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Trypanosoma cruzi: In vivo and in vitro correlation between T-cell activation and susceptibility in inbred strains of mice
EXPERIMENTAI.
PARASITOI.OGY
51,
325-334
(1981)
11rypanosoma cruzi: In Vivo and in Vitro Correlation between T-Cell Activation and Susceptibility in Inbred Strains of Mice N.NOGUEIRA, The Rockefeller
J. ELLIS, S.CHAPLAN, University,
(Accepted
AND Z.COHN
New York, New York 10021, U.S.A
for publication
5 May 1980)
NOGUEIRA,N.,ELLIS,J.,CHAPLAN,S.,ANDCOHN,Z. 1981.Tr~~panosomn cruzi:In viva and in vifro correlation between T-cell activation and susceptibility in inbred strains of mice. Experimental Parasitology 51, 325-334. The in viva susceptibility of several inbred strains of mice to the Y and CL strains of Trypunasoma cruzi was compared to the in vitro ability of spleen cells from infected mice to generate factor(s) able to activate macrophages to a trypanocidal state. Spleen cells from resistant immune mice generate higher levels of the factor(s) and do so at earlier times during infection than those of susceptible mice. The spleen cells capable of generating the in virro factor(s) are also capable of conferring resistance upon passive transfer. Removal of immunoglobulin-bearing cells from the immune spleen cell population did not affect either transfer of protection in viva or generation of the factor(s) in vitro. The cellular basis underlying the differences between susceptible and resistant mouse strains has not yet been determined. INDEX DESCRIPTORS: Trypanos~ma cruzi; Protozoa, parasitic; Hemoflagellate; Mouse, inbred strains; Natural resistance; Protective immunity; Spleen cells, passive transfer; Spleen cells, T cell enriched; Macrophage activation by spleen cell factors.
INTRODUCTION The murine model of Chagas’s disease has provided a large body of information on some of the immune phenomena involved in infections with Trypanosoma cruzi. It has long been known that various mouse strains differ markedly in their resistance to T. cruzi (Pizzi et al. 1949). The basis of natural resistance has not yet been defined, but the immune system has been implicated in the control of parasitemia and the ability of the host to survive after infection. The genetic differences are not primarily related to the mouse H-2 haplotype (Trishman et al. 1978). The protective effect of acquired immunity has been convincingly demonstrated (Seah and Marsden 1969). In vivo (Roberson et al. 1973; Kierszembaum et al. 1974) and in vitro (Hoff 1975; Nogueira et al. 1977; Nogueira and Cohn 1978) experiments have indicated the involvement of cell-mediated effector mechanisms in resistance to infection. In previous work we
have shown that macrophages become trypanocidal during the course of an infection with T. cruzi or by exposure to factor(s) derived from antigen- or mitogenstimulated lymphocytes (Nogueira et al. 1977; Nogueira and Cohn 1978). Since macrophages from both susceptible and resistant strains of mice are equally capable of supporting growth of T. cruzi (Nogueira and Cohn 1976), we have looked for differences in their ability to generate the lymphocyte factor(s) responsible for macrophage activation to a trypanocidal state. In this paper, we describe that spleen cells from various strains of infected mice differ in their ability to generate the lymphocyte factor(s) upon stimulation by the specific antigen. Differences were found in both the time course and in the quantity of macrophage activating factor(s) generated. No differences were found in the ability of macrophages from inbred strains to become activated by the lymphocyte factor(s). We describe the characteristics of our two strains of T. cruzi
325 0014.4894/81/030325-10$02.00/O Copyright @ 1981 by Academic Ress, Inc. AU rights of reproduction in any form reserved.
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in several inbred mouse strains, and show that a T-cell-enriched population, capable of generating in vitro the macrophage activating factor is also capable of conferring protection when passively transferred in vivo. MATERIALS
AND METHODS
Host animals. C57Bl110, Balb/c, C3H/He, A/J, and CBA mice were obtained from The Jackson Laboratory, Bar Harbor, Maine. C3H/He mice were also purchased from Charles River Laboratories (CR). Swiss mice (NCS) were from The Rockefeller University. For in vivo challenge female mice were infected at age 6 weeks. For immunization experiments, g-week-old female mice were used. Parasites. The Y and CL strains of Trypanosoma cruzi were obtained from Dr. Ruth Nussenzweig (New York University School of Medicine, N.Y.). Parasites for in vivo experiments were maintained by weekly passage in 18- to 20-g male A/J mice. For in vitro experiments, blood form trypomastigotes was obtained from infected mice and grown in liver-infusion tryptose (LIT) medium (Fernandes and Castellani 1966). Parasites were harvested from 30-day-old cultures in LIT and metacylic trypomastigotes obtained as previously described (Nogueira and Cohn 1978). Macrophages. Inflammatory macrophages were obtained from NCS mice (unless stated otherwise) 4 days after an intraperitoneal injection of 1 ml of a 1% solution of proteose-peptone (PP) (Difco Laboratories, Detroit, Mich.). Macrophages were harvested according to the methods of Cohn and Benson (1965), and cultivated on 13-mm round glass coverslips in 16-mm Linbro plates (Linbro Chemical CO., New Haven, Conn.) in Dulbecco’s modified Eagle’s medium (Grand Island Biological Co., Grand Island, N.Y.) containing 10% heat-inactivated fetal bovine
serum (FBS) (Grand Island) 100 U/ml penicillin, and 100 &ml streptomycin. Trypanosoma cruzi-primed spleen cells. Mice were infected intraperitoneally with 5 x lo6 live culture forms of T. cruzi in 1 ml phosphate-buffered saline (PBS), as described previously (Nogueira ef al. 1977). At different times after infection, spleens were removed, placed in ice-cold Dulbecco’s medium, teased with forceps, and passed through a sterile stainless-steel wire mesh. The screen was washed with cold medium, and the cells were dispersed by pipetting the suspension several times. The cells were then washed once in cold Dulbecco’s, and either used to prepare spleen cell factor (SCF) or for transfer experiments. Preparation of T-cell-enriched populations. Enriched populations of T cells were
