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Cytokine responses of bovine dendritic cells and T cells following exposure to live or inactivated bovine respiratory syncytial virus Dirk Werling, Robert A. Collins, Geraldine Taylor, and Chris J. Howard Institute for Animal Health, Compton, Berks, United Kingdom

Abstract: We compared the effects of live or inactivated bovine respiratory syncytial virus (BRSV) on cytokine production by bovine monocyte-derived dendritic cells (MoDC). We also investigated the response of resting memory CD4ⴙ T cells to MoDC exposed to both viral preparations. Although BRSV did not appear to replicate in MoDC or to affect expression of major histocompatibility complex (MHC) class I, MHC class II, or CD80/86, a higher percentage of cells exposed to live virus appeared to undergo apoptosis/necrosis. To investigate how the interaction of BRSV with MoDC affects the immune response, a multiplex, realtime, polymerase chain reaction was established to analyze transcription of bovine cytokines. Exposure of MoDC to live BRSV induced more interleukin (IL)-10 mRNA and markedly less IL-12p40 and IL-15 mRNA than did heat-inactivated virus. To determine whether these differences might influence the T cell response, CD4ⴙ memory T cells primed in vivo were restimulated in vitro by MoDC pulsed with heat-inactivated or live BRSV. Stimulation of CD4ⴙ T cells induced similar levels of IL-2-and IL-4-like activity and interferon-␥. These observations suggest that while IL-10, produced by MoDC as a result of exposure to live BRSV, may affect IL-12 and IL-15 synthesis by MoDC, it does not appear to affect the cytokine response of BRSV-specific memory CD4ⴙ T cells. It is possible, however, that differences in the pattern of cytokines produced by MoDC exposed to live or inactivated virus may influence the development of the primary CD4ⴙ T cell response in vivo. J. Leukoc. Biol. 72: 297–304; 2002.

INTRODUCTION

developed. These vaccines include live, attenuated, coldadapted, and inactivated virus, but with little success [1]. In the 1960s, vaccination of infants with formalin-inactivated (FI), tissue culture grown HRSV (FI-HRSV) induced more severe respiratory disease following natural exposure to HRSV than that seen in infants given a FI-parainfluenza virus-3 vaccine. Subsequent experiments in a murine model demonstrated that priming with live RSV infection induces a T helper cell type 1 (Th1) response, whereas priming with inactivated virus induces a Th2 response when mice were subsequently challenged with live RSV [2]. These studies also showed that the vaccineaugmented pulmonary pathology associated with inactivated HRSV is mediated by Th2 cells [2– 4]. The two major protective antigens (Ags), F and G glycoproteins, which play a crucial role in RSV uptake/penetration by the cell [5], also appear to prime different T cell responses. Thus, BALB/c mice immunized with recombinant vaccinia viruses expressing the F or the G protein of HRSV developed a type 1 or a type 2 cytokine response, respectively, when subsequently challenged with live RSV [6, 7]. Dendritic cells (DC) are the only antigen-presenting cells (APC) recognized as having the ability to prime naı¨ve T cells and to initiate primary T cell-mediated responses [8]. DC also dictate the development of T cell-mediated immune responses into Th1 or Th2 [9], and a given DC subset can induce a Th1 or Th2 response depending on the type of stimulation and the pathogen [10, 11]. Subsequently, cytokines secreted by T cells activated by DC can determine whether protective immunity develops. In some diseases, such as AIDS, human leprosy, or murine leishmaniasis [12–14], the ability of the host to mount a Th1 response has been associated with the capacity to eliminate pathogens, whereas a Th2-biased immune response to these pathogens is associated with disease progression. The mechanisms by which biased immune responses are induced are not yet completely understood. However, products of the cells that take up, process, and present Ag can affect the initiation and outcome of the immune response. The ability of DC to stimulate a primary and a memory T cell response has been related to their unique capacity to retain Ag

Respiratory syncytial viruses (RSV) are the most common causes of severe, lower respiratory tract infections in human infants and young calves. Human (H) and bovine (B) RSV are closely related, but antigenically distinct. There is still a clear medical and veterinary requirement for effective vaccines against these viruses, and several different vaccines have been

Correspondence: Chris J. Howard, Institute for Animal Health, Compton, Newbury Berks, RG20 7NN, UK. E-mail: [email protected] Current address of Dirk Werling: Institute of Veterinary Virology, University of Bern, La¨nggass Str. 122, CH-3012 Bern, Switzerland Received January 21, 2002; revised April 4, 2002; accepted April 8, 2002.

