Acta path. microbiol. scand. Sect. C, 84: 124 150, 1976
MACROPHAGE PROLIFERATION AND ACTIVATION DURING T O X O P L A S M A GONDZZ INFECTION IN MICE: RELATIONSHIP TO LYMPHOCYTE STIMULATION ASMUND REIKVAM Norwegian Defence Microbiological Laboratory, Oslo and Institute of Physiology, University of Oslo, Oslo, Norway
Reikvam, A. Macrophage proliferation and activation during Toxoplusma gondii infection in mice: Relationship to lymphocyte stimulation. Acta path. microhiol. scand. Sect. C, 84: 124130, 1976. Macrophage proliferation and activation as well as lymphocyte stimulation in the peritoneal cavities of mice were investigated during the course of a Toxoplasma gondii (Beverly strain) infection. Macrophage proliferation had started already after day one and reached a first peak on day 2 (3H-thymidine labelling index -6 per cent). This proliferation was not accompanied by any notable lymphocyte stimulation. An equally high L.I. for macrophages was found after injection of 0.9 per cent saline. From day 3-4 and peaking on day 7-11, a considerable blastoid transformation of lymphocytes occurred (maximum L.I. for lymphoid cells -20 per cent on day 7). In parallel with this blastoid response, a substantial macrophage proliferation took place (L.I. -8 per cent). Large numbers of activated macrophages also appeared during this period. DNA synthesizing cells were found even among the most highly activated macrophages. The results indicated that the early and the late macrophage proliferations were stimulated by different mechanisms. Key words: Toxoplusma gondii infection; macrophage proliferation ; macrophage activation; lymphocyte stimulation; mice.
A. Reikvam, Institute of Physiology, University of Oslo, Karl Johansgt. 47, Oslo 1, Norway.
Received 15.x.75
Accepted 8.xi.75
The importance of macrophages for the resistance to infections and neoplasms has been clearly demonstrated ( 2 2 ) . In the combat of intracellular micro-organisms, macrophages are particularly important since they are the ultimate killer cells in such infections. I n order to perform this task the macrophages must, however, be activated (9, 16,
18, 26, 27). 124
Toxoplasma gondii is an obligate intracellular protozoon which can be suppressed only if an adequate cellular immune response ensues (7, 12, 1 3 ) . I n a previous study it was shown that infection with this protozoon (Beverly strain) killed 15-30 per cent of the mice, deaths usually occurring between day 7 and 14 (26). The macrophages from mice surviving a Toxoplasma infection for 14 days were highly activated and had a n increased
capacity to kill another Toxoplasma strain (RH strain) and a strain of Listeria monocytogenes. The immune apparatus is thus subjected to a most severe test in mice going through this type of infection. The present study was undertaken in order to examine macrophage proliferation and activation in the course of this critical infection. Another aim was to investigate the temporal relationship between the macrophage response and lymphocyte stimulation. This again might elucidate the process of macrophage proliferation, the controlling of which is poorly understood. MATERIALS AND METHODS
Animals Male NMRI/Bom mice, aged 1-4 months, weighing 20-30 gram, were used in all experiments. Infection Procedure Toxoplasma gondii, strain Beverly, was passed to new mice every third month. Brains of infected mice were dissected out, and homogenized with 0.9 per cent NaCl in a mortar. The number of brain cysts per drop was counted and by dilution adjusted so as to give 5 cysts per 0.3 ml, which was the volume injected intraperitoneally to each new mouse. Harvesting of Peritoneal Cells At various times of Toxoplasma infection, the mice were killed and the peritoneal cells harvested. Cells destined for biochemical assays were harvested into medium 199 (Flow laboratories) containing 20 per cent foetal calf serum (Gibco), 50 I.U./ml of crystalline penicillin (Glaxo) and 50 pgram/ml of streptomycin (Glaxo). Cells to be used for evaluation of DNA-synthesis were harvested into the same type of medium containing in addition 3Hthymidine, 3 pCi/ml (Specific activity SCi/m-mol). Cells for biochemical assay were cultured on the bottom of Leighton tubes, while cells for autoradiography were cultured on 9 x 35 mm “flying cover slips” within the Leighton tubes. Further details have been given elsewhere (25, 26). The cells were incubated at 37” C in an atmosphere of 5 per cent CO, in air. After 1% hours, the cells for autoradiography were handled in the following manner: The Leighton tubes were agitated vigorously, the nonadherent cells were harvested and the adherent cells were washed twice with 2 ml medium 199. The non-adherent cells
from each Leighton tube were centrifuged, a drop of bovine albumine was added to the cell sediment, and smears were made. These smears, as well as the cover slips containing the adherent cells, were processed for autoradiography. The cells for biochemical assays were cultured for one hour. The non-adherent cells were then discarded and the adherent ones washed three times with 2 ml of 0.9 per cent NaCI. Subsequently the cells were wiped off the glass into 2 ml saline with a rubber policeman. These cell suspensions were stored at -20” C.
Autoradiography Autoradiography was performed by the dipping technique using an Ilford L 4 size “A” film. After exposure for 7 days, the films were developed and stained with Giemsa. Cells with 5 or more grains overlaying the nucleus were scored as labelled. In each coded smear, 300 cells were scored. The percentages of labelled cells of the respective cell populations are expressed as the labelling index (L.I.)
.
