Clin. exp. Immunol. (1990) 82, 369-372
Antibody repertoire against culture filtrate antigens in wild house mice infected with Mycobacterium bovis BCG K. HUYGEN, K. PALFLIET, F. JURION, C. LENOIR & J-P. VAN VOOREN* Instituut Pasteur Brussels, and *H6pital Erasme, Brussels, Belgium
van
Brabant,
(Acceptedfor publication 6 July 1990)
SUMMARY Wild house mice (Mus domesticus) captured in a Flemish pigsty were infected intravenously with 4 x 106 viable units of Mycobacterium bovis BCG and examined by Western blot analysis for IgG secretion against BCG culture filtrate (CF) antigens. Wild mice showed a marked individual variation in antibody pattern when tested 4,6 and 8 weeks after infection. Some animals reacted to a wide range of antigens and others only to a limited number. Most wild mice recognized preferentially antigens with molecular weight of 24 kD, 32 kD, 37-38-40 kD, 65 kD and 82 kD, i.e. the major CF antigens known to be recognized by sera from BCG-infected inbred laboratory strains, BALB/c, DBA/2, CBA/Ca and C57BL/6. The 32-kD fibronectin-binding protein and the 65-kD heat-shock protein appeared as very immunodominant in wild mice. Furthermore, about 20-25% of the mice reacted strongly with a unique antigen of 35 kD estimated molecular weight, to which the tested inbred laboratory mice did not respond. Monitoring the size of the bacterial population in the spleen indicated that the BCG inoculum did not replicate in wild mice, suggesting that the Bcgr allele is expressed in this population.
Keywords wild mice BCG filtrate antibody repertoire
INTRODUCTION
Following i.v. infection with live Mycobacterium bovis BCG, inbred laboratory mice produce substantial amounts of IgG antibodies against BCG culture filtrate (CF) antigens and the repertoire of these antibodies is dependent on the mouse strain (Huygen et al., 1990). Thus, BALB/c mice produce antibodies against a wide range of CF antigens, with a preferential recognition of the 65-kD heat-shock protein (Thole et al., 1988) and of the 32-kD fibronectin-binding protein (De Bruyn et al., 1987; Abou-Zeid et al., 1988), whereas C57BL/6 mice sera show a more restricted antibody pattern, almost exclusively directed against three antigens with mol. wts of 37, 38 and 40 kD (Huygen et al., 1990). The preferential recognition of certain CF antigens seems to be controlled by a gene (or genes) in the K-IA region of the MHC. A similar genetic variation has also been found in mice infected intraperitoneally with M. tuberculosis and tested for antibodies against soluble extract antigens (Brett & Ivanyi, 1990). Although these findings may eventually be relevant for the development of an efficient serodiagnostic test for mycobacterial infections, extrapolation of results in inbred laboratory strains to the human outbred population must be made with Correspondence: Kris Huygen, Department of Virology, Instituut Pasteur van Brabant, 642 Engelandstraat, 1180 Brussels, Belgium.
369
much caution. In this respect, the use of wild house mice (Mus domesticus) from which all laboratory mouse strains have been derived (Morse, 1981), may allow a more accurate estimate of genetic diversity than can be obtained by examination of laboratory strains (Falus et al., 1987). We have analysed the repertoire of antibodies against BCG CF antigens in a population of wild house mice infected intravenously with BCG. We conclude that, with the exception of a unique 35-kD CF antigen, all the immunodominant CF antigens for laboratory mouse strains are also immunodominant in wild mice. Furthermore, monitoring of BCG replication in the spleen is suggestive of the expression of the resistant allele of the Bcg gene that controls innate resistance to M. bovis BCG (Skamene et al., 1982) by affecting early mycobacterial replication in the macrophage (Stach et al., 1984).
MATERIALS AND METHODS Mice Wild mice, identified as Mus domesticus Schwarz & Schwarz 1943, or West-European long-tailed house mouse, were captured alive in a pigsty in Tienen, near Brussels, Belgium. Karyotype analysis has revealed a Robertsonian translocation Rb4.12 in this population (Hubner & Koulischer, 1990). BALB/c, DBA/2, CBA/Ca, C57BL/6 and Swiss A mice were bred in the animal facilities of the Pasteur Institute van Brabant.
