinikum Ulm, Germany
1 Correspondence: Karolinska Institutet, Center for Molecular Medicine, L8:03, Karolinska Hospital, SE-171 76 Stockholm, Sweden. E-mail: cecilia.soderberg.naucler{at}cmm.ki.se
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(MIP-1), CCL4/MIP-1ß, and CCL5/regulated on activation, normal T expressed and secreted (RANTES). Neutralizing antibodies for these chemokines reduced the effects of HCMV on chemokine receptor expression and on DC migration by 
60%. Interestingly, the surface expression of the lymphoid chemokine receptor CCR7 was not up-regulated after HCMV infection on immature DC, and immature-infected DC did not migrate in response to CCL19/MIP-3ß. These findings suggest that blocking the migratory ability of DC may be a potent mechanism used by HCMV to paralyze the early immune response of the host.
Key Words: viral infection • antigen-presenting cells • chemotaxis • chemokines
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DC have peculiar trafficking properties. Immature DC arise from blood precursors and migrate to inflamed tissues in response to chemotactic stimuli [i.e., inflammatory chemokines such as CC chemokine ligand 3 (CCL3)/macrophage inflammatory protein-1 (MIP-1), CCL4/MIP-1ß, and CCL5/regulated on activation, normal T expressed and secreted (RANTES)]. Immature DC express receptors for inflammatory chemokines, including CC chemokine receptor 1 (CCR1), CCR2, and CCR5, which guide them to inflammatory sites where antigen sampling can take place [4
]. After antigen uptake, DC undergo functional maturation, enter the afferent lymph system, and home to the T cell areas of lymphoid organs, where they await interaction with antigen-specific T cells to prime the T cell response. Maturation results in rapid changes in the migratory behavior of DC and in their expression of chemokine receptors, including up-regulation of CCR7 and loss of receptors for inflammatory chemokines. The up-regulation of CCR7 is critical for the homing of mature DC, as the CCR7 ligands CCL19/MIP-3ß and CCL21/secondary lymphoid tissue chemokine are produced in lymphoid organs [4
5
6
7
].
Human cytomegalovirus (HCMV) can cause a permissive and lytic infection of immature DC [8 ]. In vitro infection of human monocyte-derived DC by an endothelial cell (EC)-adapted strain of HCMV impaired their maturation, reduced their secretion of interleukin (IL)-12 in response to maturation stimuli, and inhibited their ability to stimulate T cell proliferation and cytotoxicity [9 10 11 ]. In mice, splenic DC can be infected with murine CMV (MCMV), which can also productively infect long-term cultures of murine DC. MCMV infection inhibited DC maturation induced with lipopolysaccharide (LPS), reduced IL-2 and IL-12 secretion, and impaired the priming of T cell activation [12 ].
These findings suggest that HCMV can interfere with the host immune response by hampering several functions of DC. It has been recently shown that HCMV impairs the migration of monocyte-derived DC matured by LPS, tumor necrosis factor , and interferon-
toward lymphoid chemokines [13
]. In this study, we assessed the effects of HCMV infection on the migratory behavior of immature monocyte-derived DC in response to inflammatory and lymphoid chemokines.
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DC culture
Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by density gradient centrifugation with Lymphoprep (Axis-Shield PoC AS, Oslo, Norway). Monocyte-derived DC were generated as described previously [14
]. Briefly, isolated PBMC (5–10x106/ml) were plated in complete RPMI containing 10% FCS and allowed to adhere for 2 h at 37°C. Nonadherent cells were removed, and the adherent cells were washed three times with phosphate-buffered saline (PBS). Complete RPMI containing 10% FCS, 1000 IU/ml IL-4, and 100 ng/ml GM-CSF was added, and the cells were cultured for 7 days. During the last 1 or 2 days of culture, 100 ng/ml LPS was added to stimulate maturation. Cell differentiation was monitored by light microscopy and flow cytometry. After gating on size and scatter parameters to exclude lymphocytes, 80–90% of the cells were of the DC phenotype [CD1a+, human leukocyte antigen (HLA)-DR+, CD14–]. Nonadherent and loosely adherent DC were collected and used after 5–7 days in culture. Immature DC were collected on day 5 before exposure to LPS, and mature DC were harvested on day 6 or 7 after incubation with LPS. DC cultured with LPS for 48 h expressed typical maturation markers. Mature DC were >50% CD83+, >70% CD80+, and HLA-DR bright.