Key Words: MHC classes I/II 䡠 real time PCR 䡠 IL-2 䡠 IL-4 䡠 IFN-␥

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and their high levels of expression of major histocompatibility complex (MHC) class II molecules and of costimulatory molecules (e.g., CD80/86) and early adhesion molecules (e.g., intercellular adhesion molecule-1) [15–17]. An additional feature could be the level or spectrum of cytokines produced by DC as compared with other APC, which might play an important role in the generation of effective immune responses and modulation of the Th1/Th2 balance. There are reports that live, attenuated, or inactivated viruses affect DC function in different ways [18 –20]. Thus, induction of cytotoxic lymphocytes (CTL) in an influenza model required infectious virus and processing onto MHC class I molecules by DC. In contrast, ultraviolet-inactivated or bromelain-treated viruses induced only a poor induction of CTL, but were presented efficiently to class II-restricted CD4⫹ T cells [18]. This study was undertaken to determine if differences in T cell priming by live and inactivated RSV could be mediated by differences in the effects of these virus preparations on DC function. We investigated the effects of live and heat-inactivated BRSV on DC survival, changes in phenotype, cytokine production by DC, and the CD4⫹ memory T cells response to BRSV presented by DC.

MATERIALS AND METHODS Preparation of cells Conventionally reared Bos Taurus (Channel Island or British Holstein-Friesian cattle) were immunized subcutaneously with glutaraldehyde-fixed bovine nasal mucosal cells (NM7) persistently infected with BRSV in Quil-A adjuvant [21]. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation (1.083 g ml⫺1 Histopaque, Sigma Chemical Co., Poole, Dorset, UK). If not otherwise stated, cells were resuspended in tissue culture medium (TCM) consisting of RPMI-1640 medium with Glutamax I (Life Technologies Ltd., Paisley, Renfrewshire, UK), supplemented with 10% heatinactivated fetal calf serum, 5 ⫻ 10⫺5 M 2-mercaptoethanol, and 100 IU ml⫺1 gentamycin (Sigma Chemical Co.). DC were generated from the adherent cells in PBMC as monocyte-derived DC (MoDC) [22]. B cells (Bc) and CD4⫹ T cells were isolated from PBMC after staining with monoclonal antibody (mAb) to B immunoglobulin (Ig) light chain (IL-A58) or mAb specific for B CD4 (CC8) [23]. Thereafter, cells were incubated with anti-mouse IgG1 superparamagnetic particles (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany), and labeled cells were isolated using MiniMacs威 columns (Miltenyi Biotech) following the manufacturer’s instructions. The purity of the cells was evaluated by flow cytometry (FCM) and shown to be ⬎98%. Cell viability, assessed by exclusion of trypan blue, was greater than 95%. Purified Bc were activated by incubation in TCM with 200 U ml⫺1 recombinant B IL-4 (rbIL-4) for 18 h before use. All incubations were at 37°C in 5% CO2 in air.

Virus The Snook strain of BRSV has been described previously [24] and was propagated in fetal calf kidney (FCK) cells. Cell lysates from infected or uninfected FCK cells were stored at ⫺70°C. For live BRSV, freshly thawed samples were always used. Heat inactivation of virus or control cell lysates was carried out at 56°C for 30 min. Virus titres were determined by plaque assay on FCK monolayers as described previously [25].

