INFMTON AND IMMUNITY, Nov. 1975, p. 1037-1043 Copyright © 1975 American Society for Microbiology
Vol. 12, No. 5
Printed in USA.
Dichotomy Between Macrophage Activation and Degree of Protection Against Listeria monocytogenes and Toxoplasma gondii in Mice Stimulated with Corynebacterium parvum JOHN E. SWARTZBERG, JAMES L. KRAHENBUHL, AND JACK S. REMINGTON* Division of Allergy, Immunology, and Infectious Diseases, Palo Alto Medical Research Foundation, Palo Alto, California 94301, and Department of Medicine, Stanford University School of Medicine, Stanford, California 94305* Received for publication 24 June 1975
In vivo and in vitro experiments were conducted to determine the effect of Corynebacterium parvum treatment of mice on resistance ofListeria monocytogenes and Toxoplasma gondii. Intravenous immunization with C. parvum conferred transient protection against intravenous challenge with Listeria or an avirulent strain of Toxoplasma but did not protect against a virulent strain of Toxoplasma. Compared with the level of protection conferred by C. parvum, a higher degree of resistance was noted when mice infected with Listeria or Toxoplasma were challenged with the homologous infecting organism. Peritoneal macrophages from mice immunized intravenously with C. parvum were activated to kill Toxoplasma in vitro. Whereas resistance to challenge in vivo was transient, this population of activated macrophages persisted. Peritoneal macrophages from C. parvum mice also markedly inhibited [3H]thymidine uptake by L cells. Mice chronically infected with Toxoplasma possess macrophages with an enhanced capacity to kill intracellular Toxoplasma (25) and are resistant to challenge with >1,000 100% lethal doses (LD,oo) of a virulent strain of the parasite (24). Similarly, specific immunization with dead antigens of Toxoplasma (3, 16, 28) as well as nonspecific immunization using Freund complete adjuvant (3), macrophage ribonucleic acid (3), or polynucleotides (3) confers resistance to challenge with Toxoplasma. Despite the fact that macrophage activation occurs in vivo with these dead vaccines, the resistance they confer is of a lesser magnitude than that which results from vaccinations with living Toxoplasma organisms. Corynebacterium parvum, a potent stimulator of the lymphoreticular system, is among the agents being employed to enhance, nonspecifically, host resistance. Injection of a suspension of killed C. parvum into experimental animals results in an associated increase in resistance to certain bacterial (1, 7) and protozoal (20) organisms and transplantable tumor cells (34). C. parvum treatment is associated with massive proliferation of the cells of the lymphoreticular system (10), increased antibody production to subsequently injected antigens (19), an increase in the threshold for tolerance induction (22), induction of autoimmunity (17), and
induction of delayed hypersensitivity to unrelated antigens (19). Because of its effects on the immune system, C. parvum is increasingly being employed as a nonspecific immunoenhancer in patients with cancer (8). As sjch patients are frequently predisposed to activation of latent Toxoplasma infection (J. Ruskin and J. S. Remington, submitted for publication; 6, 35), we considered it of interest to determine whether C. parvum confers resistance to this protozoan and, if so, whether the resistance might be effected by activated macrophages. MATERIALS AND METHODS Mice. Mice were outbred Swiss Webster females (obtained from Simonsen Laboratories, Gilroy, Calif.) weighing 20 to 25 g. Immunization. Mice were injected intravenously (i.v.) with 0.1 ml of a suspension of killed C. parvum (supplied by Wellcome Laboratories, Beckenham, Kent, United Kingdom) containing 7 mg (dry weight) of cells/ml. Sublethal infection of mice with Listeria monocytogenes type 4a was accomplished by i.v. injection of 5 x 105 bacteria prepared as previously described (26). Three days prior to challenge with L. monocytogenes, the mice were boosted with 1 x 104 L. monocytogenes intraperitoneally (i.p.). The method employed for establishing chronic infection with Toxoplasma gondii has previously been described (26). In vivo challenge with Listeria or Toxoplasma.
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Immunized and control mice were challenged i.v. with either 1 x 107 L. monocytogenes, 1 x 103 strain C56 T. gondii, or 5 x 102 or 5 x 103 strain RH T. gondii. The inoculum of Toxoplasma was prepared as previously described (16). Deaths were recorded daily. Evaluation of macrophages in vitro. Peritoneal macrophages from immunized and control mice were harvested as previously described (26). Peritoneal cells, 1.5 x 106 in 0.5 ml of medium 199 (M199; Grand Island Biological Co., Grand Island, N. Y.), containing 100 U of penicillin/ml, 100 ,ug of streptomycinlml, and 20% heat-inactivated fetal calf serum (M199-20% FCS), were seeded into four-well LabTek slide chambers (Lab-Tek Products, Naperville, Ill.) and incubated for 1 h at 37 C in an atmosphere containing 5% C02. Thereafter, the chambers were washed with Hanks balanced salt solution (HBSS) to remove nonadherent cells and reincubated with M199-20% FCS. After 2 h, the chambers were washed with HBSS and 3 x 106 strain RH T. gondii, obtained from the peritoneal fluid of 2-day-infected mice and filtered free of host cells (23), were placed on the monolayers in a volume of 0.5 ml of M199-20% FCS. After 1 h of incubation, the chambers were rinsed thoroughly with HBSS to remove extracellular organisms and then reincubated with fresh M199-20% FCS. After the 1-h incubation with T. gondii and at various intervals thereafter, medium was decanted from the slide chambers, and 1 ml of M199-20% FCS containing 1 A&Ci of tritium-labeled uridine ([3H]UdR; 28 Ci/mmol specific activity; New England Nuclear Corp., Boston, Mass.) was added to each chamber. After a 60-min pulse, the slides were washed thoroughly with HBSS and processed for autoradiography (25). Criteria used in evaluation of the effect of macrophages on Toxoplasma in the autoradiographs were as described previously (2, 25). Briefly, organisms with .5 grains were considered viable. Other criteria for viability of Toxoplasma included their staining characteristics, morphological appearance, and whether or not they divided within cytoplasmic vacuoles. Effect of macrophages on tumor cells. Studies on the effect of macrophages on tumor cells were performed by methods previously described (14) as an additional test of activation of macrophages. Briefly, peritoneal cells (4 x 106) from immunized or unimmunized mice were added to each well of tissue culture plates (16 mm Fb-16-24-TC, Linbro Chemical Company, Inc., Los Angeles, Calif.) and allowed to adhere for 3 h. Nonadherent cells were removed by washing, and 1 x 105 L-929 cells (L cells) (obtained from Jorgen Fogh, Sloan-Kettering Institute, New York, N.Y., and maintained in tissue culture in M199-10% FCS) were added to each well. Duplicate wells were pulsed for 6-h intervals with 5 ACi of tritium-labeled thymidine ([3H]TdR; 6 Ci/mmol specific activity; Schwarz/Mann, Orangeburg, N.Y.). At the conclusion of each pulse period, extracellular [3HITdR was removed by washing, and uptake of [3H]TdR by the L cells was determined by liquid scintillation counting.