obtained by “panning” on bacteriologic Petri dishes (Fisher Scientific No. 8-757-12) coated with 6- 10 Fg/ml affinity purified sheep anti-mouse Ig exactly as described by Wysocki and Sato (1978). Preparation of spleen cell factor(s). Cells were resuspended in Dulbecco’s medium containing 2% fresh FBS, 5 x lO-5 M mercaptoethanol (ME), 100 U/ml penicillin, 100 pg/ml streptomycin, and glutamine in the presence of heat-killed trypanosomes (10Vml). These were prepared as previously described (Nogueira et al. 1977). Spleen cells, IO*, were incubated in a loo-mm tissue culture dish (Nucn, Denmark) in 6.5 ml of the above medium at 37 C in a CO, atmosphere for 48 hr. The supernatants were then collected, centrifuged at 750g for 15 min to remove cells and debris, and filtered through a 0.45~pm Millex filter (Millipore Corp., Bedford, Mass.). Control supernatants consisted of normal spleen cells incubated with heat-killed trypanosomes for 48 hr. Supernatants were stored in small aliquots at -20 C. Cell transfer experiments. Spleen cells from T. cruzi-infected or noninfected mice
were resuspended in Dulbecco’s medium at the desired concentration and injected intraperitoneally simultaneously with a challenge dose of blood form trypomastigotes. Challenge of mice. Groups of 5- 10 mice were infected intraperitoneally with the desired amount of blood form trypomastigotes in 1 ml PBS. Determination of parasitemia. Ten microliters of blood was collected from the tail vein at 3- to 4-day intervals and added to 190 ~1 of 0.17 M ammonium chloride (Hoff 1974). Parasites were counted in a hemocytometer and parasitemia expressed as number of parasites per milliliter of blood. Numbers shown are arithmetic means of parasitemia values for two to three mice. Induction and evaluation of macrophage trypanocidal activity. Monolayers of proteose -peptone induced macrophages were prepared and nonadherent cells were removed after a 2-hr incubation at 37 C. Spleen cell factor was then added at the desired concentrations in the culture medium. Eighteen hours later, purified trypomastigotes were added in a volume of 100 ~1 to yield an organism/cell ratio of 2: 1, and then incubated for 120- 180 min at 37 C. At the end of the exposure period, coverslips were washed extensively to remove all extracellular parasites, and they were either fixed for microscopic observation or replenished with complete medium in the presence or absence of the appropriate dilution of SCF, and incubation was continued at 37 C for the desired time. The percentage of macrophages infected, number of parasites/100 macrophages, and total cell number were counted in Giemsa-stained samples as previously described (Nogueira and Cohn 1976). Pathology. Heart, liver, and spleens were removed from mice which died from the acute infection or from resistant mice killed at similar time intervals. The organs were fixed in a solution of 3.7% formaldehyde in Ca2+ and Mg2+-free PBS. Fol-
lowing processing, sections were stained with hematoxylin-eosin and screened. RESULTS
Relative Resistance of Mouse Strains to the Y and CL Strains of Trypanosoma cruzi To compare the relative resistance of mouse strains, we challenged animals with lo4 blood form trypomastigotes of the Y or CL strains of T. cruzi. A previous study had shown a spectrum of resistance for the Brazil strain, ranging from highly susceptible mouse strains (e.g., C3H), intermediate types (e.g., Balb/c and CBA), to highly resistant (e.g., C57Bl/lO) strains. Table I shows that C3WHe mice were very susceptible to both Y and CL strains, as measured by cumulative mortality. Balb/C and CBA were as susceptible as C3IWHe mice. BlO mice were resistant to both strains, but were found to be less resistant to the CL strain. Figure 1 shows the pattern of parasitemia for both T. cruzi strains in these mice. In agreement with the mortality data, no significant difference was found in the course of parasitemia in C3H/He and CBA (or Balb/C, not shown). In both cases, 100% of the animals died within 18 days. Daily assessment of parasitemia revealed that blood parasite levels decreased before death in mice infected with the Y strain. Individual mice exhibited variations of a few days in the time of peak parasitemia and death. The asCumulative
TABLE I Mortality at 5 Weeks Strains of Ttypanosoma cruzi
Strains of mice”
Y
CL
CBA C3H/He (Jax) C3H/He C57BUlO BalblC
20120 38138 515 5/23 (20%) lO/lO
U/15 1.5/15 ND 5/10 (50%) lO/lO
a Mice were infected ip with lo4 trypomastigotes.
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NOGUEIRA
Y stran
ET AL.
CL strain
1
C57BVlOJ
“OF C3H/HeJ
5 10 15 20 25 30
Days Days FIG. 1. Course of parasitemia in inbred strains of mice infected intraperitoneally with lo4 blood form trypomastigotes of the Y and CL strains of Trypunosoma cruzi.
2. Course of parasitemia for three challenge doses of Y strain trypomastigotes of Ttypunosoma cruzi in susceptible and resistant mice. FIG.
under similar conditions. Figure 3 shows the parasitemia in mice challenged 4 weeks tending curve shown resulted from the after sublethal infection with lo5 bloodsampling of surviving mice at 3- to 4-day stream forms. All susceptible controls intervals. This masks the descending part of died within 10 days of the challenge with the curve for dying mice. BlO mice showed very high parasitemia, while “immunized” much lower parasitemias (10’Vml or lower) mice showed either no detectable parasites and were able to clear their blood com- in the blood (CBA) or very low levels pletely by 3 weeks of infection. (C3H). Mortality was also reduced to 20% Figure 2 shows the course of parasitemia in both groups. BlO-immunized mice for three challenge doses of the Y strain in susceptible and resistant mice. It is seen that even a challenge dose of lo3 blood form trypomastigotes induced high parasite levels in susceptible mice, and led to the death of 100% of the animals by 4 weeks after infection. In contrast, BlO mice 9 10 showed a lower parasitemia, proportional COnlrOl :: A C~H/H~ to the challenge dose, and no mortality was g 0 C57B1/10 a observed within this 4-week period. 0 CBA/J b
Acquired Immunity Following Sublethal Infection
Having characterized the behavior of the two strains of T. cruzi in inbred mice, we investigated the ability of susceptible and resistant mice to display protective immunity after recovery from a sublethal infection with T. cruzi. Inbred mice were infected intraperitoneally with 5 x lo6 culture forms of the CL or Y strains and at 2-4 weeks after infection rechallenged with lo4 to lo5 bloodstream forms of the parasite. Control mice (age and sex matched) were housed
I
F $ 6 b a
I Immune
n-h
h 5
A .
c3b+/ne C5781/10
X
CBA/J
4a-WmJ
IO
15
20
25
30
FIG. 3. Course of parasitemia in control and immune mice challenged ip with IO5 trypomastigotes of the Y strain of Trypunosomu cruzi. Immune mice were obtained by sublethal infection 4 weeks earlier with 5 x 10” culture forms of the CL strain of T. cruzi.
