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Originally published online as doi:10.1189/jlb.0504301 on November 2, 2004

Published online before print November 2, 2004
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(Journal of Leukocyte Biology. 2005;77:219-228.)
© 2005 by Society for Leukocyte Biology

Human cytomegalovirus inhibits the migration of immature dendritic cells by down-regulating cell-surface CCR1 and CCR5

Stefania Varani*,{dagger}, Giada Frascaroli{dagger},{ddagger}, Mohammed Homman-Loudiyi*, Sari Feld*, Maria Paola Landini{dagger} and Cecilia Söderberg-Nauclér*,1

* Department of Medicine, Karolinska Systems Biomedicine Center, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden;
{dagger} Department of Clinical and Experimental Medicine, University of Bologna, Italy; and
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) play a key role in the host immune response to infections. Human cytomegalovirus (HCMV) infection can inhibit the maturation of DC and impair their ability to stimulate T cell proliferation and cytotoxicity. In this study, we assessed the effects of HCMV infection on the migratory behavior of human DC. The HCMV strain TB40/E inhibited the migration of immature monocyte-derived DC in response to inflammatory chemokines by 95% 1 day after infection. This inhibition was mediated by early viral replicative events, which significantly reduced the cell-surface expression of CC chemokine receptor 1 (CCR1) and CCR5 by receptor internalization. HCMV infection also induced secretion of the inflammatory chemokines CC chemokine ligand 3 (CCL3)/macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are potent antigen-presenting cells (APC), which initiate a cellular immune response by stimulating naive T cells [1 ]. Through their release of specific cytokines and chemokines, activated DC also influence the type of T cell response and participate in the recruitment and activation of other effector cells, including natural killer (NK) cells, macrophages, and B cells [2 , 3 ]. DC therefore play a key role in generating and modulating the host immune response to viral infections, and their migration from the site of antigen capture to secondary lymphoid organs is a crucial event in the early host response to inflammation and infection.

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{alpha} (MIP-1{alpha}), 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 {alpha}, and interferon-{gamma} 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media and reagents
Human fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 10% fetal calf serum (FCS; Gibco-BRL, Grand Island, NY). DC were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% pyruvate, 100 U/ml penicillin, 100 U/ml streptomycin, 10% FCS, and 5 x 10–5 M 2-mercaptoethanol (Merck, Darmstadt, Germany). Human recombinant IL-4, granulocyte macrophage-colony stimulating factor (GM-CSF), CCL3, CCL5, and goat polyclonal neutralizing anti-human CCL3, anti-human CCL4, and anti-human CCL5 were purchased from R&D Systems (Minneapolis, MN). CCL19 was from Peprotech (London, UK), LPS and formylated peptides [formyl-Met-Leu-Phe (fMLP)] were from Sigma-Aldrich (St. Louis, MO), and foscarnet (Foscavir) was from Astra (Södertälje, Sweden).

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).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Infection efficiency
In agreement with published data [8 ], the EC-adapted HCMV strain TB40/E infected immature DC more efficiently than the fibroblast-adapted strains AD169 or Towne or the clinical isolate PO (data not shown). At a MOI of 5 PFU/cell, TB40/E infected 10–15% of immature DC, as detected by HCMV IEA expression 1 day after infection. At the same MOI, less than 1% of cells was infected with AD169, Towne, or PO. Only immature DC infected with TB40/E supported the complete viral replication cycle, as demonstrated by the expression of a late viral antigen (pp150) on day 5. The maximum infection rate of mature DC was 1%, regardless of strain used.

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.

