1 Microbiology and Tumor Biology Center, Karolinska Institutet, Nobels väg 16, S-17177 Stockholm, Sweden
2 Center for Infectious Medicine, Huddinnge Hospital, Karolinska Institutet, Nobels väg 16, S-17177 Stockholm, Sweden
3 Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, UK
4 Department of Microbiology and Immunology, Louisiana State University, Health Science Center, Shreveport, LA, USA
Correspondence
Victor Levitsky
Victor.Levitsky{at}mtc.ki.se
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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Specific T lymphocytes inhibit growth of EBV-transformed B cells in vitro. In vivo, T cells suppress virus reactivation and EBV-induced tumorigenesis, as demonstrated by the effects that they mediate upon adoptive transfer to immunodeficient patients (Rooney et al., 1995; Heslop et al., 1996
; Heslop & Rooney, 1997
; Gustafsson et al., 2000
). Little is known, however, about the mechanisms that are responsible for the induction of EBV-specific immunity and the capacity of the virus to interfere with this process.
Antigen presentation by dendritic cells (DCs) is believed to be required for primary T-cell stimulation. Consistent with the critical function of DCs in anti-virus immunity, many viruses are known to infect different subsets of DCs and affect their differentiation, survival, migration and/or T cell-stimulatory capacity (reviewed by Becker, 2003; Kobelt et al., 2003
; Schneider-Schaulies et al., 2003
; Sevilla et al., 2003
; Steinman et al., 2003
). DCs were recently shown to cross-present virus antigens that are derived from EBV-infected cells and to be necessary for inhibition of EBV-induced B-cell transformation in lymphocyte cultures that were established from EBV-seronegative, but not -seropositive, individuals (Subklewe et al., 2001
; Bickham et al., 2003
). These findings support a critical role for DCs in the induction of primary T-cell anti-virus responses. Whilst other human herpesviruses, such as cytomegalovirus (CMV) and herpes simplex virus, have been shown to inhibit the T cell-stimulatory function of DCs by a number of mechanisms, the capacity of EBV to interact with, and affect the function of, DCs or their precursors is poorly characterized. We have recently shown that EBV infection inhibits DC development from monocyte precursors in the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL4), in a manner that is apparently independent of de novo expression of viral genes (Li et al., 2002
). It remains unclear, however, whether the virus is capable of entering monocytes or developing DCs and how the tropism of the virus for these cells is regulated at the molecular level.
Cellular tropism of herpesviruses is determined by virus-encoded proteins in the virion envelope that interact with various cellular receptors (reviewed by Spear & Longnecker, 2003). Recent studies have shown that the canonical EBV receptor, CD21, which interacts with the major glycoprotein of the EBV envelope, gp350, is neither sufficient nor essential for B-cell infection and that fusion of the EBV envelope with the cell membrane requires components of the trimolecular complex, which includes glycoproteins gp85, gp42, and gp25 (Li et al., 1997
; Wang & Hutt-Fletcher, 1998
; Haan et al., 2000
). Abundance of another EBV envelope glycoprotein, gB (also referred to as gp110), has recently been shown to influence EBV tropism for different cell types (Neuhierl et al., 2002
). Whilst major histocompatibiliy complex (MHC) class II molecules have been identified as the cellular ligand of gp42, the interaction partners of gp85 and gp25 are currently unknown. These EBV proteins are homologous, respectively, to the gH and gL components of the herpes simplex virus and CMV membrane fusion complexes (Yaswen et al., 1993
), are non-covalently associated in the virus envelope and are often referred to as the gH/gL heterodimer. The importance of the trimolecular assembly in EBV infection is demonstrated by the inability of recombinant viruses that are devoid of gp42 or gH to infect B cells (Wang & Hutt-Fletcher, 1998
; Oda et al., 2000
). However, the presence of gp42 in the virus envelope impedes infection of epithelial cells that are infected efficiently by gp42-deficient virions (Wang et al., 1998
). These data suggest that different modes of receptorligand interactions are operational during EBV infection of different cell types (Wang et al., 1998
). Thus, the bimolecular gH/gL complex mediates epithelial cell infection, whereas the trimolecular gp42/gH/gL complex is required for infection of B cells. The content of the bi- versus trimolecular complexes in the EBV envelope and the tropism of EBV virions can be affected by the type of cell that is supporting virus replication. Expression of human leukocyte antigen class II results in reduced levels of gp42 in the virion, due to association of these two proteins inside the cell and degradation of gp42 in the endosomal/lysosomal compartment. This may play an important role in the virus life cycle. Thus, EBV replication in epithelial cells generates virions that infect B cells preferentially, thereby establishing the EBV-carrier state in the infected individual, whereas EBV that is produced in B cells may be more efficient at mediating virus spread, due to more efficient infection of epithelial cells (Borza & Hutt-Fletcher, 2002
). The effect of EBV envelope composition on its interaction with monocytes or DCs has not been analysed.
