INFICTION AND IMMUNITY, Aug. 1977, p. 263-267 Copyright © 1977 American Society for Microbiology
Vol. 17, No. 2
Printed in U.S.A.
Enhancement ofthe Antibody Response In Vitro by Adherent Cells from Mice Infected with Mycobacterium lepraemurium SUSAN R. WATSON,' IVOR N. BROWN, AND VOJIN S. SLJIVIC* Departments of Immunology* and Bacteriology, St. Mary's Hospital Medical School, London W2 IPG, England
Received for publication 24 January 1977
Spleen cells from mice systemically infected 4 to 6 weeks previously with Mycobacterium lepraemurium gave an enhanced primary antibody response in vitro to sheep erythrocytes, but responded normally to dinitrophenylated polymerized flagellin. The ability to enhance the response was associated with the glass-adherent spleen cell population and with peritoneal cells. Similar cells obtained from infected mice depleted of T lymphocytes failed to enhance the antibody response. These studies suggest that macrophages that become activated during the development of cell-mediated immunity to infection can also stimulate antibody responses to thymus-dependent antigens. Chronic bacterial disease, such as that caused by pathogenic Mycobacteria, Brucella, Salmonella, or Listeria, is associated particularly with persistence and multiplication of the infective organisms within host macrophages. The control of infection is thought to result from the activation of infected macrophages by lymphocyte-released mediators during the development of cell-mediated immunity (8, 17). In addition to being more efficient in the killing of intracellular organisms, macrophages activated by T lymphocytes differ from resting macrophages in a number of other properties. They show enhanced adherence to surfaces, phagocytosis of certain particles, and tumoricidal activity, as well as a variety of metabolic and enzymatic activities (10). Mycobacterium lepraemiurum is an obligate intracellular organism; it is nontoxic to macrophages in which it is commonly found and has an average generation time of 7. days (3). The infection is characterized by the development of granulomata of bacilli-laden macrophages in the various tissues. In resistant strains of mice the course of infection is prolonged, and these granulomata are surrounded by numerous lymphocytes. Also, the number of bacilli within macrophages is smaller than in more susceptible strains in which the lymphocytic reaction is lacking, and the disease progresses more rapidly (6). The greater susceptibility to infection has been related to the absence of the development of cell-mediated reaction to M. lepraemurium (6, 7), and the spectrum of the disease in I Present address: Tumor Antigen Section, Biology Branch, National Cancer Institute, Bethesda, MD 20014.
mice has been compared with that found in human leprosy (6). Macrophages are required for optimal antibody responses in vitro to a variety of antigens (18, 22). We have studied this function of macrophages during the course of systemic M. lepraemurium infection and have previously described a defect of macrophage function at later stages of the infection (25). Here we show that adherent spleen cells and peritoneal cells from animals at an earlier stage of infection enhance the antibody response to sheep erythrocytes (SRBC) in vitro and that T lymphocytes are required for the activation of these cells. MATERIALS AND METHODS Mice. Female CBA mice, aged 2 to 3 months, were used throughout. Mice depleted of T cells (TXB mice) were prepared by adult thymectomy followed 10 days later by lethal whole-body irradiation (850 R) from a Siemens X-ray machine operating at 10 mA and 240 kV, with a 1-mm Cu filter at a target distance of 50 cm and a dose rate in air of 43.1 R/min. Within 4 h of irradiation the mice were reconstituted with 5 x 106 syngenic bone marrow cells given intravenously. Control mice (XB) were not thymectomized, but were otherwise treated in the same way. Infection. Mice were infected with an intravenous injection of 109 M. lepraemurium organisms freshly isolated from the heavily infected livers and spleens of donor mice (3). In one experiment smaller doses were also used. At 4 to 6 weeks after infection, when the present experiments were carried out, the mice showed enlargement of liver and spleen. ZiehlNielsen staining of sections of these organs revealed the presence of moderate numbers of mycobacteria within macrophages. 263
264
INFECT. IMMUN. WATSON, BROWN, AND SLJIVICT
Preparation and culture of spleen cells. Suspensions of spleen cells were prepared and fractionated into nonadherent and adherent cells as described before (24). Peritoneal cells were obtained by lavage of the peritoneal cavity of unstimulated mice. For all experiments cells from three to six mice were pooled. The cells were cultured in RPMI 1640 medium for 4 days in Marbrook chambers (16) in the presence of either 2 x 106 SRBC or 0.1 u.g of dinitrophenylated polymerized flagellin (DNP-POL), in a volume of 1 ml. Unfractionated spleen cells were cultured at a concentration of 20 x 106/ml, and nonadherent cells were cultured at a concentration of 13 x 106 to 14 x 106/ml. Peritoneal or adherent spleen cells were added to some cultures at the rate of 106 viable cells. Differential counts on adherent spleen cells stained with hematological stains showed that approximately 90% of cells had a moderate or large amount of cytoplasm, mostly with indented nuclei, the remaining cells being small lymphocytes and polymorphs. Cells stained for 30 min with acridine orange (0.2 mg/100 ml of medium) showed the following approximate proportions of cell types: 80% mononuclear cells with strong red cytoplasmic fluorescence characteristic of lysosomes (2), 13% small mononuclear cells with minimal or no cytoplasmic staining, and 7% polymorphs. About 75% of adherent cells took up neutral red (5 mg/100 ml of medium) after a 10-min incubation period. Measurement of the antibody response. At the end of the culture period the cells were harvested, washed once, and resuspended in 1 ml of medium. The antibody responses to SRBC and DNP were measured in terms of plaque-forming cells (PFC) by the method of Cunningham and Szenberg (9), using untreated SRBC and SRBC treated with DNP-anti-SRBC Fab fragments (21), respectively. The results are expressed as the number of PFC per culture and given as the mean of quadruplicate cultures + standard error.
