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

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Differential effects of bovine viral diarrhoea virus onmonocytes and dendritic cells

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

Correspondence

Bryan Charleston

[email protected]

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

Received 7 November 2002

Accepted 11 February 2003

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 DCswere 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)-a/b 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-a/b, as proposed for influenza virus, but is by a distinct mechanism.

INTRODUCTION

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 aclosely related group of small enveloped viruses, theFlaviviridae, with a single-stranded, positive-sense RNAgenome of approximately 12?5 kb (Meyers & Thiel, 1996).They all have a similar genomic structure and proteincomposition, the virus particles comprising a single capsidprotein surrounded by an envelope containing two or threeglycoproteins. While primary infection with many of theseviruses causes acute clinical disease, some also give riseto persistent infection associated with immunopathology(Solomon & Mallewa, 2001).

BVDV has a worldwide distribution and readily estab-lishes endemic infection in cattle populations. Disease isassociated with both acute and persistent infections and,depending on epidemiological circumstances, may manifestas outbreaks affecting large numbers of animals or a con-tinual low incidence of cases within endemically infectedherds. Both disease patterns have a major impact on theproductivity of affected cattle populations (Houe, 1999).The virus occurs in two biotypic forms, cytopathic (cp) andnon-cytopathic (ncp), and different isolates of both formscommonly exhibit antigenic differences (Hamers et al., 2001).

Non-cytopathic BVDV is the most prevalent form of thevirus. Acute, self-limiting infections with BVDV are associated

with a period of generalized immunosuppression andincreased susceptibility to secondary infection (Potgieter,1995). The fatal clinical syndrome mucosal disease is theresult of a complex, unique immunopathological event.Infection of the foetus prior to immunocompetence resultsin a persistent lifelong infection of the calf. The animal isspecifically immunotolerant to the persisting virus and ifsuperinfected by a cp strain of BVDV that is antigenicallysufficiently homologous, will die within a few weeks withextensive destruction of the organized immune tissuesbeing evident (Brownlie et al., 1984; Teichmann et al., 2000).These persistently infected cattle are reservoirs of infec-tion for naı̈ve animals. Thus, many of the most importantclinical consequences occur as a result of infection ofpregnant animals with ncp BVDV.

BVDV infects a wide variety of cell types but has apredilection for cells of the immune system. The virusinfects T cells, B cells and antigen-presenting cells (APCs)in vivo (Sopp et al., 1994). Monocytes, macrophages anddendritic cells (DCs) constitute the majority of APCsinvolved in the initial uptake of virus or their antigens andpresentation to the immune system. Of these APCs, DCsare the most effective and the only population that isrecognized as having the ability to initiate primary immuneresponses in naive animals (Banchereau et al., 2000).

Infection of APCs by viruses can have a marked effect onthe cells and important consequences for the generationof the subsequent immune response. The consequences of

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Journal of General Virology (2003), 84, 1771–1780 DOI 10.1099/vir.0.18964-0


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infection include the death of the APC as an extreme event,or more subtle effects on cytokine expression and synthesisof co-stimulatory molecules (Rescigno & Borrow, 2001;Schneider-Schaulies et al., 2002). Studying the interactionof DCs and monocytes with specific viruses and how thismight influence the primary immune response is criticalfor understanding disease pathogenesis and immunity toinfection. We have used an antigenically homologous pairof ncp and cp BVDV isolates to investigate how bovineAPCs, namely DCs and monocytes, respond to infectionwith the different biotypes of virus.

METHODS

Animals. Calves (Bos taurus) were conventionally reared BritishHolstein Friesians bred at the Institute for Animal Health. Eachanimal was of a known MHC class I haplotype (Ellis et al., 1998).The immune status of each animal was determined by assaying serafor antibody to BVDV and by testing for persistent viraemia (Frayet al., 1999). Some of the animals had been previously immunizedwith ovalbumin, as reported by Howard et al. (1997). All experi-ments were approved by the Institute’s ethical review process andwere in accord with national guidelines on animal use.

Cells. Monocytes were isolated from peripheral blood mononuclearcells (PBMCs) following incubation with anti-human CD14-labelledsuperparamagnetic particles (Miltenyi-Biotech) and labelled cellswere isolated from a Midimacs column (Miltenyi-Biotech) accordingto the manufacturer’s instructions. The purity of the cells evaluatedby flow cytometry was shown in each case to be >98 %. Cell viabi-lity was >95 %. For some experiments monocytes were purified byflow cytometry; the purity of the monocytes was >99 %.