Biochemical Assay The cells were frozen and thawed 10 times on a methanol dry ice bath in order to destroy the cells. Acid phosphatase (E.C. 3.1.3.2.) was assayed with p-glycerophosphate as substrate (2) using an incubation period of 4 hours. The liberated phosphate was quantitated by the method of Fiske & Subbarrow ( 5 ) . Protein was measured by the method of Lowry et al. using crystalline bovine serum albumin as a standard (15). More details have been given previously (26). Cytochemistry and Autoradiography In order to evaluate the acid phosphatase content of the individual DNA-synthesizing cells, the following double-labelling technique was performed: The cover slips containing macrophages were fixed in acetone and then stained, using the naphtol-AS-BI-phosphate method ( 3 ) . The cells were exposed to the substrate for 20 minutes. After staining the cells were submitted to the ordinary procedure for autoradiography. Colloidal Carbon In order to identify phagocytic cells, the ability to take up colloidal carbon ( C 11/1432 a, Gunther Wagner, Pelikan, dilution 1:50) was examined. RESULTS
All mice had signs of a severe infection during the second week. More than 50 per cent died and were not included in the present study. 125
ance of activated cells, predominated (Fig. 2A). The peritoneal lymphocytes had the appearance of small lymphocytes until days 3-4 (Fig. 1B). Notable numbers of lymphoblasts then appeared and on day 7 and 11 a strong blastoid response was observed (Fig. 2C), the majority of the lymphocytes being blasts. Thereafter the blast response abated. In addition, granulocytes were seen, particularly during the first days. They were mainly present in the non-adherent cell population. Only a minor proportion was glassadherent after the vigorous shaking of the Leighton tubes. Proliferation of Lymphocytes and Macrophages The macrophage population showed a considerable proliferation already after one day
Fig. 1. Peritoneal macrophages and lymphocytes 2 days after i.p. immunization with Toxoplasma cysts. A ) Macrophages. B ) Lymphocytes. Only small lymphocytes are seen.
Morphology of the Peritoneal Lymphocytes and Macrophages The macrophages were identified by the functional test of their ability to adhere to glass, instead of relying merely on morphological criteria. In addition, the phagocytic ability of the adherent cells was examined in some experiments, and virtually 100 per cent of them were heavily phagocytosing carbon. The macrophages did not change in appearance during the first two days (Fig. 1A). On day 3 many of the adhesive cells had a more monocyte-like appearance and less cytoplasm than the macrophages observed the preceding days. Later on, particularly on day 7 and 11, a more heterogeneous picture was found, but large macrophages, the appear126
Fig. 2. Peritoneal macrophages and lymphocytes 1 1 days after i.p. immunization with Toxoplasma cysts. A ) Macrophages. Considerable variation in cell size. Note large cells. B ) DNA-synthesizing macrophages. Macrophages differing markedly in cell size were synthesizing DNA. C) Lymphocytes. Note DNA synthesizing blasts.
10
phages, DNA-synthesizing cells were found (Fig. 2B).
%
8
Acid Phosphatase
I
i
2
4 DAYS
6
8
10
12
14
21
OF INFECTION
Fig. 3. Labelling indices (L.I.) of peritoneal macrophages after i.p. immunization with T. gondii. Each mouse received approximately 5 brain cysts suspended in 0.9 per cent saline intraperitoneally. The peritoneal macrophages were adhered to cover slips and thereby separated from the non-adherent cells by culturing for 1% hours in vitro (3H-thymidine containing medium). Control mice received 1 ml 0.9 per cent saline i.p. Means f 1 SE are given. At least 4 mice in each point. -.@ T . gondii. 0- -0NaC1.
of Macrophages
Acid phosphatase content of macrophages, measured biochemically on a per mg protein basis, was markedly and significantly increased on day 11 (Table 1). A slight but not significant increase was found on day 4 and 21. Cytochemical staining for acid phosphatase, performed on day 11, showed that the macrophages stained rather strongly. The staining intensity varied somewhat from cell to cell. Autoradiography performed on these smears did not reveal any particular pattern as to the acid phosphatase content of DNAsynthesizing cells. Even cells which were most intensely stained were found to synthesize
DNA. DISCUSSION
(Fig. 3 ) . On day 2, a further increase was found, whereafter a drop in the L.I. was observed from day 2 to 3. The macrophage proliferation observed on the first two days was not accompanied by any obvious lymphocyte stimulation (Fig. 4). From days 3-4, an increasing L.I. was found in the lymphocyte population and also in the macrophage population (Fig. 3 and 4). Even 3 weeks after the start of the infection, a considerable degree of proliferation was found in both cell populations. In control mice injected with NaCl (sterile and pyrogen-free), a substantial macrophage proliferation was also found. Indeed, the initial proliferation rate in this group was as high as in the Toxoplasma infected mice. However, the proliferative response in the control mice fell back to negligible levels on day 4.This macrophage proliferation was not accompanied by any notable lymphocyte stimulation. The tritium-labelled macrophages did not show any particular morphological characteristics as compared with the non-labelled macrophages in a given culture. Even among the largest, most typically activated macro-
The final aim of the immune response against the obligate intracellular T. gondii is to provide macrophages capable of killing, or at least restricting, its intracellular growth. However, the mice surviving the acute infection have not totally eliminated the protozoon. In these mice, the protozoon goes into
21
-
% 18 v)
2 15E 12I
3
8
9-
3p ,
-*4
2
NaCl
,
,
,
4 6 8 10 U Y S OF INFECTION
,
12
, ,:
14
: 21
Fig. 4. Labelling indices of lymphocytes in the same peritoneal cell populations as in Fig. 2.