requirement for macrophages for the response to this antigen (19). In contrast, the response to DNP-POL, a macrophage-independent antigen (11), was not affected by the removal of adherent spleen cells (cultures 3 and 4).
That macrophage might be involved in the enhancement of the antibody response in this system was suggested by the ability of peritoneal cells from infected mice to enhance the response of normal spleen cells (culture 7). This was confirmed by the results obtained with cultures containing various combinations of cells from infected and control mice. Thus, normal nonadherent spleen cells supplemented with either peritoneal cells (culture 13) or adherent spleen cells (culture 10) from infected animals gave enhanced responses. Conversely, nonadherent cells from infected mice supplemented with normal peritoneal cells or adherent spleen cells gave normal responses (cultures 14 versus 12 and 11 versus 5), indicating that lymphocytes alone were not responsible for the enhancement of the antibody response to SRBC. M. lepraemurium infection did not alter the number of background PFC among spleen cells cultured in the absence of SRBC (cultures 16 and 17). The enhancement of the antibody response in vitro to SRBC, as measured 4 weeks after infection, was directly proportional to the number of M. lepraemurium organisms injected (Table 2). To elucidate the role of T lymphocytes in the generation of cells capable of enhancing the antibody response during M. lepraemurium infection, experiments were done with mice depleted of T cells. TXB and XB mice were infected with 109 organisms 4 weeks after irradiation together with untreated mice of a similar RESULTS age. Cells from these animals were harvested 6 The results of one of several experiments on weeks after infection and were cultured in varthe antibody response in vitro of spleen cells ious combinations (Table 3). Unfractionated from control mice and mice infected 4 weeks spleen cells from infected but otherwise unpreviously are given in Table 1. The various treated mice gave an enhanced PFC response sets have been numbered in the table for ease of (culture 2) similar to that found at 4 weeks after reference. The response of unfractionated infection. Cells from TXB-infected mice gave a spleen cells from infected mice to SRBC, a thy- greatly reduced response to SRBC, but a normus-dependent antigen (5), was increased mal response to DNP-POL (culture 3), indicatnearly threefold, but the response to DNP- ing that T but not B cell function was deficient POL, a T cell-independent antigen in vitro (13), in these animals. Cells from XB-infected mice was unaffected (cultures 1 and 2). The antibody (culture 4) gave a response that was signifiresponse of nonadherent cells to SRBC (cul- cantly (P < 0.02, Student's t test) higher than tures 3 and 4) was greatly reduced compared that of normal cells (culture 1), but this enwith that of unfractionated cells (culture 1) and hancement was considerably less than that obwas close to the number of background PFC tained with cells from infected control mice found (cultures 16 and 17). The response to (culture 2). When cultures of normal nonadherSRBC could be largely reconstituted by supple- ent cells were reconstituted with either peritomenting nonadherent cells with either perito- neal cells or adherent spleen cells, those coming neal cells or adherent spleen cells (cultures 5 from infected control mice gave enhanced reand 12 versus 1), a result in agreement with the sponses (cultures 6 and 10), TXB-infected mice
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265
TABLE 1. Antibody response in vitro of spleen cells from mice infected with M. lepraemurium Culture no.
Spleen cells Unfractionated
1 Normal 2 Infected 3 4 5 6 Normal 7 Normal 8 Infected 9 Infected 10 11 12 13 14 15 16 Normal backgroundb 17 Infected backgroundb a ND, Not determined. b Cultured without SRBC.
Nonadherent
Normal Infected Normal
Adherent
Peritoneal cells
Normal Normal Infected Normal Infected
Normal Infected Normal Normal Infected Infected
Infected Normal
TABLE 2. Effect of different doses of M.
