Research in Veterinary Science 1991, 50, 152-156
Depression of lymphocyte responses to phytohaemagglutinin in lambs experimentally infected with bovine respiratory syncytiai virus R. SHARMA, Z. WOLDEHIWET*, University of Liverpool, Department of Veterinary Pathology,
Veterinary FieM Station, Leahurst, Neston, Wirral L64 7TE
Eight lambs were experimentally infected with bovine respiratory syncytial virus (BRSV) and the responses of their peripheral blood lymphocytes to the mitogen phytohaemagglutinin and HRSV antigen compared with that of control lambs injected with tissue culture fluid. The lymphocyte transformation responses to phytohaemagglutinin were significantly reduced five and 10 days after experimental infection with BRSV (P 0.05).
R E S P I R A T O R Y syncytial virus is a m a j o r respiratory pathogen in human infants and calves (Chanock et al 1970, Stott et al 1980). Attempts to reproduce clinical disease by experimental infection of calves have been fraught with difficulties but several workers have shown that lambs are consistently susceptible to experimental infection with bovine respiratory syncytial virus (BRSV) (Lehmkuhl and Cutlip 1979, Al-Darraji et al 1982, Trigo et al 1984a, Sharma and Woldehiwet 1990). Therefore, lambs appear to be a g o o d model for studying the immunopathogenesis of the BRSV infection because of low cost, easy management and availability of immunological reagents such as the recently developed monoclonal antibodies against lymphocyte surface markers (Mackay et al 1987). Lambs experimentally infected with BRSVare more susceptible to secondary infection with Pasteurella haemolytica (Al-Darraji et al 1982, Trigo et al 1984b). The mechanisms of this predisposition are not known. Significant alterations in lymphocyte subpopulations in lambs experimentally infected with BRSVwere recently demonstrated (Sharma et al 1990). *Reprint requests to Dr Z. Woldehiwet
In the present study, the authors examined the possible functional significance of these alterations. Materials and methods BRS V
A strain of bovine respiratory syncytial virus (BRSV 66), passaged in lamb testis cells three times, was maintained and used as previously described (Sharma and Woldehiwet 1990).
Lambs Sixteen, six- to eight-week-old conventionally reared Suffolk crossbred lambs, free of neutralising antibodies to respiratory syncytial virus, were used. Eight lambs were infected with BRSV as previously described and eight controls were injected with uninfected tissue culture fluid (Sharma and Woldehiwet 1990).
Collection of blood samples Peripheral blood samples were collected from eight BRsv-infected and eight control lambs in heparincoated tubes on 0, 5, 10, 14 and 21 days after inoculation. Mononuclear cells were separated from diluted blood on a Ficoll-paque column (Pharmacia). Cell viability was determined by the trypan blue exclusion technique and the cells resuspended in RPMI 1640 medium to give a concentration of 1 × 107 viable cells ml-l.
Monoclonal antibodies Monoclonal antibodies (Mab) were kindly provided by Dr M. R. Brandon, University of Melbourne, Australia. The Mabs used in the present study were 44-38, 38-65, 20-96 which recognise the CD5 (T helper), CD8 (cytotoxic/suppressor) and LCA p220 (B cell) surface antigens, respectively (Mackay et al 1987). The optimum dilution of each antibody was determined using checkerboard titration.
i52
153
Lymphocyte responses in BRSv-infected lambs Negative selection o f CD4 + cells and CD8 + cells CD4-enriched and CD8-enriched cells were prepared by selective depletion using Mabs and rabbit complement as described by Ezaki et al (1987). Briefly, CD8-enriched lymphocytes were prepared by treating peripheral blood mononuclear cells with antiCD4 and anti-LCA p220 Mab followed by rabbit complement (C'). CD4-enriched lymphocytes were prepared by treating peripheral blood mononuclear cells with anti-CD8 and anti-LCA p220 Mab followed by rabbit complement. Depletions were verified by flow cytometry.
BRSV antigen The BRSV antigen used in transformation assays was prepared from lambs' testis cell cultures infected with BRSV as described earlier (Sharma and Woldehiwet 1990). Virus-infected fluid was purified by sucrose density gradient centrifugation as described by Trepanier et al (1981). The purified virus was inactivated at 56°C for 30 minutes and stored at - 7 0 ° C until required. Control antigen was prepared from uninfected lambs' testis cells.
