International Journalfor Primed in Great Brirarn
Parasttology
Vol. 21, No. 6, pp 7 19-72 I, 1991
0
Oil2&7519/91 $3.00 + 0.00 PtTgamon Press p/c 199 I Au.srrn/inn Societyfor Parmimlogy
RESEARCH NOTE MOLECULAR ANALYSIS OF T AND B CELL REPERTOIRES IN MICE IMMUNIZED WITH OPISTHORCHIS VIVERRINI ANTIGENS SURASAKDI
WONGRATANACHEEWIN,*~
MICHAEL
MELISSA *Departments $The
Tropical
of Microbiology Health
Program,
and
F. GOOD,$
PAIBOON
SParasitology,
Queensland
and
SITHITHAWORN~
R. HASWELL-ELKINS~
Faculty of Medicine, Khon Kaen University, Thailand Institute of Medical Research, Bramston Terrace, Queensland 4006, Australia
Khon
Kaen
Herston,
40002,
Brisbane,
(Received 1 May 199 1; accepted 2 July 199 1) Abstract-WoNcRArANAcHEEWrN S., GOOD M. F., SITHITHAWORNP. and HASWELL-ELKINS M. R. 1991. Molecular analysis of T and B cell repertoires in mice immunized with Opisthorchis viverrini antigens. International Journal for Parasitology 21: 719-721. BlO mice were immunized with an Opisthorchis viverrini somatic extract and then their responses were analyzed. The antigenic fractions of the extract were separated by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose membranes and solubilized for use in lymphocyte culture. Antibody specificity was also visualized by immunoblotting using immunized mouse sera. The M, of the main immunogenic fractions for T cells ranged from 28 to 46 kDa, whereas those recognized by antibodies were 45, 52, 56, 59, 65, 69, 75 and 81 kDa. The results indicate a striking difference in the antigenic recognition pattern of T and B cells which may be important for selecting antigen molecules for immunological studies of this trematode infection in man. INDEX KEY WORDS: proliferation; immunity.
Opisthorchis viverrini: T aud
LIVER
Western
blot;
lymphocyte
immune response. An understanding of the immune response to individual antigenic molecules is the basis of current strategies to identify protective antigens and/or those involved in immunopathogenesis. The purpose of the present study was to investigate the T and B cell immune repertoires in BlO mice immunized with 0. viverrini soluble crude extract antigens. Adult 0. viverrini were obtained from experimentally infected hamsters and a soluble aqueous extract was prepared as previously described (Wongratanacheewin, Chawengkirttikul, Bunnag & Sirisinha, 1988). The same preparation was used throughout the study for immunization, T cell proliferation assays and immunoblotting. Antigens were resolved by SDS-PAGE using the discontinuous buffer system of Laemmli (1970) on S-20% acrylamide gradient gels (Bio-Rad, U.S.A.). The proteins were electrophoretically transferred to nitrocellulose paper using a semi-dry electroblotter (Bio-Rad, U.S.A.) as described by Kyhse-Andersen (1984) at 4°C for 1.5 h using 0.8 mA cm-2. Nitrocellulose membranes were cut into 32 horizontal sections, each 4 mm wide, corresponding to 14-200 kDa M, fractions. Strips were then solubilized and precipitated into particles as previously described (Abou-Zeid, Filley, Steele & Rook, 1987). After repeated washing and centrifugation with 0.15
fluke infection is an endemic disease in many parts of the world, especially among people residing in Southeast Asia, the Far East and parts of Europe. Recently, the infection has been reported from several non-endemic areas including North America and Western Europe (Anderson & Moser, 1985). In Thailand alone, it has been estimated by Preuksaraj, Jeradit, Sathitayathai, Kijvanee & Sridonrusmi (1982) that at least 7 million people are infected with Opisthorchis viverrini. This infection is associated with hepatobiliary diseases and cholangiocarcinoma, and its pathogenesis may be immunological, in part (Haswell-Elkins, Sithithaworn, Mairiang, Elkins, Wongratanacheewin, Kaewkes & Mairiang, in press). Although several lines of evidence suggest that the infection can stimulate both systemic humoral and cell-mediated immune responses during the course of infection, the possible significance of these immune responses to protective immunity is, presently, controversial. Immunological studies of this infection have been largely concerned with immunodiagnosis (Wongratanacheewin, Chawengkirttikul, Bunnag & Sirisinha, 1988; Wongratanacheewin, Bunnag, Vaeusorn & Sirisinha, 1988) and there is, thus, limited information regarding the cell-mediated
t To whom all correspondence
B cell repertoires;
should be addressed. 719
720
S. WONGRATANA(.HEEWIN et al
-02
-6a
.
