EXPERIMENTAL PARASITOLOGY 75,240-241(1992)
Plasmodium chabaudi: Estradiol Suppresses Acquiring, but Not Once-Acquired Immunity W.P.M. *Division
BENTEN,* F. WUNDERLICH,* AND H. MOSSMANN?
of Parasitology, Institute of Zoology, Heinrich-Heine-University, 4000 Duesseldorf and thfax-Planck-Institute of Immunobiology, 7800 Freiburg, Germany
1, Germany;
BENTEN, W. P. M., WUNDERLICH, F., AND MOSSMANN, H. 1992.Plasmodium chabaudi: Estradiol suppresses acquiring, but not once-acquired immunity. Experimental Parasitology 75,240-247. This study investigates the effect of estradiol (E) on self-healing of Plasmodium chabaudi malaria in mice of the inbred strain C57BL/lO. Our data show: (1) Female mice and male castrates are capable of self-healing infections when challenged with lo6 P. chabaudiinfected erythrocytes. Self-healing is completely suppressed after pretreatment of mice with 12 ug E injected SCtwice a week for 3 weeks. (2) The suppressive effect of E is prevented by the estrogen receptor blockers tamoxifen and clomiphene. (3) The nonsteroidal E-agonist diethylstilbestrol (DES) also suppresses self-healing. This suppressive DES effect is prevented by tamoxifen. (4) In mice immune to P. chabaudi, neither survival rate nor the course of parasitemia is affected by E, even at IO-fold higher E doses. Our data suggest that the immunosuppressive action of E is a specific genomic effect, i.e., E-induced gene products prevent the development of protective immunity against P. chabaudi. 8 IWZ Academic Ress,
Inc.
chabaudi; Malaria; Immunosuppression; Sex hormones; Androgen receptor; Clomiphene; Tamoxifen; Estradiol (E); Testosterone (T); Estrogen receptor (ER); Diethylstilbestrol (DES). INDEX DESCRIPTORSAND ABBREVIATIONS: Plasmodium
INTRODUCTION There is overwhelming evidence for the existence of a sex dimorphism concerning onset, course, and outcome of numerous disease processes, including infectious diseases caused by a wide variety of protozoan and helminthic endoparasites (Alexander and Stimson 1988; Bundy 1988). Very recently, a spectacular gender dependence has been shown for the outcome of the murine malaria Plasmodium chabaudi (Wunderlich et al. 1988) and Plasmodium berghei (Kamis and Ibrahim 1989). For instance, female B10 mice are able to selfheal P. chabaudi infections and, thus, to develop long-lasting immunity against homologous rechallenge. By contrast, male BlO mice cannot develop immunity and, therefore, succumb to P. chabaudi infections (Wunderlich et al. 1988, 1991; Benten et al. 1991). Sex hormones are important environ-
mental factors modulating the immune system of vertebrates (Ansar Ahmed et al. 1985; Grossman and Roselle 1986; Schuurs and Verheul 1990). For instance, T is known to suppress diverse immune reactions both in vivo and in vitro (Stimson 1987; Schuurs and Verheul 1990). This appears to be of advantage in autoimmune and/or immunopathological disorders, but is disadvantageous in infectious diseases. Indeed, T increases the susceptibility toward numerous parasitic diseases (Alexander and Stimson 1988). This also holds true for P. chabaudi infections. For instance, female BlO mice normally resistant to P. chabaudi become susceptible after T treatment, i.e., T suppresses the development of protective immune mechanisms against P. chabaudi (Wunderlich et al. 1988, 1991; Benten et al. 1991). However, it is possible that, besides T, E is also involved in the control of female resistance to P. chabaudi. At least, there is 240
0014-4894192$5.00 Copyright 6 1992 by Academic Press, Inc. ALI rights of reproduction in any form reserved.
ESTRADIOL-SUPPRESSEDP. chabaudi
some information available that E increases murine resistance to other protozoan parasites such as Leishmania mexicana (Alexander and Stimson 1988), Trypanosoma cruzi (Kierszenbaum et al. 1974), Trypanosoma lewisi (Mankau 1975), and Babesia microti (Cottrell et al. 1977). We therefore wondered if the suppressive effect of T against P. chabaudi malaria may be counterregulated by E. To our surprise, however, E also suppresses the development of immunity, even at much lower concentrations than T. This suppressive E effect is specific since E does not affect existing immunity to P. chabaudi.
MALARIA
241
passages (Wunderlich et al. 1982). Parasitemia was evaluated in Giemsa-stained blood smears. Erythrocytes were counted in a Neubauer chamber. Immune mice. Female and castrated male BlO mice normally resolve infections with P. chabaudi. Those mice which were still alive at 9 weeks postinfection, were used as immune mice (Wunderlich and Helwig, 1987).
E-induced
RESULTS suppression of self-healing.