Assessment of BRSV replication in bovine MoDC To determine whether BRSV replicated in MoDC, cells were incubated with virus at a multiplicity of infection (MOI) of 1 for 2 h at 37°C. Cells were extensively washed with phosphate-buffered saline (PBS) and were left in culture for 8 h, 24 h, and 48 h at 37°C. Thereafter, cells and medium were harvested together and stored at ⫺70°C. The titres of BRSV in thawed lysates

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of MoDC were determined by plaque assay [25]. To assess the effect of live BRSV on MoDC survival, MoDC were incubated at a concentration of 106 cells per well with live BRSV at a MOI of 1 and incubated at 37°C in 5% CO2 in air. Apoptosis was analyzed after different times in culture using a mixture of fluorescein isothiocyanate (FITC)-labeled annexin V (Ax; Boehringer Mannheim, Mannheim, Germany) and propidium iodine (PI; 50 ␮g ml⫺1, Sigma Chemical Co.) in HEPES-binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) to distinguish between apoptotic and dead cells. MoDC were pelleted by centrifugation (300 g, 2min), resuspended in 25 ␮l Ax/PI mix, and incubated for 5 min. The volume was increased to 300 ␮l with HEPES-binding buffer, and the cells were analyzed by FCM using the FL-1 and FL-2 channels to measure bound FITC-labeled Ax and PI fluorescence, respectively. Expression of MHC class I, MHC class II, and the costimulatory molecule CD80/86 was monitored as described previously [22]. The sources of mouse mAb and their isotypes, fusion proteins, secondary reagents, and methods for FCM have been described in detail [22, 26]. mAb 19 is specific for the F protein of RSV and was used to detect expression of the BRSV F protein [27]. Bound antibody was detected with FITC-labeled anti-mouse IgG. Immunofluorescent staining was analyzed using PCLysys威 software (Becton Dickinson, San Jose, CA).

Multiplex TaqMan real time-polymerase chain reaction for bovine cytokines The relative amount of cytokine transcribed by MoDC in response to live BRSV, heat-inactivated BRSV, cell lysate from uninfected cells, or medium was assessed using the TaqMan™ RT-PCR technology [28]. Samples were assayed after 2-h incubation with the Ags to examine messenger RNA for the cytokines investigated, synthesized as an early consequence of their effect on the MoDC. Total RNA was extracted from lysed MoDC using the RNeasy mini kit (Qiagen, Chatsworth, CA). The extracted total RNA was treated with RNase-free DNase I [DNA-free™, Ambion (Europe) Ltd., Cambridge, UK] to remove contaminating genomic DNA, and eluted total RNA was subsequently transcribed to cDNA using the Reverse Transcription System™ (Promega, Southampton, UK) according the manufacturer’s protocol. The cDNA was analyzed immediately. The primers and TaqMan probes were designed as described [29] using Primer Express software (Applied Biosystems, Foster City, CA), and sequences of the primers and probes are listed in Table 1. TaqMan PCR for the 18-s ribosomal RNA control (Applied Biosystems) and B cytokines were run as multiplex PCR in the same well and calculated using the comparative CT method (User Manual 2, Applied Biosystems). The PCR reactions contained 300 nM each primer, 200 nM TaqMan probe, and commercially available PCR Mastermix (TaqMan Universal PCR Mastermix, Applied Biosystems), 1.25 ␮l 18-s control, and 2.5 ␮l diluted cDNA sample in a total volume of 25 ␮l. The samples were placed in 96-well plates and amplified in an automated fluorometer (ABI Prism 7700 Sequence Detection System, Applied Biosystems). Amplification conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C, and 60 s at 60°C.

Proliferation assays To compare the effectiveness of MoDC to induce specific proliferative responses, cells were pulsed for 2 h with live or heat-inactivated BRSV Ag and incubated with autologous CD4⫹ T cells from immune calves. The optimum dilution of the Ag was determined by titration and incubation with PBMC from immunized cattle. Lysate of uninfected cell cultures failed to induce a proliferative response. MoDC (104) were incubated for 2 h at 37°C with BRSV Ag (diluted 1:50 in TCM), washed three times in PBS, irradiated (20 Gy from a 137 Cs source; Gammacell 1000 Elite, Nordion International Inc., Ontario, Canada), and added in triplicate to 105 purified CD4⫹ T cells in 96-well U-bottomed, microtiter plates (Becton Dickinson) to give a 1:10 ratio. This ratio has been shown to induce the most effective CD4⫹ T cell-proliferative response (unpublished observation). Previous experiments have shown that the proliferative response of CD4⫹ T cells plateaus after 5 days in culture (unpublished data), and cultures were incubated for 5 days to monitor cytokine response and the associated proliferative response. Before proliferation was assessed by the addition of 1 ␮Ci [3H]-thymidine for the final 12 h of culture, 150 ␮l supernatant was carefully removed and replaced with 150 ␮l fresh TCM. These culture supernatants were stored at ⫺20°C to analyze cytokines. Incorporated radioactivity was determined by liquid scintillation counting.