Lymphocyte transformation assays Lymphocyte transformation assays were performed as described by Taylor et al (1987) with slight modification. Briefly, peripheral blood mononuclear cells or CD4- or CD8-enriched fractions (105 per well) were dispensed in 100 #1 volume to the wells of 'V' bottomed microtitre plates containing 20 /d of purified phytohaemagglutinin (40 /~g m1-1) (Wellcome reagents) or 20 t~l of optimum concentration of BRSV antigen or 20 /zl of RPM[ 1640 medium. After incubation for three days, cultures were pulsed overnight with 1/~Ci tritiated (3H)-thymidine (Amersham International), in 50 #1 per well and harvested on to filtermats (Skatron) by multiple sample harvester (Skatron). The radioactivity in the filters was counted in a scintillation counter (Packard). The degree of transformation was expressed as the ratio of tritiated thymidine incorporation in cultures stimulated with phytohaemagglutinin or BRSV antigen to that of the control cultures (stimulation index).
There were no significant differences in absolute counts per minutes of tritiated thymidine uptake between cultures from control and BRsv-infected lambs when incubated without phytohaemagglutinin or BRSV antigen. There were animal to animal and day to day variations in the transformation responses of lymphocytes to phytohaemagglutinin in both control and aRsv-infected lambs. The variations observed in the control lambs were not statistically significant (Fig 1) (P>0.05). However, the mean stimulation index in phytohaemagglutinin-stimulated cultures of BRSv-infected Iambs was significantly decreased from 28.2 ± 6.56 to 9"25 ± 2.7 five days after infection ( P < 0 " 0 5 ) (Fig 1). The lymphocyte transformation responses in this group remained significantly depressed until 10 days after infection compared to uninfected control lambs (P < 0.05). The lymphocyte transformation responses returned to normal levels 14 days after infection. When mononuclear cells obtained before experimental infection with BRSV were treated with anti° CD4 Mab and C' (CD8-enriched fraction), their lymphocyte transformation responses to phyto-
5o
4o
~ 30
-~ E ~ 20
10,
Analysis o f data The stimulation indices obtained in Bgsvstimulated or phytohaemagglutinin-stimulated cultures in Bgsv-infected iambs were compared with those obtained in control lambs on different days after inoculation by Student's t test. Results
All the results are the mean of triplicate cultures.
BRSV
~- Control
0
I
0
'
I
5
i
I
'
10
|
15
'
t 2O
Days after inoculation
FIG 1 : Lymphocyte transformation responses of peripheral blood mononuclear cells to phytohaemagglutinin in BRSV-infected and control iambs. Each point represents the mean stimulation index of eight animals ( ± SE)
R. Sharma, Z. Woldehiwet
154 70-
cyte transformation responses of the CD4-enriched cells obtained from the control group on the same day of sampling ( P < 0 . 0 5 ) (Fig 2). The lymphocyte transformation responses of mononuclear cells treated with C ' alone remained unaltered. BRSV antigen-induced transformations were not observed in any of the mononuclear cell cultures obtained from BRSv-infected lambs and control lambs on different days after inoculation (P > 0"05) (Fig 3).
(a)
T
60.
50,
]
BRSVgroup
]
Control group
-T
40,
g
]
Virological studies
._~ 30,
All the BRSv-infected lambs had a significant rise in virus-neutralising antibodies to BRSVon day t4 after infection.
20
Discussion 10,
0
'
The role of cell-mediated immune responses of BRSVinfection in calves is not well established. Taylor et al (1987) recently reported that only one of 21 conventionally reared calves developed lymphocytic transformation responses to BRSV antigen after
5
Days after inoculation
407
25t
(b) ]
BRSVgroup
]
Control group
x 30 "o
20 -
~
T
10-
0
®
0
I
'
®
5
,
Days after inoculation FIG 2: Lymphocyte transformation responses of CD4-enriched cells (a) and CDS-enriched cells (b) to phytohaemagglutinin in BRSV-infected and control lambs. Each point represents the mean stimulation index of eight animals ( ± SE)
haemagghtinin were significantly reduced (P < 0" 01) (Fig 2) but treatment of these cells with anti-CD8 Mab and C' (CD4-enriched fraction) did not significantly affect the lymphocyte transformation responses to phytohaemagglutinin (P > 0" 05) (Fig 2). The phytohaemagglutinin-induced responses of CD4-enriched cells obtained five days after experimental infection with BRSV were significantly lower than those of the CD4-enriched cell fractions obtained from the same group on the day of experimental infection (P < 0" 01) (Fig 2). The phytohaemagghtinin-induced lymphocyte transformation responses of CD4-enriched ceils obtained five days after experimental infection with BRSV were also significantly lower than the lympho-
1"5
x
.