2
3
-21
4
FIG. 1. Western blot pattern of 0. viverrini antigens reacted with normal mice sera (lane 2), sera from mice immunized with 0. viverrini for antibody production (lane 3) and mice immunized with 0. viverrini for use in T cell proliferation assays (lane 4). Lane 1 represents the profiles of SDSPAGE stained with Coomassie blue. Molecular weight markers are shown on the right. Arrows indicate a 16-17 kDa doublet.
M-phosphate buffer saline pH 7.4 (PBS), the particles were resuspended in 400 ~1 of Minimum essential medium (MEM) (Gibco, U.S.A.) and stored at - 20°C for use in proliferation assays. BIO mice were immunized subcutaneously with 0. viverrini antigens (20 pug per mouse), emulsified in an equal volume of Complete Freund’s adjuvant. After 8-10 days, lymph node cells were removed and suspended (4 x 10’ cells per well) in MEM, supplemented with 2% normal mouse serum, 5 X lo-’ M-2-mercaptoethanol, 10 pmole ml-’ of HEPES, 100 U ml-’ of penicillin, 0.1 mg ml-’ of streptomycin and 2 mg of sodium bicarbonate in 96-well microtiter plates (Nunc, Denmark). Twenty microliters of the 32 separate fractions were added to each well in triplicate at the initiation of the cell cultures. After 4
days incubation, the cultures were pulsed with 18.5 kBq of ‘H-methyl thymidine (Amersham. U.K.) for 8-16 h before harvesting onto glass filters. ‘HThymidine incorporation was measured by liquid scintillation spectroscopy (LKB, Sweden) and antigen-specific proliferation was expressed as a stimulation index (mean c.p.m. fraction well/mean c.p.m. control well with nitrocellulose alone). For antibody assays, a second group of mice was immunized with the same antigens by a subcutaneous route. Antigens given were emulsified in an equal volume of Complete Freund’s adjuvant. The animals were boosted in the third week and the sera were collected I week later. Nitrocellulosc membranes bearing electrophoretically separated antigens were cut vertically into 5 mm strips and washed with 0.05% Tween 20 in 0.02 M-Tris-HCI and 0.5 M-NaCl pH 7.5 (TBS). Non-specific binding was blocked by incubation with 3% skimmed milk in TBS at 4°C overnight. Strips were incubated with mouse sera diluted I:50 in TBS containing 1% skimmed milk at 37°C for 2 h on a shaker. After washing, alkaline phosphatase-conjugated goat antimouse immunoglobulin (Bio-Rad. U.S.A.). diluted I:2000 in TBS, was added to the individual strips and incubated at 37°C for 2 h. The color was developed by using 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium as substrates. More than 30 polypeptides with M, ranging from 12 to > 1 I6 kDa could be detected by Coomassie blue staining of the adult somatic extract. Major bands were located with M, of I6 and I7 kDa (Fig. I, lane I, arrows). When each nitrocellulose fraction was tested in T cell proliferation assays, the main immunogenic fractions had M, ranging from 28 to 46 kDa (Fig. 2). Conversely, sera from a second group of mice immunized with the same antigen using an appropriate schedule for antibody assay were shown to react with components of 45, 52, 56, 59, 65, 69. 75 and 81 kDa as well as the 16-17 kDa doublet (Fig. I. lane 3). In order to determine whether T and B cells in the same mice showed different recognition profiles, sera from mice immunized for use in T cell proliferation assays were also tested for antibody activity and shown to react with antigens of M, 69. 75 and 81 kDa (Fig. 1, lane 4). The results demonstrate that T and B cells were recognizing different antigenic molecules of 0. viverrini. It is known that T and B cell epitopes differ for most antigens, so that our results obtained may not be surprising. Since mechanisms of protective immunity and immunopathology of opisthorchiasis are still unknown, the search for protective antigen(s) by antibody recognition alone may thus be inappropriate. The techniques used in this study will enable us to examine the T cell immune repertoire for the identification of the immunodominant antigens in patients with parasite-associated diseases and also to study the role of T cells in immunity to infection.
Research
Stimulation
721
Note
Index
-11 1
3
5
t
200
7
9
11
13
15
t 91
t 69
FIG. 2. T lymphocyte
stimulation pattern of antigens were separated by SDS-PAGE and membrane was cut into small strips, solubilized assay. T cells showed a strong response to corresponded to M, ranging from 28 to 46 kDa.