Normal Infected Normal Infected
Antibody response (PFC/culture) Anti-SRBC
Anti-DNP
± 114 +131 31 +30 + 41 ± 104 ± 218 ± 178 6,596 ± 229 4,686 ± 386 1,680 + 50 1,826 ± 105 4,810 + 82 1,710 - 91 5,236 + 102 94 17 80 29
1,570 ± 54 1,400 54 1,606 41
2,096 5,666 150 190 1,886 2,596 5,360 5,470
1,470 97
NDa ± 139 ± 89 ± 87 ± 61 1,446 ± 113 1,440 ± 79 1,336 ± 116 1,436 ± 67 1,526 ± 61 1,360 ± 79 ND ND
1,564 1,456 1,346 1,506
dition, the enhancing activity of adherent cells from C. parvum-treated mice was found to be response of spleen cells to SRBC radioresistant (24) and could be abrogated by carrageenan (20), a polysaccharide toxic to No. of organisms injected PFC/culture macrophages (1) but not to lymphocytes (15). None 2,190 ± 99 Finally, adherent cells alone are responsible for 103 3,376 ± 216 the defect of the antibody response in vitro 106 4,910 ± 304 during later stages of M. lepraemurium infec109 6,090 ± 58 tion (25) when macrophages are overloaded gave responses that were similar to those ob- with bacilli. The development of delayed-hypersensitivity tained with normal mouse cells (cultures 7 versus 5 and 11 versus 9), and XB-infected mice reactions during the course of infection with gave intermediate responses (cultures 8 and various intracellular organisms is associated with the appearance of macrophages whose ac12). tivation is dependent on T lymphocytes (8, 17). DISCUSSION The time when the antibody response in vitro to The in vitro experiments described here show SRBC was enhanced in the present experithat spleen cells from mice infected with M. ments corresponded to the time when delayedlepraemurium give an enhanced antibody re- hypersensitivity reactions to M. lepraemurium sponse to SRBC but not to DNP-POL. The abil- were fully developed (7; Brown, unpublished ity to non-specifically enhance the antibody re- data), i.e., when the presence of activated macsponse to SRBC was associated with peritoneal rophages would be expected. The generation of cells and adherent spleen cells rather than with cells capable of enhancing the antibody rethe nonadherent cells from infected mice. Simi- sponse in the present experiments was found to lar results have also been obtained with cells require T lymphocytes. Thus, only peritoneal from mice pretreated with killed Corynebacte- cells and adherent spleen cells from infected XB rium parvum organisms (24). The nature of mice, but not cells from infected TXB animals, adherent cells capable of enhancing antibody enhanced the response to SRBC. These obserresponses in vitro under the present conditions vations together with the above conclusion that has not yet been directly demonstrated. How- the most likely cells responsible for the enever, a number of observations indicate that hancement of the antibody response are macrothe most likely cell type responsible for this phages strongly suggest that macrophage actiphenomenon is the macrophage. Thus, the ma- vation takes place during M. lepraemurium jority of adherent spleen cells in the present infection and that, once activated, macroexperiments had the morphology and staining phages can stimulate antibody production. A properties characteristic of macrophages. In ad- similar conclusion was reached about the cellulepraemurium given to mice on the in vitro antibody
266
WATSON, BROWN, AND SLJIVIIINFECT. IMMUN. M
TABLE 3. Antibody response in vitro of spleen cells from T lymphocyte-depleted mice infected with M. lepraemurium Spleen cellsa
Antibody response (PFC/culture)
Culture
Peritoneal cells"
no.
Unfractionated
1 2 3 4 5 6 7 8 9 10 11 12
Normal Infected TXB infected XB infected
Nonadherent
Adherent
Anti-SRBC
2,065 5,970 425 2,890 1,770 4,265 1,610 2,740 1,900 5,575 1,765 3,485
Normal Normal Normal Infected Normal TXB infected Normal XB infected Normal Normal Normal Infected Normal TXB infected Normal XB infected a TXB, Thymectomized, lethally irradiated, bone marrow-reconstituted mice; bone marrow-reconstituted mice.
