Trypanosoma rhodesiense: Variable effects of cyclophosphamide on antibody production, survival, and parasitemia in infected mice

EWERIMENTAL

PARASITOLOGY

61,

26 l-269

( 1986)

Trypanosoma rhodesiense: Variable Effects of Cyclophosphamide on Antibody Production, Survival, and Parasitemia in Infected Mice JOYE Department

F. JONES

of Microbiology, Jefferson Medical College, Thomas Jefferson Philadelphia, Pennsylvania 19107, U.S.A. (Accepted

for publication

University,

8 October 1985)

JONES, J. F. 1986. Trypanosoma rhodesiensrt Variable effects of cyclophosphamide on antibody production, survival, and parasitemia in infected mice. Experimental Parasitology 61, 261-269. Mice of the CBA/&J strain, infected with Trypanosoma rhodesiense. were injected with a single high dose (approximately 200 mg/kg) of the immunosuppressive drug cyclophosphamide to determine if an induced, transient inability to make antibody affected survival or parasitemia. When given on the day of infection, the drug had no significant effect on survival. It delayed, but did not prevent, the appearance of specific antibodies and the clearance of the infecting trypanosome variants. When cyclophosphamide was injected 1 week after infection, survival was significantly decreased. Antibody production to specific variant antigens and to common trypanosome antigens was terminated, but the mice were able to eliminate the infecting trypanosomes. These findings suggest that a temporary inability to make antibody to trypanosomes does not result in more rapid death when only the infecting trypanosome variant is present. However. immunosuppression may accelerate death if it occurs when there are many different types of trypanosomes present. o 1986 Academic

Press, Inc.

DESCRIPTORS AND ABBREVIATIONS: Ttypanosoma rhodesiense; Trypanosomiasis, African; Hemoflagellate; Kinetoplastidae; Protozoa, parasitic; Mouse; Survival, host; Antibody production; lmmunosuppression; Cyclophosphamide; Salmonella typhimuvium; Bacterial clearance, mouse; Diethylaminoethyl (DEAE); Variant antigenic type (VAT); Variantspecific surface glycoprotein (VS.%). INDEX

Mice infected with Trypanosoma rhodethe cause of acute African trypanosomiasis, develop a fatal illness and have fluctuating parasitemias and variations in trypanosomal antigens and morphology (Roelants and Pinder 1984). The parasites change their surface antigens, giving rise to different VATS. Although untreated trypanosomiasis caused by T. rhodesiense or T. brucei is always fatal in mice, the course of infection varies greatly among different mouse strains (Levine and Mansfield 1981) and is affected by host-determined factors such as macrophage corn1letence (Jones and Hancock 1983) and antibody production (Campbell ef al. 1977). The clearance of siense,

trypanosomes of a given VAT is mediated by hepatic macrophages which remove from the circulation those parasites coated with antibody specific for the VSSG (MacAskill et al. 1980; Dempsey and Mansfield 1983; Levine and Mansfield 1984). Thus, antibody plays a crucial role in selecting which VATS will predominate in a population. Even though the ability to make antibody against the infecting clone is necessary for survival for any significant length of time (Levine and Mansfield 1984), there are conflicting reports as to the effects of immunosuppression induced by drugs or irradiation on survival with trypanosomiasis. Some investigators found no effect (Ashcroft 1957; Balber 1972) but others reported decreased survival times (Petana 1964; 261 0014.4894/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form rerervcd.