Trypanosoma
cruzi: T-CELL
ACTIVATION
329
showed no detectable parasitemia and 100% of them survived when challenge doses of lo4 parasitemia were used (not shown); even susceptible mice were fully protected as early as 2 weeks after the immunizing infection. It appears, therefore, that sublethal infection results in the generation of protective immunity in vivo and, as we previously reported (Nogueira and Cohn 1978), macrophage activating factors in vitro, in both susceptible and resistant strains of mice. It is important to emphasize that the immunizing dose of culture forms can be lethal to all strains of mice if animals younger than 6 weeks of age are used.
tion against a virulent challenge with T. when passively transferred into syngeneic nonimmune recipients. Susceptible C3H/He mice receiving one normal spleen equivalent of cells from syngenic mice 2-4 weeks after a sublethal infection had a very low parasitemia and cleared their blood of parasites. Mortality was lowered to levels similar to those found in resistant mice to the homologous strain of T. cruzi. Transfer of similar number of spleen cells from nonimmune mice resulted in no protective effect. Lower numbers of immune spleen cells did not result in any significant protection.
Passive Transfer of Resistance by Spleen Cells Generating the Macrophage Activating Factor(s)
Role of T-Cell-Enriched Immune Cells in Passive Transfer of Resistance in vivo and Lymphokine Production in vitro
Figure 4 is the result of experiments designed to show that the population of cells generating in vitro macrophage activating factor(s) was capable of conferring protec-
In order to test the role of T cells in passive transfer of protection, we depleted immune spleen cells of immunoglobulinbearing cells by “panning” in an antiimmunoglobulin plate. After panning, 40% of the total spleen cells are recovered, 80% of which can be killed by anti-0 sera and complement, and are therefore T cells. This number of cells was as effective as the whole starting spleen cell population in both the ability to confer in vivo protection upon passive transfer (Fig. 5A) and to generate in vitro macrophage activating factor(s) (Fig. 5B).
cruzi,
Relative Ability of Spleen Cells from Different Strains of Mice to Generate Macrophage Activating Factor(s) during Trypanosoma cruzi Infection
FIG. 4. Course of parasitemia in susceptible C3WHe mice passively transferred with spleen cells from control or immune mice. Immune spleen cells were from syngeneic mice given a sublethal infection with 5 x lo6 culture forms of the Y strain of Trypanosoma cruzi 3 weeks before transfer. Control spleen cells were obtained from noninfected C3H/He littermates. Cells were injected intraperitoneally simultaneously with a challenge dose of lo4 Y strain trypomastigotes.
As we have previously reported (Nogueira and Cohn 1976), normal macrophages from both susceptible and resistant strains of mice supported the intracellular growth of T. cruzi equally well. Therefore, the differences in their susceptibility to infection were not traceable to the growth environment provided by normal macrophages. These differences might be due to the ability of lymphoid spleen ceils to generate the activating factor(s) or in the
330
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ET AL.
Days after infection
FIG. 5. (A) Course of parasitemia in C3H/He mice infected with IO4 Y strain Ttypunosomu cruzi trypomastigotes alone (x), plus IOr’ total immune spleen cells (m) or 4 x IO7 T-cell-enriched (immunoglobulin-bearing depleted) immune spleen cells (0). (B) Trypanocidal activity induced in proteosepeptone elicited mouse peritoneal macrophages by lymphokines derived from lo8 total immune spleen cells (x) or 4 x IO’ T-cell-enriched (immunoglobulin-bearing depleted) immune spleen cells (m). See Materials and Methods for details.
capacity of macrophages to mount a microbicidal response. We therefore tested the ability of macrophages from inbred strains to respond to lymphokines generated by homologous immune spleen cells, in response to antigen. As shown in Fig. 6, immune spleen cells from all strains, when collected and tested at optimal conditions (i.e., 2% weeks after infection, 25% concentration of factor(s) in culture medium), were able to activate macrophages to a trypanocidal state. Therefore, macrophages
C3HIHeJ
C57WlOJ
Source of macrophages
FIG. 6. Lymphokine generation and macrophage activation in susceptible and resistant mouse strains. Proteose-peptone induced macrophages from inbred mouse strains were exposed to culture medium alone (open bars); medium plus 25% experimental supernatant (homologous strain immune spleen cells plus heat-killed trypanosomes (solid bars); or medium plus 25% control supernatant (spleen cells from homologous strain noninfected mice plus heat-killed Ttypunosoma cruzi) (dotted bars). See Materials and Methods for details.
from both susceptible and resistant mouse strains could be activated to a trypanocidal state by the active lymphokines. Similarly, no qualitative difference was found among spleen cells of these strains of mice in their response to specific antigen by generating macrophage activating factor(s). However, when the spleen cell factor(s) (SCF) from the different strains were titrated, it became apparent that there were marked quantitative distinctions between resistant and susceptible mouse strains (Fig. 7). Spleen cells from immune resistant mice were found to generate much higher levels of the macrophage activating factor(s) on a per cell basis than any of the susceptible strains. In addition, as can be seen in Fig. 8, these mice were also able to generate higher levels of spleen cell factor(s) at earlier times after infection than susceptible strains. Proliferative responses of these spleen cells to specific antigen and concanavalin A (Con A) were completely suppressed both in susceptible and resistant strains (data not shown). Histological Appearance from Infected Mice
of Organs
Histological examination of organs from mice infected with lo4 blood forms of Y and CL T. cruzi showed that both susceptible
Trypanosoma
331
cruzi: T-CELL ACTIVATION
3
6
9
12
15
18
21
Doys after mfectlon 1/dilution
FIG. 7. Titration of macrophage activating factor generated by inbred strains of mice infected with Ttypunosoma cruzi, in response to the specific antigen (HKT). Supematants were tested on proteose-peptone induced mouse peritoneal macrophages at different concentrations and trypanocidal activity was measured as described under Materials and Methods. These results represent the pattern obtained in six different experiments.