 
Immediate-early or early viral genes are involved in the impairment of DC migration
To evaluate the kinetics of the HCMV-induced inhibition of DC migration, we assessed the migratory response to CCL5 at different times after infection. The migration of infected cells was not affected at 3 h but was reduced by 65% at 6 h and by 95% at 24 h compared with uninfected cells (Fig. 4A ). When immature DC were infected with virus-free supernatant, obtained by filtration of the viral stock through 0.1 µm pore filters or with viral particles treated with UV to inhibit replication, the chemotactic response to CCL5 was not impaired (Fig. 4B) , indicating that intact viral particles and viral replication are required to inhibit migration. When immature DC were treated with foscarnet at 3 h after infection to block the replication of late viral genes, the inhibition of DC migration in response to CCL5 was maintained (Fig. 4B) , suggesting that late viral genes were not responsible for the impaired chemotaxis.



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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.

 
HCMV reduces surface expression of CCR1 and CCR5 by internalization
Migration of DC toward inflammatory chemokines requires the expression of CCR1, CCR2, and CCR5. As we found that HCMV inhibited DC migration in response to CCL3 and CCL5, we assessed the effects of TB40/E infection on cell-surface chemokine receptor expression by immature DC 1 day after infection. As shown by FACS analysis, TB40/E consistently down-regulated the expression of the inflammatory chemokine receptors CCR1 and CCR5 but did not affect the expression of CCR2 (inflammatory receptor) or CCR7 (lymphoid receptor; Fig. 5A ). To further evaluate CCR7 expression on infected versus uninfected, immature DC within the TB40/E-infected population, double-immunofluorescence analysis of CCR7 and a viral antigen was performed, allowing the visualization of surface CCR7 and nuclear IEA on a single-cell level. Using this technique, we found that IEA-positive DC show the same low levels of CCR7 as uninfected DC (data not shown). In flow cytometry experiments with mature DC, LPS stimulation markedly reduced inflammatory chemokine receptor expression and increased CCR7 expression; there were no differences between infected and mock-infected cells, reflecting the resistance of mature DC to HCMV infection (Fig. 5A) . Analysis of total and cell-surface expression of CCR1 and CCR5 by immature DC 1 day after infection showed reduced surface expression and no change in total expression (Fig. 5B) . This finding suggests that receptors are redistributed from the cell surface to an intracellular compartment during HCMV infection.



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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.

 
To confirm the internalization of the chemokine receptors in infected DC, we performed confocal microscopy. In mock-infected DC, CCR1 and CCR5 were mainly expressed on the cell membrane, whereas in TB40/E-infected DC, the inflammatory chemokine receptors were mostly detected intracellularly (Fig. 5C , panels A and D, and data not shown). Colocalization experiments were carried out with antibodies directed against CCR1 and CCR5 as well as markers for the following specific subcellular compartments: Rab 4 (early endosomes), Rab 7 (late endosomes), Lamp 1 (early lysosomes), and Lamp 2 (lysosomes). In uninfected DC, CCR1 and CCR5 rarely colocalized with endosome and lysosome markers, whereas in TB40/E-infected DC, the inflammatory chemokine receptors colocalized with the intracellular markers Rab 4, Rab 7, Lamp 1, and Lamp 2 (Fig. 5C , panels C and F, and data not shown), indicating that CCR1 and CCR5 may enter a degradation pathway in infected cells.

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.

 
Neutralization of CCL3, CCL4, and CCL5 secreted by infected DC strongly reduces down-regulation of chemokine receptors and inhibition of DC migration
Chemokine secretion may be responsible for auto-desensitization and internalization of cognate receptors [16 ]. Therefore, we determined whether the down-regulation of CCR1 and CCR5 was mediated by secretion of CCL3, CCL4, and CCL5. As shown by enzyme-linked immunosorbent assay (ELISA), TB40/E-infected DC began secreting CCL3, CCL4, and CCL5 by 6 h after infection (Fig. 7 ). We then blocked the excess of inflammatory chemokines in mock-infected and infected DC cultures by treating cells with neutralizing anti-CCL3, anti-CCL4, and anti-CCL5 antibody, and we evaluated the chemokine receptor expression and cell migration in response to CCL5 at 24 h postinfection. Individually, neither of these antibodies nor the irrelevant antibody had any effect (data not shown). However, in combination, the three neutralizing antibodies strongly reduced the effect of HCMV on chemokine receptor expression (Fig. 8A ). In addition, we found that the cocktail of neutralizing antibody decreased the effect of the virus on DC migration (Fig. 8B) by ~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 ({circ}) or infected with TB40/E (MOI, 5 PFU/cell; {blacksquare}) 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As a result of HCMV infection, patients frequently suffer from dysfunction of the immune system. CMV infection can cause immunosuppression by inducing leukopenia, depressing CD4+ lymphocyte responses to T cell mitogens, and decreasing cytotoxic and NK activities [17 18 19 20 ]. The HCMV genome contains homologues of cellular genes that may interfere with the host immune response, including an HLA class I, an Fc receptor, four chemokine receptors, and two {alpha}-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).

