Immunology, 1975, 28, 401.
The Immunology of Experimental Chagas' Disease II. DELAYED HYPERSENSITIVITY TO TRYPANOSOMA CRUZI ANTIGENS A. R. L. TEIXEIRA*
AND
C. A. SANTOS-BUCH
Department of Pathology, Cornell University Medical College, New York, New York, U.S.A.
(Received 9th March 1974; acceptedfor publication 6th June 1974)
Summary. Homogenates of suspensions of the trypomastigote and amastigote forms of the Ernestina strain of Trypanosoma cruzi, derived from tissue cultures, yielded two subcellular fractions which elicited strong delayed hypersensitivity reactions in rabbits. The 100,000 g x 90 minute fraction of T. cruzi homogenates gave rise to marked cell-mediated immunity. The 30,000 g x 35 minute fraction of these homogenates was also capable of eliciting a marked cell-mediated immune response. Cell-mediated immunity was assayed by experiments which established passive transfer, inhibition of blood mononuclear cell migration and blast transformation by T. cruzi-sensitized lymphocytes. Sensitized lymphocytes did not have observable effects on trypomastigotes of T. cruzi. The results of the experiments described here strongly suggest that constituents of intracytoplasmic particles of trypomastigotes and amastigotes of T. cruzi are involved in eliciting cell-mediated immunity in rabbits. INTRODUCTION In a previous report (Teixeira and Santos-Buch, 1974), a method for the partition of homogenates of trypomastigote and amastigote forms of Trypanosome cruzi into subcellular fractions was described. The soluble substances present in the cytosol fraction of T. cruzi homogenates appeared to possess antigenic properties principally related to the humoral antibody response in rabbits (Teixeira and Santos-Buch, 1974). Cell-mediated immune responses in Chagas' disease have not been fully explored (Seah, 1970). Studies have been based on morphological lesions formed in various tissues, which followed the systemic injection of viable T. cruzi forms (Pizzi and Rubio, 1955; Taliaferro and Pizzi, 1955). The findings reported in this paper indicate that cytoplasmic particles, derived from homogenates of T. cruzi elicited delayed hypersensitivity in rabbits. These particulate fractions also reacted strongly with lymphocytes derived from rabbits chronically infected with T. cruzi. Further, passive transfer of cellular immunity to normal rabbits was accomplished with lymphocytes collected from the peripheral blood of these chronically infected rabbits. * Present address: Departamento de Patologia, Faculdade de Ciencias da Saude da Universidade de Brasilia, 70000Brasilia-D.F., Brasil. Correspondence: Dr C. A. Santos-Buch, Department of Pathology, New York Hospital, Cornell Medical Center, 525 East 68th Street, New York 10021, U.S.A.
401
402
A. R. L. Teixeira and C. A. Santos-Buch
MATERIALS AND METHODS Strain of T. cruzi grown in tissue culture The Ernestina strain of T. cruzi utilized in these experiments was maintained in foetal rabbit heart and skeletal muscle primary cell line cultures as described previously by Teixeira and Santos-Buch (1974).
Partition of T. cruzi forms into subcellular fractions Trypomastigotes and amastigotes of T. cruzi harvested from rabbit heart and skeletal muscle cultures were isolated and homogenized as described by Teixeira and SantosBuch (1974). The initial homogenates of parasites were partitioned into seven subcellular fractions (Fl to F7) (Teixeira and Santos-Buch, 1974). The speed of centrifugation for the different fractions are shown in Table 2. Previous partial characterization had shown that the F5, 30,000 g x 35 minute, fraction was rich in lysosomal bodies, whereas the F6, 100,000 g x 90 minute, fraction was rich in ribosomes. The supernatant of the F6 pellet, the cytosol F7 fraction, contained about 50 per cent of the protein of the initial homogenate (Teixeira and Santos-Buch, 1974). Immunization of rabbits with homogenates and subcellular fractions of T. cruzi Two groups of eight, 6- to 12-month-old New Zealand white rabbits were each immunized with T. cruzi homogenates (TH) and subcellular antigens derived from differential centrifugation. The initial dose consisted of 0-1 ml of antigen injected intravenously and this was followed by two consecutive, weekly, subcutaneous injections of 1 ml of the antigen homogenized in 1 ml of complete and incomplete Freund's adjuvants, respectively. Subsequent courses of 0-2 ml of the antigen suspensions intravenously were spaced 1 week apart. Each rabbit of the first group (group I) received injections of antigen in the following concentrations: TH, 220 ug/ml; F1, 460 pg/ml; F2, 280 jug/ml; F3, 240 ug/ml; F4, 180 ig/ml; F5, 40 ug/ml; F6, 90 ug/ml; F7, 120 ug/ml. Each rabbit of the second group (group II) received higher dosage of antigen with the following concentrations: TH, 320 pg/ml; Fl, 780 ug/ml; F2, 670 ug/ml; F3, 460 ug/ml; F4, 250 ug/ml; F5, 70 pg/ml; F6, 115 ug/ml; and F7, 180 pg/ml. Controls consisted of rabbit C-1, immunized with homogenates of cultured allogeneic heart cells; rabbit C-2, immunized with culture medium with 5 per cent (v/v) horse serum, and normal rabbit C-3 and C-4 (Teixeira and Santos-Buch, 1974).
