Trypanosoma cruzi: Correlation of resistance and susceptibility in infected inbred mice with the in vivo primary antibody response to sheep red blood cells

EXPERIMENTAL

PARASITOLOGY

Trypanosoma Infected

Departments Albert

cruzi: Correlation

inbred

HERBERT

52,233-242(1981)

of Resistance and Susceptibility in Mice with the in Vivo Primary Antibody Response to Sheep Red Blood Cells

B. TANOWITZ,’

NAGAHIRO MINATO, MURRAY WITTNER

of Medicine (Division of Infectious Disease) Pathology Einstein College of Medicine, 1300 Morris Park Avenue,

(Accepted for publication

RICHARD

LALONDE,

and Microbiology Bronx, New York

AND

and Immunology, 10461, U.S.A.

25 August 1980)

H. B., MINATO,N.,LALONDE, R., AND WIITNER, M. 1981. Trypanosoma Correlation of resistance and susceptibility in infected inbred mice with the in primary response to sheep red blood cells. Experimental Parasitology 52, 233-242. Nonspecific immune responses during the course of murine Trypanosoma cruzi infection were examined in mouse strains genetically resistant or susceptible to the Brazil strain of T. cruzi. Spleen cells from infected susceptible (C3H) or resistant [C57 BVlO and Fl (C3H x C57)] mice at various points during the course of infection exhibited a reduced response to concanavalin A and lipopolysaccharide in vitro. .Since this reduced response occurred in both susceptible and resistant mice, it was not predictive of resistance or susceptibility in vivo. We next examined the kinetics of in vivo primary antibody response to sheep red blood cells (SRBC) in infected C3H and C57 mice. C3H mice exhibited inhibition of the direct plaque-forming cell assay (d-PFC) which persisted until death. In contrast C57 mice exhibited no inhibition of the response at Day 5 and subsequently a markedly augmented response was observed. Other strains of mice were similarly investigated: all the susceptible mice examined (A/J, BALB/c) showed inhibition or depression of the primary antibody response and resistant mice [BlOBr, C57BU10, SJL, Fl (C3H x C57)] demonstrated either no inhibition or considerable augmentation of this response. C57 mice resistant to the Brazil strain were susceptible to the Tulahuen strain. The mice in this latter group exhibited a markedly significant inhibition of the in vivo primary antibody response to SRBC. Culture forms of the Brazil strain protected C3H mice from a virulent challenge. This immunization resulted in a markedly augmented antibody response. The data reported herein are consistent with the notion that inhibition of the primary antibody response to SRBC correlates with susceptibility whereas no inhibition or, indeed, augmentation of the response correlates with natural as well as acquired resistance. INDEX DESCRIPTORS: Trypanosoma cruzi; Protozoa, parasitic; Hemoflagellate; Chagas’ disease; Immunosuppression; Sheep erythrocytes, primary antibody response; Mitogens; Lipopolysaccharide; Concanavalin A. TANOWITZ,

cruzi: vivo

INTRODUCTION

Among the adaptive mechanisms have evolved for eluding the normal response nomenon

to parasitic infection of immunosuppression

al. 1979). Immunosuppression, during

the course

originally

of parasitic

described in clinical

that host is the phe(Bloom et occurring

infection,

was

diseases by

1 To whom reprint requests should be sent at the Department of Pathology.

McGregor and Barr (1962) who reported that malarious Gambian children had poor antibody response to tetanus toxoid. Subsequently, other investigators reported suppression of delayed hypersensitivity with skin test antigens, such as purified protein derivative (PPD), Can&da sp., and dinitrochlorobenzene (DNCB), as well as poor antibody response to some Salmonella spp. antigens in patients with African 233 0014-4894/811050233-10$02.00/O Copyright @ 1981 by Academic Ress, Inc. All rights of reproduction in any form reserved.