+ 90 ± ± ± ± ± ± ± ± ± ± ±
189 102 342
Anti-DNP
1,605 1,730 1,550 1,735
± 38 + 93 ± 93 + 113
98 319 73 125 141 270 137 175
XB, lethally irradiated,
lar basis of the enhancement of the antibody activation of these cells is known to take place. response in vitro with cells from C. parvum- This conclusion is supported by the findings treated mice (20, 24). that macrophages from mice treated with C. The way in which activated macrophages parvum (20, 24) or infected with BCG (Watson, stimulate the antibody response in vitro is un- Brown, and Sljivi6, unpublished data) or Salcertain at this time. One aspect that should be monella typhimurium (Watson, Plant, Barrett remembered, however, when considering the and Sljivi6, unpublished) have all the properpossible mechanisms is the finding that only ties described above. The significance of these the antibody response to SRBC, a macrophage observations as well as the mechanisms (19) and thymus-dependent (5) antigen, was whereby activated macrophages influence lymenhanced, whereas the response to DNP-POL, phocyte function remains to be elucidated. which is independent of these two cell types (11, ACKNOWLEDGMENTS 13), was not affected. This would indicate that, We thank Peter Church and Maureen Philbert for their whatever mechanism is responsible for the enin these experiments. S.R.W. was in receipt of a hancement of the response, it is not operating assistanceResearch Council scholarship. I.N.B. is supported directly via B lymphocytes. One possibility is Medical by the Wellcome Trust. that activated macrophages present the macroLITERATURE CITED phage-dependent antigen in a manner that is more efficient for the induction of the response 1. Allison, A. C., J. S. Harington, and M. Birbeck. 1966. An examination of the cytotoxic effects of silica on by lymphocytes. A difference in the handling of macrophages. J. Exp. Med. 124:141-154. keyhole limpet hemocyanin by macrophages 2. Allison, A. C., and M. R. Young. 1969. Vital staining from mice treated with C. parvum has been and fluorescence microscopy of lysosomes, p. 600-628. In J. T. Dingle and H. B. Fell (ed.), Lysosomes in described (26), but not in the case of macrobiology and pathology, vol. 2. North-Holland Publishphages stimulated by other adjuvants (23). Aning Co., Amsterdam. other possibility is related to the production 3. Brown, I. N., and H.-N. Krenzien. 1976. Systemic Myand release by macrophages of soluble factors cobacterium lepraemurium infection in mice: differences in doubling time in liver, spleen, and bone that can affect various lymphocyte functions (4, marrow, and a method for measuring the proportion 12, 14). Activated macrophages may be either of viable organisms in an inoculum. Infect. Immun. releasing a greater amount of such factors or 13:480-486. producing factors that are more effective in con- 4. Calderon, J., J.-M. Kiely, J. L. Lefko, and E. R. Unanue. 1975. The modulation of lymphocyte functions trolling the in vitro responses of lymphocytes. by molecules secreted by macrophages. I. Description Any such factors would have to act on T lymand partial biochemical analysis. J. Exp. Med. phocytes in view of the lack of enhancement of 142:151-164. the response to DNP-POL. 5. Claman, H. N., and E. A. Chaperon. 1969. Immunologic complementation between thymus and marrow If activated macrophages have the general cells -a model for the two cell theory of immunocomability to enhance the antibody response to cerpetence. Transplant. Rev. 1:92-113. tain antigens, one would expect a similar en- 6. Closs, 0., and 0. A. Haugen. 1974. Experimental muhancement in other situations in which the rine leprosy. 2. Further evidence for varying suscepti-
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bility of outbred mice and evaluation of the response of 5 inbred mouse strains to infection with 4ycobactrium lepraemurium. Acta Pathol. Microbol. Scand. Sect. A 82:459-474. 7. Closs, O., and 0. A. Haugen. 1975. Experi9ental murine leprosy. 3. Early local reaction to MycQbacterium lepraemurium in C3H and C57/B1 mice. Aota Pathol. Microbiol. Scand. Sect. A 83:51-58. 8. Collins, F. M. 1974. Vaccines and cell-mediated immunity. Bacteriol. Rev. 38:371-402. 9. Cunningham, A. J., and A. Szenberg. 1968. Further improvements in the plaque technique for detecting single antibody forming cells. Immunology 14:599600. 10. David, J. R. 1975. Macrophage activation by lymphocyte mediators. Fed. Proc. 34:1730-1736. 11. Diener, E., K. Shortman, and P. Russell. 1970. Induction of immunity and tolerance in vitro in the absence of phagocytic cells. Nature (London) 225:731-732. 12. Erb, P., and M. Feldmann. 1975. The role of macrophages in the generation of T helper cells. Ifl. Influence of macrophage-derived factors in helper cell induction. Eur. J. Immunol. 5:759-766. 13. Feldmann, M., and A. Basten. 1971. The relationship between antigenic structure and the requirement for thymus-derived cells in the immune response. J. Exp. Med. 134:103-119. 14. Gery, I., and B. H. Waksman. 1972. Potentiation of the T-lymphocyte response to mitogens. II. The cellular source of potentiating mediator(s). J. Exp. Med. 136:143-155. 15. Hanna, N., and S. Leskowitz. 1973. Structural requirements for in vivo and in vitro immunogenicity in hapten-specific delayed hypersensitivity. Cell. Immunol. 7:189-197. 16. Marbrook, J. 1967. Primary immune response in cultures of spleen cells. Lancet ii:1279-1281. 17. North, R. J. 1974. Cell mediated immunity and the
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response to infection, p. 185-219. In R. T. McCluskey and S. Cohen (ed.), Mechanisms of cell-mediated immunity. John Wiley and Sons, New York. 18. Pierce, C. W., and J. A. Kapp. 1976. The role of macro-
10.