and resistant mouse strains generated a strong mononuclear infiltrate in parasitized organs, but susceptible strains were not able to clear them of parasites (Fig. 9A, arrow). In contrast, resistant strains, sacrificed at similar times (21 days), showed a marked elimination of parasites from the affected organs and macrophages were prominent around the disintegrating pseudocysts (Fig. 9B, arrow). In both cases parasites are completely cleared from spleens. We found no major differences in the distribution of parasites in tissues of inbred mice infected with Y and CL of T. cruzi, under the experimental conditions here described, in contrast to what has been previously reported by Brener (1977). DISCUSSION
The present study provides a direct correlation between in vitro cell-mediated effector mechanisms and protection to Trypanosoma cruzi infections in the intact host. We established the in vivo pattern of resistance in inbred mice of the Y and CL strains of T. cruzi. We showed that the experimental protocol which provides in vivo induced
FIG. 8. Time course of the generation of macrophage activating factor by immune spleen cells from different inbred strains of mice. Supematants were tested on proteose-peptone induced macrophages at a concentration of 25% in the culture medium.
microbicidal macrophages (Nogueira ef al. 1977), as well as activated lymphocytes capable of generating factor(s) which induce macrophage trypanocidal activity under in vitro conditions (Nogueira and Cohn 1978), also results in a protection of these animals from challenge doses of blood forms of the parasite capable of killing 100% of nonimmunized mice, and a population of spleen cells able to confer protection upon adoptive transfer. The cells responsible for the transfer of protection were found in a Tcell-enriched population. The generation of macrophage activating factor(s) was also found in the T-cell-enriched population. Therefore, T cells are required for conferring in vivo protection upon adoptive transfer as well as for the generation of macrophage activating factor(s) in vitro. B cells do not seem to be involved since their removal did not decrease, and actually enhanced the effectiveness of the population, on a per cell basis, in generating the two functions described. The possibility that the protection conferred by in vivo transfer is mediated by helper T cells involved in antibody production is not very likely since protective antibodies are absent at the time of passive transfer experiments (Krettli and Brener 1976). These results indicate that cell-mediated mechanisms are responsible for the ac-
332
NOGUEIRA
ET
AL.
FIG. 9. Histologic sections through the heart of C3H (A) and C57BUlO (B) mice 21 days after challenge with Trypanosoma cruzi. 600x.
quired immunity observed in the acute phase of a T. cruzi infection in the mouse. They support a role for activated T cells, which in turn enhance the microbicidal ef-
fector role of macrophages through the formation of lymphokines (Mackan ess 1969). The finding that both naturally resisl :ant
Trypanosoma
cruzi: T-CELL
and susceptible mice are able to mount protective immunity when given a nonlethal infection suggests that the difference between these strains is not due to an inherent inability of the susceptible mouse strains to mount an immune response to the infective agent. The difference may be the result of a slower and less vigorous immune response which is insufficient to handle a larger and/or more virulent challenge dose of organisms. This suggestion is supported by our findings that the levels of lymphokine generated and the speed with which immune spleen cells respond to antigen are both reduced in suscentible mouse strains. This correlates with o;r findings that resistant strains show a marked clearance of parasites from the tissues and persistence of tissue infection is very low. The cellular basis of these variations is not yet clear. We know that they are not dependent on the effector cell (the macrophage). They could reside in the ability of lymphocytes to respond appropriately to T. cruzi antigen, to respond nonspecifically to generalized stimulation by antigen or mitogen, in the relative numbers of the relevant T-cell subsets in the lymphoid organs, or else in differences in suppressor activity. Differences in lymphocyte responses to antigen or mitogens cannot be assessed through the examination of proliferative responses, since these are completely suppressed during the acute phase of the infection. These results suggest that the cells responsible for lymphokine production are not proliferating. Concanavalin A stimulation of normal or T. cruzi-immune spleen cells from different mouse strains also result in lymphokine production (the latter in the absence of a proliferative response), but no results are yet available as to the relative amounts generated by each strain. In a previous report, Rowland and Kuhn 1978 could not find any relationship between resistance and suppressor activity in inbred T. cruzi-infected mice. We are presently examining in more detail the nature of these differences at the cellular level.
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ACKNOWLEDGMENTS
This investigation received support from the Biomedical Sciences component of the UDNP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and from a Rockefeller Foundation Research Career Development Award in Geographic Medicine to the senior author. We thank M. Nussenzweig and W. Van Voorhis for providing the antimouse immunoglobulin preparation. REFERENCES
BRENER, Z. 1977. Intraspecific variations in Trypanosomn cruzi: two types of parasite populations presenting distinct characteristics. PAHU Scientific Publication 347, 11-21. COHN, Z., AND BENSON, B. 1965. The differentiation of mononuclear phagocytes. Morphology, cytochemistry and biochemistry. Journal of Experimental Medicine
121, 153.
FERNANDES, J. F., AND CASTELLANI, 0. 1966. Growth characteristics and chemical composition of Trypanosomu 195-202.
cruzi. Experimentul
Parasitology
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HOFF, R. 1974. A method for counting and concentrating living Trypanosoma cruzi in blood lysed with ammonium chloride. Journal of Parasitology 60, 527-528.
HOFF, R. 1975. Killing in vitro of Trypunosoma cruzi by macrophages from mice immunized with T. cruzi or BCG, and absence of cross-immunity on challenge in vivo. Journal of Experimental Medicine 142, 299-311. KIERSZEMBAUM, F. KNECHT, E., BUDZKO, D. B., AND PIZZIMENTI, M. C. 1974. Phagocytosis: A defense mechanism against infection with Trypanosoma cruzi. Journal of Immunology
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1844.
KRETTLI, A., AND BRENER,Z. 1976.Protective effects of specific antibodies in Trypanosoma cruzi infections. The Journal of Immunology 116, 755-760. MACKANESS, M. B. 1969. The influence of immunologically committed lymphoid cells on macrophage activity in vivo. Journal of Experimentul Medicine
129, 973-992.
NOGUEIRA, N. AND COHN, Z. 1976. Trypanosoma cruzi: Mechanism of entry and intracellular fate in mammalian cells. Journal of Experimental Medicine 143, 1402- 1420.
NOGUEIRA, N., GORDON, S., AND COHN, Z. 1977. Trypanosoma cruzi: Modification of macrophage function during infection. Journal of Experimentul Medicine 146, 157- 171. NOGUEIRA, N., AND COHN, Z. 1978. Trypanosomu cruzi: In vitro induction of macrophage microbicidal activity. Journal of Experimental Medicine 148, 288-300.