DCs, or more accurately monocyte-derived dendritic cells, weregenerated from CD14+ monocytes as previously described (Hopeet al., 2000). Monocytes were adjusted to 86105 cells ml21 in tissueculture medium (TCM) [RPMI 1640 containing Glutamax-1 (LifeTechnologies), 10 % heat inactivated foetal calf serum, 561025 Mb-mercaptoethanol, 50 mg gentamicin ml21], plus 200 U COS-7 cell-derived bovine rIL-4 ml21 and 0?2 U bovine rGMCSF ml21 (unitsbased on induction of half maximal proliferation in bone marrowprecursor cells). DCs were harvested and infected after incubation for6 days at 37 C̊ in 5 % CO2.

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

Virus isolation. Virus was isolated from Cte cells cultured on cover-slips, as previously described (Brownlie et al., 1984). Five replicatecoverslips were used for each sample. Virus-neutralizing antibodytitres were determined using a microtitre-based assay, as previouslydescribed (Brownlie et al., 1984).

To determine whether the production of infectious ncp BVDV dif-fered between monocytes and DCs, virus replication was assayed atthe same m.o.i. (2). Supernatants were removed from cultures ofmonocytes and DCs at 24 h time intervals and titrated to assayextracellular virus. Cell-associated virus was obtained by freeze-thawing monocytes and DCs. Titres of extracellular virus wereobtained from six experiments with monocytes and DCs isolatedfrom four animals. Titres of cell-associated virus were obtained fromfour 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 grownin 96-well plates (Costar) until 80–90 % confluent. After removal ofmedium, cells were washed with sterile PBS and 50 ml viral growthmedia (VGM) (Brownlie et al., 1984). Serial tenfold dilutions of thetest samples were made in VGM and 50 ml added to each well. After30 min, a further 100 ml VGM was added and the plates incubated for5 days. After 5 days the medium was removed from each well andthe cells fixed with ice-cold 80 % acetone and air-dried. Each well wasincubated with PBS/0?05 % Tween for 30 min at room temperature.After washing, each well was incubated overnight at 4 ˚C with 25 mlof a 1 : 500 dilution of monoclonal antibody (mAb) WB103 (Edwardset al., 1991) or isotyped matched control. Antibody staining wasrevealed using horseradish peroxidase-conjugated goat anti-mouse IgGfollowed by tetramethylbenzidine (ICN). Virus titres (TCID50) werecalculated from replicate wells.

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

Cell viability. Monocytes and DCs were stained with propidiumiodide (PI; Sigma) and Annexin V (Boehringer Mannheim), whichin combination indicate cells that are dying by both necrosis andapoptosis (Cella et al., 1999). Total cell counts were made beforestaining, and similar numbers of cells were analysed. Staining wasanalysed immediately by flow cytometry. Live cells were negative forPI and/or Annexin V. The mean survival of ncp/cp BVDV-infectedmonocytes and DCs was determined in four separate experimentswith cells isolated and generated from three animals.

Type 1 interferon assay. Levels of biologically active type 1 IFNwere assayed in duplicate in samples of culture media using achloramphenicol transferase (CAT) reporter assay that detects, butdoes not distinguish between, IFN-a and IFN-b (Fray et al., 2001).

T-cell proliferation assays. Purified monocytes or DCs wereinfected by adding BVDV strain Pec515ncp (m.o.i. 2) and incubat-ing for 2 days (experiments were done 2 and 3 days after infectionwith BVDV; the results were similar). These cells and mock-infectedcells were used as APCs with allogeneic CD4+ T lymphocytes orMHC-identical CD4+ T lymphocytes from ovalbumin-immunizedanimals. CD4+ T lymphocytes were purified from PBMCs by stain-ing with mAb CC8 and purifying cells with anti-mouse IgG labelledsuperparamagnetic beads (Miltenyi-Bitech), as described previously(Hope et al., 2000). In some experiments the APCs were incubatedwith ovalbumin (125 mg ml21) for 1 h before they were washedtwice with TCM. In some cases monocytes were sorted from PBMCson a FACStar-plus (Becton Dickinson) after CD14 staining toprovide cells of ¢99 % purity. APCs were irradiated (20 Gy from a137Cs source) and dilutions were incubated with 105 CD4+ Tlymphocytes. Triplicate cultures were incubated for 5 days and37 Bq [3H]thymidine (DuPont) was added for 16 h (overnight)before harvesting. Incorporated radioactivity was determined byliquid scintillation counting. The results of ten separate experimentsusing cells from three different animals are presented.

