Vol. 14, No. 4 Printed in U.S.A.
INFECTION AND IMMUNITY, OCt. 1976, P. 1103-1105 Copyright © 1976 American Society for Microbiology
NOTES Cyclophosphamide Pretreatment and Protection Against Malaria JOHN F. FINERTY* AND EDWARD P. KREHL Laboratory ofMicrobial Immunity, National Institute ofAllergy and Infectious Diseases, Bethesda, Maryland 20014,* and Trudeau Institute, Saranac Lake, New York 12983 Received for publication 14 May 1976
Mice pretreated with cyclophosphamide were able to overcome infection from lethal malarial strain. The development of resistance was preceded by increased hypersensitivity to malarial antigens. Hypersensitivity was demonstrable by a delayed footpad swelling technique. a
Cyclophosphamide (Cy) is known to potentiate infections caused by pathogenic organisms when administered simultaneously with the organisms or after the infection has been established (1, 8, 9). Although Cy is cytotoxic to cells undergoing mitosis and has been shown to deplete B-cells from spleen and lymphoid tissues (12), the manner in which this drug suppresses immunity during infection remains uncertain. Cy can also increase the immune response to nonviable antigens when given before immunization (4, 5, 10, 11). In particular, T-cell reactivity was enhanced as evidenced by increased delayed-hypersensitivity reaction to sheep erythrocytes in animals that were treated by Cy before sensitization (4). In this report, we present data on the effects of Cy on the immune response to malarial antigens and to a lethal strain of Plasmodium berghei subsp. yoelii, 17XL. Mice pretreated with Cy developed delayed footpad swelling (DFS) to P. berghei subsp. yoelii antigens, an indication of delayed hypersensitivity (4). Female CD-1 mice, 8 weeks old (Charles River Breeding Laboratories, Wilmington, Mass.), were used in this study. The parasite used was a lethal strain of P. berghei subsp. yoelii XL. This strain was isolated, in our laboratory, from a nonlethal P. berghei subsp. yoeii 17X, by continuous blood passage into normal mice. The lethal strain, XL, had a 100% lethal dose when 104 parasitized erythrocytes were injected intraperitoneally (i.p.) into mice; death followed in 8 + 1 days. A freeze-thawed parasite antigen (FT) was prepared from infected mice; the parasites were first treated as previously described (6). They were then adjusted to 2 x 109/ml in phosphatebuffered saline, frozen at - 70°C, and thawed at
37°C. This process was repeated four times. The FT antigen was stored at -70°C. Cy (Mead Johnson Co., Evansville, Ind.), dissolved in saline, was injected via a tail vein, at a dose of 200 mg/kg. Cy was given 48 h before immunization or infection. Cy-treated and nontreated mice were immunized i.p. with FT antigen, equivalent to 107 parasites/0.1 ml. DFS was measured as the increase in footpad thickness, 23 h after the infection, into the left hind footpad, of an eliciting dose of FT antigen in 50 ,ul, equivalent to 108 parasites. Measurements were made with a dial micrometer (Schnelltaster, H. C. Kroplin, Postfach, Germany; 4). The mean and range of footpad swellings were derived from a minimum of 5 mice/ group per day; no animal was tested twice. When separate groups of mice were tested for DFS on days 2, 4, and 6 after immunization, the Cy-treated group showed a response that peaked on day 4 and then decreased sharply on day 6 (Fig. 1). In contrast, the non-Cy-treated group revealed a DFS response that peaked on day 2 and remained essentially unchanged through day 6. To study the same effects upon infection, mice were treated with Cy, inoculated i.p. with 104 P. berghei subsp. yoelii XL parasites/0.1 ml, and separate groups were assayed for DFS responses 2, 4, 6, and 8 days after infection. The results (Fig. 2) revealed a DFS response that peaked on day 2, in the Cy mice, decreased sharply on day 4, and remained unchanged thereafter. The non-Cy group revealed a slight response on day 2, but a greater response on day 6. No further tests were done on the non-Cy mice, since they died of fulminat-
ing parasitemia.
Parasitemia was measured (2) in two other groups, treated as above, but not tested for
1103
1104
INFECT. IMMUN.
NOTES
14
12
The intense DFS response to malarial anti-cY 0-0 gens in either immunized or infected mice sug+ c' C C gests that an increased T-cell activity was responsible, in part, for preventing a lethal infec14
10
E
12
0
I-
Cy *-O
z a I-
10
6
Non-Cy
0-0
0 0
U-4
E
8
0
6
E
2
-
0n
o
2 4 6 8 DAYS AFTER IMMUNIZATION FIG. 1. Delayed footpad responses in CD-I mice, pretreated (@) or nontreated (0) with cyclophosphamide and then immunized with Plasmodium berghei subsp. yoelii XL antigens. Range of values is shown
4 0
U.