127
TABLE 1. Acid Phosphatase Activity in Peritoneal Macrophages after Immunization with Toxoplasma qondii
the possibility must be considered that a nonproliferative activated macrophage population coexisted with a proliferative nonactivated population. However, even among Enzyme activity Days after the largest and most typically activated mac(mU*/mg protein) immunization Mean** f SE rophages, DNA-synthesizing cells were found. Hence, proliferation seems to be a charac0 (non-immun.) 2.2 f 0.2 teristic feature of the activated macrophages 4 2.8 zk 0.6 themselves. 11 11.3 f 2.6 21 3.2 f 0.7 How then is macrophage proliferation and activation initiated and regulated? Activa* One unit of activity will hydrolyse one pmole tion has been assumed to be due to soluble of substrate in one minute. ** The enzyme activity in macrophages from 4-10 mediators (lymphokines) released from the lymphoblasts. In uitro experiments have conmice was determined on each of the days. firmed this assumption and it has clearly been demonstrated that supernatants from stima cystic stage and remains in the body, prob- ulated lymphocytes can induce the typical ably for life (8, Reikvam (unpublished re- features associated with activation, namely sults) ). The present study shows that local morphological changes (20, 2 1) , increased macrophage proliferation is a prominent, and content of lysosomes (20), and increased miprobably important, event in the response crobicidal capacity (9, l l , 27). of the animals to this infection. It also shows Stimulation of macrophage proliferation that this macrophage proliferation as well has also been suggested to be caused by lymas the lymphocyte stimulation are protracted phokines (19, 24), but this has been more responses in toxoplasma-infected mice. The difficult to demonstrate in vitro since macropeak of the responses occurred during or phages are reluctant to divide when adhered slightly before the most critical post-infection to glass or plastic surfaces. However, Coda1 period when many of the mice succumbed. et al. (9) found an increase in the number The time course of the macrophage proli- of adherent macrophages after addition of feration observed after 3-4 days shows some lymphocyte supernatants and recently, DNA resemblance to that reported to occur in mice synthesis was demonstrated in adherent macinfected with BCG (23), but is considerably rophages after addition of inflammation exmore protracted. However, in that study udate (1, 29). The exact nature of the mito(23), no distinction between peritoneal lym- genic factor was not explored in the latter phocytes and macrophages was made and studies. The present experiments showed that the BCG-infection was established by the the macrophage proliferation observed after intravenous route. about 3 days occurred in parallel with a Activated macrophages, as judged by mor- strong lymphocyte stimulation, and thus phology and by an increased content of acid might indicate a causal role for mediators phosphatase as well as by microbicidal capa- from lymphoblasts. city (26), were present in the second week The macrophage proliferation found durof the infection. At this time, a considerable ing the first two days of Toxoplasma infecmacrophage proliferation also took place. tion does not, however, fit in with this Macrophage activation might be regarded as scheme. I t was not accompanied by any nota cytoplasmic specialization designed to im- able lymphocyte stimulation and equally high prove the microbicidal and cytotoxic capa- proliferation rates could be obtained by the city of the cells (10, 17). In many systems, mere injection of saline. Such a rapid onset specialization is accompanied by a loss of of proliferation in liver macrophages (Kupfproliferative capacity. I n the present study, fer cells) has also been reported (4,14, 28). 128
Moreover, oestrogens, both natural and synthetic ones, are potent stimulators of the mononuclear phagocytes and can induce DNA synthesis in the liver macrophages after a lag phase of 16 hours (28, 14). Forbes reported proliferation of peritoneal macrophages of mice after repeated punctures of the peritoneal cavities (6). I t is difficult to see that lymphokines should be operating under these circumstances. Furthermore, lymphocyte transfer experiments, using Listeria monocytogenes as infecting agents, have demonstrated that lymphocytes from infected mice can confer resistance to normal mice, acting mainly by stimulating macrophages. However, such sensitized lymphocytes were not generated until 3 to 5 days after the start of the infection (16, 30). This time interval corresponds to that seen in the present study for onset of lymphocyte stimulation. Consequently, it seems unlikely that in the present experiments a primary stimulation of lymphocytes resulting in the production of a mitogenic factor, the subsequent release of this factor and finally stimulation of macrophages to demonstrable DNA synthesis can have occurred within 24 hours. In conclusion therefore, it seems pertinent to suggest that macrophage proliferation may be controlled in different ways. First, a shortlatency macrophage proliferation can be induced by appropriate, but yet not welI-defined, stimulators. The primary target for these stimulators seems to be the monocyte/ macrophage cell line. Second, macrophage proliferation can take place in the effector phase of a cellular immune response and, in this situation, presumably as a result of the influence of mediators from stimulated lymphocytes. During this type of proliferation activated macrophages appear, and the activated cells themselves are capable of cell division. The skilful technical assistance of Eric Toogood is gratefully appreciated. The work was supported by grants from T h e Normegian Research Council f o r Science and the Humanities, Anders Jahre’s Fund f or the promotion of Science, Crosserer Thor Dahl‘s fund and
from Direktrr Gotfred Lie and Hustru Marie Lie’s fund. This support is gratefully acknowledged.
REFERENCES 1. Adolphe, M., Fontagne, J., Pelletier, M . &
2.
3.
4.
5. 6.
7. 8.
9.
10.