Flow cytometric analysis of surface molecule expression byBVDV-infected APCs. To determine the effect of virus infectionon expression of a range of co-stimulatory surface molecules, APCswere infected with cp or ncp BVDV as described above and surface

1772 Journal of General Virology 84

E. J. Glew and others


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molecule expression was assessed at 48 and 72 h post-infection(p.i.). The surface molecules assessed were MHC class I (mAbIL-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 pre-viously (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; providedby N. MacHugh, Centre for Tropical Medicine, Roslin, UK). ControlmAbs used within the study were AV20 (mouse IgG1), AV29(mouse IgG2b) and AV37 (mouse IgG2a), directed against chickenbursal 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 predeterminedoptimal concentrations for 10 min, then washed extensively. BoundmAb was detected with FITC-labelled anti-mouse IgG (SouthernBiotechnologies). Following this, cells were fixed and assessed forintracellular expression of NS3 (p80) as described above. The cellswere analysed on a FACSCalibur (Becton Dickinson). Immuno-fluorescent staining was analysed using FCS express. Cells expressingNS3 were gated and the mean fluorescent intensity of surface mole-cule staining on the infected (gated) cells was expressed.

RESULTS

Monocytes and monocyte-derived dendritic cellsare susceptible to infection with ncp BVDV

Fig. 1(a) shows the mean percentage of NS3-stainedmonocytes and DCs at various time points p.i. The effectsof time after infection and cell type were investigated usingan analysis of variance (ANOVA) with a blocked design.Unsurprisingly, the percentage of ncp BVDV-infected cellswas significantly affected by the time interval betweeninfection and the time of observation (P<0?001). Cell typealso had a significant effect on the percentage of infectedcells (P<0?001), as the percentage of infected cells washigher for monocytes than for DCs at all time points. Therewas also a significant cell type/time interaction (P=0?035)as the magnitude of the difference between the numbersof infected cells in the two populations increased with time.At 24 h p.i., the percentage of infected monocytes wasonly 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 amarked increase in the magnitude of this difference(90?5±1?5 % and 68?5±7?0 % at 72 h; 93?8±1?1 % and53?7±5?3 % at 96 h for monocytes and DCs, respectively).

The titres of infectious virus in either the supernatant oras cell-associated virus are shown in Fig. 1(b, c). It wasapparent from visual inspection of the data that there wasno significant difference in the titres of extracellular virusbetween populations of monocytes or DCs at time pointsup to and including 48 h. This indicated that virus entryand binding did not differ significantly between the twocell types. A blocked ANOVA was performed on log-transformed extracellular virus titres from monocytes andDCs, incorporating the effects of experiment and time (72and 96 h p.i. only) into the analysis. This analysis revealed

a highly significant effect of cell type (P<0?001), as extra-cellular virus titres were higher for monocytes than for DCs.The cell-associated virus titres were analysed in a similarmanner. It was apparent from visual inspection of the datarecorded at 1 and 24 h p.i. that there was no significantdifference between cell type at these times (Fig. 1c). Hence,the analysis was restricted to observations made at 48, 72and 96 h. This analysis revealed a significant effect ofcell type (P=0?01) on the cell-associated virus titre, asvirus titres were higher for monocytes than for DCs atthe analysed time points.

Although ncp BVDV has not been associated with thedeath of cultured cells, survival of ncp BVDV-infectedmonocytes and DCs was assessed to determine whethervirus-induced cell death was responsible for the decreasein virus titre seen in populations of DCs. It was apparentfrom visual inspection of the data that there was a similarproportion of viable cells in each population at each timepoint p.i. (Fig. 1d). The pattern of the changes in theproportion of viable monocytes and DCs over time inculture was similar for ncp BVDV-infected and mock-infected cells (data not shown).