234

TANOWITZ

trypanosomiasis (Greenwood et al. 1967) or malaria (Greenwood et al. 1972). Immunosuppression has only been explored in Chagas’ disease within the past 5 years (Clinton et al. 1975). Several possible mechanisms of the immunosuppression described during the course of Trypunosoma cruzi infection have been reported including suppressor T cells (Ramos et al. 1979), suppressor macrophages (Rowland and Kuhn 1978), and a serum suppressor factor (Cunningham et al. 1978; Cunningham and Kuhn 1980b). However, the precise mechanisms of this suppression as well as its significance in the infection have not been clarified. Previous studies in our laboratory have established that in various inbred strains of mice there exists a spectrum of resistance and susceptibility to the Brazil strain of T. cruzi, a so-called myotropic strain (Trischmann et al. 1978). It was found that in susceptible mouse strains infected with a 104 inoculum, death usually occurred between Days 21 and 25 of infection and was invariably associated with high parasitemia and large numbers of intracellular organisms. Resistant mice survived the acute period, had a low transient parasitemia, and few intracellular forms were observed. In an attempt to clarify the significance of the depression of nonspecific immune responses during T. cruzi infection we examined the in vivo as well as the in vitro immune responses in a variety of mouse strains infected with the Brazil strain whose genetic susceptibility was well established (Trischmann et al. 1978). The results described herein clearly establish the positive correlation between susceptibility and a depressed in vivo primary antibody response to sheep red blood cells (SRBC) and resistance with either no depression or an augmented response. MATERIALS

AND METHODS

Host animals. Inbred used in these experiments

strains of mice were either ob-

ET AL.

tained from Jackson Laboratories, Bar Harbor, Maine, or bred in our own animal facilities. Parasite. The Brazil strain of Trypanosomu cruzi was obtained from Dr. William Hanson, University of Georgia, Athens, Georgia, and the Tulahuen strain was obtained from Drs. George Healy and Lois Norman, Center for Disease Control, Atlanta, Georgia. Strains were maintained in C3H/HeJ or A/J mice by syringe passage of blood as previously described (Trischmann et al. 1978). Epimastigotes were maintained in LIT media at 27 C on a shaker platform (Tanowitz et al. 1975). Infection of mice. Various inbred strains of mice used (C3H/HeJ, C57 Bl/lO or C57 Bl/6 (C57), BlOBr, SJL, BALB/c, A/J, Fl (C3H x C57) were inoculated intraperitoneally (ip) with lo4 trypomastigotes of either the Brazil or Tulahuen strain. Female mice 6-8 weeks of age were used. Mice given “culture forms” (>85% epimastigotes) were inoculated ip with IO-20 x lo6 organisms. Six C3H mice so immunized were examined for parasitemia at Day 14 and then subsequently challenged with lo4 trypomastigotes. Purusitemiu. Parasitemia was evaluated during the course of infection as previously described (Trischmann et al. 1978). Mitogens and isotopes. Concanavalin A (Con A) was obtained from Miles Laboratories, Elkhart, Indiana, and lipopolysaccharide (LPS) from Difco Laboratories, Detroit, Michigan. Sheep red blood cells (SRBC) were obtained from Scott Laboratories, Fiskville, Rhode Island, and [3H]thymidine (sp act, 3-6.7 Ci/mmole) New England Nuclear, Boston, Massachusetts. Mitogenic response of spleen cells. Spleen cells from either normal or infected mice were removed at varying intervals during infection. Only occasionally was a rare trypomastigote found in the spleen cell cultures by microscopic examination. The spleen cells were cultured in triplicate in Dulbecco’s minimal essential media with

Trypanosoma

cruzi:

RESPONSE TO SHEEPERYTHROCYTES

10% fetal calf serum (DME- 10% FCS) in flat bottom 96well Linbro culture plates (1 x lo6 cells/well) together with either 1 pg Con A or 1 pg LPS for 48 hr. One PCi of [3H]thymidine was added to each well for an additional 16 hr, and the cells were harvested with a multiple cell harvester (Skatron AS, Lierbyen, Norway). Direct

hemolytic

plaque-forming

assay.

