Differential effects of bovine viral diarrhoea virus on monocytes and dendritic cells

E. J. Glew, B. V. Carr, L. S. Brackenbury, J. C. Hope, B. Charleston and C. J. Howard

Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN UK

Correspondence
Bryan Charleston
bryan.charleston{at}bbsrc.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Various pathogens have been shown to infect antigen-presenting cells and affect their capacity to interact with and stimulate T-cell responses. We have used an antigenically identical pair of non-cytopathic (ncp) and cytopathic (cp) bovine viral diarrhoea virus (BVDV) isolates to determine how the two biotypes affect monocyte and dendritic cell (DC) function. We have shown that monocytes and DCs are both susceptible to infection with ncp BVDV and cp BVDV in vitro. In addition, monocytes infected with ncp BVDV were compromised in their ability to stimulate allogeneic and memory CD4+ T cell responses, but DCs were not affected. This was not due to down-regulation of a number of recognized co-stimulatory molecules including CD80, CD86 and CD40. Striking differences in the response of the two cell types to infection with cytopathic virus were seen. Dendritic cells were not susceptible to the cytopathic effect caused by cp BVDV, whereas monocytes were killed. Analysis of interferon (IFN)-{alpha}/{beta} production showed similar levels in monocytes and DCs exposed to cp BVDV, but none was detected in cells exposed to ncp BVDV. We conclude that the prevention of cell death in DCs is not associated with enhanced production of IFN-{alpha}/{beta}, as proposed for influenza virus, but is by a distinct mechanism.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The pestiviruses – bovine viral diarrhoea virus (BVDV), classical swine fever virus and border disease virus of sheep, together with the flaviviruses and hepatitis C virus – are a closely related group of small enveloped viruses, the Flaviviridae, with a single-stranded, positive-sense RNA genome of approximately 12·5 kb (Meyers & Thiel, 1996). They all have a similar genomic structure and protein composition, the virus particles comprising a single capsid protein surrounded by an envelope containing two or three glycoproteins. While primary infection with many of these viruses causes acute clinical disease, some also give rise to persistent infection associated with immunopathology (Solomon & Mallewa, 2001).

BVDV has a worldwide distribution and readily establishes endemic infection in cattle populations. Disease is associated with both acute and persistent infections and, depending on epidemiological circumstances, may manifest as outbreaks affecting large numbers of animals or a continual low incidence of cases within endemically infected herds. Both disease patterns have a major impact on the productivity of affected cattle populations (Houe, 1999). The virus occurs in two biotypic forms, cytopathic (cp) and non-cytopathic (ncp), and different isolates of both forms commonly exhibit antigenic differences (Hamers et al., 2001).

Non-cytopathic BVDV is the most prevalent form of the virus. Acute, self-limiting infections with BVDV are associated with a period of generalized immunosuppression and increased susceptibility to secondary infection (Potgieter, 1995). The fatal clinical syndrome mucosal disease is the result of a complex, unique immunopathological event. Infection of the foetus prior to immunocompetence results in a persistent lifelong infection of the calf. The animal is specifically immunotolerant to the persisting virus and if superinfected by a cp strain of BVDV that is antigenically sufficiently homologous, will die within a few weeks with extensive destruction of the organized immune tissues being evident (Brownlie et al., 1984; Teichmann et al., 2000). These persistently infected cattle are reservoirs of infection for naïve animals. Thus, many of the most important clinical consequences occur as a result of infection of pregnant animals with ncp BVDV.

BVDV infects a wide variety of cell types but has a predilection for cells of the immune system. The virus infects T cells, B cells and antigen-presenting cells (APCs) in vivo (Sopp et al., 1994). Monocytes, macrophages and dendritic cells (DCs) constitute the majority of APCs involved in the initial uptake of virus or their antigens and presentation to the immune system. Of these APCs, DCs are the most effective and the only population that is recognized as having the ability to initiate primary immune responses in naive animals (Banchereau et al., 2000).