 
A recent study has reported a lack of expression of CCR7 in HCMV-infected DC upon LPS stimulation, whereas bystander, HCMV-negative DC in the same cell culture up-regulated CCR7 following LPS treatment. The result was an overall lower CCR7 expression in HCMV-infected DC cultures than in mock-infected cell populations [13 ]. On the contrary, we did not detect any difference between mock-infected and HCMV-infected DC in terms of CCR7 expression, and we observed that mock-infected and HCMV-infected, mature DC up-regulated CCR7. A different maturation stage of the cells at the time of infection may be responsible for these divergent findings. Moutaftsi et al. [13 ] did stimulate maturation of cells that were already infected with TB40/E, whereas we explored CCR7 expression on DC, which were first stimulated with LPS and then infected with HCMV. It has been demonstrated previously [12 ], and also, we report here that mature DC are resistant to CMV infection, which is the most likely reason why the virus could not exert its effect on CCR7 expression in our experiments.

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.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Swedish Research Council (K2004-16X-12615-07A), the Swedish Society of Medicine (2003-633), the Tobias Foundation (33/02 and 28/03), the Swedish Children Cancer Research Foundation (PROJ01/046), the Emil and Wera Cornells Foundation, and the Heart and Lung Foundation (20010486, 20030830). We thank G. Jahn for providing the endothelial-adapted HCMV strain TB40/E.