In this study, we demonstrate that, compared to B lymphocyte-produced virions, EBV from epithelial cells exhibits a significantly increased capacity to infect monocytes and inhibit their development into DCs. These findings may have important implications for our understanding of the immunoregulation and pathogenesis of EBV infection.
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METHODS |
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Quantification of EBV virions.
DNA slot-blot analysis and measurement of cell-associated virus by fluorescence-activated cell sorting (FACS) analysis or real-time PCR were used to equalize virus stocks for virion content. Details of the virus-binding assay and FACS analysis for both virus quantification and comparison of envelope glycoproteins are described below. Slot-blot analysis was performed as described previously (Wang & Hutt-Fletcher, 1998). The quantity of EBV DNA in virion particles was measured by hybridization with a 32P-labelled BamHI W fragment of EBV DNA by means of a random-primed DNA labelling kit (Boehringer Mannheim). Samples (20 µl of a 100x concentrated virus stock) were digested for 10 min at room temperature with DNase I. 0·5 M EDTA (10 µl) was added to stop digestion and virus particles were sedimented for 1 h at 16 000 g. Sedimented virus was digested overnight at 56 °C with proteinase K (2·5 mg ml1 in 0·2 M EDTA) and serial dilutions were made in PBS. Sample DNA was denatured by the addition of 0·1 vol. 5 N NaOH, neutralized with 2 M ammonium acetate, applied to a nylon membrane and cross-linked by UV irradiation. Hybridizations were carried out as described previously (Mackett et al., 1990
) and quantified by scanning with a Storm PhosphorImager (Molecular Dynamics).
EBV-negative Akata cells were incubated with different dilutions of virus stocks for 1 h at 4 °C to determine the number of virions that were capable of binding to CD21-positive cells. Cells were washed three times in cold PBS and DNA was extracted with a QIAamp DNA blood mini kit (Qiagen), eluted in sterile water and stored at 20 °C. The TaqMan PCR reagent system and ABI PRISM 7700 sequence detection system (Applied Biosystems) were used to measure EBV DNA (Heid et al., 1996). The EBV target gene chosen was latent membrane protein (LMP)1 and the amplification primer sequences were: forward primer, 5'-AAGGTCAAAGAACAAGGCCAAG-3'; reverse primer, 5'-GCATCGGAGTCGGTGGG-3'; probe, 5'-AGGAGCGTGTCCCCGTGGAGG-3'. The probe was labelled with 6-carboxyfluorescein (FAM; reporter) and 6-carboxytetramethylrhodamine (TAMRA; quencher). A standard curve was created to calculate the number of EBV genomes (3) by using serial dilutions of DNA extracted from the EBV-positive human cell line Namalwa, which carries two integrated EBV copies per cellular genome (Klein et al., 1972
). The standard and the DNA samples were run in triplicate. Amplification mixtures (25 µl) contained 1x TaqMan Universal PCR master mix (Applied Biosystems) and 900 nM of each EBV primer, 200 nM EBV probe, water and 5 µl sample DNA. Cycling parameters were as follows: 50 °C for 2 min, 95 °C for 10 min and 45 cycles of 95 °C for 15 s and 60 °C for 1 min.