Male mice of the inbred strain C57BL/lO do not survive a challenge with lo6 P. chabaudi-infected erythrocytes (Table I). Males also succumb to infection when male mice are pretreated with E for 2 or even 6 weeks before infection (Table I). It may be therefore that E does not counterregulate MATERIAL AND METHODS the suppressive effect of T. Conversely, E Mice. Mice of the strains C57BL/lO and NMRI were may be suppressive itself. We therefore exbred under specific pathogen-free conditions in our amined this possibility in female and cascolony at the Heinrich-Heine-University. They were trated male B 10 mice. B 10 females and cashoused in plastic cages with standard diet (Nohrlin, Bad Salzuflen, Germany) and water ad libitum. The trated BIO males are normally self-healers, mice were 9-12 weeks old when beginning with the i.e., about 95% of mice resolve and, subseexperiments. quently, survive infections with P. chaOrchidectomy. Male mice were anesthetized under baudi (Table I, Fig. 1). However, this selfKetavet (Park-Davis, Berlin, Germany) and Rompun healing capacity is impaired and even com(Bayer, Leverkusen, Germany). The testes and epididymis were pulled off through a scrotal incision and pletely abrogated by E, depending on the conditions of E treatment (Table I). For inremoved by electrocautery (Wunderlich et al. UBl). After operation, the mice were kept under standard stance, only a slight-if any-reduction occonditions for 3 weeks before further experimental curs in survival when mice are treated with use. E only during infection. However, female Treatment with estrogens. The mice were treated and male castrates succumb to infection with E-benzoate (Progynon B oleosum, Schering, when they are pretreated with an E dose of Berlin, Germany), or DES (Steraloids Inc., Wilton, 12 kg twice a week for 3 weeks before inU.S.A.) at different doses. They received E or DES in 200~pl subcutaneous injections twice per week for 3 fection and when this treatment is continweeks, before infecting with P. chabaudi-if not oth- ued during infection (Fig. 1). Under these erwise stated. The estrogens were suspended in sesexperimental conditions, E does not essename oil (Roth, Karlsruhe, Germany). The treatment tially affect the course of parasitemia (Fig. was continued during infections-if not otherwise stated. Controls received sesame oil alone. 1). Mice can even decease after parasite Treatment with ER blockers. Mice were treated with clearance has begun and the number of intamoxifen (Nolvadex, ICI Pharma, Heidelberg, Ger- fected erythrocytes has been already signifmany) or clomiphene (Merrell Dow Research Institute, Winnersh, U.K.) suspended in sesame oil. The icantly reduced in the peripheral blood. Remice received subcutaneous injections of 200~pl por- markably, castrated males are more sensitions twice per week for 3 weeks and the treatment tive to E than females. This becomes was continued during infections-if not otherwise evident, for example, when the treatment stated. with 12 p,g E is not continued during infecParasite infections. The C57BWlO mice were intion: About 60% females recover from the fected by ip injection with lo6 P. chabaudi-infected suppressive E effect, whereas castrated erythrocytes. The latter were obtained from NMRI mice, in which P. chabaudi was maintained by weekly males remain suppressed (Table I).
242
BENTEN, WUNDERLICH,
AND MOSSMANN
TABLE I Effect of E on the Outcome of P. chabaudi Infections in C57BIJlO Mice Mice infected with P. chnbaudi E pretreatment Sex of mice Male Male Male Female Female Female Female Female Female Female Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated
male male male male male male male male
Concentration (I.4 120 120 12 120 60 12 12 12 12 120 60 60 12 12 12
Period (weeks)
E treatment during infection
Number of surviving/ total mice
Day of death of succumbing mice (days p.i. f SD)
Average survival rate (%)
+ + + + + + + + + + -
l/13 o/15 0124 32134 10/12 O/8 o/7 o/10 6/10 315 38140 10/12 o/9 O/16 o/5 o/5 o/15 o/5
11.1 * 1.7 12.1 * 3.5 11.7 ” 1.8 11.5 12.0 10.3 2 1.0 9.9 f 1.1 11.8 f 3.7 12.0 f 1.2 11.0 12.0 10.0 9.8 f 0.8 11.4 f 1.7 13.8 f 3.9 10.4 ” 1.3 12.5 f 2.3 11.0 f 1.7
8 0 0 95 83 0 0 0 60 60 95 83 0 0 0 0 0 0
2 6 3 3 3 3 1 3 3 3 3 3 1
Prevention of the E-induced suppression by ER blockers. Tamoxifen and clomiphene
are known to bind to ERs, thus blocking the E action. Table II shows that tamoxifen at a dose of 0.1 mg per injection does not affect self-healing of P. chabaudi in both females and male castrates. Also, clomiphene has no influence on self-healing in castrated BlO males (Table II). Intriguingly, however, these ER blockers prevent the suppressive effect of E. Treatment of E together with either tamoxifen or clomiphene does not result in death of mice which is typical for E treatment alone (Table II). EfSect of DES on self-healing. DES, one of the first nonsteroidal estrogens with estrogenic activity, also impairs self-healing. When castrated males are pretreated with 74 kg DES twice a week for 3 weeks before infection, the survival rate declines to 43% (Table III). Survival is completely abolished’when the DES dose is doubled to 148 pg (Table III). The DES-induced suppression of self-healing can be prevented by tamoxifen (Table III).
!
I I -1 tE (10)
t 0
2
4
6
8
10
12
14
16
18
20
22
24
Days pi.
FIG. 1. Effect of E on P. chabaudi infections in female C57BWlO mice. Ten mice were treated with 12 ug E for 3 weeks (+ E), before these and 10 untreated mice (-E) were challenged with lo6 P. chabaudiinfected erythrocytes. The E treatment was continued during infection. Means are given with either + or SD for clarity reasons.