Received May 21, 2004; revised September 4, 2004; accepted October 6, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., Palucka, K. (2000) Immunobiology of dendritic cells Annu. Rev. Immunol. 18,767-811[CrossRef][Medline]
  2. Fernandez, N. C., Lozier, A., Flament, C., Ricciardi-Castagnoli, P., Bellet, D., Suter, M., Perricaudet, M., Tursz, T., Maraskovsky, E., Zitvogel, L. (1999) Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo Nat. Med. 5,405-411[CrossRef][Medline]
  3. Rescigno, M., Granucci, F., Ricciardi-Castagnoli, P. (1999) Dendritic cells at the end of the millennium Immunol. Cell Biol. 77,404-410[CrossRef][Medline]
  4. Sozzani, S., Allavena, P., Vecchi, A., Mantovani, A. (1999) The role of chemokines in the regulation of dendritic cell trafficking J. Leukoc. Biol. 66,1-9[Abstract]
  5. Sallusto, F., Schaerli, P., Loetscher, P., Schaniel, C., Lenig, D., Mackay, C. R., Qin, S., Lanzavecchia, A. (1998) Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation Eur. J. Immunol. 28,2760-2769[CrossRef][Medline]
  6. Lin, C. L., Suri, R. M., Rahdon, R. A., Austyn, J. M., Roake, J. A. (1998) Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation Eur. J. Immunol. 28,4114-4122[CrossRef][Medline]
  7. Sozzani, S., Allavena, P., D’Amico, G., Luini, W., Bianchi, G., Kataura, M., Imai, T., Yoshie, O., Bonecchi, R., Mantovani, A. (1998) Differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties J. Immunol. 161,1083-1086[Abstract/Free Full Text]
  8. Riegler, S., Hebart, H., Einsele, H., Brossart, P., Jahn, G., Sinzger, C. (2000) Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus J. Gen. Virol. 81,393-399[Abstract/Free Full Text]
  9. Moutaftsi, M., Mehl, A. M., Borysiewicz, L. K., Tabi, Z. (2002) Human cytomegalovirus inhibits maturation and impairs function of monocyte-derived dendritic cells Blood 99,2913-2921[Abstract/Free Full Text]
  10. Beck, K., Meyer-Konig, U., Weidmann, M., Nern, C., Hufert, F. T. (2003) Human cytomegalovirus impairs dendritic cell function: a novel mechanism of human cytomegalovirus immune escape Eur. J. Immunol. 33,1528-1538[CrossRef][Medline]
  11. Grigoleit, U., Riegler, S., Einsele, H., Laib Sampaio, K., Jahn, G., Hebart, H., Brossart, P., Frank, F., Sinzger, C. (2002) Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocyte-derived immature dendritic cells Br. J. Haematol. 119,189-198[CrossRef][Medline]
  12. Andrews, D. M., Andoniou, C. E., Granucci, F., Ricciardi-Castagnoli, P., Degli-Esposti, M. A. (2001) Infection of dendritic cells by murine cytomegalovirus induces functional paralysis Nat. Immunol. 2,1077-1084[CrossRef][Medline]
  13. Moutaftsi, M., Brennan, P., Spector, S. A., Tabi, Z. (2004) Impaired lymphoid chemokine-mediated migration due to a block on the chemokine receptor switch in human cytomegalovirus-infected dendritic cells J. Virol. 78,3046-3054[Abstract/Free Full Text]
  14. Sallusto, F., Lanzavecchia, A. (1994) Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor {alpha} J. Exp. Med. 179,1109-1118[Abstract/Free Full Text]
  15. Sato, K., Kawasaki, H., Nagayama, H., Serizawa, R., Ikeda, J., Morimoto, C., Yasunaga, K., Yamaji, N., Tadokoro, K., Juji, T., Takahashi, T. A. (1999) CC chemokine receptors, CCR-1 and CCR-3, are potentially involved in antigen-presenting cell function of human peripheral blood monocyte-derived dendritic cells Blood 93,34-42[Abstract/Free Full Text]
  16. Sallusto, F., Palermo, B., Lenig, D., Miettinen, M., Matikainen, S., Julkunen, I., Forster, R., Burgstahler, R., Lipp, M., Lanzavecchia, A. (1999) Distinct patterns and kinetics of chemokine production regulate dendritic cell function Eur. J. Immunol. 29,1617-1625[CrossRef][Medline]