Infection of APCs by viruses can have a marked effect on the cells and important consequences for the generation of the subsequent immune response. The consequences of infection include the death of the APC as an extreme event, or more subtle effects on cytokine expression and synthesis of co-stimulatory molecules (Rescigno & Borrow, 2001; Schneider-Schaulies et al., 2002). Studying the interaction of DCs and monocytes with specific viruses and how this might influence the primary immune response is critical for understanding disease pathogenesis and immunity to infection. We have used an antigenically homologous pair of ncp and cp BVDV isolates to investigate how bovine APCs, namely DCs and monocytes, respond to infection with the different biotypes of virus.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Animals.
Calves (Bos taurus) were conventionally reared British Holstein Friesians bred at the Institute for Animal Health. Each animal was of a known MHC class I haplotype (Ellis et al., 1998). The immune status of each animal was determined by assaying sera for antibody to BVDV and by testing for persistent viraemia (Fray et al., 1999). Some of the animals had been previously immunized with ovalbumin, as reported by Howard et al. (1997). All experiments were approved by the Institute's ethical review process and were in accord with national guidelines on animal use.

Cells.
Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) following incubation with anti-human CD14-labelled superparamagnetic particles (Miltenyi-Biotech) and labelled cells were isolated from a Midimacs column (Miltenyi-Biotech) according to the manufacturer's instructions. The purity of the cells evaluated by flow cytometry was shown in each case to be >98 %. Cell viability was >95 %. For some experiments monocytes were purified by flow cytometry; the purity of the monocytes was >99 %.

DCs, or more accurately monocyte-derived dendritic cells, were generated from CD14+ monocytes as previously described (Hope et al., 2000). Monocytes were adjusted to 8x105 cells ml-1 in tissue culture medium (TCM) [RPMI 1640 containing Glutamax-1 (Life Technologies), 10 % heat inactivated foetal calf serum, 5x10-5 M {beta}-mercaptoethanol, 50 µg gentamicin ml-1], plus 200 U COS-7 cell-derived bovine rIL-4 ml-1 and 0·2 U bovine rGMCSF ml-1 (units based on induction of half maximal proliferation in bone marrow precursor cells). DCs were harvested and infected after incubation for 6 days at 37 °C in 5 % CO2.

Virus.
The experiments utilized a homologous pair of ncp and cp viruses (Pe515ncp and Pe515cp) originally isolated from a case of mucosal disease. The viruses were propagated and titrated in a primary cell line derived from calf testis (Cte), as previously described (Brownlie et al., 1984). Cte cells, monocytes and DCs were incubated for 1 h at 37 °C with BVDV at an m.o.i. of 2.

Virus isolation.
Virus was isolated from Cte cells cultured on coverslips, as previously described (Brownlie et al., 1984). Five replicate coverslips were used for each sample. Virus-neutralizing antibody titres were determined using a microtitre-based assay, as previously described (Brownlie et al., 1984).

To determine whether the production of infectious ncp BVDV differed between monocytes and DCs, virus replication was assayed at the same m.o.i. (2). Supernatants were removed from cultures of monocytes and DCs at 24 h time intervals and titrated to assay extracellular virus. Cell-associated virus was obtained by freeze-thawing monocytes and DCs. Titres of extracellular virus were obtained from six experiments with monocytes and DCs isolated from four animals. Titres of cell-associated virus were obtained from four experiments with monocytes and DCs isolated from three animals.

Virus titres were assayed by microtitre assay as previously described (Brownlie et al., 1984) with some modifications. Cte cells were grown in 96-well plates (Costar) until 80–90 % confluent. After removal of medium, cells were washed with sterile PBS and 50 µl viral growth media (VGM) (Brownlie et al., 1984). Serial tenfold dilutions of the test samples were made in VGM and 50 µl added to each well. After 30 min, a further 100 µl VGM was added and the plates incubated for 5 days. After 5 days the medium was removed from each well and the cells fixed with ice-cold 80 % acetone and air-dried. Each well was incubated with PBS/0·05 % Tween for 30 min at room temperature. After washing, each well was incubated overnight at 4 °C with 25 µl of a 1 : 500 dilution of monoclonal antibody (mAb) WB103 (Edwards et al., 1991) or isotyped matched control. Antibody staining was revealed using horseradish peroxidase-conjugated goat anti-mouse IgG followed by tetramethylbenzidine (ICN). Virus titres (TCID50) were calculated from replicate wells.