Sheep red blood cells were washed three times in phosphate-buffered saline (PBS, pH 7.2) and 108 cells were injected intravenously (iv) into the tail vein of uninfected (control) or infected mice during the course of infection with either T. cruzi strain. Four days later, spleens from infected or uninfected control mice were removed and teased in serum-free DME. Unfractionated spleen cells were washed three times in serum-free DME and filtered through gauze cloth to remove fat. The spleen cells were brought up to a volume of 10 ml in serumfree DME and incubated at 4 C for 5 min and subsequently diluted 1:9 with serumfree DME. The final reaction mixture contained 200 ~1 of spleen cell suspension, 100 ~1 of a 10% SRBC suspension, and 50 ~1 of guinea pig serum (complement source). A modified Cunningham hemolytic plaque assay was performed utilizing standard microscope slides that were incubated at 37 C for 2 hr after they were loaded with the final reaction mixture (Cunningham and Szenberg 1968). There was a wide daily and weekly variation of direct plaque-forming cells per spleen (d-PFC/spleen) in both infected and uninfected control animals due to differences in the source of mice, lots of SRBC, and complement source (guinea pig serum). The data, on a given day, however, were always internally consistent, when normalized as percentage of control. Preliminary experiments on the kinetics of the primary antibody response to SRBC in various inbred mouse strains were performed after ip inoculation of 108 SRBC. The data was not consistent or reproducible and, in fact, often revealed a suppressed

235

response in both infected resistant and susceptible mice similar to that reported by others (Cunningham et al. 1978). Consequently, our experiments were performed with SRBC administered by the iv route. Using this protocol, it was observed that the maximal response was obtained 4 days after immunization, and the data were always consistent and reproducible. Statistical tests. The mean, standard deviation, and standard error were determined for all experiments. Subsequently a two-tailed t test was performed and significance levels were ascertained. Significance was accepted at the P < 0.05 level. RESULTS of spleen cells in vitro. It was already evident by Day 10 of infection with Trypanosoma cruzi that the mitogenic response to Con A and LPS was significantly lower in both resistant and susceptible mice (Table I, II). The Fl (C3H x C57) mouse is a strain considered to be more resistant than the resistant parent (Trischmann, personal communication). Spleen cells from the Fl mice, infected with a lo4 inoculum of the Brazil strain, were also unresponsive to Con A (Table I). In vivo primary antibody response to Mitogenic

response

SRBC in inbred mice infected with the Brazil strain of T. cruzi. The kinetics of the

primary response to SRBC in infected C3H and C57 mice are shown in Fig. 1. There was a reduction in d-PFC/spleen in C3H mice by Day 5 of infection and this depression persisted until death. In contrast, C57 mice similarly infected, exhibited no depression in d-PFC/spleen by Day 5, however, an augmented response to SRBC was always observed subsequently, most significantly at Day 10. The primary antibody response to SRBC was examined in several other inbred strains of mice infected with the Brazil strain. The mouse strains chosen were known to be either resistant or susceptible to a 1O4inoculum of T. cruzi (Trischmann et

236

TANOWITZ

ET AL.

al. 1978). Day 15 was chosen to study the response. At this time even susceptible mice were clinically well and had a parasitemia ranging from lo5 to 10Yml. As shown in Table III all susceptible mice showed a depression of the primary antibody response to SRBC (-60% depression), whereas all the resistant mice exhibited no depression or significant augmentation of the response. Additionally, the d-PFC/ spleen in C3H mice infected with lo4 inoculum of the Brazil strain but not previously immunized with SRBC was significantly elevated at Day 25 of infection (data not shown). This suggested polyclonal Bcell activation during infection even in susceptible mice. These results are consistent with those of Ortiz-Ortiz et al. (1980) and Schmunis et al. (1977). In vivo antibody response to SRBC in mice infected with the Tulahukn strain. Studies in our laboratory have shown that the Tulahuen strain is more virulent than the Brazil strain. In fact, mice ordinarily highly resistant to the Brazil strain at lo4 inoculum are susceptible even to a lo3 inoculum of the Tulahuen strain (unpublished observation). Figure 2 shows the parasitemia in C57 BY10 mice infected with a lo4 inoculum of the Brazil and Tulahuen strains. The five mice infected with the Brazil strain had a transient parasitemia which was eventually cleared and no mortality was observed in this gorup even after 56 days of observation. However, all five mice inoculated with the Tulahuen strain died; death began as early as Day 20. These mice achieved high parasitemia before death. In order to pursue the possible correlation between susceptibility to the Brazil strain and depression of the antibody response we compared the primary antibody response to SRBC after infection with the Brazil or Tulahuen strain in both the C3H and C57 mice. The results presented in Table IV shows that in the C3H mouse given a lo4 inoculum of Brazil or Tulahuen

