The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 7525 -7536
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reske, A.
Right arrow Articles by Chain, B. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reske, A.
Right arrow Articles by Chain, B. M.
Right arrowPubmed/NCBI databases
*Substance via MeSH

Glycoprotein-Dependent and TLR2-Independent Innate Immune Recognition of Herpes Simplex Virus-1 by Dendritic Cells1

Adi Reske*, Gabriele Pollara*, Claude Krummenacher{dagger}, David R. Katz* and Benjamin M. Chain2,*

* Department of Immunology and Molecular Pathology, University College London, Windeyer Institute of Medical Sciences, London, United Kingdom; and {dagger} Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Innate immune recognition is an important early event in the host response to herpes simplex virus-1 (HSV-1) infection. Dendritic cells (DC) play an important sentinel role in this recognition. Previous studies have shown that monocyte-derived DC (MDDC) respond to HSV-1 by up-regulation of costimulatory molecules and type I IFN release, but the molecular targets on the virus recognized by the DC have not been defined. In this study we show that MDDC recognize and respond to the four essential viral glycoproteins, gB, gD, and gHgL, independent of other viral proteins or nucleic acids. DC recognition of these four glycoproteins leads to the up-regulation of CD40, CD83, CD86, and HLA-DR and to the production of IFN-{alpha} and IL-10, but not IL-12p70. Glutaraldehyde-fixation and nonfunctional gH mutants were used to show that recognition of glycoproteins does not require membrane fusion. The nature of the recognition event was probed further by transfecting glycoproteins individually or in combination, by blocking individual proteins with Abs, or by using mutant gD constructs unable to bind to their known cognate receptors. Unexpectedly, MDDC responses were found to require expression of all four glycoproteins. Furthermore, gD mutants that cannot bind nectin-1 and/or herpesvirus entry mediator can still induce DC maturation. Finally, although HSV-1 can signal via the TLR2 receptor, this receptor does not mediate recognition of glycoproteins. Thus, the complex of the four essential HSV-1 entry glycoproteins on the cell surface can provide a target for innate immune recognition of this virus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Herpes simplex virus-1 (HSV-1)3 is a common human pathogen. Usually primary infection with the virus is limited, but HSV-1 can infect sensory neurons where it can establish lifelong latency, with sporadic reactivation and virus shedding. Viral replication during both primary and secondary reactivation is controlled by the host, and both innate and adaptive immunity are important in this control. The release of type I IFNs are thought to be an important component of the innate immune response to HSV-1 (1, 2), and people who have genetic defects in type I IFN production show increased susceptibility to HSV-1 encephalitis (3).

HSV-1 interaction with dendritic cells (DC), sentinel cells of the innate system, is an important early step in the natural history of infection, and therefore this interaction has been examined in several studies (4, 5, 6, 7, 8, 9, 10). Monocyte-derived DC (MDDC) are readily infected by HSV-1. Ultimately, infection leads to impairment of Ag-presenting function and eventual apoptosis (4, 5, 6). However, the initial effect of HSV-1 on MDDC is maturation, as well as release of type I IFNs (11). IFN in turn induces paracrine maturation of further uninfected bystander DC. HSV-1 also activates macrophages, leading to release of IFNs and proinflammatory cytokines (12).

Despite this strong evidence for involvement of innate immunity in the host response to HSV-1, the recognition mechanisms leading to innate immune activation remain incompletely understood. Some, but not all, HSV-1 strains are recognized by TLR2 (10). Additionally, DC (and other cells involved in innate immunity) have multiple TLR-dependent and TLR-independent recognition mechanisms for viral nucleic acids, some of which have been implicated in HSV recognition (13, 14, 15, 16).

In this study we analyze the interaction between MDDC and HSV-1 virion proteins, focusing on the four glycoproteins B (gB), D (gD), H (gH), and L (gL), which are essential for viral attachment/entry (17). To study the interaction between MDDC and these proteins independently of any other viral components, we develop a model in which the HSV-1 glycoproteins are expressed by transient transfection of Cos7 cells. Remarkably, a collaborative interaction involving all four entry glycoproteins (gB, gD, and the heterodimer gHgL) are both necessary and sufficient for the induction of DC maturation and for the release of cytokines. Thus, the complex of the four HSV-1 glycoproteins on the surface of Cos7 cells mediates MDDC recognition via a nucleic acid-independent and TLR2-independent pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Antibodies

The following mAbs were used: CD3 (supernatant mouse mAb UCH T1, IgG1, gift of Prof. P. Beverley, Edward Jenner Institute for Vaccine Research, Newbury, U.K.); CD2 (clone RPA-2.10, IgG1; eBioscience); CD19 (supernatant mouse mAb BU12, IgG1, gift of D. Hardie, Birmingham University, Birmingham, U.K.); HLA-DR (supernatant mouse mAb L243, IgG2a, gift of Prof. P. Beverley); CD14 (supernatant mouse mAb HB246, IgG2b, gift of Prof. P. Beverley); CD1a (supernatant mouse mAb NA1/34, IgG2a, gift of Prof. A. McMichael, John Radcliffe Hospital, Oxford, U.K.); CD86 (supernatant mouse mAb, BU63, IgG1, gift of D. Hardie); CD83 (clone HB15e; eBioscience); CD40 (clone 5C3; eBioscience); CD13-PE (mouse mAb WM15, IgG1; BD Pharmingen); IgG1 isotype control Ab to Aspergillus niger glucose oxidase (mouse mAb DAK-GO1, IgG1; DakoCytomation); PE-conjugated goat anti-mouse Ig (DakoCytomation); FITC-conjugated rabbit anti-mouse Ig (DakoCytomation); Alexa Fluor-conjugated goat anti-mouse IgG (Invitrogen); HSV-gD (supernatant mouse mAb LP2, IgG1, gift of Prof. T. Minson, University of Cambridge, Cambridge, U.K.); HSV-gD (supernatant mouse mAb AP7, IgG1, gift of Prof. T. Minson); HSV-gHgL (supernatant mouse mAb LP11, IgG1, gift of Prof. T. Minson); HSV-gB (purified IgG 2153, gift of Prof. T. Minson).

Plasmids

The following plasmids were used for transfection of Cos7 cells: HSV-gBgD and HSV-gHgL (gifts of Prof. T. Minson); HSV-gB (pSR175), HSV-gD (pSR390), HSV-gH (pHC138), and HSV-gL (pCMV3gL-1) (gifts of Prof. G. H. Cohen and Prof. R. J. Eisenberg, University of Pennsylvania, Philadelphia, PA); HSV-gH {sigma}Leu (L382P, GLL384–386WPP) (18), HSV-gH HR1-EL (E450G, L453A) (19), and HSV-gH HR2-down2 (LARA570–573AVPQ) (20) (gifts of Prof. G. Campadelli-Fiume, University of Bologna, Bologna, Italy); and HSV-pDL485 gD(37C–302C) (21), HSV-pDL449 gD(D30A) (22), and HSV-pDL490 gD(3C–38C) (23) (gifts of Prof. G. H. Cohen and Prof. R. J. Eisenberg).

MDDC preparation

MDDC were prepared from 120 ml fresh whole blood from healthy volunteers. Mononuclear cells separated on Lymphoprep (Nycomed Pharma, Oslo, Norway) (400 g, 30 min) were incubated in six-well tissue culture plates (BD Falcon from BD Biosciences) at 37°C/5% CO2 in RPMI 1640 (Invitrogen) supplemented with 10% FCS (PAA Laboratories), 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine (all from Clare Hall Laboratories, Cancer Research U.K., London, U.K.) as complete medium. After 2 h, nonadherent cells were removed and the adherent cells were cultured in fresh medium with 100 ng/ml human recombinant GM-CSF and 50 ng/ml IL-4 (both gifts from Schering-Plough Research Institute, Kenilworth, NJ). After a 4-day incubation, loosely adherent cells were collected and any remaining lymphocytes depleted by incubation with CD3, CD2, and CD19 mAb, followed by anti-mouse IgG-coated immunomagnetic Dynabeads (Dynal Biotech). The DC were cultured for a further 3 days in complete medium with fresh GM-CSF and IL-4 at a concentration of 5 x 105 DC/ml and then used as immature DC for stimulation. Throughout this report these cells will be referred to as MDDC.

Monocyte-derived Langerhans cells (MDLC) were prepared similarly to MDDC, but with an addition of 10 ng/ml TGF-β (R&D Systems) on day 0 and day 4 of culture.