Detection of viral glycoprotein by flow cytometry.
Monocytes or DCs were stained for intracellular BVDV NS3 (p80) protein using a slight modification of procedures described previously (Glew & Howard, 2001; Sopp et al., 1994). Mouse mAb WB103 (Edwards et al., 1991) was used to detect intracellular viral antigen with optimally diluted goat anti-mouse secondary antibody conjugated to FITC (Southern Biotechnology Associates). Immunofluorescent staining was analysed using a FACScan (Becton Dickinson) and data were analysed by using WinMDI (obtained from Joseph Trotter, Scripps Research Institute, San Diego, CA, USA) and FCS Express (De novo Software).

Cell viability.
Monocytes and DCs were stained with propidium iodide (PI; Sigma) and Annexin V (Boehringer Mannheim), which in combination indicate cells that are dying by both necrosis and apoptosis (Cella et al., 1999). Total cell counts were made before staining, and similar numbers of cells were analysed. Staining was analysed immediately by flow cytometry. Live cells were negative for PI and/or Annexin V. The mean survival of ncp/cp BVDV-infected monocytes and DCs was determined in four separate experiments with cells isolated and generated from three animals.

Type 1 interferon assay.
Levels of biologically active type 1 IFN were assayed in duplicate in samples of culture media using a chloramphenicol transferase (CAT) reporter assay that detects, but does not distinguish between, IFN-{alpha} and IFN-{beta} (Fray et al., 2001).

T-cell proliferation assays.
Purified monocytes or DCs were infected by adding BVDV strain Pec515ncp (m.o.i. 2) and incubating for 2 days (experiments were done 2 and 3 days after infection with BVDV; the results were similar). These cells and mock-infected cells were used as APCs with allogeneic CD4+ T lymphocytes or MHC-identical CD4+ T lymphocytes from ovalbumin-immunized animals. CD4+ T lymphocytes were purified from PBMCs by staining with mAb CC8 and purifying cells with anti-mouse IgG labelled superparamagnetic beads (Miltenyi-Bitech), as described previously (Hope et al., 2000). In some experiments the APCs were incubated with ovalbumin (125 µg ml-1) for 1 h before they were washed twice with TCM. In some cases monocytes were sorted from PBMCs on a FACStar-plus (Becton Dickinson) after CD14 staining to provide cells of >=99 % purity. APCs were irradiated (20 Gy from a 137Cs source) and dilutions were incubated with 105 CD4+ T lymphocytes. Triplicate cultures were incubated for 5 days and 37 Bq [3H]thymidine (DuPont) was added for 16 h (overnight) before harvesting. Incorporated radioactivity was determined by liquid scintillation counting. The results of ten separate experiments using cells from three different animals are presented.

Flow cytometric analysis of surface molecule expression by BVDV-infected APCs.
To determine the effect of virus infection on expression of a range of co-stimulatory surface molecules, APCs were infected with cp or ncp BVDV as described above and surface molecule expression was assessed at 48 and 72 h post-infection (p.i.). The surface molecules assessed were MHC class I (mAb IL-A88) and MHC class II (CC158), CD11a (IL-A99), CD11b (CC94), CD11c (IL-A16), CD1b (CC14), CD14 (CCG-33), MyD-1 (CC149), CD62L (CC32), mannose receptor (3.29B1) and CD32 (CCG36). The isotypes and sources of these mAbs have been described previously (Brooke et al., 1998; Howard et al., 1997). In addition CD40, CD80 and CD86 were assessed following staining with IL-A156, IL-A159 and IL-A190, respectively (all mAbs were IgG1; provided by N. MacHugh, Centre for Tropical Medicine, Roslin, UK). Control mAbs used within the study were AV20 (mouse IgG1), AV29 (mouse IgG2b) and AV37 (mouse IgG2a), directed against chicken bursal B cells, chicken CD4+ cells and a chicken spleen cell subset, respectively, all provided by T. F. Davison (IAH, Berkshire, UK). The APCs were incubated with primary mAb at predetermined optimal concentrations for 10 min, then washed extensively. Bound mAb was detected with FITC-labelled anti-mouse IgG (Southern Biotechnologies). Following this, cells were fixed and assessed for intracellular expression of NS3 (p80) as described above. The cells were analysed on a FACSCalibur (Becton Dickinson). Immunofluorescent staining was analysed using FCS express. Cells expressing NS3 were gated and the mean fluorescent intensity of surface molecule staining on the infected (gated) cells was expressed.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Monocytes and monocyte-derived dendritic cells are susceptible to infection with ncp BVDV
Fig. 1(a) shows the mean percentage of NS3-stained monocytes and DCs at various time points p.i. The effects of time after infection and cell type were investigated using an analysis of variance (ANOVA) with a blocked design. Unsurprisingly, the percentage of ncp BVDV-infected cells was significantly affected by the time interval between infection and the time of observation (P<0·001). Cell type also had a significant effect on the percentage of infected cells (P<0·001), as the percentage of infected cells was higher for monocytes than for DCs at all time points. There was also a significant cell type/time interaction (P=0·035) as the magnitude of the difference between the numbers of infected cells in the two populations increased with time. At 24 h p.i., the percentage of infected monocytes was only slightly higher than the percentage of infected DCs (44·6±8·1 % and 33·8±5·4 % for monocytes and DCs, respectively). However, by 72 and 96 h there had been a marked increase in the magnitude of this difference (90·5±1·5 % and 68·5±7·0 % at 72 h; 93·8±1·1 % and 53·7±5·3 % at 96 h for monocytes and DCs, respectively).



