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FactorRelease of Macrophage Migration InhibitoryReorganization of the Cytoskeleton, and Down-Regulation of Chemokine Receptors,Macrophage Motility through Human Cytomegalovirus Paralyzes

Paola Landini and Thomas MertensPretsch, Michael Bacher, Lin Leng, Richard Bucala, Maria Giada Frascaroli, Stefania Varani, Nina Blankenhorn, Robert

http://www.jimmunol.org/content/182/1/477doi: 10.4049/jimmunol.182.1.477

2009; 182:477-488; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/182/1/477.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Human Cytomegalovirus Paralyzes Macrophage Motilitythrough Down-Regulation of Chemokine Receptors,Reorganization of the Cytoskeleton, and Release ofMacrophage Migration Inhibitory Factor1

Giada Frascaroli,* Stefania Varani,† Nina Blankenhorn,* Robert Pretsch,* Michael Bacher,‡

Lin Leng,§ Richard Bucala,§ Maria Paola Landini,† and Thomas Mertens2*

Macrophages contribute to host defense and to the maintenance of immune homeostasis. Conversely, they are important targetsof human cytomegalovirus (HCMV), a herpesvirus that has evolved many strategies to modulate the host immune response.Because an efficient macrophage trafficking is required for triggering an adequate immune response, we investigated the effectsexerted by HCMV infection on macrophage migratory properties. By using endotheliotropic strains of HCMV, we obtained highrates of productively infected human monocyte-derived macrophages (MDM). Twenty-four hours after infection, MDM showedreduced polar morphology and became unable to migrate in response to inflammatory and lymphoid chemokines, bacterialproducts and growth factors, despite being viable and metabolically active. Although chemotactic receptors were only partiallyaffected, HCMV induced a dramatic reorganization of the cytoskeleton characterized by rupture of the microtubular network,stiffness of the actin fibers, and collapse of the podosomes. Furthermore, supernatants harvested from infected MDM containedhigh amounts of macrophage migration inhibitory factor (MIF) and were capable to block the migration of neighboring uninfectedMDM. Because immunodepletion of MIF from the conditioned medium completely restored MDM chemotaxis, we could show forthe first time a functional role of MIF as an inhibitor of macrophage migration in the context of HCMV infection. Our findingsreveal that HCMV uses different mechanisms to interfere with movement and positioning of macrophages, possibly leading to animpairment of antiviral responses and to an enhancement of the local inflammation. The Journal of Immunology, 2009, 182:477–488.

M acrophages with their numerous biologic functionshave long been recognized as key cells in the mam-malian host defense. Due to their capacity to phago-

cyte foreign materials and dying cells, to digest bacteria and vi-ruses, to present Ags, and to secrete inflammatory mediators, thesecells are essential for the reactivity of the immune system (1). Incontrast, macrophages have been implicated in the onset and pro-gression of various chronic diseases, such as certain forms of can-cers and vascular and autoimmune diseases (2). Interestingly, hu-man cytomegalovirus (HCMV),3 a widespread herpesvirus that has

evolved multiple immune evasion strategies to persist and replicateeven in a fully immunocompetent host (3), infects both macro-phages and their precursor monocytes. Circulating monocytes andtissue macrophages are believed to be the predominant cell typesharboring HCMV in the peripheral blood (4) and in the infectedorgans (5), respectively. Additionally, monocytes and macro-phages significantly contribute to HCMV pathogenesis serving asvehicles for viral dissemination and as reservoirs for persistentviral infection.

Though it is not known whether HCMV plays a causative role,active HCMV infection has been observed in certain solid tumors(6), in atherosclerotic plaques (7), and in autoimmune diseases(8–10). Intriguingly, these pathologic conditions are often associ-ated with an accumulation of infiltrating activated macrophages,which represent at the same time a major regulator of local in-flammation and a target of HCMV infection.

The composition and size of macrophage infiltrates is deter-mined by the balance of migration into tissues, retention, andegress. The movements of macrophages are directed by gradientsof chemotactic factors that bind to G protein-coupled receptorswhich expression on the cell surface highly regulated to assure aprecise tuning of the cellular flux (11). As a result of the engage-ment of these receptors, macrophages are stimulated to rearrangetheir cytoskeleton, to polarize, and consequently to migrate (12).One mechanism for retaining cells in a tissue is to generate “stop”signals that arrest cell migration and position cells to carry outeffector functions. A fine regulation of the macrophage traffickingis crucial to avoid chronic inflammation, impaired tissue healingand recurrent infection (13). Because it has become clear that

*Institute for Virology, University of Ulm, Ulm, Germany; †Department of Hema-tology and Oncology “L. and A. Seragnoli”, University of Bologna, Bologna, Italy;‡Department of Neurology, Philipps-University of Marburg, Marburg, Germany; and§Section of Rheumatology, Department of Internal Medicine, Yale University Schoolof Medicine, New Haven, CT 06520

Received for publication April 17, 2007. Accepted for publication October 28, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by Project SFB 451-A2 from the Deutsche Forschungs-gemeinschaft (to T.M.), by AIDS Project from the Ministry of Public Health, by BasicTarget Research, ex Fundus 60% from the University of Bologna (to M.P.L. andS.V.), and by the National Institutes of Health (to R.B. and L.L.).2 Address correspondence and reprint requests to Dr. Thomas Mertens, Institute forVirology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.E-mail address: thomas.mertens@uniklinik-ulm.de3 Abbreviations used in this paper: HCMV, human cytomegalovirus; MDM, mono-cyte-derived macrophage; MOI, multiplicity of infection; p.i., postinfection; MIF, mac-rophage migration inhibitory factor; hrMIF, human recombinant MIF; MFI, mean fluo-rescence intensity; PI, propidium iodide; VEGF, vascular endothelial growth factor.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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HCMV has evolved specific strategies to interfere with the orderedmovements of other myeloid cell types, such as dendritic cells andmonocytes (14–16), we decided to investigate whether HCMVinfection could disturb macrophage motility.

In this study, we demonstrate that HCMV infection causes astriking and almost complete paralysis of cell migration associatedwith a complete reorganization of the cytoskeleton in macrophagesthat remain viable and metabolically active. HCMV-infected mac-rophages secrete macrophage migration inhibitory factor (MIF), apowerful proinflammatory factor thus suggesting that HCMV canpromote chronic inflammation in the area of infection.

It is likely that the macrophage unresponsiveness to chemoat-tractants together with the “stop” signal provided by MIF deter-mine the described accumulation of macrophage-like cells in thosetissues where HCMV replicates. The biologic consequences ofsuch inhibition in relation to HCMV pathology in chronic inflam-matory diseases still remain to be defined.