Females Females Females Females Females Females Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated
mates males males males males males males males males males males males
Sex of mice
-
3 -
-
12 12 12 12 60 60 12 60 12 12 12 60 12
i
T 3 3 3 3 3 3 -
T -
Period (weeks)
Concentration (lud
E
100 100 100 100 100 100 100 100 -
-
Concentration bd
Tamoxifen
T 3 3 3 3 3 3 -
-
Period (weeks)
Pretreatment
100 100 100
-
-
-
-
Concentration OLB)
5 3 3
-
-
Period (weeks)
Clomiphene
-
+ + -
+ + + -
+ + + f + + + + + -
Tamoxifen
E
-
-
-
Clomiphene
Treatment during infection
1l/l2 o/10 IO/l2 10112 6l8 5/s 19/21 O/l6 O/5 o/15 819 s/9 617 6112 S/5 416 415 8/11
Number of surviving/ total mice
TABLE II Effect of Tamoxifen and Clomiphene on the E-Induced Susceptibility of BlO Mice to P. chubaudi
-9.5 11.012.0 f 1.0
11.5 11.0 12.0 11.4 ‘- 1.7 13.8 2f 2.3 3.9 12.5 10.0 12.9 2 1.6 17.0 10.7 + 1.6
11.0 11.7 f 3.2 12.0
Day of death of succumbing mice (days p.i. t SD)
Mice infected with P. chabaudi
67 loo 80 73
0 89 56 86 50
83 75 100 91 i
92 0 83
Average survival rate (%)
E s
$
5 g P -.a
P
g
s
z
in
m
244
BENTEN, WUNDERLICH,
AND MOSSMANN
TABLE III Effect of DES on Self-Healing of P. chubaudi in Castrated Male BlO Mice Pretreatment
Mice infectedwith P. Tamoxifen
DES
Concentration Period (weeks) (I%) 74 148 148
3 3 3
-
-
Treatmentduring infection Concentration Period ~ (weeks) DES Tamoxifen (l&d 100 100
3 3
+ -
Effect of E on existing immunity. After self-healing, both female and castrated male BlO mice have acquired long-lasting immunity. Thus, reinfection with P. chabaudi at 9 weeks after the first infection does not cause any symptoms of sickness. Parasitized erythrocytes can be detected only on Days 8-12 postreinfection, but the parasitemia does not regularly exceed 0.1%. When mice have once acquired immunity, this cannot be suppressed by E (Table IV). Neither outcome nor course of infections are changed by treatment with 12 kg E (Table IV). Even if the E dose is increased to 120 p,g, no change in survival rates can be observed (Table IV). Also, parasitemia is not raised. DISCUSSION
Our data show that E can impair and even completely abolish self-healing of P. chabaudi malaria. Self-healing obviously
Number of surviving/ total mice
+
22.l23 3l7 o/10 7l7 819
chaboudi
Day of death of succumbingmice (daysp.i. 2 SD)
Average survival rate (%)
14.0
96 43 0
10.5f 0.6 10.7 f 2.0 10.0
100 89
reflects the successful mounting of protective immune mechanisms against P. chabaudi (Wunderlich and Helwig 1987). Thus, it is reasonable to assume that E acts as an immunosuppressant. This view is consistent with numerous other studies showing that E suppresses a series of immune reactions both in vivo and in vitro (Grossman and Roselle 1986; Schuurs and Verheul 1990). The immunosuppressive activity of E may also explain why E is known to increase the susceptibility of vertebrate hosts toward diverse infectious diseases caused, for example, by parasites such as Toxoplasma (Grossman 1984; Pung and Luster 1986), Trichomonas (Alexander and Stimson 1988), and Trichinella (Dean et al. 1980; Luebke et al. 1984), as well as by bacteria such as Listeria (Dean et al. 1980; Pung et al. 1984), Chlamydia (Rank et al. 1982), and Staphylococcus (Toivanen 1967). However, the immunosuppressive action of E does not necessarily contrast to those tind-
TABLE IV Effect of E on the Outcome of P. chabaudi Infections in Immune C57BL/lO Mice Immune mice infected with P. chabaudi E pretreatment of mice
Concentration (IJd
Period (weeks)
E treatment during infection
Number of surviving/ total mice
Average survival rate (%)
Females Females Females Castrated males Castrated males
60 120 60
3 3 3
+ + +
1909 515 12/12 lO/lO 717
100 100 100 100 100
Sex
ESTRADIOL-SUPPRESSEDP. chabaudi MALARIA
245