20.
21.
22. 23. 24.
25.
26.
phages in antibody responses in vitro, p. 1-33. In D. S. Nelson (ed.), Immunobiology of the macrophage. Academic Press Inc., New York. Sjoberg, O., J. Anderson, and G. Moller. 1972. Requirement for adherent cells in the primary and secondary immune response in vitro. Eur. J. Immunol. 2:123126. Sljivic, V. S., and S. R. Watson. 1977. The adjuvant effect of Corynebacterium parvum: T-cell dependence of macrophage activation. J. Exp. Med. 145:45-57. Strausbach, P., A. Sulica, and D. Givol. 1970. General method for the detection of cells producing antibodies against haptens and proteins. Nature (London) 227:68-69. Unanue, E. R. 1972. The regulatory role of macrophages in antigenic stimulation. Adv. Immunol. 15:95-165. Unanue, E. R., B. A. Askonas, and A. C. Allison. 1969. A role of macrophages in the stimulation of immune responses by adjuvants. J. Immunol. 103:71-78. Watson, S. R., and V. S. Slivic. 1976. The role of macrophage in the adjuvant effect on antibody production of Corynebacterium parvum. Clin. Exp. Immunol. 23:149-153. Watson, S. R., V. S. Sljivic, and I. N. Brown. 1975. Defect of macrophage function in the antibody response to sheep erythrocytes in systemic Mycobacterium lepraemurium infection. Nature (London) 256:206-208. Wiener, E., and A. Bandieri. 1975. Modification in the handling in vitro of 125I-labelled keyhole limpet haemocyanin by peritoneal macrophages from mice pretreated with the adjuvant Corynebacterium parvum. Immunology 29:265-274.
Vol. 17, No. 2
Printed in U.S.A.
Enhancement ofthe Antibody Response In Vitro by Adherent Cells from Mice Infected with Mycobacterium lepraemurium SUSAN R. WATSON,' IVOR N. BROWN, AND VOJIN S. SLJIVIC* Departments of Immunology* and Bacteriology, St. Mary's Hospital Medical School, London W2 IPG, England
Received for publication 24 January 1977
Spleen cells from mice systemically infected 4 to 6 weeks previously with Mycobacterium lepraemurium gave an enhanced primary antibody response in vitro to sheep erythrocytes, but responded normally to dinitrophenylated polymerized flagellin. The ability to enhance the response was associated with the glass-adherent spleen cell population and with peritoneal cells. Similar cells obtained from infected mice depleted of T lymphocytes failed to enhance the antibody response. These studies suggest that macrophages that become activated during the development of cell-mediated immunity to infection can also stimulate antibody responses to thymus-dependent antigens. Chronic bacterial disease, such as that caused by pathogenic Mycobacteria, Brucella, Salmonella, or Listeria, is associated particularly with persistence and multiplication of the infective organisms within host macrophages. The control of infection is thought to result from the activation of infected macrophages by lymphocyte-released mediators during the development of cell-mediated immunity (8, 17). In addition to being more efficient in the killing of intracellular organisms, macrophages activated by T lymphocytes differ from resting macrophages in a number of other properties. They show enhanced adherence to surfaces, phagocytosis of certain particles, and tumoricidal activity, as well as a variety of metabolic and enzymatic activities (10). Mycobacterium lepraemiurum is an obligate intracellular organism; it is nontoxic to macrophages in which it is commonly found and has an average generation time of 7. days (3). The infection is characterized by the development of granulomata of bacilli-laden macrophages in the various tissues. In resistant strains of mice the course of infection is prolonged, and these granulomata are surrounded by numerous lymphocytes. Also, the number of bacilli within macrophages is smaller than in more susceptible strains in which the lymphocytic reaction is lacking, and the disease progresses more rapidly (6). The greater susceptibility to infection has been related to the absence of the development of cell-mediated reaction to M. lepraemurium (6, 7), and the spectrum of the disease in I Present address: Tumor Antigen Section, Biology Branch, National Cancer Institute, Bethesda, MD 20014.
