1 Department of Dermatology, University Hospital Erlangen, Hartmannstrasse 14, D-91052 Erlangen, Germany
2 Department of Immunology and Molecular Pathology, University College London, London W1P 6DB, UK
3 BioVex Ltd, Oxford OX14 4RX, UK
4 Max Delbruck Center for Molecular Medicine, MDC, Robert-Rossle-Str. 10, 13092 Berlin, Germany
5 Institute for Biomedical Technology, Department of Cell Biology, University Hospital Aachen, Aachen, Germany
Correspondence
Alexander T. Prechtel
alexander.prechtel{at}derma.imed.uni-erlangen.de
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ABSTRACT |
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INTRODUCTION |
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Immature DCs differ significantly from mature DCs in their response to specific chemokines, their ability to migrate and their capacity to stimulate immune responses (Gunn, 2003). Whilst immature DCs respond to many CC and CXC chemokines, such as CCL3 (MIP-1
), CCL4 (MIP-1
), CCL5 (RANTES) and CCL20 (MIP-3
), the expression or activity of the corresponding receptors is reduced or abolished completely in mature DCs (Delgado et al., 1998
; Lin et al., 1998
; Sallusto et al., 1998
; Sozzani et al., 1998
). As a consequence of the enhanced expression of CCR7 and CXCR4, mature DCs show an increased response to CCL19 (MIP-3
) and CXCL12 (SDF-1
) (Cavanagh & Von Andrian, 2002
; Delgado et al., 1998
; Förster et al., 1999
; Gunn et al., 1999
). The necessity of CCR7 expression for the induction of a primary immune response (Förster et al., 1999
; Parlato et al., 2001
) has also been demonstrated in vivo by studies with CCL19-knockout mice, showing that a lack of CCR7 leads to defects in lymphocyte homing and DC location (Gunn et al., 1999
). Furthermore, the possibility of enhancing an immune response against a viral infection with HSV-1 by providing the CCR7 ligands (CCL19 and CCL21) has been reported (Toka et al., 2003
).
HSV-1 belongs to the family Herpesviridae, which is divided into the subfamilies Alpha-, Beta- and Gammaherpesvirinae, depending on their tropism and pathogenicity. The life cycle of herpesviruses is usually bipartite and starts with the infection of epidermal or mucosal tissues, followed by a replication cycle. During this primary infection, the viral genes of the lytic programme are expressed, allowing the virus to enter cutaneous sensory axons. After the acute phase, additional genes are expressed that enable the virus to establish a latent infection in the neurons of dorsal root ganglia. Reactivation of the latent virus leads again to lytic replication, accompanied by the typical herpes lesions (Daheshia et al., 1998; Whitley & Roizman, 2001
). Although DCs are able to stimulate protective antiviral immune responses (Ludewig et al., 1998
) and are well-known as the most effective mediators of cytotoxic T-lymphocyte (CTL) responses to influenza virus (Nonacs et al., 1992
), Sendai virus, Moloney murine leukemia virus (Kast et al., 1988
) and HSV (Hengel et al., 1987
) in the murine model, in the case of HSV-1, the immune system fails to eliminate the virus particles generated during the acute stage, which leads in consequence to the establishment of a persistent infection (Becker, 2002
, 2003
; Whitley & Roizman, 2001
). In order to escape immune responses, gene products of many viruses interfere with DC function (Kobelt et al., 2003
). Well-known examples of viruses causing defects in DC maturation or function include HSV-1 (Kruse et al., 2000
; Mikloska et al., 2001
; Mossman et al., 2001
; Samady et al., 2003
), measles virus (Fugier-Vivier et al., 1997
), vaccinia virus (Engelmayer et al., 1999
; Jenne et al., 2001
) and human immunodeficiency virus (HIV) (Knight & Patterson, 1997
).
For HSV-1, several mechanisms that suppress a specific immune response have been identified (Pollara et al., 2004a): the immediate-early gene product ICP47 has been characterized to interfere with the transporter associated with antigen presentation (TAP) and inhibits the transport of peptides into the endoplasmic reticulum (Ahn et al., 1996
; Goldsmith et al., 1998
; Hill et al., 1995
). Furthermore, infection of mature DCs with HSV-1 leads to a significant downregulation of CD83 surface expression, resulting in a reduced capacity to stimulate T cell-mediated immunity (Kruse et al., 2000
). Recently, the RNase activity of the HSV-1 virion host shutoff protein (vhs) has been postulated to be involved in the mechanism of immune evasion by global destabilization of cellular mRNAs (Smiley, 2004
).