RESULTS Effect of C. parvum on resistance to Listeria and Toxoplasma. The results of an i.v. challenge with Listeria in mice immunized with C. parvum or Listeria are shown in Fig. 1. Both groups of immunized mice were significantly (P < 0.01) protected as compared to control animals. Although fewer Listeria-immunized mice died than C. parvum-immunized mice, the differences were not statistically significant. The results of an experiment in which mice immunized with C. parvum were challenged i.v. with strain C56 T. gondii are shown in Fig. 2. Protection was seen in those animals given C. parvum 1 and 2 weeks prior to challenge. This protection was significant when compared with the results in control mice (P < 0.03 for group A and P < 0.01 for groups B and C in Fig. 2) solely as a prolongation in time to death. There was no significant difference in overall mortality between any of the groups. In Fig. 3 are the results of an experiment in which mice immunized 1 week previously with C. parvum were challenged i.v. with strain RH T. gondii. The experiments were performed in parallel with mice chronically infected with Toxoplasma for 6 months to compare homolo100
I
.
90
* CONTROL
80 _
C parvum IMMUNIZED MICE 0 LISTERIA IMMUNIZED MICE 0
70-
60
-
)50
L
w
S 40 30-
20
-
* INOCULATION
5
10
I5
20
nAY OF )FATW
25
30
35
FIG. 1. Results of Listeria challenge of control mice and mice immunized with C. parvum or Listeria. There were 15 mice in each group.
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a
w C-) 0
w
.R 0
INOCULATION
FIG. 2. Results of Toxoplasma (strain C56) challenge of control mice immunized with C. parvum 3,2, and 1 week prior to challenge. There were 15 mice in the control groups, 9 mice in the group immunized 1 week prior to challenge, and 10 mice in the other two groups.
gous resistance. Groups of Toxoplasma-infected and C. parvum-immunized mice were challenged with two doses of strain RH Toxoplasma (500 and 5,000 LD,oo). There was no demonstrable protection in those animals immunized with C. parvum; 100% died by 1 week. There were no deaths in the mice chronically infected with T. gondii. Effect of peritoneal macrophages from C. parvum-immunized mice on Toxoplasma. The results of an experiment in which peritoneal macrophages from mice immunized with C. parvum 21, 17, 10, and 3 days prior to harvest of the macrophages were challenged with strain RH T. gondii are shown in Fig. 4. At zero time, the number of macrophages infected from C. parvum-immunized mice and the numbers of Toxoplasma per macrophage were the same as or less than observed in normal macrophages. At 8 h, 70% of Toxoplasma organisms within normal macrophages actively incorporated [3H]UdR as indicated by the presence of -5 grains per organism. In contrast, less than 5% of Toxoplasma in macrophages from C. parvum-immunized mice were labeled with .5
-
DAY
OF
DEATH
FIG. 3. Results of i.v. strain RH T. gondii challenge of 10 control mice, 19 mice immunized with C. parvum, and 20 mice chronically infected with Toxoplasma. The control, C. parvum-immunized mice, and 10 of the chronically infected mice were challenged with 5 x 102 T. gondii. The other 10 chronically infected mice were challenged with 5 x 103 T. gondii.
grains at 8 h. After 18 h of incubation, approximately 90% of the Toxoplasma in normal macrophages had incorporated .5 grains; all of these had divided, forming pairs, tetrads, or rosettes. In contrast, after 18 h, less than 2% of Toxoplasma in macrophages from C. parvum-immunized mice had .5 grains and none had divided. Results did not vary with the length of time from immunization with C. parvum to challenge with Toxoplasma (3 to 21 days). Effect of macrophage culture supernatants on Toxoplasma and Listeria. As Bast et al. (4) and Sethi et al. (32) have demonstrated evidence for a listericidal factor in supernatants from monolayers of peritoneal macrophages derived from BCG-immunized guinea pigs and Listeria-immunized mice, respectively, we attempted to establish if such a factor might be operative in our experimental model. In three separate experiments, the supernatants from cultures of peritoneal macrophages derived from mice immunized with L. monocytogenes type 4a had no influence upon the growth
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SWARTZBERG, KRAHENBUHL, AND REMINGTON
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TABLE 1. Effects of macrophages from C. parvumimmunized mice on [3H]TdR uptake by L cells Day of macrophage harvesta
*17 DAY (D 80 *21 DAY I/
0 u
3 70
Mean counts/min 0 to 6 h
12 to 18 h
4,283 (+877) (P < 0.2)c
12,399 (±6,493) (P < 0.01) 1,274 (+0) (P < 0.05) 1,574 (±737) (P < 0.001) 948 (+552) (P < 0.0001) 1,709 (+738) (P < 0.001) 81,697 (±13,216) 347,873 (±13,300)
7 /
@ 60
10
z o0 50
17 21
v
40
Normal
0~
a
1,054 (+278) (P < 0.001) 1,036 (±+48) (P < 0.01) 1,852 (±+239) (P < 0.002) 5,764 (+618) 8,363 (±323)
None Number of days since immunization with C.
x
0
(±SD)b during
pulse interval
30
a
20_/_ I0 0
parvum. bSD, Standard deviation. c P value refers to differences between groups with macrophages from C. parvum-immunized mice
and normal macrophages (Student's t test).
