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The onset of immune protection in acute experimental chagas' disease in C3H(HE) mice
International
Journalfor
Parasitology. 1915. Vol. 5. pp. 241-244. Pcrgamon Press. Printed in Great Britain.
THE ONSET OF IMMUNE PROTECTION IN ACUTE EXPERIMENTAL CHACAS’ DISEASE IN C3H(HE) MICE R. E. KU~IN and S. K. DURUM Department
of Biology, Wake Forest University, Winston-Salem,
North Carolina 27109, U.S.A.
(Received 14 May; amended 29 July 1974) Abstract-KUHN R. E. and DURUMS. K. 1975. The onset of immune protection in acute experimental Chagas’ disease in C3H(He) mice, ~nfer~tiona~ JournaZ~r Puras~to~ogy 5: 24-244. The onset of protective immunity against ~~y~u~~~o~u crzczi in mice was determined by adoptively immunizing newly infected recipients with spleen cells from normal or infected donor mice. It was found that spleen cells from animals with 3 day and 6 day infections did not provide protection but that spleen cells from infections of 9, 12, 15 and 18 days significantly increased longevity in infected recipient animals. The protective capacity per spleen cell was found to increase in proportion to the duration of infection of donor mice. It was further noted that immune protection, as reflected in increased longevity, did not result in decreased development of parasitemia. Immunized mice which demonstrated the greatest longevity developed parasitemias over twice that observed in control groups. INDEX KEY WORDS: ~ry~ff~~~o~~ crrrzi; Chagas’ disease; spleen cells; adoptive immunity; mice; immune protection,
INTRODUCTION KOEBERLE (1968) has suggested that most of the damage done to host tissues during Trypanasoma cruri infections occurs in the acute stage of the
disease. It is also during this initial period of the host-parasite relationship that one would expect the mammalian host to recognize the antigenicity of the parasite and to begin the development of protective immunity. It is appropriate, therefore, to examine the onset of immunity during acute experimental Chagas’ disease. We report here the results of studies on the adoptive transfer of immunity against T. cruzi by C3H(He) mouse spleen cells which demonstrate an early and progressive development of immune protection. MATERIALS
AND METHODS
Animals All mice in this study were C3H(He) females, weighing 20-25 g and were purchased from Flow Research Laboratories, Inc. (Dublin, Va.). Animals were maintained in our laboratory for 2 weeks prior to use in experiments and were housed 6 to a cage. Purina rat chow and water were supplied ad libitum. Infections Mice were infected by intraperitoneal (IP) injection of 5 x 104 trypomastigotes (7’. cruzi, Brazil strain) in 0.1 ml of phosphate buffered saline (PBS). Trypomastigotes were taken from stock mice which had been infected with 5 x 104 trypomastigotes 14 days earlier. Donor mice were bled from the orbital plexus into cold RPMI-1640 (Gibco, Grand Island, N.Y.) containing 10 fzg of heparin per ml, 241
and the blood subsequently was centrifuged and washed 3 times in cold PBS. The concentration of parasites was determined by hemacytometer counting and adjusted to 5 x 104 trypomastigotes per 0.1 ml of PBS. C3H(He) female mice receiving this inoculum usually die on about day 18. Parasitemia determinations Parasitemia in infected animals was determined by placing a 4 pl sample of tail vein blood uniformly under an 18 mm circular glass coverslip, counting the parasites in 100 fields, and converting this figure to the number of parasites per ml of blood. Preparation of spleen ceils Animals to be used as spleen cell donors received IP injections of 0.1 ml PBS containing 5 x 104 thrice washed trypomastigotes. At specified times the donors were bled to death and their spleens were removed. Spleen cells were harvested by gently pressing the spleen through a 32 gauge stainless steel screen into cold RPMI-1640 medium; cell clumping was reduced by repeated passage through a 26 gauge needle. Spleen cells from at least 2 animals were pooled, washed 3 times and the concentration adjusted to 25 x 106 viable, nucleated cells in O-2 ml of RPMI-1~0. All s&en cell nrenarations used contained at least 90 per cent viable ceils as determined by the technique of trypan blue exclusion (Sullivan, Berke & Amos, 1972).