Viruses
Viral stocks of the fibroblast-adapted HCMV strains AD169 and Towne, the EC-adapted strain TB40/E (kindly provided by Prof. Gerhard Jahn, University of Tubingen, Germany), and a clinical isolate (PO) were prepared by infecting human lung fibroblasts at a low multiplicity of infection [MOI, 0.1–1 plaque forming unit (PFU)/cell]. The cells were cultured in DMEM with 10% FCS until the cytopathic effects became advanced (5–7 days). Supernatants were collected and centrifuged at 1200 g for 10 min to remove cell debris and stored at –80°C in small aliquots. Viral titers, determined in a plaque assay from dilution of virus on fibroblasts grown in 24-well plates for 2 weeks, were 2 x 106–1 x 107 PFU/ml.
HCMV infection of DC
DC were incubated with HCMV at a MOI of 0.1–5 PFU/cell for 3–4 h at 37°C. The virus was then removed, the cells were washed once with PBS, and fresh medium containing cytokines was added. HCMV infection was detected with an indirect immunofluorescence assay. Briefly, DC were stained with monoclonal antibodies (mAb) against HCMV immediate-early antigen (IEA; clone E13, Argene, Varilhes, France), diluted 1:40 in PBS, or against a late viral antigen [antiphosphoprotein 150 (anti-pp150), clone F3, Abbott Diagnostics Division, Chicago, IL], diluted 1:200 in PBS, followed by a second incubation with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin G (IgG; Dako, Glostrup, Denmark).
Flow cytometry and immunofluorescence analyses
Antibodies for DC immunophenotyping were anti-CD1a, anti-CD14, anti-CD80, anti-HLA-DR, mouse isotype controls (BD PharMingen, San Diego, CA), and anti-CD83 (Immunotech, Marseilles, France). All antibodies were mouse mAb conjugated to FITC or phycoerythrin (PE). Antibodies for chemokine receptor studies were unconjugated isotype controls, anti-CCR1, anti-CCR2, anti-CCR5 (R&D Systems), or anti-CCR7 antibody (BD PharMingen). FITC-conjugated rabbit anti-mouse IgG (Dako) was used after anti-CCR1, -CCR2, and -CCR5 staining, and biotinylated anti-mouse IgM (BD PharMingen) and streptavidin-PE (BD PharMingen) were used after anti-CCR7 staining. For determination of the total (cell-surface and intracellular) levels of chemokine receptors, DC were fixed and permeabilized with the Cytofix/Cytoperm kit (BD PharMingen) according to the manufacturer’s instructions. Data were acquired and analyzed with FACSCalibur (BD PharMingen) using CellQuest software. Antigen expression was measured as the percentage of positive cells and as the mean channel fluorescence value of the respective antibody compared with the isotype-matched control.
At 1 day postinfection, double-immunofluorescence staining was performed for simultaneous detection of CCR7 and IEA. Intact, immature-infected DC were incubated with anti-CCR7 antibody (BD PharMingen, 1:20 in PBS), followed by incubation with biotinylated anti-mouse IgM (BD PharMingen) and streptavidin-PE (BD PharMingen). DC were then fixed and permeabilized with the Cytofix/Cytoperm kit (BD PharMingen) and stained with anti-IEA mAb (clone E13, Argene), diluted 1:25 in PBS, followed by a second incubation with FITC-rabbit anti-mouse IgG (Dako). All incubations were performed on ice. Subsequently, cells were cytocentrifuged on a glass slide and read under a Nikon Eclipse E600W microscope.
For colocalization experiments, mock-infected or TB40/E-infected, immature DC were collected at 1 day postinfection, cytocentrifuged on glass slides, fixed with the Cytofix solution of the Cytofix/Cytoperm kit (BD PharMingen) for 20 min, washed, and stained with different mAb. Antibodies specific for the following markers were used: Rab 4 (early endosome marker) and Rab 7 (late endosome marker, Santa Cruz Biotechnology, Santa Cruz, CA), Lamp 1 (early lysosome marker) and Lamp 2 (lysosome marker, Protégé Corp., Madison, WI). These antibodies were used as 1:100 dilutions from stocks of 200 µg/ml in Cytoperm solution and were detected with Cy3 AffiniPure donkey anti-goat IgG (Jackson Immunoresearch Europe Ltd., Cambridgeshire, UK). Anti-CCR1 and -CCR5 (R&D Systems) were used at 1:50 diluition in Cytoperm solution and were detected with FITC AffiniPure F(ab')2 fragment donkey anti-mouse IgG (Jackson Immunoresearch Europe Ltd.).