Identification of further mechanisms of immune evasion is of special interest as HSV-1 can cause severe medical problems, such as primary and recurrent infections of mucous membranes (e.g. herpes labialis, genital infections and encephalitis) and complications including lid, conjunctival, corneal and intraocular infections and retinitis (Dwyer & Cunningham, 2002; Liesegang, 2001
; Stumpf et al., 2002
). The exact characterization of immune-escape mechanisms is even more important for individuals undergoing chemotherapy, organ- or bone marrow-transplant recipients and patients suffering from HIV infection. These patients can develop multiple and extensive lesions and, in some cases, visceral spread may occur (Liesegang, 2001
; Stewart et al., 1995
).
In this study, we have provided evidence that DCs infected with HSV-1 show a significant change in their expression profiles of the CCR7 and CXCR4 chemokine receptors. These data were initially achieved by gene-chip analyses and confirmed by RT-PCR. Furthermore, we have demonstrated that, although the surface expression of these two chemokine-receptor molecules was affected, the observed downregulation was not due to virus-induced apoptosis. As a consequence, DCs lost their migration capability towards their respective chemokines, CCL19 and CXCL12. Finally, by using a vhs mutant virus lacking the vhs gene, we were able to demonstrate that the decreased expression was not due to vhs-induced degradation of cellular mRNA. Thus, this represents a novel viral immune-escape mechanism for HSV-1.
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METHODS |
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Generation of mature DCs.
Peripheral blood mononuclear cells (PBMCs) were isolated from a single healthy donor by sedimentation using Lymphoprep (Nycomed Pharma AS) and cultured in RPMI 1640 medium (BioWhittaker) supplemented with 1 % autologous serum, 10 mM HEPES pH 7·5 (Sigma-Aldrich), 2 mM L-glutamine (Cambrex BioScience), 100 U penicillin ml1 and 100 µg streptomycin ml1 (Sigma). PBMCs were seeded on to standard tissue-culture flasks (Nunc) for 1 h. The non-adherent fraction was washed off after 1 h with RPMI 1640 without any supplements. Immature DCs were generated in RPMI 1640 medium supplemented with 1 % autologous serum, 10 mM HEPES, 2 mM L-glutamine, 800 U granulocytemacrophage colony-stimulating factor (GM-CSF) ml1 (Wyeth) and 250 U interleukin 4 (IL4) ml1 (Strathmann). The non-adherent cells were collected after 4 days cultivation, counted and transferred into new flasks. Maturation was induced by adding 10 ng tumour necrosis factor alpha ml1 (Strathmann), 1 µg prostaglandin E2 ml1 (Sigma), 200 U IL1 (Strathmann) ml1, 40 U GM-CSF ml1 and 250 U IL4 ml1 to the medium. Maturation was completed 2 days later.
HSV-1 infection of mature DCs.
Mature DCs were collected, washed in RPMI 1640 medium without any supplements and counted. Cells were incubated with HSV-1 at an m.o.i. of 1 in RPMI 1640 medium supplemented with 20 mM HEPES pH 7·5 (Sigma) for 1 h. Cells were then washed and cultured in RPMI 1640 medium containing 1 % autologous serum, 10 mM HEPES pH 7·5, 2 mM L-glutamine, 100 U penicillin ml1 and 100 µg streptomycin ml1 until they were harvested for further experiments.
Expression analysis using Affymetrix gene chips.
Synthesis of biotin-labelled RNA, hybridization, washing and scanning of gene chips (Human Genome U133A) were performed according to the manufacturer's technical manual (Affymetrix). Briefly, total RNA was isolated from DCs and first-strand cDNA synthesis was performed by using a T7-(dT)24 primer [5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24], followed by second-strand cDNA synthesis. After cleaning up the double-stranded cDNA, biotin-labelled cRNA was produced in an in vitro transcription reaction. The resulting RNA was fragmented and used for hybridization to a gene chip. Hybridization in a gene-chip hybridization oven 640 (Affymetrix) was followed by washing and scanning steps. A first data analysis was performed with the help of the gene-chip Analysis Suite software (Affymetrix).
RT-PCR.