Experimentalprotocol Each group of 5 experimental animals received 25 x 106spleen cells from animals with 3.6. 9. 12. 15 or 18 day infections of T. crazi. Sensitized 'normal spleen cells were injected into the left side of the peritoneal cavity. Three hours later, 5 x 104 washed t~pomastigotes
or
242
R. E. KUHN and S. K. DURUM
were inoculated into the right side of the peritoneal cavity. One control group received normal spleen cells followed by trypomastigotes, the other control group received trypomastigotes alone. All animals were infected at the same time with the same preparation of blood-form trypomastigotes to insure similar infective doses. Parasitemia was determined on alternate days and time of host death was recorded. (Parasitemia values were not included when there were fewer than three survivors in an experimental or control group.) RESULTS
The mean interval between time of infection and death is shown for each group in Fig. 1. Mean Iongevity was identical for groups receiving either normal cells or cells from 3- or &day infections; the mean for animals receiving trypomastigotes alone was slightly greater (0.6 days). However, recipients of spleen cells from mice harboring a 9-day infection lived an average of 1.2 days longer than controls. After the ninth day of infection, the data show that the longer the duration of infection of the cell donor, the greater the longevity of the cell recipients. Thus, 12-, 15- and 18-day cell recipients lived an average of 2.6, 3.6 and 4.0 days longer than controls, respectively. The mean number of parasites per ml of blood on days subsequent to experimental infection is shown for each group in Fig. 2. As can be seen, recipients of 18-day splenocytes developed the highest parasitemias, and all spleen cell recipients, whether
VOL.
5.
1975
from normal or infected donors, show higher parasitemias just prior to death than are observed in animals receiving trypomastigotes alone. The data indicate that immunity to T. cruzi in C3H(He) female mice is developed during acute lethal infections. Immune protection by spleen cells, here defined as an increase in longevity of the recipient, is restricted to those cells from donors infected for 9 days or more. The protective capacity per cell was found to increase in proportion to the duration of the previous parasitic infection until day 18 at which time these mice usually die of the stock infection (5~ 104 trypomastigotes). DISCUSSION
The results of this study do not provide an elucidation of the mechanism of immunity in experimental Chagas’ disease. No interpretation of the data can be made to favor either a cell-mediated or a humoral (antibody) mediated response as being responsible for the observed increase in longevity. It is apparent, however, that these mice, which are incapable of surviving the disease, do develop an early and progressive, if inadequate, immunity to the parasite. Rubio (1959) has suggested that parasitemia is not a good index of the pathologic state in experimental Chagas’ disease. it was noted during the course of our experiments also that parasitemia could not be used as an indicator of immune
I
I
8
L
0
3
6
9
Days duration
I.J.P.
of spleen
1
12
I
15
I
I6
cet I donors
FIG. 1. The effect of adoptive immuni~tion on longevity of mice infected with ~~~~u~u~Q~u cruzi. Each point represents the mean longevity (~s.D.) of 5 mice infected with 5 x 104 trypomastigotes and receiving 25 x 106 spleen cells from normal or infected donors. Donor cells were taken from mice with infections of 3, 6, 9, 12, 15 or 18 days.
I.I.P.
VOL.
Immunity to Trypanosoma cruzi
5. 1975
243
12
IO
a6 w
2
FIG. 2. The effect of adoptive immunization
on parasitemia of mice infected with Trypunosomu cruzi.