ings showing an increased resistance under ously, the E-activated genes ultimately E influence (Grossman 1984; Alexander prevent the functional expression of those and Stimson 1988). For it may be that-E genes of the mouse MHC, the H-2 comalso suppresses infection-caused immuno- plex, and those genes of the non-H-2 backpathological reactions. Interestingly, there ground, which normally control the develis some information available that protec- opment of protective immunity against P. tive immunity against P. chabaudi malaria chabaudi (Wunderlich et al. 1988; cf. also is not only directed against parasites but Sayles and Wassom, 1988). also appears to neutralize infection-caused Finally, it is conspicuous that there exist reactions primarily responsible for the le- a parallelism in the actions of E and T. Both thal outcome of P. chabaudi infections hormones suppress the development of (Wunderlich et al. 1988, 1992; cf. also Play- protective immunity (Benten et al. 1991; fair et al. 1990; Riley et al. 1991). This view Wunderlich et al. 1991), and both hormones is consistent with our present finding that are ineffective to suppress existing immusome of the E-treated mice succumb to P. nity against P. chabaudi (Wunderlich et al. chabaudi only after parasite clearance has 1992). Moreover, the immunosuppressive begun and parasitemia has been largely re- effect of E can be attained with doses which duced (cf. Fig. 1). are only about l-2% of that used for T. ReThe immunosuppressive action of E in P. markably, about l-5% of T is normally mechabaudi malaria is rather specific as indi- tabolized to E in vivo (Wilson 1990). Such cated by several of our findings. First, ex- an aromatization of T to E is indeed a way isting immunity to P. chabaudi is not af- to mediate T effects through E receptors fected by E even if the E concentration is (Callard et al. 1978; Martini 1982; Sheridan raised lo-fold. Specifically, E neither re- 1991). It is therefore attractive to speculate duces survival rate nor causes any signifi- that the immunosuppressive effect of T is cant rise in parasitemia of immune BlO also mediated via ERs. This is currently inmice reinfected with P. chabaudi. This also vestigated in our laboratory. indicates that E does not induce reactions ACKNOWLEDGMENTS which are toxic/lethal to mice. Second, E We thank Mrs. A. Grunwald and L. Langenstrassen does not act as a general immunosuppressant as, for example, corticosteroids. The for technical assistance and Prof. Dr. P. W. Jungblut latter can indeed suppress preexisting im- for critical reading of the manuscript. munity to malaria. Thus, raised levels of REFERENCES corticosteroids have been previously found to be associated with a loss of protective ALEXANDER, J., AND STIMSON, W. H. 1988. Sex hormones and the course of parasitic infection. Par&immunity to P. berghei in mice during pregtology Today 4, 18!+193. nancy (van Zon et al. 1982, 1986). Even in ANSAR AHMED, S., PENHALE, W. J., AND TALAL, N. nonpregnant mice, an increase in the en1985. Sex hormones, immune responses, and autoimmune diseases: Mechanisms of sex hormone acdogenous levels of corticosterone results in tion. American Journal of Pathology 121,53l-55 1. a loss of malaria immunity against P. BENTEN, W. P. M., BETTENHAELJSER, U., WUNDERberghei (van Zon et al. 1985). Third, the LICH, F., VAN VLIET, E., AND MOSSMANN, H. immunosuppressive E effect can be pre1991. Testosterone-induced abrogation of selfvented by ER blockers such as tamoxifen healing of PIasmodium chabaudi malaria in BlO mice: Mediation by spleen cells. Infection and Zmand clomiphene. Tamoxifen also inhibits munity 59, 44864490. the immunosuppressive activity of the nonBUNDY, D. A. P. 1988. Gender-dependent patterns of steroidal E agonist DES. These latter tindinfection and diseases. Parasitology Today 4, 186ings show that the E-induced immunosup189. pression is a specific genomic effect. Obvi- CALLARD, G. V., PETRO, Z., AND RYAN, K. J. 1978.
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on chlamydial genital infection in female guinea pigs. Infection and Zmmunity 38, 699-705. RILEY, E. M., JAKOBSEN, P. H., ALLEN, S. J., WHEELER, J. G., BENNETT, S., JEPSEN, S., AND GREENWOOD,B. M. 1991. Immune response to soluble exoantigens of Plasmodium falciparum may contribute to both pathogenesis and protection in clinical malaria: Evidence from a longitudinal, prospective study of semi-immune African children. European Journal of Immunology 21, 1019-1025. SAYLES, P. C., AND WASSOM, D. L. 1988. Immunoregulation in murine malaria: Susceptibility of inbred mice to infection with Plasmodium yoellii depends on the dynamic interplay of host and parasite genes. Journal of Immunology 141, 241-248. SCHUURS, A. H. W. M., AND VERHEUL, H. A. M. 1990. Effects of gender and sex steroids on the immune response. Journal of Steroid Biochemistry 35, 157-172.