11.
2. 3. 4.
15.
Giroud, J . P.: Induction of DNA synthesis in rat macrophages in vitro by inflammatory exudate. Nature (Lond.) 253: 637, 1975. Appelmans, F., Wattiaux, R . & DeDuve, C.: Tissue fractionating studies. 5. The association of acid phosphatase with a special class of cytoplasmic granules in rat liver. Biochem. J. 59: 438-445, 1955. Bancroft, J . D.: An introduction to histochemical techniques. Butterworth, London 1967, pp. 192-194. Donald, K . J . & Pound, A . W.: Proliferation and migration of reticuloendothelial cells following injection of a tubercle bacillary lipid. Br. J. exp. Path. 54: 79-89, 1973. Fiske, C. H . & Subbarrow, Y.: The colorimetric determination of phosphorus. J. biol. Chem. 66: 375-400, 1925. Forbes, I . J.: Mitoses in mouse peritoneal macrophages. J. Immunol. 96: 734-743, 1966. Frenkel, J. K.: Adoptive immunity to intracellular infection. J. Immunol. 98: 1309-1318, 1967. Frenkel, 1. K.: Models for infectious diseases. Fed. Proc. 28: 179-190, 1969. Godal, T., Rees, R . J . W . & Lamuik, J . 0.: Lymphocyte-mediated modification of bloodderived macrophage function in vitro; inhibition of growth of intracellular mycobacteria with lymphokines. Clin. exp. Immunol. 8: 625-637, 1971. Hibbs, J . B. jr,, Lambert, L. H . jr. & Remington, J . S.: Possible role of macrophage mediated nonspecific cytotoxicity in tumour resistance. Nature (Lond.) 235: 48-50, 1972. lones. T . & Youmans. G . P.: The in vitro inhibition of growth of intracellular Listeria monoctogenes by lymphocyte products. Cell. Immunol. 9: 353-362, 1973. Jones, T . C.: Macrophages and intracellular parasitism. J. reticulwndoth. SOC. 15: 439450, 1974. Jones, T . C., Len, L . & Hirsch, J . G.: Assessment in vitro of immunity against Toxoplasma gondii. J. exp. Med. 141: 466-482, 1975. Kelly, L. S., Dobson, E. L., Finney, C. R . & Hirsch, J . D.: Proliferation of the reticuloendothelid system in the liver. Amer. J. Physiol. 198: 1134-1138, 1960. Lowry, 0. H . , Rosebrough, N . J , , Farr, A . L . & Randall, R . J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193: 265-275, 1951.
. ,
129
16. Mackaness, G . B.: The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. exp. Med. 129: 973-992, 1969. 17. Mackaness, G . B.: Cellular immunity. In: Furth, R. van (Ed.) : Mononuclear phagocytes. Blackwell Scientific Publications, Oxford 1970, pp. 461-476. 18. McGregor, D . D . & Koster, F. T.: The mediator of cellular immunity. IV Cooperation between lymphocytes and mononuclear phagocytes. Cell. Immunol. 2: 317-325, 1971. 19. More, D . G., Penrose, I. M . , Kearney, R . & Nelson, D . S.: Immunological induction of DNA synthesis in mouse peritoneal macrophages. Int. Arch. Allergy 44: 611-630, 1973. 20. Nath, I., Poulter, L. W . & Turk, J . L.: Effect of lymphocyte mediators on macrophages in vitro. A correlation of morphological and cytochemical changes. Clin. exp. Immunol. 13: 455-466, 1973. 21. Nathan, C. F., Karnovsky, M . L, & David, I. R.: Alterations of macrophage functions by mediators from lymphocytes. J. exp. Med. 133: 1356-1376, 1971. 22. NeZson, D . S.: Immunity to infection, allograft immunity and tumour immunity: parallels and contrasts. Transplant. Rev. 19: 226254, 1974. 23. North, R . 1.: Cellular kinetics associated with the development of acquired cellular resistance. J. exp. Med. 130: 299-314, 1969.
24. North, R . 1.: The mitotic potential of fixed phagocytes in the liver as revealed during the development of cellular immunity. J. exp. Med. 130: 315-326, 1969. 25. Reikvam, A . & Hoiby, E. A.: Phagocytosis and rnicrobicidal capacity of mouse macrophages non-specifically activated in vitro. Acta path. microbiol. scand. Sect. C, 83: 121-128, 1975. 26. Reikvam, A., Grammeltvedt, R . & Hoiby, E. A . : Activated mouse macrophages: morphology, lysosomal biochemistry and microbicidal properties of in vivo and in vitro activated cells. Acta path. microbiol. scand. Sect. C , 83: 129-138, 1975. 27. Simon, H . B. & Sheagren, J . N.: Cellular immunity in vitro. I. Immunologically mediated enhancement of macrophage bactericidal capacity. J. exp. Med. 133: 1377-1389, 1971. 28. Warr, G. W . & SljiviC, V . S.: Origin and division of liver macrophages during stimulation of the mononuclear phagocyte system. Cell Tissue Kinet. 7: 559-565, 1974. 29. Wynne, K . M . , Spector, W . G . & Willoughby, D . A.: Macrophage proliferation in vitro induced by exudates. Nature (Lond.) 253: 636-637, 1975. 30. Zinkernagel, R. M., Blanden, R . V . & Langman, R . E.: Early appearance of sensitized lymphocytes in mouse infected with Listeria monocytogenes. J. Immunol. 112: 496-501, 1974.