Dendritic cells are resistant to lysis with cpBVDV but monocytes are susceptible

Cytopathic BVDV has been shown to kill cells by theinduction of apoptosis (Zhang et al., 1996); hence, PI andAnnexin V staining were used to determine the percentageof dead cells in the populations of monocytes and DCsexposed to cp BVDV. Fig. 1(h) shows the percentage ofviable monocytes and DCs at various time points after cpBVDV challenge. A blocked ANOVA showed that both celltype and time point had a highly significant effect on thepercentage of viable monocytes (P<0?001). There wasalso a highly significant interaction between these twoparameters (P<0?001), the magnitude of the differenceincreasing with time. This was apparent from Fig. 1(h). Thenumber of viable monocytes decreased from 76±2?1 % at1 h p.i. to 26±10?1 % at 96 h p.i. In contrast, viabilityof DCs did not deviate markedly from approximately 75 %at each time point.

To establish whether cp BVDV was able to replicateefficiently in monocytes and DCs, cells were incubatedwith cp BVDV and stained for viral NS3. Staining wasanalysed by flow cytometry. Dead cells were distinguishedfrom live cells by their decreased forward and side scatterprofile on the dot plots and were not included in theanalysis of NS3 expression. Fig. 1(e) shows mean stainingfor NS3 in populations of live cells from four experiments.The data were analysed using a blocked ANOVA, whichrevealed a significant effect of cell type (P<0?001) and time(P=0?001) and a significant interaction between the twoparameters. This was because both monocytes and DCsexpressed 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

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Differential effects of BVDV on APCs


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and 96 h p.i., a significantly lower proportion of DCsexpressed NS3 compared with monocytes.

Virus replication curves were undertaken to quantify theproduction of infectious virus from populations of mono-cytes and DCs. Fig. 1(f) and (g) show the titres of extra-cellular and cell-associated virus, respectively, at 24, 48, 72and 96 h p.i. Similar quantities of cp BVDV were producedat each time point after infection except that there wasless cell-associated virus present in the DC cultures at24 h p.i. (P<0?05, Student’s t-test).

The effect of cp BVDV infection on Cte cells, monocytesand DCs was determined (Fig. 2). Cte cells, monocytes

and DCs incubated with ncp BVDV (Fig. 2d–f) appearedsimilar to mock-infected cells (Fig. 2a–c). CytopathicBVDV caused an extensive cytopathic effect in Cte cellsand monocytes (Fig. 2g, h); however, no cytopathic effectwas seen in DCs (Fig. 2i).

Monocytes infected with ncp BVDV arecompromised in their ability to stimulate T cellresponses but dendritic cells are not affected

The effect of BVDV infection on the ability of APCs tostimulate allogenic CD4+ T cells was determined (Fig. 3).The proliferation induced by infected monocytes was lower

Fig. 1. Monocytes (&) and dendritic cells (%) 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. Thetitres (TCID50) of extracellular virus (b, f) and cell-associated virus (c, g) were determined. The percentage of viable cellspresent in each culture is shown in (d) and (h). The values represented in all the graphs are the means of replicateexperiments; error bars are±SEM.




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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 experimentswas the proliferation induced by infected DCs significantlylower than the proliferation induced by mock-infectedDCs. For all experiments, the incorporation of thymidineinto APCs alone or CD4+ T cells alone was <800 c.p.m.Representative data from monocyte- or DC-induced allo-geneic proliferation are shown in Fig. 3 (upper graphs).

It was possible that monocytes, which were isolated withparamagnetic beads from PBMCs of BVDV-immune

animals and were >97 % pure for CD14+ cells, werecontaminated with T or B cells. These lymphocytes mayhave been stimulated by the ncp BVDV-infected mono-cytes to produce immumodulatory cytokines, which wereresponsible for the reduction in allogeneic proliferation seenwith BVDV-infected monocytes. Thus, monocytes werepurified by flow cytometry before titration with allogeneicT cells. The purity of the monocytes was greater than99 %, and in three separate experiments the ncp BVDV-infected monocytes stimulated an allogeneic T cell responsethat was significantly lower than the mock-infected cells(data not shown), as was the case with CD14+ monocytes

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) orinfected with non-cytopathic BVDV (NCP BVDV) or cytopathic BVDV (CP BVDV). After 96 h in culture, cytopathic effect wasonly visible in cultures of monocytes and Cte cells. Representative photomicrographs of each culture are shown.




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that had been purified with magnetic beads. Staining ofthe allogeneic CD4+ T cells with mAb to NS3 and analysisby flow cytometry after 5 days culture with ncp BVDVinfected monocytes indicated that the CD4+ T cells werenot infected with ncp BVDV (data not shown).

