Institute for Animal Health, Compton, Berks, United Kingdom
Correspondence: Chris J. Howard, Institute for Animal Health, Compton, Newbury Berks, RG20 7NN, UK. E-mail: chris.howard{at}bbsrc.ac.uk
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. 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 CD4+ 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 CD4+ T cell response in vivo.
Key Words: MHC classes I/II • real time PCR • IL-2 • IL-4 • IFN-
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dendritic cells (DC) are the only antigen-presenting cells (APC) recognized as having the ability to prime naïve 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 13 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 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 16 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 19 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.
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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 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 µg 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 µl Ax/PI mix, and incubated for 5 min. The volume was increased to 300 µl 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® 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 TaqManTM 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-freeTM, Ambion (Europe) Ltd., Cambridge, UK] to remove contaminating genomic DNA, and eluted total RNA was subsequently transcribed to cDNA using the Reverse Transcription SystemTM (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 µl 18-s control, and 2.5 µl diluted cDNA sample in a total volume of 25 µl. 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.
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Table 1. Sequences for Primer and Probes used for TaqMan Real-Time PCR
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Measurement of bovine IL-2, IL-4, and interferon- (IFN-) proteins
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 µg ml-1) for 4 days at 37°C. Con A lymphoblasts (105) in 100 µl 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 µCi [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 µl 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.
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Figure 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).
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(TNF-) mRNA (P<0.05), IL-1ß 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-1ß 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-1ß, and IL-6 mRNA produced by MoDC exposed to live BRSV were similar to those of MoDC exposed to heat-inactivated 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).
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Figure 2. Multiplex TaqMan® 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.
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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. 4A
4B
4C
).
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Figure 3. Proliferative responses of CD4+ T cells induced by MoDC, exposed to live or heat-inactivated 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.
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Figure 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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 heat-inactivated 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
35
36
37
], and elevated levels of IL-1ß, IL-6, and TNF- are evident after exposure to 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 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-1ß 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 IL-10-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.
Received January 21, 2002; revised April 4, 2002; accepted April 8, 2002.
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