J
~
I
J
-~ ~
1-01
J
1
0.5-
BRSV 41- C o n t r o l
0.0 0
5
10 15 Days after inoculation
20
FIG 3~ Lymphocyte transformation responses of peripheral blood mononuclear cells to BRSV antigen in BRSV-infected and control lambs. Each point represents the mean stimulation index of eight animals ( ± SE)
Lymphocyte responses in BRSv-infected lambs experimental infection, compared to six of six calves exposed to an inactivated vaccine. In the present study, virus-specific transformation responses were not detected up to 21 days after infection in ~RSVinfected lambs. Schauf et al (1979) also suggested that a single infection with human respiratory syncytial virus does not always confer enough cellular responses to the virus to be demonstrable in assay systems. BRSV has been implicated as a predisposing factor to secondary bacterial infections in natural respiratory disease in calves and experimental infections in lambs (Bryson et al 1979, Thomas et al 1980, Pirie et al 1981, A1-Darraji et al 1982, Trigo et al 1984b) but the mechanisms of this predisposition have not been explored. In the present study, the authors demonstrated that lymphocytes from BRSVinfected lambs had depressed reactivity to phytohaemagglutinin as early as five days after infection. Roberts (1982) also demonstrated reduced lymphocyte t r a n s f o r m a t i o n responses to p h y t o haemagglutinin in human lymphocytes infected with respiratory syncytial virus in vitro. Decreased lymphocyte blastogenesis to mitogens is a feature of many viral infections (Carney et al 1981, Hyypia et al 1984). The immunosuppression caused by some viral infections has been attributed to either direct infection of i m m u n o c o m p e t e n t cells or the imbalances of immunoregulatory T cells (Hyypia et al 1984). A number of viruses including human respiratory syncytial virus, pestiviruses and rubella virus are capable of infecting lymphocytes (Muscoplat et al 1973, Chantler and Tingle 1982, Roberts 1982). It is not clear whether respiratory syncytial virus selectively infects specific subsets of lymphocytes and thereby affects their competence but there is some evidence to suggest that human respiratory syncytial virus can infect human lymphocytes and monocytesmacrophages in vitro (Domurat et al 1985, Bangham and McMichael 1986). Furthermore, circulating mononuclear leucocytes obtained from symptomatic children infected with human respiratory syncytial virus frequently express viral antigens (Domurat et al 1985). In the present study, the authors found that lymphocyte transformation responses to phytohaemagglutinin were significantly reduced when mononuclear cells were treated with anti-CD4 Mab and complement but the responses to phytohaemagglutinin were not affected when mononuclear cells were treated with anti-CD8 Mab and complement. This is in agreement with the findings of Ezaki et al (1987) who suggested that, in sheep, lymphoproliferative responses to phytohaemagglutinin were mediated by T lymphocytes bearing T1 and T4 (CD4) surface antigens and not by lymphocytes bearing T8 (CD8) antigen.
155
The lymphocyte transformation responses of CD4-enriched lymphocytes obtained five days after experimental infection with BRSV were significantly lower than those obtained before infection or those obtained from control lambs on the same day (five days after infection) of sampling. This suggests that the depressed lymphocyte transformation responses of mononuclear cells to phytohaemagglutinin due to BRSV infection is associated with CD4+ lymphocytes. The present results also indicate that the depressed response to phytohaemagglutinin attributable to CD4 + T-lymphocytes were not due to the reduction in the number of circulating CD4+ lymphocytes because the lymphocyte numbers in cultures were kept constant. It appears, therefore, that BRSV may depress immune responses both by reducing the number of circulating CD4 + T-lymphocytes (Sharma et al 1990) and by depressing their reactivity.
Acknowledgements The authors thank Dr M. R. Brandon, University of Melbourne for providing the monoclonal antibodies. R.S. is a Commonwealth scholar supported by the Association of Commonwealth Universities.