Infection of rabbits with T. cruzi Eight, 1-month-old, New Zealand white rabbits were injected intraperitoneally with trypomastigotes of T. cruzi derived from tissue culture. The inoculum was calculated by multiplying the body weight in grams x I05 trypomastigotes. Wet preparations showed parasites in the peripheral blood between days 21 and 30 following inoculation. NNN media inoculated with 0-5 to 1 ml of peripheral blood from these rabbits were positive by day 15 and they became repeatedly negative after the 3rd month following inoculation. Skin response Each rabbit immunized with its subcellular fraction of T. cruzi was skin tested with specific antigen 1 week after the 5th and 1 week after the 10th immunizing dose. The chronically infected rabbits were skin tested with each fraction during the period of
403 Experimental Chagas' Disease demonstrable negative blood cultures. 01 ml of antigen was injected intradermally. The protein concentration of antigens was the same used for immunization. Control injections consisted of 0 1 ml of the solution used for suspension of the antigens. Readings were taken after 6, 12, 24, 48, and 72 hours. A positive, immediate type of reaction was considered an area of redness measuring at least 1 cm in diameter which developed within 24 hours, and the degree of reaction was assayed as follows: one plus (+) was equivalent to an area of redness of 1-1 5 cm, and three plus ( + + + ) was an area of redness of over 2*5 cm. An intermediate reading was considered as (+ +). A delayed-type reaction was considered an area of induration persisting for 72 hours after antigen injection, and a similar scale of one zero (0) to three zeros (000) was used to assay the degree of induration.
Collection of blood mononuclear cells Seven to 10 days after the last injection of antigen, the rabbits were bled from a marginal ear vein. The first collection of blood was obtained after the twelfth immunizing dosage, and later collections were made at other times, following further courses of antigen
injection. Peripheral mononuclear cells were isolated from the blood of eight rabbits with T. cruzi infection in a similar fashion, after negative blood culture tests had been obtained.
Assay of inhibition of monocyte migration A modification of the capillary tube method for studying the inhibition of migration of blood monocytes was used. This method was reviewed recently by Bloom and Glade (1971). Monocytes and lymphocytes were isolated from 35-40 ml of fresh heparinized rabbit venous blood (sodium heparin, Upjohn, 10 i.u./ml) by the Ficoll-Hypaque (Pharmacia, Uppsala, Sweden; Winthrop Laboratories, New York) density gradient centrifugation technique of Boyum (1968). For convenience, the Hypaque concentration was used at full strength (50 per cent w/v) before it was mixed with Ficoll at 9 per cent (w/v) in proportions of 2*4 volumes of Ficoll: 1 volume of Hypaque. The blood was layered on top of the Ficoll-Hypaque mixture (3 parts of blood: 2 parts Ficoll-Hypaque), and centrifuged at 1000 g for 50 minutes at 200. The layer of mononuclear cells at the interface was collected with a sterile Pasteur pipette. This modification of the method gave good yields of mononuclear cells. The suspension of cells revealed an average differential count of 87-90 per cent lymphocytes and 10-13 per cent monocytes. More than 90 per cent of the cells were viable as judged by trypan blue exclusion. The mononuclear cells were washed once with phosphate-buffered saline+0 003 M ethylenediaminotetraacetate (PBS-EDTA) and three times in Hanks's buffered saline solution (HBSS). The lymphocyte and monocyte pellet was then suspended in 50 p1 Hanks's minimal essential medium (MEM) (Flow Laboratories, Maryland, U.S.A.) to a concentration of approximately 60 x 106 cells/ml and were sucked into capillary tubes attached to a rubber nipple of an Eppendorff microlitre pipette (Sargent-Welch Scientific, Springfield, New Jersey). The capillary tips were anchored in a drop of sterile silicone high vacuum grease (DowCorning, Midland, Michigan) in the wells of a microchamber which were sealed with glass coverslips. The culture medium used in the well was MEM with 10 per cent horse serum (HS). All the antigens were made up to a concentration of 150 jg/ml, except for F5 and F6 which were made up to 100 ug/ml concentration. All tests were run in triplicate. The largest diameters of the migration areas were measured, after 20 hours incubation at
A. R. L. Teixeira and C. A. Santos-Buch 370, with a magnifying comparator with a scale in mm (ACE Scientific Supply, Linden, New Jersey). The migration in the presence and absence of antigen was then compared.