Plasmacytoid DC (pDC) preparation

pDC were isolated from PBMC by immunomagnetic cell sorting (MACS CD304 microbead separation kit, Miltenyi Biotec). In brief, 120 ml fresh peripheral blood was separated on Lymphoprep (400 g, 30 min) and the PBMC were incubated with CD304 Abs (Miltenyi Biotec) (15 min, 4°C). Bead-bound cells were then separated by magnetic separation, counted, and cultured for analysis.

Virus preparation

The HSV-1 construct used was derived from strain 17+ containing a cassette of a CMV immediate early promoter that drives expression of GFP (Clontech Laboratories) and a respiratory syncitial virus promoter that drives expression of β-galactosidase. This cassette is inserted into the UL43 gene, as described elsewhere (24). The virus was propagated on confluent baby hamster kidney cells in DMEM supplemented with 10% FCS and 100 U/ml penicillin/streptomycin (complete DMEM). Forty-eight hours after infection, cellular debris was removed by low-speed centrifugation (3000 x g, 30 min, 4°C). The supernatant was removed immediately and filtered through a double filter (0.65 and 0.45 µm, respectively) before being centrifuged at 16,000 x g for 2 h at 4°C. The supernatant was discarded and the viral pellet was resuspended gently in 1 ml DMEM. The prepared aliquots were stored at –70°C until use. The virus stock used in this entire study had a titer of 1 x 109 PFU/ml as determined by plaque assay on baby hamster kidney cells. This virus preparation is referred to as HSV/GFP.

HSV/GFP fixed virus was inactivated by incubating virus in 0.05% glutaraldehyde (Sigma-Aldrich) in PBS for 2 min. Quenching was performed by diluting the virus in complete medium before addition to the cells. This virus is referred to as Fix-HSV/GFP.

Cos7 cell culture transfection

Cos7 cells were grown in complete DMEM. Cultured cells were transfected transiently using FuGENE 6 transfection reagent (Roche Diagnostics) with vectors expressing gB, gD, gH, and gL and cultured for 48 h to allow expression at the cell surface. Glycoprotein expression on transfected Cos7 cells was measured by flow cytometry and confocal microscopy using glycoprotein-specific mAbs.

Alternatively, transfected Cos7 cells were inactivated by washing in PBS and then fixing in 0.05% glutaraldehyde (Sigma-Aldrich) in PBS for 2 min. Excess glutaraldehyde was washed off with PBS before addition of complete DMEM. These cells are referred to as Fix-Cos7.

DC stimulation

Day 7 purified immature DC (2 x 105 to 5 x 105) were stimulated with LPS (100 ng/ml; E. coli 0111:B4, InvivoGen).

Immature DC were infected with HSV/GFP at a multiplicity of infection (MOI) of 5 or with Fix-HSV/GFP at an equivalent MOI of 10. GFP expression upon infection was determined by fluorescence microscopy or flow cytometry. Alternatively, transfected Cos7 cells and Fix-Cos7 cells were overlayed with immature DC 48 h posttransfection. Preliminary dose-response experiments have shown a maximum DC response with 3 x 104 Cos7 cells per 3 x 105 DC (ratio 1:10) (data not shown). Recombinant soluble truncated gD (gD285t) (25) was used to determine whether gD was solely responsible for activating DC. All DC for analysis were harvested 24 h poststimulation.

Flow cytometry

Nonspecific Ab binding was blocked with 10% goat serum or 10% rabbit serum (20 min, 4°C). DC were then stained for specific surface markers by incubation first with the relevant mAb (30 min, 4°C) followed by 1/20 diluted PE-conjugated goat anti-mouse Ig or FITC-conjugated rabbit anti-mouse Ig (30 min, 4°C). After removal of secondary conjugate, DC/Cos7 cell mixtures were incubated in 1% mouse serum (10 min, 4°C) and then further stained with PE-conjugated anti-CD13 (20 min, 4°C) to differentiate between Cos7 cells and DC. All stained cells were immediately examined by flow cytometry using a FACScan flow cytometer (BD Biosciences) and analyzed with WinMDI software.

T cell proliferation assay

Allogeneic T cells from HLA-mismatched donors were obtained from the nonadherent population of peripheral blood mononuclear cell fraction and cryopreserved in FCS containing 10% DMSO (Sigma-Aldrich) at –70°C. Cells were thawed rapidly (37°C), and activated T cells, B cells, monocytes, and macrophages were depleted by incubation with CD19, HLA-DR, and CD14 monoclonal Abs for 30 min on ice. Cells were washed and then mixed with magnetic microbeads and separated on magnetic columns. T cells were used immediately after purification.

Titrations of purified DC, either untreated, HSV-1 infected, cocultured with transfectd Cos7 cells, or LPS treated were incubated at 37°C, 5% CO2 with allogeneic T cells in flat-bottom 96-well microtiter plates for 6 days and then pulsed with 1 µCi of [3H]thymidine (ICN Biomedicals) for the final 18 h of culture. Cells were harvested, and T cell proliferation was measured by liquid scintillation counting (Microbeta Systems). All assays were performed in triplicate. Results were expressed as counts per minute.

ELISA

After 18–24 h stimulation, supernatant was harvested and used to assay for cytokine secretion. IFN-{alpha} was measured in cell supernatant using ELISA according to the manufacturer's protocol (PBL Biomedical). Sandwich ELISA was set up for the detection of IL-12p70, IL-10, and TNF-{alpha}. ELISA plates were coated overnight with 1 µg/ml IL-12p70, IL-10, or TNF-{alpha} capture Abs (clones BT21, JES3–9D7, or MAb1, respectively, eBioscience) in PBS at 4°C. Next, the plates were blocked with 1% BSA in PBS for 2 h at room temperature (RT). After washing 3x with PBS + 0.05% Tween 20, the plates were incubated with 50 µl sample for 60 min at RT. Standards (eBioscience) were diluted in culture medium. Again, after washing 3x with PBS-Tween, the plates were incubated with 50 µl of 1 µg/ml biotin anti-human IL-12, anti-human IL-10, or anti-human TNF-{alpha} detection Asb (clones C8.6, JES3–12G8, or MAb11, respectively, eBioscience) in PBS-Tween for 60 min at RT. Next, the plates were washed 3x with PBS-Tween and incubated with 50 µl streptavidin HRP (R&D Systems) conjugated in PBS-Tween for 30 min at RT. After washing 3x with PBS-Tween, HRP presence was detected using TMB substrate (eBioscience) and MRX Revelation plate reader (450 nm) and the Revelation 4.21 software (Dynex Technologies). Concentrations were determined in accordance with the standards.

Confocal microscopy

Fixed Cos cells and DC were examined by confocal microscopy using a Leica confocal microscope. Cells were fixed with 3.7% paraformaldehyde for 15 min before incubation with Abs. Incubation with primary Ab anti-gD (LP2) was performed at RT for 1 h. Incubation with secondary Abs (FITC-conjugated rabbit anti-mouse Ig or Alexa Fluor-conjugated goat anti-mouse IgG) was performed at RT for 1 h. All cells stained with the DNA stain DAPI to identify the nucleus. The images were analyzed using the Leica confocal software.

TLR2 activation

Engineered HEK293 cells stably transfected with the TLR2 and secreted alkaline phosphatase gene placed under the control of NF-{kappa}B (InvivoGen) were used to test for viral glycoprotein triggering of the TLR. Cells were grown in complete medium (DMEM supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, and 100 µg/ml Normocin). At 80% confluence, cells were removed and cocultured in HEK-Blue detection medium (5 x 104 cells) (InvivoGen) with Cos7 fibroblasts expressing either all four HSV-1 entry glycoproteins (gBgD/gHgL) or just gBgD, gHgL, gB, gD, mock transfection, or no transfection (Cos7). After an overnight coculture in 37°C, 5% CO2, cells were analyzed for secreted alkaline phosphatase using the MRX Revelation plate reader (633 nm) and the Revelation 4.21 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HSV-1 entry glycoproteins induce MDDC maturation

MDDC prepared according to the protocol described were a homogeneous population of cells expressing high levels of CD1a and CD13 (Fig. 1A). The population contained <1% CD19+ B lymphocytes (Fig. 1A) or CD3+ T lymphocytes (not shown) and expressed low levels of CD14 (Fig. 1A, right panel), consistent with the MDDC phenotype.