Materials and MethodsEstablishment of monocyte-derived macrophage (MDM) cultures

PBMC were isolated from buffy coats of HCMV-seronegative blood do-nors (Institut fur Klinische Transfusionsmedizin und Immungenetik UlmGmbH, Ulm, Germany) by centrifugation over Ficoll-Paque. Monocyteswere isolated by negative selection with magnetic microbeads (MonocyteIsolation Kit II; Miltenyi Biotec) according to the manufacturer’s instruc-tions and their purity was �95% as assessed by CD14 detection by flowcytometry. A total of 3 � 106 monocytes/ml were then cultured for 7 daysin RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, antibiotics,

and 100 ng/ml M-CSF (R&D Systems) on hydrophobic lumox dishes(Greiner Bio-one) as described (17). The differentiation of MDM occurredover a period of 7 days and was evaluated by morphological criteria,phagocytosis, and flow cytometric analysis (see Table I).

Assessment of apoptosis, necrosis, and metabolic activity

Flow cytometric evaluation of Annexin V/propidium iodide (PI) stainingwas used to determine the percentage of viable (Annexin�/PI�), apoptotic(Annexin�/PI�), and necrotic (PI�) cells. At the indicated time point, 1 �105 MDM were resuspended in 100 �l of Annexin V binding buffer (10mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) and stained with 5�l of Annexin V-FITC (Caltag Laboratories) and 5 �g/ml PI (Sigma-Al-drich). Cellular metabolic activity was quantified by the MTS metaboliza-tion tetrazolium salt assay (18) using the CellTiter 96 Aqueous One So-lution (Promega) according to the manufacturer’s instruction.

Preparation of viral stocks and infection of MDM cultures

Different strains of HCMV were used for the infection of macrophages: thefibroblast adapted strain AD169 and the endotheliotropic strains TB40Eand VHLE provided by Dr. C. Sinzger (University of Tubingen, Tubingen,Germany) and Dr. J Waldman (Ohio State University, Columbus, OH),respectively. Mycoplasma-negative cell-free viral stocks were preparedfrom supernatants of infected fibroblasts, frozen at �80°C, titrated andthawed before single use as previously described (16). UV-inactivated vi-rus was prepared as described (16) and was used in the same manner asviable virus. For infection of macrophage cultures, MDM were counted,resuspended in fresh RPMI 1640 supplemented with all but M-CSF andinoculated with viral stocks by using a multiplicity of infection (MOI) of5 PFU/cell. After 12 h, cells were washed with citrate buffer (40 nM so-dium citrate, 10 mM KCl, 135 mM NaCl (pH 3.0)) to inactivate unab-sorbed virus (19). For single-cycle viral growth curves, MDM were in-fected as described and, at the indicated time point after infection, cellularfractions and supernatants were collected separately and stored at �80°Cfor successive determination of the infectious titer (20).

Immunofluorescence

MDM were seeded in 8-well Lab-Tek Chamber Slides (Nalge Nunc Inter-national) before mock or HCMV infection. For detection of viral proteins,mAbs reactive against the immediate-early proteins 1 and 2 (IE 1–2,pUL122 and pUL123, mAb E13; Argene-Biosoft), the early protein p52(pUL44, mAb CCH2; DakoCytomation), and the late tegument proteinpp150 (ppUL32, mAb XP1; Dade Behring) were used. MDM were fixedwith ice-cold methanol/acetone and probed with mAbs against viral Ags,followed by incubation with FITC-conjugated goat anti-mouse Ig (ICNBiomedical). For visualization of the cytoskeleton, MDM were fixed with4% formaldehyde, permeabilized with 0.2% Triton X-100 and incubatedwith 0.1 �g/ml FITC-labeled phalloidin (Sigma-Aldrich) or with mAbsanti-�-tubulin (Molecular Probes) and anti-vimentin (Oncogene ResearchProduct) followed by TRITC-conjugated anti-mouse Ig (DakoCytoma-tion). Intracellular MIF was visualized using rabbit anti-human MIF serum

FIGURE 1. MDM support lytic and productive infection by the HCMV endotheliotropic strain TB40E. A, Viral Ags characteristic for the three phasesof viral gene expression are detected by indirect immunofluorescence in MDM infected with the strain TB40E at an MOI of 5. MDM were stained withmAbs (green) specific for the viral immediate-early proteins 1 and 2 (IE 1–2 Ags, pUL122/123), early protein p52 (E Ag, pUL44) and late phosphoproteinpp150 (L Ag, ppUL32) and counterstained with Evans blue (red). All photographs are at original magnification of �60 and are from one donor repre-sentative of 10. Mock-infected MDM (inset) were negative for viral Ags as shown. B, MDM were inoculated with TB40E at an MOI of 5 (time point 0)and washed with acid buffer 12 h p.i. to remove unabsorbed input virus. At the indicated time points, supernatants and cells were differentially collectedfor measurement of infectivity by plaque assay.

Table I. Phenotypical characterization of MDM from HCMV blood a

Ag

Percentage of PositiveCells MFI

Monocytes MDM Monocytes MDM

CD14 93 � 6 96 � 2 107 � 13 107 � 11CD68 10 � 6 95 � 3 9 � 4 69 � 22CD64 91 � 3 92 � 3 78 � 13 30 � 12MHC class I 99 � 1 99 � 2 130 � 9 100 � 12MHC class II 94 � 1 94 � 3 24 � 5 32 � 12CD80 95 � 1 87 � 7 11 � 1 38 � 5CD86 18 � 9 55 � 16 3 � 1 19 � 5CD71 2 � 1 41 � 17 2 � 1 12 � 5

a MDM were generated from human monocytes after 7 days of stimulation withM-CSF.

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as described (21). Nuclei were counterstained with DAPI (4�,6-diamidino-2-phenylindole). Staining was evaluated using a Zeiss Axioskop2 fluores-cence microscope.

Flow cytometry

Samples were acquired using a FACSCalibur (BD Biosciences). For im-munophenotyping, MDM were incubated in blocking buffer (10% humanIgL (Flebogamma), 3% FCS, and 0.01% sodium azide in PBS) containinganti-CD14, anti-CD80, anti-CD86, anti-HLA-DR, anti-HLA-A, -B, -C (BDPharmingen)) or CD68 (DakoCytomation). All mAbs as well as the match-ing isotypic controls (Immunotech) were FITC- or PE-conjugated. Theexpression of chemokine receptors was evaluated using anti-CR1, anti-CCR2, anti-CCR5, anti-CXCR1, anti-CXCR2, and anti-CXCR4 (R&DSystems), anti-CCR7 (BD Pharmingen), anti-CX3CR (MBL), or matchingisotypic controls (IgG1, IgG2a, and IgG2b; DakoCytomation), followed byincubation with PE-conjugated rabbit anti-mouse Ig (DakoCytomation).MDM were permeabilized by using Cytofix/Cytoperm kit (BD Pharmin-gen) according to the manufacturer’s instructions. Data were analyzed us-ing CellQuest software (BD Immunocytometry Systems), and for each Agthe expression level was measured as a percentage of positive cells as wellas channel mean fluorescence intensity (MFI) of the respective Ab com-pared with the isotype-matched control.