Preparation of monocytes and DCs and EBV infection.
Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by centrifugation on Ficoll density gradients. Monocytes were purified from PBMCs by centrifugation on Percoll density gradients, as described by Kouwenhoven et al. (2001). Alternatively, CD14+ monocytes were purified by using a monocyte-negative isolation kit (Dynal Biotech) or MACS CD14 MicroBeads (Miltenyi Biotec) according to the manufacturers' instructions. The negatively selected population contained >98 % CD14+ cells, as determined by staining and FACS analysis.
Monocytes were resuspended in culture medium (RPMI 1640 medium supplemented with 10 % heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U penicillin ml1 and 100 µg streptomycin ml1) at a density of 1x106 cells ml1 and cultured for the indicated time in medium supplemented with 1000 U recombinant GM-CSF ml1 and 1000 U recombinant IL4 ml1 (a kind gift from the Schering-Plough Research Institute, New Jersey, USA). Changes in cell morphology were monitored daily by visual inspection using an inverted microscope.
EBV infection was performed by resuspending 1x106 monocytes in culture medium containing 1 arbitrary unit (AU) (see below) of the indicated EBV preparation. Cells were incubated for 2 h at 37 °C, washed and cultured in medium that contained IL4 and GM-CSF to induce DC development.
EBV-specific antibodies and binding assays.
The mAbs F-2-1 (Strnad et al., 1982), which is specific for the EBV envelope glycoprotein gp42 (Li et al., 1995
), E1D1 (Oba & Hutt-Fletcher, 1988
) [reacting with the EBV gH/gL complex (Li et al., 1995
)], 72A1 (Hoffman et al., 1980
), specific for the EBV gp350/220 glycoprotein, and CL59 (Molesworth et al., 2000
), specific for gH, were purified from culture supernatants of the corresponding hybridoma cell lines. The mAb 5B2, which is specific for gB, was purchased from Virusys. FACS analysis of virus bound to the surface of Burkitt's lymphoma Raji cells, which express high levels of the CD21 molecule, was used to measure virus in different preparations and analyse the content of different glycoproteins in the EBV envelope. To compare virus stocks, Raji cells were fixed in 0·1 % paraformaldehyde, resuspended in 100 µl RPMI medium that contained the indicated EBV preparation and incubated for 1 h. Cells were washed in PBS and incubated in 50 µl 2L10 gp350-specific mAb or isotype control antibody diluted in PBS. Antibody binding was revealed by allophycocyanin (APC)-conjugated goat anti-mouse antibody (BD Biosciences) or R-phycoerythrin (R-PE)-conjugated rabbit anti-mouse antibody (Dako). All procedures were carried out on ice. Cells that had not been exposed to EBV were used as a control. The amount of virus that generated a mean fluorescence intensity (MFI) value with gp350-specific antibody in the binding assay that was comparable to that obtained with 1 ml B95.8 supernatant was expressed as 1 AU of EBV virions. B95.8 virus (0·5 AU) was used in binding assays that were performed for equalization of virus stocks. To analyse the protein composition of the envelope, EBV was reduced to 0·25 AU, virus glycoprotein-specific antibodies were used at 10 µg per sample (2·5 µg per sample for mAb 5B2) and the biotinylated anti-mouse antibody and PE- or APC-conjugated streptavidin were used at a 1 : 100 dilution (all from BD Biosciences).
Detection of apoptosis and GFP expression.