T., AGOSIN, M., CHRISTEN, R., HOECKER, G., NEGHME, A. 1949. Estudios sobre immunobiologia de las enfermedades parasitarias: 1.
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Influencia de la constituiccion genetica en la resistencia de las lauchas a la infection experimental por Trypnnosomu cruzi. Boletin Informuciones Porasitorias Chilenus 4, 48-49. ROBERSON, E. L., HANSON, W. L., AND CHAPMAN, W. L. 1973. Trypanosomu cruzi: Effects of antithymocyte serum in mice and neonatal thymectomy in rats. Experimentul Parasitology 34, 168- 180. ROWLAND, E. C., AND KUHN, R. 1978. Suppression of cellular responses in mice during Trypanosomu cruzi infections. Infection und Immunity 20, 393-397. SEAH, S., AND MARSDEN, P. D. 1969. The protection
ET
AL.
of mice against a virulent strain of Trypunosomu cruzi by previous inoculation with an avirulent strain. Annuls of Tropicul Medicine und Purusitology 63, 211-214. TRISHMAN, BLOOM,
T.,
TANOWITZ,
H.,
WITTNER,
M.,
AND
B. 1978. Trypunosomu cruzi: Role of the immune response in the natural resistance of inbred strains of mice. Experimentul Prrrasitology 45, 160- 168. WYSOCKI, L. J. AND SATO, V. L. 1978. “Panning” for lymphocytes: A method for cell selection. Proceedings of the Nutionul Academy of Sciences USA 75, 2844-2848.
PARASITOI.OGY
51,
325-334
(1981)
11rypanosoma cruzi: In Vivo and in Vitro Correlation between T-Cell Activation and Susceptibility in Inbred Strains of Mice N.NOGUEIRA, The Rockefeller
J. ELLIS, S.CHAPLAN, University,
(Accepted
AND Z.COHN
New York, New York 10021, U.S.A
for publication
5 May 1980)
NOGUEIRA,N.,ELLIS,J.,CHAPLAN,S.,ANDCOHN,Z. 1981.Tr~~panosomn cruzi:In viva and in vifro correlation between T-cell activation and susceptibility in inbred strains of mice. Experimental Parasitology 51, 325-334. The in viva susceptibility of several inbred strains of mice to the Y and CL strains of Trypunasoma cruzi was compared to the in vitro ability of spleen cells from infected mice to generate factor(s) able to activate macrophages to a trypanocidal state. Spleen cells from resistant immune mice generate higher levels of the factor(s) and do so at earlier times during infection than those of susceptible mice. The spleen cells capable of generating the in virro factor(s) are also capable of conferring resistance upon passive transfer. Removal of immunoglobulin-bearing cells from the immune spleen cell population did not affect either transfer of protection in viva or generation of the factor(s) in vitro. The cellular basis underlying the differences between susceptible and resistant mouse strains has not yet been determined. INDEX DESCRIPTORS: Trypanos~ma cruzi; Protozoa, parasitic; Hemoflagellate; Mouse, inbred strains; Natural resistance; Protective immunity; Spleen cells, passive transfer; Spleen cells, T cell enriched; Macrophage activation by spleen cell factors.
INTRODUCTION The murine model of Chagas’s disease has provided a large body of information on some of the immune phenomena involved in infections with Trypanosoma cruzi. It has long been known that various mouse strains differ markedly in their resistance to T. cruzi (Pizzi et al. 1949). The basis of natural resistance has not yet been defined, but the immune system has been implicated in the control of parasitemia and the ability of the host to survive after infection. The genetic differences are not primarily related to the mouse H-2 haplotype (Trishman et al. 1978). The protective effect of acquired immunity has been convincingly demonstrated (Seah and Marsden 1969). In vivo (Roberson et al. 1973; Kierszembaum et al. 1974) and in vitro (Hoff 1975; Nogueira et al. 1977; Nogueira and Cohn 1978) experiments have indicated the involvement of cell-mediated effector mechanisms in resistance to infection. In previous work we
have shown that macrophages become trypanocidal during the course of an infection with T. cruzi or by exposure to factor(s) derived from antigen- or mitogenstimulated lymphocytes (Nogueira et al. 1977; Nogueira and Cohn 1978). Since macrophages from both susceptible and resistant strains of mice are equally capable of supporting growth of T. cruzi (Nogueira and Cohn 1976), we have looked for differences in their ability to generate the lymphocyte factor(s) responsible for macrophage activation to a trypanocidal state. In this paper, we describe that spleen cells from various strains of infected mice differ in their ability to generate the lymphocyte factor(s) upon stimulation by the specific antigen. Differences were found in both the time course and in the quantity of macrophage activating factor(s) generated. No differences were found in the ability of macrophages from inbred strains to become activated by the lymphocyte factor(s). We describe the characteristics of our two strains of T. cruzi
325 0014.4894/81/030325-10$02.00/O Copyright @ 1981 by Academic Ress, Inc. AU rights of reproduction in any form reserved.
326
NOGUEIRA ET AL.