Cell migration

Chemotaxis was evaluated using 48-well Boyden chambers (Neuroprobe)with 5-�m pore polycarbonate filters as previously described (22). Humanrecombinant MCP-1/CCL2, RANTES/CCL5, Gro-�/CXCL1, IL-8/CXCL8, VEGF, M-CSF (R&D Systems), MIP-3�/CCL19, SDF-1/CXCL12, and Fractalkine/CX3CL1 (PeproTech) were used at the finalconcentration of 100 ng/ml in migration medium (RPMI 1640 supple-mented with 1% FCS). fMLP (Sigma-Aldrich) was used at the final con-centration of 10�8 M. MDM were resuspended in migration medium andseeded 7.5 � 104 cells/well. For each well, the number of migrated cellswas calculated as the number of cells counted in five consecutive highpower fields. All stimuli were assayed in triplicates, and results were ex-pressed as mean � SD.

Actin polymerization assay

Uninfected and HCMV-infected MDM were resuspended in RPMI 1640supplemented with 1% FCS at a concentration of 1.5 � 106 cells/ml. Fol-lowing a preincubation of 30 min at 37°C, cells were stimulated with either200 ng/ml RANTES/CCL5 or 500 ng/ml VEGF or 10�7 M fMLP for 0, 15,30, 60, 300, or 900 s. Reactions were stopped by fixing the cells with 4%paraformaldehyde. Following permeabilization with 0.1% ice-cold TritonX-100, cells were stained with 1.5–2.0 mg/ml FITC-phalloidin (Sigma-Aldrich). At each time point, MFI values were measured by FACS instimulated and unstimulated MDM. The relative content of F-actin wasobtained by subtracting the MFI values of unstimulated cells from the MFIvalues of stimulated cells.

Polarization assay

Macrophage polarization assay was performed as described (23) with mi-nor modification. Briefly, prewarmed MDM (106/ml) were stimulated, induplicates, with chemoattractants or medium alone for 10 min. The reac-tion was stopped by adding ice-cold phosphate-buffered formaldehyde(10% v/v (pH 7.2)) and the percentage of cells with a bipolar configuration(front-tail) was determined in at least 200 cells for each tube by phase-contrast microscopy.

Measurements of NO and MIF

Due to the very high nitrate levels in RPMI 1640 medium, for measurementof NO, macrophages were seeded at the concentration of 1 � 106 cell/mlin MEM 10% FCS before mock or HCMV infection. At the indicated time

FIGURE 2. HCMV alters morphology, immunophenotype, and viabil-ity of MDM. A, MDM were mock-infected or infected with TB40E at anMOI of 5 and observed by light microscopy at 1 and 7 days p.i. All pho-tographs are at original magnification of �60 and are from one donorrepresentative of 10. B, MDM were mock-infected or infected withTB40E at an MOI of 5 and the immunophenotype was investigated byFACS at 1 and 7 days p.i. Surface expression of the indicated molecules

(thickline histograms) and staining with isotype-matched controls (thin linehistograms) are shown. Representative data from one of five experimentsare shown. C, HCMV infection induces cell death at late time points afterinfection in a dose-dependent manner. Levels of apoptosis (percentage ofAnnexin V� cells) and necrosis (percentage of PI� cells) were evaluated atday 1 and 7 p.i. in mock- and TB40E-infected MDM, using decreasingMOI from 5 to 0.05. Representative data from one of five experiments areshown.

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point after infection, culture supernatants were collected and assayed usinga Nitrate/Nitrite Colorimetric Assay kit (Cayman Chemical) following themanufacturer’s instruction. NO production was quantified by measuringthe levels of nitrite and nitrate, the stable oxidation products of NO, in thecell supernatants using the Greiss reaction. For the measurement of MIF,1 � 106 MDM/ml were seeded in RPMI 1640 with 10% FCS before mockor HCMV infection. At the indicated time point, culture supernatants werecollected and analyzed by a sandwich ELISA, using Quantikine kit forhuman MIF (R&D Systems) according to the manufacturer’s instruction.

Western blot analysis

MDM were seeded in RPMI 1640 supplemented with 1% FCS at a con-centration of 1 � 106 cells/ml and mock- or HCMV-infected with TB40E(MOI of 5). At the indicated time point, culture supernatants were col-lected, clarified by centrifugation, and concentrated in Centricon-10 con-centrators (Amicon) according to the operating manual. Concomitantly, thecells were lysed with 100 �l of lysis buffer (50 mM Tris-HCl (pH 7.4), 150mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, DTT and protease inhibi-tors). Concentrated supernatants and cell-associated proteins were sepa-rated by SDS-PAGE and blotted onto nitrocellulose membranes (Bio-Rad).Human recombinant MIF (hrMIF, 10 ng/lane; R&D Systems) was used aspositive control. Membranes were blocked with PBS containing 0.1%Tween 20 and 3% dry milk powder and incubated first with anti-MIF goatIg (diluted 1/500; R&D Systems) and then with rabbit anti-goat IgG per-oxidase conjugated (Pierce). A chemiluminescence detection kit (Super-Signal West Dura; Pierce) was used according to the manufacturer’sinstructions.

Northern blot analysis

Total RNA was isolated as previously described (16). A total of 5 �g ofRNA/lane were electrophoresed and transferred onto a positively chargednylon membrane (Boehringer Mannhein). RNA levels were equalized onthe basis of GAPDH levels. Probes for human GAPDH were amplified and

labeled with digoxigenin using commercially available primers (Biomol)and the PCR DIG Probe Synthesis kit (Roche). Similarly, a 348-bp frag-ment of human MIF cDNA (GenBank accession no. BC022414) clonedinto pENTR (Invitrogen) was digoxigenin-labeled using the primers(sense) 5�-ACAGAATATGCCGATGTTCATCGTAAACACC-3� and(antisense) 5�-ATCGAATTCTTAGGCGAAGGTGGAGTTGTTCCAGC-3�. Chemiluminescence detection was performed according to the instruc-tion for the use of the CDP-Star chemiluminescence substrate for alkalinephosphatase (Roche).