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JOYE F. JONES

Hudson and Terry 1979). Since immunosuppressive drugs are important tools in understanding how the immune system functions to modulate the course of trypanosome infection, we have further examined the effects of immunosuppression on trypanosomiasis, using the immunosuppressive drug cyclophosphamide to investigate whether the differences reported by other workers could be related to the time of drug treatment relative to the time of infection. Cyclophosphamide affects many immunologic functions, including the capacity of the host to develop antibodies following antigenic challenge (reviewed bv Shand 1979). We have used cyclophosphamide to examine how an artificially induced, transient inability to make antibody affects the ability of mice to survive trypanosomiasis, and how this immunosuppression affects parasitemia. The advantage of this immunosuppressive regimen is that within 1 week of receiving a single dose of cyclophosphamide, mice recover immunocompetence (Shand 1979). Thus, it is possible to examine the effects of a temporary loss of immune function on trypanosomiasis. In the studies reported here, we have found that the time of drug treatment has a decided effect on the outcome. Thus. when given on the day of infection, the drug delayed the antibody response and consequently delayed the clearance of the infecting clone but had no effect on survival. However, when given a week after infection, the drug had no effect on the parasitemia profile but inhibited antibody production and significantly decreased survival. MATERIALSANDMETHODS CBA/CaJ male mice (Jackson Laboratories, Bar Harbor, ME, USA) were 8-12 weeks old at the time of infection. CD-I male mice (Charles River Breeding Laboratories, Wilmington, MA, USA) were used to produce antisera against typanosomes and to obtain large numbers of parasites for antibody analysis. All mice were housed in a facility fully accredited by the

American Association of Laboratory Animal Sciences and received food and water ad libitum. Immunosuppression was induced in mice by injetting 4 mg (200 mg/kg) cyclophosphamide (Cytoxan; Mead Johnson & CO., Evansville, IN, USA) intraperitoneally on the indicated day. In early experiments, control mice received an equal volume of saline. However, this was discontinued following the demonstration that these saline injected mice did not differ from uninjected mice. No mice died following treatment with cyclophosphamide alone. Mice were routinely infected on Day 0 with IO4TITpunosoma rhodesiense trypanosomes clone Jeffat I .O derived from strain EATRO 1886. This infection kills CBA/Cal mice in approximately 29 days (Jones and Hancock 1983). Clone Jeffat 1.45 was used as a specifICI‘ty control in some experiments. This clone was derived by triply cloning trypanosomes from C57Bl16J mice infected 45 days earlier with 10“ Jeffat I.0 try-

panosomes.

At various days after infection, two to four mice per group were bled from the retroorbital plexus into heparinized capillarv . _..pipets. A portion of each sample was diluted with a carbol fuchsin staining reagent (Murray and Morrison 1979), and the trypanosomes were counted in a hemacytometer. The limit of detectability by this method is approximately lo5 trypanosomes per milliliter. The rest of the blood was centrifuged to separate the cells from the plasma, and the plasma was stored at -20 C until analyzed for antibody activity. Antisera were obtained from CD-l mice infected with 104trypanosomes of the appropriate clone. Beginning 7 days after infection, each mouse was cured by intraperitoneal injection with 125 )*g diminazene aceturate (Berenil; Calbiochem Behring Corp., La Jolla, CA, USA) per day for 3 days. Seven days after the final injection, mice were exsanguinated, and the sera were pooled and stored frozen until used. Control sera were obtained from mice injected with the drug but not infected with the parasites. Antiserum against Jeffat I.0 was made specific for that clone by absorbing twice with glutaraldehyde fixed trypanosomes of clone Jeffat 1.45. This procedure removes antibodies against common antigens exposed by glutaraldehyde as well as antibodies against the VSSG of Jeffat 1.45. The antigenic variation of the parasites from individual mice was determined on trypanosomes isolated from diethylaminoethyl cellulose (DE52, Whatman Ltd., Maidstone, Kent, UK) columns (Lanham and Godfrey 1970). Live trypanosomes were incubated in wells of round-bottom microtiter plates (106 per well) on ice either with normal mouse serum or with an antiserum against either Jeffat 1.0 or Jeffat 1.45. The antibody binding to the parasites was detected with a fluorescein-conjugated goat anti-mouse IgG (specific