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BCG infection Mice were inoculated intravenously (wild mice under mild ether anaesthesia) with 0 5 mg (±4 x 106 colony-forming units (CFU)) of M. bovis BCG strain GL2 (grown for 2 weeks as a surface pellicle in Sauton medium and prepared freshly every week at the Pasteur Institute). Mice were anaesthesized with ether and exsanguinated after various time intervals. Sera from wild mice were collected individually, sera from inbred laboratory mice were collected as pools from at least four animals/ group. Sera were kept frozen at -20'C until assay. For the boost injection, a second i.v. inoculation (0 5 mg) was given 2 months after the initial one, and mice were bled 4 weeks later. Antigens CF antigens were obtained from 14-day-old cultures of M. bovis BCG GL2 (the same as the one used for infection). Proteins were concentrated by precipitation with ammonium sulphate (80% saturation). After centrifugation, the pellet was dissolved in and dialysed against phosphate-buffered saline (PBS). The filtrate was passed through a 0-22-pm sterilizing membrane (Flow) and stored at -20'C until use. The same batch of CF was used in all experiments. SDS-PA GE and Western blot analysis SDS-PAGE of BCG culture filtrate was performed in reducing conditions on 12-5 % acrylamide gels. After completion of SDSPAGE, the proteins were electrophorectically transferred to nitrocellulose sheets (Towbin, Staehelin & Gordon, 1979) and sera from BCG-infected mice were used for the Western blot analysis as described elsewhere (Huygen et al., 1990). Briefly, nitrocellulose was incubated with bovine serum albumin to prevent non-specific absorption of proteins. Strips of nitrocellulose (containing ± 30 pg of CF) were then incubated with mouse sera (1: 50) overnight and revealed with peroxidase-conjugated rabbit anti-mouse IgG and oe-chloronaphthol in the presence of
hydrogen peroxide. Enumeration of BCG CFU in the spleen of infected mice Following ether anaesthesia and exsanguination, spleens were removed aseptically and spleen cells isolated using a loosely fitting Dounce homogenizer. Serial three-fold dilutions were made in PBS without antibiotics and 200-pl volumes were plated on Middlebrook 7HI 1 Bacto agar (Difco). Each dilution was tested in triplicate. Petri dishes were incubated in sealed plastic bags at 37°C and the number of colonies was counted after 18 days of culture.
RESULTS Repertoire of antibodies against CF antigens in wild mice infected with M. bovis BCG As shown in Fig. 1, a marked individual variation was observed in Western blot (immunoblot) patterns of anti-CF IgG antibodies in sera from wild mice infected with BCG. Some sera reacted with numerous antigens, other sera only with one or a few antigens. Most sera contained antibodies against the 65-kD mycobacterial heat-shock protein (Thole et al., 1988) and against the 32-kD fibronectin-binding protein (De Bruyn et al., 1987; Abou-Zeid et al., 1988). Furthermore, there was a marked variation in immunodominance of the CF antigens, and depending on the individual serum tested, the most intense
staining could be observed with antigens of respective mol. wts of 65, 35, 32 and 24 kD. Comparison of the repertoire in wild mice with the repertoire in inbred laboratory strains Figure 2 shows the immunoblot pattern ofanti-CF antibodies in sera from three wild mice, compared with sera from four laboratory strains. As described elsewhere (Huygen et al., 1990), laboratory strains showed a marked variation in anti-CF antibody repertoire. BALB/c sera reacted with a wide range of CF antigens and preferentially with the 65-kD and 32-kD antigens. C57BL/6 antibodies were directed against a 40-kD antigen and an 82-kD antigen. CBA/Ca sera reacted very weakly with the 32-kD and 65-kD antigens, and DBA/2 sera reacted very weakly with the 65-kD antigen only. All these antigens were also stained by sera from wild mice. The 35-kD antigen was not recognized by sera from C57BL/6, CBA/Ca or DBA/2 mice. BALB/c sera reacted faintly with an antigen of more or less the same mol. wt, but the intense staining so typical for wild mice was never observed. b
a
826538-
35*3224-
Fig. 1. Western blot analysis against a 14-day-old BCG culture filtrate of sera from wild mice infected intravenously with BCG 4 weeks (a), 6 weeks (b) or 8 weeks (c) previously.
b -82
65-
-40
35* 32
-
2 3 4 Figure 2. Comparison of Western blot patterns in sera from three wild mice (a) and four inbred laboratory strains (b) infected with BCG 4 weeks previously. 1, BALB/c; 2, C57BL/6; 3, CBA/Ca; and 4, DBA/2. *The unique antigen with an estimated mol. wt of 35 kD.