Immunodepletion of MIF from the conditioned medium

For each experiment, a total of 100 �g of goat anti-human MIF or goat Ig(R&D Systems) was added to 100 �l of protein A-dynabeads (Invitrogen),and incubated for 30 min at room temperature. The beads were thenwashed, resuspended in the conditioned medium, and incubated on slowrotation for 90 min at 4°C. By placing the sample tubes on the magnet, thebeads-Ig-MIF complexes were retained and the conditioned medium couldbe recovered. One aliquot of each conditioned medium was then subjectedto Western blot to confirm that MIF had been depleted.

RhoA activation assay

A commercially available ELISA-based RhoA activity assay (G-LISA; Cy-toskeleton) was used to measure the relative RhoA activity of MDM eitherleft untreated, infected with TB40E by using an MOI of 5, or stimulatedwith hrMIF (1 �g/ml). According to the manufacturer’s protocol, thewhole cell lysates were extracted and the protein concentrations were de-termined. The 25 �g of cell lysates were incubated in microwells to whichthe rhotekin binding domain peptide was bound, and active RhoA wasrevealed using indirect immunodetection followed by a colorimetric reac-tion measured by absorbance at 490 nm (24).

FIGURE 3. HCMV infection induces a potent inhibition of MDM motility. Macrophage chemotaxis in response to chemokines (RANTES/CCL5,MCP-1/CCL2, Fractalkine/CX3CL1, MIP-3�/CCL19, Gro-�/CXCL1, IL-8/CXCL8 and SDF-1/CXCL12; 100 ng/ml), bacterial product (fMLP; 10�8 M),and growth factors (VEGF and M-CSF, 100 ng/ml) was evaluated by using a Boyden chamber as described in Materials and Methods. MDM were seededin three wells and for each well the number of migrated cells was calculated as number of cells counted in five consecutive high power fields (HPF). A,The migration of mock- or TB40E-infected MDM (MOI of 5) was assessed at 24 h p.i. The number of migrated cells per five consecutive high power fieldsis obtained as mean � SD of independent experiments performed with macrophages obtained from 10 different blood donors. �, p � 0.05 between the twogroups. B, At 24 h p.i., the migration of MDM incubated with UV-inactivated TB40E (UV-TB40E) or AD169 (MOI of 5) was compared with the migrationof mock-infected MDM. Macrophages were obtained from five different blood donors. C, MDM were infected with decreasing MOI of TB40E, and at 24 hp.i. chemotaxis was evaluated. Macrophages were obtained from three different blood donors. D, MDM were infected with TB40E at an MOI of 5, andat 7 days p.i. cells were washed, counted, and tested for their migratory capacity. Results of four independent experiments as mean � SD are shown.�, p � 0.05 between the two groups.

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Statistical analysis

Statistical analysis of the results was performed using an unpaired, two-tailed Student’s t test. Differences were considered significant withp � 0.05.

ResultsMDM are highly susceptible to infection by endotheliotropicstrains of HCMV and support the lytic viral replicative cycle

MDM were obtained after 7 days of stimulation of monocytes withM-CSF, which in vivo regulates growth, differentiation, and func-tion of many types of tissue macrophages (25). According toYoung et al. (17), at the end of the differentiation period, MDMacquire the typical macrophage size and morphology, i.e., giantcells having an elongated or stellate morphology, abundant cyto-plasm with granules and vacuoles, and exhibit the expected phe-notype concerning expression of surface markers (26) (Table I).

Initiation of the viral replicative cycle was evaluated by detectionof immediate early Ags (IE 1–2, pUL122/123) in MDM infectedwith endotheliotropic (TB40E and VHLE) or fibroblast adapted(AD169) strains of HCMV using an MOI of 5. In agreement withfindings obtained with other cell types of the myeloid lineage (16,27, 28), the infection was efficient with both endotheliotropicstrains of HCMV, and at 1 day postinfection (p.i.) up to 90% ofTB40E-infected MDM were positive for IE Ags (Fig. 1A). MDMwere poorly susceptible to infection by the fibroblast-adaptedstrain AD169 and less than 5–10% of MDM expressed IE Ags(data not shown). The course of infection was further characterizedby kinetic analysis of viral Ag expression, electron microscopy andrelease of viral progeny. As shown in Fig. 1A, viral products char-acteristic for the subsequent replicative phases (early Ag, p52/UL44 and late Ag, pp150/UL32) were expressed and progressivelyaccumulated in TB40E-infected MDM. The successful completion

FIGURE 4. HCMV infection selectively modulates the surface expression of the chemokine receptors CCR1 and CCR5. At 1 day p.i., mock- andTB40E-infected MDM (MOI of 5) were harvested, stained for the indicated markers, and examined by FACS. A, Cell surface expression (thick linehistogram, intact cells) and total expression (gray-filled histogram, permeabilized cells) were evaluated for each chemokine receptor. Staining withisotype-matched control Abs (thin line histogram) in intact or permeabilized cells is shown. Representative data from one of ten experiments are shown.B, The expression of chemokine receptors was measured in mock- and TB40E-infected MDM as a percentage of positive cells. Results are mean � SDof 10 separate experiments. �, p � 0.05. C, Expression of receptors VEGF-R1 and M-CSF-R (thick line histograms) on the cell surface of mock- andTB40E-infected MDM. Staining with isotypic control (thin line histogram) is shown. Representative data from one of five experiments are shown.

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of the viral cycle was confirmed by detection of an increasinginfectivity in both supernatants and cellular fractions of TB40E-infected MDM (Fig. 1B), as well as by electron microscopic de-tection of abundant viral particles in the nucleus and in the cyto-plasm of TB40E-infected MDM at 7 days p.i. (data not shown).

Effect of HCMV infection on the morphology, immunophenotype,and viability of MDM

Following HCMV infection, progressive changes were observed inboth macrophage morphology and immunophenotype. During theobservation period, HCMV-infected MDM enlarged, reduced theirsubstrate adhesion, and tended to round up and float in the me-dium. The viral cytopathic effect became detectable 4 days p.i.with substrate detachment of macrophages and proceeded to thelysis of infected cultures at day 7 after infection (Fig. 2A). In ad-dition, although at day 1 p.i. only MHC class I and CD80 wereclearly down-modulated on the surface of the majority of TB40E-infected MDM, 7 days p.i. all tested molecules were stronglydown-regulated as compared with mock-infected cells (Fig. 2B).By Annexin V-PI staining, we observed that HCMV infection in-duced MDM death in a dose-dependent manner at late time pointsafter infection. As shown in Fig. 2C, although at day 1 p.i. mock-and TB40E-infected MDM exhibited the same levels of PI andAnnexin V staining, at day 7 p.i. the viability of TB40E-infectedMDM decreased proportionally to the MOI used (7 days p.i. MDMinfected with an MOI of 0.05, 0.5, or 5 exhibited a survival of63 � 15%, 50 � 8%, and 31 � 10%, respectively).