Trypanusoma

rhodesiense: EFFECTSOFCYCLOPHOSPHAMIDE

for both heavy and light chains; Cappel Labs., Cochranville, PA, USA). The clone specific antisera were used at dilutions which bound to, but did not agglutinate, parasites from a 4-day infection with Jeffat 1.0. Samples were considered positive if any fluorescent parasites were seen by fluorescent microscopy under high power. Although this technique cannot establish the number of different variants present in the sample, it does allow us to determine when the infecting VATS have been eliminated. Plasma samples obtained from retroorbital bleedings were stored frozen until tested for antibodies against Jeffat 1.O VSSG or common antigens. All samples from one experiment were tested at the same time following the procedure described by Dempsey and Mansfield (1983). Briefly, individual plasma samples were serially diluted in phosphatebuffered saline with glucose (Lanham and Godfrey 1970) in wells of round-bottom microtiter plates. In tests for antibody specific for clone Jeffat 1.0, trypanosomes from CD-I mice infected 4 days earlier with Jeffat 1.O were eluted from DEAE ion exchange columns (Lanham and Godfrey 1970) and suspended in ice cold phosphate-buffered saline with glucose; lo6 viable parasites were added to each well. The plates were incubated on ice for 30-45 min, then washed with ice cold phosphate-buffered saline with glucose. The trypanosomes were fixed with 1% glutaraldehyde for 30 min in the cold, stained with fluorescein-conjugated goat anti-mouse IgG, and examined microscopically for fluorescence. This procedure identifies VSSGs (Dempsey and Mansfield 1983). In tests for antibodies against common antigens, trypanosomes of clone Jeffat 1.45 were fixed with glutaraldehyde, and 106of these washed, fixed parasites were mixed with the diluted plasma. These were subsequently stained with the fluorescent reagent and examined microscopically. By fixing the parasites first, common antigens are exposed which can then react with antibody. Titers are the reciprocal of the highest plasma dilutions given uniform fluorescence. In all experiments, controls with plasma from uninfected mice were negative. Jeffat 1.0 trypanosomes bound specific antiserum to Jeffat 1.0 but not antiserum to Jeffat 1.45 and vice versa.

263

200 mg/kg cyclophosphamide on the same day (Day 0 group) or 1 week later (Day 7 group), or gave them no drug at all (control group). We then collected plasma samples from individual mice at various times after infection and tested the plasma for the presence of antibodies specific for Jeffat 1.O(Fig. 1) or for common parasite antigens (Fig. 2), using indirect immunofluorescence. Control mice made a typical antibody response against Jeffat 1.0 VSSG, with a maximal response around 10 days after infection and a slight decrease until the mice died about a month after infection (Fig. 1). Whether this decrease in antibody levels is real due perhaps to the appearance of antibodies to the idiotype (Kelsoe et al. 1980) or only apparent due to the nature of the test for antibody, is not known. Day 0 mice also made antibodies which bound to the Jeffat 1.0 parasites, but the appearance of these antibodies was delayed by approximately 1 week. In spite of this delayed appearance, the kinetics of production and the levels achieved were very similar to that seen in the control group. However, in Day 0 mice, the amount of antibody remained at the peak level until the mice died. This may simply reflect the fact that the mice died before the normal control mechanisms would reduce antibody levels. It may, however, be due to altered control of antibody perhaps due to abnormal antiidiotype production (Kelsoe et al. 1980) or to residual effects of cyclophosphamide on regulatory T cells, particularly suppressor T cells (LaGrange et al. 1974). In contrast, the Day 7 mice, like the controls, had RESULTS begun to produce antibody at the time they Since we were interested in the effects of received the drug; after cyclophosphamide treatment, they no longer produced antiimmunosuppression on trypanosomiasis, we first determined whether our treatment body, and the antibody they had produced regimen suppressed the production of anti- dropped to a very low (but still detectable) level. bodies against antigens of Trypanosoma To determine if this immunosuppression rhodesiense. We infected mice with 104 Jeffat 1.0 parasites and injected them with affected the ability to make antibodies to

264

JOYE F. JONES

20

10 DAYS

AFTER

30

INFECTION

FIG. 1. Antibody specific for Ttypunosomu rhodesiense clone Jeffat 1.O in plasma of mice infected with IO4Jeffat 1.Oparasites on Day 0 and receiving no further treatment (0). 4 mg cyclophosphamide on Day 0 (O), or cyclophosphamide on Day 7 (A). Binding to live Jeffat 1.0 parasites was determined by indirect immunofluorescence. Mean of two plasma samples per time point. Data are from one of six similar experiments.