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Fig. 1. Monocytes ({blacksquare}) and dendritic cells ({square}) were infected with either non-cytopathic (NCP; a–d) or cytopathic (CP; e–h) BVDV at an m.o.i. of 2. In (a) and (e) the percentage of cells expressing the BVDV non-structural protein NS3 are shown. The titres (TCID50) of extracellular virus (b, f) and cell-associated virus (c, g) were determined. The percentage of viable cells present in each culture is shown in (d) and (h). The values represented in all the graphs are the means of replicate experiments; error bars are±SEM.

 
The titres of infectious virus in either the supernatant or as cell-associated virus are shown in Fig. 1(b, c). It was apparent from visual inspection of the data that there was no significant difference in the titres of extracellular virus between populations of monocytes or DCs at time points up to and including 48 h. This indicated that virus entry and binding did not differ significantly between the two cell types. A blocked ANOVA was performed on log-transformed extracellular virus titres from monocytes and DCs, incorporating the effects of experiment and time (72 and 96 h p.i. only) into the analysis. This analysis revealed a highly significant effect of cell type (P<0·001), as extracellular virus titres were higher for monocytes than for DCs. The cell-associated virus titres were analysed in a similar manner. It was apparent from visual inspection of the data recorded at 1 and 24 h p.i. that there was no significant difference between cell type at these times (Fig. 1c). Hence, the analysis was restricted to observations made at 48, 72 and 96 h. This analysis revealed a significant effect of cell type (P=0·01) on the cell-associated virus titre, as virus titres were higher for monocytes than for DCs at the analysed time points.

Although ncp BVDV has not been associated with the death of cultured cells, survival of ncp BVDV-infected monocytes and DCs was assessed to determine whether virus-induced cell death was responsible for the decrease in virus titre seen in populations of DCs. It was apparent from visual inspection of the data that there was a similar proportion of viable cells in each population at each time point p.i. (Fig. 1d). The pattern of the changes in the proportion of viable monocytes and DCs over time in culture was similar for ncp BVDV-infected and mock-infected cells (data not shown).

Dendritic cells are resistant to lysis with cp BVDV but monocytes are susceptible
Cytopathic BVDV has been shown to kill cells by the induction of apoptosis (Zhang et al., 1996); hence, PI and Annexin V staining were used to determine the percentage of dead cells in the populations of monocytes and DCs exposed to cp BVDV. Fig. 1(h) shows the percentage of viable monocytes and DCs at various time points after cp BVDV challenge. A blocked ANOVA showed that both cell type and time point had a highly significant effect on the percentage of viable monocytes (P<0·001). There was also a highly significant interaction between these two parameters (P<0·001), the magnitude of the difference increasing with time. This was apparent from Fig. 1(h). The number of viable monocytes decreased from 76±2·1 % at 1 h p.i. to 26±10·1 % at 96 h p.i. In contrast, viability of DCs did not deviate markedly from approximately 75 % at each time point.