3
6
12 9 TIME (hours)
15
18
FIG. 4. Effect ofpertitoneal macrophages from normal and C. parvum-immunized mice on Toxoplasma. The macrophages were harvested and challenged with Toxoplasma on days 21, 17, 10, and 3
after i.v. C. parvum immunization of the mice.
curve of Listeria. In two of these experiments, the supernatants were also evaluated in the tumor cell assay system; in both cases, the supernatants had no effect upon the uptake of [3H]TdR by the tumor cells. In a fourth experiment, numerous supernatants from monolayers of peritoneal macrophages obtained from mice immune to T. gondii were incubated with viable extracellular Toxoplasma. In no case was detectable killing (as judged by dye exclusion [29] and morphology) of Toxoplasma by the supernatants observed. When Listeria were treated with these same supernatants, there was no detectable effect on the growth curve. Effect of peritoneal macrophages from C. parvum-immunized mice on [3H]TdR uptake by L cells. Peritoneal macrophages from the same animals used in the in vitro macrophage challenge study described above were simultaneously employed in an experiment to determine their effect on uptake of [3H]TdR by L cells. The results revealed that macrophages from C. parvum-immunized mice markedly inhibited [3H]TdR uptake by L cells (Table 1). Slight inhibition of [3H]TdR uptake was also
noted in L cells cultured in the presence of normal macrophages when compared with [3H]TdR uptake by these target cells cultured alone. The effectiveness of activated macrophages in inhibiting [3H]TdR uptake by L cells is readily demonstrable by comparison of the data obtained at the 18-h pulse period. In the presence of macrophages from C. parvum-immunized mice, the uptake of [3H]TdR was only 1.2 to 15.2% of the amount incorporated in L cells cultured with normal macrophages. With one exception, the length of time between immunization of the mice and in vitro challenge of their macrophages with the tumor cells had no influence on the uptake of [3H]TdR by L cells. This exception was seen with those macrophages obtained from animals immunized with C. parvum 3 days earlier. Although they did inhibit [3H]TdR uptake by L cells, they were less effective in this inhibition than were macrophages from those groups of mice immunized for longer periods of time. DISCUSSION Peritoneal and lung macrophages from mice immunized i.v. with C. parvum have been demonstrated to have a marked cytostatic and/or cytocidal effect on tumor target cells in vitro (21, 30, 31). The role of these macrophages against living organisms has not previously been delineated. The present study demonstrates that peritoneal macrophages from mice
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immunized with killed C. parvum are activated immunized i.v. with C. parvum and thereafter to kill Toxoplasma. This activation is present challenged i.p. with C56 strain T. gondii. within 3 days of i.v. C. parvum injection and Separate studies in our laboratory (J. L. Krapersists for at least 21 days. In a parallel experi- henbuhl and J. S. Remington, submitted for ment, these activated macrophages produced a publication) have shown that adminstration of cytotoxic effect on tumor cells in vitro which C. parvum i.v. results in significantly less actiwas evident over the same time period. These vation of peritoneal macrophages to inhibit or observations provide additional support for kill tumor cells than i.p. inoculation; subcutathose data which indicate that C. parvum is an neous inoculation ofC. parvum did not activate activator of the cellular immune response. peritoneal macrophages to inhibit or kill tumor Pretreatment of mice with C. parvum in- cells. Thus, the route of C. parvum immunizacreases their resistance to infection with such tion appears critical. This is in contrast to obintracellular parasites as Salmonella enteriti- servations with live Toxoplasma infection dis (7), Bordetella pertussis (1), Brucella abor- where the route of original infection has previtus (1), and Plasnodium berghei (20). The pres- ously been shown to be unimportant in conent study was designed to determine if C. par- ferring resistance against heterologous orgavum immunization confers protection against nisms (27) and tumors (11). challenge with L. monocytogenes or T. gondii, Collins and Scott have demonstrated enand, if so, whether the protection is comparable hanced clearing of Salmonella in the spleens to that in mice immunized with the homologous and livers of mice immunized i.v. with C. parvum (7). Mice given C. parvum cleared Salmoorganism. Immunization with C. parvum conferred pro- nella enteritidis from their livers and spleens tection against challenge with L. monocyto- significantly more rapidly than control mice. genes (an inoculum of approximately 1 LD6O) or Although macrophages from these organs were an avirulent strain of T. gondii (an inoculum of not tested in their study or in ours, the results approximately 1 LD6O). When these mice were obtained by Collins and Scott (7) would suggest challenged with the more virulent strain RH T. that in our experimental model, macrophages gondii, there was no evidence of protection. A in these organs were also activated. Because of the remarkable ability of peritohigher degree of resistance was noted when mice infected with Listeria or Toxoplasma were neal macrophages from mice immunized with challenged with the homologous infecting orga- C. parvum to kill Toxoplasma, no differentianism. For instance, mice chronically infected tion could be made with the methodology we with Toxoplasma resisted completely an inocu- employed between the relative efficacy in killlum containing 5,000 LD1oo strain RH T. gon- ing by these macrophages and those from Toxodii. Furthermore, the poor protection conferred plasma-infected mice. The actual degree of actiby C. parvum against the avirulent C56 strain vation of macrophages in vivo may vary considT. gondii appeared to be transient, whereas the erably and thereby result in differences in their Toxoplasma-infected mice were resistant to ability to kill or inhibit various organisms, and challenge with the virulent