were qualitatively similar to those observed with specific antibodies. Control mice and Day 0 mice made similar amounts of antibody with similar kinetics, but the appearance of antibodies in the Day 0 mice was delayed by about 1 week; Day 7 mice stopped producing antibody and the antibodies that they had produced disappeared. Cyclophosphamide has numerous effects on various components of the immune system. We therefore tested its effects on phagocytosis by measuring the capacity of mice to clear Salmonella typhimurium from the blood (Glick and Jones 1984) on various days after drug treatment. At all times, J I I I drug treated and control animals were able I I 20 IO to remove the bacteria from the blood INFECTION DAYS AFTER equally well (data not shown). FIG. 2. Antibody against common trypanosome anSince our treatment regimen clearly suptigens in plasma of mice infected with lo4 T~ypanopressed antibody production, we then decoma rhodesiense clone Jeffat 1.0 on Day 0 and receiving no further treatment (O), 4 mg cyclophosphatermined the effect of this drug induced immide on Day 0 (0), or cyclophosphamide on Day 7 munosuppression on host survival. We (A). Binding to glutaraldehyde fixed Jeffat 1.45 try- monitored the survival of infected mice repanosomes was determined by indirect immunofluorescence. Mean of two to three plasma samples per ceiving either no drug or cyclophosphamide on Day 0 or Day 7 (Fig. 3). Mice intime point. Data are from one of two similar experiments. jected with cyclophosphamide on the day

other parasites which might arise during the course of infection, we measured the ability of the infected mice to produce antibodies which would recognize antigens on glutaraldehyde-fixed Jeffat 1.45 parasites, antigens which would (presumably) be common to a number of variants of the Jeffat 1.0 serodeme. The findings (Fig. 2)

Trypanosoma

rhodesiense: EFFECTSOFCYCLOPHOSPHAMIDE

20

”

DAYS

AFTER

265

40 INFECTION

FIG. 3. Survival after infection with Trypanosoma rhodesicnse clone Jeffat 1.0 on Day 0 in mice injected with 4 mg cyclophosphamide on the day of infection (Day 0), a week after infection (Day 7), or mice receiving no drug (None). Combined data from six experiments. In each experiment, each group contained four to seven mice. Horizontal bars represent geometric mean survival time. Each circle represents the death of one mouse. Survival was significantly less than control in groups receiving cyclophosphamide on Day 7 (P < 0.025 by Student’s t test).

of infection lived as long as control mice, whereas those receiving the drug a week later died significantly sooner than controls (P < 0.025 by Student’s t test). In fact, if mice received the drug any time during the first week of infection, they died sooner than controls (data not shown). Because cyclophosphamide affected the survival of infected mice differently, depending on when it was administered, we monitored the concentration of blood parasites throughout the course of infection to determine if survival was related to parasitemia. Although Day 0 mice lived as long as control mice, they were unable to clear the first peak of parasitemia, i.e., there was no decrease in the number of parasites on Day 10, which in control mice, is the low point (Fig. 4). In contrast, Day 7 mice cleared the first wave as well as controls, and their parasitemia profiles were not different from controls. Seven days after infection, the trypanosomes were predominantly of the infecting Jeffat 1.0 clone, regardless of whether the mice had received cyclophosphamide (Table I); Day 0 mice also had low numbers of Jeffat 1.45 parasites. By the tenth day, control mice and Day 7 mice had no detect-

able Jeffat 1.0 trypanosomes, whereas this VAT was still detectable in Day 0 mice. By Day 13, Jeffat 1.0 parasites were undetectable in all groups. DISCUSSION

Since immunosuppressive drugs can be important tools in understanding the role that immunity plays in modulating the course of a parasitic infection, we have used cyclophosphamide to determine how a transient loss of the ability to make antibody affects infection with Trypanosoma rhodesiense, and whether the effects of this immunosuppression are related to when it occurs. We found that a single high dose of cyclophosphamide given on the day of infection delayed the appearance of antibody and hence delayed the clearance of the infecting VAT, but had no effect on survival. When given a week later, when antibodies to the infecting VAT were already present, cyclophosphamide had no effect on parasitemia, but abolished antibody production and significantly shortened survival time. The dose of cyclophosphamide used in these experiments affects both T- and Bcell function @hand 1979) but it is the Bcell function that is probably most critical

JOYE F. JONES

266

TABLE I Detection of Trypanosoma rhodesiense Clone Jeffat I .Oin Control and Cyclophosphamide Treated Mice

0 00

108--

Antiserum0 (dilution)

: -1 I m :

; 0 m 0 : :