To establish whether cp BVDV was able to replicate efficiently in monocytes and DCs, cells were incubated with cp BVDV and stained for viral NS3. Staining was analysed by flow cytometry. Dead cells were distinguished from live cells by their decreased forward and side scatter profile on the dot plots and were not included in the analysis of NS3 expression. Fig. 1(e) shows mean staining for NS3 in populations of live cells from four experiments. The data were analysed using a blocked ANOVA, which revealed a significant effect of cell type (P<0·001) and time (P=0·001) and a significant interaction between the two parameters. This was because both monocytes and DCs expressed NS3 throughout the 96 h period. At 24 h p.i., similar numbers of monocytes and DCs were stained (26±14 % and 22±2 %, respectively). However, at 48, 72 and 96 h p.i., a significantly lower proportion of DCs expressed NS3 compared with monocytes.

Virus replication curves were undertaken to quantify the production of infectious virus from populations of monocytes and DCs. Fig. 1(f) and (g) show the titres of extracellular and cell-associated virus, respectively, at 24, 48, 72 and 96 h p.i. Similar quantities of cp BVDV were produced at each time point after infection except that there was less cell-associated virus present in the DC cultures at 24 h p.i. (P<0·05, Student's t-test).

The effect of cp BVDV infection on Cte cells, monocytes and DCs was determined (Fig. 2). Cte cells, monocytes and DCs incubated with ncp BVDV (Fig. 2d–f) appeared similar to mock-infected cells (Fig.  2a–c). Cytopathic BVDV caused an extensive cytopathic effect in Cte cells and monocytes (Fig. 2g, h); however, no cytopathic effect was seen in DCs (Fig. 2i).



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Fig. 2. Calf testis cells (Cte; a, d, g), monocytes (Mo; b, e, h) and dendritic cells (DC; c, f, i) were mock infected (mock) or infected with non-cytopathic BVDV (NCP BVDV) or cytopathic BVDV (CP BVDV). After 96 h in culture, cytopathic effect was only visible in cultures of monocytes and Cte cells. Representative photomicrographs of each culture are shown.

 
Monocytes infected with ncp BVDV are compromised in their ability to stimulate T cell responses but dendritic cells are not affected
The effect of BVDV infection on the ability of APCs to stimulate allogenic CD4+ T cells was determined (Fig. 3). The proliferation induced by infected monocytes was lower than proliferation induced by mock-infected monocytes. The reduction was significant (P<0·05, Student's t-test) in 7/10 experiments. However, in none of six experiments was the proliferation induced by infected DCs significantly lower than the proliferation induced by mock-infected DCs. For all experiments, the incorporation of thymidine into APCs alone or CD4+ T cells alone was <800 c.p.m. Representative data from monocyte- or DC-induced allogeneic proliferation are shown in Fig. 3 (upper graphs).



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Fig. 3. Effect of ncp-BVDV infection on antigen presentation. Purified monocytes or DCs were infected by adding ncp BVDV (m.o.i. of 2) and incubating for 2 days (results after 3 days were similar). These cells and mock-infected cells were used as APCs with allogeneic CD4+ T lymphocytes or MHC-identical CD4+ T lymphocytes from ovalbumin-immunized animals. In some experiments the APCs were incubated with ovalbumin (125 µg ml-1) for 1 h before washing. APCs were irradiated and dilutions (10–0·1x103 cells) were incubated with 105 CD4+ T lymphocytes. Triplicate cultures were incubated for 5 days and 37 Bq [3H]thymidine was added for 16 h (overnight) before harvesting. Incorporated radioactivity was determined by liquid scintillation counting and presented as counts x103 min-1±SD.

 
It was possible that monocytes, which were isolated with paramagnetic beads from PBMCs of BVDV-immune animals and were >97 % pure for CD14+ cells, were contaminated with T or B cells. These lymphocytes may have been stimulated by the ncp BVDV-infected monocytes to produce immumodulatory cytokines, which were responsible for the reduction in allogeneic proliferation seen with BVDV-infected monocytes. Thus, monocytes were purified by flow cytometry before titration with allogeneic T cells. The purity of the monocytes was greater than 99 %, and in three separate experiments the ncp BVDV-infected monocytes stimulated an allogeneic T cell response that was significantly lower than the mock-infected cells (data not shown), as was the case with CD14+ monocytes that had been purified with magnetic beads. Staining of the allogeneic CD4+ T cells with mAb to NS3 and analysis by flow cytometry after 5 days culture with ncp BVDV infected monocytes indicated that the CD4+ T cells were not infected with ncp BVDV (data not shown).