Trypanosoma

cruzi:

RESPONSE

TO

SHEEP

TABLE II Response to LPS of Spleen Cells from Mice Infected with Ttypanosoma C3H/He mice”

Day of infection

Response* 112,333 158,521 15,122 15,924 5,369

0

5 10 15 20

(?23,877) (?34,921) (2 802) (k 691) (2 773)

237

ERYTHROCYTES

cruzi

C57 mice” P

0.01 0.001 0.001

Responseb 43,925 10,211 7120 3588 6988

(23522) (51511) (k 708) (k 488) (” 52)

P

0.001 0.01 0.001 0.0005

(2Three mice were studied at each time point. b The mean mitogenic response to LPS (1 cLg/loP spleen cells) of triplicate cultures obtained from mice infected with a 10’ inoculum of the Brazil strain. The results were determined by the incorporation of [3H]thymidine and expressed as mean counts per minute (f SE).

strain, there is a more significant inhibition of the primary antibody response to SRBC in the latter group at Day 14 of infection (-60% depression vs 80% depression). However, C57 mice which are resistant to the Brazil strain and have an augmented in vivo response to SRBC (-170% augmentation) were susceptible to a lo* inoculum of the Tulahuen strain and had a marked inhibition of the in vivo primary antibody re-

160

P’OI

P’OI

p:oo5

p=oo1

5 zoog

6

-4c57

pso1

LLchl 3 p=o1

100

P’OI

6

5

3

Control

5

lo

3

15

20

Days of infection

1. Kinetics of the in vivo primary antibody response to sheep red blood cells in C3H and C57 mice infected with a 10’ inoculum of the Brazil strain of Trypanosoma cruzi. At intervals during the infection mice were given 108 SRBC iv and spleens were removed 4 days later and assayed for direct plaqueforming cells per spleen *SE. Numbers in the bars refer to mice in each exoeriment. FIG.

sponse to SRBC at Day 14 of infection (-60% depression). In vivo antibody response to SRBC in C3HIHeJ mice inoculated with culture forms. Administration of avirulent forms of T. cruzi protects otherwise susceptible mice

when challenged with virulent trypomastigotes (Seah and Marsden 1969). Consequently, we examined the effect of immunization of susceptible mice with culture forms on the in vivo primary antibody response to SRBC. Twelve C3H mice were given 10-20~10~ culture forms (Brazil strain) ip. Fourteen days later, when no parasites were observed in the circulation, half the group were challenged with IO4 trypomastigotes and the remaining mice were injected with lo8 SRBC iv. All the mice in the former group were followed for 120 days and all survived. The mice given SRBC were sacrificed 4 days later and the d-PFC response was assayed. As shown in Table V the d-PFC response was not depressed in the group which received culture forms but rather slightly augmented (-130%). DISCUSSION

Immunosuppression is associated with many parasitic infections. Although this phenomenon has been studied extensively in man and animals its significance remains unclear. Recently a World Health Organization scientific group (WHO 1978) consid-

238

TANOWITZ

ET AL.