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CGG CAA GTT CAA CGG CAC AGT CA CCGG TCA TCG TGG CCA TGG AGA AG AAG CGC CTT CAC TCC ATT CGC TGT C CAC GGG CCT GAC ATC AAG GAG CA TGC CAA CGT CCG CGT GCA A TTT GGG CTG TAT CAG TGC AAG TCT TCC C CAA TGC CCT CAT GGC CAA CGG TCT CTT TCG AGG CCG GAG AGC ATC A TTC G CTG TTT TCA CG

Recombinant bovine IL-2 and IL-4 were produced in the baculovirus-insect cell system as described elsewhere [30, 31]. Peripheral blood cells were used as a source of concanavalin A (Con A) blasts for the IL-2 bioassay and purified Bc for the IL-4-like bioassay [30, 31]. Con A lymphoblasts to assay bIL-2 were generated by incubating PBMC with Con A (5 ␮g ml⫺1) for 4 days at 37°C. Con A lymphoblasts (105) in 100 ␮l TCM were added to dilutions of supernatants from the stimulated CD4⫹ T cells or to dilutions of rbIL-2 used as a standard. Cultures were incubated for an additional 24 h and pulsed with 1 ␮Ci [3H]-thymidine for the final 12 h of culture. Values were derived by comparison with the standard curve for which 1 U was defined as the amount of rbIL-2, giving a half-maximal response [30]. B Bc were labeled with mAb to IgM and purified on paramagnetic columns. Bc (105) in 100 ␮l TCM were incubated with dilutions of supernatants for 24 h, and proliferation was assayed in a similar manner by comparison with a rbIL-4 [31]. The activity is referred to as IL-2 or IL-4 for simplicity, although strictly, it should be termed IL-2- and IL-4-like. Bovine IFN-␥ was measured in culture supernatants using a commercial kit (CSL Ltd., Parkville, Victoria, Australia) as per the manufacturer’s instructions. Data were read on a Spectral Max 250 enzyme-linked immunosorbent assay plate reader (Molecular Devices Corporation, Sunnyvale, CA) at 650 nm, and results were expressed as ng ml⫺1 based on rbIFN-␥ standard received from Ciba Geigy (Switzerland).

Statistical analysis Statistical analysis of the data was performed in Excel and GraphPad Prism software package version 2.0. For multiplex PCR, samples of three different animals were analyzed in duplicate, and data are expressed as means ⫾ SD. Differences between cytokine transcription in each treatment were analyzed by a two-way analysis of variance (ANOVA), followed by a Bonferroni t-test in case of a significant ANOVA. Differences were considered significant if P ⬍ 0.05. For proliferation assay and cytokine assays, data are presented for analyses performed on the same day using the same batch of cells.

CTT GTA AGC TTC TAG ACT GGT GCA

CCC GGC AGC TCC TAG GCC TGT GGA

GTT ACT CCC CCC CGG AGT CTT GGA

CTC GTT AGG AGT TCC TTG CAA CCA

TGC CCT G GAG CGG CTT GCT TTA

C CA

Probe Reverse primer

RESULTS

GAG TGC AGG AGC CTG CAT GCT CAG

Incubation of DC with BRSV did not alter surface-Ag expression, but induced apoptosis

GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

GTA TGA TTC CAC CC GGC AGG TGG TG ACT CCC GCT TCA ACA GGC TGA G CATGCC ACA AGG TCA TGT CTT CA GAC TCG TAT G GAA GAG AAG TCA GA CCA TCA TGA GCC TCA CAT TGG GCG GTT GCT CTA GAT AAG TGG TGG AAA CAT AAA CAG GGT CCA GGC CGG CAG GAPDH IL-1␤ IL-6 IL-10 IL-12 p40 IL-15 TNF-␣ INF-␥

Forward primer

TABLE 1.