Acknowledgements-We
are grateful to Dr D. P. McManus for his valuable comments and suggestions. This work was supported by the Australian National Health and Medical Research Council (NH&MRC) and the Tropical Health Program, Queensland Institute of Medical Research. Brisbane, Australia. REFERENCES ABOU-ZEID C., FILLEY E., STEELEJ. & ROOK G. A. W. 1987. A simple new method for using antigens separated by polyacrylamide electrophoresis to stimulate gel lymphocytes in vitro after converting bands cut from Western blots into antigen bearing particles. Journal q/ Immunological ANDERSON
Methods
17
Fraction
98: 5-10.
J. P. & MOSER R. J. 1985. Parasite screening and treatment among Indochinese refugees. Cost-benefit utility and the general health policy model. Journal of the American Medical Association 253: 2229-2235. HASWELL-ELKINS M. R.. SITHITHAWORNP.. MAIRIANG E., ELKINS D. B., WONGRATANACHEEWINS., KAEWKES S. & MAIRIANC‘I P. (in press) Immune responsiveness and parasite-specific antibody levels in human hepato-biliary disease associated with Opisthorchis viverrini infection.
19
21
23
25
27
29
ji
number
t 46
t
t 21
t 14
BlO mice immunized with 0. viverrini. The transferred to nitrocellulose membrane. The and precipitated for lymphocyte proliferation antigenic fractions numbered 18-21 which The average background c.p.m. value was 510.
Clinical and Experimental
Immunology.
KYHSE-ANDERSONJ. 1984. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. Journnl of Biochemical and Biophysical Methods 10 : 203-209. LAEMMLIU. K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature (London) 227: 680685. PREUKSARAJS., JERADIT C., SATHITAYATHAIA., KIJVANEE S. & SRI~NRUSMI T. 1982. Studies on prevalence and intensity of intestine1 helminthic infection in rural populations of Thailand. Communication Disease Journal 8: 245-269.
WONGRATANACHEEWINS., BUNNAC D., VAEUSORN N. & SIRISINHA S. 1988. Characterization of humoral immune response in the serum and bile of patients with opisthorchiasis and its application in immunodiagnosis. American 356-362.
Journal
qf Tropical
Medicine
and Hygiene
38:
WONGRATANACHEEWINS., CHAWENGKIRTTIKULR., BUNNAG D. & SIRISINHAS. 1988. Analysis of Opisthorchis viverrini antigens by immunoprecipitation and polyacrylamide gel electrophoresis. Parasifofogy 96: 119-128.
Parasttology
Vol. 21, No. 6, pp 7 19-72 I, 1991
0
Oil2&7519/91 $3.00 + 0.00 PtTgamon Press p/c 199 I Au.srrn/inn Societyfor Parmimlogy
RESEARCH NOTE MOLECULAR ANALYSIS OF T AND B CELL REPERTOIRES IN MICE IMMUNIZED WITH OPISTHORCHIS VIVERRINI ANTIGENS SURASAKDI
WONGRATANACHEEWIN,*~
MICHAEL
MELISSA *Departments $The
Tropical
of Microbiology Health
Program,
and
F. GOOD,$
PAIBOON
SParasitology,
Queensland
and
SITHITHAWORN~
R. HASWELL-ELKINS~
Faculty of Medicine, Khon Kaen University, Thailand Institute of Medical Research, Bramston Terrace, Queensland 4006, Australia
Khon
Kaen
Herston,
40002,
Brisbane,
(Received 1 May 199 1; accepted 2 July 199 1) Abstract-WoNcRArANAcHEEWrN S., GOOD M. F., SITHITHAWORNP. and HASWELL-ELKINS M. R. 1991. Molecular analysis of T and B cell repertoires in mice immunized with Opisthorchis viverrini antigens. International Journal for Parasitology 21: 719-721. BlO mice were immunized with an Opisthorchis viverrini somatic extract and then their responses were analyzed. The antigenic fractions of the extract were separated by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose membranes and solubilized for use in lymphocyte culture. Antibody specificity was also visualized by immunoblotting using immunized mouse sera. The M, of the main immunogenic fractions for T cells ranged from 28 to 46 kDa, whereas those recognized by antibodies were 45, 52, 56, 59, 65, 69, 75 and 81 kDa. The results indicate a striking difference in the antigenic recognition pattern of T and B cells which may be important for selecting antigen molecules for immunological studies of this trematode infection in man. INDEX KEY WORDS: proliferation; immunity.