404
Interaction between T. cruzi and peripheral blood lymphocytes in vitro Fresh mononuclear cells were obtained from rabbit peripheral blood as described above. After the first washing in PBS-EDTA and three more washes in HBSS, the cells were resuspended in MEM with 10 per cent (v/v) horse serum and transferred into a culture plastic vessel in order to allow monocytes to stick onto its surface. The supernatant population of lymphocytes was collected after 4 hours incubation at 36-370. These lymphocytes were transferred into sterile 5-ml plastic tubes and adjusted to concentrations of 1, 2, and 3 x 105 cells/ml in MEM. Trypomastigotes (1 x 105 parasites/ml) were added to the tubes to make 1: 1, 1: 2, and 1: 3 parasite to lymphocyte ratios. One millilitre of each of the lymphocyte/living parasite mixtures was transferred to wells of a microchamber and sealed with coverslips. The tubes and the microchambers were incubated at 36-370. The wells of the microchamber were inspected periodically with a microscope. At the end of 72 hours incubation, the tubes were centrifuged, and the cells were washed with PBS and resuspended to the original volume. A sample of the suspension was smeared on a slide and stained by the Giemsa-May-Grunwald method. Lymphocytes were quantitated by counting the number of cells in three medium power fields of the smear and blast transformation was determined by standard morphological criteria (Ling, 1968).
Transfer of cell-mediated immunity from chronically infected rabbits to normal rabbits Mononuclear cells were isolated from T. cruzi-infected rabbits with negative blood cultures. Two male, 8-10-month-old, New Zealand white rabbits were used as recipients. The first rabbit received six intramuscular injections of approximately 3 x 107 intact lymphocytes (Landsteiner and Chase, 1942). The second rabbit received six intramuscular injections of the homogenate derived from equal numbers of disrupted cells following repeated freezing and thawing (Lawrence, Al-Askari, David, Franklin and Zweiman, 1963). RESULTS SKIN RESPONSE TO ANTIGENS OF
T. cruzi
Skin reactivity of the actively immunized and chronically infected rabbits infected with the antigens derived from the initial homogenate of T. cruzi was tested. The results of the experiment are given in Table 1. Both immediate- and delayed-type skin reactions occurred in the specifically immunized rabbits after the intradermal injection of homologous antigen. The initial homogenate contained antigens capable of eliciting a strong immediate-type reaction and also marked delayed-type skin response. The cytosol F7 fraction elicited an immediate- rather than a delayed-type response. In contrast, the particulate antigens gave rise to a greater degree of induration and delayed-type of skin reaction. Also, delayed skin reactivity was more marked in chronically infected rabbits E, F and G when the particulate fractions F4, F5 and F6 were used, than when the cytosol F7 fraction was used.