References AI-DARRAJI, A. M., CUTLIP, R. C., LEHMKUL, H. D., GRAHAM, D. L., KLUGE, J. P. & FRANK, G. H. (1982) Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: clinical and microbiologic studies. American Journal of Veterinary Research 43,236-240 BANGHAM, C. R. M. & McMICHAEL, A. J. (1986) Specific human cytotoxic T cells recognise B cell lines persistently infected with respiratory syncytial virus. Proceedings of the National Academy of Sciences of the United States of America (Washington) 83, 9183-9187 BRYSON, D. G , McFERRAN, J. B., BALL, H. J. & NEILL, S. D. (1979) Observations on outbreaks of respiratory disease in calves associated with parainfluenza type 3 virus and respiratory syncytial virus infection. Veterinary Record 104, 45-49 CARNEY, W. P., RUBIN, R. H., HOFFMAN, R. A., HANSEN, W. P., HEALEY, K. & HIRCH, M. S. (1981) Analysis of Tlymphocyte subsets in cytomegalovirus mononucleosis. Journal of Immunology 126, 2114-2116 CHANOCK, R. M., KAPIKIAN, A. Z., MILLS, J., KIM, H. W. & PARROTT, R. H. (1970) Influence of immunological factors in respiratory syncytial virus disease of the lower respiratory tract. Archives of Environmental Health 21,347-355 CHANTLER, J. K. & TINGLE, A. J. (1982) Isolation of rubella virus from human lymphocytes after acute natural infection. Journal of Infectious Diseases 145,673-677 DOMURAT, F., ROBERTS, N. J. Jr, WALSH, E. E. & DAGAN, R. (1985) Respiratory syncytial virus infection of human mononuclear leucocytes in vitro and in vivo. Journal of Infectious Diseases 152, 895-902 EZAKI, T., MIYASAKA, M., BEYA, M. F., DUDLER, L. & TRNKA, Z. (1987) A murine anti-sheep T8 monoclonal antibody, ST-8, that defines the cytotoxic T lymphocyte population. International Archives of Allergy and Applied Immunology 82, 168-177
156
R . S h a r m a , Z. W o l d e h i w e t
HYYPIA, T., ESKOLA, J., LA1NE, M. & MEURMAN, O. (1984) B cell function in vitro during rubella infection. Infection and Immunity 43, 589-592 LEHMKUHL, H. D. & CUTLIP, R. C. (1979) Experimentally induced respiratory syncytial virus infection in lambs. American Journal of Veterinary Research 40, 512- 514 MACKAY, C. R., MADDOX, J. F. & BRANDON, M. R. (1987) Lymphocyte antigens of sheep: Identification and characterisation using a panel of monoclonal antibodies. Veterinary Immunology and Immunopathology 17, 91-102 MUSCOPLAT, C. C., JOHNSON, D. W. & TEUSCHER, E. (1973) Surface immunoglobulins of circulating lymphocytes in chronic bovine diarrhoea: Abnormalities in cell populations and cell function. American Journal of Veterinary Research 34, 1101-1104 PIRIE, H. M., PETRIE, L., PRINGLE, C. R., ALLAN, E. M. & KENNEDY, G. J. (1981) Acute fatal pneumonia in calves due to respiratory syncytial virus. Veterinary Record 108, 411-416 ROBERTS, N. J. Jr (1982) Different effects of influenza virus, respiratory syncytial virus, and Sendai virus on human lymphocytes and macrophages. Infection and Immunity 35, 1142-1146 SCHAUF, V., PURCELL, C., MIZEN, M. & MIZEN, S. (1979) Lymphocyte transformation in response to antigens of respiratory syncytial virus. Proceedings of the Society for Experimental Biology and Medicine 161,564-569 SHARMA, R. & WOLDEHIWET, Z. (1990) Pathogenesis of bovine respiratory syncytial virus in experimentally infected lambs. Veterinary Microbiology 23, 267-272 SHARMA, R., WOLDEHIWET, Z., SPILLER, D. G. & WARENIUS, H. M. (1990) Lymphocyte subpopulations in
peripheral blood of lambs experimentally infected with bovine respiratory syncytial virus. Veterinary Immunology and Immunopathology 24, 383-391 STOTT, E. J., THOMAS, L. H., COLLINS, A. P., CROUCH, S., JEBBETT, J., SMITH, G. S., LUTHER, P. D. & CASWELL, R. (1980) A survey of virus infections of the respiratory tract of cattle and their association with disease. Journal of Hygiene, Cambridge 85, 257-270 TAYLOR, G., STOTT, E. J. &THOMAS, L. H. (1987) Lymphocyte transformation response of calves to respiratory syncytial virus. Journal of Medical Virology 22, 333-344 THOMAS, L. H., STOTT, E. J., JONES, P. W., JEBBETT, J. & COLLINS, A. P. (1980) The possible role of respiratory syncytial virus and Pasteurella spp in calf respiratory disease. Veterinary Record 107, 304-307 TREPANIER, P., PAYMENT, P. & TRUDEL, M. (1981) Concentration of human respiratory syncytial virus using ammonium sulphate, polyethylene glycol or honow fibre ultrafiltration. Journal of Virological Methods 3 , 2 0 1 - 2 l 1 TR1GO, F. J., BREEZE, R. G., EVERMANN, J. F. & GALLINA, A. M. (1984a) Pathogenesis of experimental bovine respiratory syncytial virus infection in sheep. American Journal of Veterinary Research 45, 1663-1670 TRIGO, F. J., BREEZE, R. G., LIGG1TT, H. D., EVERMANN, J. F. & TRIGO, E. (1984b) Interaction of bovine respiratory syncytial virus and Pasteurella haemolytica in the ovine lung. American Journal of Veterinary Research 45, 1671-1678
Received March 12, 1990 Accepted September 18, 1990
Depression of lymphocyte responses to phytohaemagglutinin in lambs experimentally infected with bovine respiratory syncytiai virus R. SHARMA, Z. WOLDEHIWET*, University of Liverpool, Department of Veterinary Pathology,
Veterinary FieM Station, Leahurst, Neston, Wirral L64 7TE
Eight lambs were experimentally infected with bovine respiratory syncytial virus (BRSV) and the responses of their peripheral blood lymphocytes to the mitogen phytohaemagglutinin and HRSV antigen compared with that of control lambs injected with tissue culture fluid. The lymphocyte transformation responses to phytohaemagglutinin were significantly reduced five and 10 days after experimental infection with BRSV (P 0.05).