2
by bracketed lines.
4 2 6 8 DFS. Parasites were detected in the Cy and non-Cy mice on day 2 (Fig. 3). The parasitemia DAYS AFTER INFECTION in the Cy animals did not increase until day 4, 2. FIG. Delayed footpad responses in CD-I mice, peaked on day 11, but then decreased and bepretreated (a) or nontreated (0) with cyclophosphacame subpatent by day 24. In contrast, the mide and then infected with Plasmodium berghei parasitemia of the non-Cy group constantly in- subsp. yoelii XL parasites. D, Died, non-Cy mice. creased, maximum levels occurred on day 8, and all of the mice were dead by day 9. The effect of immunosuppressive drugs on the immune response varies according to the time of administration (7). Cy given before immunization enhances the response to nonliving / 100 antigens (4, 5, 10). This same effect was ob10._ , served with the FT malarial antigen. d g{.. o ~ The data from the present study suggest that Cy altered the host immune response in a manner so as to protect the host from a lethal infection. Mice treated with Cy before P. berghei subsp. yoelii XL inoculation revealed an increased DFS response and protection from infection. All but one of these mice survived. The mechanism involved remains to be determined. DAYS AFTER INFECTION A previous report showed that Cy has no direct effect on malarial parasites (9); thus, we believe FIG. 3. Parasitemia in CD-I mice, pretreated that the protection afforded was the result of a ( ) or nontreated (-----) with cyclophosphamide heightened immune response, and not to resid- and then infected with Plasmodium berghei subsp. ual drug activity. yoelii XL parasites.
NOTES
VOL. 14, 1976
tion. In fact, on day 2 when the DFS of infected mice was high, the parasitemia was low in the Cy group; on day 4 when the response had decreased, the parasitemia increased, suggesting a vital role for T-cells early in infection. It is also possible that pretreatment with Cy depleted a B-cell response (3) that normally could interfere with the development of immunity to the parasites. Regardless of the mechanism(s) responsible, these data demonstrate that protection in CD-1 mice against a lethal malarial infection can be achieved by pretreatment with Cy. Whether this holds true for other P. berghei strains in other mice is open to study. Further, the data indicate that host immunity can be manipulated by the use of chemical agents in a situation in which immunization fails to protect the host. We are now investigating the mechanisms involved. LITERATURE CITED 1. Cozad, G. C., and T. J. Lindsey. 1974. Effect of cyclophosphamide on Histopiasma capsulatum. Infect. Immun. 9:261-265. 2. Finerty, J. F., J. E. Tobie, and C. B. Evans. 1972. Antibody and immunoglobulin synthesis in germfree and conventional mice infected with Plasmodium berghei. Am. J. Trop. Med. Hyg. 21:499-505.
1105
3. Katz, S. I., D. Parker, and J. L. Turk. 1974. B-cell suppression of delayed hypersensitivity reactions. Nature (London) 251:550-551. 4. Lagrange, P. H., G. B. Mackaness, and T. E. Miller. 1974. Potentiation of T-cell mediated immunity by selective suppression of antibody formation with cyclophosphamide. J. Exp. Med. 139:1529-1539. 5. Maguire, H. C., and V. L. Ettore. 1967. Enhancement of dinitrochlorobene (DNCB) contact sensitization by cyclophosphamide in the guinea pig. J. Invest. Dermatol. 48:39-43. 6. Martin, W. J., J. F. Finerty, and A. Rosenthal. 1971. Isolation of Plasmodium berghei (malaria) parasites by ammonium chloride lysis of infected erythrocytes. Nature (London) New Biol. 233:260-261. 7. Santos, G. W. 1967. Immunosuppressive drugs. I. Fed. Proc. 26:907-913. 8. Singer, S. H., M. Ford, and R. L. Kerchstein. 1972. Respiratory diseases in cyclophosphamide-treated mice. I. Increased virulence of Mycoplasma pulmonis. Infect. Immun. 5:953-956. 9. Spira, D. T., P. H. Silverman, and G. Gaines. 1972. Multiple modes of action of cyclophosphamide on plasmodial infections in rats. Trans. R. Soc. Trop. Med. Hyg. 66:921-930. 10. Turk, J. L., and D. Parker. 1973. Further studies on Blymphocyte suppression in delayed hypersensitivity, indicating a possible mechanism for Jones-Mate hypersensitivity. Immunology 24:751-758. 11. Turk, J. L., D. Parker, and L. W. Poulter. 1972. Functional aspects of the selective depletion of lymphoid tissue by cyclophosphamide. Immunology 23:493-501. 12. Turk, J. L., and L. W. Poulter. 1972. Selective depletion of lymphoid tissue by cyclophosphamide. Clin. Exp. Immunol. 10:285-296.