  17. Farrell, H. E., Degli-Esposti, M. A., Davis-Poynter, N. J. (1999) Cytomegalovirus evasion of natural killer cell responses Immunol. Rev. 168,187-197[CrossRef][Medline]
  18. Odeberg, J., Cerboni, C., Browne, H., Karre, K., Moller, E., Carbone, E., Soderberg-Naucler, C. (2002) Human cytomegalovirus (HCMV)-infected endothelial cells and macrophages are less susceptible to natural killer lysis independent of the downregulation of classical HLA class I molecules or expression of the HCMV class I homologue, UL18 Scand. J. Immunol. 55,149-161[CrossRef][Medline]
  19. Schrier, R. D., Rice, G. P., Oldstone, M. B. (1986) Suppression of natural killer cell activity and T cell proliferation by fresh isolates of human cytomegalovirus J. Infect. Dis. 153,1084-1091[Medline]
  20. Timon, M., Arnaiz-Villena, A., Ruiz-Contreras, J., Ramos-Amador, J. T., Pacheco, A., Regueiro, J. R. (1993) Selective impairment of T lymphocyte activation through the T cell receptor/CD3 complex after cytomegalovirus infection Clin. Exp. Immunol. 94,38-42[Medline]
  21. Loenen, W. A., Bruggeman, C. A., Wiertz, E. J. (2001) Immune evasion by human cytomegalovirus: lessons in immunology and cell biology Semin. Immunol. 13,41-49[CrossRef][Medline]
  22. Michelson, S. (2004) Consequences of human cytomegalovirus mimicry Hum. Immunol. 65,465-475[CrossRef][Medline]
  23. Streblow, D. N., Soderberg-Naucler, C., Vieira, J., Smith, P., Wakabayashi, E., Ruchti, F., Mattison, K., Altschuler, Y., Nelson, J. A. (1999) The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration Cell 99,511-520[CrossRef][Medline]
  24. Vieira, J., Schall, T. J., Corey, L., Geballe, A. P. (1998) Functional analysis of the human cytomegalovirus US28 gene by insertion mutagenesis with the green fluorescent protein gene J. Virol. 72,8158-8165[Abstract/Free Full Text]
  25. Kledal, T. N., Rosenkilde, M. M., Schwartz, T. W. (1998) Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28 FEBS Lett. 441,209-214[CrossRef][Medline]
  26. Penfold, M. E., Dairaghi, D. J., Duke, G. M., Saederup, N., Mocarski, E. S., Kemble, G. W., Schall, T. J. (1999) Cytomegalovirus encodes a potent {alpha} chemokine Proc. Natl. Acad. Sci. USA 96,9839-9844[Abstract/Free Full Text]
  27. Gredmark, S., Soderberg-Naucler, C. (2003) Human cytomegalovirus inhibits differentiation of monocytes into dendritic cells with the consequence of depressed immunological functions J. Virol. 77,10943-10956[Abstract/Free Full Text]
  28. McColl, S. R. (2002) Chemokines and dendritic cells: a crucial alliance Immunol. Cell Biol. 80,489-496[CrossRef][Medline]
  29. Lagneaux, L., Delforge, A., Snoeck, R., Bosmans, E., Schols, D., De Clereq, E., Stryckmans, P., Bron, D. (1996) Imbalance in production of cytokines by bone marrow stromal cells following cytomegalovirus infection J. Infect. Dis. 174,913-919[Medline]
  30. Michelson, S., Dal Monte, P., Zipeto, D., Bodaghi, B., Laurent, L., Oberlin, E., Arenzana-Seisdedos, F., Virelizier, J. L., Landini, M. P. (1997) Modulation of RANTES production by human cytomegalovirus infection of fibroblasts J. Virol. 71,6495-6500[Abstract]
  31. Craigen, J. L., Yong, K. L., Jordan, N. J., MacCormac, L. P., Westwick, J., Akbar, A. N., Grundy, J. E. (1997) Human cytomegalovirus infection up-regulates interleukin-8 gene expression and stimulates neutrophil transendothelial migration Immunology 92,138-145[CrossRef][Medline]
  32. Cheeran, M. C., Hu, S., Yager, S. L., Gekker, G., Peterson, P. K., Lokensgard, J. R. (2001) Cytomegalovirus induces cytokine and chemokine production differentially in microglia and astrocytes: antiviral implications J. Neurovirol. 7,135-147[CrossRef][Medline]