in several inbred mouse strains, and show that a T-cell-enriched population, capable of generating in vitro the macrophage activating factor is also capable of conferring protection when passively transferred in vivo. MATERIALS
AND METHODS
Host animals. C57Bl110, Balb/c, C3H/He, A/J, and CBA mice were obtained from The Jackson Laboratory, Bar Harbor, Maine. C3H/He mice were also purchased from Charles River Laboratories (CR). Swiss mice (NCS) were from The Rockefeller University. For in vivo challenge female mice were infected at age 6 weeks. For immunization experiments, g-week-old female mice were used. Parasites. The Y and CL strains of Trypanosoma cruzi were obtained from Dr. Ruth Nussenzweig (New York University School of Medicine, N.Y.). Parasites for in vivo experiments were maintained by weekly passage in 18- to 20-g male A/J mice. For in vitro experiments, blood form trypomastigotes was obtained from infected mice and grown in liver-infusion tryptose (LIT) medium (Fernandes and Castellani 1966). Parasites were harvested from 30-day-old cultures in LIT and metacylic trypomastigotes obtained as previously described (Nogueira and Cohn 1978). Macrophages. Inflammatory macrophages were obtained from NCS mice (unless stated otherwise) 4 days after an intraperitoneal injection of 1 ml of a 1% solution of proteose-peptone (PP) (Difco Laboratories, Detroit, Mich.). Macrophages were harvested according to the methods of Cohn and Benson (1965), and cultivated on 13-mm round glass coverslips in 16-mm Linbro plates (Linbro Chemical CO., New Haven, Conn.) in Dulbecco’s modified Eagle’s medium (Grand Island Biological Co., Grand Island, N.Y.) containing 10% heat-inactivated fetal bovine
serum (FBS) (Grand Island) 100 U/ml penicillin, and 100 &ml streptomycin. Trypanosoma cruzi-primed spleen cells. Mice were infected intraperitoneally with 5 x lo6 live culture forms of T. cruzi in 1 ml phosphate-buffered saline (PBS), as described previously (Nogueira ef al. 1977). At different times after infection, spleens were removed, placed in ice-cold Dulbecco’s medium, teased with forceps, and passed through a sterile stainless-steel wire mesh. The screen was washed with cold medium, and the cells were dispersed by pipetting the suspension several times. The cells were then washed once in cold Dulbecco’s, and either used to prepare spleen cell factor (SCF) or for transfer experiments. Preparation of T-cell-enriched populations. Enriched populations of T cells were
obtained by “panning” on bacteriologic Petri dishes (Fisher Scientific No. 8-757-12) coated with 6- 10 Fg/ml affinity purified sheep anti-mouse Ig exactly as described by Wysocki and Sato (1978). Preparation of spleen cell factor(s). Cells were resuspended in Dulbecco’s medium containing 2% fresh FBS, 5 x lO-5 M mercaptoethanol (ME), 100 U/ml penicillin, 100 pg/ml streptomycin, and glutamine in the presence of heat-killed trypanosomes (10Vml). These were prepared as previously described (Nogueira et al. 1977). Spleen cells, IO*, were incubated in a loo-mm tissue culture dish (Nucn, Denmark) in 6.5 ml of the above medium at 37 C in a CO, atmosphere for 48 hr. The supernatants were then collected, centrifuged at 750g for 15 min to remove cells and debris, and filtered through a 0.45~pm Millex filter (Millipore Corp., Bedford, Mass.). Control supernatants consisted of normal spleen cells incubated with heat-killed trypanosomes for 48 hr. Supernatants were stored in small aliquots at -20 C. Cell transfer experiments. Spleen cells from T. cruzi-infected or noninfected mice
were resuspended in Dulbecco’s medium at the desired concentration and injected intraperitoneally simultaneously with a challenge dose of blood form trypomastigotes. Challenge of mice. Groups of 5- 10 mice were infected intraperitoneally with the desired amount of blood form trypomastigotes in 1 ml PBS. Determination of parasitemia. Ten microliters of blood was collected from the tail vein at 3- to 4-day intervals and added to 190 ~1 of 0.17 M ammonium chloride (Hoff 1974). Parasites were counted in a hemocytometer and parasitemia expressed as number of parasites per milliliter of blood. Numbers shown are arithmetic means of parasitemia values for two to three mice. Induction and evaluation of macrophage trypanocidal activity. Monolayers of proteose -peptone induced macrophages were prepared and nonadherent cells were removed after a 2-hr incubation at 37 C. Spleen cell factor was then added at the desired concentrations in the culture medium. Eighteen hours later, purified trypomastigotes were added in a volume of 100 ~1 to yield an organism/cell ratio of 2: 1, and then incubated for 120- 180 min at 37 C. At the end of the exposure period, coverslips were washed extensively to remove all extracellular parasites, and they were either fixed for microscopic observation or replenished with complete medium in the presence or absence of the appropriate dilution of SCF, and incubation was continued at 37 C for the desired time. The percentage of macrophages infected, number of parasites/100 macrophages, and total cell number were counted in Giemsa-stained samples as previously described (Nogueira and Cohn 1976). Pathology. Heart, liver, and spleens were removed from mice which died from the acute infection or from resistant mice killed at similar time intervals. The organs were fixed in a solution of 3.7% formaldehyde in Ca2+ and Mg2+-free PBS. Fol-
lowing processing, sections were stained with hematoxylin-eosin and screened. RESULTS
Relative Resistance of Mouse Strains to the Y and CL Strains of Trypanosoma cruzi To compare the relative resistance of mouse strains, we challenged animals with lo4 blood form trypomastigotes of the Y or CL strains of T. cruzi. A previous study had shown a spectrum of resistance for the Brazil strain, ranging from highly susceptible mouse strains (e.g., C3H), intermediate types (e.g., Balb/c and CBA), to highly resistant (e.g., C57Bl/lO) strains. Table I shows that C3WHe mice were very susceptible to both Y and CL strains, as measured by cumulative mortality. Balb/C and CBA were as susceptible as C3IWHe mice. BlO mice were resistant to both strains, but were found to be less resistant to the CL strain. Figure 1 shows the pattern of parasitemia for both T. cruzi strains in these mice. In agreement with the mortality data, no significant difference was found in the course of parasitemia in C3H/He and CBA (or Balb/C, not shown). In both cases, 100% of the animals died within 18 days. Daily assessment of parasitemia revealed that blood parasite levels decreased before death in mice infected with the Y strain. Individual mice exhibited variations of a few days in the time of peak parasitemia and death. The asCumulative
TABLE I Mortality at 5 Weeks Strains of Ttypanosoma cruzi
Strains of mice”
Y
CL
CBA C3H/He (Jax) C3H/He C57BUlO BalblC
20120 38138 515 5/23 (20%) lO/lO
U/15 1.5/15 ND 5/10 (50%) lO/lO
a Mice were infected ip with lo4 trypomastigotes.
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NOGUEIRA
Y stran
ET AL.
CL strain
1
C57BVlOJ
“OF C3H/HeJ
5 10 15 20 25 30
Days Days FIG. 1. Course of parasitemia in inbred strains of mice infected intraperitoneally with lo4 blood form trypomastigotes of the Y and CL strains of Trypunosoma cruzi.