INFECTION AND IMMUNITY, OCt. 1976, P. 1103-1105 Copyright © 1976 American Society for Microbiology
NOTES Cyclophosphamide Pretreatment and Protection Against Malaria JOHN F. FINERTY* AND EDWARD P. KREHL Laboratory ofMicrobial Immunity, National Institute ofAllergy and Infectious Diseases, Bethesda, Maryland 20014,* and Trudeau Institute, Saranac Lake, New York 12983 Received for publication 14 May 1976
Mice pretreated with cyclophosphamide were able to overcome infection from lethal malarial strain. The development of resistance was preceded by increased hypersensitivity to malarial antigens. Hypersensitivity was demonstrable by a delayed footpad swelling technique. a
Cyclophosphamide (Cy) is known to potentiate infections caused by pathogenic organisms when administered simultaneously with the organisms or after the infection has been established (1, 8, 9). Although Cy is cytotoxic to cells undergoing mitosis and has been shown to deplete B-cells from spleen and lymphoid tissues (12), the manner in which this drug suppresses immunity during infection remains uncertain. Cy can also increase the immune response to nonviable antigens when given before immunization (4, 5, 10, 11). In particular, T-cell reactivity was enhanced as evidenced by increased delayed-hypersensitivity reaction to sheep erythrocytes in animals that were treated by Cy before sensitization (4). In this report, we present data on the effects of Cy on the immune response to malarial antigens and to a lethal strain of Plasmodium berghei subsp. yoelii, 17XL. Mice pretreated with Cy developed delayed footpad swelling (DFS) to P. berghei subsp. yoelii antigens, an indication of delayed hypersensitivity (4). Female CD-1 mice, 8 weeks old (Charles River Breeding Laboratories, Wilmington, Mass.), were used in this study. The parasite used was a lethal strain of P. berghei subsp. yoelii XL. This strain was isolated, in our laboratory, from a nonlethal P. berghei subsp. yoeii 17X, by continuous blood passage into normal mice. The lethal strain, XL, had a 100% lethal dose when 104 parasitized erythrocytes were injected intraperitoneally (i.p.) into mice; death followed in 8 + 1 days. A freeze-thawed parasite antigen (FT) was prepared from infected mice; the parasites were first treated as previously described (6). They were then adjusted to 2 x 109/ml in phosphatebuffered saline, frozen at - 70°C, and thawed at
37°C. This process was repeated four times. The FT antigen was stored at -70°C. Cy (Mead Johnson Co., Evansville, Ind.), dissolved in saline, was injected via a tail vein, at a dose of 200 mg/kg. Cy was given 48 h before immunization or infection. Cy-treated and nontreated mice were immunized i.p. with FT antigen, equivalent to 107 parasites/0.1 ml. DFS was measured as the increase in footpad thickness, 23 h after the infection, into the left hind footpad, of an eliciting dose of FT antigen in 50 ,ul, equivalent to 108 parasites. Measurements were made with a dial micrometer (Schnelltaster, H. C. Kroplin, Postfach, Germany; 4). The mean and range of footpad swellings were derived from a minimum of 5 mice/ group per day; no animal was tested twice. When separate groups of mice were tested for DFS on days 2, 4, and 6 after immunization, the Cy-treated group showed a response that peaked on day 4 and then decreased sharply on day 6 (Fig. 1). In contrast, the non-Cy-treated group revealed a DFS response that peaked on day 2 and remained essentially unchanged through day 6. To study the same effects upon infection, mice were treated with Cy, inoculated i.p. with 104 P. berghei subsp. yoelii XL parasites/0.1 ml, and separate groups were assayed for DFS responses 2, 4, 6, and 8 days after infection. The results (Fig. 2) revealed a DFS response that peaked on day 2, in the Cy mice, decreased sharply on day 4, and remained unchanged thereafter. The non-Cy group revealed a slight response on day 2, but a greater response on day 6. No further tests were done on the non-Cy mice, since they died of fulminat-
ing parasitemia.
Parasitemia was measured (2) in two other groups, treated as above, but not tested for
1103
1104
INFECT. IMMUN.
NOTES
14
12
The intense DFS response to malarial anti-cY 0-0 gens in either immunized or infected mice sug+ c' C C gests that an increased T-cell activity was responsible, in part, for preventing a lethal infec14
10
E
12
0
I-
Cy *-O
z a I-
10
6
Non-Cy
0-0
0 0
U-4
E
8
0
6
E
2
-
0n
o
2 4 6 8 DAYS AFTER IMMUNIZATION FIG. 1. Delayed footpad responses in CD-I mice, pretreated (@) or nontreated (0) with cyclophosphamide and then immunized with Plasmodium berghei subsp. yoelii XL antigens. Range of values is shown
4 0
U.