To assess the effect of infection by BVDV on the ability of the APCs to process and present exogenous antigen, monocytes and DCs were pulsed with ovalbumin. In 7/8 experiments, BVDV-infected monocytes were compromised in their ability to stimulate ovalbumin-specific CD4+ T cell proliferation when compared with mock-infected, ovalbumin-pulsed monocytes. The reduction of proliferation was significantly lower in six experiments and a representative sample of data is shown in Fig. 3 (lower graphs). In contrast, DCs were not compromised in their ability to stimulate ovalbumin-specific T cells. This was confirmed by four experiments and a representative sample of data is shown in Fig. 3. Control wells of CD4+ T cells or APCs alone had thymidine incorporation of <500 c.p.m. These data together show that ncp BVDV infection of monocytes, but not of DCs, compromised their stimulatory capacity and possibly their processing and presentation capacity.

The expression of a range of co-stimulatory molecules was compared for DCs infected with ncp and cp BVDV and monocytes infected with ncp BVDV (Table 1). The effect of cp BVDV on surface antigen expression by monocytes could not be examined, as it was lytic for these cells. No evidence for a down-regulatory effect of infection on expression of this range of molecules was evident. Thus, down-regulation of co-stimulatory molecules was not the explanation for the lower stimulatory capacity of infected monocytes compared with controls.


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Table 1. Surface molecule expression by monocytes and DCs infected with cp or ncp BVDV

Monocytes and monocyte-derived dendritic cells (DCs) were infected with cp or ncp BVDV and surface molecule expression was assessed at 48 h p.i. (results at 72 h p.i. were similar). The APCs were incubated with primary mAb at predetermined optimal concentrations for 10 min, and then washed extensively. Bound mAb was detected with FITC-labelled anti-mouse IgG. Following this, the cells were fixed and assessed for intracellular expression of BVDV non-structural protein NS3 (p80). The cells were analysed on a FACSCalibur. Cells expressing NS3 were gated and the mean fluorescence intensity of surface molecule staining on the infected (gated) cells was expressed. The average value from two separate experiments is shown.

 
The ability of dendritic cells to resist death induced by cp BVDV is not related to the production of IFN-{alpha}/{beta} by infected cells
It has been reported previously that activated human DCs can avoid death induced by influenza virus by the rapid induction of IFN-{alpha}/{beta} (Cella et al., 1999). Thus, it was necessary to determine whether DCs infected with cp BVDV were avoiding death by the production of IFN-{alpha}/{beta}. Control cells (Cte), monocytes and DCs were infected with ncp BVDV or cp BVDV, or treated with an equivalent volume of mock antigen or 10 µg dsRNA (poly I:C) ml-1 for 48 h. The supernatant was removed from the cells and added to an assay that uses a CAT reporter gene to detect the presence of bovine IFN-{alpha}/{beta} (Fray et al., 2001). The results from monocytes and DCs isolated or generated from four animals and control Cte cells showed that neither Cte cells, monocytes nor DCs incubated with mock antigen or ncp BVDV produced IFN-{alpha}/{beta} after 48 h of culture (Fig. 4). However, Cte cells, monocytes and DCs incubated with cp BVDV produced IFN-{alpha}/{beta} (5·3±1·79, 11·1±3·0 and 12·1±5·3 international units, respectively), with monocytes and DCs producing similar quantities (P<0·05, Student's t-test). Cytopathic BVDV consistently induced more IFN-{alpha}/{beta} than dsRNA in all three cell types examined.