To assess the effect of infection by BVDV on the abilityof the APCs to process and present exogenous antigen,monocytes and DCs were pulsed with ovalbumin. In 7/8experiments, BVDV-infected monocytes were compromisedin their ability to stimulate ovalbumin-specific CD4+

T cell proliferation when compared with mock-infected,ovalbumin-pulsed monocytes. The reduction of prolifera-tion was significantly lower in six experiments and arepresentative sample of data is shown in Fig. 3 (lowergraphs). In contrast, DCs were not compromised in theirability to stimulate ovalbumin-specific T cells. This wasconfirmed by four experiments and a representativesample 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 BVDVinfection of monocytes, but not of DCs, compromised theirstimulatory capacity and possibly their processing andpresentation capacity.

The expression of a range of co-stimulatory moleculeswas compared for DCs infected with ncp and cp BVDV andmonocytes infected with ncp BVDV (Table 1). The effectof cp BVDV on surface antigen expression by monocytescould not be examined, as it was lytic for these cells. Noevidence for a down-regulatory effect of infection onexpression of this range of molecules was evident. Thus,down-regulation of co-stimulatory molecules was not theexplanation for the lower stimulatory capacity of infectedmonocytes compared with controls.

The ability of dendritic cells to resist deathinduced by cp BVDV is not related to theproduction of IFN-a/b by infected cells

It has been reported previously that activated human DCscan avoid death induced by influenza virus by the rapidinduction of IFN-a/b (Cella et al., 1999). Thus, it wasnecessary to determine whether DCs infected with cp BVDVwere avoiding death by the production of IFN-a/b. Controlcells (Cte), monocytes and DCs were infected with ncpBVDV or cp BVDV, or treated with an equivalent volumeof mock antigen or 10 mg dsRNA (poly I:C) ml21 for48 h. The supernatant was removed from the cells andadded to an assay that uses a CAT reporter gene to detectthe presence of bovine IFN-a/b (Fray et al., 2001). Theresults from monocytes and DCs isolated or generatedfrom four animals and control Cte cells showed that neitherCte cells, monocytes nor DCs incubated with mock antigenor ncp BVDV produced IFN-a/b after 48 h of culture(Fig. 4). However, Cte cells, monocytes and DCs incubatedwith cp BVDV produced IFN-a/b (5?3±1?79, 11?1±3?0and 12?1±5?3 international units, respectively), with mono-cytes and DCs producing similar quantities (P<0?05,Student’s t-test). Cytopathic BVDV consistently inducedmore IFN-a/b than dsRNA in all three cell types examined.

DISCUSSION

We have performed a series of experiments to determinewhether monocytes and DCs, derived in vitro by culturingmonocytes with GMCSF and IL-4, respond differently toinfection with ncp and cp BVDV. The ability to supportvirus replication, control cytopathic effect and maintainthe ability to present antigens was assessed.

Fig. 3. Effect of ncp-BVDV infection onantigen presentation. Purified monocytes orDCs were infected by adding ncp BVDV(m.o.i. of 2) and incubating for 2 days(results after 3 days were similar). Thesecells and mock-infected cells were used asAPCs with allogeneic CD4+ T lymphocytesor MHC-identical CD4+ T lymphocytesfrom ovalbumin-immunized animals. In someexperiments the APCs were incubated withovalbumin (125 mg ml21) for 1 h beforewashing. APCs were irradiated and dilutions(10–0?16103 cells) were incubated with105 CD4+ T lymphocytes. Triplicate cultureswere incubated for 5 days and 37 Bq[3H]thymidine was added for 16 h (over-night) before harvesting. Incorporated radio-activity was determined by liquid scintillationcounting and presented as counts6103 min21±SD.




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Our results showed that DCs are more resistant to infec-tion with ncp BVDV than monocytes. This conclusion wasbased on experiments in which the level of virus replica-tion was assessed by staining for the presence of thenon-structural protein NS3 in the APCs and by titrationof infectious virus that was either cell-associated or in

supernatants. No evidence was obtained that this differ-ence in susceptibility of the two APCs to infection wasdependent on a cytopathogenic effect of ncp BVDV formonocytes. However, despite the clear difference betweenthe virus titres in monocytes and DCs, more than 40 % ofthe DCs became infected, demonstrating that replicationof ncp BVDV was not completely blocked in DCs.