4,6-Diamide-2-phenylindol (DAPI; Sigma-Aldrich) at 4 ng/ml was used to stain the nucleus blue. Fluorescence microscopy analysis was performed with a Leica DMRXA microscope (Leica Microsystems Gmbh, Wetzlar, Germany) equipped with a cooled charged-coupled device camera (Model S/N 370 KL 0565, Cooke Corp., Auburn Hills, MI). Filter sets for DAPI/Hoechst, FITC, Cy3, and Cy5 were obtained from Chroma Technology (Brattleboro, VT). The images were acquired and analyzed using the image processing software SlideBook 2.1.5 (Intelligent Imaging Innovations, Denver, CO). Images were postprocessed and mounted in Adobe Photoshop CS (Adobe Systems, San Jose, CA) and printed on a Tektronix Phaser 860DP printer (Xerox, Stamford, CT).
Chemotaxis assay
Chemotaxis assays were performed as described [15
] using transwell cell-culture chambers (24 wells) with gelatin-coated polycarbonate filters (pore size, 8 µm; Costar, Cambridge, MA). DC (105 in 100 µl FCS-free RPMI) were added to the upper wells, and 600 µl FCS-free medium containing 100 ng/ml CCL3, 100 ng/ml CCL5, or 500 ng/ml CCL19 was added to the lower wells. Formylated peptides (final concentration, 10–7 M) served as a control. After 2 h of incubation at 37°C, the filters were washed three times in PBS, fixed in methanol/acetone (1:1) for 5 min at –20°C, and stained with Mayer’s hematoxylin and eosin (Histolab Products, Gothenburg, Sweden). Cells remaining on the upper side of the filters were scraped off with a cotton swab. DC on the lower side of the filters were counted by light microscopy at 40x magnification in 10 randomly selected fields per well. The experiments were performed in triplicate.
Migrated and nonmigrated TB40/E-infected DC were analyzed for the expression of IEA by indirect immunofluorescence assay in separate experiments. After fixation, migrated cells were stained on the filters with an anti-HCMV IEA mAb (clone E13, Argene) followed by a second incubation with FITC-rabbit anti-mouse IgG (Dako). DAPI (Sigma-Aldrich) at 4 ng/ml was used to stain the nucleus blue. Nonmigrated DC were collected from the upper wells of the chambers after migration, cytocentrifuged on glass slides, and fixed with methanol/acetone 1:1 for 5 min. Cytospots were incubated with anti-IEA mAb (Argene) and subsequently with a FITC-conjugated goat anti-mouse antibody (Dako). Evans blue was used as unspecific contrast staining.
Quantification of CCL3, CCL4, and CCL5
Supernatants from HCMV- and mock-infected DC (106 cells/ml) were collected 6, 24, and 72 h after infection, clarified by centrifugation at 1500 rpm for 10 min, and frozen at –80°C until tested. CCL3, CCL4, and CCL5 levels were measured with commercially available kits (R&D Systems).
Neutralization of CCL3, CCL4, and CCL5
DC were mock-infected or infected with HCMV at a MOI of 5 PFU/cell for 3 h at 37°C. The virus was then removed, the cells were washed once with PBS, and fresh medium containing cytokines was added. Goat anti-human CCL3, CCL4, and CCL5 polyclonal neutralizing antibodies were added individually or in combination at concentrations of 15, 20, and 10 µg/ml, respectively. Normal goat IgG (R&D Systems) served as a negative control. After 1 day of incubation, cells were washed with PBS. Migration assay in response to CCL5 (100 ng/ml) was then performed, and surface expression of chemokine receptors was analyzed by fluorescein-activated cell sorter (FACS).