effectiveness in this system. Control animals receiving 5 x 104 trypomastigotes alone, died with approximately 4-5~ 106 trypanosomes per ml of blood on about day 19. One might have predicted that parasitemia would be depressed in immune animals and that death would have occurred when parasitemia reached 4-5~ 106/ml in all cases. As Fig. 2 demonstrates, however, this was clearly not the case. Mice which survived the longest also developed the highest parasitemias. In other experiments in our laboratory we have observed gross disparities between parasitemia and predicted time of death in immune manipulated mice (unpublished observations). If as suggested by our studies the number of parasites in the blood of mice with experimental Chagas’ disease does not reflect the pathologic state of the host, one must question the nature of acquired immunity to T. cruzi. While significant protection by immune serum has been demonstrated by passive immunizations (Culbertson & Kolodny, 1938; Kolodny, 1940; Roberson, Hanson & Chapman, 1973), the intracellular stage of the parasite, being sterically inaccessible, is apparently unaffected by antibody (Goble, 1970). Also, Pizzi & Knierim (1955) reported that the level of gamma globulin during T. cruzi infections was not significant in altering the course of infection in normal and splenectomized mice. These observations would indicate that a specific antibody response to a
particular component or product of the trypanosome may be more important in protective immunity than the production of a wide spectrum of antibodies to several antigens. Koeberle (1968) has postulated that the invasion of trypanosomes into cells is mediated by a lytic enzyme elaborated by the parasite. If a qualitatively distinct antibody is produced against a parasiteelaborated factor which facilitates invasion, this might explain the observed lack of correlation between parasitemia and pathologic state in adoptively immunized mice. Circulating antibody to this ‘lytic invasion factor’ could inhibit invasion and, thereby, cause an accumulation of parasites in the blood. The result of this would be an increased longevity and an increase in parasitemia in these immune manipulated mice. The increased longevity would then be due to decreased tissue damage since fewer cells would be invaded and destroyed. Demonstration of the onset of immunity following the sixth day of infection may be correlated to the release of parasites from host cells and the reinvasion of these trypomastigotes into other host cells. Dvorak & Hyde (1973) have shown in vitro that mammalian cells can be invaded within minutes of the introduction of the parasite. Kuhn, Vaughn & Iannuzzi (1974) have reported that epismastigotes are cleared very rapidly from the blood with less than 3 per cent remaining in the blood 2 h post intravenous injection of 10X 106 parasites in mice.
244
R. E.
and S. K. DURUM
KUHN
Dvorak cellular
& Hyde (1973) also reported that intraproliferation and subsequent escape of T. cruzi took 6.5 days in vitro and resulted in approximately 512 parasites/invaded host cell. If the sum of these observations can be applied to the events leading to the development of immunity in vivo, then one might assume that the first extensive interaction of the host’s immune system with the parasite and its elaborated products, if any, would not occur at the time of infection, but rather at the time of the release of parasites following the first intracellular cycle. Acknowledgement-This research was supported Brown-Hazen Grant from Research Corporation.
by
a
REFERENCES CULBERTSON J. T. & KOLODNY M. H. 1938. Acquired immunity in rats against Trypanosomu cruzi. Journal ofParasitology 24: 83-90. DVORAK J. A. & HYDE T. P. 1973. Trypanosoma cruzi: Interaction with vertebrate cells in vitro. I. Individual interactions at the cellular and subcellular level. Experimental Parasitology 34: 268-283. GOBLE F. 1970. In Immunity to Parasitic Animals (Edited by JACKSON G. J., HERMAN R. & SINGER I.) Vol. 2,
pp. 597-689. Appleton, New York.
I.J.P.
VOL.
5.
1975
KOEBERLE F. 1968. Chagas’ disease and Chagas’ syndrome: The pathology of American trypanosomiasis. Advances in Parasitology 6: 63-l 16. KOLODNY M. H. 1940. Studies on age resistance against trypanosome infections. VII. The influence of age upon the immunological response of rats to infection with Trypanosoma cruzi. American Journal of Hygiene 31: l-8. KUHN R. E., VAUGHN R. T. & IANNUZZI N. P. 1974. The in vivo distribution of 5iCr-labeled Trypanosoma cruzi in mice. International Journal for Parasitology. 4: 585588. PIZZI T. & KNIERIM F. 1955. Modificaciones de1 bazo en relation con la tasa de anticuerpos circulantes en ratones experimentalmente infectados con Trypanosoma cruzi. Boletin Chileno de Parasitologh 10: 4249. ROBERSON E. L., HANSON W. L. & CHAPMAN W. L. 1973. Trypanosoma cruzi: Effects of anti-thymocyte serum in mice and neonatal thymectomy in rats. Enperimental Parasitology 34: 168-180. RUBIO M. 1959. Natural and acquired immunity against Trypanosoma cruzi in the hamster (Cricetus auratus). Bioldgica, Santiago 27-28: 95-l 16. SULLIVAN K.. BERKE G. & AMOS D. B. 1972. Chromium51 leakage’ from and uptake of trypan blue by target cells undergoing cell-mediated destruction. Transplantation 13: 627-628.