R E S P I R A T O R Y syncytial virus is a m a j o r respiratory pathogen in human infants and calves (Chanock et al 1970, Stott et al 1980). Attempts to reproduce clinical disease by experimental infection of calves have been fraught with difficulties but several workers have shown that lambs are consistently susceptible to experimental infection with bovine respiratory syncytial virus (BRSV) (Lehmkuhl and Cutlip 1979, Al-Darraji et al 1982, Trigo et al 1984a, Sharma and Woldehiwet 1990). Therefore, lambs appear to be a g o o d model for studying the immunopathogenesis of the BRSV infection because of low cost, easy management and availability of immunological reagents such as the recently developed monoclonal antibodies against lymphocyte surface markers (Mackay et al 1987). Lambs experimentally infected with BRSVare more susceptible to secondary infection with Pasteurella haemolytica (Al-Darraji et al 1982, Trigo et al 1984b). The mechanisms of this predisposition are not known. Significant alterations in lymphocyte subpopulations in lambs experimentally infected with BRSVwere recently demonstrated (Sharma et al 1990). *Reprint requests to Dr Z. Woldehiwet
In the present study, the authors examined the possible functional significance of these alterations. Materials and methods BRS V
A strain of bovine respiratory syncytial virus (BRSV 66), passaged in lamb testis cells three times, was maintained and used as previously described (Sharma and Woldehiwet 1990).
Lambs Sixteen, six- to eight-week-old conventionally reared Suffolk crossbred lambs, free of neutralising antibodies to respiratory syncytial virus, were used. Eight lambs were infected with BRSV as previously described and eight controls were injected with uninfected tissue culture fluid (Sharma and Woldehiwet 1990).
Collection of blood samples Peripheral blood samples were collected from eight BRsv-infected and eight control lambs in heparincoated tubes on 0, 5, 10, 14 and 21 days after inoculation. Mononuclear cells were separated from diluted blood on a Ficoll-paque column (Pharmacia). Cell viability was determined by the trypan blue exclusion technique and the cells resuspended in RPMI 1640 medium to give a concentration of 1 × 107 viable cells ml-l.
Monoclonal antibodies Monoclonal antibodies (Mab) were kindly provided by Dr M. R. Brandon, University of Melbourne, Australia. The Mabs used in the present study were 44-38, 38-65, 20-96 which recognise the CD5 (T helper), CD8 (cytotoxic/suppressor) and LCA p220 (B cell) surface antigens, respectively (Mackay et al 1987). The optimum dilution of each antibody was determined using checkerboard titration.
i52
153
Lymphocyte responses in BRSv-infected lambs Negative selection o f CD4 + cells and CD8 + cells CD4-enriched and CD8-enriched cells were prepared by selective depletion using Mabs and rabbit complement as described by Ezaki et al (1987). Briefly, CD8-enriched lymphocytes were prepared by treating peripheral blood mononuclear cells with antiCD4 and anti-LCA p220 Mab followed by rabbit complement (C'). CD4-enriched lymphocytes were prepared by treating peripheral blood mononuclear cells with anti-CD8 and anti-LCA p220 Mab followed by rabbit complement. Depletions were verified by flow cytometry.
BRSV antigen The BRSV antigen used in transformation assays was prepared from lambs' testis cell cultures infected with BRSV as described earlier (Sharma and Woldehiwet 1990). Virus-infected fluid was purified by sucrose density gradient centrifugation as described by Trepanier et al (1981). The purified virus was inactivated at 56°C for 30 minutes and stored at - 7 0 ° C until required. Control antigen was prepared from uninfected lambs' testis cells.