Figure 1
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 1. Phenotype of immature DC and glycoprotein expression by Cos7 cells. MDDC were prepared as described in Materials and Methods and analyzed by flow cytometry. A, The forward/side scatter (FSC/SSC) profile is shown (left panel). Subsequent analysis is always gated on the major live cell population shown as R1. R1 cells almost all expressed both the myeloid marker CD13 and the MDDC marker CD1a. R1 cells contained few B cells (stained with CD19) and express low levels of CD14 (two right panels). Filled profile shows staining with IgG control. B, Cos7 fibroblasts were transiently transfected with vectors expressing gB, gD, gH, and gL and cultured for 24, 48, or 72 h to allow expression of the glycoproteins at the cell surface. Expression of each glycoprotein was monitored by flow cytometry using Ab 2153 for gB, Ab LP2 for gD, and Ab LP11 for gH/gL. A representative of three or more experiments is shown in each panel.

 
To investigate the interactions of HSV-1 surface glycoproteins with DC independently of the effects of other virus components, Cos7 fibroblasts were transfected to express HSV-1 glycoproteins (gB, gD, and the heterodimer gHgL). Expression was monitored using Abs specific for each glycoprotein either by flow cytometry (Fig. 1B) or by confocal microscopy (data not shown). Optimum expression levels were observed 48 h after transfection. Therefore, Cos7 cells that had been transfected for 48 h were used to stimulate DC in subsequent experiments. Cos7 cells expressing the four glycoproteins were frequently multinucleate (see below and Fig. 5A), presumably as a result of cell–cell fusion induced by the glycoproteins (26).


Figure 5
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 5. Glutaraldehyde fixation inactivates HSV-1, but does not block glycoprotein-mediated MDDC maturation. A, Immature MDDC were cultured with LPS (100 ng/ml), viable HSV/GFP (MOI of 5), 0.05% glutaraldehyde-fixed HSV/GFP, or both LPS and fixed HSV/GFP. Expression of CD86 was analyzed after 18 h (open histograms). The inserted histograms show GFP expression to confirm that the HSV/GFP has been inactivated by the concentration of glutaraldehyde used in these experiments. B, Immature DC were cocultured with mock-transfected Cos7 cells, viable Cos7 cells transfected with all four glycoproteins, Cos7 cells transfected with all four glycoproteins and then fixed with 0.05% glutaraldehyde, and fixed transfected Cos7 cells and LPS (100 ng/ml). Expression of CD86 was analyzed after 18 h coculture (open histograms). Filled histograms represent isotype controls for staining. Figures show one representative of three experiments. C, As in B, but figure shows the MFI values relative to MFI of MDDC cocultured with all four glycoproteins from three independent experiments. Error bars represent SEM. Statistical analysis was performed using Student's t test. *, p < 0.01; **, p < 0.05.

 
DC were cocultured with HSV-1 (a MOI of 5 PFU/cell was used, which gave infection rates of around 80%), with the TLR4 ligand LPS as a positive control for the induction of maturation, or with Cos7 cells transfected previously with all four glycoproteins (gB, gD, and gHgL) or with mock-transfected Cos7 cells. After 24 h of coculture, all cells were collected and stained for CD13 to differentiate the MDDC from Cos7 cells (Fig. 2A, left two panels) because Cos7 cells were negative for this marker. CD13+ cells were gated (Fig. 2A, right two panels) and the expression of maturation markers CD86, CD83, and HLA-DR on MDDC was measured. These markers were selected because they exemplify the primary Ag signal (HLA-DR), the second costimulatory signal (CD86), and the final step of DC activation (CD83) and because we have found them to be the most reliable markers of MDDC activation/maturation. Both representative flow cytometry histograms from individual experiments (Fig. 2B) and mean fluorescence levels obtained from several experiments (Fig. 2C) are shown. In some experiments CD40 was also included and showed changes that mirrored those of the markers shown.


Figure 2
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 2. Cos7 cells transfected with all four HSV-1 surface glycoproteins induce DC maturation. A, Flow cytometric analysis of MDDC/Cos7 cell cocultures. Live cells present in DC/Cos7 cocultures were identified by gating on the FSC/SSC region R1. These cells were further divided into CD13+ MDDC and CD13– Cos7 cells (second panel). The levels of maturation markers CD86, CD83, and HLA-DR were analyzed on MDDC alone by gating on the CD13+ region R2 (right two panels). B, Immature DC were cultured in medium (top row) or stimulated with either whole virus (HSV/GFP, MOI of 5), LPS (100 ng/ml), or Cos7 cells transfected with all four glycoproteins. The levels of CD86, CD83, and HLA-DR were measured by flow cytometry as in Fig. 1 (for top three rows) or as described above in A (for bottom row). Filled histograms show staining using Ig control Ab. The vertical dotted line indicates the mode of the distribution in unstimulated immature MDDC. The figure shows one representative experiment from at least three independent experiments from three different donors. C, As in B, but figure shows the mean fluorescent intensity (MFI) values from three independent experiments. An additional group of MDDC cocultured with mock-transfected Cos7 cells is included, but no significant differences were seen between mock-transfected Cos7 cells, untransfected Cos7 cells, or MDDC alone. The experiments shown in the left and right panels are shown separately because they were conducted using different batches of secondary Ab, and hence the absolute values of fluorescence cannot be directly compared. Error bars represent SEM. Statistical analysis was performed using Student's t test. *, p < 0.05.

 
In agreement with previous studies, HSV-1 virions induced up-regulation of all three surface markers, consistent with the induction of MDDC maturation, albeit the levels were statistically lower than those seen in the presence of LPS (Fig. 2). However, Cos7 cells (and CHO cells, data not shown) transfected with all four essential HSV glycoproteins also induced strong up-regulation of all three surface markers (Fig. 2B, bottom panels, compared with top panel (unstimulated); Fig. 2C). Levels of CD86 detected on MDDC cocultured with transfected Cos7 cell were similar to those on LPS-matured MDDC (Fig. 2, B and C) and higher than those obtained by stimulation with HSV/GFP (Fig. 2, B and C). CD83 and HLA-DR expression on MDDC varies over a broader range after exposure to transfected Cos7 and up-regulation is mostly similar to that induced by HSV virions (Fig. 2B). Mock-transfected cells (Fig. 2C) did not mature MDDC significantly. Thus, the four HSV-1 glycoproteins are sufficient to mature MDDC independently of any other viral components.

Having shown that the four glycoproteins are able to up-regulate DC maturation markers, the ability of these mature DC to induce T cell proliferation was measured in an allogeneic T cell proliferation assay. Similar to previous studies, HSV-1 virions impaired the ability to induce T cell proliferation. However, compared with LPS-stimulated DC, which showed a significant increase in T cell proliferation, DC matured by the glycoproteins expressing Cos7 showed no increase in allogeneic T cell proliferation similar to unstimulated and mock-transfected DC (Fig. 3A).


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 3. Cos7 cells transfected with all four glycoproteins do not induce allogeneic T cell activation. A, MDDC cocultured with Cos7 cells expressing the four glycoproteins mock transfected or infected with HSV (MOI of 1), stimulated with LPS (100 ng/ml), or unstimulated were used to stimulate allogeneic T cell proliferation. Shown are the mean cpm of triplicate wells of T cells. Error bars represent SEM. Statistical analysis was performed using Student's t test. *, p < 0.05. The figure is one representative experiment from three independent experiments from three different donors. B, Immature MDDC were cocultured with Cos7 cells expressing four glycoproteins, mock transfection, or infected with HSV, stimulated with CD40L, or unstimulated. CCR7 expression was measured by flow cytometry 18 h poststimulation. Dotted histograms represent the isotype control. The figure represents one representative experiment from two independent experiments from two different donors.

 
Similar results were seen when the DC were stained for CCR7, a chemokine thought to control the migration of mature DC to the lymph nodes. Unstimulated DC, HSV-1-infected DC, and DC cocultured with transfected Cos7 cells did not show an up-regulation of the CCR7 expression. As a positive control, CD40L induced up-regulation of CCR7 on DC as expected (Fig. 3B).