HCMV infection inhibits MDM motility

To evaluate the chemotactic responsiveness of HCMV-infectedMDM, we measured macrophage migration in response to a widerange of chemoattractants, such as inflammatory and homeostaticchemokines, growth factors (e.g., VEGF and M-CSF) and aformylpeptide of bacterial origin (fMLP). As shown in Fig. 3A, at24 h p.i. TB40E-infected MDM were unable to migrate in responseto all tested stimuli and exhibited a significantly reduced sponta-neous basal migration as compared with mock-infected cells. Asmentioned, at this time point after infection, TB40E-infected

MDM were neither apoptotic nor necrotic. However, to furtherensure that macrophage cellular functionality was intact, wecompared the metabolic activity of uninfected with HCMV-infected MDM. By using the MTS metabolization tetrazoliumsalt assay, we found similar levels of dehydrogenase activityand superoxide formation in mock- and TB40E-infected MDM(absorbance 0.35 � 0.04 vs 0.32 � 0.02, respectively), thusexcluding that the effect of HCMV on cell motility was a con-sequence of cell damage. MDM exposed for 24 h to AD169 or

FIGURE 5. HCMV infection impairs cellular polarization and actin polymerization in MDM. A, At 1 day p.i., MDM were stimulated for 10 min withfMLP (10�7 M), RANTES/CCL5 (200 ng/ml), or VEGF (500 ng/ml) to induce cell polarization. The phase contrast images are representative examplesof a polarized and a nonpolarized cell, exhibiting a front-tail organization and a round shape, respectively. The percentage of polarized cells was determinedin mock- and TB40E-infected MDM (MOI of 5 and 0.05) by differential count as described in Materials and Methods. Results of four independentexperiments as mean � SD are shown. �, p � 0.05. B, Time course of changes in F-actin content in mock- and TB40E-infected MDM (MOI of 5 and 0.05)stimulated with RANTES/CCL5 (200 ng/ml), fMLP (10�7 M), and VEGF (500 ng/ml) for the indicated time points. The relative F-actin content wasevaluated by FITC-phalloidin staining and FACS analysis as described in Materials and Methods.

FIGURE 6. HCMV infection induces a complete reorganization of theMDM cytoskeleton. The effect of TB40E infection (MOI of 5) on thestructural organization of MDM cytoskeleton was investigated by indirectimmunofluorescence at 1 day p.i. Staining for actin filaments (A), micro-tubules (B), and intermediate filaments (C) in mock- and TB40E-infectedMDM is shown. Results shown are from one donor representative of five.

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to UV-inactivated TB40E (Fig. 3B), which can enter the mac-rophages but cannot replicate, exhibited similar or even in-creased chemotactic responsiveness than mock-infected MDM.Thus, de novo viral gene expression and not simply virus-cellcontact was necessary to block macrophage motility. In detail,the chemotactic responsiveness of TB40E-infected MDM tofMLP, RANTES/CCL5 and VEGF was reduced by 75 � 10%already at 6 h after infection as compared with uninfected cells(data not shown), suggesting that immediate-early viral geneswere responsible for the impairment of MDM migration. Inaddition, the migration of MDM was inhibited by HCMV in adose-dependent manner (Fig. 3C). The block of macrophagemigration was persistent and at day 7 p.i. HCMV-infectedMDM exhibited a total block of migration (Fig. 3D).

HCMV infection induces intracellular accumulation of CCR1and CCR5

Having found that HCMV inhibited migration of MDM, weanalyzed whether HCMV infection was responsible for the

down-regulation of chemokine receptors. An extensive analysiswas performed and both the cell surface and intracellular ex-pression levels of several chemokine and growth factor recep-tors were investigated in mock- and TB40E-infected MDM. Asshown in Fig. 4, A and B, at day 1 p.i. only the surface expres-sion of CCR1 and CCR5 was significantly reduced in TB40E-infected MDM as compared with mock-infected cells. Con-versely, the expression levels of CCR2, CCR7, CXCR1,CXCR2, CXCR4, and CX3CR were not affected by HCMVinfection. Similarly to the block of migration, the cell surfacedown-regulation of CCR1 and CCR5 was dependent on viralgene expression because MDM exposed to UV-inactivatedTB40E (MOI of 5) for 24 h exhibited the same levels of CCR1and CCR5 us mock-infected cells (data not shown). The totalamount of chemokine receptors detectable after cell permeabi-lization was similar in mock- and TB40E-infected MDM, indi-cating that upon HCMV infection CCR1 and CCR5 underwentredistribution from the cell surface to an intracellular compart-ment without being degraded (Fig. 4A). Finally, although the

FIGURE 7. HCMV infection stimulates the secretion of soluble inhibitors in MDM. A, Uninfected MDM were treated for 24 h with conditioned medium(CM) obtained from mock- and TB40E-infected MDM as well as from MDM treated with UV-inactivated TB40E, and migration experiments wereperformed. CM were collected at 1 day p.i. and prior incubation with uninfected MDM were filtered to remove the viral particles. Results of five independentexperiments as mean � SD are shown. �, p � 0.05. B, Total nitrate and nitrite production from mock- and TB40E-infected (MOI of 5 and 0.5) MDM.Results from four different experiments as mean � SD are shown. C, MIF release was analyzed by sandwich ELISA of supernatants collected at theindicated time points from mock- and TB40E-infected MDM (MOI of 5, 0.5, and 0.05). Results of five independent experiments as mean � SD are shown.�, p � 0.05 between mock- and TB40E-infected cells. D, Northern blot analysis of MIF. MDM were either mock- or TB40E-infected (MOI of 5) for theindicated time point. Blots were probed for MIF (a single 0.6-kb transcript was found) and then reprobed for GAPDH. Results show MDM from one donorrepresentative of three studied. E, Western blot analysis of intracellular and extracellular content of MIF in MDM. Cells were mock- or TB40E-infectedwith an MOI of 5 and collected at the indicated time points after infection. Concentrated supernatants and cell lysates were separated by SDS-PAGE andblotted onto nitrocellulose membranes. The hrMIF (10 ng/lane) was used as positive control. F, At 24 h p.i., the intracellular distribution of MIF wasvisualized by indirect immunofluorescence (green) in mock- and TB40E-infected (MOI of 5) MDM. DAPI staining was used to detect the nuclei.Photographs at original magnification of �60 are from one donor representative of five studied.