Toxoplasma strain thus in host resistance. In addition, as differfor more than 6 months. Of interest is that both ences in activation occur at different sites of the groups of mice (C. parvum immunized and Tox- body (5), the site at which challenge occurs will oplasma infected) had peritoneal macrophages be critical to the outcome (12). The findings in this study support previous which were activated to kill Toxoplasma in vitro. Both the relatively low order of in vivo results and conclusions from this laboratory: protection conferred by C. parvum and the loss resistance in the homologous system (Toxoof that protection at a time when the host mac- plasma-Toxoplasma) is far greater than that rophages were activated to kill Toxoplasma conferred by a heterologous system (in the presraise a question as to the role of activated mac- ent study, C. parvum-Toxoplasma). The activarophages in resistance. The apparent dichot- tion of macrophages in immunized mice is the omy remains to be explained. Why, in animals result of a specific interaction between the antiwith a population of macrophages highly acti- gen and sensitized lymphocytes. The activation vated to kill Toxoplasma in vitro, is there rela- is then mediated by these lymphocytes (2, 9, 13, tively poor protection when these same animals 15, 18, 33). The effect of this activation is to are challenged with Toxoplasma in vivo? That confer resistance which can be expressed both the route of challenge could not explain the specifically (against homologous organisms) observed differences is clear from pilot studies and nonspecifically (against heterologous orgain our laboratory which revealed no significant nisms). In no system which we have employed difference in mortality or time to death in mice have we been able to confer resistance to Toxo-
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SWARTZBERG, KRAHENBUHL, AND REMINGTON
plasma with heterologous live or dead organisms or other agents (e.g., antigen ribonucleic acid, polynucleotides) to the degree that is conferred by infection with Toxoplasma itself. Although C. parvum confers relatively little resistance to Toxoplasma in vivo in the mouse model we studied, it was effective in activating host macrophages to kill this parasite. Whether macrophage activation occurs in vivo in humans receiving C. parvum is not yet known. If such activation does occur, some protection against dissemination of Toxoplasma might be conferred. ACKNOWLEDGMENTS This work was supported by Public Health Service research grant AI 04717 and training grant Al 00260, both from the National Insitute of Allergy and Infectious Diseases. J. L. K. is the recipient of Public Health Service Research Career Devleopment Award 1K04AICA70931 from the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Adlam, C., E. S. Broughton, and M. T. Scott. 1972. Enhanced resistance of mice to infection with bacteria following pretreatment with Corynebacterium parvum. Nature (London) New Biol. 235:219-220. 2. Anderson, S. E., and J. S. Remington. 1974. Effect of normal and activated human macrophages on Toxoplasma gondii. J. Exp. Med. 139:1154-1174. 3. Araujo, F. G., and J. S. Remington. 1974. Protection against Toxoplasma gondii in mice immunized with Toxoplasma cell fractions, RNA, and synthetic polyribonucleotides. Immunology 27:711-721. 4. Bast, R. C., Jr., R. P. Cleveland, B. H. Littman, B. Zbar, and H. J. Rapp. 1974. Acquired cellular immunity: extracellular killing of Listeria monocytogenes by a product of immunologically activated macrophages. Cell. Immunol. 10:248-259. 5. Blanden, R. V., M. J. Lefford, and G. B. Mackaness. 1969. The host response to Calmette-Guerin bacillus infection in mice. J. Exp. Med. 129:1079-1101. 6. Carey, R. M., A. C. Kimball, D. Armstrong, and P. H. Lieberman. 1973. Toxoplasmosis: clinical experiences in a cancer hospital. Am. J. Med. 54:30-38. 7. Collins, F. M., and M. T. Scott. 1974. Effect of Corynebacterium parvum treatment on the growth ofSalmonella enteritidis on mice. Infect. Immun. 9:863-869. 8. Editorial. 1975. Immunological control of cancer. Lancet 1:502-503. 9. Fowles, R. E., I. M. Fajardo, J. L. Leibowitch, and J. R. David. 1973. The enhancement of macrophage bacteriostasis by products of activated lymphocytes. J. Exp. Med. 138:952-964. 10. Halpern, B. N., A. R. Prevot, G. Biozzi, C. Stiffel, D. Mouton, J. C. Morard, Y. Bouthillier, and C. Decreusefond. 1964. Stimulation of the phagocytic activity of the reticuloendothelial system provoked by Corynebacterium parvum. RES J. Reticuloendothel. Soc. 1:77-96. 11. Hibbs, J. B. Jr., L. H. Lambert, Jr., and J. S. Remington. 1971. Resistance to murine tumors conferred by chronic infection with intracellular protozoa, Toxoplasma gondii and Besnoitia jellisoni. J. Infect. Dis. 124:587-592. 12. Krahenbuhl, J. L., L. Levy, and J. S. Remington. 1974. Resistance to Mycobacterium leprae in mice infected
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with Toxoplasma gondii and Besnoitia jellisoni. Infect. Immun. 10:1068-1071. Krahenbuhl, J. L., and J. S. Remington. 1971. In vitro induction of nonspecific resistance in macrophages by specifically sensitized lymphocytes. Infect. Immun. 4:337-343. Krahenbuhl, J. L., and J. S. Remington. 1974. The role of activated macrophages in specific and nonspecific cytostasis of tumor cells. J. Immunol. 113:507-516. Krahenbuhl, J. L., L. T. Rosenberg, and J. S. Remington. 1973. The role of thymus-derived lymphocytes in the in vitro activation of macrophages to kill Listeria monocytogenes. J. Immunol. 111:992-995. Krahenbuhl, J. L., J. Ruskin, and J. S. Remington. 1972. The use of killed vaccines in immunization against an intracellular parasite: Toxoplasma gondii. J. Immunol. 108:425-431. McCracken, A., W. H. McBride, and D. M. Weir. 1971. Adjuvant-induced anti-red blood cell activity in CBA mice. Clin. Exp. Immunol. 8:949-955. Mackaness, G. B. 1969. The influence of immunologically committed lymphoid cells on macrophage activation in vivo. J. Exp. Med. 129:973-992. Neveu, T., A. Brannellec, and G. Biozzi. 