SHERIDAN,P. J. 1991. Can a single androgen receptor fill the bill? Molecular and Cellular Endocrinology 76, C3!9-C45. STIMSON, W. H. 1987. Sex steroids, steroid receptors and immunity. In “Hormone and Immunity” (I. Berczi and K. Kovacs, Eds.), pp. 43-53. MTP Press, Lancaster. TOIVANEN, P. 1%7. Enhancement of staphylococcal infection in mice by estrogens. I. Effect of timing, quantity ad quality of the hormone. Annales Medicinae Experimentalis et Biologiae Fenniae 45, 138146. VAN ZON, A. A. J. C., ELING, W. M. C., HERMSEN, C. C., AND KOEKOEK, A. A. G. M. 1982. Corticosterone regulation of the effector function of malarial immunity during pregnancy. Infection and Zmmunity 36, 484-491. VAN ZON, A. A. J. C., ELING, W. M. C., SCHETTERS, T. P. M., AND HERMSEN, C. C. 1985. ACTHdependent modulation of malaria immunity in mice. Parasite Immunology 7, 107-117. VAN ZON, A. A. J. C., TERMAAT, R.-M., SCHETTERS, T. P. M., AND ELING, W. M. C. 1986.Plasmodium berghei: Reduction of the mouse’s specific lymphoproliferative response in relation to corticosterone and pregnancy. Experimental Parasitology 62, 71-78. WILSON, J. D. 1990. Androgens. In “Goodman and
Gilman’s The pharmacological Basis of Therapeutics” (A. G. Gilman, Ed.), pp. 1413-1430.Pergamon Press, New York. WUNDERLICH, F., BENTEN, W. P. M., BETTENHAEUSER, U., SCHMITT-WREDE, H.-P., AND MossMANN, H. 1992. Testosterone-unresponsiveness of existing immunity against Plasmodium chabaudi malaria. Parasite Zmmunology 14, 307-320. WUNDERLICH, F., BRENNER, H.-H., AND HELWIG, M. 1988. Plasmodium chabaudi malaria: Protective
ESTRADIOL-SUPPRESSED hnrnunization with surface membranes of infected erythrocytes. Infection and Immunity 56, 33263328. WUNDERLICH, F., AND HELWIG, M. 1987. Plasmodium chabaudi malaria: Red blood cells with altered membrane proteins in immune mice. European Journal of Ceil Biology 43, 4%500. WUNDERLICH, F., MARINOVSKI, P., BENTEN,
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chabaudi in BlO mice: Intluence of the H-2 complex and testosterone. Znfection and Immunity 56, 24OCL 2406. WUNDERLICH, F., STUEBIG, H., AND KOENIGK, E. 1982. Development of Plasmodium chabaudi in mice red blood cells: Structural properties of the host and parasite membranes. Journal of Protozoology 29, 60-M.
tor(s) restrict the effkacy of genes controlling resistance to Plasmodium chabaudi malaria. Parasite Immunology 13, 357-367.
Received 3 March 1992; accepted with revision 30 June 1992
Plasmodium chabaudi: Estradiol Suppresses Acquiring, but Not Once-Acquired Immunity W.P.M. *Division
BENTEN,* F. WUNDERLICH,* AND H. MOSSMANN?
of Parasitology, Institute of Zoology, Heinrich-Heine-University, 4000 Duesseldorf and thfax-Planck-Institute of Immunobiology, 7800 Freiburg, Germany
1, Germany;
BENTEN, W. P. M., WUNDERLICH, F., AND MOSSMANN, H. 1992.Plasmodium chabaudi: Estradiol suppresses acquiring, but not once-acquired immunity. Experimental Parasitology 75,240-247. This study investigates the effect of estradiol (E) on self-healing of Plasmodium chabaudi malaria in mice of the inbred strain C57BL/lO. Our data show: (1) Female mice and male castrates are capable of self-healing infections when challenged with lo6 P. chabaudiinfected erythrocytes. Self-healing is completely suppressed after pretreatment of mice with 12 ug E injected SCtwice a week for 3 weeks. (2) The suppressive effect of E is prevented by the estrogen receptor blockers tamoxifen and clomiphene. (3) The nonsteroidal E-agonist diethylstilbestrol (DES) also suppresses self-healing. This suppressive DES effect is prevented by tamoxifen. (4) In mice immune to P. chabaudi, neither survival rate nor the course of parasitemia is affected by E, even at IO-fold higher E doses. Our data suggest that the immunosuppressive action of E is a specific genomic effect, i.e., E-induced gene products prevent the development of protective immunity against P. chabaudi. 8 IWZ Academic Ress,
Inc.
chabaudi; Malaria; Immunosuppression; Sex hormones; Androgen receptor; Clomiphene; Tamoxifen; Estradiol (E); Testosterone (T); Estrogen receptor (ER); Diethylstilbestrol (DES). INDEX DESCRIPTORSAND ABBREVIATIONS: Plasmodium
INTRODUCTION There is overwhelming evidence for the existence of a sex dimorphism concerning onset, course, and outcome of numerous disease processes, including infectious diseases caused by a wide variety of protozoan and helminthic endoparasites (Alexander and Stimson 1988; Bundy 1988). Very recently, a spectacular gender dependence has been shown for the outcome of the murine malaria Plasmodium chabaudi (Wunderlich et al. 1988) and Plasmodium berghei (Kamis and Ibrahim 1989). For instance, female B10 mice are able to selfheal P. chabaudi infections and, thus, to develop long-lasting immunity against homologous rechallenge. By contrast, male BlO mice cannot develop immunity and, therefore, succumb to P. chabaudi infections (Wunderlich et al. 1988, 1991; Benten et al. 1991). Sex hormones are important environ-
mental factors modulating the immune system of vertebrates (Ansar Ahmed et al. 1985; Grossman and Roselle 1986; Schuurs and Verheul 1990). For instance, T is known to suppress diverse immune reactions both in vivo and in vitro (Stimson 1987; Schuurs and Verheul 1990). This appears to be of advantage in autoimmune and/or immunopathological disorders, but is disadvantageous in infectious diseases. Indeed, T increases the susceptibility toward numerous parasitic diseases (Alexander and Stimson 1988). This also holds true for P. chabaudi infections. For instance, female BlO mice normally resistant to P. chabaudi become susceptible after T treatment, i.e., T suppresses the development of protective immune mechanisms against P. chabaudi (Wunderlich et al. 1988, 1991; Benten et al. 1991). However, it is possible that, besides T, E is also involved in the control of female resistance to P. chabaudi. At least, there is 240
0014-4894192$5.00 Copyright 6 1992 by Academic Press, Inc. ALI rights of reproduction in any form reserved.