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expression of VEGF receptor 1 (VEGF-R1 or flt-1) (29) was notaffected by HCMV infection, the levels of MCSF receptor(MCSF-R or CD115) were slightly reduced in TB40E-infectedMDM as compared with mock-infected cells (Fig. 4C).

HCMV infection impairs macrophage polarization and dynamicactin polymerization

Chemotaxis is preceded by cell polarization, an event that occurswithin minutes after chemoattractant stimulation and that ismarked by formation of cell protrusions and pseudopodia (12). Weobserved that the capacity of TB40E-infected MDM to polarize inresponse to stimulation by fMLP, RANTES/CCL5 and VEGF wasimpaired in a dose-dependent manner (Fig. 5A). Following polar-ization, oriented migration requires rapid assembly and disassem-bly of actin filaments (12) a phenomenon that can be experimen-tally followed by measuring the intracellular amount of F-actinupon stimulation with chemoattractants. As shown in Fig. 5B, theinfection caused a significant impairment of actin assembly andMDM infected with TB40E by using an MOI of 5 showed noincrease of F-actin content at any time until 15 min after stimula-tion. The block of actin polymerization caused by the virus wasdose-dependent and MDM infected with an MOI of 0.05 exhibiteda mild inhibition as compared with MDM infected with an MOIof 5.

All components of the MDM cytoskeleton are reorganized uponHCMV infection

The impairment of MDM cell polarization and actin polymeriza-tion caused by HCMV was consistent with the inhibition of cellmotility and prompted us to investigate the organization of thecellular cytoskeleton. Although in mock-infected MDM, actin wasorganized in abundant and extended podosomes, defined as dot-like cores of actin fibers essential for adhesion and movements(30), in TB40E-infected MDM these structures disappeared andactin aggregated in dense perinuclear cores (Fig. 6A). The micro-tubules and the vimentin network were also changed upon infec-tion (Fig. 6, B and C, respectively). As compared with uninfectedcells, TB40E-infected MDM exhibited a reduced extension andcomplexity of both the tubular network and the vimentin filaments.However, by Western blot we detected similar amounts of actin,tubulin, and vimentin in uninfected and infected MDM (data notshown), suggesting that the dramatic structural reorganization ofthe MDM cytoskeleton was not paralleled by degradation of themain components of the cytoskeleton.

MDM release soluble inhibitors of migration upon HCMVinfection

To test whether soluble factors might contribute to the inhibition ofmigration, freshly prepared MDM were incubated with virus-freeconditioned medium obtained from mock- and TB40E-infectedMDM. As shown in Fig. 7A, conditioned medium obtained fromTB40E-infected MDM blocked cell responsiveness to all testedchemoattractants, whereas conditioned medium obtained from un-infected MDM as well as from MDM treated with UV-inactivatedvirus (UV-TB40E) did not inhibit cell migration. These data indi-cated that soluble inhibitors were secreted by MDM upon activeHCMV infection. We first focused our attention on NO, which isknown to inhibit cytoskeletal assembly and pseudopodia formationin macrophages (31). By measuring identical low levels of nitriteand nitrate in the conditioned medium obtained from mock- andHCMV-infected MDM (Fig. 7B), we could exclude the NO in-volvement in the block of cell migration. We then measured thesecretion of MIF, a soluble product that blocks the random andchemokine-driven migration of monocytes/macrophages (32–34)

by a mechanism that is still unknown. As shown in Fig. 7C,HCMV infection stimulated the secretion of MIF into the super-natants in a dose- and time-dependent manner. We then investi-gated the gene expression, the total protein amount, and the spe-cific localization of MIF in mock- and TB40E-infected MDM.HCMV infection was not accompanied by an increased MIFgene transcription (Fig. 7D) but induced a redistribution of thisfactor between the intracellular and the extracellular compart-ment. Indeed, in TB40E-infected MDM the intracellular MIFwas reduced, whereas the extracellular MIF accumulated (Fig.7, E and F).

MIF is the soluble inhibitor of migration secretedby HCMV-infected MDM

To investigate whether secreted MIF acts as soluble inhibitor ofmacrophage chemotaxis in our experimental setting, we immu-nodepleted this protein from TB40E-infected conditioned mediumby using beads coated with anti-MIF Abs (35). The specific re-moval of MIF was confirmed by Western blot analysis (Fig. 8A).Conditioned medium incubated with beads coated with anti-MIFIg were depleted from MIF, whereas conditioned medium incu-bated with beads coated with control Ig as well as conditioned

FIGURE 8. MIF is the soluble factor responsible for the inhibition ofMDM migration. A, Dynabeads were coated with goat Igs raised againstMIF (Anti-MIF Ig) or unspecific (Control Ig) and then incubated withconditioned medium (CM) using ratios for beads to conditioned medium of1:5 and 1:10. Depletion of MIF from conditioned medium was proven byWestern blot analysis as described in Materials and Methods. To verifyequal loading and transfer the Poinceau staining is shown. B, MDM wereincubated for 24 h with virus-free conditioned medium obtained frommock- and TB40E-infected cultures and then used for migration experi-ments. The conditioned medium were left nontreated (n.t.) or were treatedwith beads coated with anti-MIF or control Ig. The number of migratedcells per five consecutive high power fields (HPF) are obtained as mean �SD of independent experiments performed with macrophages obtainedfrom five different blood donors.

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medium left untreated exhibited intact amounts of MIF. Unin-fected MDM were then incubated for 24 h with MIF-depleted con-ditioned medium and tested for their migratory capacity. Althoughthe depletion of MIF from mock-infected conditioned medium didnot significantly affect the MDM chemotaxis (Fig. 8B, left), byremoving MIF from TB40E-infected conditioned medium we ob-tained a complete restoration of the MDM migratory property (Fig.8B, right). As expected, TB40E-infected conditioned mediumtreated with beads coated with control Ig inhibited the migration ofMDM in the same extent than untreated TB40E-infected condi-tioned medium.