Journalfor
Parasitology. 1915. Vol. 5. pp. 241-244. Pcrgamon Press. Printed in Great Britain.
THE ONSET OF IMMUNE PROTECTION IN ACUTE EXPERIMENTAL CHACAS’ DISEASE IN C3H(HE) MICE R. E. KU~IN and S. K. DURUM Department
of Biology, Wake Forest University, Winston-Salem,
North Carolina 27109, U.S.A.
(Received 14 May; amended 29 July 1974) Abstract-KUHN R. E. and DURUMS. K. 1975. The onset of immune protection in acute experimental Chagas’ disease in C3H(He) mice, ~nfer~tiona~ JournaZ~r Puras~to~ogy 5: 24-244. The onset of protective immunity against ~~y~u~~~o~u crzczi in mice was determined by adoptively immunizing newly infected recipients with spleen cells from normal or infected donor mice. It was found that spleen cells from animals with 3 day and 6 day infections did not provide protection but that spleen cells from infections of 9, 12, 15 and 18 days significantly increased longevity in infected recipient animals. The protective capacity per spleen cell was found to increase in proportion to the duration of infection of donor mice. It was further noted that immune protection, as reflected in increased longevity, did not result in decreased development of parasitemia. Immunized mice which demonstrated the greatest longevity developed parasitemias over twice that observed in control groups. INDEX KEY WORDS: ~ry~ff~~~o~~ crrrzi; Chagas’ disease; spleen cells; adoptive immunity; mice; immune protection,
INTRODUCTION KOEBERLE (1968) has suggested that most of the damage done to host tissues during Trypanasoma cruri infections occurs in the acute stage of the
disease. It is also during this initial period of the host-parasite relationship that one would expect the mammalian host to recognize the antigenicity of the parasite and to begin the development of protective immunity. It is appropriate, therefore, to examine the onset of immunity during acute experimental Chagas’ disease. We report here the results of studies on the adoptive transfer of immunity against T. cruzi by C3H(He) mouse spleen cells which demonstrate an early and progressive development of immune protection. MATERIALS
AND METHODS
Animals All mice in this study were C3H(He) females, weighing 20-25 g and were purchased from Flow Research Laboratories, Inc. (Dublin, Va.). Animals were maintained in our laboratory for 2 weeks prior to use in experiments and were housed 6 to a cage. Purina rat chow and water were supplied ad libitum. Infections Mice were infected by intraperitoneal (IP) injection of 5 x 104 trypomastigotes (7’. cruzi, Brazil strain) in 0.1 ml of phosphate buffered saline (PBS). Trypomastigotes were taken from stock mice which had been infected with 5 x 104 trypomastigotes 14 days earlier. Donor mice were bled from the orbital plexus into cold RPMI-1640 (Gibco, Grand Island, N.Y.) containing 10 fzg of heparin per ml, 241
and the blood subsequently was centrifuged and washed 3 times in cold PBS. The concentration of parasites was determined by hemacytometer counting and adjusted to 5 x 104 trypomastigotes per 0.1 ml of PBS. C3H(He) female mice receiving this inoculum usually die on about day 18. Parasitemia determinations Parasitemia in infected animals was determined by placing a 4 pl sample of tail vein blood uniformly under an 18 mm circular glass coverslip, counting the parasites in 100 fields, and converting this figure to the number of parasites per ml of blood. Preparation of spleen ceils Animals to be used as spleen cell donors received IP injections of 0.1 ml PBS containing 5 x 104 thrice washed trypomastigotes. At specified times the donors were bled to death and their spleens were removed. Spleen cells were harvested by gently pressing the spleen through a 32 gauge stainless steel screen into cold RPMI-1640 medium; cell clumping was reduced by repeated passage through a 26 gauge needle. Spleen cells from at least 2 animals were pooled, washed 3 times and the concentration adjusted to 25 x 106 viable, nucleated cells in O-2 ml of RPMI-1~0. All s&en cell nrenarations used contained at least 90 per cent viable ceils as determined by the technique of trypan blue exclusion (Sullivan, Berke & Amos, 1972).