106--

oc I-

10 DAYS

20 AFTER

INFECTION

FIG. 4. Parasitemia in mice infected with IO4 T/y’panosoma rhodesiense clone Jeffat I .O on Day 0 and receiving no further treatment (O), 4 mg cyclophosphamide on Day 0 (0), or cyclophosphamide on Day 7 (A). Mean of two mice per time point. Data from one of five similar analyses.

groupb

anti-Jeffat I .O (1512)

Control Day 0 Day 7

anti-Jeffat 1.45 (1:128)

Control Day 0 Day 7

lO7--

m

Treatment

Day after infection 7

10

+ + NDd + ND

+ + -

11Antiserum binding to live trypanosomes detected by indirect immunofluorescence. The antiserum against Jeffat I .O was used at a dilution which bound but did not agglutinate Jeffat 1.0 parasites and did not bind to Jeffat 1.45 parasites. The antiserum to Jeffat 1.45 did not bind to Jeffat 1.Oparasites at this dilution. Serum from uninfected, Berenil treated mice did not bind to any trypanosomes when tested at dilutions of I:4 or greater. One of three similar analyses. b Mice infected with clone Jeffat I.0 were not treated (control) or were injected with 4 mg of cyclophosphamide on the day of infection (Day 0) or 1 week later (Day 7). c Trypanosomes isolated from the blood on the indicated day. Two mice per group per day. + = uniform fluorescence; - = no fluorescence. Trypanosomes from mice infected for 13 days were also tested. In all cases, none of these parasites bound either of the antiserum probes. d ND = Not done.

in these studies. There is little evidence that T cells play a significant role in modulating the course of trypanosomiasis since nude mice survive as well, have the same against the parasites, they recovered from levels of parasitemia, and make as much the immunosuppression (Fig. 1) and were antibody as euthymic controls (Campbell et ultimately able to remove the infecting VAT al. 1978; Finerty et al. 1982). Although the (Table I) and produce antibodies against effects of cyclophosphamide on macro- new clones (Fig. 2). This delay in antibody phages are unclear (Buhles and Shifrine production had no effect on survival. In 1977), we found no evidence of impaired contrast, mice receiving the drug a week phagocytosis. after infection died before they had recovThe effects of cyclophosphamide are ered the ability to make antibody (Figs. 1 transient, and mice recover from its im- and 2). We do not know why they died, but munosuppressive effects in about a week one possible explanation is that a more vir(Shand 1979). It is this transient effect ulent clone(s) appeared during the second which makes this drug particularly useful in week of infection, and an inability to conthese studies. Although mice injected with trol this particular clone(s) results in more cyclophosphamide on the day of infection rapid death. had a delay in the appearance of antibodies Other investigators who have examined

Trypanosoma

rhodesiense: EFFECTSOFCYCLOPHOSPHAMIDE

the effect of immunosuppression on survival with trypanosomiasis have had mixed results. In rats that were injected daily with cortisone, Ashcroft (1957) found no effect on survival, with both experimental and control animals surviving for approximately 1 month after infection with T. rhodesiense. In contrast, Petana (1964) found that cortisone treated rats lived only half as long as controls. His controls, however, lived only about 2 weeks after infection with T. rhodesiense. Indeed, a critical difference between Ashcroft’s studies and Petana’s is the difference in virulence of the two strains of T. rhodesiense. An important difference in both of those studies from the one reported here is that the immunosuppressive drug cortisone affects primarily Tcell function (Rogers and MatossianRogers 1982), and those authors did not measure the capacity of the drug treated animals to make antibody to trypanosomes. Balber (1972) found that immunosuppression induced by irradiating mice the day before infection with T. brucei inhibited antibody production but had little effect on survival. In his experiments, infected mice survived only lo-11 days. In the studies reported here, even when mean survival was reduced by cyclophosphamide (Day 7 drug treatment, Fig. 3), the mice still lived 7-10 days after receiving the drug. It may be that the clone of trypanosomes used in Balber’s study caused death so rapidly that an effect on survival would not be detectable. Hudson and Terry (1979) studied the effect of a single high dose of cyclophosphamide (300 mg/kg) on mice infected with a clone of T. brucei which causes death in 60-90 days. Although they did not show that this drug regimen inhibited the ability to make antibody to the trypanosomes, the mice were unable to make antibody to a Tdependent antigen. When the drug was given 30 days after infection, it decreased survival significantly, with drug treated