Lymphocyte transformation assays Lymphocyte transformation assays were performed as described by Taylor et al (1987) with slight modification. Briefly, peripheral blood mononuclear cells or CD4- or CD8-enriched fractions (105 per well) were dispensed in 100 #1 volume to the wells of 'V' bottomed microtitre plates containing 20 /d of purified phytohaemagglutinin (40 /~g m1-1) (Wellcome reagents) or 20 t~l of optimum concentration of BRSV antigen or 20 /zl of RPM[ 1640 medium. After incubation for three days, cultures were pulsed overnight with 1/~Ci tritiated (3H)-thymidine (Amersham International), in 50 #1 per well and harvested on to filtermats (Skatron) by multiple sample harvester (Skatron). The radioactivity in the filters was counted in a scintillation counter (Packard). The degree of transformation was expressed as the ratio of tritiated thymidine incorporation in cultures stimulated with phytohaemagglutinin or BRSV antigen to that of the control cultures (stimulation index).
There were no significant differences in absolute counts per minutes of tritiated thymidine uptake between cultures from control and BRsv-infected lambs when incubated without phytohaemagglutinin or BRSV antigen. There were animal to animal and day to day variations in the transformation responses of lymphocytes to phytohaemagglutinin in both control and aRsv-infected lambs. The variations observed in the control lambs were not statistically significant (Fig 1) (P>0.05). However, the mean stimulation index in phytohaemagglutinin-stimulated cultures of BRSv-infected Iambs was significantly decreased from 28.2 ± 6.56 to 9"25 ± 2.7 five days after infection ( P < 0 " 0 5 ) (Fig 1). The lymphocyte transformation responses in this group remained significantly depressed until 10 days after infection compared to uninfected control lambs (P < 0.05). The lymphocyte transformation responses returned to normal levels 14 days after infection. When mononuclear cells obtained before experimental infection with BRSV were treated with anti° CD4 Mab and C' (CD8-enriched fraction), their lymphocyte transformation responses to phyto-
5o
4o
~ 30
-~ E ~ 20
10,
Analysis o f data The stimulation indices obtained in Bgsvstimulated or phytohaemagglutinin-stimulated cultures in Bgsv-infected iambs were compared with those obtained in control lambs on different days after inoculation by Student's t test. Results
All the results are the mean of triplicate cultures.
BRSV
~- Control
0
I
0
'
I
5
i
I
'
10
|
15
'
t 2O
Days after inoculation
FIG 1 : Lymphocyte transformation responses of peripheral blood mononuclear cells to phytohaemagglutinin in BRSV-infected and control iambs. Each point represents the mean stimulation index of eight animals ( ± SE)
R. Sharma, Z. Woldehiwet
154 70-
cyte transformation responses of the CD4-enriched cells obtained from the control group on the same day of sampling ( P < 0 . 0 5 ) (Fig 2). The lymphocyte transformation responses of mononuclear cells treated with C ' alone remained unaltered. BRSV antigen-induced transformations were not observed in any of the mononuclear cell cultures obtained from BRSv-infected lambs and control lambs on different days after inoculation (P > 0"05) (Fig 3).
(a)
T
60.
50,
]
BRSVgroup
]
Control group
-T
40,
g
]
Virological studies
._~ 30,
All the BRSv-infected lambs had a significant rise in virus-neutralising antibodies to BRSVon day t4 after infection.
20
Discussion 10,
0
'
The role of cell-mediated immune responses of BRSVinfection in calves is not well established. Taylor et al (1987) recently reported that only one of 21 conventionally reared calves developed lymphocytic transformation responses to BRSV antigen after
5
Days after inoculation
407
25t
(b) ]
BRSVgroup
]
Control group
x 30 "o
20 -
~
T
10-
0
®
0
I
'
®
5
,
Days after inoculation FIG 2: Lymphocyte transformation responses of CD4-enriched cells (a) and CDS-enriched cells (b) to phytohaemagglutinin in BRSV-infected and control lambs. Each point represents the mean stimulation index of eight animals ( ± SE)
haemagghtinin were significantly reduced (P < 0" 01) (Fig 2) but treatment of these cells with anti-CD8 Mab and C' (CD4-enriched fraction) did not significantly affect the lymphocyte transformation responses to phytohaemagglutinin (P > 0" 05) (Fig 2). The phytohaemagglutinin-induced responses of CD4-enriched cells obtained five days after experimental infection with BRSV were significantly lower than those of the CD4-enriched cell fractions obtained from the same group on the day of experimental infection (P < 0" 01) (Fig 2). The phytohaemagghtinin-induced lymphocyte transformation responses of CD4-enriched ceils obtained five days after experimental infection with BRSV were also significantly lower than the lympho-
1"5
x
.