Sequences for Primer and Probes used for TaqMan Real-Time PCR

Measurement of bovine IL-2, IL-4, and interferon-␥ (IFN-␥) proteins

Incubation of MoDC with live BRSV did not lead to an increase in viral titre, as analyzed by plaque formation assay, and only low levels of virus could be recovered 48 h after infection (data not shown). To test the effect of incubation with BRSV on the expression of MHC classes I and II and the costimulatory molecules CD80/86, bovine MoDC were incubated for 2 h, 1 day, and 2 days with the live BRSV or medium. Thereafter, cells were harvested, stained, and analyzed by FCM for the expression of these molecules. Differences in the number of cells expressing MHC class I, MHC class II, or CD80/86 or the number of molecules expressed per cell, as judged by the intensity of staining, were not detected. No staining of MoDC was detected with mAb 19 directed against the F protein of RSV. However, a higher percentage of MoDC reacting with PI and Ax was detected following exposure to live BRSV (about 21%) compared with MoDC incubated with medium (about 12%) after 2 days of incubation (Fig. 1).

The cytokine response of MoDC was influenced by exposure to live or heat-inactivated BRSV To investigate further how BRSV might affect MoDC and subsequently influence the T cell response, a Multiplex TaqWerling et al. Interaction of DC and BRSV

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Fig. 1. Discrimination of dead and apoptotic DC incubated with live BRSV for the time periods indicated. MoDC were incubated with live BRSV (MOI of 1) or the medium alone. At times indicated, MoDC were harvested, stained, and analyzed by FCM. Data are presented as histograms of 10,000 cells (ungated). Controls (clear histograms) were incubated with medium alone and are plotted against the histograms derived from MoDC cultures incubated with BRSV (filled histograms). For the 0 h value, controls (filled histograms) are plotted against the autofluorescence of the cells (clear histograms).

Man威 PCR was developed to measure mRNA isolated from MoDC (Fig. 2). Following exposure to heat-inactivated BRSV, MoDC produced significantly more tumor necrosis factor ␣ (TNF-␣) mRNA (P⬍0.05), IL-1␤ mRNA (P⬍0.05), IL-6 (P⬍0.001), IL-12p40 (P⬍0.05), and IL-15 mRNA (P⬍0.05) than MoDC incubated with heated, uninfected FCK lysate (Fig. 2). After being exposed to live BRSV, MoDC produced significantly more TNF-␣ mRNA (P⬍0.05), IL-1␤ mRNA (P⬍0.05), IL-6 (P⬍0.001), and IL-10 (P⬍0.001) than MoDC incubated with uninfected FCK lysate (Fig. 2). The amounts of TNF-␣, IL-1␤, and IL-6 mRNA produced by MoDC exposed to live BRSV were similar to those of MoDC exposed to heatinactivated BRSV. However, heat-inactivated BRSV induced more IL-12p40 and IL-15 mRNA (P⬍0.05) than did live BRSV, whereas live BRSV induced significantly more IL-10 mRNA (P⬍0.05) than did heat-inactivated BRSV. IFN-␥ mRNA could not be detected in MoDC exposed to either BRSV Ag preparation (data not shown).