HSV-1 surface glycoprotein interaction with MDDC induces release of type I IFN, IL-10, but not IL-12 or TNF-{alpha}

The release of type I IFN is another important consequence of interaction between HSV-1 and MDDC (11). Therefore, the secretion of IFN-{alpha} by DC exposed to entry glycoproteins was investigated. Supernatants from MDDC cocultured with Cos7 cells transfected with all four glycoproteins, with mock-transfected Cos7 cells, or from MDDC cocultured with HSV-1 or poly(I:C) (50 µg/ml) for 16 h were collected and IFN-{alpha} was measured by ELISA (Fig. 4A). MDDC cocultured with the four glycoproteins produced IFN-{alpha}, as did DC cocultured with whole virus, or with poly(I:C). Neither DC cultured with mock-transfected Cos7 cells, nor Cos7 cells alone, produced any detectable IFN-{alpha}. In contrast, neither HSV-1 virions nor the glycoproteins induced detectable IL-12p70 (Fig. 4B), although the MDDC were capable of releasing large amounts of this cytokine under appropriate stimulation with LPS. Both MDDC cocultured with the four glycoproteins and with whole virus produced low amounts of IL-10, compared with the mock-transfected Cos7 cells or the transfected Cos7 cells on their own, which produced no detectable IL-10 (Fig. 4C). Neither the MDDC cocultured with Cos7 cells transfected with the four glycoproteins nor the MDDC exposed to HSV-1 virions produced significant levels of TNF-{alpha} (Fig. 4D).


Figure 4
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 4. ELISA for IFN-{alpha}, IL-12p70, IL-10, and TNF-{alpha}. Supernatant from TLR3- (poly(I:C)) or TLR4- (LPS) stimulated MDDC, HSV-infected MDDC, and MDDC cocultured with transfected Cos7 cells were harvested 16 h after stimulation and the cytokine secretion (IFN-{alpha} (A), IL-12p70 (C), IL-10 (B), and TNF-{alpha} (D)) was determined by ELISA. All ELISA experiments were performed in duplicates. The figure shows one representative experiment from at least three independent experiments.

 
Cell fusion is not the trigger for DC maturation

Because the complex of all four glycoproteins induces extensive cell fusion (27) (see below and Fig. 6A), it was important to determine whether the fusion event per se was the trigger for induction of DC maturation. One way to block fusion is to fix the glycoproteins, thus blocking the conformational changes required. As described previously, HSV-1/GFP virions fixed in 0.05% glutaraldehyde still induced CD86 up-regulation (11) (Fig. 5A). The DC exposed to Fix-HSV/GFP matured further in response to LPS, confirming their viability. Cos7 cells transfected with all four glycoproteins, and then fixed in 0.05% glutaraldehyde, also induced DC maturation as efficiently as did unfixed cells (Fig. 5, B and C). Similar results were observed with CD83 expression (data not shown). Thus, fixation does not block the ability of the glycoproteins to induce DC maturation.


Figure 6
View larger version (45K):
[in this window]
[in a new window]

  		FIGURE 6. gH mutants lacking fusogenic activity do not induce DC maturation. A, Control-transfected Cos7 cells (gBgD/gL), Cos7 cells transfected with gBgD/gHgL, or wild-type gB, gD, and gL together with mutants of gH as shown were cultured for 48 h and stained for gD (to identify transfected cells; LP2 and Alexa Fluor-conjugated secondary Ab) and DAPI (to identify nuclei). Presence of multinucleated cells was detected by confocal microscopy. The figures show a high magnification view of a single field, but examination of many fields showed that in the cultures containing Cos7 cells expressing all four wild-type glycoproteins, >90% of cells expressing gD were multinucleate. In contrast, in cultures of control-transfected cells or cells transfected with mutant gH, no multinucleated cells were observed. B, MDDC were cocultured for 18 h with Cos7 cells transfected with either all four wild-type glycoproteins gBgD/gHgL or with wild-type gBgD/gL and mutant gH as shown. Expression of CD86 on CD13+ DC was measured by flow cytometry as described in Fig. 2A (open histogram). The filled histograms represent immature MDDC. The vertical dotted lines represent the levels of these markers in mature DC. Shown is one representative experiment from at least three independent experiments. C, As in B, but figure shows the MFI values relative to MFI of MDDC cocultured with all four glycoproteins from three independent experiments. Error bars represent SEM. Statistical analysis was performed using Student's t test. *, p < 0.01; **, p < 0.1.

 
Various reports have suggested that gHgL is one of the components required for viral fusion during HSV-1 entry, and the fusion domain within gH has been studied extensively (18, 28). Therefore, we confirmed that fusion was not necessary for the induction of DC maturation by HSV glycoproteins by using gH mutants (see Materials and Methods) that lacked fusogenic activity (18, 19, 20). We transfected these mutants together with gB, gD, and gL into Cos7 cells. In the Cos7 cells expressing all four wild-type glycoproteins, >90% of cells expressing gD were observed to be multinucleated as a result of glycoprotein-induced Cos7 cell fusion (Fig. 6A, second panel). In contrast, Cos7 cells transfected with gB, gD, gL, and any of the mutant gH glycoproteins showed no evidence of multinucleate cells (Fig. 6A, panels 3–5), confirming that these mutants were unable to induce cell fusion. However, Cos7 cells transfected with the mutated gH were similar to wild-type gH transfection in their ability to induce DC maturation (Fig. 6, B and C; CD83 data not shown). In agreement with the data obtained using fixed transfected Cos7 cells, these data suggest that membrane fusion is not required for HSV-1 glycoprotein-induced MDDC activation.

All four of the major HSV-1 surface glycoproteins are necessary for DC maturation

Having shown that Cos7 cells expressing all four glycoproteins are able to induce DC maturation, we transfected Cos7 cells with either one, two, or three glycoproteins. Because gL on its own is not present on the cell surface and because gH requires gL for cell surface expression (29, 30), we always used gHgL together and investigated its role as a complex. After coculture flow cytometry (Fig. 7) showed that neither gBgD (Fig. 7A, third row), nor gHgL (Fig. 7A, fourth row), nor gB or gD alone (not shown) induced significant up-regulation of either CD86 or CD83 or the induction of IFN-{alpha} (not shown). Furthermore, DC formed distinct clusters around gB/gD/gH/gL-transfected Cos7 cells resembling the features noted postinfection in our previous study (30), while no clustering was observed when mock or three or less glycoproteins were transfected (Fig. 7A). To confirm the requirement for all four glycoproteins in inducing DC maturation, glycoprotein-specific Abs were added to the cocultures. Abs to gHgL (LP11) and gD (AP7) inhibited DC maturation independently in the cocultures (Fig. 7, A and B), as well as infection of DC by HSV-1 (data not shown). We also tested one Ab to gB (2153), which did not block either DC activation or viral infection. Finally, because soluble gD has recently been shown to induce cell signaling (31), we tested the ability of recombinant gD to induce DC maturation. No effect was seen at any concentration tested (Fig. 7D).


Figure 7
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 7. All four HSV-1 surface glycoproteins are necessary for phenotypic changes in DC. A, MDDC were cocultured for 24 h with mock-transfected Cos7 fibroblasts or with Cos7 cells transfected with either gBgD/gHgL, gBgD only, or gHgL only. Expression of CD86 and CD83 on CD13+ MDDC was measured by flow cytometry as described in Fig. 2A (open histogram). The filled histograms represent IgG control staining. The dotted lines indicate the mode of the distribution in unstimulated immature MDDC. Shown is one representative experiment from at least three independent experiments. MFI values (data not shown) show statistically significant differences (p < 0.01, Student's t test) between Cos7 cells transfected with all four glycoproteins and Cos7 cells transfected with either gBgD or gHgL alone. Images of the cells in culture after 24 h were obtained by inverted phase contrast microscopy. Filled arrows indicate MDDC; dashed arrows indicate Cos7 cells. B, MDDC were cocultured for 24 h with Cos7 cells transfected with gBgD/gHgL and anti-gD- (AP7) or anti-gHgL- (LP11) specific Abs or IgG2a control Abs. The Abs were added to the Cos7 cells 1 h before the addition of MDDC. CD86 was measured by flow cytometry (open histograms). Filled histograms represent IgG staining control. Results are representative of three or more independent experiments. C, As in B, but figure shows the MFI values relative to MFI of MDDC cocultured with all four glycoproteins from three independent experiments. Error bars represent SEM. Statistical analysis was performed using Student's t test. *, p < 0.05. D, Immature MDDC were stimulated with increasing concentrations of soluble gD (12.5 g/ml and 25 µg/ml) or LPS (100 ng/ml). Expression of CD86 was measured by flow cytometry. Filled histogram represents immature unstimulated MDDC.