Efficiency of Annexin V binding to the surface of EBV-infected and control cells was measured by using an Annexin VPE apoptosis detection kit I (Pharmingen). GFP expression in cells infected by the recombinant EBV strains was monitored either by measuring the intensity of green fluorescence in infected cells using FACS analysis or by immunoblotting of total cell lysates using a mixture of two GFP-specific mAbs, 7.1 and 13.1 (Roche).
gH/gL binding assay.
gH was co-expressed with gL in SF9 insect cells and precipitated as a gH/gL heterodimer. SF9 cells were infected at an m.o.i. of 3 with baculovirus expressing gH/gL (Pulford et al., 1995). Five days later, the culture medium was clarified by low-speed centrifugation to remove cells and then centrifuged at 16 000 g for 90 min to remove virus. PEG 3500 was added to a final concentration of 20 % (w/v), the solution was stirred for 1 h at 40 °C and then centrifuged at 14 000 g for 20 min. The pellet was resuspended in 0·1 vol. PBS and the precipitated gH/gL was dialysed against PBS in dialysis tubing with a molecular mass cut-off of 25 000 Da, as described previously (Pulford et al., 1995
). The indicated cells were resuspended at 3x105 cells in 200 µl PBS containing 10 % serum from an EBV-seronegative individual and were incubated on ice for 1 h in the presence of 50 µg purified gH/gL. Where indicated, 100 µg E1D1 mAb or isotype control antibody were added to the reaction. Cells were then washed in PBS and gH/gL binding was revealed by CL59 mAb staining, followed by incubation with anti-mouse IgG fluorescein isothiocyanate (FITC)-labelled secondary antibody. Cells were washed and analysed by FACS.
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RESULTS |
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Purified monocytes were infected for 2 h (1 AU in 1x106 monocytes) with E-EBV, E-II-EBV or B-EBV and cultured in the presence of IL4 and GM-CSF to stimulate their differentiation into DCs. To assess the pro-apoptotic activity of different EBV preparations, cells were stained with Annexin V and analysed by flow cytometry. After infection with E-EBV, the proportion of apoptotic cells in developing DC cultures reached 50 % on day 3 and >70 % on day 5 post-infection. In contrast, only 9·9 and 6·4 % of cells were apoptotic in control cultures at the same time points. Infection with B-EBV only slightly increased the proportion of Annexin V-positive cells, whilst E-II-EBV exhibited an intermediate activity, as compared with B-EBV and E-EBV (Fig. 1). Similar relative pro-apoptotic activities of different EBV virions were revealed by monitoring the recovery of viable cells in DC cultures at day 6 or 7 post-infection (Table 1
).
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Immature DCs remain permissive for EBV entry
In our previous study, we showed that immature DCs become resistant to the pro-apoptotic effect of the virus at day 56 of their development in culture in vitro (Li et al., 2002). To analyse whether the capacity of EBV virions to inhibit DC development correlates with the capacity of EBV virions to enter cells, we infected monocytes or developing DCs with B-EBV, E-EBV, or E-II-EBV at days 0, 3 or 6 of culture in vitro in the presence of GM-CSF and IL4. Efficiency of EBV entry was assessed by monitoring green fluorescence intensity in monocytes 48 h after infection. Infection with 1 AU B-EBV resulted in only a slight increase in the MFI of infected cells, compared to controls (Fig. 4
). In contrast, the majority of cells that were infected with either E-EBV or E-II-EBV became positive for green fluorescence. Infection with E-EBV was more efficient than with E-II-EBV, as judged by GFP signal intensity in infected cells (Figs 3 and 4
), which was consistent with the differences in their capacity to inhibit DC development. This pattern was observed in cells that were infected at different time points.