MoDC pulsed with live or heat-inactivated BRSV stimulated similar proliferative responses and cytokine synthesis in memory CD4⫹ T cells A series of experiments was performed to compare the responses of resting memory CD4⫹ T cells with MoDC exposed to live or heat-inactivated BRSV. Although CD4⫹ T cells from one calf had a greater proliferative response to live compared with heat-inactivated virus, the responses of CD4⫹ T cells from two other calves to live or inactivated virus were similar (Fig. 3). To examine whether the cytokine response of CD4⫹ T cells was influenced by MoDC exposed to different Ags, supernatants from cultures were collected, and the levels of IL-2, IL-4, and IFN-␥ were determined for two of the calves. There were no consistent differences in the levels of IL-2 activity, IL-4 activity, or IFN-␥ in supernatants of CD4⫹ T cells incubated with MoDC pulsed with either viral Ag (Fig. 4, A–C). 300

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DISCUSSION In the present experiments, we investigated the effect of BRSV on MoDC phenotype and cytokine production and on the in vitro immunostimulatory potential of MoDC exposed to live or heat-inactivated BSRV. Although there was no evidence that BSRV replicated in bovine MoDC or influenced the expression of MHC class I, class II, or CD80/86, which are critical for the induction and stimulation of T cells, the virus did appear to affect MoDC survival. The absence of altered surface expression of viral protein or costimulatory molecules is in contrast to observations with influenza virus that infects human DC efficiently, as judged by the expression of the viral HA and NS1 proteins [32]; however, it is in line with data published on the infection of bovine DC with bovine herpesvirus-1 [33]. The bias of T cell responses is influenced by the cytokines produced by the APC. To investigate whether differences in cytokine synthesis by DC following exposure to and interaction with live or killed virus might occur that could influence the T cell response subsequently induced by the APC, levels of cytokine transcripts in the MoDC exposed to live or heatinactivated BRSV were assessed with a quantitative PCR. Differences in the synthesis of cytokines by MoDC incubated with live or heat-inactivated RSV were evident, which have the potential to influence any T cell response subsequently induced. Thus, following exposure to live BRSV, increased synthesis of IL-10 mRNA, and reduced synthesis, notably of IL-15 and IL-12p40, were evident compared with MoDC exposed to heat-inactivated virus. These differences in MoDC response would be expected to lead to a down-regulation of the T cell response induced by DC exposed to live compared with killed virus. They could also affect the bias of the T cell response, as reduced levels of IL-12 might result in less IFN-␥ synthesis and a less intense Th1 bias. Our results are similar to data published on the production of cytokines by monocytes/macrophages exposed to live HRSV in vitro [34 –37], and elevated levels of IL-1␤, IL-6, and TNF-␣ are evident after exposure to http://www.jleukbio.org

Fig. 2. Multiplex TaqMan威 RT-PCR analysis of mRNA isolated from MoDC pulsed with live or heat-inactivated BRSV. MoDC (106 per well) were pulsed for 2 h with live or heat-inactivated BRSV. Total RNA was extracted and analyzed for the presence of the indicated cytokines. Each sample was analyzed in duplicate, and data are expressed as n-fold difference of expression compared with values obtained for unstimulated MoDC. Samples were analyzed in duplicates from three different animals, and data are expressed as mean ⫾ SD. *, P ⬍ 0.05.

Fig. 3. Proliferative responses of CD4⫹ T cells induced by MoDC, exposed to live or heatinactivated BRSV Ag. Resting memory CD4⫹ T cells (105 per well) purified from PBMC were incubated with MoDC (104 per well) pulsed with live or heat-inactivated (Hi) BRSV Ag. Proliferative responses were measured by the incorporation of [3H]-thymidine. Data are shown of three animals, and for each animal, data are expressed as mean cpm ⫾ SD of the triplicate samples. Highest background value was 1225 ⫾ 58 cpm for DC pulsed with FCK lysate as control and incubated with CD4⫹ T cells.