Infected and mock-infected DCs (106 cells) were harvested and washed at the indicated time points. Total RNA was isolated by using an RNeasy Mini kit and QIAshredder spin columns (both from Qiagen). Traces of genomic DNA were removed by DNase digestion with an RNase-free DNase set (Qiagen). Subsequently, 1 µg of each RNA was reverse-transcribed into a single-stranded cDNA, using avian myeloblastosis virus reverse transcriptase as specified by the manufacturer (Promega). Equal amounts of the resulting cDNAs were used as PCR templates to amplify the transcripts CCR7, CXCR4, -actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and to amplify the HSV-1 gene transcript ICP27.
The following PCR primers were used: CCR7 sense (5'-CTCAAGACCATGACCGATACC-3') and antisense (5'-TGAAGAGCTTGAAGAGATCGTTGC-3'), CXCR4 sense (5'-TTCTACCCCAATGACTTGTG-3') and antisense (5'-ATGTAGTAAGGCAGCCAACA-3'), GAPDH sense (5'-CACCACCATGGAGAAGGCTGG-3') and antisense (5'-GAAGTCAGAGGAGACCACCTG-3'), ICP27 sense (5'-CGAGACCAGACGGGTCTCCTGG-3' and antisense (5'-GCAGACACGACTCGAACACTCCTG-3'). The PCR cycling profile (30 cycles) used was 94 °C for 60 s, 60 °C for 60 s and 72 °C for 2 min. The reaction products were visualized by ethidium bromide staining on 2 % agarose gels. Previously described PCR primers (Cebulla et al., 2002) were used for
-actin amplification.
Fluorescence-activated cell sorting (FACS) analysis.
The phenotype of the DCs was analysed by FACS. For flow-cytometry analyses, the following mAbs were used according to the manufacturer's instructions: major histocompatibility complex (MHC) class II (HLA-DR, clone G46-6), CXCR4 (clone 12G5) and CCR7 (clone 150503), together with the isotype control IgG2a (clone G155-178) (all obtained from BD Biosciences). All antibodies were labelled with phycoerythrin (PE). Annexin V and propidium iodide (PI) staining were performed by using an Annexin V/PE/PI detection kit (Bender MedSystems) according to the manufacturer's instructions.
Migration assay.
DCs were counted, matured and resuspended in migration medium (RPMI 1640 supplemented with 500 U GM-CSF ml1, 250 U IL4 ml1, 1 % autologous serum and glutamine). Transwell inserts (Costar) with a pore size of 5 µm and 24-well plates (Nunc) were used as follows: inserts were pre-incubated with 100 µl migration medium in 24-well plates, each well containing 600 µl of the same medium. Cells (2x105) were seeded in the upper compartment. To analyse migration towards the gradient, CCL19 or CXCL12 (100 ng ml1; Tebu-Bio GmbH) was added to the lower wells. To analyse migration against a CCL19 gradient, the chemokine (100 ng ml1) was added to the upper well. DCs were allowed to migrate for 2 h. After this time period, DCs were harvested from the lower chamber and collected by brief centrifugation. The supernatant was removed and the cells were lysed by adding 25 µl PBS (BioWhittaker) and 5 µl 1 % Triton X-100 (Roche Diagnostics). The -glucuronidase activity in the lysates was determined photometrically by using p-nitrophenyl-
-D-glucuronide (Sigma) as a substrate according to the manufacturer's instructions. The resultant A405 was measured by using a Wallac Reader (Wallac Oy) and the number of migrated cells was calculated by using a separate standard curve for each cell population.
Statistical methods.
To determine the significance of variance in the experiments, data were analysed by using Student's t-test. Significance was accepted for P<0·01.
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RESULTS |
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Surface expression of CCR7 and CXCR4 is reduced in HSV-1-infected mature DCs, but not as a consequence of virus-induced cell death
The transport of processed antigens to the secondary lymphoid organs by APCs, particularly DCs, is a crucial step during the induction of primary immune responses and depends strictly on expression of the migration-mediating chemokine receptors CCR7 and CXCR4 (Cavanagh & Von Andrian, 2002; Delgado et al., 1998
; Gunn, 2003
). Thus, in the next experiments, the surface expression of CCR7 and CXCR4 molecules was analysed. Mature DCs were infected with HSV-1 wt EGFP or mock-infected and surface expression was analysed by FACS analysis at various time points after infection. Fig. 4(a)
shows the EGFP expression profile of infected mature DCs. In mock-infected DCs, no expression of EGFP was detectable (data not shown), whilst in infected cells, as early as 4 h post-infection, approximately 50 % of the cells were EGFP-positive, rising to a level of about 90 % EGFP-positive cells by 8 h post-infection. Fig. 4(b)
shows CCR7 surface expression: 4 h after infection (and simultaneous with the increase in EGFP expression), a slight loss of CCR7 surface expression was observed, but a significant downregulation was detectable by 8 h post-infection. This effect increased over time, resulting in a cell population with only approximately 40 % CCR7-positive cells after 24 h. A similar behaviour was observed for the surface expression of CXCR4 (Fig. 4c
), with downregulation of CXCR4 expression on the surface of DCs. In this case, however, the first significant decrease appeared 16 h after infection. To provide evidence that infection of mature DCs with HSV-1 wt EGFP did not result in a general loss of surface molecules, we examined MHC class II (HLA-DR) surface expression. Fig. 4(d)
demonstrates that the expression of MHC class II was not affected at any time point during HSV-1 infection.