Opisthorchis viverrini: T aud
LIVER
Western
blot;
lymphocyte
immune response. An understanding of the immune response to individual antigenic molecules is the basis of current strategies to identify protective antigens and/or those involved in immunopathogenesis. The purpose of the present study was to investigate the T and B cell immune repertoires in BlO mice immunized with 0. viverrini soluble crude extract antigens. Adult 0. viverrini were obtained from experimentally infected hamsters and a soluble aqueous extract was prepared as previously described (Wongratanacheewin, Chawengkirttikul, Bunnag & Sirisinha, 1988). The same preparation was used throughout the study for immunization, T cell proliferation assays and immunoblotting. Antigens were resolved by SDS-PAGE using the discontinuous buffer system of Laemmli (1970) on S-20% acrylamide gradient gels (Bio-Rad, U.S.A.). The proteins were electrophoretically transferred to nitrocellulose paper using a semi-dry electroblotter (Bio-Rad, U.S.A.) as described by Kyhse-Andersen (1984) at 4°C for 1.5 h using 0.8 mA cm-2. Nitrocellulose membranes were cut into 32 horizontal sections, each 4 mm wide, corresponding to 14-200 kDa M, fractions. Strips were then solubilized and precipitated into particles as previously described (Abou-Zeid, Filley, Steele & Rook, 1987). After repeated washing and centrifugation with 0.15
fluke infection is an endemic disease in many parts of the world, especially among people residing in Southeast Asia, the Far East and parts of Europe. Recently, the infection has been reported from several non-endemic areas including North America and Western Europe (Anderson & Moser, 1985). In Thailand alone, it has been estimated by Preuksaraj, Jeradit, Sathitayathai, Kijvanee & Sridonrusmi (1982) that at least 7 million people are infected with Opisthorchis viverrini. This infection is associated with hepatobiliary diseases and cholangiocarcinoma, and its pathogenesis may be immunological, in part (Haswell-Elkins, Sithithaworn, Mairiang, Elkins, Wongratanacheewin, Kaewkes & Mairiang, in press). Although several lines of evidence suggest that the infection can stimulate both systemic humoral and cell-mediated immune responses during the course of infection, the possible significance of these immune responses to protective immunity is, presently, controversial. Immunological studies of this infection have been largely concerned with immunodiagnosis (Wongratanacheewin, Chawengkirttikul, Bunnag & Sirisinha, 1988; Wongratanacheewin, Bunnag, Vaeusorn & Sirisinha, 1988) and there is, thus, limited information regarding the cell-mediated
t To whom all correspondence
B cell repertoires;
should be addressed. 719
720
S. WONGRATANA(.HEEWIN et al
-02
-6a
.
2
3
-21
4
FIG. 1. Western blot pattern of 0. viverrini antigens reacted with normal mice sera (lane 2), sera from mice immunized with 0. viverrini for antibody production (lane 3) and mice immunized with 0. viverrini for use in T cell proliferation assays (lane 4). Lane 1 represents the profiles of SDSPAGE stained with Coomassie blue. Molecular weight markers are shown on the right. Arrows indicate a 16-17 kDa doublet.
M-phosphate buffer saline pH 7.4 (PBS), the particles were resuspended in 400 ~1 of Minimum essential medium (MEM) (Gibco, U.S.A.) and stored at - 20°C for use in proliferation assays. BIO mice were immunized subcutaneously with 0. viverrini antigens (20 pug per mouse), emulsified in an equal volume of Complete Freund’s adjuvant. After 8-10 days, lymph node cells were removed and suspended (4 x 10’ cells per well) in MEM, supplemented with 2% normal mouse serum, 5 X lo-’ M-2-mercaptoethanol, 10 pmole ml-’ of HEPES, 100 U ml-’ of penicillin, 0.1 mg ml-’ of streptomycin and 2 mg of sodium bicarbonate in 96-well microtiter plates (Nunc, Denmark). Twenty microliters of the 32 separate fractions were added to each well in triplicate at the initiation of the cell cultures. After 4
days incubation, the cultures were pulsed with 18.5 kBq of ‘H-methyl thymidine (Amersham. U.K.) for 8-16 h before harvesting onto glass filters. ‘HThymidine incorporation was measured by liquid scintillation spectroscopy (LKB, Sweden) and antigen-specific proliferation was expressed as a stimulation index (mean c.p.m. fraction well/mean c.p.m. control well with nitrocellulose alone). For antibody assays, a second group of mice was immunized with the same antigens by a subcutaneous route. Antigens given were emulsified in an equal volume of Complete Freund’s adjuvant. The animals were boosted in the third week and the sera were collected I week later. Nitrocellulosc membranes bearing electrophoretically separated antigens were cut vertically into 5 mm strips and washed