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371
Effect of a boost injection of BCG Mice were infected with BCG, boosted 2 months later and examined 4 weeks after the second infection (Fig. 3). In wild mice, Western blot patterns became more pronounced, but sera reacted essentially with the same antigens as during a primary infection, i.e. 32, 35, 65 and 82 kD. Response to the 37-38-40kD antigens (dominant in C57BL/6 mice) was more pronounced in wild mice and in CBA/Ca mice after a second infection with BCG than after a primary infection. The 35-kD antigen was again found to be very immunoreactive in wild mice orly (two out of eight sera, 25%).
the number of CFU had decreased about 100-fold (initial number of CFU/spleen at day 1 of infection: + 106 CFU; data not shown). In C57BL/6 mice, known to carry the susceptible Bcgs allele, the reduction in number of CFU was not even 10fold. Outbred Swiss albino mice showed a decrease similar to wild mice. Additional experiments showed that of 108 wild mice tested, only one showed the susceptible phenotype with more than 106 CFU/spleen after 4 weeks of infection (data not shown).
Bacterial replication in wild mice As shown in Table 1, wild mice showed a very rapid clearance of the BCG inoculum from the spleen. After 4 weeks of infection,
DISCUSSION
b
a
82 -
38-
35* -
1
2
3
4
Fig. 3. Comparison of Western blot patterns in wild mice (a) and inbred laboratory mice (b) in sera from mice infected twice with BCG with a 2month interval, and killed 4 weeks after the second injection. 1, BALB/c; 2, C57BL/6; 3, CBA/Ca; and 4, DBA/2. *The unique antigen with an estimated mol.wt of 35 kD.
Table 1. Enumeration of colony-forming units (CFU) BCG in the spleen from wild mice and two laboratory strains infected intravenously with M. bovis BCG
Weeks after BCG 2 4 6 8
Wild mice 5 37+0-62 (12) 4-17+0 52 (11) 3-69 + 0-82 (23) 3-57+049 (13)
Swiss A 5 46+0-32* 3-81 +0.44* 3.48 + 0.40* 3-43 +0.37*
C57BL/6
(5) (5) (5) (5)
5.69+0.10* (9) 5 25+0 09t (10) 4-72+0 30t (9) 3-87+0 1St (10)
Number of CFU/spleen expressed in Loglo units+s.d. (the number of animals is in parentheses). * Statistically not significantly different from mean values in wild mice (Student's f-test). t Statistically significantly different (P < 0-05) from mean values in wild mice (Student's I-test).
Protective immunity against mycobacteria is mediated by acquired populations of specifically sensitized T cells which activate macrophages by secretion of lymphokines (Kaufmann & Flesh, 1988). Antibodies do not confer protection (Reggiardo & Middlebrook, 1974), and passive transfer of anti-mycobacterial antibodies may even enhance the bacterial replication in the spleen of infected animals (Forget et al., 1976). Both humoral and cellular acquired immunity towards mycobacteria seem to be regulated to some extent by genes from the MHC. Thus, the intensity of the granulomatous response to M. lepraemurium is dependent on the H-2 haplotype (Closs et al., 1983), as is the amount of interferon-gamma (IFN-y) produced by spleen cells from BCG-sensitized mice challenged with a purified 32-kD protein antigen from M. bovis BCG (Huygen et al., 1988). Murine antibody responses following immunization with killed M. tuberculosis extract (Ivanyi & Sharp, 1986) or M. tuberculosis filtrate (Ljungqvist, Worsaae & Heron, 1988) are affected by H-2-linked genes. Finally, production of antibodies to filtrate or extract antigens following infection with live mycobacteria seems to be controlled to some extent by genes from the MHC (Huygen et al., 1990; Brett & Ivanyi, 1990). The MHC is characterized by its extreme polymophism. Studies on H-2 polymorphism in wild mouse populations from Europe and Africa have allowed estimates of allelic variants that ranged from 100 different alleles for class I K and D antigens to 20-50 alleles for class II H-21 antigens (Gotze et al., 1980; Klein, 1981). Our report that the wild mouse population in this study demonstrated an antibody repertoire to CF antibodies that