MACROPHAGE PROLIFERATION AND ACTIVATION DURING T O X O P L A S M A GONDZZ INFECTION IN MICE: RELATIONSHIP TO LYMPHOCYTE STIMULATION ASMUND REIKVAM Norwegian Defence Microbiological Laboratory, Oslo and Institute of Physiology, University of Oslo, Oslo, Norway
Reikvam, A. Macrophage proliferation and activation during Toxoplusma gondii infection in mice: Relationship to lymphocyte stimulation. Acta path. microhiol. scand. Sect. C, 84: 124130, 1976. Macrophage proliferation and activation as well as lymphocyte stimulation in the peritoneal cavities of mice were investigated during the course of a Toxoplasma gondii (Beverly strain) infection. Macrophage proliferation had started already after day one and reached a first peak on day 2 (3H-thymidine labelling index -6 per cent). This proliferation was not accompanied by any notable lymphocyte stimulation. An equally high L.I. for macrophages was found after injection of 0.9 per cent saline. From day 3-4 and peaking on day 7-11, a considerable blastoid transformation of lymphocytes occurred (maximum L.I. for lymphoid cells -20 per cent on day 7). In parallel with this blastoid response, a substantial macrophage proliferation took place (L.I. -8 per cent). Large numbers of activated macrophages also appeared during this period. DNA synthesizing cells were found even among the most highly activated macrophages. The results indicated that the early and the late macrophage proliferations were stimulated by different mechanisms. Key words: Toxoplusma gondii infection; macrophage proliferation ; macrophage activation; lymphocyte stimulation; mice.
A. Reikvam, Institute of Physiology, University of Oslo, Karl Johansgt. 47, Oslo 1, Norway.
Received 15.x.75
Accepted 8.xi.75
The importance of macrophages for the resistance to infections and neoplasms has been clearly demonstrated ( 2 2 ) . In the combat of intracellular micro-organisms, macrophages are particularly important since they are the ultimate killer cells in such infections. I n order to perform this task the macrophages must, however, be activated (9, 16,
18, 26, 27). 124
Toxoplasma gondii is an obligate intracellular protozoon which can be suppressed only if an adequate cellular immune response ensues (7, 12, 1 3 ) . I n a previous study it was shown that infection with this protozoon (Beverly strain) killed 15-30 per cent of the mice, deaths usually occurring between day 7 and 14 (26). The macrophages from mice surviving a Toxoplasma infection for 14 days were highly activated and had a n increased
capacity to kill another Toxoplasma strain (RH strain) and a strain of Listeria monocytogenes. The immune apparatus is thus subjected to a most severe test in mice going through this type of infection. The present study was undertaken in order to examine macrophage proliferation and activation in the course of this critical infection. Another aim was to investigate the temporal relationship between the macrophage response and lymphocyte stimulation. This again might elucidate the process of macrophage proliferation, the controlling of which is poorly understood. MATERIALS AND METHODS
Animals Male NMRI/Bom mice, aged 1-4 months, weighing 20-30 gram, were used in all experiments. Infection Procedure Toxoplasma gondii, strain Beverly, was passed to new mice every third month. Brains of infected mice were dissected out, and homogenized with 0.9 per cent NaCl in a mortar. The number of brain cysts per drop was counted and by dilution adjusted so as to give 5 cysts per 0.3 ml, which was the volume injected intraperitoneally to each new mouse. Harvesting of Peritoneal Cells At various times of Toxoplasma infection, the mice were killed and the peritoneal cells harvested. Cells destined for biochemical assays were harvested into medium 199 (Flow laboratories) containing 20 per cent foetal calf serum (Gibco), 50 I.U./ml of crystalline penicillin (Glaxo) and 50 pgram/ml of streptomycin (Glaxo). Cells to be used for evaluation of DNA-synthesis were harvested into the same type of medium containing in addition 3Hthymidine, 3 pCi/ml (Specific activity SCi/m-mol). Cells for biochemical assay were cultured on the bottom of Leighton tubes, while cells for autoradiography were cultured on 9 x 35 mm “flying cover slips” within the Leighton tubes. Further details have been given elsewhere (25, 26). The cells were incubated at 37” C in an atmosphere of 5 per cent CO, in air. After 1% hours, the cells for autoradiography were handled in the following manner: The Leighton tubes were agitated vigorously, the nonadherent cells were harvested and the adherent cells were washed twice with 2 ml medium 199. The non-adherent cells
from each Leighton tube were centrifuged, a drop of bovine albumine was added to the cell sediment, and smears were made. These smears, as well as the cover slips containing the adherent cells, were processed for autoradiography. The cells for biochemical assays were cultured for one hour. The non-adherent cells were then discarded and the adherent ones washed three times with 2 ml of 0.9 per cent NaCI. Subsequently the cells were wiped off the glass into 2 ml saline with a rubber policeman. These cell suspensions were stored at -20” C.
Autoradiography Autoradiography was performed by the dipping technique using an Ilford L 4 size “A” film. After exposure for 7 days, the films were developed and stained with Giemsa. Cells with 5 or more grains overlaying the nucleus were scored as labelled. In each coded smear, 300 cells were scored. The percentages of labelled cells of the respective cell populations are expressed as the labelling index (L.I.)
.