J
~
I
J
-~ ~
1-01
J
1
0.5-
BRSV 41- C o n t r o l
0.0 0
5
10 15 Days after inoculation
20
FIG 3~ Lymphocyte transformation responses of peripheral blood mononuclear cells to BRSV antigen in BRSV-infected and control lambs. Each point represents the mean stimulation index of eight animals ( ± SE)
Lymphocyte responses in BRSv-infected lambs experimental infection, compared to six of six calves exposed to an inactivated vaccine. In the present study, virus-specific transformation responses were not detected up to 21 days after infection in ~RSVinfected lambs. Schauf et al (1979) also suggested that a single infection with human respiratory syncytial virus does not always confer enough cellular responses to the virus to be demonstrable in assay systems. BRSV has been implicated as a predisposing factor to secondary bacterial infections in natural respiratory disease in calves and experimental infections in lambs (Bryson et al 1979, Thomas et al 1980, Pirie et al 1981, A1-Darraji et al 1982, Trigo et al 1984b) but the mechanisms of this predisposition have not been explored. In the present study, the authors demonstrated that lymphocytes from BRSVinfected lambs had depressed reactivity to phytohaemagglutinin as early as five days after infection. Roberts (1982) also demonstrated reduced lymphocyte t r a n s f o r m a t i o n responses to p h y t o haemagglutinin in human lymphocytes infected with respiratory syncytial virus in vitro. Decreased lymphocyte blastogenesis to mitogens is a feature of many viral infections (Carney et al 1981, Hyypia et al 1984). The immunosuppression caused by some viral infections has been attributed to either direct infection of i m m u n o c o m p e t e n t cells or the imbalances of immunoregulatory T cells (Hyypia et al 1984). A number of viruses including human respiratory syncytial virus, pestiviruses and rubella virus are capable of infecting lymphocytes (Muscoplat et al 1973, Chantler and Tingle 1982, Roberts 1982). It is not clear whether respiratory syncytial virus selectively infects specific subsets of lymphocytes and thereby affects their competence but there is some evidence to suggest that human respiratory syncytial virus can infect human lymphocytes and monocytesmacrophages in vitro (Domurat et al 1985, Bangham and McMichael 1986). Furthermore, circulating mononuclear leucocytes obtained from symptomatic children infected with human respiratory syncytial virus frequently express viral antigens (Domurat et al 1985). In the present study, the authors found that lymphocyte transformation responses to phytohaemagglutinin were significantly reduced when mononuclear cells were treated with anti-CD4 Mab and complement but the responses to phytohaemagglutinin were not affected when mononuclear cells were treated with anti-CD8 Mab and complement. This is in agreement with the findings of Ezaki et al (1987) who suggested that, in sheep, lymphoproliferative responses to phytohaemagglutinin were mediated by T lymphocytes bearing T1 and T4 (CD4) surface antigens and not by lymphocytes bearing T8 (CD8) antigen.
155
The lymphocyte transformation responses of CD4-enriched lymphocytes obtained five days after experimental infection with BRSV were significantly lower than those obtained before infection or those obtained from control lambs on the same day (five days after infection) of sampling. This suggests that the depressed lymphocyte transformation responses of mononuclear cells to phytohaemagglutinin due to BRSV infection is associated with CD4+ lymphocytes. The present results also indicate that the depressed response to phytohaemagglutinin attributable to CD4 + T-lymphocytes were not due to the reduction in the number of circulating CD4+ lymphocytes because the lymphocyte numbers in cultures were kept constant. It appears, therefore, that BRSV may depress immune responses both by reducing the number of circulating CD4 + T-lymphocytes (Sharma et al 1990) and by depressing their reactivity.
Acknowledgements The authors thank Dr M. R. Brandon, University of Melbourne for providing the monoclonal antibodies. R.S. is a Commonwealth scholar supported by the Association of Commonwealth Universities.