Cytopathic BVDV caused cell death of Cte cells and bovinemonocytes, but the same dose of virus had no visiblecytopathic effect on DCs. When death of cells was detectedby staining and flow cytometry, the same difference in abilityof cp BVDV to kill monocytes but not DCs was evident. Ahigher percentage of monocytes became infected with cpBVDV compared with DCs; however, similar quantities ofvirus were associated with the cells or in the supernatants ofmonocyte and DC cultures. Clearly, a definitive interpreta-tion of these complex cell/viral infection culture systems isdifficult, but approximately 60 % of DCs became infectedand produced viable cp BVDV. Despite infection of themajority of DCs, no cell death was detected. When a limitednumber of studies were performed using a high m.o.i. of10, the percentage of infected cells did not increase signifi-cantly and the cells remained viable (J. Glew, unpublishedobservation). Overall, the virus titres in the supernatantsof cp BVDV-infected monocytes and DCs were lower thanin ncp BVDV-infected cultures. The most likely reason forthe different virus titres is the induction of IFN in the cpBVDV cultures.

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 incu-

bated with primary mAb at predetermined optimal concentrations for 10 min, and then washed exten-

sively. 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 ana-

lysed 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.

Dendritic cells Monocytes

ncp BVDV cp BVDV Mock-infected ncp BVDV Mock-infected

MHC I 408?5 331?0 378?3 1359?3 1431?1

MHC II 187?0 171?5 196?6 282?0 233?0

CD11a 264?0 160?4 205?1 93?5 98?3

CD40 68?0 56?2 56?4 48?0 43?5

CD80 70?5 64?0 57?0 21?0 21?0

CD86 68?0 58?9 60?6 27?0 26?0

CD11b 75?2 54?4 53?9 840?3 997?6

CD11c 61?5 51?4 48?4 ND ND

CD14 52?2 50?9 50?8 961?0 1087?6

CD62L 48?3 51?0 47?2 ND ND

MyD-1 61?4 51?6 51?0 1977?2 2025?0

MR 55?1 50?7 48?9 ND ND

CD32 64?0 59?5 60?4 ND ND

CD1 59?1 60?5 77?3 ND ND

Fig. 4. IFN-a/b production after 48 h by monocytes and den-dritic cells. Control cells (Cte), monocytes and DCs wereinfected with ncp BVDV or cp BVDV at an m.o.i. of 2, ortreated with an equivalent volume of mock antigen or 10 mgdsRNA (poly I:C) ml21 for 48 h. The supernatant was removedfrom the cells and added to an assay that uses a CAT reportergene to detect the presence of bovine IFN-a/b. Values are themean IFN-a/b concentrations from replicate experiments; errorbars are±SEM.




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Studies with influenza virus and human macrophages andDCs have shown similar differences in susceptibility of thetwo types of APC to the cytolytic action of the virus (Benderet al., 1998). Thus, while 90 % of DCs exposed to m.o.i.sof between 2 and 4 became infected with virus, as judgedby staining for viral proteins, the infection was non-toxic.In contrast, the majority of macrophages died within24–36 h and synthesized tenfold higher levels of virusthan DCs. Substantial induction of the antiviral cytokineIFN-a by the DCs was noted. Subsequent studies withhuman DCs and influenza (Cella et al., 1999) have alsoreported that cells were activated by exposure to the virus,as well as to dsRNA. It was proposed that the productionof IFN-a/b and up-regulation of MxA, a protein that isinduced by type-1 IFN and mediates resistance to severalviruses, protected the DCs from the lethal effects ofinfluenza virus. Our results with cp and ncp BVDV indi-cated that the difference in resistance to lysis of monocytesand DCs or replication of the viruses was not related todifferences in synthesis of IFN-a/b by the two cell types ofAPC. Synthesis of IFN-a/b was evident in both monocytesand DCs exposed to cp BVDV but not in the two types ofAPC following exposure to ncp BVDV. Thus, differences ininduction of IFN-a/b in the two APC populations is relatedto the genomic structure of the virus, as has been shown innon-myeloid cells (Adler et al., 1997). DCs are not able toresist lysis by all cytopathogenic viruses, and measlesvirus (Servet-Delprat et al., 2000) and parainfluenza virus(Plotnicky-Gilquin et al., 2001) have been shown to induceapoptosis of DCs. Therefore, the survival factors producedby DCs must be specific to certain virus types.