Biochemical Assay The cells were frozen and thawed 10 times on a methanol dry ice bath in order to destroy the cells. Acid phosphatase (E.C. 3.1.3.2.) was assayed with p-glycerophosphate as substrate (2) using an incubation period of 4 hours. The liberated phosphate was quantitated by the method of Fiske & Subbarrow ( 5 ) . Protein was measured by the method of Lowry et al. using crystalline bovine serum albumin as a standard (15). More details have been given previously (26). Cytochemistry and Autoradiography In order to evaluate the acid phosphatase content of the individual DNA-synthesizing cells, the following double-labelling technique was performed: The cover slips containing macrophages were fixed in acetone and then stained, using the naphtol-AS-BI-phosphate method ( 3 ) . The cells were exposed to the substrate for 20 minutes. After staining the cells were submitted to the ordinary procedure for autoradiography. Colloidal Carbon In order to identify phagocytic cells, the ability to take up colloidal carbon ( C 11/1432 a, Gunther Wagner, Pelikan, dilution 1:50) was examined. RESULTS
All mice had signs of a severe infection during the second week. More than 50 per cent died and were not included in the present study. 125
ance of activated cells, predominated (Fig. 2A). The peritoneal lymphocytes had the appearance of small lymphocytes until days 3-4 (Fig. 1B). Notable numbers of lymphoblasts then appeared and on day 7 and 11 a strong blastoid response was observed (Fig. 2C), the majority of the lymphocytes being blasts. Thereafter the blast response abated. In addition, granulocytes were seen, particularly during the first days. They were mainly present in the non-adherent cell population. Only a minor proportion was glassadherent after the vigorous shaking of the Leighton tubes. Proliferation of Lymphocytes and Macrophages The macrophage population showed a considerable proliferation already after one day
Fig. 1. Peritoneal macrophages and lymphocytes 2 days after i.p. immunization with Toxoplasma cysts. A ) Macrophages. B ) Lymphocytes. Only small lymphocytes are seen.
Morphology of the Peritoneal Lymphocytes and Macrophages The macrophages were identified by the functional test of their ability to adhere to glass, instead of relying merely on morphological criteria. In addition, the phagocytic ability of the adherent cells was examined in some experiments, and virtually 100 per cent of them were heavily phagocytosing carbon. The macrophages did not change in appearance during the first two days (Fig. 1A). On day 3 many of the adhesive cells had a more monocyte-like appearance and less cytoplasm than the macrophages observed the preceding days. Later on, particularly on day 7 and 11, a more heterogeneous picture was found, but large macrophages, the appear126
Fig. 2. Peritoneal macrophages and lymphocytes 1 1 days after i.p. immunization with Toxoplasma cysts. A ) Macrophages. Considerable variation in cell size. Note large cells. B ) DNA-synthesizing macrophages. Macrophages differing markedly in cell size were synthesizing DNA. C) Lymphocytes. Note DNA synthesizing blasts.
10
phages, DNA-synthesizing cells were found (Fig. 2B).
%
8
Acid Phosphatase
I
i
2
4 DAYS
6
8
10
12
14
21
OF INFECTION
Fig. 3. Labelling indices (L.I.) of peritoneal macrophages after i.p. immunization with T. gondii. Each mouse received approximately 5 brain cysts suspended in 0.9 per cent saline intraperitoneally. The peritoneal macrophages were adhered to cover slips and thereby separated from the non-adherent cells by culturing for 1% hours in vitro (3H-thymidine containing medium). Control mice received 1 ml 0.9 per cent saline i.p. Means f 1 SE are given. At least 4 mice in each point. -.@ T . gondii. 0- -0NaC1.
of Macrophages
Acid phosphatase content of macrophages, measured biochemically on a per mg protein basis, was markedly and significantly increased on day 11 (Table 1). A slight but not significant increase was found on day 4 and 21. Cytochemical staining for acid phosphatase, performed on day 11, showed that the macrophages stained rather strongly. The staining intensity varied somewhat from cell to cell. Autoradiography performed on these smears did not reveal any particular pattern as to the acid phosphatase content of DNAsynthesizing cells. Even cells which were most intensely stained were found to synthesize
DNA. DISCUSSION
(Fig. 3 ) . On day 2, a further increase was found, whereafter a drop in the L.I. was observed from day 2 to 3. The macrophage proliferation observed on the first two days was not accompanied by any obvious lymphocyte stimulation (Fig. 4). From days 3-4, an increasing L.I. was found in the lymphocyte population and also in the macrophage population (Fig. 3 and 4). Even 3 weeks after the start of the infection, a considerable degree of proliferation was found in both cell populations. In control mice injected with NaCl (sterile and pyrogen-free), a substantial macrophage proliferation was also found. Indeed, the initial proliferation rate in this group was as high as in the Toxoplasma infected mice. However, the proliferative response in the control mice fell back to negligible levels on day 4.This macrophage proliferation was not accompanied by any notable lymphocyte stimulation. The tritium-labelled macrophages did not show any particular morphological characteristics as compared with the non-labelled macrophages in a given culture. Even among the largest, most typically activated macro-
The final aim of the immune response against the obligate intracellular T. gondii is to provide macrophages capable of killing, or at least restricting, its intracellular growth. However, the mice surviving the acute infection have not totally eliminated the protozoon. In these mice, the protozoon goes into
21
-
% 18 v)
2 15E 12I
3
8
9-
3p ,
-*4
2
NaCl
,
,
,
4 6 8 10 U Y S OF INFECTION
,
12
, ,:
14
: 21
Fig. 4. Labelling indices of lymphocytes in the same peritoneal cell populations as in Fig. 2.