References AI-DARRAJI, A. M., CUTLIP, R. C., LEHMKUL, H. D., GRAHAM, D. L., KLUGE, J. P. & FRANK, G. H. (1982) Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: clinical and microbiologic studies. American Journal of Veterinary Research 43,236-240 BANGHAM, C. R. M. & McMICHAEL, A. J. (1986) Specific human cytotoxic T cells recognise B cell lines persistently infected with respiratory syncytial virus. Proceedings of the National Academy of Sciences of the United States of America (Washington) 83, 9183-9187 BRYSON, D. G , McFERRAN, J. B., BALL, H. J. & NEILL, S. D. (1979) Observations on outbreaks of respiratory disease in calves associated with parainfluenza type 3 virus and respiratory syncytial virus infection. Veterinary Record 104, 45-49 CARNEY, W. P., RUBIN, R. H., HOFFMAN, R. A., HANSEN, W. P., HEALEY, K. & HIRCH, M. S. (1981) Analysis of Tlymphocyte subsets in cytomegalovirus mononucleosis. Journal of Immunology 126, 2114-2116 CHANOCK, R. M., KAPIKIAN, A. Z., MILLS, J., KIM, H. W. & PARROTT, R. H. (1970) Influence of immunological factors in respiratory syncytial virus disease of the lower respiratory tract. Archives of Environmental Health 21,347-355 CHANTLER, J. K. & TINGLE, A. J. (1982) Isolation of rubella virus from human lymphocytes after acute natural infection. Journal of Infectious Diseases 145,673-677 DOMURAT, F., ROBERTS, N. J. Jr, WALSH, E. E. & DAGAN, R. (1985) Respiratory syncytial virus infection of human mononuclear leucocytes in vitro and in vivo. Journal of Infectious Diseases 152, 895-902 EZAKI, T., MIYASAKA, M., BEYA, M. F., DUDLER, L. & TRNKA, Z. (1987) A murine anti-sheep T8 monoclonal antibody, ST-8, that defines the cytotoxic T lymphocyte population. International Archives of Allergy and Applied Immunology 82, 168-177
156
R . S h a r m a , Z. W o l d e h i w e t
HYYPIA, T., ESKOLA, J., LA1NE, M. & MEURMAN, O. (1984) B cell function in vitro during rubella infection. Infection and Immunity 43, 589-592 LEHMKUHL, H. D. & CUTLIP, R. C. (1979) Experimentally induced respiratory syncytial virus infection in lambs. American Journal of Veterinary Research 40, 512- 514 MACKAY, C. R., MADDOX, J. F. & BRANDON, M. R. (1987) Lymphocyte antigens of sheep: Identification and characterisation using a panel of monoclonal antibodies. Veterinary Immunology and Immunopathology 17, 91-102 MUSCOPLAT, C. C., JOHNSON, D. W. & TEUSCHER, E. (1973) Surface immunoglobulins of circulating lymphocytes in chronic bovine diarrhoea: Abnormalities in cell populations and cell function. American Journal of Veterinary Research 34, 1101-1104 PIRIE, H. M., PETRIE, L., PRINGLE, C. R., ALLAN, E. M. & KENNEDY, G. J. (1981) Acute fatal pneumonia in calves due to respiratory syncytial virus. Veterinary Record 108, 411-416 ROBERTS, N. J. Jr (1982) Different effects of influenza virus, respiratory syncytial virus, and Sendai virus on human lymphocytes and macrophages. Infection and Immunity 35, 1142-1146 SCHAUF, V., PURCELL, C., MIZEN, M. & MIZEN, S. (1979) Lymphocyte transformation in response to antigens of respiratory syncytial virus. Proceedings of the Society for Experimental Biology and Medicine 161,564-569 SHARMA, R. & WOLDEHIWET, Z. (1990) Pathogenesis of bovine respiratory syncytial virus in experimentally infected lambs. Veterinary Microbiology 23, 267-272 SHARMA, R., WOLDEHIWET, Z., SPILLER, D. G. & WARENIUS, H. M. (1990) Lymphocyte subpopulations in
peripheral blood of lambs experimentally infected with bovine respiratory syncytial virus. Veterinary Immunology and Immunopathology 24, 383-391 STOTT, E. J., THOMAS, L. H., COLLINS, A. P., CROUCH, S., JEBBETT, J., SMITH, G. S., LUTHER, P. D. & CASWELL, R. (1980) A survey of virus infections of the respiratory tract of cattle and their association with disease. Journal of Hygiene, Cambridge 85, 257-270 TAYLOR, G., STOTT, E. J. &THOMAS, L. H. (1987) Lymphocyte transformation response of calves to respiratory syncytial virus. Journal of Medical Virology 22, 333-344 THOMAS, L. H., STOTT, E. J., JONES, P. W., JEBBETT, J. & COLLINS, A. P. (1980) The possible role of respiratory syncytial virus and Pasteurella spp in calf respiratory disease. Veterinary Record 107, 304-307 TREPANIER, P., PAYMENT, P. & TRUDEL, M. (1981) Concentration of human respiratory syncytial virus using ammonium sulphate, polyethylene glycol or honow fibre ultrafiltration. Journal of Virological Methods 3 , 2 0 1 - 2 l 1 TR1GO, F. J., BREEZE, R. G., EVERMANN, J. F. & GALLINA, A. M. (1984a) Pathogenesis of experimental bovine respiratory syncytial virus infection in sheep. American Journal of Veterinary Research 45, 1663-1670 TRIGO, F. J., BREEZE, R. G., LIGG1TT, H. D., EVERMANN, J. F. & TRIGO, E. (1984b) Interaction of bovine respiratory syncytial virus and Pasteurella haemolytica in the ovine lung. American Journal of Veterinary Research 45, 1671-1678
Received March 12, 1990 Accepted September 18, 1990
