B00005Cytokine mRNA expression in spleen of mice immunized with Bovine Respiratory Syncytial Virus
P. Lestrate, I. Knott, C. Clavareau, K. Walravens, J.P. Matheise and J.J. Letesson
Lab. Immunologie, UR Biologie Moléculaire, FUNDP, B-5000 Namur, Belgium
Abstract:
Bovine respiratory syncytial virus (BRSV) can lead to immunopathological phenomenons in calves, similar to those described in case of HRSV in infants. This pathogenesis may be mediated by cytokines produced by type 2 T helper cells. We have used a mouse model to explore the pathogenesis of BRSV infection. Replication of BRSV in mice was established. In this study, cytokine mRNA expression was examined using RT-PCR technique. Mice were immunized with live BRSV or killed BRSV (virus adjuvanted with QuilA). After two immunizations, spleen of mice were analyzed for cytokine mRNA. IL-4 mRNA was detected at the days 1 and 4. IL-2 and INF-g mRNA were detected at days 4 and 8. At day 12, no mRNA was detected. These preliminary results show that the cytokine pattern was the same for BRSV aduvanted or not. Thus, in our model, although Il-4 was detected in the first days, the immune response towards BRSV seems to be of the TH1 type.
Introduction:
Bovine Respiratory Syncytial Virus (BRSV), as well as Human RSV, belongs to the Paramyxoviridae family. BRSV is a major cause of respiratory disease in calves, as HRSV in infants. Immunopathological phenomenons were observed after infection or different kinds of immunization for RSV in bovine, humans and laboratory animal models. Concerning HRSV, Balb/c mouse model was used to study this enhanced pathology. Graham et al. (1993) observed that priming immunization determines T helper cytokine mRNA expression patterns in lungs of mice challenged with HRSV. Here, we used a Balb/c mouse model where BRSV was demonstrated to replicate in lungs of mice (Walravens et al., 1996). We compared the cytokine patterns in spleen after intranasal BRSV infection or killed BRSV immunization.
Materials and Methods:
Virus, mice and immunization
BRSV (strain RB94) was grown on Vero cell monolayers (ATCC, CCL 81) in DMEM (Dubelcco´s minimal Eagle medium) (BioWhittaker, Belgium) with 2 % of fetal calf serum (Gibco, Belgium).
Balb/c mice were housed at the animal facilities of the FUNDP. Before the immunization, the mice were anesthetized. Each mice was immunized with 106 TCID50 diluted in a final volume of 20 µl containing PBS or Quil A (0.33mg/ml of PBS) (Spikoside, Isotec product, Sweden). The immunizations were performed by the intranasal route twice 15 days apart.
mRNA extraction and RT-PCR
The RNA was extract from fresh spleen of mice with Ultraspec (Biotecx, USA) upon the recommendations of the manufacturer. Moloney Murine Virus Reverse Transcriptase (M-MLV RT) (Gibco, USA) was used for the reverse transcriptase and the Thermus Brokianus DNA polymerase (DynazymeTM, Sweden) was used for the PCR reactions. Primers used for the detection of IL-2, IL-4, IFN-g and b2microglobulin were described by Legoux et al. (1992). PCR reactions were performed in a Thermal Cycler Gene E (Techne, New Brunswick Scientific, Netherlands) under the following conditions : 5 min at 94 °C followed by 30 cycles : 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. Reaction products were visualized on ethidium bromide stained 2% agarose gel.
In vitro expression of cytokines
Spleen cells were isolated by use of a loosely fitting Dounce homogenizer. Red blood cells were eliminated and spleen cells were washed and adjusted to a concentration of 2 X 105 cells per well. Cells were grown in U-bottom microwell plates (Nunc, Denmark), in RPMI 1640 medium supplemented with glutamine, HEPES, b-mercaptoethanol, antibiotics and 10 % fetal calf serum. Antigens were RPMI medium alone, mock-infected cells purified on sucrose gradient, BRSV purified on sucrose gradient at a concentration of 1 or 0.1 TCID50/cell, concanavalin A at 2 µg/ml or pokeweed at 2.5 µg/ml. Cells were incubated at 37°C in a humidified CO2 incubator, and supernatants were harvested after 24 h (IL-2) and 72 h (IFN-g and IL-4). Supernatants were stored frozen at -20°C until assay. IL-2 assay is based on CTLL-2 cells as described by Huygen et al. (1992). IFN-g was assayed by ELISA (Genzyme, UK) following the instructions of the manufacturer. IL-4 was assayed by Elisa (Boehringer, Belgium) following the instructions of the manufacturer.