with 0.05% Tween 20 in 0.02 M-Tris-HCI and 0.5 M-NaCl pH 7.5 (TBS). Non-specific binding was blocked by incubation with 3% skimmed milk in TBS at 4°C overnight. Strips were incubated with mouse sera diluted I:50 in TBS containing 1% skimmed milk at 37°C for 2 h on a shaker. After washing, alkaline phosphatase-conjugated goat antimouse immunoglobulin (Bio-Rad. U.S.A.). diluted I:2000 in TBS, was added to the individual strips and incubated at 37°C for 2 h. The color was developed by using 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium as substrates. More than 30 polypeptides with M, ranging from 12 to > 1 I6 kDa could be detected by Coomassie blue staining of the adult somatic extract. Major bands were located with M, of I6 and I7 kDa (Fig. I, lane I, arrows). When each nitrocellulose fraction was tested in T cell proliferation assays, the main immunogenic fractions had M, ranging from 28 to 46 kDa (Fig. 2). Conversely, sera from a second group of mice immunized with the same antigen using an appropriate schedule for antibody assay were shown to react with components of 45, 52, 56, 59, 65, 69. 75 and 81 kDa as well as the 16-17 kDa doublet (Fig. I. lane 3). In order to determine whether T and B cells in the same mice showed different recognition profiles, sera from mice immunized for use in T cell proliferation assays were also tested for antibody activity and shown to react with antigens of M, 69. 75 and 81 kDa (Fig. 1, lane 4). The results demonstrate that T and B cells were recognizing different antigenic molecules of 0. viverrini. It is known that T and B cell epitopes differ for most antigens, so that our results obtained may not be surprising. Since mechanisms of protective immunity and immunopathology of opisthorchiasis are still unknown, the search for protective antigen(s) by antibody recognition alone may thus be inappropriate. The techniques used in this study will enable us to examine the T cell immune repertoire for the identification of the immunodominant antigens in patients with parasite-associated diseases and also to study the role of T cells in immunity to infection.
Research
Stimulation
721
Note
Index
-11 1
3
5
t
200
7
9
11
13
15
t 91
t 69
FIG. 2. T lymphocyte
stimulation pattern of antigens were separated by SDS-PAGE and membrane was cut into small strips, solubilized assay. T cells showed a strong response to corresponded to M, ranging from 28 to 46 kDa.
Acknowledgements-We
are grateful to Dr D. P. McManus for his valuable comments and suggestions. This work was supported by the Australian National Health and Medical Research Council (NH&MRC) and the Tropical Health Program, Queensland Institute of Medical Research. Brisbane, Australia. REFERENCES ABOU-ZEID C., FILLEY E., STEELEJ. & ROOK G. A. W. 1987. A simple new method for using antigens separated by polyacrylamide electrophoresis to stimulate gel lymphocytes in vitro after converting bands cut from Western blots into antigen bearing particles. Journal q/ Immunological ANDERSON
Methods
17
Fraction
98: 5-10.
J. P. & MOSER R. J. 1985. Parasite screening and treatment among Indochinese refugees. Cost-benefit utility and the general health policy model. Journal of the American Medical Association 253: 2229-2235. HASWELL-ELKINS M. R.. SITHITHAWORNP.. MAIRIANG E., ELKINS D. B., WONGRATANACHEEWINS., KAEWKES S. & MAIRIANC‘I P. (in press) Immune responsiveness and parasite-specific antibody levels in human hepato-biliary disease associated with Opisthorchis viverrini infection.
19
21
23
25
27
29
ji
number
t 46
t
t 21
t 14
BlO mice immunized with 0. viverrini. The transferred to nitrocellulose membrane. The and precipitated for lymphocyte proliferation antigenic fractions numbered 18-21 which The average background c.p.m. value was 510.
Clinical and Experimental
Immunology.
KYHSE-ANDERSONJ. 1984. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. Journnl of Biochemical and Biophysical Methods 10 : 203-209. LAEMMLIU. K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature (London) 227: 680685. PREUKSARAJS., JERADIT C., SATHITAYATHAIA., KIJVANEE S. & SRI~NRUSMI T. 1982. Studies on prevalence and intensity of intestine1 helminthic infection in rural populations of Thailand. Communication Disease Journal 8: 245-269.
WONGRATANACHEEWINS., BUNNAC D., VAEUSORN N. & SIRISINHA S. 1988. Characterization of humoral immune response in the serum and bile of patients with opisthorchiasis and its application in immunodiagnosis. American 356-362.
Journal
qf Tropical
Medicine
and Hygiene
38:
WONGRATANACHEEWINS., CHAWENGKIRTTIKULR., BUNNAG D. & SIRISINHAS. 1988. Analysis of Opisthorchis viverrini antigens by immunoprecipitation and polyacrylamide gel electrophoresis. Parasifofogy 96: 119-128.