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Fig. 4. IFN-{alpha}/{beta} production after 48 h by monocytes and dendritic cells. Control cells (Cte), monocytes and DCs were infected with ncp BVDV or cp BVDV at an m.o.i. of 2, or treated with an equivalent volume of mock antigen or 10 µg dsRNA (poly I:C) ml-1 for 48 h. The supernatant was removed from the cells and added to an assay that uses a CAT reporter gene to detect the presence of bovine IFN-{alpha}/{beta}. Values are the mean IFN-{alpha}/{beta} concentrations from replicate experiments; error bars are±SEM.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have performed a series of experiments to determine whether monocytes and DCs, derived in vitro by culturing monocytes with GMCSF and IL-4, respond differently to infection with ncp and cp BVDV. The ability to support virus replication, control cytopathic effect and maintain the ability to present antigens was assessed.

Our results showed that DCs are more resistant to infection with ncp BVDV than monocytes. This conclusion was based on experiments in which the level of virus replication was assessed by staining for the presence of the non-structural protein NS3 in the APCs and by titration of infectious virus that was either cell-associated or in supernatants. No evidence was obtained that this difference in susceptibility of the two APCs to infection was dependent on a cytopathogenic effect of ncp BVDV for monocytes. However, despite the clear difference between the virus titres in monocytes and DCs, more than 40 % of the DCs became infected, demonstrating that replication of ncp BVDV was not completely blocked in DCs.

Cytopathic BVDV caused cell death of Cte cells and bovine monocytes, but the same dose of virus had no visible cytopathic effect on DCs. When death of cells was detected by staining and flow cytometry, the same difference in ability of cp BVDV to kill monocytes but not DCs was evident. A higher percentage of monocytes became infected with cp BVDV compared with DCs; however, similar quantities of virus were associated with the cells or in the supernatants of monocyte and DC cultures. Clearly, a definitive interpretation of these complex cell/viral infection culture systems is difficult, but approximately 60 % of DCs became infected and produced viable cp BVDV. Despite infection of the majority of DCs, no cell death was detected. When a limited number of studies were performed using a high m.o.i. of 10, the percentage of infected cells did not increase significantly and the cells remained viable (J. Glew, unpublished observation). Overall, the virus titres in the supernatants of cp BVDV-infected monocytes and DCs were lower than in ncp BVDV-infected cultures. The most likely reason for the different virus titres is the induction of IFN in the cp BVDV cultures.

Studies with influenza virus and human macrophages and DCs have shown similar differences in susceptibility of the two types of APC to the cytolytic action of the virus (Bender et al., 1998). Thus, while 90 % of DCs exposed to m.o.i.s of between 2 and 4 became infected with virus, as judged by staining for viral proteins, the infection was non-toxic. In contrast, the majority of macrophages died within 24–36 h and synthesized tenfold higher levels of virus than DCs. Substantial induction of the antiviral cytokine IFN-{alpha} by the DCs was noted. Subsequent studies with human DCs and influenza (Cella et al., 1999) have also reported that cells were activated by exposure to the virus, as well as to dsRNA. It was proposed that the production of IFN-{alpha}/{beta} and up-regulation of MxA, a protein that is induced by type-1 IFN and mediates resistance to several viruses, protected the DCs from the lethal effects of influenza virus. Our results with cp and ncp BVDV indicated that the difference in resistance to lysis of monocytes and DCs or replication of the viruses was not related to differences in synthesis of IFN-{alpha}/{beta} by the two cell types of APC. Synthesis of IFN-{alpha}/{beta} was evident in both monocytes and DCs exposed to cp BVDV but not in the two types of APC following exposure to ncp BVDV. Thus, differences in induction of IFN-{alpha}/{beta} in the two APC populations is related to the genomic structure of the virus, as has been shown in non-myeloid cells (Adler et al., 1997). DCs are not able to resist lysis by all cytopathogenic viruses, and measles virus (Servet-Delprat et al., 2000) and parainfluenza virus (Plotnicky-Gilquin et al., 2001) have been shown to induce apoptosis of DCs. Therefore, the survival factors produced by DCs must be specific to certain virus types.