was not essentially different from patterns of inbred laboratory strains was somewhat surprising. These results may therefore suggest that despite the high degree of polymorphism of such outbred populations, the number of immunodominant antigens is limited. Only one antigen, the 35-kD protein, was exclusively recognized by wild mouse sera and not by the sera from the four laboratory strains. It remains to be established whether the immunoreactivity to this antigen is really exclusive for wild mice, or associated an H-2 haplotype other than b, d or k that were tested here. Studies on mice with other H-2 haplotypes are in progress to elucidate that matter. The innate resistance of inbred strains of mice to M. bovis BCG is controlled by a single autosomal gene on chromosome 1 (Beg gene), which segregates for dominant resistant (Bcgr) and recessive susceptible (Bcgr) alleles (Skamene, 1982). Natural resistance to Salmonella typhimurium and Leishmania donovani are under control of respectively the Ity and Lsh gene, also located on chromosome 1 and probably identical to the Bcg gene (Skamene, 1986). Macrophages expressing the Bcgr allele
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demonstrate a higher intracellular bactericidal potential (Stach et al., 1984) and Ta expression than Bcgs macrophages (Zwilling, Vespa & Massie, 1987). Little is known about the innate susceptibility to intracellular parasites in wild mice. Screening of wild mice for susceptibility to S. typhimurium indicated that three out of four populations were resistant (O'Brien et al., 1986). From our results it may be concluded that the resistant allele is expressed in this population of wild mice also for the Bcg gene. As the BCgr allele is dominant over the recessive Bcgs allele, it is impossible to conclude for the moment whether wild mice carry the resistant allele in homozygous or in heterozygous form. Of 25 commonly used inbred strains, about one-half are known to segregate as resistant and one-half as susceptible strains (Skamene, 1986). This could be an indication that both alleles were indeed present in the wild mice populations from which they were originally derived. Homozygous Bcgs susceptible animals, however, seem to be very rare and in this study we could type only one mouse out of more than 100 as susceptible. Crossing of wild mice to inbred strains with known Bcg allele may help to answer this question. ACKNOWLEDGMENTS The authors are very grateful to M. Weckx for the generous gift of BCG and BCG culture filtrate. This work was supported in part by grant no. 3.4554.86 and 3.0020.89 of the FRSM-NFWO (Belgian National Research Council).
REFERENCES ABOU-ZEID, C., RATLIFF, T.L., WIKER, H.G., HARBOE, M., BENNEDSEN,
J. & ROOK, G.A.W. (1988) Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG. Infect. Immun. 56, 3046. BRETT, S.J. & IVANYI, J. (1990) Genetic influences on the immune repertoire following tuberculous infection in mice. Immunology, 71, 113. CLOSS, O., LovIK, M., WIGZELL, H. & TAYLOR, B.A. (1983) H-2 linked gene(s) influence the granulomatous reaction to viable Mycobacterium lepraemurium in the mouse. Scand. J. Immunol. 18, 59. DE BRUYN, J., HUYGEN, K., BOSMANS, R., FAUVILLE, M., LIPPENS, R., VAN VOOREN, J-P., FALMAGNE, P., WIKER, H.G., HARBOE, M. &
TURNEER, M. (1987) Purification, characterization and identification of a 32 kDa protein antigen of Mycobacterium bovis BCG. Microbiol. Pathogen. 2, 351. FALUS, A., WAKELAND, E.K., MCCONNELL, T.J., GITLIN, J., WHITEHEAD, A.S. & COLTEN, H.R. (1987) DNA polymorphism of MHC. III. Genes in inbred and wild mouse strains. Immunogenetics, 25, 290. FORGET, A., BENOIT, J.C., TURCOTTE, R. & GUSEW-CHARTRAND, N.
(1976) Enhancement activity of anti-mycobacterial sera in experimental M. bovis BCG infection in mice. Infect. Immun. 13, 1301. GOTZE, D., NADEAU, J., WAKELAND, EK., BERRY, R.J., BONHOMME, F.,
EGOROV, I., HJORTH, J.P., HOOGSTRAAL, H., VIVES, J., WINKING, H. & KLEIN, J. (1980) Histocompatibility-2 system in wild mice. X.