Experimentalprotocol Each group of 5 experimental animals received 25 x 106spleen cells from animals with 3.6. 9. 12. 15 or 18 day infections of T. crazi. Sensitized 'normal spleen cells were injected into the left side of the peritoneal cavity. Three hours later, 5 x 104 washed t~pomastigotes
or
242
R. E. KUHN and S. K. DURUM
were inoculated into the right side of the peritoneal cavity. One control group received normal spleen cells followed by trypomastigotes, the other control group received trypomastigotes alone. All animals were infected at the same time with the same preparation of blood-form trypomastigotes to insure similar infective doses. Parasitemia was determined on alternate days and time of host death was recorded. (Parasitemia values were not included when there were fewer than three survivors in an experimental or control group.) RESULTS
The mean interval between time of infection and death is shown for each group in Fig. 1. Mean Iongevity was identical for groups receiving either normal cells or cells from 3- or &day infections; the mean for animals receiving trypomastigotes alone was slightly greater (0.6 days). However, recipients of spleen cells from mice harboring a 9-day infection lived an average of 1.2 days longer than controls. After the ninth day of infection, the data show that the longer the duration of infection of the cell donor, the greater the longevity of the cell recipients. Thus, 12-, 15- and 18-day cell recipients lived an average of 2.6, 3.6 and 4.0 days longer than controls, respectively. The mean number of parasites per ml of blood on days subsequent to experimental infection is shown for each group in Fig. 2. As can be seen, recipients of 18-day splenocytes developed the highest parasitemias, and all spleen cell recipients, whether
VOL.
5.
1975
from normal or infected donors, show higher parasitemias just prior to death than are observed in animals receiving trypomastigotes alone. The data indicate that immunity to T. cruzi in C3H(He) female mice is developed during acute lethal infections. Immune protection by spleen cells, here defined as an increase in longevity of the recipient, is restricted to those cells from donors infected for 9 days or more. The protective capacity per cell was found to increase in proportion to the duration of the previous parasitic infection until day 18 at which time these mice usually die of the stock infection (5~ 104 trypomastigotes). DISCUSSION
The results of this study do not provide an elucidation of the mechanism of immunity in experimental Chagas’ disease. No interpretation of the data can be made to favor either a cell-mediated or a humoral (antibody) mediated response as being responsible for the observed increase in longevity. It is apparent, however, that these mice, which are incapable of surviving the disease, do develop an early and progressive, if inadequate, immunity to the parasite. Rubio (1959) has suggested that parasitemia is not a good index of the pathologic state in experimental Chagas’ disease. it was noted during the course of our experiments also that parasitemia could not be used as an indicator of immune
I
I
8
L
0
3
6
9
Days duration
I.J.P.
of spleen
1
12
I
15
I
I6
cet I donors
FIG. 1. The effect of adoptive immuni~tion on longevity of mice infected with ~~~~u~u~Q~u cruzi. Each point represents the mean longevity (~s.D.) of 5 mice infected with 5 x 104 trypomastigotes and receiving 25 x 106 spleen cells from normal or infected donors. Donor cells were taken from mice with infections of 3, 6, 9, 12, 15 or 18 days.
I.I.P.
VOL.
Immunity to Trypanosoma cruzi
5. 1975
243
12
IO
a6 w
2
FIG. 2. The effect of adoptive immunization
on parasitemia of mice infected with Trypunosomu cruzi.