Exogenous MIF only partially mimics the effect induced byHCMV infection on MDM migration and chemokine receptorexpression

To determine whether exogenous MIF could reproduce the effectsinduced by HCMV infection on MDM migration, fresh MDMwere incubated for 24 h with hrMIF (produced as described in Ref.32 and containing not more than 32 endotoxin U/mg of protein)and then were tested for chemotaxis and chemokine receptor ex-pression. As shown in Fig. 9A, hrMIF inhibited MDM migration inresponse to RANTES, IL-8 and SDF-1, but did not affect randommigration. Although the dose-dependent inhibition of migrationshowed some differences between stimuli, in agreement with pub-lished data (36) at the dose of 100 ng/ml hrMIF efficiently blockedthe chemokine-driven migration of MDM. The necessity of RhoGTPases for macrophage chemotaxis has been extensively proven(37). Therefore, we tested the effect of hrMIF and HCMV infectionon RhoA activation of MDM. We observed a reduced RhoA ac-tivation in both MDM infected with HCMV and treated withhrMIF (Fig. 9B). Because recent evidence shows that MIF canbind CXCR2 and CXCR4 and cause down-regulation of their ex-pression (38), we analyzed the expression levels of these receptorsin MDM either incubated with TB40E-infected conditioned me-dium or with hrMIF. As shown in Fig. 9C, both treatments induceda profound down-regulation of CXCR2 and CXCR4 on the sur-faces of treated MDM as compared with untreated cells. Con-versely, HCMV infection did not affect the expression of CXCR2and CXCR4 on MDM. Moreover, the expression levels of CCR1and CCR5, that were significantly down-regulated during HCMVinfection (Fig. 3A), were not affected by hrMIF at any dose (datanot shown).

FIGURE 9. Effects of sustained treatment with hrMIF on MDM. A, In-hibition of MDM migration by hrMIF. Uninfected MDM were incubatedfor 24 h with increasing amounts (1–500 ng/ml) of hrMIF before measure-ment of their chemotaxis by using a Boyden chamber. Each symbol depictsmean � SD for independently performed experiments with cells obtainedfrom three blood donors. �, p � 0.05 between mock and treated cells. B,HCMV infection and hrMIF reduce RhoA activation in MDM. MDM ob-tained from three different donors were incubated in starvation medium(RPMI 1640 medium supplemented with 1% FCS) and either left untreated(mock), or infected with TB40E (MOI of 5) or stimulated with hrMIF (1�g/ml). After 24 h, 25 �g of cell lysates were subjected to the G-LISARhoA activation assay Biochem kit. �, p � 0.05 between mock-infectedand either TB40E-infected or hrMIF-treated cells. C, The expression of thechemokine receptors CXCR2 and CXCR4 was evaluated by FACS on thecell surface of MDM incubated for 24 h either with 100 ng/ml hrMIF orwith virus-free conditioned medium obtained from TB40E-infected mac-rophages. As control, the expression of these receptors was measured onmock- and TB40E-infected MDM. Staining with specific Ab (gray linehistogram) or isotype-matched control Ab (black line histogram) is shown.Representative data from one of five experiments are shown.

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Thus, hrMIF can only partially mimic the effects induced byHCMV infection on chemokine receptor expression and migrationin MDM.

DiscussionOur study shows that HCMV inhibits the chemotaxis of humanMDM in response to a number of stimuli by using different strat-egies. Immediately after entry of HCMV into MDM and onset ofviral gene expression, some cellular chemotactic receptors undergosurface down-regulation. Furthermore cytoskeleton componentsare reorganized in dense and rigid structures. Finally, through thesecretion of soluble inhibitors, the block of migration is also as-sured in the neighboring uninfected macrophages. Importantly,HCMV inhibition of macrophage migration takes place in cellsthat are viable and metabolically active. The physiological conse-quences of this inhibition may lead to an impairment of the anti-viral response and to a deregulation of the inflammatorymicroenvironment.

These data complete the analysis of the effects exerted byHCMV on the migratory ability of APCs. In our view it is startlingthat upon HCMV infection not only monocytes (16) and dendriticcells (14, 15) but also macrophages undergo an impairment ofmigration. The molecular mechanisms adopted by HCMV appearto be different in the three cell types and most likely they reflect thehigh level of HCMV adaptation to its host.

Although it is well accepted that viral and microbial pathogenscan manipulate the macrophage chemotactic machinery for theirown benefit, so far few reports describe the effects exerted bypathogens on the general migratory properties of macrophages.Besides HCMV, infection of macrophages with influenza and HSVis associated to impaired chemotactic responses of these cells (39,40). Similarly, macrophages infected with Salmonella (41) or Tox-oplasma (42) exhibit a reduced motility. However, the diminishedability of infected macrophages to respond to chemotactic stimuliis not a universal effect. While vaccinia virus and poliovirus do notaffect macrophage motility, HIV, or Mycobacterium tuberculosiscan even enhance the chemotactic response of macrophages (43,44).

It is generally accepted that HCMV undermines different hostimmune functions and that virus-induced immunomodulation con-tributes to persistence and spread of HCMV. A well-documentedexample is the infection and resulting functional deregulation ofdendritic cell maturation, cytokine production, and lymphocytestimulation capability (45, 46). The mechanisms that HCMV ex-ploits to avoid immune eradication often lead to increased inflam-mation that in turn plays a central role in the viral pathogenesis. Anextensive literature reports that HCMV induces the secretion of anumber of inflammatory mediators that may enhance viral repli-cation (47) and reactivation from latency (48). As key cells in thelocal inflammation (49) and as important site of HCMV reactiva-tion and replication (5, 50), macrophages represent an element inwhich the host inflammatory response and HCMV infection dis-play a synergistic relationship. Macrophages, in contrast to mono-cytes, exhibit the potential to support HCMV reactivation fromlatency and viral replication as well as to sustain the host inflam-matory response through secretion of proinflammatory mediators.Because their regulated trafficking and distribution in response toenvironmental signals are prerequisites for mounting an effectiveantiviral immune response as well as for the limitation of damageand the healing after immune response (51), we wanted to inves-tigate whether HCMV infection can alter the migratory propertiesof macrophages.

In our experimental system, MDM were obtained from mono-cytes stimulated with M-CSF, which in vivo is one of the main

regulators of growth, differentiation and function of tissue macro-phages (25). Consistent with findings of Sinzger et al. (28), whohowever used macrophages differentiated in the presence of GM-CSF, we found that MDM were highly susceptible to HCMV in-fection and supported the complete replicative viral cycle. As aresult of HCMV infection, MDM underwent a complete block ofcell migration and became unresponsive to inflammatory and con-stitutive chemokines, bacterial products and growth factors.