mice has been compared with that found in human leprosy (6). Macrophages are required for optimal antibody responses in vitro to a variety of antigens (18, 22). We have studied this function of macrophages during the course of systemic M. lepraemurium infection and have previously described a defect of macrophage function at later stages of the infection (25). Here we show that adherent spleen cells and peritoneal cells from animals at an earlier stage of infection enhance the antibody response to sheep erythrocytes (SRBC) in vitro and that T lymphocytes are required for the activation of these cells. MATERIALS AND METHODS Mice. Female CBA mice, aged 2 to 3 months, were used throughout. Mice depleted of T cells (TXB mice) were prepared by adult thymectomy followed 10 days later by lethal whole-body irradiation (850 R) from a Siemens X-ray machine operating at 10 mA and 240 kV, with a 1-mm Cu filter at a target distance of 50 cm and a dose rate in air of 43.1 R/min. Within 4 h of irradiation the mice were reconstituted with 5 x 106 syngenic bone marrow cells given intravenously. Control mice (XB) were not thymectomized, but were otherwise treated in the same way. Infection. Mice were infected with an intravenous injection of 109 M. lepraemurium organisms freshly isolated from the heavily infected livers and spleens of donor mice (3). In one experiment smaller doses were also used. At 4 to 6 weeks after infection, when the present experiments were carried out, the mice showed enlargement of liver and spleen. ZiehlNielsen staining of sections of these organs revealed the presence of moderate numbers of mycobacteria within macrophages. 263
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INFECT. IMMUN. WATSON, BROWN, AND SLJIVICT
Preparation and culture of spleen cells. Suspensions of spleen cells were prepared and fractionated into nonadherent and adherent cells as described before (24). Peritoneal cells were obtained by lavage of the peritoneal cavity of unstimulated mice. For all experiments cells from three to six mice were pooled. The cells were cultured in RPMI 1640 medium for 4 days in Marbrook chambers (16) in the presence of either 2 x 106 SRBC or 0.1 u.g of dinitrophenylated polymerized flagellin (DNP-POL), in a volume of 1 ml. Unfractionated spleen cells were cultured at a concentration of 20 x 106/ml, and nonadherent cells were cultured at a concentration of 13 x 106 to 14 x 106/ml. Peritoneal or adherent spleen cells were added to some cultures at the rate of 106 viable cells. Differential counts on adherent spleen cells stained with hematological stains showed that approximately 90% of cells had a moderate or large amount of cytoplasm, mostly with indented nuclei, the remaining cells being small lymphocytes and polymorphs. Cells stained for 30 min with acridine orange (0.2 mg/100 ml of medium) showed the following approximate proportions of cell types: 80% mononuclear cells with strong red cytoplasmic fluorescence characteristic of lysosomes (2), 13% small mononuclear cells with minimal or no cytoplasmic staining, and 7% polymorphs. About 75% of adherent cells took up neutral red (5 mg/100 ml of medium) after a 10-min incubation period. Measurement of the antibody response. At the end of the culture period the cells were harvested, washed once, and resuspended in 1 ml of medium. The antibody responses to SRBC and DNP were measured in terms of plaque-forming cells (PFC) by the method of Cunningham and Szenberg (9), using untreated SRBC and SRBC treated with DNP-anti-SRBC Fab fragments (21), respectively. The results are expressed as the number of PFC per culture and given as the mean of quadruplicate cultures + standard error.
requirement for macrophages for the response to this antigen (19). In contrast, the response to DNP-POL, a macrophage-independent antigen (11), was not affected by the removal of adherent spleen cells (cultures 3 and 4).
That macrophage might be involved in the enhancement of the antibody response in this system was suggested by the ability of peritoneal cells from infected mice to enhance the response of normal spleen cells (culture 7). This was confirmed by the results obtained with cultures containing various combinations of cells from infected and control mice. Thus, normal nonadherent spleen cells supplemented with either peritoneal cells (culture 13) or adherent spleen cells (culture 10) from infected animals gave enhanced responses. Conversely, nonadherent cells from infected mice supplemented with normal peritoneal cells or adherent spleen cells gave normal responses (cultures 14 versus 12 and 11 versus 5), indicating that lymphocytes alone were not responsible for the enhancement of the antibody response to SRBC. M. lepraemurium infection did not alter the number of background PFC among spleen cells cultured in the absence of SRBC (cultures 16 and 17). The enhancement of the antibody response in vitro to SRBC, as measured 4 weeks after infection, was directly proportional to the number of M. lepraemurium organisms injected (Table 2). To elucidate the role of T lymphocytes in the generation of cells capable of enhancing the antibody response during M. lepraemurium infection, experiments were done with mice depleted of T cells. TXB and XB mice were infected with 109 organisms 4 weeks after irradiation together with untreated mice of a similar RESULTS age. Cells from these animals were harvested 6 The results of one of several experiments on weeks after infection and were cultured in varthe antibody response in vitro of spleen cells ious combinations (Table 3). Unfractionated from control mice and mice infected 4 weeks spleen cells from infected but otherwise unpreviously are given in Table 1. The various treated mice gave an enhanced PFC response sets have been numbered in the table for ease of (culture 2) similar to that found at 4 weeks after reference. The response of unfractionated infection. Cells from TXB-infected mice gave a spleen cells from infected mice to SRBC, a thy- greatly reduced response to SRBC, but a normus-dependent antigen (5), was increased mal response to DNP-POL (culture 3), indicatnearly threefold, but the response to DNP- ing that T but not B cell function was deficient POL, a T cell-independent antigen in vitro (13), in these animals. Cells from XB-infected mice was unaffected (cultures 1 and 2). The antibody (culture 4) gave a response that was signifiresponse of nonadherent cells to SRBC (cul- cantly (P < 0.02, Student's t test) higher than tures 3 and 4) was greatly reduced compared that of normal cells (culture 1), but this enwith that of unfractionated cells (culture 1) and hancement was considerably less than that obwas close to the number of background PFC tained with cells from infected control mice found (cultures 16 and 17). The response to (culture 2). When cultures of normal nonadherSRBC could be largely reconstituted by supple- ent cells were reconstituted with either peritomenting nonadherent cells with either perito- neal cells or adherent spleen cells, those coming neal cells or adherent spleen cells (cultures 5 from infected control mice gave enhanced reand 12 versus 1), a result in agreement with the sponses (cultures 6 and 10), TXB-infected mice
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TABLE 1. Antibody response in vitro of spleen cells from mice infected with M. lepraemurium Culture no.