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HCMV strain TB40/E inhibits the migration of immature DC
As mentioned above, HCMV has been shown to affect the DC behavior in different manners. To evaluate the effect of HCMV infection on the DC migratory ability, immature DC were infected with different strains of HCMV, and chemotaxis assays in response to CCL3, CCL5, and CCL19 were performed. Immature DC infected with TB40/E exhibited impaired migration in response to CCL3 and CCL5 1 day after infection (Fig. 1A
). Infection with AD169, Towne, or PO had no effect on the migratory response to these inflammatory chemokines (Fig. 1A
and 1B)
. In addition, immature, mock-infected and HCMV-infected DC responded chemotactically to fMLP. This indicates that the failure of TB40/E-infected DC to migrate in response to inflammatory chemokines did not reflect a general impairment of cell locomotion. Only a few TB40/E-infected and mock-infected, immature DC migrated in response to the lymphoid chemokine CCL19 (Fig. 1A)
. As expected, mature DC did not migrate in response to CCL3, CCL5, or fMLP (data not shown). When DC were infected with TB40/E at different MOI (0.1–5 PFU/cell), migration in response to CCL5 was inhibited in a dose-dependent manner (Fig. 2
). TB40/E-infected, immature DC, which migrated in response to CCL5, were analyzed for the expression of IEA. Interestingly, that only 1–2% of migrated cells were IEA-positive (Fig. 3
). As we detected approximately 10% of infected DC in the cell population, which we used for the migration experiments, 90% of infected DC failed to migrate in response to CCL5.
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Figure 1. Infection with an EC-adapted strain of HCMV, but not with fibroblast-adapted strains or a clinical isolate, impairs DC migration. Immature DC were infected with the fibroblast-adapted strains AD169 or Towne, the EC-adapted strain TB40/E, or the clinical isolate PO. One day after infection, DC were seeded in the upper compartments of transwell chambers, and CCL3, CCL5 (100 ng/ml), CCL19 (500 ng/ml), or fMLP (10–7 M) was added to the lower compartments. Medium without FCS was used as control of background migration. Cells that migrated to the lower compartment were counted in 10 representative fields for each well. (A) Migration of mock-infected (open bars), AD169-infected (shaded bars), and TB40/E-infected (solid bars) DC 2 h after incubation. Values are mean ± SEM of four separate experiments performed in triplicate. (B) Migration of immature DC in response to CCL5 1 day after infection with TB40/E, AD169 (AD), Towne (To), or PO. Mock-infected DC were used as control. Values are mean ± SEM of three separate experiments performed in triplicate.
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Figure 2. HCMV infection of DC inhibits migration in a dose-dependent manner. Immature DC were infected with TB40/E at different MOI (0.1, 1, and 5 PFU/cell). Mock-infected DC were used as control. One day after infection, the cells were placed in the upper compartments of transwell chambers, and migration in response to CCL5 (100 ng/ml) was assessed. Cells that migrated were counted in 10 representative fields for each well. Values are mean ± SEM of three separate experiments performed in triplicate.
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Figure 3. Viral antigen expression in migrated and nonmigrated, TB40/E-infected DC. Immature DC were infected with TB40/E (MOI, 5 PFU/cell), and migration in response to CCL5 was assessed at day 1 after infection. Migrated and nonmigrated DC were analyzed for the expression of IEA by an indirect immunofluorescence assay. (A) After fixation in methanol/acetone (1:1), migrated cells were stained on the filters with an anti-HCMV IEA mAb followed by a second incubation with FITC-rabbit anti-mouse IgG. DAPI was used to stain the nucleus blue. (B) Nonmigrated DC were collected from the upper wells of the chambers after migration, cytocentrifuged on glass slides, and fixed with methanol/acetone (1:1). Cytospots were incubated with an anti-IEA mAb and subsequently, with FITC-rabbit anti-mouse IgG. Evans blue was used as unspecific contrast-staining.
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Figure 4. Early events in viral replication are involved in the inhibition of DC migration. (A) Immature DC were mock-infected or infected with TB40/E (MOI, 5 PFU/cell), and migration in response to CCL5 (100 ng/ml) was assessed at different times after infection. Inhibition of DC migration was calculated as 1 – (infected cells that migrated/mock-infected cells that migrated), expressed as a percentage. Mean values from three separate experiments are shown. (B) Immature DC were mock-infected, infected with TB40/E, incubated with virus-free supernatant (TB40-sup) or with virus inactivated by ultraviolet (TB40-UV), or treated with foscarnet (Foscavir; final concentration, 0.5 mM) 3 h after TB40/E infection, and migration in response to CCL5 (100 ng/ml) was assayed in triplicate 1 day after infection. Values are mean ± SEM of three separate experiments.