  33. Redman, T. K., Britt, W. J., Wilcox, C. M., Graham, M. F., Smith, P. D. (2002) Human cytomegalovirus enhances chemokine production by lipopolysaccharide-stimulated lamina propria macrophages J. Infect. Dis. 185,584-590[CrossRef][Medline]
  34. Fischer, F. R., Luo, Y., Luo, M., Santambrogio, L., Dorf, M. E. (2001) RANTES-induced chemokine cascade in dendritic cells J. Immunol. 167,1637-1643[Abstract/Free Full Text]
  35. Saederup, N., Lin, Y. C., Dairaghi, D. J., Schall, T. J., Mocarski, E. S. (1999) Cytomegalovirus-encoded ß chemokine promotes monocyte-associated viremia in the host Proc. Natl. Acad. Sci. USA 96,10881-10886[Abstract/Free Full Text]
  36. Saederup, N., Aguirre, S. A., Sparer, T. E., Bouley, D. M., Mocarski, E. S. (2001) Murine cytomegalovirus CC chemokine homolog MCK-2 (m131–129) is a determinant of dissemination that increases inflammation at initial sites of infection J. Virol. 75,9966-9976[Abstract/Free Full Text]
  37. Ludewig, B., Junt, T., Hengartner, H., Zinkernagel, R. M. (2001) Dendritic cells in autoimmune diseases Curr. Opin. Immunol. 13,657-662[CrossRef][Medline]
  38. Legge, K. L., Gregg, R. K., Maldonado-Lopez, R., Li, L., Caprio, J. C., Moser, M., Zaghouani, H. (2002) On the role of dendritic cells in peripheral T cell tolerance and modulation of autoimmunity J. Exp. Med. 196,217-227[Abstract/Free Full Text]
  39. Cravens, P. D., Lipsky, P. E. (2002) Dendritic cells, chemokine receptors and autoimmune inflammatory diseases Immunol. Cell Biol. 80,497-505[CrossRef][Medline]
  40. Turley, S. J. (2002) Dendritic cells: inciting and inhibiting autoimmunity Curr. Opin. Immunol. 14,765-770[CrossRef][Medline]
  41. Thomas, R. (2001) Dendritic cells and autoimmunity Lotze, M. Thomson, A. eds. Dendritic Cells ,459-471 Academic San Diego, CA.
  42. Pettit, A. R., Ahern, M. J., Zehntner, S., Smith, M. D., Thomas, R. (2001) Comparison of differentiated dendritic cell infiltration of autoimmune and osteoarthritis synovial tissue Arthritis Rheum. 44,105-110[CrossRef][Medline]
  43. Murayama, T., Jisaki, F., Ayata, M., Sakamuro, D., Hironaka, T., Hirai, K., Tsuchiya, N., Ito, K., Furukawa, T. (1992) Cytomegalovirus genomes demonstrated by polymerase chain reaction in synovial fluid from rheumatoid arthritis patients Clin. Exp. Rheumatol. 10,161-164[Medline]
  44. Rahbar, A., Bostrom, L., Lagerstedt, U., Magnusson, I., Soderberg-Naucler, C., Sundqvist, V. A. (2003) Evidence of active cytomegalovirus infection and increased production of IL-6 in tissue specimens obtained from patients with inflammatory bowel diseases Inflamm. Bowel Dis. 9,154-161[CrossRef][Medline]
  45. Berlin, B. S., Chandler, R., Green, D. (1977) Anti-"i" antibody and hemolytic anemia associated with spontaneous cytomegalovirus mononucleosis Am. J. Clin. Pathol. 67,459-461[Medline]
  46. Pass, R. (2001) Cytomegalovirus Knipe, D. Howley, P. eds. Fields Virology ,2675-2705 Lippincott Williams and Wilkins Philadelphia, PA.
  47. Soderberg, C., Sumitran-Karuppan, S., Ljungman, P., Moller, E. (1996) CD13-specific autoimmunity in cytomegalovirus-infected immunocompromised patients Transplantation 61,594-600[CrossRef][Medline]
  48. Toyoda, M., Galfayan, K., Galera, O. A., Petrosian, A., Czer, L. S., Jordan, S. C. (1997) Cytomegalovirus infection induces anti-endothelial cell antibodies in cardiac and renal allograft recipients Transpl. Immunol. 5,104-111[CrossRef][Medline]
  49. Varani, S., Muratori, L., De Ruvo, N., Vivarelli, M., Lazzarotto, T., Gabrielli, L., Bianchi, F. B., Bellusci, R., Landini, M. P. (2002) Autoantibody appearance in cytomegalovirus-infected liver transplant recipients: correlation with antigenemia J. Med. Virol. 66,56-62[CrossRef][Medline]



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