Spleen cells Unfractionated
1 Normal 2 Infected 3 4 5 6 Normal 7 Normal 8 Infected 9 Infected 10 11 12 13 14 15 16 Normal backgroundb 17 Infected backgroundb a ND, Not determined. b Cultured without SRBC.
Nonadherent
Normal Infected Normal
Adherent
Peritoneal cells
Normal Normal Infected Normal Infected
Normal Infected Normal Normal Infected Infected
Infected Normal
TABLE 2. Effect of different doses of M.
Normal Infected Normal Infected
Antibody response (PFC/culture) Anti-SRBC
Anti-DNP
± 114 +131 31 +30 + 41 ± 104 ± 218 ± 178 6,596 ± 229 4,686 ± 386 1,680 + 50 1,826 ± 105 4,810 + 82 1,710 - 91 5,236 + 102 94 17 80 29
1,570 ± 54 1,400 54 1,606 41
2,096 5,666 150 190 1,886 2,596 5,360 5,470
1,470 97
NDa ± 139 ± 89 ± 87 ± 61 1,446 ± 113 1,440 ± 79 1,336 ± 116 1,436 ± 67 1,526 ± 61 1,360 ± 79 ND ND
1,564 1,456 1,346 1,506
dition, the enhancing activity of adherent cells from C. parvum-treated mice was found to be response of spleen cells to SRBC radioresistant (24) and could be abrogated by carrageenan (20), a polysaccharide toxic to No. of organisms injected PFC/culture macrophages (1) but not to lymphocytes (15). None 2,190 ± 99 Finally, adherent cells alone are responsible for 103 3,376 ± 216 the defect of the antibody response in vitro 106 4,910 ± 304 during later stages of M. lepraemurium infec109 6,090 ± 58 tion (25) when macrophages are overloaded gave responses that were similar to those ob- with bacilli. The development of delayed-hypersensitivity tained with normal mouse cells (cultures 7 versus 5 and 11 versus 9), and XB-infected mice reactions during the course of infection with gave intermediate responses (cultures 8 and various intracellular organisms is associated with the appearance of macrophages whose ac12). tivation is dependent on T lymphocytes (8, 17). DISCUSSION The time when the antibody response in vitro to The in vitro experiments described here show SRBC was enhanced in the present experithat spleen cells from mice infected with M. ments corresponded to the time when delayedlepraemurium give an enhanced antibody re- hypersensitivity reactions to M. lepraemurium sponse to SRBC but not to DNP-POL. The abil- were fully developed (7; Brown, unpublished ity to non-specifically enhance the antibody re- data), i.e., when the presence of activated macsponse to SRBC was associated with peritoneal rophages would be expected. The generation of cells and adherent spleen cells rather than with cells capable of enhancing the antibody rethe nonadherent cells from infected mice. Simi- sponse in the present experiments was found to lar results have also been obtained with cells require T lymphocytes. Thus, only peritoneal from mice pretreated with killed Corynebacte- cells and adherent spleen cells from infected XB rium parvum organisms (24). The nature of mice, but not cells from infected TXB animals, adherent cells capable of enhancing antibody enhanced the response to SRBC. These obserresponses in vitro under the present conditions vations together with the above conclusion that has not yet been directly demonstrated. How- the most likely cells responsible for the enever, a number of observations indicate that hancement of the antibody response are macrothe most likely cell type responsible for this phages strongly suggest that macrophage actiphenomenon is the macrophage. Thus, the ma- vation takes place during M. lepraemurium jority of adherent spleen cells in the present infection and that, once activated, macroexperiments had the morphology and staining phages can stimulate antibody production. A properties characteristic of macrophages. In ad- similar conclusion was reached about the cellulepraemurium given to mice on the in vitro antibody
266
WATSON, BROWN, AND SLJIVIIINFECT. IMMUN. M
TABLE 3. Antibody response in vitro of spleen cells from T lymphocyte-depleted mice infected with M. lepraemurium Spleen cellsa
Antibody response (PFC/culture)
Culture
Peritoneal cells"
no.