2
by bracketed lines.
4 2 6 8 DFS. Parasites were detected in the Cy and non-Cy mice on day 2 (Fig. 3). The parasitemia DAYS AFTER INFECTION in the Cy animals did not increase until day 4, 2. FIG. Delayed footpad responses in CD-I mice, peaked on day 11, but then decreased and bepretreated (a) or nontreated (0) with cyclophosphacame subpatent by day 24. In contrast, the mide and then infected with Plasmodium berghei parasitemia of the non-Cy group constantly in- subsp. yoelii XL parasites. D, Died, non-Cy mice. creased, maximum levels occurred on day 8, and all of the mice were dead by day 9. The effect of immunosuppressive drugs on the immune response varies according to the time of administration (7). Cy given before immunization enhances the response to nonliving / 100 antigens (4, 5, 10). This same effect was ob10._ , served with the FT malarial antigen. d g{.. o ~ The data from the present study suggest that Cy altered the host immune response in a manner so as to protect the host from a lethal infection. Mice treated with Cy before P. berghei subsp. yoelii XL inoculation revealed an increased DFS response and protection from infection. All but one of these mice survived. The mechanism involved remains to be determined. DAYS AFTER INFECTION A previous report showed that Cy has no direct effect on malarial parasites (9); thus, we believe FIG. 3. Parasitemia in CD-I mice, pretreated that the protection afforded was the result of a ( ) or nontreated (-----) with cyclophosphamide heightened immune response, and not to resid- and then infected with Plasmodium berghei subsp. ual drug activity. yoelii XL parasites.
NOTES
VOL. 14, 1976
tion. In fact, on day 2 when the DFS of infected mice was high, the parasitemia was low in the Cy group; on day 4 when the response had decreased, the parasitemia increased, suggesting a vital role for T-cells early in infection. It is also possible that pretreatment with Cy depleted a B-cell response (3) that normally could interfere with the development of immunity to the parasites. Regardless of the mechanism(s) responsible, these data demonstrate that protection in CD-1 mice against a lethal malarial infection can be achieved by pretreatment with Cy. Whether this holds true for other P. berghei strains in other mice is open to study. Further, the data indicate that host immunity can be manipulated by the use of chemical agents in a situation in which immunization fails to protect the host. We are now investigating the mechanisms involved. LITERATURE CITED 1. Cozad, G. C., and T. J. Lindsey. 1974. Effect of cyclophosphamide on Histopiasma capsulatum. Infect. Immun. 9:261-265. 2. Finerty, J. F., J. E. Tobie, and C. B. Evans. 1972. Antibody and immunoglobulin synthesis in germfree and conventional mice infected with Plasmodium berghei. Am. J. Trop. Med. Hyg. 21:499-505.
1105
3. Katz, S. I., D. Parker, and J. L. Turk. 1974. B-cell suppression of delayed hypersensitivity reactions. Nature (London) 251:550-551. 4. Lagrange, P. H., G. B. Mackaness, and T. E. Miller. 1974. Potentiation of T-cell mediated immunity by selective suppression of antibody formation with cyclophosphamide. J. Exp. Med. 139:1529-1539. 5. Maguire, H. C., and V. L. Ettore. 1967. Enhancement of dinitrochlorobene (DNCB) contact sensitization by cyclophosphamide in the guinea pig. J. Invest. Dermatol. 48:39-43. 6. Martin, W. J., J. F. Finerty, and A. Rosenthal. 1971. Isolation of Plasmodium berghei (malaria) parasites by ammonium chloride lysis of infected erythrocytes. Nature (London) New Biol. 233:260-261. 7. Santos, G. W. 1967. Immunosuppressive drugs. I. Fed. Proc. 26:907-913. 8. Singer, S. H., M. Ford, and R. L. Kerchstein. 1972. Respiratory diseases in cyclophosphamide-treated mice. I. Increased virulence of Mycoplasma pulmonis. Infect. Immun. 5:953-956. 9. Spira, D. T., P. H. Silverman, and G. Gaines. 1972. Multiple modes of action of cyclophosphamide on plasmodial infections in rats. Trans. R. Soc. Trop. Med. Hyg. 66:921-930. 10. Turk, J. L., and D. Parker. 1973. Further studies on Blymphocyte suppression in delayed hypersensitivity, indicating a possible mechanism for Jones-Mate hypersensitivity. Immunology 24:751-758. 11. Turk, J. L., D. Parker, and L. W. Poulter. 1972. Functional aspects of the selective depletion of lymphoid tissue by cyclophosphamide. Immunology 23:493-501. 12. Turk, J. L., and L. W. Poulter. 1972. Selective depletion of lymphoid tissue by cyclophosphamide. Clin. Exp. Immunol. 10:285-296.