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Figure 5. HCMV infection of immature DC down-regulates the surface expression of CCR1 and CCR5 by internalization. (A) Immature (iDC) and mature (mDC) DC were infected with TB40/E (MOI, 5 PFU/cell; solid bars) or were mock-infected (open bars). One day after infection, the cells were stained with mAb against CCR1, CCR2, CCR5, or CCR7, and the surface expression of each receptor was analyzed by FACS. The expression of chemokine receptors was measured as the mean fluorescence intensity (MFI). Values are mean ± SEM of five separate experiments. *, P < 0.001. (B) Immature DC were infected with TB40/E (MOI, 5 PFU/cell) or were mock-infected. Cells were stained with anti-CCR1, anti-CCR5, or an isotype-matched control 1 day after infection. FACS analysis of cell-surface and total (intracellular and surface) expression of CCR1 and CCR5 in mock-infected (solid lines) and TB40/E-infected (shaded histograms) DC. For assessment of total receptor expression, cells were fixed, permeabilized, and stained with mAb against CCR1 and CCR5. Representative results from one of three experiments are shown. Intact DC, Unfixed cells; Perm DC, fixed and permeabilized DC. (C) Confocal microscopy analyses demonstrate internalization of CCR1 in TB40/E-infected DC. Expression of CCR1 (green fluorescence, panels A and D) and the early lysosome marker Lamp 1 (red staining, panels B and E) in mock-infected (I) and TB40/E-infected (II), immature DC at day 1 postinfection. Colocalization (panels C and F) was performed to identify whether inflammatory chemokine receptors were redistributed intracellularly in infected DC. Lamp 1 showed only weak colocalization with CCR1 in mock-infected DC, whereas colocalization for these two markers was demonstrated in TB40/E-infected DC. DAPI was used to stain the nucleus blue.
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HCMV-mediated down-regulation of CCR1 and CCR5 expression is dependent on the presence of infectious virus and on early events of HCMV replication
To determine if infectious virus caused the down-regulation of inflammatory chemokine receptors, we incubated immature DC with virus-free supernatant and with UV-inactivated viral particles. Surface expression of CCR1 and CCR5 was not impaired (Fig. 6A
and 6B
). However, when infected DC were treated with foscarnet 3 h after infection, CCR1 and CCR5 were down-regulated (Fig. 6C) . These findings suggest that infectious virions, viral replication, and immediate-early or early gene expression are required to down-regulate cell-surface CCR1 and CCR5 and to inhibit migration in response to CCR1 and CCR5 ligands.
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Figure 6. Down-regulation of CCR1 and CCR5 in immature, TB40/E-infected DC is related to early replicative events during HCMV infection. (A) To determine if the virus or soluble factors in the supernatant caused down-regulation of CCR1 and CCR5, immature DC were mock-infected (solid lines), infected with TB40/E (MOI, 5 PFU/cell; shaded histograms), or incubated with virus-free supernatant (light blue lines). Cells were stained with anti-CCR1, anti-CCR5, or an isotype-matched control 1 day after infection. CCR1 and CCR5 cell-surface expression was analyzed by FACS. Representative data from one of three experiments are shown. (B) To determine if down-regulation of CCR1 and CCR5 was dependent on viral replication, immature DC were mock-infected (solid lines), infected with TB40/E (MOI, 5 PFU/cell; shaded histograms), or infected with UV-inactivated virus (red lines). Cells were stained with anti-CCR1, anti-CCR5, or an isotype-matched control 1 day after infection. CCR1 and CCR5 cell-surface expression was analyzed by FACS. Representative data from one of three experiments are shown. (C) To determine if early or late viral genes were involved in the down-regulation of CCR1 and CCR5, immature DC were mock-infected (solid lines), infected with TB40/E (MOI, 5 PFU/cell; shaded histograms), or infected with TB40/E and treated with foscarnet (final concentration, 0.5 mM; red dotted lines) 3 h after infection. Cells were stained with anti-CCR1, anti-CCR5, or an isotype-matched control 1 day after infection. Cell-surface expression was analyzed by FACS. Representative data from one of three experiments are shown.
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60%. These observations suggest that HCMV-induced secretion of inflammatory chemokines by DC results in auto-desensitization and internalization of the cognate receptors and inhibits the ability of DC to migrate.