1964. Adjuvant effect of Corynebacterium parvum on antibody production and on induction of delayed hypersensitivity to conjugated proteins. Ann. Inst. Pasteur Paris 106:771-777. Nussenzweig, R. S. 1967. Increased nonspecific resistance to malaria produced by administration of killed Corynebacterium parvum. Exp. Parasitol. 21:224231. Olivotto, M., and R. Bomford. 1974. In vitro inhibition of tumor cell growth and DNA synthesis by peritoneal and lung macrophages from mice injected with Corynebacterium parvum. Int. J. Cancer 13:478-488. Pinckard, R. N., D. M. Weir, and W. H. McBride. 1967. Factors influencing the immune response. Clin. Exp. Immunol. 3:331-341 Remington, J. S., M. M. Bloomfield, E. Russel, Jr., and W. S. Robinson. 1970. The RNA of Toxoplasma gondii. Proc. Soc. Exp. Biol. Med. 133:623-626. Remington, J. S., L. Jacobs, and M. Melton. 1961. Congenital transmission of toxoplasmosis from mother animals with acute and chronic infections. J. Infect. Dis. 108:163-173. Remington, J. S., J. L. Krahenbuhl, and J. W. Mendenhall. 1972. A role for activated macrophages in resistance to infection with Toxoplasma. Infect. Immun. 6:829-834. Ruskin, J., J. McIntosh, and J. S. Remington. 1969. Studies on the mechanisms of resistance to phylogenetically diverse intracellular organisms. J. Immunol. 103:252-259. Ruskin, J., and J. S. Remington. 1968. Immunity and intracellular infection: resistance to bacteria in mice infected with a protozoan. Science 160:72-74. Ruskin, J., and J. S. Remington. 1971. Resistance to intracellular infection in mice immunized with Toxoplasma vaccine and adjuvant. J. Reticuloendothel. Soc. 9:465-479. Sabin, A. B., and H. A. Feldman. 1948. Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoan parasite (Toxoplasma). Science 108:660-663. Scott, M. T. 1974. Corynebacterium parvum as a therapeutic antitumor agent in mice. I. Systemic effects from intravenous injection. J. Natl. Cancer Inst. 53:855-860. Scott, M. T. 1974. Corynebacterium parvum as a therapeutic antitumor agent in mice. II. Local injection. J. Natl. Cancer Inst. 53:861-865.
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Vol. 12, No. 5
Printed in USA.
Dichotomy Between Macrophage Activation and Degree of Protection Against Listeria monocytogenes and Toxoplasma gondii in Mice Stimulated with Corynebacterium parvum JOHN E. SWARTZBERG, JAMES L. KRAHENBUHL, AND JACK S. REMINGTON* Division of Allergy, Immunology, and Infectious Diseases, Palo Alto Medical Research Foundation, Palo Alto, California 94301, and Department of Medicine, Stanford University School of Medicine, Stanford, California 94305* Received for publication 24 June 1975
In vivo and in vitro experiments were conducted to determine the effect of Corynebacterium parvum treatment of mice on resistance ofListeria monocytogenes and Toxoplasma gondii. Intravenous immunization with C. parvum conferred transient protection against intravenous challenge with Listeria or an avirulent strain of Toxoplasma but did not protect against a virulent strain of Toxoplasma. Compared with the level of protection conferred by C. parvum, a higher degree of resistance was noted when mice infected with Listeria or Toxoplasma were challenged with the homologous infecting organism. Peritoneal macrophages from mice immunized intravenously with C. parvum were activated to kill Toxoplasma in vitro. Whereas resistance to challenge in vivo was transient, this population of activated macrophages persisted. Peritoneal macrophages from C. parvum mice also markedly inhibited [3H]thymidine uptake by L cells. Mice chronically infected with Toxoplasma possess macrophages with an enhanced capacity to kill intracellular Toxoplasma (25) and are resistant to challenge with >1,000 100% lethal doses (LD,oo) of a virulent strain of the parasite (24). Similarly, specific immunization with dead antigens of Toxoplasma (3, 16, 28) as well as nonspecific immunization using Freund complete adjuvant (3), macrophage ribonucleic acid (3), or polynucleotides (3) confers resistance to challenge with Toxoplasma. Despite the fact that macrophage activation occurs in vivo with these dead vaccines, the resistance they confer is of a lesser magnitude than that which results from vaccinations with living Toxoplasma organisms. Corynebacterium parvum, a potent stimulator of the lymphoreticular system, is among the agents being employed to enhance, nonspecifically, host resistance. Injection of a suspension of killed C. parvum into experimental animals results in an associated increase in resistance to certain bacterial (1, 7) and protozoal (20) organisms and transplantable tumor cells (34). C. parvum treatment is associated with massive proliferation of the cells of the lymphoreticular system (10), increased antibody production to subsequently injected antigens (19), an increase in the threshold for tolerance induction (22), induction of autoimmunity (17), and
induction of delayed hypersensitivity to unrelated antigens (19). Because of its effects on the immune system, C. parvum is increasingly being employed as a nonspecific immunoenhancer in patients with cancer (8). As sjch patients are frequently predisposed to activation of latent Toxoplasma infection (J. Ruskin and J. S. Remington, submitted for publication; 6, 35), we considered it of interest to determine whether C. parvum confers resistance to this protozoan and, if so, whether the resistance might be effected by activated macrophages. MATERIALS AND METHODS Mice. Mice were outbred Swiss Webster females (obtained from Simonsen Laboratories, Gilroy, Calif.) weighing 20 to 25 g. Immunization. Mice were injected intravenously (i.v.) with 0.1 ml of a suspension of killed C. parvum (supplied by Wellcome Laboratories, Beckenham, Kent, United Kingdom) containing 7 mg (dry weight) of cells/ml. Sublethal infection of mice with Listeria monocytogenes type 4a was accomplished by i.v. injection of 5 x 105 bacteria prepared as previously described (26). Three days prior to challenge with L. monocytogenes, the mice were boosted with 1 x 104 L. monocytogenes intraperitoneally (i.p.). The method employed for establishing chronic infection with Toxoplasma gondii has previously been described (26). In vivo challenge with Listeria or Toxoplasma.