effectiveness in this system. Control animals receiving 5 x 104 trypomastigotes alone, died with approximately 4-5~ 106 trypanosomes per ml of blood on about day 19. One might have predicted that parasitemia would be depressed in immune animals and that death would have occurred when parasitemia reached 4-5~ 106/ml in all cases. As Fig. 2 demonstrates, however, this was clearly not the case. Mice which survived the longest also developed the highest parasitemias. In other experiments in our laboratory we have observed gross disparities between parasitemia and predicted time of death in immune manipulated mice (unpublished observations). If as suggested by our studies the number of parasites in the blood of mice with experimental Chagas’ disease does not reflect the pathologic state of the host, one must question the nature of acquired immunity to T. cruzi. While significant protection by immune serum has been demonstrated by passive immunizations (Culbertson & Kolodny, 1938; Kolodny, 1940; Roberson, Hanson & Chapman, 1973), the intracellular stage of the parasite, being sterically inaccessible, is apparently unaffected by antibody (Goble, 1970). Also, Pizzi & Knierim (1955) reported that the level of gamma globulin during T. cruzi infections was not significant in altering the course of infection in normal and splenectomized mice. These observations would indicate that a specific antibody response to a
particular component or product of the trypanosome may be more important in protective immunity than the production of a wide spectrum of antibodies to several antigens. Koeberle (1968) has postulated that the invasion of trypanosomes into cells is mediated by a lytic enzyme elaborated by the parasite. If a qualitatively distinct antibody is produced against a parasiteelaborated factor which facilitates invasion, this might explain the observed lack of correlation between parasitemia and pathologic state in adoptively immunized mice. Circulating antibody to this ‘lytic invasion factor’ could inhibit invasion and, thereby, cause an accumulation of parasites in the blood. The result of this would be an increased longevity and an increase in parasitemia in these immune manipulated mice. The increased longevity would then be due to decreased tissue damage since fewer cells would be invaded and destroyed. Demonstration of the onset of immunity following the sixth day of infection may be correlated to the release of parasites from host cells and the reinvasion of these trypomastigotes into other host cells. Dvorak & Hyde (1973) have shown in vitro that mammalian cells can be invaded within minutes of the introduction of the parasite. Kuhn, Vaughn & Iannuzzi (1974) have reported that epismastigotes are cleared very rapidly from the blood with less than 3 per cent remaining in the blood 2 h post intravenous injection of 10X 106 parasites in mice.
244
R. E.
and S. K. DURUM
KUHN
Dvorak cellular
& Hyde (1973) also reported that intraproliferation and subsequent escape of T. cruzi took 6.5 days in vitro and resulted in approximately 512 parasites/invaded host cell. If the sum of these observations can be applied to the events leading to the development of immunity in vivo, then one might assume that the first extensive interaction of the host’s immune system with the parasite and its elaborated products, if any, would not occur at the time of infection, but rather at the time of the release of parasites following the first intracellular cycle. Acknowledgement-This research was supported Brown-Hazen Grant from Research Corporation.
by
a
REFERENCES CULBERTSON J. T. & KOLODNY M. H. 1938. Acquired immunity in rats against Trypanosomu cruzi. Journal ofParasitology 24: 83-90. DVORAK J. A. & HYDE T. P. 1973. Trypanosoma cruzi: Interaction with vertebrate cells in vitro. I. Individual interactions at the cellular and subcellular level. Experimental Parasitology 34: 268-283. GOBLE F. 1970. In Immunity to Parasitic Animals (Edited by JACKSON G. J., HERMAN R. & SINGER I.) Vol. 2,
pp. 597-689. Appleton, New York.
I.J.P.
VOL.
5.
1975
KOEBERLE F. 1968. Chagas’ disease and Chagas’ syndrome: The pathology of American trypanosomiasis. Advances in Parasitology 6: 63-l 16. KOLODNY M. H. 1940. Studies on age resistance against trypanosome infections. VII. The influence of age upon the immunological response of rats to infection with Trypanosoma cruzi. American Journal of Hygiene 31: l-8. KUHN R. E., VAUGHN R. T. & IANNUZZI N. P. 1974. The in vivo distribution of 5iCr-labeled Trypanosoma cruzi in mice. International Journal for Parasitology. 4: 585588. PIZZI T. & KNIERIM F. 1955. Modificaciones de1 bazo en relation con la tasa de anticuerpos circulantes en ratones experimentalmente infectados con Trypanosoma cruzi. Boletin Chileno de Parasitologh 10: 4249. ROBERSON E. L., HANSON W. L. & CHAPMAN W. L. 1973. Trypanosoma cruzi: Effects of anti-thymocyte serum in mice and neonatal thymectomy in rats. Enperimental Parasitology 34: 168-180. RUBIO M. 1959. Natural and acquired immunity against Trypanosoma cruzi in the hamster (Cricetus auratus). Bioldgica, Santiago 27-28: 95-l 16. SULLIVAN K.. BERKE G. & AMOS D. B. 1972. Chromium51 leakage’ from and uptake of trypan blue by target cells undergoing cell-mediated destruction. Transplantation 13: 627-628.