ESTRADIOL-SUPPRESSEDP. chabaudi
some information available that E increases murine resistance to other protozoan parasites such as Leishmania mexicana (Alexander and Stimson 1988), Trypanosoma cruzi (Kierszenbaum et al. 1974), Trypanosoma lewisi (Mankau 1975), and Babesia microti (Cottrell et al. 1977). We therefore wondered if the suppressive effect of T against P. chabaudi malaria may be counterregulated by E. To our surprise, however, E also suppresses the development of immunity, even at much lower concentrations than T. This suppressive E effect is specific since E does not affect existing immunity to P. chabaudi.
MALARIA
241
passages (Wunderlich et al. 1982). Parasitemia was evaluated in Giemsa-stained blood smears. Erythrocytes were counted in a Neubauer chamber. Immune mice. Female and castrated male BlO mice normally resolve infections with P. chabaudi. Those mice which were still alive at 9 weeks postinfection, were used as immune mice (Wunderlich and Helwig, 1987).
E-induced
RESULTS suppression of self-healing.
Male mice of the inbred strain C57BL/lO do not survive a challenge with lo6 P. chabaudi-infected erythrocytes (Table I). Males also succumb to infection when male mice are pretreated with E for 2 or even 6 weeks before infection (Table I). It may be therefore that E does not counterregulate MATERIAL AND METHODS the suppressive effect of T. Conversely, E Mice. Mice of the strains C57BL/lO and NMRI were may be suppressive itself. We therefore exbred under specific pathogen-free conditions in our amined this possibility in female and cascolony at the Heinrich-Heine-University. They were trated male B 10 mice. B 10 females and cashoused in plastic cages with standard diet (Nohrlin, Bad Salzuflen, Germany) and water ad libitum. The trated BIO males are normally self-healers, mice were 9-12 weeks old when beginning with the i.e., about 95% of mice resolve and, subseexperiments. quently, survive infections with P. chaOrchidectomy. Male mice were anesthetized under baudi (Table I, Fig. 1). However, this selfKetavet (Park-Davis, Berlin, Germany) and Rompun healing capacity is impaired and even com(Bayer, Leverkusen, Germany). The testes and epididymis were pulled off through a scrotal incision and pletely abrogated by E, depending on the conditions of E treatment (Table I). For inremoved by electrocautery (Wunderlich et al. UBl). After operation, the mice were kept under standard stance, only a slight-if any-reduction occonditions for 3 weeks before further experimental curs in survival when mice are treated with use. E only during infection. However, female Treatment with estrogens. The mice were treated and male castrates succumb to infection with E-benzoate (Progynon B oleosum, Schering, when they are pretreated with an E dose of Berlin, Germany), or DES (Steraloids Inc., Wilton, 12 kg twice a week for 3 weeks before inU.S.A.) at different doses. They received E or DES in 200~pl subcutaneous injections twice per week for 3 fection and when this treatment is continweeks, before infecting with P. chabaudi-if not oth- ued during infection (Fig. 1). Under these erwise stated. The estrogens were suspended in sesexperimental conditions, E does not essename oil (Roth, Karlsruhe, Germany). The treatment tially affect the course of parasitemia (Fig. was continued during infections-if not otherwise stated. Controls received sesame oil alone. 1). Mice can even decease after parasite Treatment with ER blockers. Mice were treated with clearance has begun and the number of intamoxifen (Nolvadex, ICI Pharma, Heidelberg, Ger- fected erythrocytes has been already signifmany) or clomiphene (Merrell Dow Research Institute, Winnersh, U.K.) suspended in sesame oil. The icantly reduced in the peripheral blood. Remice received subcutaneous injections of 200~pl por- markably, castrated males are more sensitions twice per week for 3 weeks and the treatment tive to E than females. This becomes was continued during infections-if not otherwise evident, for example, when the treatment stated. with 12 p,g E is not continued during infecParasite infections. The C57BWlO mice were intion: About 60% females recover from the fected by ip injection with lo6 P. chabaudi-infected suppressive E effect, whereas castrated erythrocytes. The latter were obtained from NMRI mice, in which P. chabaudi was maintained by weekly males remain suppressed (Table I).