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Immunized and control mice were challenged i.v. with either 1 x 107 L. monocytogenes, 1 x 103 strain C56 T. gondii, or 5 x 102 or 5 x 103 strain RH T. gondii. The inoculum of Toxoplasma was prepared as previously described (16). Deaths were recorded daily. Evaluation of macrophages in vitro. Peritoneal macrophages from immunized and control mice were harvested as previously described (26). Peritoneal cells, 1.5 x 106 in 0.5 ml of medium 199 (M199; Grand Island Biological Co., Grand Island, N. Y.), containing 100 U of penicillin/ml, 100 ,ug of streptomycinlml, and 20% heat-inactivated fetal calf serum (M199-20% FCS), were seeded into four-well LabTek slide chambers (Lab-Tek Products, Naperville, Ill.) and incubated for 1 h at 37 C in an atmosphere containing 5% C02. Thereafter, the chambers were washed with Hanks balanced salt solution (HBSS) to remove nonadherent cells and reincubated with M199-20% FCS. After 2 h, the chambers were washed with HBSS and 3 x 106 strain RH T. gondii, obtained from the peritoneal fluid of 2-day-infected mice and filtered free of host cells (23), were placed on the monolayers in a volume of 0.5 ml of M199-20% FCS. After 1 h of incubation, the chambers were rinsed thoroughly with HBSS to remove extracellular organisms and then reincubated with fresh M199-20% FCS. After the 1-h incubation with T. gondii and at various intervals thereafter, medium was decanted from the slide chambers, and 1 ml of M199-20% FCS containing 1 A&Ci of tritium-labeled uridine ([3H]UdR; 28 Ci/mmol specific activity; New England Nuclear Corp., Boston, Mass.) was added to each chamber. After a 60-min pulse, the slides were washed thoroughly with HBSS and processed for autoradiography (25). Criteria used in evaluation of the effect of macrophages on Toxoplasma in the autoradiographs were as described previously (2, 25). Briefly, organisms with .5 grains were considered viable. Other criteria for viability of Toxoplasma included their staining characteristics, morphological appearance, and whether or not they divided within cytoplasmic vacuoles. Effect of macrophages on tumor cells. Studies on the effect of macrophages on tumor cells were performed by methods previously described (14) as an additional test of activation of macrophages. Briefly, peritoneal cells (4 x 106) from immunized or unimmunized mice were added to each well of tissue culture plates (16 mm Fb-16-24-TC, Linbro Chemical Company, Inc., Los Angeles, Calif.) and allowed to adhere for 3 h. Nonadherent cells were removed by washing, and 1 x 105 L-929 cells (L cells) (obtained from Jorgen Fogh, Sloan-Kettering Institute, New York, N.Y., and maintained in tissue culture in M199-10% FCS) were added to each well. Duplicate wells were pulsed for 6-h intervals with 5 ACi of tritium-labeled thymidine ([3H]TdR; 6 Ci/mmol specific activity; Schwarz/Mann, Orangeburg, N.Y.). At the conclusion of each pulse period, extracellular [3HITdR was removed by washing, and uptake of [3H]TdR by the L cells was determined by liquid scintillation counting.
RESULTS Effect of C. parvum on resistance to Listeria and Toxoplasma. The results of an i.v. challenge with Listeria in mice immunized with C. parvum or Listeria are shown in Fig. 1. Both groups of immunized mice were significantly (P < 0.01) protected as compared to control animals. Although fewer Listeria-immunized mice died than C. parvum-immunized mice, the differences were not statistically significant. The results of an experiment in which mice immunized with C. parvum were challenged i.v. with strain C56 T. gondii are shown in Fig. 2. Protection was seen in those animals given C. parvum 1 and 2 weeks prior to challenge. This protection was significant when compared with the results in control mice (P < 0.03 for group A and P < 0.01 for groups B and C in Fig. 2) solely as a prolongation in time to death. There was no significant difference in overall mortality between any of the groups. In Fig. 3 are the results of an experiment in which mice immunized 1 week previously with C. parvum were challenged i.v. with strain RH T. gondii. The experiments were performed in parallel with mice chronically infected with Toxoplasma for 6 months to compare homolo100
I
.
90
* CONTROL
80 _
C parvum IMMUNIZED MICE 0 LISTERIA IMMUNIZED MICE 0
70-
60
-
)50
L
w
S 40 30-
20
-
* INOCULATION
5
10
I5
20
nAY OF )FATW
25
30
35
FIG. 1. Results of Listeria challenge of control mice and mice immunized with C. parvum or Listeria. There were 15 mice in each group.