 
The DC response to HSV-1 glycoproteins does not require herpesvirus entry mediator (HVEM), nectin-1, or TLR2

The gD receptor HVEM is a member of the TNF receptor family and is known to transduce activation signals in response to its ligand LIGHT (32, 33, 34). Although gD alone failed to induce DC maturation, it remained possible that gD/HVEM interaction was one essential component in the overall activation process. To test this possibility we made use of three gD mutants, one of which cannot bind HVEM (gD(D30A)), one that cannot bind to nectin-1 (gD(A3C, Y38C)), and one that binds neither receptor (gD(V37C–A302C)) (21, 22, 23). Remarkably, all three mutants induced MDDC maturation when expressed together with gB and gHgL, although levels of CD86 were consistently lower than those observed with wild-type gD (Fig. 8).


Figure 8
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 8. DC maturation response to viral glycoproteins does not require binding to HVEM and/or nectin-1. MDDC were cocultured for 24 h with Cos7 cells transfected with either all four wild-type glycoproteins gBgD/gHgL or with wild-type gBgHgL and mutant gD as shown. Expression of CD86 and HLA-DR on CD13+ DC was measured by flow cytometry as described in Fig. 2A (white histogram). The filled histograms represent IgG control staining. The vertical dotted lines represent the mode of the distribution for immature (left lines) and LPS matured (right lines) MDDC. Shown is one representative experiment from at least three independent experiments from three different donors.

 
HSV-1 has previously been shown to activate innate immune responses via the TLR2 receptor, although the target of the receptor on the virion remains undefined (10). To test whether the glycoprotein-induced DC activation described in this paper is mediated via TLR2, we made use of an indicator line in which secreted alkaline phosphatase (sAP) is driven by a reporter construct in a cell line expressing TLR2. As shown in Fig. 9, the TLR2 ligand Pam3Cys was a powerful activator of sAP expression in this system. HSV-1 also induced a smaller, but consistent response, in agreement with previous reports (10). In contrast, Cos7 cells expressing all four glycoproteins failed to stimulate any significant sAP activity.


Figure 9
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 9. DC response to HSV-1 glycoproteins is independent of TLR2. HEK-293 cells stably expressing TLR2 were stimulated with whole virus (MOI of 5), Pam3Cys (100 ng/ml), or H2O or were cocultured with Cos7 cells expressing either all four HSV-1 entry glycoproteins (gBgD/gHgL) or just gBgD, gHgL, gB, gD, mock transfection, or no transfection (Cos7). After an overnight coculture, cells were analyzed for secreted alkaline phosphatase on a plate reader at 633 nm. Histograms represent the mean values of triplicate readings. Shown is one representative experiment from three independent experiments. Error bars represent SEM. B, Monocyte-derived Langerhans cells were prepared as described in Materials and Methods. Cells were infected with HSV (MOI of 5), left unstimulated, or stimulated with TLR2 ligand, Pam3Cys, and analyzed by flow cytometry for CD86. Dotted vertical line represents the unstimulated immature MDLC phenotype. The small histograms represent HSV infectivity as measured by GFP expression. C, Monocyte-derived Langerhans cells were cocultured with Cos7 cells transfected with all four glycoproteins, mock transfected, or they were cultured in medium only. CD86 was measured by flow cytometry. Vertical line represents the phenotype of immature MDLC. Shown is one representative experiment from at least three independent experiments.

 
To confirm that glycoprotein activation was TLR2 independent, we used MDLC, which have been shown previously to express no or very low levels of TLR2 (35, 36). We confirmed that these cells did not respond to the TLR2 ligand, Pam3Cys (Fig. 9B). MDLC, however, showed up-regulation of CD86 in response both to HSV-1 infection (Fig. 9B) and in response to Cos7 cells transfected with all four glycoproteins (Fig. 9C).

HSV-1 glycoproteins do not induce maturation in plasmacytoid DC

Having shown that the four glycoprotein are necessary and sufficient for the maturation of MDDC, we next studied the glycoprotein effect on peripheral CD303+CD304+ plasmacytoid DC (pDC). Although HSV-1 virion induced the up-regulation of the maturation marker CD80 in the pDC (Fig. 10A), neither the four glycoproteins nor the single glycoproteins induced any maturation (Fig. 10B). Interestingly, in HSV-1-infected pDC, the activation marker CD83 was not up-regulated. Supernatants from pDC cocultured with Cos7 cells expressing the four glycoproteins, mock-transfected Cos7 cells, HSV-1-infected pDC, and CpG-stimulated pDC were then used for the measurement of type I IFN and TNF-{alpha}. In accordance with the flow cytometry results, both the CpG-stimulated and the HSV-1-infected pDC produced both cytokines, while the pDC cocultured with the mock-transfected and the four glycoprotein-transfected Cos7 cells did not (Fig. 10C).


Figure 10
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 10. HSV-1 entry glycoproteins do not induce pDC maturation. Primary pDC were isolated from peripheral blood as described in Materials and Methods. A, Cells were infected with HSV-1 or stimulated with CpG, and the expression of CD80 and CD83 was measured by flow cytometry. Open histograms represent the unstimulated pDC. B, Cells were cocultured with Cos7 cells expressing the four HSV-1 entry glycoproteins or single glycoproteins, and the expression of CD80 and CD83 was measured. Dotted histograms represent pDC cocultured with mock-transfected Cos7 cells. Shown is a representative of two independent experiments from two different donors. C, Supernatants from stimulated DC were used to measure TNF-{alpha} and IFN-{alpha} by ELISA. All ELISA experiments were performed in duplicates. Shown is one representative experiment from at least two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
During the past few years there has been a rapid increase in the number of recognized pathogen-associated molecular pattern (PAMP)/pattern recognition receptor (PRR) interactions that mediate activation of innate immunity. Several of these studies have looked at recognition of HSV (10, 16, 37, 38), but they have primarily focused on detection nucleic acids within the cell. In this study we have focused on analyzing the interaction between DC and a set of viral proteins found on the surface of the HSV-1 envelope.

We have shown previously that UV-inactivated, or formaldehyde-fixed virus, unable to express any viral gene products de novo, is still able to induce DC to mature and to activate T cells (11). This suggested that activation is mediated by components of the viral particle itself. The viral particle, however, contains many components that could in principle mediate DC activation. The first hypothesis tested in this study was therefore whether the four entry glycoproteins, gB, gD, gH, and gL, that are involved in entry and membrane fusion, function as HSV-1 PAMPs responsible for activation of DC.

To establish the role of HSV glycoproteins in the absence of other viral components, we have adapted a model developed initially for the study of glycoprotein-mediated attachment and fusion (27). In this model, DC are stimulated by Cos7 cells expressing HSV-1 glycoproteins on their surface as a surrogate for exposure to intact viral particles. This strategy has proved robust, being much less susceptible to LPS contamination than is seen when using purified recombinant glycoproteins, and allows a very flexible structure/function analysis of the role of individual glycoproteins and their combinations. This model system was then used to show clearly that the four entry glycoproteins were able to induce DC phenotypic maturation without requirement for any other viral component. This recognition pathway may be important in vivo, because glycoproteins are expressed at high levels on infected keratinocytes within HSV lesions (39). Interaction between DC (or Langerhans cells) and infected keratinocytes may provide one important signal for the initiation of adaptive immunity.

Our study showed, however, that the even though the viral entry glycoproteins were able to induce phenotypic maturation in MDDC, these glycoproteins did not enhance the ability of the DC to activate T cells, nor did they induce expression of the chemokine receptor CCR7. The glycoproteins therefore induce "partial maturation", stimulating an innate antiviral response (i.e., type I IFN) without enhancing T cell activation. Several examples of partial maturation have been reported previously (40, 41, 42, 43, 44, 45) (46). In certain cases partial maturation has been associated with the induction of tolerance or regulatory T cells (41, 42), and further studies will be necessary to determine whether this is the case with HSV glycoproteins. The interaction with glycoproteins leads, however, to the release of IFN-{alpha} by the MDDC, independent of viral nucleic acid recognition and therefore presumably independently of pathways such as retinoic acid-inducible gene-I (RIG-I) (38) or DAI (16). Type I IFNs are key effector molecules of innate anti-HSV-1 immunity (1, 2), and indeed susceptibility to HSV-1 encephalitis has recently been linked to defects in the IFN signaling pathway (47). DC release of type I IFNs will also induce maturation and viral resistance in neighboring DC within and immediately around the zone of infection, and this paracrine pathway may be a key step in triggering adaptive immunity (11).