2. Course of parasitemia for three challenge doses of Y strain trypomastigotes of Ttypunosoma cruzi in susceptible and resistant mice. FIG.
under similar conditions. Figure 3 shows the parasitemia in mice challenged 4 weeks tending curve shown resulted from the after sublethal infection with lo5 bloodsampling of surviving mice at 3- to 4-day stream forms. All susceptible controls intervals. This masks the descending part of died within 10 days of the challenge with the curve for dying mice. BlO mice showed very high parasitemia, while “immunized” much lower parasitemias (10’Vml or lower) mice showed either no detectable parasites and were able to clear their blood com- in the blood (CBA) or very low levels pletely by 3 weeks of infection. (C3H). Mortality was also reduced to 20% Figure 2 shows the course of parasitemia in both groups. BlO-immunized mice for three challenge doses of the Y strain in susceptible and resistant mice. It is seen that even a challenge dose of lo3 blood form trypomastigotes induced high parasite levels in susceptible mice, and led to the death of 100% of the animals by 4 weeks after infection. In contrast, BlO mice 9 10 showed a lower parasitemia, proportional COnlrOl :: A C~H/H~ to the challenge dose, and no mortality was g 0 C57B1/10 a observed within this 4-week period. 0 CBA/J b
Acquired Immunity Following Sublethal Infection
Having characterized the behavior of the two strains of T. cruzi in inbred mice, we investigated the ability of susceptible and resistant mice to display protective immunity after recovery from a sublethal infection with T. cruzi. Inbred mice were infected intraperitoneally with 5 x lo6 culture forms of the CL or Y strains and at 2-4 weeks after infection rechallenged with lo4 to lo5 bloodstream forms of the parasite. Control mice (age and sex matched) were housed
I
F $ 6 b a
I Immune
n-h
h 5
A .
c3b+/ne C5781/10
X
CBA/J
4a-WmJ
IO
15
20
25
30
FIG. 3. Course of parasitemia in control and immune mice challenged ip with IO5 trypomastigotes of the Y strain of Trypunosomu cruzi. Immune mice were obtained by sublethal infection 4 weeks earlier with 5 x 10” culture forms of the CL strain of T. cruzi.
Trypanosoma
cruzi: T-CELL
ACTIVATION
329
showed no detectable parasitemia and 100% of them survived when challenge doses of lo4 parasitemia were used (not shown); even susceptible mice were fully protected as early as 2 weeks after the immunizing infection. It appears, therefore, that sublethal infection results in the generation of protective immunity in vivo and, as we previously reported (Nogueira and Cohn 1978), macrophage activating factors in vitro, in both susceptible and resistant strains of mice. It is important to emphasize that the immunizing dose of culture forms can be lethal to all strains of mice if animals younger than 6 weeks of age are used.
tion against a virulent challenge with T. when passively transferred into syngeneic nonimmune recipients. Susceptible C3H/He mice receiving one normal spleen equivalent of cells from syngenic mice 2-4 weeks after a sublethal infection had a very low parasitemia and cleared their blood of parasites. Mortality was lowered to levels similar to those found in resistant mice to the homologous strain of T. cruzi. Transfer of similar number of spleen cells from nonimmune mice resulted in no protective effect. Lower numbers of immune spleen cells did not result in any significant protection.
Passive Transfer of Resistance by Spleen Cells Generating the Macrophage Activating Factor(s)
Role of T-Cell-Enriched Immune Cells in Passive Transfer of Resistance in vivo and Lymphokine Production in vitro
Figure 4 is the result of experiments designed to show that the population of cells generating in vitro macrophage activating factor(s) was capable of conferring protec-
In order to test the role of T cells in passive transfer of protection, we depleted immune spleen cells of immunoglobulinbearing cells by “panning” in an antiimmunoglobulin plate. After panning, 40% of the total spleen cells are recovered, 80% of which can be killed by anti-0 sera and complement, and are therefore T cells. This number of cells was as effective as the whole starting spleen cell population in both the ability to confer in vivo protection upon passive transfer (Fig. 5A) and to generate in vitro macrophage activating factor(s) (Fig. 5B).
cruzi,
Relative Ability of Spleen Cells from Different Strains of Mice to Generate Macrophage Activating Factor(s) during Trypanosoma cruzi Infection
FIG. 4. Course of parasitemia in susceptible C3WHe mice passively transferred with spleen cells from control or immune mice. Immune spleen cells were from syngeneic mice given a sublethal infection with 5 x lo6 culture forms of the Y strain of Trypanosoma cruzi 3 weeks before transfer. Control spleen cells were obtained from noninfected C3H/He littermates. Cells were injected intraperitoneally simultaneously with a challenge dose of lo4 Y strain trypomastigotes.
As we have previously reported (Nogueira and Cohn 1976), normal macrophages from both susceptible and resistant strains of mice supported the intracellular growth of T. cruzi equally well. Therefore, the differences in their susceptibility to infection were not traceable to the growth environment provided by normal macrophages. These differences might be due to the ability of lymphoid spleen ceils to generate the activating factor(s) or in the
330
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ET AL.
Days after infection
FIG. 5. (A) Course of parasitemia in C3H/He mice infected with IO4 Y strain Ttypunosomu cruzi trypomastigotes alone (x), plus IOr’ total immune spleen cells (m) or 4 x IO7 T-cell-enriched (immunoglobulin-bearing depleted) immune spleen cells (0). (B) Trypanocidal activity induced in proteosepeptone elicited mouse peritoneal macrophages by lymphokines derived from lo8 total immune spleen cells (x) or 4 x IO’ T-cell-enriched (immunoglobulin-bearing depleted) immune spleen cells (m). See Materials and Methods for details.
capacity of macrophages to mount a microbicidal response. We therefore tested the ability of macrophages from inbred strains to respond to lymphokines generated by homologous immune spleen cells, in response to antigen. As shown in Fig. 6, immune spleen cells from all strains, when collected and tested at optimal conditions (i.e., 2% weeks after infection, 25% concentration of factor(s) in culture medium), were able to activate macrophages to a trypanocidal state. Therefore, macrophages
C3HIHeJ
C57WlOJ
Source of macrophages
FIG. 6. Lymphokine generation and macrophage activation in susceptible and resistant mouse strains. Proteose-peptone induced macrophages from inbred mouse strains were exposed to culture medium alone (open bars); medium plus 25% experimental supernatant (homologous strain immune spleen cells plus heat-killed trypanosomes (solid bars); or medium plus 25% control supernatant (spleen cells from homologous strain noninfected mice plus heat-killed Ttypunosoma cruzi) (dotted bars). See Materials and Methods for details.