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a
w C-) 0
w
.R 0
INOCULATION
FIG. 2. Results of Toxoplasma (strain C56) challenge of control mice immunized with C. parvum 3,2, and 1 week prior to challenge. There were 15 mice in the control groups, 9 mice in the group immunized 1 week prior to challenge, and 10 mice in the other two groups.
gous resistance. Groups of Toxoplasma-infected and C. parvum-immunized mice were challenged with two doses of strain RH Toxoplasma (500 and 5,000 LD,oo). There was no demonstrable protection in those animals immunized with C. parvum; 100% died by 1 week. There were no deaths in the mice chronically infected with T. gondii. Effect of peritoneal macrophages from C. parvum-immunized mice on Toxoplasma. The results of an experiment in which peritoneal macrophages from mice immunized with C. parvum 21, 17, 10, and 3 days prior to harvest of the macrophages were challenged with strain RH T. gondii are shown in Fig. 4. At zero time, the number of macrophages infected from C. parvum-immunized mice and the numbers of Toxoplasma per macrophage were the same as or less than observed in normal macrophages. At 8 h, 70% of Toxoplasma organisms within normal macrophages actively incorporated [3H]UdR as indicated by the presence of -5 grains per organism. In contrast, less than 5% of Toxoplasma in macrophages from C. parvum-immunized mice were labeled with .5
-
DAY
OF
DEATH
FIG. 3. Results of i.v. strain RH T. gondii challenge of 10 control mice, 19 mice immunized with C. parvum, and 20 mice chronically infected with Toxoplasma. The control, C. parvum-immunized mice, and 10 of the chronically infected mice were challenged with 5 x 102 T. gondii. The other 10 chronically infected mice were challenged with 5 x 103 T. gondii.
grains at 8 h. After 18 h of incubation, approximately 90% of the Toxoplasma in normal macrophages had incorporated .5 grains; all of these had divided, forming pairs, tetrads, or rosettes. In contrast, after 18 h, less than 2% of Toxoplasma in macrophages from C. parvum-immunized mice had .5 grains and none had divided. Results did not vary with the length of time from immunization with C. parvum to challenge with Toxoplasma (3 to 21 days). Effect of macrophage culture supernatants on Toxoplasma and Listeria. As Bast et al. (4) and Sethi et al. (32) have demonstrated evidence for a listericidal factor in supernatants from monolayers of peritoneal macrophages derived from BCG-immunized guinea pigs and Listeria-immunized mice, respectively, we attempted to establish if such a factor might be operative in our experimental model. In three separate experiments, the supernatants from cultures of peritoneal macrophages derived from mice immunized with L. monocytogenes type 4a had no influence upon the growth
INFECT. IMMUN.
SWARTZBERG, KRAHENBUHL, AND REMINGTON
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TABLE 1. Effects of macrophages from C. parvumimmunized mice on [3H]TdR uptake by L cells Day of macrophage harvesta
*17 DAY (D 80 *21 DAY I/
0 u
3 70
Mean counts/min 0 to 6 h
12 to 18 h
4,283 (+877) (P < 0.2)c
12,399 (±6,493) (P < 0.01) 1,274 (+0) (P < 0.05) 1,574 (±737) (P < 0.001) 948 (+552) (P < 0.0001) 1,709 (+738) (P < 0.001) 81,697 (±13,216) 347,873 (±13,300)
7 /
@ 60
10
z o0 50
17 21
v
40
Normal
0~
a
1,054 (+278) (P < 0.001) 1,036 (±+48) (P < 0.01) 1,852 (±+239) (P < 0.002) 5,764 (+618) 8,363 (±323)
None Number of days since immunization with C.
x
0
(±SD)b during
pulse interval
30
a
20_/_ I0 0
parvum. bSD, Standard deviation. c P value refers to differences between groups with macrophages from C. parvum-immunized mice
and normal macrophages (Student's t test).
3
6
12 9 TIME (hours)
15
18
FIG. 4. Effect ofpertitoneal macrophages from normal and C. parvum-immunized mice on Toxoplasma. The macrophages were harvested and challenged with Toxoplasma on days 21, 17, 10, and 3
after i.v. C. parvum immunization of the mice.