DC stimulated with HSV-1 glycoproteins also produced small amounts of the anti-inflammatory cytokine IL-10, but they did not produce any detectable IL-12, a key cytokine in induction of TH1 adaptive immune response. A similar high IL-10–IL-12 ratio has been found in recurrent HSV lesions in vivo (48). A previous study has reported IL-12 mRNA but no IL-12 protein production in HSV-1-infected blood DC (49). The lack of IL-12 response during HSV-1 infection can be attributed to the viral virion host shutoff protein, which suppresses the host's cellular protein synthesis (50). However, the lack of response to Cos7-transfected cells cannot be attributed to viral virion host shutoff protein. Down-regulation of IL-12 may also be the result of autocrine IL-10 feedback, because Cos7 cells expressing the four glycoproteins do stimulate IL-10 release, and IL-10 blocks transcription of both IL-12 subunit genes (51, 52).

The complex of four glycoproteins on Cos7 cells is in principle sufficient to allow fusion between the Cos cells and the DC (17). Fusion in itself could provide a trigger for DC maturation, for example by introducing Cos cell DNA into the DC cytoplasm (16) rather than the interaction between individual glycoproteins and their receptors. Two sets of experiments were conducted to test this hypothesis. In the first set, the transfected Cos7 cells were briefly fixed with glutaraldehyde, which completely blocks fusion, before coculture with the DC. In the second, we used a set of gH mutants with nonfunctional fusion domains (18, 19, 20). Both sets of experiments demonstrated unequivocally that DC maturation induced by the glycoproteins is independent of membrane fusion.

DC recognition of HSV-1 can therefore occur in the absence of other viral components and the absence of fusion. These results strongly suggested the existence of a receptor or receptors on the DC surface that are able to interact with the glycoproteins and transmit an activating signal to the DC. Several receptors of the individual HSV-1 glycoproteins have already been described (53), with different distribution on different cell types. Thus, we next tested whether any of the glycoproteins individually could induce DC maturation via interaction with their respective receptors. Unexpectedly, DC maturation was found to require the cooperative interaction of all four glycoproteins. The requirement for cooperative interactions was demonstrated both by transfection of each glycoprotein alone or in various combinations, as well as by the use of blocking Abs to gD and gH. Both approaches confirmed the synergism between gB, gD, and gH in triggering DC maturation.

One of the known receptors for gD is HVEM (54, 55), a TNF receptor family member that is known to signal via NF-{kappa}B in response to its natural ligand LIGHT (32, 33, 34, 56). Signaling via this receptor might therefore be an essential, although clearly not a sufficient event for DC maturation in our model. To test this hypothesis we adopted two complementary approaches. We used a set of three gD mutants that are respectively unable to bind to HVEM, nectin-1, or either receptor. All three mutants induced DC activation when used in conjunction with the other glycoproteins, albeit to a slightly lesser extent than did the wild-type protein. We also showed that recombinant gD, which can bind to both HVEM and nectin-1 (55, 57), did not induce maturation, even at concentrations of 25 µg/ml. Thus, the direct interaction between gD and HVEM or nectin-1 on the DC surface does not appear to be essential or sufficient for initiating DC maturation. We cannot, however, rule out an indirect role for gD in induction of DC maturation. For example, gD may interact in cis- with receptor on the Cos7 cells, inducing a conformational change in gD, and hence in gB and/or gH (17). Alternatively, glycoprotein interactions and complex formation may occur before receptor binding (Ref. (52) and C. Krummenacher, unpublished observations).

We next addressed the role of TLR2 in glycoprotein recognition, because this receptor has a well-established role in innate responses to HSV-1 (10, 58). We used a very sensitive bioassay for TLR2 ligands, which as expected responded strongly to the TLR2 ligand Pam3Cys. The bioassay gave a small but consistent signal to HSV-1, consistent with the previous reports of HSV-1 as a TLR2 agonist (10, 58). However, there was no response to the Cos7 cells expressing HSV-1 glycoproteins under any conditions tested. Consistent with this finding, Abs to TLR2 failed to block DC maturation (not shown) and MDLC, which did not respond to a TLR2 ligand, Pam3Cys, induced maturation when cocultured with the four glycoproteins expressing Cos7 cells. The unusual selective pattern of cytokine release observed further highlights the differences between the glycoprotein PAMP/PRR recognition pathway and classical TLR-dependent viral sensors. Our results are therefore consistent with a model of two parallel pathways, one mediated via TLR2 and an undefined viral component, and another mediated by a still uncharacterized receptor on the DC interacting with a glycoprotein complex on the HSV-1 envelope.

Finally, we studied the effect of the glycoproteins on pDC, a distinct subtype of DC expressing TLR9 with a potent ability to produce high levels of type I IFN in response to viral infection (59, 60, 61). High levels of IFN-{alpha} and TNF-{alpha} were detected in pDC stimulated with CpG, a TLR9 ligand, and with pDC infected with the HSV-1 virions. HSV-1 DNA has been shown to carry CpG motifs, hence rendering the virus highly immunostimulatory by TLR9 (62). These results are consistent with studies on murine pDC showing TLR9-dependent recognition of HSV DNA (13, 14, 15). However, no cytokine production was detected in pDC cocultured with the Cos7 cells expressing the four glycoproteins, suggesting that the viral entry glycoproteins do not trigger TLR9.

In conclusion, the data presented in this paper demonstrate that myeloid DC and monocyte- derived Langerhans cells, but not plasmacytoid DC, can recognize a PAMP that is dependent on the four major entry glycoproteins of HSV. As it is not yet possible to reconstitute the complex of the four glycoproteins in a cell-free system, we cannot rule out that some other cell protein on Cos and CHO cells may also contribute to recognition. The recognition event triggers up-regulation of several maturation markers on DC and the release of type I IFN, but it fails to up-regulate T cell activation or CCR7 expression. The implications of this recognition event in terms of HSV infection require further study. On the one hand, release of type I IFN will enhance the antiviral state. In contrast, partial maturation of DC can also result in tolerance. Finally, we cannot rule out the possibility that the signaling cascade triggered in the target cell by the HSV glycoproteins plays some role in enhancing HSV infection. Further studies are in progress to define the key elements of the signaling pathway triggered by the glycoproteins, which may provide some clues as to the receptor on the DC involved in the recognition process.


   Acknowledgments
 
We thank Prof. T. Minson and Dr. H. Browne at the Department of Pathology, University of Cambridge, and Prof. G. H. Cohen and Prof. R. J. Eisenberg at the University of Pennsylvania for providing the HSV-1 surface glycoprotein plasmids and the soluble gD285t. We also thank Prof. G. Campadelli-Fiume at the University of Bologna for providing us with the gH mutations.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Barclay Family Cancer Research Foundation and by the Lewis Foundation. C.K. is supported by Public Health Service Grant AI-0733 84 from the National Institute of Allergy and Infectious Diseases. Back

2 Address correspondence and reprint requests to Dr. Benjamin M. Chain, University College London, Windeyer Building, 46 Cleveland Street, London W1T 4JF, U.K. E-mail address: b.chain{at}ucl.ac.uk Back

3 Abbreviations used in this paper: HSV-1, herpes simplex virus-1; DC, dendritic cell; FSC/SSC, forward/side scatter; HVEM, herpesvirus entry mediator; MDDC, monocyte-derived DC; MDLC, monocyte-derived Langerhans cell; MFI, mean fluorescence intensity; MOI, multiplicity of infection; PAMP/PRR, pathogen-associated molecular pattern/pattern recognition receptor; pDC, plasmacytoid DC; RT, room temperature. Back