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DCs infected with HSV-1 lose their ability to migrate towards CCL19 and CXCL12 chemokine gradients
As migration of mature DCs to secondary lymphoid organs is mediated by the chemokine receptors CCR7 and CXCR4 and their corresponding chemokines CCL19 and CXCL12, we next tried to obtain an insight into whether migration of mature DCs towards these chemokines was influenced by HSV-1 infection.
At the indicated time points, DCs infected with HSV-1 wt EGFP or mock-infected DCs were transferred to the upper well of a Transwell migration chamber. CCL19 or CXCL12 was added to the lower well of the chamber, except for the control experiment, where no chemokine was added, to check for spontaneous migration. The converse experiment (Fig. 5a and b, column 2), where the respective chemokine was added to the upper well of the migration chamber, demonstrated the influence of the cytokine on the direction of migration.
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Following the results of these experiments, CCL19 chemokine-mediated migration was analysed further. We investigated whether the observed loss of migration capability was due to the active expression of viral genes or whether inactivated viral particles could induce the same phenomenon. To address this question, viral particles were inactivated by using UV radiation and the standard infection procedure was carried out by using infectious or UV-inactivated virus at an m.o.i. of 1. As shown in Fig. 5(c, column 3), UV-inactivated HSV-1 wt EGFP was no longer able to block migration and cells infected with the inactivated virus migrated with equal efficiency when compared with mock-infected DCs.
In addition, we wanted to exclude the possibility that the insertion of an EGFP-expressing cassette into the viral genome (see Methods) might have influenced viral gene expression and functionality. Thus, we performed additional migration experiments towards a CCL19 chemokine gradient, using a standard laboratory wild-type strain of HSV-1 (not expressing GFP). Fig. 5(c, column 4) shows that, 16 h after infection with the non-EGFP wild-type strain (HSV-1 wt ang), the DCs lost their ability to migrate. This effect was comparable to DCs infected with HSV-1 wt EGFP (Fig. 5c
, column 2). The migration assays were performed with cells from at least three different donors and the results show the mean±SD.
Infection of DCs with HSV-1 prior to maturation also leads to reduced surface expression of CCR7 and CXCR4 and to reduced migration
As HSV-1 would also interact with immature DCs at sites of inflammation, we next investigated the effect of HSV-1 infection on the ability of immature DCs to express CCR7 and CXCR4 in the presence of appropriate maturation stimuli.
Immature DCs were infected at an m.o.i. of 1 with HSV-1 wt EGFP. Maturation stimuli were then added (see Methods) and DCs were allowed to mature for 24 h. After this maturation time, the surface expression of CCR7, CXC4 and MHC II was analysed by FACS and compared with that of mock-infected cells. The expression level on DCs that were left uninfected prior to maturation was taken as 100 % and the expression on infected cells was normalized to this value to give relative surface expression. As shown in Fig. 6(a), DCs that were infected with HSV-1 before adding the maturation stimuli showed an almost equal level of surface expression of MHC class II as uninfected cells (Fig. 6a
, shaded column). However, CCR7 (Fig. 6a
, filled column) and CXCR4 (Fig. 6a
, open column) expression was strongly reduced. The infection rate of these DCs was determined by EGFP expression: 24 h post-infection, more than 90 % of the DCs were EGFP-positive (data not shown). To ensure that the observed effects were not due to virus-induced cell death, the viability of the cells was determined by PI and Annexin V staining. In the case of immature DCs that were not infected prior to maturation, only approximately 5 % were Annexin V-positive and approximately 4 % were PI-positive. Similarly, 6 % of cells infected prior to maturation were Annexin V-positive and approximately 4 % were PI-positive 24 h after infection (data not shown). These experiments were repeated at least three times with cells from different donors.