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Figure 7. HCMV infection of DC induces secretion of CCL3, CCL4, and CCL5 early after infection. Immature DC were mock-infected ( ) or infected with TB40/E (MOI, 5 PFU/cell;  ) for 3 h. The virus was removed, and the cells were washed and seeded (106 cells/ml) in fresh medium containing IL-4 and GM-CSF. Supernatants were collected at 6, 24, and 72 h after infection from the same wells, and the concentrations of CCL3 (A), CCL4 (B), and CCL5 (C) were measured by ELISA.
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Figure 8. Neutralization of CCL3, CCL4, and CCL5 produced by infected DC strongly decreases the effect of HCMV on chemokine receptor expression and on DC migration. DC were mock-infected or infected with TB40/E (MOI, 5 PFU/cell) for 3 h, washed, and cultured with or without anti-CCL3, anti-CCL4, and anti-CCL5 neutralizing antibodies or an irrelevant antibody (goat IgG). After 1 day of incubation, DC were collected and washed with PBS. (A) DC were stained with anti-CCR1, anti-CCR5, or an isotype-matched control. Cell-surface expression was analyzed by FACS. Solid lines, mock-infected DC; filled histograms, TB40/E-infected DC; red lines, TB40/E-infected DC treated with anti-CCL3, anti-CCL4, and anti-CCL5. Results of one representative experiment of three are shown. (B) Migration of DC in response to CCL5 (100 ng/ml) 1 day after infection. Values are mean ± SEM of three separate experiments performed in triplicate. Mock, Mock-infected DC; TB40+goat IgG, TB40/E-infected DC treated with an irrelevant antibody; TB40+Ab, TB40/E-infected DC treated with anti-CCL3, anti-CCL4, and anti-CCL5.
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-chemokine homologues [21
, 22
]. The viral chemokine receptor US28, for example, influences the migration of infected cells to host ß chemokines and selectively recognizes CXC chemokine ligand 1 (CX3CL1)/fractalkine [21
, 23
24
25
], and the viral chemokine UL146 mimics the action of CXCL8/IL-8 on neutrophil migration [26
]. Thus, HCMV appears to disrupt normal chemokine signaling and alter leukocyte migration and trafficking behavior. HCMV can also suppress and modulate the host immune response by impairing the function of DC. It has been found that HCMV infection of DC reduced IL-12 secretion in response to maturation stimuli and inhibited their ability to stimulate T cell proliferation and cytotoxicity [9
10
11
]. In addition, data provided by our group showed that in vitro HCMV infection of monocytes blocks the cytokine-induced differentiation into functionally active DC, which also had signs of severely depressed immunological functions [27
]. To fulfill their immunologic function as APC that potently stimulate T cell responses, DC must be able to migrate from the circulation to the sites of inflammation and infection and then to the lymphoid organs. A recent report has shown that HCMV blocks the migration of mature DC toward lymphoid chemokines CCL19 and CCL21 [13 ]. In this study, we demonstrate that HCMV inhibited the migration of immature DC in response to the inflammatory chemokines CCL3 and CCL5. One day after infection with an EC-adapted strain, migration of infected DC in response to inflammatory chemokines was reduced by 95%, and cell-surface expression of CCR1 and CCR5 was markedly down-regulated. Both of these effects were related to early events during HCMV infection and were dependent on viral replication.
There are two major mechanisms for chemokine receptor regulation on the cell surface: altered gene expression and desensitization caused by phosphorylation-dependent internalization of the receptor upon ligand binding [16 , 28 ]. We found no differences in the total levels of CCR1 and CCR5 during the course of infection, and confocal microscopy analyses further showed that these receptors were internalized, which suggests that this phenomenon was responsible for the reduced cell-surface expression of chemokine receptors on HCMV-infected DC.
HCMV infection stimulates chemokine secretion from various cell types [29 30 31 32 33 ]. In this study, we show that HCMV infection induced secretion of CCL3, CCL4, and CCL5 by DC early after infection. We hypothesized that the internalization of chemokine receptors and consequent impairment of DC migration were caused by auto-desensitization of the receptors as a result of secretion of the related ligands. Consistent with this hypothesis, specific neutralizing polyclonal antibodies strongly reduced the down-regulation of chemokine receptors and the inhibition of DC migration. Thus, the secretion of inflammatory chemokines by infected DC alters the expression of the related receptors and influences the migratory properties of DC.