242
BENTEN, WUNDERLICH,
AND MOSSMANN
TABLE I Effect of E on the Outcome of P. chabaudi Infections in C57BIJlO Mice Mice infected with P. chnbaudi E pretreatment Sex of mice Male Male Male Female Female Female Female Female Female Female Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated
male male male male male male male male
Concentration (I.4 120 120 12 120 60 12 12 12 12 120 60 60 12 12 12
Period (weeks)
E treatment during infection
Number of surviving/ total mice
Day of death of succumbing mice (days p.i. f SD)
Average survival rate (%)
+ + + + + + + + + + -
l/13 o/15 0124 32134 10/12 O/8 o/7 o/10 6/10 315 38140 10/12 o/9 O/16 o/5 o/5 o/15 o/5
11.1 * 1.7 12.1 * 3.5 11.7 ” 1.8 11.5 12.0 10.3 2 1.0 9.9 f 1.1 11.8 f 3.7 12.0 f 1.2 11.0 12.0 10.0 9.8 f 0.8 11.4 f 1.7 13.8 f 3.9 10.4 ” 1.3 12.5 f 2.3 11.0 f 1.7
8 0 0 95 83 0 0 0 60 60 95 83 0 0 0 0 0 0
2 6 3 3 3 3 1 3 3 3 3 3 1
Prevention of the E-induced suppression by ER blockers. Tamoxifen and clomiphene
are known to bind to ERs, thus blocking the E action. Table II shows that tamoxifen at a dose of 0.1 mg per injection does not affect self-healing of P. chabaudi in both females and male castrates. Also, clomiphene has no influence on self-healing in castrated BlO males (Table II). Intriguingly, however, these ER blockers prevent the suppressive effect of E. Treatment of E together with either tamoxifen or clomiphene does not result in death of mice which is typical for E treatment alone (Table II). EfSect of DES on self-healing. DES, one of the first nonsteroidal estrogens with estrogenic activity, also impairs self-healing. When castrated males are pretreated with 74 kg DES twice a week for 3 weeks before infection, the survival rate declines to 43% (Table III). Survival is completely abolished’when the DES dose is doubled to 148 pg (Table III). The DES-induced suppression of self-healing can be prevented by tamoxifen (Table III).
!
I I -1 tE (10)
t 0
2
4
6
8
10
12
14
16
18
20
22
24
Days pi.
FIG. 1. Effect of E on P. chabaudi infections in female C57BWlO mice. Ten mice were treated with 12 ug E for 3 weeks (+ E), before these and 10 untreated mice (-E) were challenged with lo6 P. chabaudiinfected erythrocytes. The E treatment was continued during infection. Means are given with either + or SD for clarity reasons.
Females Females Females Females Females Females Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated Castrated
mates males males males males males males males males males males males
Sex of mice
-
3 -
-
12 12 12 12 60 60 12 60 12 12 12 60 12
i
T 3 3 3 3 3 3 -
T -
Period (weeks)
Concentration (lud
E
100 100 100 100 100 100 100 100 -
-
Concentration bd
Tamoxifen
T 3 3 3 3 3 3 -
-
Period (weeks)
Pretreatment
100 100 100
-
-
-
-
Concentration OLB)
5 3 3
-
-
Period (weeks)
Clomiphene
-
+ + -
+ + + -
+ + + f + + + + + -
Tamoxifen
E
-
-
-
Clomiphene
Treatment during infection
1l/l2 o/10 IO/l2 10112 6l8 5/s 19/21 O/l6 O/5 o/15 819 s/9 617 6112 S/5 416 415 8/11
Number of surviving/ total mice
TABLE II Effect of Tamoxifen and Clomiphene on the E-Induced Susceptibility of BlO Mice to P. chubaudi
-9.5 11.012.0 f 1.0
11.5 11.0 12.0 11.4 ‘- 1.7 13.8 2f 2.3 3.9 12.5 10.0 12.9 2 1.6 17.0 10.7 + 1.6
11.0 11.7 f 3.2 12.0
Day of death of succumbing mice (days p.i. t SD)
Mice infected with P. chabaudi
67 loo 80 73
0 89 56 86 50
83 75 100 91 i
92 0 83
Average survival rate (%)
E s
$
5 g P -.a
P
g
s
z
in
m
244
BENTEN, WUNDERLICH,
AND MOSSMANN
TABLE III Effect of DES on Self-Healing of P. chubaudi in Castrated Male BlO Mice Pretreatment
Mice infectedwith P. Tamoxifen
DES
Concentration Period (weeks) (I%) 74 148 148
3 3 3
-
-
Treatmentduring infection Concentration Period ~ (weeks) DES Tamoxifen (l&d 100 100
3 3
+ -
Effect of E on existing immunity. After self-healing, both female and castrated male BlO mice have acquired long-lasting immunity. Thus, reinfection with P. chabaudi at 9 weeks after the first infection does not cause any symptoms of sickness. Parasitized erythrocytes can be detected only on Days 8-12 postreinfection, but the parasitemia does not regularly exceed 0.1%. When mice have once acquired immunity, this cannot be suppressed by E (Table IV). Neither outcome nor course of infections are changed by treatment with 12 kg E (Table IV). Even if the E dose is increased to 120 p,g, no change in survival rates can be observed (Table IV). Also, parasitemia is not raised. DISCUSSION
Our data show that E can impair and even completely abolish self-healing of P. chabaudi malaria. Self-healing obviously
Number of surviving/ total mice
+
22.l23 3l7 o/10 7l7 819
chaboudi
Day of death of succumbingmice (daysp.i. 2 SD)
Average survival rate (%)
14.0
96 43 0
10.5f 0.6 10.7 f 2.0 10.0
100 89