Received for publication October 10, 2007. Accepted for publication March 14, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Leib, D. A., T. E. Harrison, K. M. Laslo, M. A. Machalek, N. J. Moorman, H. W. Virgin. 1999. Interferons regulate the phenotype of a wild-type and mutant herpes simplex viruses in vivo. J. Exp. Med. 189: 663-672. [Abstract/Free Full Text]
  2. Noisakran, S., I. L. Campbell, D. J. J. Carr. 2000. IFN-{alpha}1 plasmid construct affords protection against HSV-1 infection in transfected L929 fibroblasts. J. Interferon Cytokine Res. 20: 107-115. [Medline]
  3. Jouanguy, E., S. Y. Zhang, A. Chapgier, V. Sancho-Shimizu, A. Puel, C. Picard, S. Boisson-Dupuis, L. Abel, J. L. Casanova. 2007. Human primary immunodeficiencies of type I interferons. Biochimie 89: 878-883. [Medline]
  4. Salio, M., M. Cella, M. Suter, A. Lanzavecchia. 1999. Inhibition of dendritic cell maturation by herpes simplex virus. Eur. J. Immunol. 29: 3245-3253. [Medline]
  5. Kruse, M., O. Rosorius, F. Kratzer, G. Stelz, C. Kuhnt, G. Schuler, J. Hauber, A. Steinkasserer. 2000. Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity. J. Virol. 74: 7127-7136. [Abstract/Free Full Text]
  6. Pollara, G., K. Speidel, L. Samady, M. Rajpopat, Y. McGrath, J. Ledermann, R. S. Coffin, D. R. Katz, B. Chain. 2003. Herpes simplex virus infection of dendritic cells: balance among activation, inhibition, and immunity. J. Infect. Dis. 187: 165-178. [Medline]
  7. Samady, L., E. Costigliola, L. MacCormac, Y. McGrath, S. Cleverley, C. E. Lilley, J. Smith, D. S. Latchman, B. Chain, R. S. Coffin. 2003. Deletion of the virion host shutoff protein (vhs) from herpes simplex virus (HSV) relieves the viral block to dendritic cell activation: potential of vhs– HSV vectors for dendritic cell-mediated immunotherapy. J. Virol. 77: 3768-3776. [Abstract/Free Full Text]
  8. Pollara, G., D. Katz, B. Chain. 2004. The host response to herpes simplex virus infection. Infect. Dis. 17: 199-203.
  9. Novak, N., W. Peng. 2005. Dancing with the enemy: the interplay of herpes simplex virus with dendritic cells. Clin. Exp. Immunol. 142: 405-410. [Medline]
  10. Sato, A., M. M. Linehan, A. Iwasaki. 2006. Dual recognition of herpes simplex viruses by TLR2 and TLR9 in dendritic cells. Proc. Natl. Acad. Sci. USA 103: 17343-17348. [Abstract/Free Full Text]
  11. Pollara, G., M. Jones, M. Handley, M. Rajpopat, A. Kwan, R. Coffin, G. Foster, B. Chain, D. Katz. 2004. Herpes simplex virus type-1-induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion. J. Immunol. 173: 4108-4119. [Abstract/Free Full Text]
  12. Ellermann-Eriksen, S.. 2005. Macrophages and cytokines in the early defence against herpes simplex virus. Virol. J. 3: 59-90.
  13. Lund, J., A. Sato, S. Akira, R. Medzhitov, A. Iwasaki. 2003. Toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells. J. Exp. Med. 198: 513-520. [Abstract/Free Full Text]
  14. Hochrein, H., B. Schlatter, M. O'Keeffe, C. Wagner, F. Schmitz, M. Schiemann, S. Bauer, M. Suter, H. Wagner. 2004. Herpes simplex virus type-1 induces IFN-{alpha} production via Toll-like receptor 9-dependent and -independent pathways. Proc. Natl. Acad. Sci. USA 101: 11416-11421. [Abstract/Free Full Text]
  15. Krug, A., G. D. Luker, W. Barchet, D. A. Leib, S. Akira, M. Colonna. 2004. Herpes simplex virus type 1 activates murine natural interferon-producing cells through Toll-like receptor 9. Blood 103: 1433-1437. [Abstract/Free Full Text]
  16. Takaoka, A., Z. Wang, M. K. Choi, H. Yanai, H. Negishi, T. Ban, Y. Lu, M. Miyagishi, T. Kodama, K. Honda, et al 2007. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 448: 501-505. [Medline]
  17. Reske, A., G. Pollara, C. Krummenacher, B. Chain, D. R. Katz. 2007. Understanding HSV-1 entry glycoproteins. Rev. Med. Virol. 17: 205-215. [Medline]
  18. Gianni, T., P. Martelli, R. Casadio, G. Campadelli-Fiume. 2005. The ectodomain of herpes simplex virus glycoprotein H contains a membrane {alpha}-helix with attributes of an internal fusion peptide, positionally conserved in the Herpesviridae family. J. Virol. 79: 2931-2940. [Abstract/Free Full Text]
  19. Gianni, T., L. Menotti, G. Campadelli-Fiume. 2005. A heptad repeat in herpes simplex virus 1 gH, located downstream of the {alpha}-helix with attributes of a fusion peptide, is critical for virus entry and fusion. J. Virol. 79: 7042-7049. [Abstract/Free Full Text]
  20. Gianni, T., A. Piccoli, C. Bertucci, G. Campadelli-Fiume. 2006. Heptad repeat 2 in herpes simplex virus 1 gH interacts with heptad repeat 1 and is critical for virus entry and fusion. J. Virol. 80: 2216-2224. [Abstract/Free Full Text]
  21. Krummenacher, C., V. M. Supekar, J. C. Whitbeck, E. Lazear, S. A. Connolly, R. J. Eisenberg, G. H. Cohen, D. C. Wiley, A. Carfi. 2005. Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry. EMBO J. 24: 4144-4153. [Medline]
  22. Connolly, S., D. Landsburg, A. Carfi, D. Wiley, G. Cohen, R. Eisenberg. 2003. Structure-based mutagenesis of herpes simplex virus glycoprotein D defines three critical regions at the gD-HveA/HVEM binding interface. J. Virol. 77: 8127-8140. [Abstract/Free Full Text]
  23. Connolly, S., D. Landsburg, A. Carfi, J. Whitbeck, Y. Zuo, D. Wiley, G. Cohen, R. Eisenberg. 2005. Potential nectin-1 binding site on herpes simplex virus glycoprotein D. J. Virol. 79: 1282-1295. [Abstract/Free Full Text]
  24. Coffin, R. S., A. R. MacLean, D. S. Latchman, S. M. Brown. 1996. Gene delivery to the central and peripheral nervous systems of mice using HSV1 ICP34.5 deletion mutant vectors. Gene Ther. 3: 886-891. [Medline]
  25. Rux, A. H., S. H. Willis, A. V. Nicola, W. Hou, C. Peng, H. Lou, G. H. Cohen, R. J. Eisenberg. 1998. Functional region IV of glycoprotein D from herpes simplex virus modulates glycoprotein binding to the herpesvirus entry mediator. J. Virol. 72: 7091-7098. [Abstract/Free Full Text]
  26. Turner, A., B. Bruun, T. Minson, H. Browne. 1998. Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1a are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J. Virol. 72: 873-875. [Abstract/Free Full Text]
  27. Browne, H., B. Bruun, T. Minson. 2001. Plasma membrane requirements for cell fusion induced by herpes simplex virus type 1 glycoproteins gB, gD, gH and gL. J. Gen. Virol. 82: 1419-1422. [Abstract/Free Full Text]
  28. Forrester, A., H. Farrell, G. Wilkinson, J. Kaye, N. Davis-Poynter, T. Minson. 1992. Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted. J. Virol. 66: 341-348. [Abstract/Free Full Text]
  29. Hutchinson, L., H. Browne, V. Wargent, N. Davis-Poynter, S. Primorac, K. Goldsmith, A. Minson, D. Johnson. 1992. A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH. J. Virol. 66: 2240-2250. [Abstract/Free Full Text]
  30. Roop, D., L. Hutchinson, D. Johnson. 1993. A mutant herpes simplex virus type 1 unable to express glycoprotein L cannot enter cells, and its particles lack glycoprotein H. J. Virol. 67: 2285-2297. [Abstract/Free Full Text]
  31. Sciortino, M. Teresa, M. A. Medici, F. Marino-Merlo, D. Zaccaria, M. Giuffrè, A. Venuti, M. A. Grelli. 2007. Signaling pathway used by HSV-1 to induce NF-{kappa}B activation: possible role of herpes virus entry receptor A. Ann. NY Acad. Sci. 1096: 89-96. [Medline]