Unfractionated
1 2 3 4 5 6 7 8 9 10 11 12
Normal Infected TXB infected XB infected
Nonadherent
Adherent
Anti-SRBC
2,065 5,970 425 2,890 1,770 4,265 1,610 2,740 1,900 5,575 1,765 3,485
Normal Normal Normal Infected Normal TXB infected Normal XB infected Normal Normal Normal Infected Normal TXB infected Normal XB infected a TXB, Thymectomized, lethally irradiated, bone marrow-reconstituted mice; bone marrow-reconstituted mice.
+ 90 ± ± ± ± ± ± ± ± ± ± ±
189 102 342
Anti-DNP
1,605 1,730 1,550 1,735
± 38 + 93 ± 93 + 113
98 319 73 125 141 270 137 175
XB, lethally irradiated,
lar basis of the enhancement of the antibody activation of these cells is known to take place. response in vitro with cells from C. parvum- This conclusion is supported by the findings treated mice (20, 24). that macrophages from mice treated with C. The way in which activated macrophages parvum (20, 24) or infected with BCG (Watson, stimulate the antibody response in vitro is un- Brown, and Sljivi6, unpublished data) or Salcertain at this time. One aspect that should be monella typhimurium (Watson, Plant, Barrett remembered, however, when considering the and Sljivi6, unpublished) have all the properpossible mechanisms is the finding that only ties described above. The significance of these the antibody response to SRBC, a macrophage observations as well as the mechanisms (19) and thymus-dependent (5) antigen, was whereby activated macrophages influence lymenhanced, whereas the response to DNP-POL, phocyte function remains to be elucidated. which is independent of these two cell types (11, ACKNOWLEDGMENTS 13), was not affected. This would indicate that, We thank Peter Church and Maureen Philbert for their whatever mechanism is responsible for the enin these experiments. S.R.W. was in receipt of a hancement of the response, it is not operating assistanceResearch Council scholarship. I.N.B. is supported directly via B lymphocytes. One possibility is Medical by the Wellcome Trust. that activated macrophages present the macroLITERATURE CITED phage-dependent antigen in a manner that is more efficient for the induction of the response 1. Allison, A. C., J. S. Harington, and M. Birbeck. 1966. An examination of the cytotoxic effects of silica on by lymphocytes. A difference in the handling of macrophages. J. Exp. Med. 124:141-154. keyhole limpet hemocyanin by macrophages 2. Allison, A. C., and M. R. Young. 1969. Vital staining from mice treated with C. parvum has been and fluorescence microscopy of lysosomes, p. 600-628. In J. T. Dingle and H. B. Fell (ed.), Lysosomes in described (26), but not in the case of macrobiology and pathology, vol. 2. North-Holland Publishphages stimulated by other adjuvants (23). Aning Co., Amsterdam. other possibility is related to the production 3. Brown, I. N., and H.-N. Krenzien. 1976. Systemic Myand release by macrophages of soluble factors cobacterium lepraemurium infection in mice: differences in doubling time in liver, spleen, and bone that can affect various lymphocyte functions (4, marrow, and a method for measuring the proportion 12, 14). Activated macrophages may be either of viable organisms in an inoculum. Infect. Immun. releasing a greater amount of such factors or 13:480-486. producing factors that are more effective in con- 4. Calderon, J., J.-M. Kiely, J. L. Lefko, and E. R. Unanue. 1975. The modulation of lymphocyte functions trolling the in vitro responses of lymphocytes. by molecules secreted by macrophages. I. Description Any such factors would have to act on T lymand partial biochemical analysis. J. Exp. Med. phocytes in view of the lack of enhancement of 142:151-164. the response to DNP-POL. 5. Claman, H. N., and E. A. Chaperon. 1969. Immunologic complementation between thymus and marrow If activated macrophages have the general cells -a model for the two cell theory of immunocomability to enhance the antibody response to cerpetence. Transplant. Rev. 1:92-113. tain antigens, one would expect a similar en- 6. Closs, 0., and 0. A. Haugen. 1974. Experimental muhancement in other situations in which the rine leprosy. 2. Further evidence for varying suscepti-
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ANTIBODY RESPONSES IN MURINE LEPROSY
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