from both susceptible and resistant mouse strains could be activated to a trypanocidal state by the active lymphokines. Similarly, no qualitative difference was found among spleen cells of these strains of mice in their response to specific antigen by generating macrophage activating factor(s). However, when the spleen cell factor(s) (SCF) from the different strains were titrated, it became apparent that there were marked quantitative distinctions between resistant and susceptible mouse strains (Fig. 7). Spleen cells from immune resistant mice were found to generate much higher levels of the macrophage activating factor(s) on a per cell basis than any of the susceptible strains. In addition, as can be seen in Fig. 8, these mice were also able to generate higher levels of spleen cell factor(s) at earlier times after infection than susceptible strains. Proliferative responses of these spleen cells to specific antigen and concanavalin A (Con A) were completely suppressed both in susceptible and resistant strains (data not shown). Histological Appearance from Infected Mice
of Organs
Histological examination of organs from mice infected with lo4 blood forms of Y and CL T. cruzi showed that both susceptible
Trypanosoma
331
cruzi: T-CELL ACTIVATION
3
6
9
12
15
18
21
Doys after mfectlon 1/dilution
FIG. 7. Titration of macrophage activating factor generated by inbred strains of mice infected with Ttypunosoma cruzi, in response to the specific antigen (HKT). Supematants were tested on proteose-peptone induced mouse peritoneal macrophages at different concentrations and trypanocidal activity was measured as described under Materials and Methods. These results represent the pattern obtained in six different experiments.
and resistant mouse strains generated a strong mononuclear infiltrate in parasitized organs, but susceptible strains were not able to clear them of parasites (Fig. 9A, arrow). In contrast, resistant strains, sacrificed at similar times (21 days), showed a marked elimination of parasites from the affected organs and macrophages were prominent around the disintegrating pseudocysts (Fig. 9B, arrow). In both cases parasites are completely cleared from spleens. We found no major differences in the distribution of parasites in tissues of inbred mice infected with Y and CL of T. cruzi, under the experimental conditions here described, in contrast to what has been previously reported by Brener (1977). DISCUSSION
The present study provides a direct correlation between in vitro cell-mediated effector mechanisms and protection to Trypanosoma cruzi infections in the intact host. We established the in vivo pattern of resistance in inbred mice of the Y and CL strains of T. cruzi. We showed that the experimental protocol which provides in vivo induced
FIG. 8. Time course of the generation of macrophage activating factor by immune spleen cells from different inbred strains of mice. Supematants were tested on proteose-peptone induced macrophages at a concentration of 25% in the culture medium.
microbicidal macrophages (Nogueira ef al. 1977), as well as activated lymphocytes capable of generating factor(s) which induce macrophage trypanocidal activity under in vitro conditions (Nogueira and Cohn 1978), also results in a protection of these animals from challenge doses of blood forms of the parasite capable of killing 100% of nonimmunized mice, and a population of spleen cells able to confer protection upon adoptive transfer. The cells responsible for the transfer of protection were found in a Tcell-enriched population. The generation of macrophage activating factor(s) was also found in the T-cell-enriched population. Therefore, T cells are required for conferring in vivo protection upon adoptive transfer as well as for the generation of macrophage activating factor(s) in vitro. B cells do not seem to be involved since their removal did not decrease, and actually enhanced the effectiveness of the population, on a per cell basis, in generating the two functions described. The possibility that the protection conferred by in vivo transfer is mediated by helper T cells involved in antibody production is not very likely since protective antibodies are absent at the time of passive transfer experiments (Krettli and Brener 1976). These results indicate that cell-mediated mechanisms are responsible for the ac-
332
NOGUEIRA
ET
AL.
FIG. 9. Histologic sections through the heart of C3H (A) and C57BUlO (B) mice 21 days after challenge with Trypanosoma cruzi. 600x.
quired immunity observed in the acute phase of a T. cruzi infection in the mouse. They support a role for activated T cells, which in turn enhance the microbicidal ef-
fector role of macrophages through the formation of lymphokines (Mackan ess 1969). The finding that both naturally resisl :ant
Trypanosoma
cruzi: T-CELL
and susceptible mice are able to mount protective immunity when given a nonlethal infection suggests that the difference between these strains is not due to an inherent inability of the susceptible mouse strains to mount an immune response to the infective agent. The difference may be the result of a slower and less vigorous immune response which is insufficient to handle a larger and/or more virulent challenge dose of organisms. This suggestion is supported by our findings that the levels of lymphokine generated and the speed with which immune spleen cells respond to antigen are both reduced in suscentible mouse strains. This correlates with o;r findings that resistant strains show a marked clearance of parasites from the tissues and persistence of tissue infection is very low. The cellular basis of these variations is not yet clear. We know that they are not dependent on the effector cell (the macrophage). They could reside in the ability of lymphocytes to respond appropriately to T. cruzi antigen, to respond nonspecifically to generalized stimulation by antigen or mitogen, in the relative numbers of the relevant T-cell subsets in the lymphoid organs, or else in differences in suppressor activity. Differences in lymphocyte responses to antigen or mitogens cannot be assessed through the examination of proliferative responses, since these are completely suppressed during the acute phase of the infection. These results suggest that the cells responsible for lymphokine production are not proliferating. Concanavalin A stimulation of normal or T. cruzi-immune spleen cells from different mouse strains also result in lymphokine production (the latter in the absence of a proliferative response), but no results are yet available as to the relative amounts generated by each strain. In a previous report, Rowland and Kuhn 1978 could not find any relationship between resistance and suppressor activity in inbred T. cruzi-infected mice. We are presently examining in more detail the nature of these differences at the cellular level.
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ACKNOWLEDGMENTS
This investigation received support from the Biomedical Sciences component of the UDNP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and from a Rockefeller Foundation Research Career Development Award in Geographic Medicine to the senior author. We thank M. Nussenzweig and W. Van Voorhis for providing the antimouse immunoglobulin preparation. REFERENCES
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B. 1978. Trypunosomu cruzi: Role of the immune response in the natural resistance of inbred strains of mice. Experimentul Prrrasitology 45, 160- 168. WYSOCKI, L. J. AND SATO, V. L. 1978. “Panning” for lymphocytes: A method for cell selection. Proceedings of the Nutionul Academy of Sciences USA 75, 2844-2848.