curve of Listeria. In two of these experiments, the supernatants were also evaluated in the tumor cell assay system; in both cases, the supernatants had no effect upon the uptake of [3H]TdR by the tumor cells. In a fourth experiment, numerous supernatants from monolayers of peritoneal macrophages obtained from mice immune to T. gondii were incubated with viable extracellular Toxoplasma. In no case was detectable killing (as judged by dye exclusion [29] and morphology) of Toxoplasma by the supernatants observed. When Listeria were treated with these same supernatants, there was no detectable effect on the growth curve. Effect of peritoneal macrophages from C. parvum-immunized mice on [3H]TdR uptake by L cells. Peritoneal macrophages from the same animals used in the in vitro macrophage challenge study described above were simultaneously employed in an experiment to determine their effect on uptake of [3H]TdR by L cells. The results revealed that macrophages from C. parvum-immunized mice markedly inhibited [3H]TdR uptake by L cells (Table 1). Slight inhibition of [3H]TdR uptake was also
noted in L cells cultured in the presence of normal macrophages when compared with [3H]TdR uptake by these target cells cultured alone. The effectiveness of activated macrophages in inhibiting [3H]TdR uptake by L cells is readily demonstrable by comparison of the data obtained at the 18-h pulse period. In the presence of macrophages from C. parvum-immunized mice, the uptake of [3H]TdR was only 1.2 to 15.2% of the amount incorporated in L cells cultured with normal macrophages. With one exception, the length of time between immunization of the mice and in vitro challenge of their macrophages with the tumor cells had no influence on the uptake of [3H]TdR by L cells. This exception was seen with those macrophages obtained from animals immunized with C. parvum 3 days earlier. Although they did inhibit [3H]TdR uptake by L cells, they were less effective in this inhibition than were macrophages from those groups of mice immunized for longer periods of time. DISCUSSION Peritoneal and lung macrophages from mice immunized i.v. with C. parvum have been demonstrated to have a marked cytostatic and/or cytocidal effect on tumor target cells in vitro (21, 30, 31). The role of these macrophages against living organisms has not previously been delineated. The present study demonstrates that peritoneal macrophages from mice
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immunized with killed C. parvum are activated immunized i.v. with C. parvum and thereafter to kill Toxoplasma. This activation is present challenged i.p. with C56 strain T. gondii. within 3 days of i.v. C. parvum injection and Separate studies in our laboratory (J. L. Krapersists for at least 21 days. In a parallel experi- henbuhl and J. S. Remington, submitted for ment, these activated macrophages produced a publication) have shown that adminstration of cytotoxic effect on tumor cells in vitro which C. parvum i.v. results in significantly less actiwas evident over the same time period. These vation of peritoneal macrophages to inhibit or observations provide additional support for kill tumor cells than i.p. inoculation; subcutathose data which indicate that C. parvum is an neous inoculation ofC. parvum did not activate activator of the cellular immune response. peritoneal macrophages to inhibit or kill tumor Pretreatment of mice with C. parvum in- cells. Thus, the route of C. parvum immunizacreases their resistance to infection with such tion appears critical. This is in contrast to obintracellular parasites as Salmonella enteriti- servations with live Toxoplasma infection dis (7), Bordetella pertussis (1), Brucella abor- where the route of original infection has previtus (1), and Plasnodium berghei (20). The pres- ously been shown to be unimportant in conent study was designed to determine if C. par- ferring resistance against heterologous orgavum immunization confers protection against nisms (27) and tumors (11). challenge with L. monocytogenes or T. gondii, Collins and Scott have demonstrated enand, if so, whether the protection is comparable hanced clearing of Salmonella in the spleens to that in mice immunized with the homologous and livers of mice immunized i.v. with C. parvum (7). Mice given C. parvum cleared Salmoorganism. Immunization with C. parvum conferred pro- nella enteritidis from their livers and spleens tection against challenge with L. monocyto- significantly more rapidly than control mice. genes (an inoculum of approximately 1 LD6O) or Although macrophages from these organs were an avirulent strain of T. gondii (an inoculum of not tested in their study or in ours, the results approximately 1 LD6O). When these mice were obtained by Collins and Scott (7) would suggest challenged with the more virulent strain RH T. that in our experimental model, macrophages gondii, there was no evidence of protection. A in these organs were also activated. Because of the remarkable ability of peritohigher degree of resistance was noted when mice infected with Listeria or Toxoplasma were neal macrophages from mice immunized with challenged with the homologous infecting orga- C. parvum to kill Toxoplasma, no differentianism. For instance, mice chronically infected tion could be made with the methodology we with Toxoplasma resisted completely an inocu- employed between the relative efficacy in killlum containing 5,000 LD1oo strain RH T. gon- ing by these macrophages and those from Toxodii. Furthermore, the poor protection conferred plasma-infected mice. The actual degree of actiby C. parvum against the avirulent C56 strain vation of macrophages in vivo may vary considT. gondii appeared to be transient, whereas the erably and thereby result in differences in their Toxoplasma-infected mice were resistant to ability to kill or inhibit various organisms, and challenge with the virulent Toxoplasma strain thus in host resistance. In addition, as differfor more than 6 months. Of interest is that both ences in activation occur at different sites of the groups of mice (C. parvum immunized and Tox- body (5), the site at which challenge occurs will oplasma infected) had peritoneal macrophages be critical to the outcome (12). The findings in this study support previous which were activated to kill Toxoplasma in vitro. Both the relatively low order of in vivo results and conclusions from this laboratory: protection conferred by C. parvum and the loss resistance in the homologous system (Toxoof that protection at a time when the host mac- plasma-Toxoplasma) is far greater than that rophages were activated to kill Toxoplasma conferred by a heterologous system (in the presraise a question as to the role of activated mac- ent study, C. parvum-Toxoplasma). The activarophages in resistance. The apparent dichot- tion of macrophages in immunized mice is the omy remains to be explained. Why, in animals result of a specific interaction between the antiwith a population of macrophages highly acti- gen and sensitized lymphocytes. The activation vated to kill Toxoplasma in vitro, is there rela- is then mediated by these lymphocytes (2, 9, 13, tively poor protection when these same animals 15, 18, 33). The effect of this activation is to are challenged with Toxoplasma in vivo? That confer resistance which can be expressed both the route of challenge could not explain the specifically (against homologous organisms) observed differences is clear from pilot studies and nonspecifically (against heterologous orgain our laboratory which revealed no significant nisms). In no system which we have employed difference in mortality or time to death in mice have we been able to confer resistance to Toxo-
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SWARTZBERG, KRAHENBUHL, AND REMINGTON
plasma with heterologous live or dead organisms or other agents (e.g., antigen ribonucleic acid, polynucleotides) to the degree that is conferred by infection with Toxoplasma itself. Although C. parvum confers relatively little resistance to Toxoplasma in vivo in the mouse model we studied, it was effective in activating host macrophages to kill this parasite. Whether macrophage activation occurs in vivo in humans receiving C. parvum is not yet known. If such activation does occur, some protection against dissemination of Toxoplasma might be conferred. ACKNOWLEDGMENTS This work was supported by Public Health Service research grant AI 04717 and training grant Al 00260, both from the National Insitute of Allergy and Infectious Diseases. J. L. K. is the recipient of Public Health Service Research Career Devleopment Award 1K04AICA70931 from the National Institute of Allergy and Infectious Diseases.
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