We focused our investigations on the early phase of viral rep-lication, when the levels of apoptosis/necrosis were very low andthe metabolic activities were comparable in mock- and HCMV-infected MDM. In this way we excluded that the inhibition of cellmigration was simply due to impending cell death or cell injury. Ingeneral, cell migration is a highly integrated multistep process thatis triggered by the presence of chemoattractants. Chemokines, bac-terial products, and growth factors are sensed by receptors ex-pressed on the cell surface. In accordance with the high motilityexhibited by mock-infected MDM, we measured high levels ofchemokine (i.e., CCR1, CCR2, CXCR4) and growth factor recep-tors (VEGF-R and MCSF-R) on the plasma membrane of unin-fected MDM. Low responsiveness of MDM to Gro-�, IL-8, andMIP-3� was paralleled by the low surface levels of CXCR1,CXCR2, and CCR7. HCMV infection did not have a profoundeffect on the expression pattern of chemotactic receptors and onlyCCR1 and CCR5 were clearly down-regulated on the surface ofTB40E-infected MDM. The restricted viral effect on the expres-sion of chemotactic receptors and the observation that the basalMDM migration was also impaired upon infection prompted us toexamine the effect of HCMV infection on the properties of thecellular cytoskeleton.

We observed that the cell polarization in response to chemotac-tic stimulation was inhibited by HCMV in a dose-dependent man-ner. Although mock-infected MDM exhibited large ruffling lamel-lipodia on one side of the cell and a conical tail at the opposite side,HCMV-infected MDM remained round and possessed only fewmembrane protrusions. The defective morphological polarizationwas mirrored by a potent inhibition of actin polymerization, whichnormally drives and maintains the formation of cell membraneprotrusions. HCMV-infected MDM did not assemble actin fila-ments in response to stimulation and presented a completely al-tered cytoskeleton architecture. These modifications were not as-sociated with cell death (52), but because microtubules and actinfilaments functionally cooperate in generating cell migration, webelieve that the observed cytoskeletal modifications in HCMV-infected MDM represent a mechanism for the inhibition of che-motaxis and chemokinesis.

Additionally, HCMV showed to impair the migration of unin-fected neighboring cells through the release of soluble inhibitors.In the supernatants of HCMV-infected MDM we measured in-creased amounts of MIF, a pleiotropic cytokine that has beenshown to influence monocyte/macrophage migration (32–34, 36,38) and to potently stimulate the production of proinflammatorymediators (53). To confirm the physiologic role of MIF as solubleinhibitor of macrophage chemotaxis we adopted different ap-proaches. Monoclonal and polyclonal Ab directed against MIF(21, 32, 54), the chemical inhibitor ISO-1 (55) and the secretioninhibitor Probenecid (56) were used to specifically inhibit MIF inthe conditioned medium produced by HCMV-infected MDM. Al-though the addition of the anti-MIF mAb NIH3D9 did not reversethe inhibitory effect exerted by TB40E-infected conditioned me-dium, both the polyclonal anti-MIF serum and the chemical com-pound ISO-1 repeatedly reverted roughly 50% of the inhibitoryeffect. Though the mAb NIH3D9 has been used by several labo-ratories, it is possible that the neutralization capacity of the mAb

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with respect to MIF-dependent migration is less complete than thatof the polyclonal Ab that recognizes multiple epitopes. Becausethe depletion of MIF from the conditioned medium induced thecomplete restoration of cell migration, we could prove that oncereleased from HCMV-infected MDM MIF is responsible for theblock of bystander macrophage migration.

The exact role of MIF in modulating monocyte/macrophage mi-gration is still controversial. Although MIF was initially recog-nized as inhibitory factor able to block random and chemokine-driven migration (32–34, 36, 57), recent evidence shows that thiscytokine can evoke monocytic chemotaxis through interactionwith CXCR2 and CXCR4 (38). Altogether, these observationssuggest that a sustained presence of MIF can inhibit cell migration,whereas a short-term stimulation or the presence of a constitutedgradient for this cytokine may induce monocyte/macrophage re-cruitment. Notably, a similar dual regulation has been demon-strated for TGF-�: short-term treatment with TGF-� stimulatesmigration of macrophages and raises the levels of GTP-RhoA,whereas long-term exposure decreases both effects (61).

Rho GTPases have been proven to be crucial for macrophagechemotaxis by regulating the cytoskeletal architecture and provid-ing traction forces (37). Interestingly, we observed reduced RhoAactivation in both MDM infected with HCMV and MDM treatedwith hrMIF for 24 h. Although HCMV-dependent reduction ofRhoA activity has already been reported (58), the MIF-dependentinhibition of RhoA activity in primary macrophages has not beenpreviously described. In contrast, MIF-dependent RhoA activationhas been proven in the murine cell line NIH3T3 (59) and in thealveolar carcinoma cell line A549 (60). Not only the cellular sys-tems are completely different from ours because these are residentconnective tissue cell lines that possess stress fibers and large focaladhesions (a cytoskeletal architecture completely different frommacrophages), but also the type and duration of MIF stimulationare different. Therefore, the results reported in these publicationsshould neither be directly compared with our results, nor consid-ered contradictory. We can speculate that similarly to TGF-�, aftera short exposure MIF acts as a chemoattractant and leads to RhoAactivation; in contrast, after a sustained exposure MIF acts as asoluble inhibitor and leads to down-regulation of active Rho.

Finally, our findings indicate that the effects induced by exog-enous MIF and by HCMV infection do not completely overlap andthat a monocausal association between HCMV-dependent secre-tion of MIF and block of MDM migration is not likely to be thecase. MDM treated with hrMIF for 24 h do not express reducedlevels of CCR1 and CCR5 as virally infected cells do, but in agree-ment with a recent study (38) undergo desensitization of CXCR2and CXCR4. Because during HCMV infection MDM becomecompletely unresponsive to SDF-1, Gro-�, and IL-8 even thoughCXCR1, CXCR2, and CXCR4 are not desensitized, we concludethat during HCMV infection various mediators and signaling path-ways simultaneously influence the biologic properties of MDMand thus lead to additional effects. The capacity of HCMV to ac-tivate redundant and sometimes contradictory cellular functionsfor its own “survival” is a peculiarity of this complex virus (3).

We propose that our in vitro findings may have important clin-ical implications in chronic inflammatory diseases with locally ac-tive HCMV infection. We suggest that once tissue macrophagesare recruited into the area of HCMV replication, they quickly looseresponsiveness to chemoattractants and are retained in situ. Thesecells can then provide sites for viral replication and MIF produc-tion that in turn triggers the secretion of proinflammatory factorsand ultimately exacerbates episodes of acute inflammation.

Hence, the interference of HCMV with the migratory ability ofMDM may alter macrophage trafficking and contribute to viral

immune evasion, but may also contribute to boost already existinginflammatory conditions and viral persistence in the organism.

AcknowledgmentsWe thank Anke Luske for the excellent viral stock preparations, KerstinMueller for support in the immunodepletion, and Ingrid Bennett for criticalreading.

DisclosuresDr. Richard Bucala is co-inventor on a patent describing the therapeuticbenefit of anti-MIF Abs. The remaining authors have no financial conflictof interest.

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