Frequencies of H-2 and Ia antigens in wild mice from Europe and Africa. J. Immunol. 124, 2675. HOBNER, R. & KOULISCHER, L. (1990) Cytogenetic studies on wild house mice from Belgium. Genetica, 80, 93. HUYGEN, K., LJUNGQVIST, L., TEN BERG, L. & VAN VOOREN, J-P. (1990) Repertoires of antibodies to culture filtrate antigens in different mouse strains infected with Mycobacterium bovis BCG. Infect. Immun. 58, 2192. HUYGEN, K., PALFLIET, K., JURION, F., HILGERS, J., TEN BERG, R., VAN VOOREN, J-P. & DE BRUYN, J. (1988) H-2 linked control of in vitro interferon-y production in response to a 32 kDa antigen (P32) of Mycobacterium bovis BCG. Infect. Immun. 56, 3196. IVANYI, J. & SHARP, K. (1986) Control by H-2 genes of murine antibody responses to protein antigens of Mycobacterium tuberculosis. Immunology, 59, 329. KAUFMANN, S.H.E. & FLESH, I. (1988) The role of T cell-macrophage interactions in tuberculosis. Springer Semin. Immunopathol. 10, 337. KLEIN, J. (1981) The Histocompatibility-2 (H-2) Complex. In The Mouse in Biomedical Research (ed. by H. L. Foster, J. D. Small & J.G. Fox) p. 119. Academic Press, New York. KLEIN, D., ZALESKA-RUTCZYNSKA, Z., DAVIS, W.C., FIGUEROA, F. & KLEIN, J. (1987) Monoclonal antibodies specific for mouse class I and class II MHC determinants. Immunogenetics, 25, 351. LJUNGQVIST, L., WORSAAE, A. & HERON, I. (1988) Antibody responses against Mycobacterium tuberculosis in 11 strains of inbred mice: novel monoclonal specificities generated by fusions, using spleens from BALB.B10 and CBA/J mice. Infect. Immun. 56, 1994. MORSE, H.C. III (1981) The laboratory mouse-a historical perspective. In The Mouse in Biomedical Research (ed. by H. L. Foster, J. D. Small & J. G. Fox) p. 7. Academic Press, New York. O'BRIEN, A.D., WEINSTEIN, D.L., D'HOOSTELAERE, L.A. & POTTER, M. (1986) Susceptibility of Mus musculus musculus (Czech I) mice to Salmonella typhimurium infection. Curr. Top. Microbiol. Immunol. 127, 309. REGGIARDO, Z. & MIDDLEBROOK, G. (1974) Failure of passive serum transfer of immunity against aerogeneic tuberculosis in guinea pigs. Proc. Soc. exp. Biol. Med. 145, 173. SKAMENE, E., GROS, P., FORGET, A., KONGSHAVN, P.A.L., ST CHARLES, C. & TAYLOR, B.A. (1982) Genetic regulation of resistance to intracellular pathogens. Nature, 297, 506. SKAMENE, E. (1986) Genetic regulation of resistance to mycobacterial infection. Curr. Top. Microbiol. Immunol. 124, 49. STACH J.L., GROS P., FORGET A. & SKAMENE E. (1984) Phenotypic expression of genetically controlled natural resistance to Mycobacterium bovis BCG. J. Immunol. 132, 888. THOLE, J.E.R., VAN SCHOOTEN, W.C.A., KEULEN, W.J., HERMANS, P.W.M., JANSON, A.A.M., DE VRIES, R.E.P., KOLK, A.M.J. & VAN EMBDEN, J.D.A. (1988) Use of recombinant antigens expressed in E. coli K- 12 to map B cell and T cell epitopes on the immunodominant 65 kDa protein of M. bovis BCG. Infect. Immun. 56, 1633. TOWBIN, H., STAEHELIN, T. & GORDON, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. natl Acad. Sci. USA, 76, 4350. ZWILLING, B., VESPA, L. & MASSIE, M. (1987) Regulation of I-A expression by murine peritoneal macrophages: differences linked to the Bcg gene. J. Immunol. 138, 1372.
Antibody repertoire against culture filtrate antigens in wild house mice infected with Mycobacterium bovis BCG K. HUYGEN, K. PALFLIET, F. JURION, C. LENOIR & J-P. VAN VOOREN* Instituut Pasteur Brussels, and *H6pital Erasme, Brussels, Belgium
van
Brabant,
(Acceptedfor publication 6 July 1990)
SUMMARY Wild house mice (Mus domesticus) captured in a Flemish pigsty were infected intravenously with 4 x 106 viable units of Mycobacterium bovis BCG and examined by Western blot analysis for IgG secretion against BCG culture filtrate (CF) antigens. Wild mice showed a marked individual variation in antibody pattern when tested 4,6 and 8 weeks after infection. Some animals reacted to a wide range of antigens and others only to a limited number. Most wild mice recognized preferentially antigens with molecular weight of 24 kD, 32 kD, 37-38-40 kD, 65 kD and 82 kD, i.e. the major CF antigens known to be recognized by sera from BCG-infected inbred laboratory strains, BALB/c, DBA/2, CBA/Ca and C57BL/6. The 32-kD fibronectin-binding protein and the 65-kD heat-shock protein appeared as very immunodominant in wild mice. Furthermore, about 20-25% of the mice reacted strongly with a unique antigen of 35 kD estimated molecular weight, to which the tested inbred laboratory mice did not respond. Monitoring the size of the bacterial population in the spleen indicated that the BCG inoculum did not replicate in wild mice, suggesting that the Bcgr allele is expressed in this population.
Keywords wild mice BCG filtrate antibody repertoire
INTRODUCTION
Following i.v. infection with live Mycobacterium bovis BCG, inbred laboratory mice produce substantial amounts of IgG antibodies against BCG culture filtrate (CF) antigens and the repertoire of these antibodies is dependent on the mouse strain (Huygen et al., 1990). Thus, BALB/c mice produce antibodies against a wide range of CF antigens, with a preferential recognition of the 65-kD heat-shock protein (Thole et al., 1988) and of the 32-kD fibronectin-binding protein (De Bruyn et al., 1987; Abou-Zeid et al., 1988), whereas C57BL/6 mice sera show a more restricted antibody pattern, almost exclusively directed against three antigens with mol. wts of 37, 38 and 40 kD (Huygen et al., 1990). The preferential recognition of certain CF antigens seems to be controlled by a gene (or genes) in the K-IA region of the MHC. A similar genetic variation has also been found in mice infected intraperitoneally with M. tuberculosis and tested for antibodies against soluble extract antigens (Brett & Ivanyi, 1990). Although these findings may eventually be relevant for the development of an efficient serodiagnostic test for mycobacterial infections, extrapolation of results in inbred laboratory strains to the human outbred population must be made with Correspondence: Kris Huygen, Department of Virology, Instituut Pasteur van Brabant, 642 Engelandstraat, 1180 Brussels, Belgium.
369
much caution. In this respect, the use of wild house mice (Mus domesticus) from which all laboratory mouse strains have been derived (Morse, 1981), may allow a more accurate estimate of genetic diversity than can be obtained by examination of laboratory strains (Falus et al., 1987). We have analysed the repertoire of antibodies against BCG CF antigens in a population of wild house mice infected intravenously with BCG. We conclude that, with the exception of a unique 35-kD CF antigen, all the immunodominant CF antigens for laboratory mouse strains are also immunodominant in wild mice. Furthermore, monitoring of BCG replication in the spleen is suggestive of the expression of the resistant allele of the Bcg gene that controls innate resistance to M. bovis BCG (Skamene et al., 1982) by affecting early mycobacterial replication in the macrophage (Stach et al., 1984).
MATERIALS AND METHODS Mice Wild mice, identified as Mus domesticus Schwarz & Schwarz 1943, or West-European long-tailed house mouse, were captured alive in a pigsty in Tienen, near Brussels, Belgium. Karyotype analysis has revealed a Robertsonian translocation Rb4.12 in this population (Hubner & Koulischer, 1990). BALB/c, DBA/2, CBA/Ca, C57BL/6 and Swiss A mice were bred in the animal facilities of the Pasteur Institute van Brabant.
370
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BCG infection Mice were inoculated intravenously (wild mice under mild ether anaesthesia) with 0 5 mg (±4 x 106 colony-forming units (CFU)) of M. bovis BCG strain GL2 (grown for 2 weeks as a surface pellicle in Sauton medium and prepared freshly every week at the Pasteur Institute). Mice were anaesthesized with ether and exsanguinated after various time intervals. Sera from wild mice were collected individually, sera from inbred laboratory mice were collected as pools from at least four animals/ group. Sera were kept frozen at -20'C until assay. For the boost injection, a second i.v. inoculation (0 5 mg) was given 2 months after the initial one, and mice were bled 4 weeks later. Antigens CF antigens were obtained from 14-day-old cultures of M. bovis BCG GL2 (the same as the one used for infection). Proteins were concentrated by precipitation with ammonium sulphate (80% saturation). After centrifugation, the pellet was dissolved in and dialysed against phosphate-buffered saline (PBS). The filtrate was passed through a 0-22-pm sterilizing membrane (Flow) and stored at -20'C until use. The same batch of CF was used in all experiments. SDS-PA GE and Western blot analysis SDS-PAGE of BCG culture filtrate was performed in reducing conditions on 12-5 % acrylamide gels. After completion of SDSPAGE, the proteins were electrophorectically transferred to nitrocellulose sheets (Towbin, Staehelin & Gordon, 1979) and sera from BCG-infected mice were used for the Western blot analysis as described elsewhere (Huygen et al., 1990). Briefly, nitrocellulose was incubated with bovine serum albumin to prevent non-specific absorption of proteins. Strips of nitrocellulose (containing ± 30 pg of CF) were then incubated with mouse sera (1: 50) overnight and revealed with peroxidase-conjugated rabbit anti-mouse IgG and oe-chloronaphthol in the presence of
hydrogen peroxide. Enumeration of BCG CFU in the spleen of infected mice Following ether anaesthesia and exsanguination, spleens were removed aseptically and spleen cells isolated using a loosely fitting Dounce homogenizer. Serial three-fold dilutions were made in PBS without antibiotics and 200-pl volumes were plated on Middlebrook 7HI 1 Bacto agar (Difco). Each dilution was tested in triplicate. Petri dishes were incubated in sealed plastic bags at 37°C and the number of colonies was counted after 18 days of culture.
RESULTS Repertoire of antibodies against CF antigens in wild mice infected with M. bovis BCG As shown in Fig. 1, a marked individual variation was observed in Western blot (immunoblot) patterns of anti-CF IgG antibodies in sera from wild mice infected with BCG. Some sera reacted with numerous antigens, other sera only with one or a few antigens. Most sera contained antibodies against the 65-kD mycobacterial heat-shock protein (Thole et al., 1988) and against the 32-kD fibronectin-binding protein (De Bruyn et al., 1987; Abou-Zeid et al., 1988). Furthermore, there was a marked variation in immunodominance of the CF antigens, and depending on the individual serum tested, the most intense
staining could be observed with antigens of respective mol. wts of 65, 35, 32 and 24 kD. Comparison of the repertoire in wild mice with the repertoire in inbred laboratory strains Figure 2 shows the immunoblot pattern ofanti-CF antibodies in sera from three wild mice, compared with sera from four laboratory strains. As described elsewhere (Huygen et al., 1990), laboratory strains showed a marked variation in anti-CF antibody repertoire. BALB/c sera reacted with a wide range of CF antigens and preferentially with the 65-kD and 32-kD antigens. C57BL/6 antibodies were directed against a 40-kD antigen and an 82-kD antigen. CBA/Ca sera reacted very weakly with the 32-kD and 65-kD antigens, and DBA/2 sera reacted very weakly with the 65-kD antigen only. All these antigens were also stained by sera from wild mice. The 35-kD antigen was not recognized by sera from C57BL/6, CBA/Ca or DBA/2 mice. BALB/c sera reacted faintly with an antigen of more or less the same mol. wt, but the intense staining so typical for wild mice was never observed. b
a
826538-
35*3224-
Fig. 1. Western blot analysis against a 14-day-old BCG culture filtrate of sera from wild mice infected intravenously with BCG 4 weeks (a), 6 weeks (b) or 8 weeks (c) previously.
b -82
65-
-40
35* 32
-
2 3 4 Figure 2. Comparison of Western blot patterns in sera from three wild mice (a) and four inbred laboratory strains (b) infected with BCG 4 weeks previously. 1, BALB/c; 2, C57BL/6; 3, CBA/Ca; and 4, DBA/2. *The unique antigen with an estimated mol. wt of 35 kD.
Anti-CF antibodies in wild mice infected with BCG
371
Effect of a boost injection of BCG Mice were infected with BCG, boosted 2 months later and examined 4 weeks after the second infection (Fig. 3). In wild mice, Western blot patterns became more pronounced, but sera reacted essentially with the same antigens as during a primary infection, i.e. 32, 35, 65 and 82 kD. Response to the 37-38-40kD antigens (dominant in C57BL/6 mice) was more pronounced in wild mice and in CBA/Ca mice after a second infection with BCG than after a primary infection. The 35-kD antigen was again found to be very immunoreactive in wild mice orly (two out of eight sera, 25%).
the number of CFU had decreased about 100-fold (initial number of CFU/spleen at day 1 of infection: + 106 CFU; data not shown). In C57BL/6 mice, known to carry the susceptible Bcgs allele, the reduction in number of CFU was not even 10fold. Outbred Swiss albino mice showed a decrease similar to wild mice. Additional experiments showed that of 108 wild mice tested, only one showed the susceptible phenotype with more than 106 CFU/spleen after 4 weeks of infection (data not shown).
Bacterial replication in wild mice As shown in Table 1, wild mice showed a very rapid clearance of the BCG inoculum from the spleen. After 4 weeks of infection,
DISCUSSION
b
a
82 -
38-
35* -
1
2
3
4
Fig. 3. Comparison of Western blot patterns in wild mice (a) and inbred laboratory mice (b) in sera from mice infected twice with BCG with a 2month interval, and killed 4 weeks after the second injection. 1, BALB/c; 2, C57BL/6; 3, CBA/Ca; and 4, DBA/2. *The unique antigen with an estimated mol.wt of 35 kD.
Table 1. Enumeration of colony-forming units (CFU) BCG in the spleen from wild mice and two laboratory strains infected intravenously with M. bovis BCG
Weeks after BCG 2 4 6 8
Wild mice 5 37+0-62 (12) 4-17+0 52 (11) 3-69 + 0-82 (23) 3-57+049 (13)
Swiss A 5 46+0-32* 3-81 +0.44* 3.48 + 0.40* 3-43 +0.37*
C57BL/6
(5) (5) (5) (5)
5.69+0.10* (9) 5 25+0 09t (10) 4-72+0 30t (9) 3-87+0 1St (10)
Number of CFU/spleen expressed in Loglo units+s.d. (the number of animals is in parentheses). * Statistically not significantly different from mean values in wild mice (Student's f-test). t Statistically significantly different (P < 0-05) from mean values in wild mice (Student's I-test).
Protective immunity against mycobacteria is mediated by acquired populations of specifically sensitized T cells which activate macrophages by secretion of lymphokines (Kaufmann & Flesh, 1988). Antibodies do not confer protection (Reggiardo & Middlebrook, 1974), and passive transfer of anti-mycobacterial antibodies may even enhance the bacterial replication in the spleen of infected animals (Forget et al., 1976). Both humoral and cellular acquired immunity towards mycobacteria seem to be regulated to some extent by genes from the MHC. Thus, the intensity of the granulomatous response to M. lepraemurium is dependent on the H-2 haplotype (Closs et al., 1983), as is the amount of interferon-gamma (IFN-y) produced by spleen cells from BCG-sensitized mice challenged with a purified 32-kD protein antigen from M. bovis BCG (Huygen et al., 1988). Murine antibody responses following immunization with killed M. tuberculosis extract (Ivanyi & Sharp, 1986) or M. tuberculosis filtrate (Ljungqvist, Worsaae & Heron, 1988) are affected by H-2-linked genes. Finally, production of antibodies to filtrate or extract antigens following infection with live mycobacteria seems to be controlled to some extent by genes from the MHC (Huygen et al., 1990; Brett & Ivanyi, 1990). The MHC is characterized by its extreme polymophism. Studies on H-2 polymorphism in wild mouse populations from Europe and Africa have allowed estimates of allelic variants that ranged from 100 different alleles for class I K and D antigens to 20-50 alleles for class II H-21 antigens (Gotze et al., 1980; Klein, 1981). Our report that the wild mouse population in this study demonstrated an antibody repertoire to CF antibodies that was not essentially different from patterns of inbred laboratory strains was somewhat surprising. These results may therefore suggest that despite the high degree of polymorphism of such outbred populations, the number of immunodominant antigens is limited. Only one antigen, the 35-kD protein, was exclusively recognized by wild mouse sera and not by the sera from the four laboratory strains. It remains to be established whether the immunoreactivity to this antigen is really exclusive for wild mice, or associated an H-2 haplotype other than b, d or k that were tested here. Studies on mice with other H-2 haplotypes are in progress to elucidate that matter. The innate resistance of inbred strains of mice to M. bovis BCG is controlled by a single autosomal gene on chromosome 1 (Beg gene), which segregates for dominant resistant (Bcgr) and recessive susceptible (Bcgr) alleles (Skamene, 1982). Natural resistance to Salmonella typhimurium and Leishmania donovani are under control of respectively the Ity and Lsh gene, also located on chromosome 1 and probably identical to the Bcg gene (Skamene, 1986). Macrophages expressing the Bcgr allele
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demonstrate a higher intracellular bactericidal potential (Stach et al., 1984) and Ta expression than Bcgs macrophages (Zwilling, Vespa & Massie, 1987). Little is known about the innate susceptibility to intracellular parasites in wild mice. Screening of wild mice for susceptibility to S. typhimurium indicated that three out of four populations were resistant (O'Brien et al., 1986). From our results it may be concluded that the resistant allele is expressed in this population of wild mice also for the Bcg gene. As the BCgr allele is dominant over the recessive Bcgs allele, it is impossible to conclude for the moment whether wild mice carry the resistant allele in homozygous or in heterozygous form. Of 25 commonly used inbred strains, about one-half are known to segregate as resistant and one-half as susceptible strains (Skamene, 1986). This could be an indication that both alleles were indeed present in the wild mice populations from which they were originally derived. Homozygous Bcgs susceptible animals, however, seem to be very rare and in this study we could type only one mouse out of more than 100 as susceptible. Crossing of wild mice to inbred strains with known Bcg allele may help to answer this question. ACKNOWLEDGMENTS The authors are very grateful to M. Weckx for the generous gift of BCG and BCG culture filtrate. This work was supported in part by grant no. 3.4554.86 and 3.0020.89 of the FRSM-NFWO (Belgian National Research Council).
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