127
TABLE 1. Acid Phosphatase Activity in Peritoneal Macrophages after Immunization with Toxoplasma qondii
the possibility must be considered that a nonproliferative activated macrophage population coexisted with a proliferative nonactivated population. However, even among Enzyme activity Days after the largest and most typically activated mac(mU*/mg protein) immunization Mean** f SE rophages, DNA-synthesizing cells were found. Hence, proliferation seems to be a charac0 (non-immun.) 2.2 f 0.2 teristic feature of the activated macrophages 4 2.8 zk 0.6 themselves. 11 11.3 f 2.6 21 3.2 f 0.7 How then is macrophage proliferation and activation initiated and regulated? Activa* One unit of activity will hydrolyse one pmole tion has been assumed to be due to soluble of substrate in one minute. ** The enzyme activity in macrophages from 4-10 mediators (lymphokines) released from the lymphoblasts. In uitro experiments have conmice was determined on each of the days. firmed this assumption and it has clearly been demonstrated that supernatants from stima cystic stage and remains in the body, prob- ulated lymphocytes can induce the typical ably for life (8, Reikvam (unpublished re- features associated with activation, namely sults) ). The present study shows that local morphological changes (20, 2 1) , increased macrophage proliferation is a prominent, and content of lysosomes (20), and increased miprobably important, event in the response crobicidal capacity (9, l l , 27). of the animals to this infection. It also shows Stimulation of macrophage proliferation that this macrophage proliferation as well has also been suggested to be caused by lymas the lymphocyte stimulation are protracted phokines (19, 24), but this has been more responses in toxoplasma-infected mice. The difficult to demonstrate in vitro since macropeak of the responses occurred during or phages are reluctant to divide when adhered slightly before the most critical post-infection to glass or plastic surfaces. However, Coda1 period when many of the mice succumbed. et al. (9) found an increase in the number The time course of the macrophage proli- of adherent macrophages after addition of feration observed after 3-4 days shows some lymphocyte supernatants and recently, DNA resemblance to that reported to occur in mice synthesis was demonstrated in adherent macinfected with BCG (23), but is considerably rophages after addition of inflammation exmore protracted. However, in that study udate (1, 29). The exact nature of the mito(23), no distinction between peritoneal lym- genic factor was not explored in the latter phocytes and macrophages was made and studies. The present experiments showed that the BCG-infection was established by the the macrophage proliferation observed after intravenous route. about 3 days occurred in parallel with a Activated macrophages, as judged by mor- strong lymphocyte stimulation, and thus phology and by an increased content of acid might indicate a causal role for mediators phosphatase as well as by microbicidal capa- from lymphoblasts. city (26), were present in the second week The macrophage proliferation found durof the infection. At this time, a considerable ing the first two days of Toxoplasma infecmacrophage proliferation also took place. tion does not, however, fit in with this Macrophage activation might be regarded as scheme. I t was not accompanied by any nota cytoplasmic specialization designed to im- able lymphocyte stimulation and equally high prove the microbicidal and cytotoxic capa- proliferation rates could be obtained by the city of the cells (10, 17). In many systems, mere injection of saline. Such a rapid onset specialization is accompanied by a loss of of proliferation in liver macrophages (Kupfproliferative capacity. I n the present study, fer cells) has also been reported (4,14, 28). 128
Moreover, oestrogens, both natural and synthetic ones, are potent stimulators of the mononuclear phagocytes and can induce DNA synthesis in the liver macrophages after a lag phase of 16 hours (28, 14). Forbes reported proliferation of peritoneal macrophages of mice after repeated punctures of the peritoneal cavities (6). I t is difficult to see that lymphokines should be operating under these circumstances. Furthermore, lymphocyte transfer experiments, using Listeria monocytogenes as infecting agents, have demonstrated that lymphocytes from infected mice can confer resistance to normal mice, acting mainly by stimulating macrophages. However, such sensitized lymphocytes were not generated until 3 to 5 days after the start of the infection (16, 30). This time interval corresponds to that seen in the present study for onset of lymphocyte stimulation. Consequently, it seems unlikely that in the present experiments a primary stimulation of lymphocytes resulting in the production of a mitogenic factor, the subsequent release of this factor and finally stimulation of macrophages to demonstrable DNA synthesis can have occurred within 24 hours. In conclusion therefore, it seems pertinent to suggest that macrophage proliferation may be controlled in different ways. First, a shortlatency macrophage proliferation can be induced by appropriate, but yet not welI-defined, stimulators. The primary target for these stimulators seems to be the monocyte/ macrophage cell line. Second, macrophage proliferation can take place in the effector phase of a cellular immune response and, in this situation, presumably as a result of the influence of mediators from stimulated lymphocytes. During this type of proliferation activated macrophages appear, and the activated cells themselves are capable of cell division. The skilful technical assistance of Eric Toogood is gratefully appreciated. The work was supported by grants from T h e Normegian Research Council f o r Science and the Humanities, Anders Jahre’s Fund f or the promotion of Science, Crosserer Thor Dahl‘s fund and
from Direktrr Gotfred Lie and Hustru Marie Lie’s fund. This support is gratefully acknowledged.
REFERENCES 1. Adolphe, M., Fontagne, J., Pelletier, M . &
2.
3.
4.
5. 6.
7. 8.
9.
10.
11.
2. 3. 4.
15.