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Reduction of CCR7- and CXCR4-mediated migration is not a consequence of mRNA degradation of HSV-1 vhs protein
HSV-1 vhs protein, an early gene product in the replication cycle, has been characterized in detail by several groups and is known to be involved in the degradation of cellular mRNAs (Everly et al., 2002; Oroskar & Read, 1989
; Taddeo et al., 2002
). Recently, the activity of this viral RNase was discussed as another mechanism for HSV-1 to evade the host's immune response (Smiley, 2004
). To exclude the possibility that degradation of essential cellular mRNAs was the reason for the migration block, we performed additional experiments using a
vhs mutant virus (Lilley et al., 2001
). The vhs gene in this virus strain was eliminated by insertion of an EGFP/LacZ expression cassette into the UL41 gene. Infection of mature DCs with this
vhs virus at an m.o.i. of 1 led to approximately 8085 % EGFP-positive cells (data not shown). First, we studied the influence of infection with this
vhs strain on cellular mRNA levels. Mature DCs were infected with the HSV-1
vhs EGFP mutant and total mRNA was extracted from the cells at the indicated time points after infection, essentially as described above. Fig. 7(a)
shows downregulation of the CCR7 (row 2) and CXCR4 (row 3) mRNA levels, which went hand in hand with increasing viral gene expression, as shown by ICP27 (row 1). However, the observed downregulation of CCR7 and CXCR4 was not as efficient as that caused by infection with a wild-type strain (as shown in Fig. 3
), suggesting an additional viral gene involved in this effect. GAPDH served as a control and was not influenced by the viral genes (Fig. 7a
, row 4).
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DISCUSSION |
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By using T cell-receptor transgenic mice, it has been demonstrated that the presence of professional APCs is crucial for the stimulation of HSV-1-specific CTLs in the draining lymph nodes (Mueller et al., 2002). As this activation event does not depend on the presence of virus in the draining lymph nodes, a transport of information by APCs from cutaneous infection sites to the sites of T-cell activation must be the reason for the induction of an immune response (Mueller et al., 2002
). These results indicate clearly that two of the most limiting factors in initiating an immune response soon after infection are the availability of professional APCs to present the viral antigens and their ability to get in direct contact with antigen-specific T lymphocytes in the secondary lymphoid organs.
In this respect, DCs that have taken up and processed antigens and then migrated to areas of T-cell stimulation represent the most potent group of APCs (Banchereau & Steinman, 1998). Inhibiting DC migration would therefore be a crucial step towards interfering with the induction of antiviral immune responses.
The question of whether viruses, especially HSV-1, are able to infect certain subgroups of APCs has been addressed by several groups. Most studies demonstrated that HSV-1 infects immature DCs efficiently, resulting in effects such as incomplete maturation, inadequate induction of T-cell responses or delayed apoptosis (Björck, 2004; Galvan & Roizman, 1998
; Mikloska et al., 2001
; Müller et al., 2004
; Pollara et al., 2003
). In addition, when immature DCs were infected efficiently with HSV-1 (>90 % EGFP-positive cells), we saw a dramatic effect on maturation, in the form of reduced CCR7 and CXCR4 expression, and reduced chemokine-mediated migration was observed.
However, as most HSV-1-infected individuals sooner or later develop a sufficient immune response, the disruption of immature DCs by viral infection may be overcome by the maturation and migration of so-called bystander DCs (Pollara et al., 2003; Salio et al., 1999
). In this respect, Pollara et al. (2004b)
recently reported that, subsequent to HSV-1 infection, both infected and uninfected DCs acquired a more mature phenotypic status. Furthermore, they also reported that myeloid DCs, which represent the group of APCs that first contact HSV-1 in vivo, can activate uninfected bystander DCs by releasing type I interferon, which subsequently leads to the secretion of elevated levels of IL12 p40 and p70. However, release of type I interferon was not the crucial factor for maturation of the DCs. In fact, direct contact of viral particles with DCs, mediating activation of NF-
B and p38 MAPK pathways, was responsible for DC maturation and the release of type I interferon. Taken together, these data demonstrate that DCs can mount an antiviral immune response. However, under the conditions of a naturally occurring HSV-1 infection, these activation effects are probably counterbalanced by the virus-induced inhibition of DCs. Thus, the balance between these two modes of actions will determine whether an adaptive immune response can be mounted (Pollara et al., 2004b
).