Immunoglobulin isotype assay
Sera were assayed in ELISA against BRSV antigen which was prepared as described by Matheise et al. (1995). BRSV antigen was diluted in PBS and adsorbed onto 96-well plates (Maxisorp, Nunc, Denmark) overnight at 4°C. After saturation with casein hydrolysate during 2 h at room temperature, serial five-fold dilutions of sera were added to the wells and the plates were incubated for 1h. Wells were then incubated with biotinylated goat anti-mouse Ig, or anti-mouse IgG1 or anti-mouse IgG2a (Amersham, UK) for 1h at room temperature. Then, streptavidine-peroxidase was added for 1h. Bound conjugates were detected by incubation with 0.4 mg/ml O-phenylendiamine (Sigma Co, USA) and 0.01 % H2O2 in substrate buffer (65 mM Na2HPO4, 17 mM citric acid, pH5).
Results:
Cytokine mRNA expression
After the second immunization, mice of each group (n=2) were killed at days 1, 4, 8 and 12. RNA from the spleen was extracted and cytokine mRNA were analyzed by RT-PCR reaction using primers for IL-2, IL-4, IFN-g and b2m. Results are showed in table 1 *See Table 1*. One day after the last immunization, only IL-4 mRNA was detected. At day 4, we detected simultaneously IL-4, IL-2 and INF-g mRNA. IFN-g and IL-2 mRNA were still observed 8 days after the second immunization while IL-4 mRNA was undetectable . After 12 days, any cytokine mRNA expression was observed.
The result we have obtained for the mice immunized with live BRSV are the same to those described with BRSV adjuvanted in Quil A.
In vitro cytokine expression
Spleen cells were isolated from individual mice in groups of days 4 and 12 represented in table 1. Cells were restimulated with Con A, pokeweed, sucrose-gradient purified BRSV or sucrose-gradient purified mock. No IL4 was detected when cells were restimulated with BRSV antigen while the pokeweed stimulated cells (positive controls) produced IL4 (between 100 and 200 pg/ml).
Table 2 *See Table 2* presents the results concerning the IFN-g and IL2 production in vitro. At day 4, no BRSV-specific IFN-g was observed whatever the in vitro restimulation or immunization conditions. But at day 12, we observed a specific IFN-g synthesis in both groups of mice whatever the in vitro restimulation dose of BRSV (data shown only for 0.1 TCID50/cell). Concerning IL-2, significant SI were observed when cells were restimulated from mice sacrificed 12 days after the second immunization.
Immunoglobulin isotype analysis
The figure 1 *See Figure 1* shows the titers of the IgG1 and IgG2a obtained for the two groups of mice. The serological response seems to be more important during the days 4 and 8 in each group. In the mice immunized with BRSV in Quil A, the titers of IgG1 seem to be more important. But these results are difficult to interpret because in both group the titer are quite low. This is probably due to the route of the immunization.
Discussion and
Conclusions:
We have observed IL4, IL2 and IFN-g mRNA expression after BRSV immunization. If the IL-4 mRBA was only observed at day 1 and 4, IL-2 and IFN-g were detected at day 4 and 8. These results are in agreement with those Waris et al. (1996) concerning the HRSV. Moreover, Hussel et al. (1996) have showed the production of IL-10 in the beginning of a HRSV infection. It could be interesting to test this in the BRSV model, the presence of IL-4 in our study indicating that we could find some other Th2 cytokine like IL-10. Graham et al. (1993) described that priming immunization determines T helper cytokine mRNA expression patterns in lungs of mice challenged with HRSV. Mice were immunized with live or killed HRSV (formalin inactivated preparation containing aluminum hydroxide). Here we didn't observe any difference between killed BRSV with QuilA and live BRSV. But our killed virus preparation was very different from the one used by Graham. The adjuvantation by Quil A didn't seem to have any effect in the orientation of the immune response. The cytokine mRNA expression patterns were completed with in vitro cytokine expression experiments. We detected only cytokines of Th1 pattern in the two groups of mice. A time shift was observed for cytokine secretion in comparison with cytokine mRNA secretion.
In conclusion, in our mice model, the immune response towards BRSV seems to be of the TH1 type.
References:
Graham et al. J. Immunol. 151, 2032-2040 (1993)..
Huygen et al. Infection Immunity, 60, 2880-2886 (1992).
Hussell et al. Gen. Virol.77, 2447-2455 (1996).
Legoux et al. European Cytokine Network 3, 553-563 (1992).
Walravens et al. Archiv. Virol., 141, 2313-2326 (1996).
Waris et al. J. Virol.70, 2852-2860 (1996).
Comments:
Address questions and comments about this abstract to Pascal Lestrate ( Isabelle.Knott@fundp.ac.be).