One can argue that CCR1 and CCR5 desensitization, as a result of inflammatory chemokine secretion, is a common signaling step in the DC maturation process. However, the surface expression of the lymphoid chemokine receptor CCR7 was not up-regulated after HCMV infection on immature DC, whereas CCR7 was highly expressed on DC stimulated with LPS (Fig. 5A) . This finding indicates that HCMV does not act as a maturation signal and that the virus affects DC function with kinetics that differ from LPS-induced maturation. In Figure 9 , we propose a model to explain the mechanism by which HCMV affects DC migration.
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Figure 9. Proposed model by which HCMV influences DC migration. HCMV infects immature DC (1), resulting in the secretion of inflammatory chemokines (2), which bind to the cognate receptors CCR1 and CCR5, leading to internalization and reducing surface expression (3). Down-regulation of CCR1 and CCR5 expression is responsible for the impaired migration of DC in response to inflammatory chemokines (4). In addition, HCMV does not affect the surface expression level of CCR7 (3a), and for this reason, DC do not migrate in response to the lymphoid chemokine CCL19 (4a).
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We detected that the neutralization of CCL3, CCL4, and CCL5 activity reduced the effects of HCMV on DC migration by 60%; therefore, other soluble factors might be involved in causing the receptor internalization. In support of this hypothesis, HCMV infection induces the secretion of a variety of cytokines and chemokines from different cell types [29
30
31
32
33
], and CCL5 itself activates a cascade of chemokines in immature DC [34
], which may take part in the receptor desensitization.
The dysregulation of DC migration during HCMV infection may have important clinical implications, as it would interfere with the normal trafficking of these cells during in vivo infections. In our experiments, immature HCMV-infected DC did not express inflammatory chemokine receptors required for homing to inflamed tissues or lymphoid chemokine receptors for homing to lymph nodes. Circulating DC, which display an immature phenotype, may become infected with virus produced by infected EC (as other peripheral leukocytes do), and after the infection, a rapid inability in their migration properties may occur. Infected DC would therefore remain in the circulatory system, and this would prevent them to execute their function to take up antigen in inflamed tissues. Conversely, the virus may infect DC, which reside in an immature stage in peripheral tissues. After infection, DC would not up-regulate CCR7 and would not be able to migrate to lymph nodes, and this would interfere with the generation of T cell responses. Both of these effects may contribute to immunosuppression. As an alternative explanation, up-regulation of chemokine secretion by infected DC may be favorable for HCMV to attract more immature DC and other inflammatory cells to the site of infection to increase viral targets and to promote viral spread. Under such circumstances, CCR1 and CCR5 down-regulation, via homologous desensitization and internalization, may represent a side-effect that would be subordinated to a viral strategy of cell recruitment. Consistent with this hypothesis, MCMV has been shown to encode ß-chemokine homologues, and the expression of these chemokines has been linked to the recruitment of mononuclear cells, which act as vehicles for dissemination during acute infection [35 , 36 ].
Through their ability to generate central and peripheral tolerance, prime immune responses, and stimulate memory and effector T cells, DC may also play a key role in autoimmune disorders [37 38 39 40 ]. Viral infections cause effects that increase susceptibility to autoimmunity, such as the presence of a high amount of apoptotic cells available for the capture by DC and the possibility of viral peptides to mimic self-peptides leading to priming of cross-reactive self-antigens [41 ]. Factors that perpetuate autoreactive responses may also be involved. In support of this hypothesis, DC infiltration at effector sites was associated with infiltration of T and B cells in several autoimmune disorders, and the number of infiltrated DC in patients with rheumatoid arthritis was related to clinical disease activity [41 , 42 ]. The down-regulation of inflammatory chemokine receptors and the lack of up-regulation of lymphoid chemokine receptors, which we observed in HCMV-infected DC, could also contribute to an accumulation of these cells in infected tissues that could generate or perpetuate autoimmune events in susceptible individuals. Interestingly, that HCMV DNA has been detected in sinovial fluids from patients with rheumatoid arthritis [43 ], and HCMV-infected cells have been observed in inflamed tissues obtained from patients with inflammatory bowel diseases [44 ]. Furthermore, HCMV infection has been associated with various autoimmune manifestations, including hemolytic anemia, cytopenia, and the development of autoantibodies, such as rheumatoid factor and anti-smooth muscle, anti-EC, anti-nuclear, and anti-CD13 antibodies [45 46 47 48 49 ]. Hence, the interference of the virus with the migratory ability of DC may alter homeostatic DC trafficking and contribute to immune dysfunction and immune abnormalities during HCMV infection.