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DISCUSSION |
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Previous studies have demonstrated that EBV infection of monocytes results in a number of biological effects (Gosselin et al., 1991, 1992
; Roberge et al., 1997
; Shimakage et al., 1999
; Savard et al., 2000a
, b
; Masy et al., 2002
; Tardif et al., 2002
) that have been attributed either to virus entry-independent effects or infection of a small proportion of cells. In this respect, our finding that E-EBV can infect virtually all cells in monocyte cultures is quite remarkable. The relatively high capacity of EBV from epithelial cells to inhibit DC development from monocyte precursors efficiently may play an important role in the regulation of virushost interactions. EBV spreads in the human population through the oral route of infection and cells of the oral or nasopharyngeal epithelium represent its most likely entry site. This hypothesis is supported by a recent demonstration of EBV replication in polarized in vitro cultures of non-transformed human epithelial cells (Tugizov et al., 2003
). In vivo, replication of the virus in epithelial cells is observed in hairy leukoplakia [reviewed by Rickinson & Kieff (1996)
], as well as in histologically normal epithelium of severely immunocompromised patients (Herrmann et al., 2002
), and is likely to occur during primary EBV infection in individuals lacking virus-specific immunity. One arbitrary unit of EBV used in our experiments corresponded to 20 virions per cell on average, an m.o.i. that seems to be easily achievable at a site of EBV replication in vivo. Although strong humoral and cellular immune responses are eventually elicited against the virus, EBV infection is asymptomatic in the majority of infected individuals. Even in rare cases of infectious mononucleosis, a lag period of several weeks is frequently observed between the infection episode and appearance of clinical symptoms of the disease, which may be caused by the pathological side effects of extensive anti-virus immunity (Peter & Ray, 1998
). DCs are critical for efficient induction of primary immune responses and inhibition of their function is likely to delay the development of both cellular and humoral specific immunity in EBV-infected individuals. In agreement with this model, sites of hairy leukoplakia lack morphological and histological signs of inflammation and have a decreased content of Langerhans cells (Daniels et al., 1987
; Walling et al., 2004
); this may be explained by the ability of EBV virions, which are produced abundantly in leukoplakia lesions, to exert an inhibitory effect on DCs. In contrast, reactivation of the virus in latently infected B cells of immune, chronic virus carriers should result in the production of EBV virions with a significantly decreased capacity to infect and modulate the function of monocytes and DCs, which would be more compatible with asymptomatic and non-pathogenic EBV persistence.
DCs that develop from monocyte precursors in the presence of IL4 and GM-CSF gradually lose their sensitivity to the pro-apoptotic effects of EBV infection. Whether this reflects decreased infectivity of immature DCs by EBV, their decreased general sensitivity to apoptosis or changes in the regulation of signalling pathways mediated by EBV infection was unclear. Here, we show that immature DCs that become resistant to EBV-induced apoptosis still support virus entry, as demonstrated by GFP expression conferred by infection with recombinant EBV strains (Fig. 4). Therefore, resistance to EBV-induced apoptosis can be accounted for by differentiation-associated changes in developing DCs. However, the ability of EBV to infect immature DCs raises the possibility that the virus can influence phenotypic and functional characteristics of these cells without affecting their lifespan.
In addition to a number of EBV-associated malignancies and infectious mononucleosis, EBV has been suggested to play a role in the pathogenesis of many other diseases, such as rheumatoid arthritis, histiocytosis, multiple sclerosis, meningitis, encephalitis and peripheral neuropathia, based primarily on the results of epidemiological studies and analysis of EBV serology (Scotet et al., 1996; Rubin & Daube, 1999
; Takeda et al., 2000
; Ascherio et al., 2001
; Kleinschmidt-DeMasters & Gilden, 2001
). Involvement of the virus in some of these pathological conditions has been difficult to prove, as EBV replication or latent infection is often not detected in the tissues or cell types that are affected in a given disease. Our results suggest that EBV replication can be involved in disease pathogenesis indirectly through effects that are mediated by EBV virions independently of viral gene expression. In addition, the cell type supporting EBV replication at, or in close proximity to, the site affected by the disease may significantly influence the activity and type of effects that are induced by EBV virions.
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ACKNOWLEDGEMENTS |
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Received 28 March 2004;
accepted 24 June 2004.