/ B 5

25 15 20 Days of infection

IO

30

35

FIG. 2. Mean parasitemia (2 SE) of five mice infected intraperitoneally with lo4 trypomastigotes of the Brazil strain (04) and five mice infected with 104 trypomastigotes of the TulahuCn strain (0- - -0) of Trypanosoma cruzi.

ered the problem and stated that parasiteassociated immunosuppression or secondary immunodeficiency may have a number of important consequences. It was suggested that as a consequence of immunosuppression parasitized hosts may be more susceptible to an unrelated infection; that following vaccination parasitized hosts may not be able to raise a proper antibody response; and that immunodepression may involve responses aimed at the homologous parasite that may, in part, explain the chronic nature of many parasitic infections. Nonspecific immunologic indicators such as Con A, LPS, and SRBC have been used to explore the nature of resistance and susceptibility to infectious agents. In the present study no correlation was found between the in vitro mitogenic response to Con A and LPS and resistance and susceptibility to Trypanosoma cruzi. This is similar to the report of Rowland and Kuhn (1978). In addition, the mitogenic response to normal spleen cells obtained from resistant and susceptible mice to T. cruzi antigens was similar (Lalonde and Tanowitz, unpub-

Trypanosoma

cruzi:

239

RESPONSE TO SHEEP ERYTHROCYTES

TABLE IV Antibody Response to Sheep Erythrocytes in C3H and C57 Mice Infected with the Brazil and Tulahuen Strains of Trypanosoma cruzi C3H/He

C57 Bl/lO

d-PFUspleen” Control Brazil strain Tulahdn strain

78,670 (k7300) 33,670 (-t6085) 15,250 (22237)

P

d-PFC/spleen

P

0.001 0.001 (0.02)b

19,800 (k 2,484) 52,570 (212,107) 7,750 (A 2,017)

0.02 0.01 (O.Ol)b

a The C3H and C57 mouse strains were infected ip with IO4 trypomastigotes of Brazil or Tulahuen strain. Fourteen days later uninfected and infected mice were given lo8 SRBC iv, spleens were removed 4 days later and assayed for d-PFC/spleen (see text for details). Results are expressed as the mean d-PFCispleen of five mice (%SE). * Tulahuen vs Brazil.

lished observations). Moreover, the in vitro primary (Cunningham and Kuhn 1980a) and secondary (Minato and Tanowitz, unpublished observations) responses to SRBC or TNP were found to be depressed in susceptible as well as resistant mouse strains. Although several in vitro studies did not distinguish between resistance and susceptibility, when the in vivo primary antibody response to SRBC was examined strong direct correlation was evident. Susceptibility was, therefore, reflected early in the infection by inhibition of the in vivo primary antibody response to SRBC whereas either no inhibition or actual augmentation was seen in resistant mice as well as those given avirulent culture forms. The reason for this observation in the latter group may be that culture forms, which contain primarily TABLE V Effect of Trypanosoma cruzi Culture Forms on the Antibody Response to Sheep Erythrocytes in C3H Mice Mouse groupsa

d-PFClspleen*

P

C3H uninfected C3H + culture forms

60,200 (211,367) 177,600 (?21,770)

0.001

a Six mice in each group. * C3H mice were inoculated ip with lo-20 x lo6 culture forms per mouse. On Day 15 following immunization, control and immunized mice were inoculated iv with 108 SRBC, spleens were removed 4 days later and assayed for d-PFC/spleen. Results are expressed as mean d-PFCispleen (&SE).

epimastigotes and relatively few avirulent trypomastigotes (Tanowitz, unpublished observations), cause low grade or perhaps chronic infection which stimulates helpercell function. We are currently investigating this phenomenon. Additionally, the studies in the C57 mouse infected with the Brazil and Tulahuen strain supports the contention that the strain of T. cruzi may be as important as the host in determining the outcome of the infection. Immunosuppression in T. cruzi infection was first reported by Clinton et al. (1975) who noted a reduction of the primary antibody response to burro erythrocytes in infected mice. Subsequent studies in man (Teixeira et al. 1978) and experimental animals (Ramos et al. 1978) revealed a depressed response to a variety of antigens during infection. Moreover, Ramos ef al. (1979) reported the presence of suppressor T cells in T. cruzi-infected mice, observations which differ with those of Reed et al. (1977, 1978) and Rowland and Kuhn (1978), who suggested that a macrophage-like cell is responsible for cellular unresponsiveness reported in these infections. Recently Cunningham and Kuhn (1980a) reported that inhibition of the in vitro primary antibody response to SRBC and TNP was mediated by a plastic-adherent macrophage-like cell. Cunningham and Kuhn (1978) reported that the in vivo primary antibody response to SRBC was depressed in both C3H (sus-