reflects the successful mounting of protective immune mechanisms against P. chabaudi (Wunderlich and Helwig 1987). Thus, it is reasonable to assume that E acts as an immunosuppressant. This view is consistent with numerous other studies showing that E suppresses a series of immune reactions both in vivo and in vitro (Grossman and Roselle 1986; Schuurs and Verheul 1990). The immunosuppressive activity of E may also explain why E is known to increase the susceptibility of vertebrate hosts toward diverse infectious diseases caused, for example, by parasites such as Toxoplasma (Grossman 1984; Pung and Luster 1986), Trichomonas (Alexander and Stimson 1988), and Trichinella (Dean et al. 1980; Luebke et al. 1984), as well as by bacteria such as Listeria (Dean et al. 1980; Pung et al. 1984), Chlamydia (Rank et al. 1982), and Staphylococcus (Toivanen 1967). However, the immunosuppressive action of E does not necessarily contrast to those tind-
TABLE IV Effect of E on the Outcome of P. chabaudi Infections in Immune C57BL/lO Mice Immune mice infected with P. chabaudi E pretreatment of mice
Concentration (IJd
Period (weeks)
E treatment during infection
Number of surviving/ total mice
Average survival rate (%)
Females Females Females Castrated males Castrated males
60 120 60
3 3 3
+ + +
1909 515 12/12 lO/lO 717
100 100 100 100 100
Sex
ESTRADIOL-SUPPRESSEDP. chabaudi MALARIA
245
ings showing an increased resistance under ously, the E-activated genes ultimately E influence (Grossman 1984; Alexander prevent the functional expression of those and Stimson 1988). For it may be that-E genes of the mouse MHC, the H-2 comalso suppresses infection-caused immuno- plex, and those genes of the non-H-2 backpathological reactions. Interestingly, there ground, which normally control the develis some information available that protec- opment of protective immunity against P. tive immunity against P. chabaudi malaria chabaudi (Wunderlich et al. 1988; cf. also is not only directed against parasites but Sayles and Wassom, 1988). also appears to neutralize infection-caused Finally, it is conspicuous that there exist reactions primarily responsible for the le- a parallelism in the actions of E and T. Both thal outcome of P. chabaudi infections hormones suppress the development of (Wunderlich et al. 1988, 1992; cf. also Play- protective immunity (Benten et al. 1991; fair et al. 1990; Riley et al. 1991). This view Wunderlich et al. 1991), and both hormones is consistent with our present finding that are ineffective to suppress existing immusome of the E-treated mice succumb to P. nity against P. chabaudi (Wunderlich et al. chabaudi only after parasite clearance has 1992). Moreover, the immunosuppressive begun and parasitemia has been largely re- effect of E can be attained with doses which duced (cf. Fig. 1). are only about l-2% of that used for T. ReThe immunosuppressive action of E in P. markably, about l-5% of T is normally mechabaudi malaria is rather specific as indi- tabolized to E in vivo (Wilson 1990). Such cated by several of our findings. First, ex- an aromatization of T to E is indeed a way isting immunity to P. chabaudi is not af- to mediate T effects through E receptors fected by E even if the E concentration is (Callard et al. 1978; Martini 1982; Sheridan raised lo-fold. Specifically, E neither re- 1991). It is therefore attractive to speculate duces survival rate nor causes any signifi- that the immunosuppressive effect of T is cant rise in parasitemia of immune BlO also mediated via ERs. This is currently inmice reinfected with P. chabaudi. This also vestigated in our laboratory. indicates that E does not induce reactions ACKNOWLEDGMENTS which are toxic/lethal to mice. Second, E We thank Mrs. A. Grunwald and L. Langenstrassen does not act as a general immunosuppressant as, for example, corticosteroids. The for technical assistance and Prof. Dr. P. W. Jungblut latter can indeed suppress preexisting im- for critical reading of the manuscript. munity to malaria. Thus, raised levels of REFERENCES corticosteroids have been previously found to be associated with a loss of protective ALEXANDER, J., AND STIMSON, W. H. 1988. Sex hormones and the course of parasitic infection. Par&immunity to P. berghei in mice during pregtology Today 4, 18!+193. nancy (van Zon et al. 1982, 1986). Even in ANSAR AHMED, S., PENHALE, W. J., AND TALAL, N. nonpregnant mice, an increase in the en1985. Sex hormones, immune responses, and autoimmune diseases: Mechanisms of sex hormone acdogenous levels of corticosterone results in tion. American Journal of Pathology 121,53l-55 1. a loss of malaria immunity against P. BENTEN, W. P. M., BETTENHAELJSER, U., WUNDERberghei (van Zon et al. 1985). Third, the LICH, F., VAN VLIET, E., AND MOSSMANN, H. immunosuppressive E effect can be pre1991. Testosterone-induced abrogation of selfvented by ER blockers such as tamoxifen healing of PIasmodium chabaudi malaria in BlO mice: Mediation by spleen cells. Infection and Zmand clomiphene. Tamoxifen also inhibits munity 59, 44864490. the immunosuppressive activity of the nonBUNDY, D. A. P. 1988. Gender-dependent patterns of steroidal E agonist DES. These latter tindinfection and diseases. Parasitology Today 4, 186ings show that the E-induced immunosup189. pression is a specific genomic effect. Obvi- CALLARD, G. V., PETRO, Z., AND RYAN, K. J. 1978.
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Received 3 March 1992; accepted with revision 30 June 1992