Received May 21, 2004; revised September 4, 2004; accepted October 6, 2004.
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 G. Frascaroli, S. Varani, N. Blankenhorn, R. Pretsch, M. Bacher, L. Leng, R. Bucala, M. P. Landini, and T. Mertens Human Cytomegalovirus Paralyzes Macrophage Motility through Down-Regulation of Chemokine Receptors, Reorganization of the Cytoskeleton, and Release of Macrophage Migration Inhibitory Factor J. Immunol., January 1, 2009; 182(1): 477 - 488. [Abstract] [Full Text] [PDF]
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M. Baron and J.-L. Davignon Inhibition of IFN-{gamma}-Induced STAT1 Tyrosine Phosphorylation by Human CMV Is Mediated by SHP2 J. Immunol., October 15, 2008; 181(8): 5530 - 5536. [Abstract] [Full Text] [PDF]
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J. L. Stern and B. Slobedman Human Cytomegalovirus Latent Infection of Myeloid Cells Directs Monocyte Migration by Up-Regulating Monocyte Chemotactic Protein-1 J. Immunol., May 15, 2008; 180(10): 6577 - 6585. [Abstract] [Full Text] [PDF]
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 C. S. Wagner, L. Walther-Jallow, E. Buentke, H.-G. Ljunggren, A. Achour, and B. J. Chambers Human cytomegalovirus-derived protein UL18 alters the phenotype and function of monocyte-derived dendritic cells J. Leukoc. Biol., January 1, 2008; 83(1): 56 - 63. [Abstract] [Full Text] [PDF]
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S. Varani, M. Cederarv, S. Feld, C. Tammik, G. Frascaroli, M. P. Landini, and C. Soderberg-Naucler Human Cytomegalovirus Differentially Controls B Cell and T Cell Responses through Effects on Plasmacytoid Dendritic Cells J. Immunol., December 1, 2007; 179(11): 7767 - 7776. [Abstract] [Full Text] [PDF]
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 V. Mayer, K. L. Hudkins, F. Heller, H. Schmid, M. Kretzler, U. Brandt, H.-J. Anders, H. Regele, P. J. Nelson, C. E. Alpers, et al. Expression of the chemokine receptor CCR1 in human renal allografts Nephrol. Dial. Transplant., June 1, 2007; 22(6): 1720 - 1729. [Abstract] [Full Text] [PDF]
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P. Sun, C. M. Celluzzi, M. Marovich, H. Subramanian, M. Eller, S. Widjaja, D. Palmer, K. Porter, W. Sun, and T. Burgess CD40 Ligand Enhances Dengue Viral Infection of Dendritic Cells: A Possible Mechanism for T Cell-Mediated Immunopathology J. Immunol., November 1, 2006; 177(9): 6497 - 6503. [Abstract] [Full Text] [PDF]
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C. A. King, J. Baillie, and J. H. Sinclair Human cytomegalovirus modulation of CCR5 expression on myeloid cells affects susceptibility to human immunodeficiency virus type 1 infection. J. Gen. Virol., August 1, 2006; 87(Pt 8): 2171 - 2180. [Abstract] [Full Text] [PDF]
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 J. Loeffler, M. Steffens, E.-M. Arlt, M.-R. Toliat, M. Mezger, A. Suk, T. F. Wienker, H. Hebart, P. Nurnberg, M. Boeckh, et al. Polymorphisms in the genes encoding chemokine receptor 5, interleukin-10, and monocyte chemoattractant protein 1 contribute to cytomegalovirus reactivation and disease after allogeneic stem cell transplantation. J. Clin. Microbiol., May 1, 2006; 44(5): 1847 - 1850. [Abstract] [Full Text] [PDF]
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G. Frascaroli, S. Varani, A. Mastroianni, S. Britton, D. Gibellini, G. Rossini, M. P. Landini, and C. Soderberg-Naucler Dendritic cell function in cytomegalovirus-infected patients with mononucleosis J. Leukoc. Biol., May 1, 2006; 79(5): 932 - 940. [Abstract] [Full Text] [PDF]
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