  32. Marsters, S., T. Ayres, M. Skubatch, C. Gray, M. Rothe, A. Ashkenazi. 1997. Herpesvirus entry mediator, a member of the tumor necrosis factor receptor (TNFR) family, interacts with members of the TNFR-associated factor family and activates the transcription factors NF-{kappa}B and AP-1. J. Biol. Chem. 272: 14029-14032. [Abstract/Free Full Text]
  33. Tamada, K., K. Shimozaki, A. Chapoval, Y. Zhai, J. Su, S. Chen, S. Hsieh, S. Nagata, J. Ni, L. Chen. 2000. LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response. J. Immunol. 164: 4105-4110. [Abstract/Free Full Text]
  34. Pollara, G., D. Katz, B. Chain. 2005. LIGHTing up dendritic cell activation: immune regulation and viral exploitation. J. Cell. Physiol. 205: 161-162. [Medline]
  35. Takeuchi, J., E. Watari, E. Shinya, Y. Norose, M. Matsumoto, T. Seya, M. Sugita, S. Kawana, H. Takahashi. 2003. Down-regulation of Toll-like receptor expression in monocyte-derived Langerhans cell-like cells: implications of low-responsiveness to bacterial components in the epidermal Langerhans cells. Biochem. Biophys. Res. Commun. 306: 674-679. [Medline]
  36. van der Aar, A. M. G., R. M. R. Sylva-Steenland, J. D. Bos, M. L. Kapsenberg, E. C. de Jong, M. B. M. Teunissen. 2007. Cutting edge: Loss of TLR2, TLR4, and TLR5 on Langerhans cells abolishes bacterial recognition. J. Immunol. 178: 1986-1990. [Abstract/Free Full Text]
  37. Herbst-Kralovetz, M. M., R. B. Pyles. 2006. Toll-like receptors, innate immunity, and HSV pathogenesis. Herpes 13: 37-41. [Medline]
  38. Yoneyama, M., T. Fujita. 2007. Function of RIG-I-like receptors in antiviral innate immunity. J. Biol. Chem. 282: 15315-15318. [Free Full Text]
  39. Cunningham, A. L., R. R. Turner, A. C. Miller, M. F. Para, T. C. Merigan. 1985. Evolution of recurrent herpes simplex lesions: an immunohistologic study. J. Clin. Invest. 75: 226-233. [Medline]
  40. Lutz, M. B., G. Schuler. 2002. Immature, semi-mature, and fully mature dendritic cells: which signals induce tolerance or immunity?. Trends Immunol. 23: 445-449. [Medline]
  41. Kleindienst, P., C. Wiethe, M. B. Lutz, T. Brocker. 2005. Simultaneous induction of CD4 T cell tolerance and CD8 T cell immunity by semimature dendritic cells. J. Immunol. 174: 3941-3947. [Abstract/Free Full Text]
  42. Verginis, P., H. S. Li, G. Carayanniotis. 2005. Tolerogenic semimature dendritic cells suppress experimental autoimmune thyroiditis by activation of thyroglobulin-specific CD4+CD25+ T cells. J. Immunol. 174: 7433-7439. [Abstract/Free Full Text]
  43. Krathwohl, M., T. Schacker, J. Anderson. 2006. Abnormal presence of semimature dendritic cells that induce regulatory T cells in HIV-infected subjects. J. Infect. Dis. 193: 494-504. [Medline]
  44. Braun, D., L. Galibert, T. Nakajima, H. Saito, V. V. Quang, M. Rubio, M. Sarfati. 2006. Semimature stage: a checkpoint in a dendritic cell maturation program that allows for functional reversion after signal-regulatory protein-{alpha} ligation and maturation signals. J. Immunol. 177: 8550-8559. [Abstract/Free Full Text]
  45. Bayry, J., F. Triebel, S. V. Kaveri, D. F. Tough. 2007. Human dendritic cells acquire a semimature phenotype and lymph node homing potential through interaction with CD4+CD25+ regulatory T cells. J. Immunol. 178: 4184-4193. [Abstract/Free Full Text]
  46. Dulphy, N., J. L. Herrmann, J. Nigou, D. Rea, N. Boissel, G. Puzo, D. Charron, P. H. Lagrange, A. Toubert. 2007. Intermediate maturation of Mycobacterium tuberculosis LAM-activated human dendritic cells. Cell. Microbiol. 9: 1412-1425. [Medline]
  47. Casrouge, A., S. Y. Zhang, C. Eidenschenk, E. Jouanguy, A. Puel, K. Yang, A. Alcais, C. Picard, N. Mahfoufi, N. Nicolas, et al 2006. Herpes simplex virus encephalitis in human UNC-93B deficiency. Science 314: 308-312. [Abstract/Free Full Text]
  48. Mikloska, Z., V. A. Danis, S. Adams, A. R. Lloyd, D. L. Adrian, A. L. Cunningham. 1998. In vivo production of cytokines and β (C-C) chemokines in human recurrent herpes simplex lesions: do herpes simplex virus-infected keratinocytes contribute to their production?. J. Infect. Dis. 177: 827-838. [Medline]
  49. Ghanekar, S., L. Zheng, A. Logar, J. Navratil, L. Borowski, P. Gupta, C. Rinaldo. 1996. Cytokine expression by human peripheral blood dendritic cells stimulated in vitro with HIV-1 and herpes simplex virus. J. Immunol. 157: 4028-4036. [Abstract]
  50. Smiley, J. R.. 2004. Herpes simplex virus virion host shutoff protein: immune evasion mediated by a viral RNase?. J. Virol. 78: 1063-1068. [Free Full Text]
  51. D'Andrea, A., M. Aste-Amezaga, N. M. Valiante, X. Ma, M. Kubin, G. Trinchieri. 1993. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178: 1041-1048. [Abstract/Free Full Text]
  52. Aste-Amezaga, M., X. Ma, A. Sartori, G. Trinchieri. 1998. Molecular mechanisms of the induction of IL-12 and its inhibition by IL-10. J. Immunol. 160: 5936-5944. [Abstract/Free Full Text]
  53. Spear, P.. 2004. Herpes simplex virus: receptors and ligands for cell entry. Cell. Microbiol. 6: 401-410. [Medline]
  54. Montgomery, R., M. Warner, B. Lum, P. Spear. 1996. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87: 427-436. [Medline]
  55. Whitbeck, J., C. Peng, H. Lou, R. Xu, S. Willis, M. Ponce de Leon, T. Peng, A. Nicola, R. Montgomery, M. Warner, et al 1997. Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry. J. Virol. 71: 6083-6093. [Abstract]
  56. Hikichi, Y., H. Matsui, I. Tsuji, K. Nishi, T. Yamada, Y. Shintani, H. Onda. 2001. LIGHT, a member of the TNF superfamily, induces morphological changes and delays proliferation in the human rhabdomyosarcoma cell line RD. Biochem. Biophys. Res. Commun. 289: 670-677. [Medline]
  57. Willis, S. H., A. H. Rux, C. Peng, J. C. Whitbeck, A. V. Nicola, H. Lou, W. Hou, L. Salvador, R. J. Eisenberg, G. H. Cohen. 1998. Examination of the kinetics of herpes simplex virus glycoprotein D binding to the herpesvirus entry mediator, using surface plasmon resonance. J. Virol. 72: 5937-5947. [Abstract/Free Full Text]
  58. Finberg, R. W., D. M. Knipe, E. A. Kurt-Jones. 2005. Herpes simplex virus and Toll-like receptors. Viral Immunol. 18: 457-465. [Medline]
  59. Siegal, F. P., N. Kadowaki, M. Shodell, P. A. Fitzgerald-Bocarsly, K. Shah, S. Ho, S. Antonenko, Y. J. Liu. 1999. The nature of the principal type 1 interferon-producing cells in human blood. Science 284: 1835-1837. [Abstract/Free Full Text]
  60. Asselin-Paturel, C., A. Boonstra, M. Dalod, I. Durand, N. Yessaad, C. Dezutter-Dambuyant, A. Vicari, A. O'Garra, C. Biron, F. Briere, G. Trinchieri. 2001. Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology. Nat. Immunol. 2: 1144-1150. [Medline]
  61. Colonna, M., A. Krug, M. Cella. 2002. Interferon-producing cells: on the front line in immune responses against pathogens. Curr. Opin. Immunol. 14: 373-379. [Medline]
  62. Lundberg, P., P. Welander, X. Han, E. Cantin. 2003. Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo. J. Virol. 77: 11158-11169. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Virol.Home page
H. Jin, Y. Ma, B. S. Prabhakar, Z. Feng, T. Valyi-Nagy, Z. Yan, D. Verpooten, C. Zhang, Y. Cao, and B. He
The {gamma}134.5 Protein of Herpes Simplex Virus 1 Is Required To Interfere with Dendritic Cell Maturation during Productive Infection
J. Virol., May 15, 2009; 83(10): 4984 - 4994.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reske, A.
Right arrow Articles by Chain, B. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reske, A.
Right arrow Articles by Chain, B. M.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS