Parasite Immunology 1979 1 , 3 0 5 3 1 6
Phagosome/lysosome fusion: a possible prerequisite for the enhancement of antibody responses in vitro by BCG, Mycobacterium Ieprae and Corynebacterium parvum CA RO L Y N A.BROWN, I.N.BROWN* & V.S.3LJIVIC Departments of Immunology and Bacteriology*, St. Mary's Hospital Medical School, London, W2 I PG Accepted for publication 12 June 1979 Summary. Primary in vitro antibody responses to SRBC were suppressed in cultures prepared from the spleens of CBA mice injected i.v. 2 lays previously with lo8 live BCG. In contrast, cultures prepared from mice ir ted with dead BCG showed enhanced responses. I n vitro spleen cell responses the mice had returned to normal levels 4-6 weeks after their injection, but if ad BCG, M . leprae or C . parvum was added to the cultures, resy----- --,--- lanced. The enhancing effect of the added bacteria could be removtu "I auu1116m r a suramin, ~ a drug known to inhibit in vitro fusion of lysosomes with phagosomes. It is suggested that the different in vivo effects of live and dead BCG may relate to differences in their handling by macrophages and more especially that the enhanced antibody forming cell response seen in the restimulated cultures of spleen cells from BCG primed mice, depends upon efficient intracellular fusion of lysosomes with the phagosomes containing the added dead bacteria.
Key words: adjuvants, BCG, leprosy, Corynebacterium parvum, phagosome/ lysosome fusion, antibody response, suramin
Introduction The organelles of mouse peritoneal macrophages cultured in vitro respond differently to dead than to live tubercle bacilli. After ingestion, most organisms, including tubercle bacilli killed by irradiation, are enclosed in phagosomes which then become fused with surrounding lysosomes to form phagolysosomes. This phagosome/lysosome fusion is, however, infrequent with viable virulent tubercle strains such as Mycobacterium microti (Hart et al. 1972) and M . tuberculosis H37Rv and, at least in the early stages of infection of cultured mouse peritoneal macrophages, with the avirulent M . bouis, BCG (Armstrong & Hart 1971). The normal fusion of lysosomes with phagosomes containing live baker's yeast, Correspondence: Dr Carolyn A. Brown, Department of Immunology, St. Mary's Hospital Medical School, Paddington, London, W2 I PG. 0141-9838/79/1200-0309%02.00
0 1979 Blackwell Scientific Publications
309
310
Carolyn A . Brown, I.N. Brown & V.S. SljiviC
Saccharomyces cerevisiae, in cultured macrophages can be inhibited (Hart & Young 1975) or otherwise altered (Pesanti 1978) if the macrophages are first incubated with suramin, a trypanocidal drug known to accumulate in lysosomes and capable of reducing breakdown of endocytosed material (Davis, Lloyd & Beck 1971). The ability of suramin treated mice to become resistant to Listeria monocytogenes challenge after immunization with dead rather than the live L. monocytogenes has been attributed to prevention of degradation of the immunizing dose of bacteria by the drug (Van der Meer, Hofhuis & Willers 1977). BCG and Corynebacterium parvum are among the most potent of adjuvants. There is growing evidence that many agents capable of stimulating antibody responses do so through their effect on macrophages (Allison 1973, sljivik & Watson 1977). It is shown here that the modulating effect of two species of mycobacteria and C. parvum on antibody responses of mouse spleen cells in vitro is related to conditions known to affect phagosome/lysosome fusion. Materials and methods BACTERIA
The Glaxo strain of BCG was used within 6 h of harvest from a 14 day bulk culture in glycerol-free medium except in the case of Experiment A in Table 1 and that shown in Figure 2, when the bacteria were obtained from a 14-day laboratory culture in Middlebrook 7H9 medium (Difco) containing Tween 80. The numbers of bacilli were estimated by opacity and by viable counts on Middlebrook 7H 10 medium (Difco). BCG organisms were killed by heating to 70°C for 1 h in a water bath. The M . leprue suspension, 1O'O bacilli/ml, was a gift from Dr R.J.W.Rees, National Institute for Medical Research, London. The bacilli had been harvested from infected armadillo tissue, freed from tissue debris and exposed to 2.5 Mrads (25 kGy) 6oCoy-irradiation. The C. parvum suspension, 7 mg dry weight/ml, was a gift from Dr C.Adlam, Wellcome Laboratories, Beckenham, Kent (C. parvum CN6 134, heat killed, no preservative, batch PX383). MICE
Female CBA mice, aged 3-6 months, were used. They were obtained from the breeding colony at St. Mary's Hospital Medical School. Mice were injected i.v. with lo8 viable or killed BCG organisms. SPLEEN CELL CULTURES
Spleen cell suspensions were cultured for 4 days in modified Marbrook chambers
Adjuvants and phagosomellysosome fusion
31 1
(Marbrook 1967). The primary antibody response was assessed in terms of the numbers of cells producing antibody in vitro to sheep erythrocytes (SRBC) and, in some experiments, to dinitrophenylated Ficoll (DNP-Ficoll), using the method of Cunningham & Szenberg (1 968). Full details of the basic method used have been described elsewhere (SljiviC & Watson 1977). Briefly, 2 x lo7 spleen cells were cultured in 1 ml RPMI 1640 medium containing 5% foetal calf serum and 10 pg/ml gentamicin. Cultures were maintained at 37°C in humidified air containing 5% COz.SRBC (2x lo6) or DNP-Ficoll(20 ng) were added to the cultures before incubation and the number of direct antibody-forming cells was determined 4 days later using untreated or trinitrophenylated-SRBC (Rittenberg & Pratt 1969) respectively. Previous treatment of the spleen-cell donors and details of the various additions of bacteria and suramin are described in the Results section. SURAMIN
This was a gift from Dr J.Williamson, National Institute for Medical Research, London (Antrypol, ICI).The drug was dissolved in distilled water, sterilized by filtration and stored at 4°C. Before addition to both the inner tube containing the cells and the outer chamber of Marbrook vessels suitable dilutions were made in RPMI 1640 without added serum.
Results B C G A N D IN VITRO A N T I B O D Y RESPONSES
The effect of i.v. administration of BCG on the subsequent in vitro antibody response of mouse spleen cells was dependent on whether live or dead bacilli were given and the time after BCG injection. Between 2 and 4 weeks after injection, live BCG caused suppression whereas dead BCG caused enhancement of anti-SRBC responses, as illustrated by a typical experiment shown in Figure 1. Mice were injected with lo8 dead or lo8 live organisms and 20 days later their spleen cells were cultured in the presence of SRBC and the responses obtained compared with the response of cells from untreated animals. Full details of this work will be presented elsewhere (Brown, Brown & SljiviC 1979). A D D I T I O N OF K I L L E D B C G M.LEPRAEORC. P A R Y U M TO C U L T U R E S
The number of direct antibody-forming cells (AFC) to SRBC detectable in spleen cell cultures prepared from mice treated with live or dead BCG returned to normal levels after 4-6 weeks (results not shown). If however, killed BCG, M . leprae or C. parvum was added to cultures of spleen cells from live BCG-primed
312
Carolyn A . Brown, I.N. Brown & V.S. SljiviC
-
18
-
16
-
14
-
12
-
m
0 3 10-
e
-u' c
\
0 LL
8 -
a
6 -
4-
2-
0
Dead
Live
None
Figure 1. Numbers of antibody-forming cells against SRBC produced in uifro by spleen cells from control mice or mice pretreated with live or dead BCG 20 days before. BCG was harvested from Glaxo glycerol free medium and part was killed by heating at 70°C for 1 h. Cells from two spleens in each group were pooled and the results show mean & s.e.m. AFC/culture for quadruplicate cultures. Pvalues in a two-tailed Student's test were for dead us untreated P < 0.001 and for live us untreated P < 0.025.
mice at this time, in vitro responses to SRBC were markedly enhanced (Table 1, Brown, Brown & Sljivik 1978). THE EFFECT OF S U R A M I N
The different systemic effects of dead and live BCG described above could be connected with the observation (Armstrong & Hart 1971) that viable BCG organisms somehow resist fusion of their enclosing phagosomes with surrounding lysosomes. To look further at this phenomenon and to examine the possibility that fusion is a prerequisite for the enhancement we have described, experiments were undertaken with the lysosornotropic drug, suramin. The highest dose of suramin chosen was the maximum tolerated in the in uitro macrophage culture system described by Hart & Young (1975), and produced a clear suppression of the normal phagosome/lysosome fusion response to yeast cells within macro-
Adjuvants andphagosome/lysosomefusion
3 13
Table 1. Effect of suramin on antibody responses in vitro and their enhancement induced by mycobacteria and C. parvum
Suramin concentration (pglrnl) Challenging Experiment antigen SRBC SRBC
Bacteria added None BCG
0
40
4400f201 11550f1664
5730k524 8655f1061
1747f268* 1740+291*
6725 f724 6585f539
5030 t_ 508 5150t_339
2007 f 157* 1593+231*
2553f273* 6235 f.347
14352306 3460 f.863
1540f305 1220+ 42
200
A
DNP-Ficoll None DNP-Ficoll BCG SRBC SRBC
None M. leprae
B DNP-Ficoll None DNP-Ficoll M. leprae C
SRBC SRBC
None C. poruum
16795f 1433 17795k2035 4275 f425 9505 1241
12850f800 1146Ok 180 17587+2296* 11710+448* n.d. n.d.
2070 f 393 1325& 424
All values are means fs.e.m. of antibody-forming cell numbers per culture for 3* or 4 cultures in each group. Spleen cells were obtained from mice which had been injected i.v. with 1081iveBCG organisms 7-28 weeks previously. Heat killed BCG or irradiated M. leprae were added at a concentration of lo8organisms per culture. C. paruwn was added at a concentration of 70pg/culture. n.d. =not done.
phages. Two types of experiment were done; the cells were either exposed to suramin before culture or cultured in the presence of suramin. Figure 2 shows an . example of the first type. BCG-primed spleen cells were pretreated with suramin (200 pg/ml in RPMI for 2 h) before cultural exposure to killed BCG and SRBC. Under these conditions, suramin treatment alone had little effect on the AFC response but suppressed significantly (P< 0.01) the enhancement induced by adding BCG. An example of the second type of experiment is shown in Table 1, where the effect of suramin at various concentrations in the culture was determined using spleen cells from BCG-primed mice with added BCG, M . leprae or C .parvum. All three additions caused an enhancement of the anti-SRBC response of over two fold, but the same concentrations of M . leprae or BCG had no effect on the response of the same cell population to DNP-Ficoll. The presence of suramin at 200 pg/ml in the cultures removed the enhancing effect and reduced the number of AFC produced. In the absence of added bacteria, AFC were reduced to approximately 50% the normal value. However, in the presence of added bacteria the reduction in AFC numbers resulting from suramin exposure was even greater (down to 12-20% of the normal value).
314
Carolyn A . Brown, i . N . Brown & V.S. SljiviC 22
2c ia
16
-
14
n
P
3 e
12
4
10
-
-a
u
LL
a
8
6 4 2
0
ICG ~
s+ Figure 2. Effect of pretreatment with suramin(s) on the adjuvant effect of BCG in uitro. The captions refer to additions to spleen cells from mice injected with live BCG, as in Table I , 8 weeks previously. None
S
Discussion These experiments show that moderately high doses of live BCG injected systemically are immunosuppressive whereas the same number of dead organisms enhance the antibody response in vitro to SRBC. Spleen cells taken from mice 2 months or more after priming with live BCG gave normal in uitro primary antibody responses but the addition of killed BCG, M . leprae or C.paruum to the cultures enhanced the AFC response. Suramin prevented the enhancing effect of BCG if used to pretreat the spleen cells to be cultured. If added to the cultures at the same time as the SRBC it had two effects; it reduced the number of AFC formed in the absence of bacteria and abolished the enhancing effect observed in the presence of bacteria. Polyclonal activation of B-lymphocytes in uitro by BCG (Sultzer 1978), or by the other two organisms, is an unlikely explanation of the enhanced AFC numbers reported here since there was selective enhancement of in uitro antiSRBC but not anti-DNP-Ficoll responses. Moreover, since the in uitro antibody
Adjuvants andphagosome/lysosomefusion
3 15
response to SRBC is T-lymphocyte and macrophage-dependent, whereas the response to DNP-Ficoll is not, the enhanced anti-SRBC responses must have been related somehow to an effect of the added bacteria on either or both of these cell types. Suramin binds readily to proteins, concentrates intralysosomally, interferes with phagosome/lysosome fusion and inhibits various proteases (Pesanti 1978, Williamson 1976). In view of its known.ability to reduce the breakdown of phagocytosed material, it is tempting to speculate that in the experiments described above, the drug was preventing some degree of degradation or ‘processing’ of the added bacteria within macrophages necessary for their adjuvant effect to be expressed. The reduction of AFC numbers in the SRBC stimulated cultures without added bacteria supports this idea because phagocytically active macrophages are a critical requirement for a primary response to SRBC in the culture system used. Another possibility is that the suramin interfered with the release from macrophages of soluble factors which are known to influence lymphocyte responses (Unanue 1978) and whose release may be enhanced after phagosomel lysosome fusion. Certain lysosome-labilizing agents are known to act as adjuvants (Spitznagel & Allison 1970) and a similar process may occur after the addition of the bacteria used in these experiments. This stimulus of lysosomelabilization may be prevented in vitro by suramin. and in vitro by sulphatides (Goren et al. 1976) or cyclic AMP (Lowrie, Jackett & Ratcliffe 1975) both of which are produced by viable tubercle bacilli and prevent phagosome/lysosome fusion. M . leprae has been shown to have adjuvant activity in vivo although it is much less active than M . tuberculosis (Stewart-Tull & Davies 1972). Table 1 shows that it will also enhance in vitrp antibody responses of mouse spleen cells and, moreover, that M . leprae is as good as BCG at stimulating responses of cells from BCG-primed mice. This latter observation may be due to direct stimulation of sensitized T-cells and thus reflect antigenic overlap between the two organisms or to direct stimulation of macrophages or both. In view of the results presented above and the known properties of suramin, we think that direct stimulation of macrophages represents the major contribution to the described adjuvant effect. If this is so, then recent reports of BCG-induced protection against leprosy in mice (Shepard, Walker & Van Landingham 1978) and BCG and killed M . leprae stimulation of a local non-specific immunity against Listeria monocytogenes in the mouse foot pad (Pate1 & Lefford 1978) could also be interpreted in this way.
Acknowledgements This work was supported by a grant from the Cancer Research Campaign. We thank Gwen Dewey for technical assistance and Glaxo Research Laboratories Limited, Greenford, for the BCG.
3 16
Carolyn A . Brown, I.N. Brown & V.S. SljiviC
References ALLISONA.C. (1973) Effects of adjuvants on different cell types and their interactions in immune responses. In Immunopotentiation, Ciba Foundation Symposium 18 p. 73. Associated Scientific Publishers, Amsterdam ARMSTRONG J.A. & HARTP.D'A. (1971) Response of cultured macrophages to Mycobacterim tuberculosis, with observations on fusion of lysosomes with phagosomes. Journal of Experimental Medicine 134,713 BROWNC.A., BROWNI.N. & SLJIVIC V.S.(1978) Enhancement of the antibody response in vitro by BCG. Developmental Biology Standard 38, 153. BROWNC.A., BROWNI.N. & SLJIVI~ V.S. (1979) Suppressed or enhanced antibody responses in vitro after BCG treatment of mice: importance of BCG viability. Immunology 38 (in press) A. (1968) Further improvements in the plaque technique for CUNMNGHAM A.J. & SZENBERG detecting single antibody forming cells. Immunology 14, 599 DAVISM., LLOYD J.B. & BECKF. (1971) The effect of trypan blue, suramin and aurothiomalate on the breakdown of '251-labelledalbumin within rat liver lysosomes. Biochemical Journal 121,21 GORENM.B., HARTP.D'A., YOUNGM.R. & ARMSTRONG J.A. (1976) Prevention of phagosomelysosome fusion in cultured macrophages by sulphatides of Mycobacterium tuberculosis. Proceedings of National Academy of Sciences 73,2510 HARTP.D'A., ARMSTRONG J.A., BROWNC.A. & DRAPERP. (1972) Ultrastructural study of the behavior of macrophages toward parasitic mycobacteria. Infection and Immunity 5,803 HARTP.D'A. & YOUNGM.R. (1975) Interference with normal phagosome-lysosome fusion in macrophages, using ingested yeast cells and suramin. Nature 256,47 LOWRIED.B., JACKETT P.S. & RKTCLIFFE N.A. (1975) Mycobacterium microti may protect itself from intracellular destruction by releasing cyclic AMP into phagosomes. Nature 254,600 MARBROOK J. (1967) Primary immune response in cultures of spleen cells. Lancet 6, 1279 PATELP.J. & LEFFORDM.J. (1978) Specific and nonspecific resistance in mice immunized with irradiated Mycobacterium leprue. Infection and Immunity 20,692 PESANTIE.L. (1978) Suramin effects on macrophage phagolysosome formation and antimicrobial activity. Infection and Immunity 20, 503 RITTENBERG M.B. & PRATTK.L. (1969) Antitrinitrophenyl (TNP) plaque assay. Primary response of BALB/c mice to soluble and particulate immunogen. Proceedings of the Society for Experimental Biology and Medicine 132, 575 SHEPARD C.C., WALKER L.L. &VANLANDINGHAM R.M. (1978) Immunity to Mycobacterium leprae infections induced in mice by BCG vaccination at different times before and after challenge. Infection and Immunity 19,391 SLJIVI~ V.S. & WATSONS.R. (1977) The adjuvant effect of Corynebacterium parvum: T-cell dependence of macrophage activation. Journal of Experimental Medicine 145,45 SPITZNAGEL J.K. & ALLISONA.C. (1970) Mode of action of adjuvants: retinol and other lysosomelabilizing agents as adjuvants. Journal of Immunology 104, 1 19 STEWART-TULL D.E.S. & DAVIES M. (1972) Adjuvant activity of Mycobacterium leprae. Infection and Immunity 6,909. SULTZER B.M. (1978) Infection with bacillus Calmette-Guerin activates murine thymus independent (B) lymphocytes. Journal of Immunology 120,254 UNANUE E.R. (1978) The regulation of lymphocyte functions by the macrophage. Immunological Reviews 40,227 VANDER MEERC., HOFHUISF.M.A. & WILLERSJ.M.N. (1977) Killed Listeria monocytogenes vaccine becomes protective on addition of polyanions. Nature 269,594 WILLIAMSON J. (1976) Chemotherapy of African trypanosomiasis. Tropical Diseases Bulletin 73,53 1
Phagosome/lysosome fusion: a possible prerequisite for the enhancement of antibody responses in vitro by BCG, Mycobacterium Ieprae and Corynebacterium parvum CA RO L Y N A.BROWN, I.N.BROWN* & V.S.3LJIVIC Departments of Immunology and Bacteriology*, St. Mary's Hospital Medical School, London, W2 I PG Accepted for publication 12 June 1979 Summary. Primary in vitro antibody responses to SRBC were suppressed in cultures prepared from the spleens of CBA mice injected i.v. 2 lays previously with lo8 live BCG. In contrast, cultures prepared from mice ir ted with dead BCG showed enhanced responses. I n vitro spleen cell responses the mice had returned to normal levels 4-6 weeks after their injection, but if ad BCG, M . leprae or C . parvum was added to the cultures, resy----- --,--- lanced. The enhancing effect of the added bacteria could be removtu "I auu1116m r a suramin, ~ a drug known to inhibit in vitro fusion of lysosomes with phagosomes. It is suggested that the different in vivo effects of live and dead BCG may relate to differences in their handling by macrophages and more especially that the enhanced antibody forming cell response seen in the restimulated cultures of spleen cells from BCG primed mice, depends upon efficient intracellular fusion of lysosomes with the phagosomes containing the added dead bacteria.
Key words: adjuvants, BCG, leprosy, Corynebacterium parvum, phagosome/ lysosome fusion, antibody response, suramin
Introduction The organelles of mouse peritoneal macrophages cultured in vitro respond differently to dead than to live tubercle bacilli. After ingestion, most organisms, including tubercle bacilli killed by irradiation, are enclosed in phagosomes which then become fused with surrounding lysosomes to form phagolysosomes. This phagosome/lysosome fusion is, however, infrequent with viable virulent tubercle strains such as Mycobacterium microti (Hart et al. 1972) and M . tuberculosis H37Rv and, at least in the early stages of infection of cultured mouse peritoneal macrophages, with the avirulent M . bouis, BCG (Armstrong & Hart 1971). The normal fusion of lysosomes with phagosomes containing live baker's yeast, Correspondence: Dr Carolyn A. Brown, Department of Immunology, St. Mary's Hospital Medical School, Paddington, London, W2 I PG. 0141-9838/79/1200-0309%02.00
0 1979 Blackwell Scientific Publications
309
310
Carolyn A . Brown, I.N. Brown & V.S. SljiviC
Saccharomyces cerevisiae, in cultured macrophages can be inhibited (Hart & Young 1975) or otherwise altered (Pesanti 1978) if the macrophages are first incubated with suramin, a trypanocidal drug known to accumulate in lysosomes and capable of reducing breakdown of endocytosed material (Davis, Lloyd & Beck 1971). The ability of suramin treated mice to become resistant to Listeria monocytogenes challenge after immunization with dead rather than the live L. monocytogenes has been attributed to prevention of degradation of the immunizing dose of bacteria by the drug (Van der Meer, Hofhuis & Willers 1977). BCG and Corynebacterium parvum are among the most potent of adjuvants. There is growing evidence that many agents capable of stimulating antibody responses do so through their effect on macrophages (Allison 1973, sljivik & Watson 1977). It is shown here that the modulating effect of two species of mycobacteria and C. parvum on antibody responses of mouse spleen cells in vitro is related to conditions known to affect phagosome/lysosome fusion. Materials and methods BACTERIA
The Glaxo strain of BCG was used within 6 h of harvest from a 14 day bulk culture in glycerol-free medium except in the case of Experiment A in Table 1 and that shown in Figure 2, when the bacteria were obtained from a 14-day laboratory culture in Middlebrook 7H9 medium (Difco) containing Tween 80. The numbers of bacilli were estimated by opacity and by viable counts on Middlebrook 7H 10 medium (Difco). BCG organisms were killed by heating to 70°C for 1 h in a water bath. The M . leprue suspension, 1O'O bacilli/ml, was a gift from Dr R.J.W.Rees, National Institute for Medical Research, London. The bacilli had been harvested from infected armadillo tissue, freed from tissue debris and exposed to 2.5 Mrads (25 kGy) 6oCoy-irradiation. The C. parvum suspension, 7 mg dry weight/ml, was a gift from Dr C.Adlam, Wellcome Laboratories, Beckenham, Kent (C. parvum CN6 134, heat killed, no preservative, batch PX383). MICE
Female CBA mice, aged 3-6 months, were used. They were obtained from the breeding colony at St. Mary's Hospital Medical School. Mice were injected i.v. with lo8 viable or killed BCG organisms. SPLEEN CELL CULTURES
Spleen cell suspensions were cultured for 4 days in modified Marbrook chambers
Adjuvants and phagosomellysosome fusion
31 1
(Marbrook 1967). The primary antibody response was assessed in terms of the numbers of cells producing antibody in vitro to sheep erythrocytes (SRBC) and, in some experiments, to dinitrophenylated Ficoll (DNP-Ficoll), using the method of Cunningham & Szenberg (1 968). Full details of the basic method used have been described elsewhere (SljiviC & Watson 1977). Briefly, 2 x lo7 spleen cells were cultured in 1 ml RPMI 1640 medium containing 5% foetal calf serum and 10 pg/ml gentamicin. Cultures were maintained at 37°C in humidified air containing 5% COz.SRBC (2x lo6) or DNP-Ficoll(20 ng) were added to the cultures before incubation and the number of direct antibody-forming cells was determined 4 days later using untreated or trinitrophenylated-SRBC (Rittenberg & Pratt 1969) respectively. Previous treatment of the spleen-cell donors and details of the various additions of bacteria and suramin are described in the Results section. SURAMIN
This was a gift from Dr J.Williamson, National Institute for Medical Research, London (Antrypol, ICI).The drug was dissolved in distilled water, sterilized by filtration and stored at 4°C. Before addition to both the inner tube containing the cells and the outer chamber of Marbrook vessels suitable dilutions were made in RPMI 1640 without added serum.
Results B C G A N D IN VITRO A N T I B O D Y RESPONSES
The effect of i.v. administration of BCG on the subsequent in vitro antibody response of mouse spleen cells was dependent on whether live or dead bacilli were given and the time after BCG injection. Between 2 and 4 weeks after injection, live BCG caused suppression whereas dead BCG caused enhancement of anti-SRBC responses, as illustrated by a typical experiment shown in Figure 1. Mice were injected with lo8 dead or lo8 live organisms and 20 days later their spleen cells were cultured in the presence of SRBC and the responses obtained compared with the response of cells from untreated animals. Full details of this work will be presented elsewhere (Brown, Brown & SljiviC 1979). A D D I T I O N OF K I L L E D B C G M.LEPRAEORC. P A R Y U M TO C U L T U R E S
The number of direct antibody-forming cells (AFC) to SRBC detectable in spleen cell cultures prepared from mice treated with live or dead BCG returned to normal levels after 4-6 weeks (results not shown). If however, killed BCG, M . leprae or C. parvum was added to cultures of spleen cells from live BCG-primed
312
Carolyn A . Brown, I.N. Brown & V.S. SljiviC
-
18
-
16
-
14
-
12
-
m
0 3 10-
e
-u' c
\
0 LL
8 -
a
6 -
4-
2-
0
Dead
Live
None
Figure 1. Numbers of antibody-forming cells against SRBC produced in uifro by spleen cells from control mice or mice pretreated with live or dead BCG 20 days before. BCG was harvested from Glaxo glycerol free medium and part was killed by heating at 70°C for 1 h. Cells from two spleens in each group were pooled and the results show mean & s.e.m. AFC/culture for quadruplicate cultures. Pvalues in a two-tailed Student's test were for dead us untreated P < 0.001 and for live us untreated P < 0.025.
mice at this time, in vitro responses to SRBC were markedly enhanced (Table 1, Brown, Brown & Sljivik 1978). THE EFFECT OF S U R A M I N
The different systemic effects of dead and live BCG described above could be connected with the observation (Armstrong & Hart 1971) that viable BCG organisms somehow resist fusion of their enclosing phagosomes with surrounding lysosomes. To look further at this phenomenon and to examine the possibility that fusion is a prerequisite for the enhancement we have described, experiments were undertaken with the lysosornotropic drug, suramin. The highest dose of suramin chosen was the maximum tolerated in the in uitro macrophage culture system described by Hart & Young (1975), and produced a clear suppression of the normal phagosome/lysosome fusion response to yeast cells within macro-
Adjuvants andphagosome/lysosomefusion
3 13
Table 1. Effect of suramin on antibody responses in vitro and their enhancement induced by mycobacteria and C. parvum
Suramin concentration (pglrnl) Challenging Experiment antigen SRBC SRBC
Bacteria added None BCG
0
40
4400f201 11550f1664
5730k524 8655f1061
1747f268* 1740+291*
6725 f724 6585f539
5030 t_ 508 5150t_339
2007 f 157* 1593+231*
2553f273* 6235 f.347
14352306 3460 f.863
1540f305 1220+ 42
200
A
DNP-Ficoll None DNP-Ficoll BCG SRBC SRBC
None M. leprae
B DNP-Ficoll None DNP-Ficoll M. leprae C
SRBC SRBC
None C. poruum
16795f 1433 17795k2035 4275 f425 9505 1241
12850f800 1146Ok 180 17587+2296* 11710+448* n.d. n.d.
2070 f 393 1325& 424
All values are means fs.e.m. of antibody-forming cell numbers per culture for 3* or 4 cultures in each group. Spleen cells were obtained from mice which had been injected i.v. with 1081iveBCG organisms 7-28 weeks previously. Heat killed BCG or irradiated M. leprae were added at a concentration of lo8organisms per culture. C. paruwn was added at a concentration of 70pg/culture. n.d. =not done.
phages. Two types of experiment were done; the cells were either exposed to suramin before culture or cultured in the presence of suramin. Figure 2 shows an . example of the first type. BCG-primed spleen cells were pretreated with suramin (200 pg/ml in RPMI for 2 h) before cultural exposure to killed BCG and SRBC. Under these conditions, suramin treatment alone had little effect on the AFC response but suppressed significantly (P< 0.01) the enhancement induced by adding BCG. An example of the second type of experiment is shown in Table 1, where the effect of suramin at various concentrations in the culture was determined using spleen cells from BCG-primed mice with added BCG, M . leprae or C .parvum. All three additions caused an enhancement of the anti-SRBC response of over two fold, but the same concentrations of M . leprae or BCG had no effect on the response of the same cell population to DNP-Ficoll. The presence of suramin at 200 pg/ml in the cultures removed the enhancing effect and reduced the number of AFC produced. In the absence of added bacteria, AFC were reduced to approximately 50% the normal value. However, in the presence of added bacteria the reduction in AFC numbers resulting from suramin exposure was even greater (down to 12-20% of the normal value).
314
Carolyn A . Brown, i . N . Brown & V.S. SljiviC 22
2c ia
16
-
14
n
P
3 e
12
4
10
-
-a
u
LL
a
8
6 4 2
0
ICG ~
s+ Figure 2. Effect of pretreatment with suramin(s) on the adjuvant effect of BCG in uitro. The captions refer to additions to spleen cells from mice injected with live BCG, as in Table I , 8 weeks previously. None
S
Discussion These experiments show that moderately high doses of live BCG injected systemically are immunosuppressive whereas the same number of dead organisms enhance the antibody response in vitro to SRBC. Spleen cells taken from mice 2 months or more after priming with live BCG gave normal in uitro primary antibody responses but the addition of killed BCG, M . leprae or C.paruum to the cultures enhanced the AFC response. Suramin prevented the enhancing effect of BCG if used to pretreat the spleen cells to be cultured. If added to the cultures at the same time as the SRBC it had two effects; it reduced the number of AFC formed in the absence of bacteria and abolished the enhancing effect observed in the presence of bacteria. Polyclonal activation of B-lymphocytes in uitro by BCG (Sultzer 1978), or by the other two organisms, is an unlikely explanation of the enhanced AFC numbers reported here since there was selective enhancement of in uitro antiSRBC but not anti-DNP-Ficoll responses. Moreover, since the in uitro antibody
Adjuvants andphagosome/lysosomefusion
3 15
response to SRBC is T-lymphocyte and macrophage-dependent, whereas the response to DNP-Ficoll is not, the enhanced anti-SRBC responses must have been related somehow to an effect of the added bacteria on either or both of these cell types. Suramin binds readily to proteins, concentrates intralysosomally, interferes with phagosome/lysosome fusion and inhibits various proteases (Pesanti 1978, Williamson 1976). In view of its known.ability to reduce the breakdown of phagocytosed material, it is tempting to speculate that in the experiments described above, the drug was preventing some degree of degradation or ‘processing’ of the added bacteria within macrophages necessary for their adjuvant effect to be expressed. The reduction of AFC numbers in the SRBC stimulated cultures without added bacteria supports this idea because phagocytically active macrophages are a critical requirement for a primary response to SRBC in the culture system used. Another possibility is that the suramin interfered with the release from macrophages of soluble factors which are known to influence lymphocyte responses (Unanue 1978) and whose release may be enhanced after phagosomel lysosome fusion. Certain lysosome-labilizing agents are known to act as adjuvants (Spitznagel & Allison 1970) and a similar process may occur after the addition of the bacteria used in these experiments. This stimulus of lysosomelabilization may be prevented in vitro by suramin. and in vitro by sulphatides (Goren et al. 1976) or cyclic AMP (Lowrie, Jackett & Ratcliffe 1975) both of which are produced by viable tubercle bacilli and prevent phagosome/lysosome fusion. M . leprae has been shown to have adjuvant activity in vivo although it is much less active than M . tuberculosis (Stewart-Tull & Davies 1972). Table 1 shows that it will also enhance in vitrp antibody responses of mouse spleen cells and, moreover, that M . leprae is as good as BCG at stimulating responses of cells from BCG-primed mice. This latter observation may be due to direct stimulation of sensitized T-cells and thus reflect antigenic overlap between the two organisms or to direct stimulation of macrophages or both. In view of the results presented above and the known properties of suramin, we think that direct stimulation of macrophages represents the major contribution to the described adjuvant effect. If this is so, then recent reports of BCG-induced protection against leprosy in mice (Shepard, Walker & Van Landingham 1978) and BCG and killed M . leprae stimulation of a local non-specific immunity against Listeria monocytogenes in the mouse foot pad (Pate1 & Lefford 1978) could also be interpreted in this way.
Acknowledgements This work was supported by a grant from the Cancer Research Campaign. We thank Gwen Dewey for technical assistance and Glaxo Research Laboratories Limited, Greenford, for the BCG.
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Carolyn A . Brown, I.N. Brown & V.S. SljiviC
References ALLISONA.C. (1973) Effects of adjuvants on different cell types and their interactions in immune responses. In Immunopotentiation, Ciba Foundation Symposium 18 p. 73. Associated Scientific Publishers, Amsterdam ARMSTRONG J.A. & HARTP.D'A. (1971) Response of cultured macrophages to Mycobacterim tuberculosis, with observations on fusion of lysosomes with phagosomes. Journal of Experimental Medicine 134,713 BROWNC.A., BROWNI.N. & SLJIVIC V.S.(1978) Enhancement of the antibody response in vitro by BCG. Developmental Biology Standard 38, 153. BROWNC.A., BROWNI.N. & SLJIVI~ V.S. (1979) Suppressed or enhanced antibody responses in vitro after BCG treatment of mice: importance of BCG viability. Immunology 38 (in press) A. (1968) Further improvements in the plaque technique for CUNMNGHAM A.J. & SZENBERG detecting single antibody forming cells. Immunology 14, 599 DAVISM., LLOYD J.B. & BECKF. (1971) The effect of trypan blue, suramin and aurothiomalate on the breakdown of '251-labelledalbumin within rat liver lysosomes. Biochemical Journal 121,21 GORENM.B., HARTP.D'A., YOUNGM.R. & ARMSTRONG J.A. (1976) Prevention of phagosomelysosome fusion in cultured macrophages by sulphatides of Mycobacterium tuberculosis. Proceedings of National Academy of Sciences 73,2510 HARTP.D'A., ARMSTRONG J.A., BROWNC.A. & DRAPERP. (1972) Ultrastructural study of the behavior of macrophages toward parasitic mycobacteria. Infection and Immunity 5,803 HARTP.D'A. & YOUNGM.R. (1975) Interference with normal phagosome-lysosome fusion in macrophages, using ingested yeast cells and suramin. Nature 256,47 LOWRIED.B., JACKETT P.S. & RKTCLIFFE N.A. (1975) Mycobacterium microti may protect itself from intracellular destruction by releasing cyclic AMP into phagosomes. Nature 254,600 MARBROOK J. (1967) Primary immune response in cultures of spleen cells. Lancet 6, 1279 PATELP.J. & LEFFORDM.J. (1978) Specific and nonspecific resistance in mice immunized with irradiated Mycobacterium leprue. Infection and Immunity 20,692 PESANTIE.L. (1978) Suramin effects on macrophage phagolysosome formation and antimicrobial activity. Infection and Immunity 20, 503 RITTENBERG M.B. & PRATTK.L. (1969) Antitrinitrophenyl (TNP) plaque assay. Primary response of BALB/c mice to soluble and particulate immunogen. Proceedings of the Society for Experimental Biology and Medicine 132, 575 SHEPARD C.C., WALKER L.L. &VANLANDINGHAM R.M. (1978) Immunity to Mycobacterium leprae infections induced in mice by BCG vaccination at different times before and after challenge. Infection and Immunity 19,391 SLJIVI~ V.S. & WATSONS.R. (1977) The adjuvant effect of Corynebacterium parvum: T-cell dependence of macrophage activation. Journal of Experimental Medicine 145,45 SPITZNAGEL J.K. & ALLISONA.C. (1970) Mode of action of adjuvants: retinol and other lysosomelabilizing agents as adjuvants. Journal of Immunology 104, 1 19 STEWART-TULL D.E.S. & DAVIES M. (1972) Adjuvant activity of Mycobacterium leprae. Infection and Immunity 6,909. SULTZER B.M. (1978) Infection with bacillus Calmette-Guerin activates murine thymus independent (B) lymphocytes. Journal of Immunology 120,254 UNANUE E.R. (1978) The regulation of lymphocyte functions by the macrophage. Immunological Reviews 40,227 VANDER MEERC., HOFHUISF.M.A. & WILLERSJ.M.N. (1977) Killed Listeria monocytogenes vaccine becomes protective on addition of polyanions. Nature 269,594 WILLIAMSON J. (1976) Chemotherapy of African trypanosomiasis. Tropical Diseases Bulletin 73,53 1