240

TANOWITZ

ET AL.

ceptible) as well as in C57 (resistant) mice. sponse to a variety of mitogens was related However, they introduced the antigen to the virulence of the rickettsial strain and (SRBC) ip. Subsequently, we were able to paralleled clinical disease. Nonpathogenic confirm these results. When SRBC were Rickettsia spp. did not cause suppression of administered by the intravenous route, lymphocyte transformation. These results however, significant differences were found differ from our observations where signifibetween susceptible and resistant mice. cant reduction in mitogenic response was Similarly, Mitchell and Handman (1977) re- always observed. ported that their results in the d-PFC assay Recent studies that have explored nonparameters of resistant and were dependent upon the route of RBC ad- immunologic susceptible mice to T. cruzi have shown ministration. marked depletion of choline acetyltransThe relationship of immunosuppression with susceptibility and resistance has been ferase in hearts of susceptible mice early explored in other parasitic and infectious in the infection (4-7 days). However, no diseases. For instance, it was observed that change in enzyme levels could be detected in T. muse&i-infected rodents in vitro unre- in infected resistant strains suggesting proresponsiveness to mitogens was maximal at found differences in the biochemical the height of parasitemia. In addition, it was sponse to T. cruzi infection between the found that the in vivo primary antibody re- mouse strains examined (Tanowitz et al. sponse to SRBC was suppressed at the 1981). height of parasitemia and clinical disease Finally, it is unclear why the responses to only to return to normal levels as the mice SRBC differ in the various infected mouse recovered and parasitemia waned (Albright strains. A possible explanation, however, is et al. 1977; Hazlett and Tizard 1978). In that the antigenic stimulus (i.e., Trypanocontrast, in the present report the in vivo soma cruzi) causes amplification or enprimary antibody response to SRBC in in- hancement of suppressor cells in suscepfected resistant animals was either aug- tible animals. These may be suppressor mented or not inhibited throughout the pe- T cells such as described by Ramos et al. riod of maximal parasitemia whereas in (1979) or another cell type suggested by susceptible animals this response was Reed et al. (1977, 1978) or Rowland and markedly inhibited. Weinbaum et al. (1978) Kuhn (1978). Further, in resistant animals, reported that the in vitro mitogenic re- the antigen may stimulate the proliferation sponses to PHA, LPS, and malaria antigen and/or amplification of helper-cell function of spleen cells derived from mice infected such as T helper cell. Work is currently in with a nonlethal strain of Plasmodium berprogress to delineate the factors responsighei yoelli were maximally suppressed at ble for the suppressed or augmented rethe height of parasitemia but returned to sponse in the different infected mice normal with declining parasitemia. In strains. murine cutaneous leishmaniasis it has been shown that susceptible animals showed ACKNOWLEDGMENTS suppressed in vitro response to homologous antigen as well as to mitogens, whereas in This work was supported in part by Grants AI 12770, AI 07183, AI 10202, and AI 09807 from the resistant mice, there was increased lymInstitutes of Health. Dr. phocyte responsiveness to mitogens (Perez United States National Richard Lalonde is a recipient of a Medical Research et al. 1978) During the course of rickettsial Council of Canada fellowship. The authors wish to exinfection in guinea pigs Oster et al. (1978) press their thanks to Drs. Barry R. Bloom and P. A. found that suppression of lymphocyte re- Neighbour for their review of the manuscript, and to

Trypanosoma

cruzi: RESPONSE TO SHEEPERYTHROCYTES

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