In this respect, the inhibition of DC migration, an absolute prerequisite for the induction of potent immune responses, would represent an interesting way to interfere with the establishment of such antiviral immune mechanisms. Indeed, here we have described the dramatic influence of HSV-1 on the chemokine-mediated migration of mature DCs. The importance of a coordinated switch in chemokine-receptor expression during maturation for the induction of T cell-mediated immune responses has been described by several groups. After maturation, DCs lose their responsiveness to the chemokines CCL3, CCL4, CCL5 and CCL20, but show a greatly increased sensitivity for CCL19 and CXCL12, a consequence of increased CCR7 and CXCR4 expression (Lin et al., 1998; Kellermann et al., 1999
; Parlato et al., 2001
; Salio et al., 1999
; Sallusto et al., 1998
). Furthermore, Förster et al. (1999)
were able to demonstrate that mice lacking the CCR7 molecule showed severely delayed kinetics in bringing DCs and lymphocytes into direct contact. Gunn et al. (1999)
provided further evidence for the importance of CCR7/CCL19-mediated migration by demonstrating that mice lacking expression of secondary lymphoid-organ chemokines (such as CCL19) have defects in T-cell homing and accumulation of DCs in spleen and lymph nodes. Interestingly, when we investigated the expression profiles of CXCR4 and CCR7 in HSV-1-infected mature DCs by Affymetrix gene-chip analyses and RT-PCR time-course experiments, we found a dramatic downregulation of these molecules that, in consequence, led to a reduced surface expression of these two molecules. A similar observation was made by Moutaftsi et al. (2004)
with HCMV-infected DCs. Salio et al. (1999)
reported that immature DCs infected with disabled infectious single-cycle (DISC) HSV-1 encoding GFP failed to respond to EpsteinBarr virus-induced molecule 1 ligand chemokine (ELC, another name for CCL19), whereas mature DCs did respond. However, our observations on mature DCs are strongly supported by the data reported by Toka et al. (2003)
. In their study, Toka and co-workers overexpressed, amongst others, the chemokine CCL19, which led to strong enhancement of the protective immune response against HSV-1, with the CCR7 ligand acting as a molecular adjuvant. It is possible that the increased expression of CCL19 simultaneously increases migration and immune-response induction by bystander DCs, but this hypothesis has yet to be proven.
A large number of HSV gene products are associated with immune-escape mechanisms: the immediate-early protein ICP47, for instance, blocks the presentation of antigens by inhibiting TAP (Ahn et al., 1996; Hill et al., 1995
; York et al., 1994
). Another immediate-early protein, ICP0, influences the host cell by inducing the degradation of specific cellular proteins and disturbing cellular structures (Hagglund & Roizman, 2004
; Parkinson & Everett, 2000
). Other viral genes such as UL41 and
134.5 influence the surface expression of MHC class II molecules (Trgovcich et al., 2002
).
Samady et al. (2003) also provided evidence that HSV-1 vhs protein plays a critical role in the inhibition of DC maturation. When this viral gene was deleted, the reduction of CD83 surface expression in infected mature DCs was almost abolished and the ability to become activated following HSV infection was retained. Furthermore, vhs has been characterized as a viral RNase, involved in the degradation of essential cellular mRNAs (Everly et al., 2002
; Oroskar & Read, 1989
), and has been suggested as an essential mediator of immune evasion (Smiley, 2004
). Therefore, we wanted to obtain an insight into whether the RNase activity of vhs was responsible for the observed downregulation of CCR7 and CXCR4 mRNA levels. By infecting DCs with a
vhs mutant virus, we were able to exclude the possibility that vhs plays a dominant role in the observed effects on DC migration. However, it is worth pointing out that the observed downregulation of CCR7 and CXCR4 mRNA levels was less than that with the wild-type EGFP virus, suggesting that vhs is at least involved, but is not the exclusive mediator of the observed effects. An additional clue towards the involvement of another viral gene product was provided by the migration experiment with UV-inactivated virions. As vhs is a virion protein, it was introduced into the DCs by attachment of the inactivated virions to the cells, but was not able to induce any effects due to the UV irradiation. Thus, the viral gene product responsible for this effect on mRNA remains to be identified.
In summary, the results presented in this paper suggest a previously undescribed strategy of HSV-1 to escape the immune system at one of the most important steps during the initiation of immune responses the migration of DCs from the periphery to areas of T-cell activation.
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ACKNOWLEDGEMENTS |
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Received 22 December 2004;
accepted 17 February 2005.
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