Werling et al. Interaction of DC and BRSV

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Fig. 4. Cytokine responses of CD4⫹ T cells induced by MoDC exposed to live or Hi BRSV Ag. Supernatants of these cultures were harvested and analyzed for the presence of IL-2 (A), IL-4 (B), and IFN-␥ (C). Values for cytokines in culture supernatants of cells stimulated with uninfected FCK lysates were less than 0.1 U ml⫺1 for IL-2, less than 1 U ml⫺1 for IL-4, and 0 pg ml⫺1 for IFN-␥. Supernatants of CD4⫹ T cells alone did not contain detectable amounts of the cytokines analyzed. Samples were analyzed in triplicates (except IFN-␥) from two different animals, and data are expressed as mean ⫾ SD of the triplicate samples.

virus. Enhanced IL-10 production following exposure to live HRSV has also been reported for human alveolar macrophages [36]. The ways in which RS viruses induce IL-10 are not clear. Other studies have shown that macrophages produce IL-10 following exposure to purified HRSV G protein [38, 39]. However, the F protein may also provide a stimulus for IL-10 production, as this protein, like lipopolysaccharides, which can directly stimulate the release of chemokines that enhance IL-10 production [40], binds to Toll-like receptor-4 and CD14 on APC [41, 42]. IL-10 may play an important role in the pathogenesis of RSV infections. Thus, IL-10 has been shown to decrease the release of chemokines in a model of lung inflammation by inhibiting nuclear factor-␬B and destabilization of mRNA, leading to a reduced accumulation of neutrophils in 302

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the lung [43]. In addition, IL-10 can down-regulate the function of alveolar macrophages and lung inflammation by inhibiting TNF-␣, IL-8, and IL-1␤ production [36, 43]. IL-10 may decrease cytokine levels through induction of endoribonucleases that degrade cytokine transcripts [44] or by direct interference with the production of these cytokines at the transcriptional level [45]. Interestingly, HRSV directly induces similar endoribonucleases [36], thus possibly contributing to the IL10-induced immunosuppression. The stimulation of endoribonucleases and IL-10 by RSV infection may lead to an autocrine loop acting on DC exposed to live RSV and the subsequent inhibition of proinflammatory cytokines and chemokines by these cells, thus reducing viral clearance. IL-10 has also been shown to have a down-regulatory effect on the production of IFN-␥ and IL-12 by CD4⫹ T cells and DC [29, 46] and may therefore influence the Th bias of the immune response. To investigate whether IL-10 synthesized as a result of exposure to live virus affects the intensity and/or bias of the CD4⫹ T cell response, we compared the response of memory CD4⫹ T cells to MoDC exposed to live or inactivated BRSV. Similar T cell-proliferative and -cytokine responses were evident with CD4⫹ T cells stimulated with MoDC exposed to live or killed BSRV. This suggests that once primed, the response of memory CD4⫹ T cells in vitro is not greatly affected by the nature of the viral Ag and the cytokines synthesized by MoDC in the B system. Similarly, the pattern of cytokines produced by CD4⫹ T cells from calves inoculated with an inactivated BRSV vaccine, which induces protection against BRSV in calves, suggests that the immune response induced by this vaccine, or in general to BRSV infection, is not markedly biased toward Th1 or Th2. The cytokine response of memory CD4⫹ T cells from the vaccinated calves in the present study was similar to that seen in pulmonary lymphocytes from BRSV-infected gnotobiotic calves and to that of PBMC from HRSV-seropositive human individuals, stimulated with HRSV in vitro [39, 47, 48]. In both cases, a Th0 or a mixed Th1/Th2 pattern of cytokine production could be detected with no clear Th1-Th2 pattern induced by a RSV infection. The findings from the present study indicate that the protective glutaraldehyde-inactivated BRSV vaccine does not prime for a biased pattern of cytokines in contrast with mice vaccinated with FI-RSV, which primes a Th2 response and induces an atypical pneumonia following challenge with live virus [2, 49, 50]. In summary, our studies indicate that the synthesis of cytokines by MoDC is affected by the nature of the RSV Ag, live virus resulted in the synthesis of more IL-10 and less IL-15 and IL-12 transcripts than did killed virus. Although these differences did not appear to influence the in vitro responses of CD4⫹ T memory cells that had been primed in vivo, they may have an effect on the induction of a primary immune response induced in vivo at the site of infection.

ACKNOWLEDGMENTS We thank S. Wyld (Institute for Animal Health, Compton, U.K.) for technical assistance. This work was supported by MAFF and the BBSRC. D. W. was supported by a Marie Curie Fellowship of the EC. http://www.jleukbio.org

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