267

mice dying 40-42 days after infection (lo-12 days after cyclophosphamide). These findings are very similar to the ones we report here and suggest that immunosuppression during an ongoing infection, even with less virulent parasites, rapidly results in death. However, Hudson and Terry found that cyclophosphamide also increased the level of parasitemia. We did not find this when we gave the drug during the infection, but this may be because, with Jeffat 1.0, the fluctuations in parasitemia are not very marked after Day 12; parasitemia is high and remains high until the mouse dies, however long that may be. We did find an effect of cyclophosphamide on parasitemia when the drug was given the day of infection, probably because there is a 2.5 log drop in parasitemia in control mice around Day 10 (Fig. 4). Mice receiving cyclophosphamide on Day 0 do not have this reduction because they are making little, if any, antibody to the trypanosomes (Fig. 1). As expected, mice which received cyclophosphamide on the day of infection did not clear the infecting VATS as early as the controls, but once they recovered from the immunosuppression, they were able to make antibody and clear these VATS. Since they had already made antibody to Jeffat 1.0, Day 7 mice could clear these trypanosomes; the drop in antibody titer in these mice was probably due to the absorption of the antibody to the trypanosomes which were then removed from the circulation. The antibodies to Jeffat 1.0 are not completely absorbed because of the loss of these parasites from circulation. One possibility for the decreased survival of Day 7 mice is the appearance of more virulent trypanosomes during the course of infection. Day 7 mice, immunosuppressed during or shortly after the appearance of these clones, may be unable to recover immune competence before they die. Day 0 mice may be able to recover from the immunosuppression in time to

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JOYE F. JONES

make antibodies to these new clones, clearing them from the circulation before they cause death. It is apparent that Day 0 mice, unlike controls, do in fact have trypanosomes that react with antiserum against Jeffat 1.45 and are able to clear these parasites. In studies on genetic control of susceptibility to trypanosomiasis, Levine and Mansfield (1984) found that C3H mice were unable to make VAT specific antibodies to the infecting clone of trypanosomes (but were able to make antibodies against common antigens) and died rapidly, whereas BlO.BR mice, which made rapid antibody responses to the infecting VAT, lived much longer. They suggested that the ability to survive with trypanosomiasis depended on a capacity to make antibody to the infecting VAT. The findings reported here are consistent with that hypothesis. However, it may be the inability of C3H mice and of Day 7 mice to make any VAT specific antibodies-whether to the infecting clone or to those that appear subsequently-that causes accelerated death. Another possibility is that the trypanosomes are inducing a profound immunosuppression, and cyclophosphamide is compounding it. Trypanosomes do suppress antibody response to unrelated T-dependent, but not T-independent, antigens (Mansfield and Bagasra 1978), and there is some evidence that more virulent trypanosomes inhibit antibody responses to less virulent trypanosomes (Inverso and Mansfield 1983). Also, late in infection, the host apparently makes less antibody to the trypanosomes than it does earlier in infection (Sacks and Askonas 1980). Since, in most studies, parasite induced immunosuppression does not occur until the second week of infection, Day 0 mice would have begun to recover from the effects of cyclophosphamide at this time, whereas Day 7 mice are most severely affected by the drug at the same time that parasite induced suppression is becoming significant.

These studies on Trypanosoma rhodesiense suggest that, although the ability to make antibody to trypanosomes is crucial to survival, the timing of this antibody production is perhaps even more crucial. These observations also confirm that continuing high levels of parasitemia do not necessarily lead to more rapid death. More important than numbers of parasites is their relative virulence, ACKNOWLEDGMENTS

This work was supported in part by the U.S. Public Health Service through Grant AI-18519 and by the Department of Microbiology of Thomas Jefferson University. 1 thank Laurie Mitchell and Gerald Hancock for excellent technical assistance, Kathleen Givens and Rita Taylor for secretarial assistance, and Dr. Catherine Calkins, Dr. John F. Finerty, and Dr. Carl Kirkpatrick for critical review and discussions. REFERENCES ASHCROFT, M. T. 1957. The polymorphism of Trypunosoma hrucei and T. rhodesiense, its relation to

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