A consequence of the infection of monocytes by ncp BVDV was a reduced capacity to stimulate T-cell proliferation. This was evident using two models, one involving antigen uptake, processing and presentation detected using CD4 T cells from ovalbumin-immune animals as the responding cells and one not dependent on uptake, processing and presentation detected using allogeneic CD4 T cells. In contrast to observations with monocytes, infection of DCs had no detectable effect on their ability to stimulate CD4 memory T cells or allogeneic CD4 T cells. We have previously noted effective stimulation of CD4 and CD8 T cells by bovine monocytes infected in vivo with BVDV (Glew and Howard, 2001), but a comparison of DCs was not made in that study. However, since monocytes in that study appeared not to be compromised, it seems likely that acute transient infections and persistent infections in specifically immunotolerant animals might have different consequences. A relationship between the altered expression of a range of co-stimulatory molecules, which included CD80, CD86 and CD40, on APCs and differences in the capacity to stimulate proliferation could not be demonstrated. Further investigations are required to determine whether cytokine production by APCs is affected by infection with BVDV. We have transferred the supernatant from ncp BVDV-infected monocytes to fresh T-cell proliferation assays to establish whether a soluble factor is responsible for the immunosuppresion. The results have been variable, suggesting a soluble factor may be present. Analysis of cells or supernatants using either RT-PCR or ELISA has failed to identify significant up-regulation of IL-10 or TGF-{beta} production (J. Glew & J. Hope, unpublished data).

Transient lymphopenia and immunosuppression characterized by loss of T-cell responses is also evident in animals infected with BVDV (Charleston et al., 2001, 2002), but whether the mechanism of this is similar to that seen with other viruses is not known. DCs transport measles virus to the lymph node and measles virus infection of DCs in vivo results in a reduced ability to stimulate allogeneic T cells, partly due to a CD40L signalling defect and reduced IL-12 synthesis and possibly also due to the effect of viral protein expressed on the surface of DCs giving a negative signal to the T cell. Rauscher leukaemia virus infects bone marrow DCs leading to a failure of T-cell activation associated with dysregulated cytokine production, specifically reduced IL-12 and IL-4 production (Rescigno and Borrow, 2001; Schneider-Schaulies et al., 2002). Another member of the Flaviviridae, hepatitis C virus, has been shown to have a specific suppressive effect in which specific viral polypeptides are implicated (Sarobe et al., 2002).

The interference by viruses with APC function can occur in various ways and is a strategy that appears to have evolved in a number of viruses to enable them to avoid immune effector mechanisms. The induction of the destruction of the APCs could have dire consequences for immunity, as shown by infection of mice with lymphocytic choriomeningitis virus (Borrow et al., 1995), and the ability of DCs to resist this destruction and control virus replication while retaining the ability to present viral antigen to T cells effectively, clearly is to the advantage of the host. There are other examples of DCs controlling virus infection and its consequence in contrast to the effects seen with monocytes, consistent with our findings for BVDV. These include bovine herpes virus 1 (Renjifo et al., 1999) and influenza virus (Bender et al., 1998). Interference with DC maturation has also been reported. For example, vaccinia virus prevents the maturation of immature DCs in response to a variety of maturation signals; the consequence is a failure to induce a CD8 T-cell response (Bonini et al., 2001).

Dissemination of BVDV throughout the host will be expedited by productively infecting migrating APCs. However, there are problems using APCs as a vehicle, as they could induce efficient presentation of viral antigens to naïve T cells via MHC class I and class II (Banchereau et al., 2000). In addition, if DCs are able to resist lysis by cytopathic viruses this would allow presentation of viral antigens. Our studies show that BVDV does not affect the capacity of DCs to present antigen to T cells in vitro; therefore, if the virus is transported by APCs, it is not surprising that alternative strategies have been exploited by the pathogen to cause immunosuppression in the host. Infection with ncp BVDV stimulates a protective immune response, although a specific T-cell proliferative response is relatively slow to develop post-exposure compared with other viruses (Collen & Morrison, 2000). Our observations suggest that DCs are able to stimulate effectively a primary immune response during acute BVDV infections, but that other factors delay the rapid development of that response. One factor causing a delay could be the ability of the virus to compromise the capacity of monocytes, and conceivably B cells, to present antigen. We have demonstrated previously that infection with BVDV transiently suppresses a T-cell memory response to a third party antigen (Charleston et al., 2002). A reduced capacity of in vivo-infected monocytes to stimulate memory T cells may explain this period of suppression.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 7 November 2002; accepted 11 February 2003.