A consequence of the infection of monocytes by ncpBVDV was a reduced capacity to stimulate T-cell prolifera-tion. This was evident using two models, one involvingantigen uptake, processing and presentation detected usingCD4 T cells from ovalbumin-immune animals as the res-ponding cells and one not dependent on uptake, processingand presentation detected using allogeneic CD4 T cells. Incontrast to observations with monocytes, infection of DCshad no detectable effect on their ability to stimulate CD4memory T cells or allogeneic CD4 T cells. We have pre-viously noted effective stimulation of CD4 and CD8 T cellsby bovine monocytes infected in vivo with BVDV (Glewand Howard, 2001), but a comparison of DCs was notmade in that study. However, since monocytes in thatstudy appeared not to be compromised, it seems likelythat acute transient infections and persistent infections inspecifically immunotolerant animals might have differentconsequences. A relationship between the altered expressionof a range of co-stimulatory molecules, which includedCD80, CD86 and CD40, on APCs and differences in thecapacity to stimulate proliferation could not be demon-strated. Further investigations are required to determinewhether cytokine production by APCs is affected byinfection with BVDV. We have transferred the supernatantfrom ncp BVDV-infected monocytes to fresh T-cell pro-liferation assays to establish whether a soluble factor is

responsible for the immunosuppresion. The results havebeen variable, suggesting a soluble factor may be present.Analysis of cells or supernatants using either RT-PCR orELISA has failed to identify significant up-regulation ofIL-10 or TGF-b production (J. Glew & J. Hope, unpub-lished data).

Transient lymphopenia and immunosuppression character-ized by loss of T-cell responses is also evident in animalsinfected with BVDV (Charleston et al., 2001, 2002), butwhether the mechanism of this is similar to that seen withother viruses is not known. DCs transport measles virus tothe lymph node and measles virus infection of DCs in vivoresults in a reduced ability to stimulate allogeneic T cells,partly due to a CD40L signalling defect and reduced IL-12synthesis and possibly also due to the effect of viral proteinexpressed on the surface of DCs giving a negative signal tothe T cell. Rauscher leukaemia virus infects bone marrowDCs leading to a failure of T-cell activation associatedwith dysregulated cytokine production, specifically reducedIL-12 and IL-4 production (Rescigno and Borrow, 2001;Schneider-Schaulies et al., 2002). Another member ofthe Flaviviridae, hepatitis C virus, has been shown to havea specific suppressive effect in which specific viral poly-peptides are implicated (Sarobe et al., 2002).

The interference by viruses with APC function can occurin various ways and is a strategy that appears to haveevolved in a number of viruses to enable them to avoidimmune effector mechanisms. The induction of thedestruction of the APCs could have dire consequences forimmunity, as shown by infection of mice with lymphocyticchoriomeningitis virus (Borrow et al., 1995), and the abilityof DCs to resist this destruction and control virus replica-tion while retaining the ability to present viral antigen toT cells effectively, clearly is to the advantage of the host.There are other examples of DCs controlling virus infec-tion and its consequence in contrast to the effects seenwith monocytes, consistent with our findings for BVDV.These include bovine herpes virus 1 (Renjifo et al., 1999) andinfluenza virus (Bender et al., 1998). Interference with DCmaturation has also been reported. For example, vacciniavirus prevents the maturation of immature DCs in responseto a variety of maturation signals; the consequence is afailure to induce a CD8 T-cell response (Bonini et al., 2001).

Dissemination of BVDV throughout the host will beexpedited by productively infecting migrating APCs.However, there are problems using APCs as a vehicle, asthey could induce efficient presentation of viral antigensto naı̈ve T cells via MHC class I and class II (Banchereauet al., 2000). In addition, if DCs are able to resist lysis bycytopathic viruses this would allow presentation of viralantigens. Our studies show that BVDV does not affect thecapacity of DCs to present antigen to T cells in vitro;therefore, if the virus is transported by APCs, it is notsurprising that alternative strategies have been exploited bythe pathogen to cause immunosuppression in the host.Infection with ncp BVDV stimulates a protective immune




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response, although a specific T-cell proliferative responseis relatively slow to develop post-exposure compared withother viruses (Collen & Morrison, 2000). Our observationssuggest that DCs are able to stimulate effectively a primaryimmune response during acute BVDV infections, but thatother factors delay the rapid development of that response.One factor causing a delay could be the ability of the virusto compromise the capacity of monocytes, and conceivablyB cells, to present antigen. We have demonstrated pre-viously that infection with BVDV transiently suppresses aT-cell memory response to a third party antigen (Charlestonet al., 2002). A reduced capacity of in vivo-infected mono-cytes to stimulate memory T cells may explain this periodof suppression.

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

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