Giroud, J . P.: Induction of DNA synthesis in rat macrophages in vitro by inflammatory exudate. Nature (Lond.) 253: 637, 1975. Appelmans, F., Wattiaux, R . & DeDuve, C.: Tissue fractionating studies. 5. The association of acid phosphatase with a special class of cytoplasmic granules in rat liver. Biochem. J. 59: 438-445, 1955. Bancroft, J . D.: An introduction to histochemical techniques. Butterworth, London 1967, pp. 192-194. Donald, K . J . & Pound, A . W.: Proliferation and migration of reticuloendothelial cells following injection of a tubercle bacillary lipid. Br. J. exp. Path. 54: 79-89, 1973. Fiske, C. H . & Subbarrow, Y.: The colorimetric determination of phosphorus. J. biol. Chem. 66: 375-400, 1925. Forbes, I . J.: Mitoses in mouse peritoneal macrophages. J. Immunol. 96: 734-743, 1966. Frenkel, J. K.: Adoptive immunity to intracellular infection. J. Immunol. 98: 1309-1318, 1967. Frenkel, 1. K.: Models for infectious diseases. Fed. Proc. 28: 179-190, 1969. Godal, T., Rees, R . J . W . & Lamuik, J . 0.: Lymphocyte-mediated modification of bloodderived macrophage function in vitro; inhibition of growth of intracellular mycobacteria with lymphokines. Clin. exp. Immunol. 8: 625-637, 1971. Hibbs, J . B. jr,, Lambert, L. H . jr. & Remington, J . S.: Possible role of macrophage mediated nonspecific cytotoxicity in tumour resistance. Nature (Lond.) 235: 48-50, 1972. lones. T . & Youmans. G . P.: The in vitro inhibition of growth of intracellular Listeria monoctogenes by lymphocyte products. Cell. Immunol. 9: 353-362, 1973. Jones, T . C.: Macrophages and intracellular parasitism. J. reticulwndoth. SOC. 15: 439450, 1974. Jones, T . C., Len, L . & Hirsch, J . G.: Assessment in vitro of immunity against Toxoplasma gondii. J. exp. Med. 141: 466-482, 1975. Kelly, L. S., Dobson, E. L., Finney, C. R . & Hirsch, J . D.: Proliferation of the reticuloendothelid system in the liver. Amer. J. Physiol. 198: 1134-1138, 1960. Lowry, 0. H . , Rosebrough, N . J , , Farr, A . L . & Randall, R . J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193: 265-275, 1951.
. ,
129
16. Mackaness, G . B.: The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. exp. Med. 129: 973-992, 1969. 17. Mackaness, G . B.: Cellular immunity. In: Furth, R. van (Ed.) : Mononuclear phagocytes. Blackwell Scientific Publications, Oxford 1970, pp. 461-476. 18. McGregor, D . D . & Koster, F. T.: The mediator of cellular immunity. IV Cooperation between lymphocytes and mononuclear phagocytes. Cell. Immunol. 2: 317-325, 1971. 19. More, D . G., Penrose, I. M . , Kearney, R . & Nelson, D . S.: Immunological induction of DNA synthesis in mouse peritoneal macrophages. Int. Arch. Allergy 44: 611-630, 1973. 20. Nath, I., Poulter, L. W . & Turk, J . L.: Effect of lymphocyte mediators on macrophages in vitro. A correlation of morphological and cytochemical changes. Clin. exp. Immunol. 13: 455-466, 1973. 21. Nathan, C. F., Karnovsky, M . L, & David, I. R.: Alterations of macrophage functions by mediators from lymphocytes. J. exp. Med. 133: 1356-1376, 1971. 22. NeZson, D . S.: Immunity to infection, allograft immunity and tumour immunity: parallels and contrasts. Transplant. Rev. 19: 226254, 1974. 23. North, R . 1.: Cellular kinetics associated with the development of acquired cellular resistance. J. exp. Med. 130: 299-314, 1969.
24. North, R . 1.: The mitotic potential of fixed phagocytes in the liver as revealed during the development of cellular immunity. J. exp. Med. 130: 315-326, 1969. 25. Reikvam, A . & Hoiby, E. A.: Phagocytosis and rnicrobicidal capacity of mouse macrophages non-specifically activated in vitro. Acta path. microbiol. scand. Sect. C, 83: 121-128, 1975. 26. Reikvam, A., Grammeltvedt, R . & Hoiby, E. A . : Activated mouse macrophages: morphology, lysosomal biochemistry and microbicidal properties of in vivo and in vitro activated cells. Acta path. microbiol. scand. Sect. C , 83: 129-138, 1975. 27. Simon, H . B. & Sheagren, J . N.: Cellular immunity in vitro. I. Immunologically mediated enhancement of macrophage bactericidal capacity. J. exp. Med. 133: 1377-1389, 1971. 28. Warr, G. W . & SljiviC, V . S.: Origin and division of liver macrophages during stimulation of the mononuclear phagocyte system. Cell Tissue Kinet. 7: 559-565, 1974. 29. Wynne, K . M . , Spector, W . G . & Willoughby, D . A.: Macrophage proliferation in vitro induced by exudates. Nature (Lond.) 253: 636-637, 1975. 30. Zinkernagel, R. M., Blanden, R . V . & Langman, R . E.: Early appearance of sensitized lymphocytes in mouse infected with Listeria monocytogenes. J. Immunol. 112: 496-501, 1974.