Experimental Chagas' Disease
405
TABLE 1
SKIN RESPONSE
Chronically infected rabbits
Actively immunized rabbits*
T. cruzi antigen TH Fl F2 F3 F4 F5 F6 F7
Group II
Group I Immediate
Immediate
typet
Delayed type:
+++ ++ ++ + + + + +++
000 00 00 0 0 0 0 0
+++ +++ ++ ++ ++ + + +++
Immediate
Delayed
typet
Rabbit
typet
Delayed type:
A B C D E F G H
+++ ++ ++ ++ + + + +++
000 00 00 0 00 00 00 0
typel 000 000 00 00 00 00 0 0
* Each specific T. cruzi antigen was injected into a correspondingly immunized rabbit intradermally. Immediate-type reaction was recorded after 24 hours and delayed-type reaction was recorded after 48 hours. t Development of hyperaemic wheal within 24 hours of injection. + = Area of redness of 1-1 5 cm. + + + = Area of redness of over 2-5 cm. + + = Intermediate values. $ Induration of skin persisting for 72 hours of injection. 0 = Area of induration of 1-1 5 cm. 000 = Induration measuring over 2-5 cm. 00 = Intermediate values. CELL-MEDIATED IMMUNITY
Inhibition of blood mononuclear cell migration (David, Al-Askari, Lawrence and Thomas, 1964; Bloom and Bennet, 1966) was used to assess cell-mediated immunity in rabbits actively immunized with subcellular fractions of T. cruzi. This test was also used to determine the responsiveness of blood mononuclear cells derived from a chronically infected rabbit (Rabbit E). The results of these experiments are given in Table 2. The TABLE 2
CELL-MEDIATED
MIGRATION* (BLOOD MONUCLEAR CELLS
IMMUNE RESPONSE: INHIBITION OF MONOCYTE
vs
T. cruzi
ANTIGENS)
T. cruzi antigen used in testt
Actively immunized rabbits of groups I and 11:
Chronically infected
T. cruzi antigen
Control rabbits C-1, C-2 and C-4§
TH (initial homogenate) F 1 (350 g x 10 minutes) F2 (700 g x 10 minutes) F3 (5000 g x 10 minutes) F4 (15,000 gx25 minutes) F5 (30,000 gx35 minutes) F6 (100,000 g x 90 minutes) F7 (cytosol)
47-8+ 9-8 52 8+ 6-9 48-2 +4-7 36-5+8-1 415+5 3 518+ 7-311 42-4+ 9 1l¶
56-7 + 9 4 46-3+ 16 53-7+ 1P6 49-1 +4-3 56-4+5-9 90-6+ 12-1** 76-9 + 3.9** 48-4+2-3**
TH
14 7+ 3*4
F5
9 7+3 1
F7
6-0+3-6
33-2+2±0¶
rabbit E
* Percentage inhibition = [100- (the average diameter of mononuclear cell migration exposed to antigen)/(the average diameter of mononuclear cell migration in the absence of antigen)] x 100. The results were recorded after 20 hours. t Blood mononuclear cells were exposed to 150 yg/ml of antigens TH, Fl, F2, F3, F4 and F7. For F5 and F6, 100 pg/ml were used. T The antigen used in the test was the antigen used to immunize the rabbit. % Controls = C-1, C-2 and C-4 rabbits (see text). ¶ F5 vs F7, t11 = 6-0609, 0 005>P>0 001; F6 vs F7, tj1 = 2-0172, 0 1 >P>0-05. ** F5 vs F7, t11 = 5-8708, 0-005>P>0-00l; F6 vs F7, t11 = 10-4790, 0-001 >P.
406
A. R. L. Teixeira and C. A. Santos-Buch blood mononuclear cells derived from rabbits immunized with the F 1 fraction and the lysosome-rich F5 fraction showed the highest degree of inhibition of migration. The degree of migration of the cells derived from rabbits immunized with the lysosome-rich F5 fraction (51 8+ 7-3 per cent) contrasted sharply with the degree of migration of those cells derived from rabbits immunized with the cytosol F7 fraction (33-2+2-0 per cent), when these sensitized blood mononuclear cells were tested with their specific antigens. This would suggest that the particulate component of the lysosome-rich F5 fraction was involved in eliciting a cell-mediated immune response. Further evidence in support of this suggestion was provided by the reactivity of the blood mononuclear cells of chronically infected rabbit E when exposed to subcellular antigens F5, F6, and F7 (Table 2). This experiment showed that the degree of inhibition elicited by the antigens present in the lysosome-rich F5 fraction and also the antigens present in the ribosome-rich F6 fraction (Fig. 1) was marked, as compared to the degree
FIG. 1. This plate shows the migration of blood mononuclear cells, obtained from a chronically infected rabbit (CIRb-E) in the absence of T. cruzi antigen (top wells), in the presence of lyosome-rich F5 fraction (middle wells) and in the presence of ribosome-rich F6 fraction (bottom wells) derived from homogenates -of T. cruzi. Note the marked inhibition of mononuclear cell migration in the presence of T. cruzi antigens (see text).
of inhibition observed when the cytosol F7 fraction was used. These differences were statistically significant. All these results contrast sharply with those obtained from control rabbits C- 1, C-2 and C-4 where the degree of inhibition obtained ranged from 14 7 + 3 4 per cent to 6-0+3 6 per cent fcr TH, F5 and cytosol. Do sensitized blood lymphocytes have a cytotoxic effect upon trypomastigotes of T. cruzi? Results of experiments that shed light on this question are given in Table 3. Peripheral blood lymphocytes from chronically infected rabbit D, and from a rabbit immunized with the lysosome-rich F5 fraction, which had shown a marked degree of inhibition of monocyte migration on survey tests, were used. Incubation of these sensitized lymphocytes with previously washed trypomastigotes showed that there was no reduction in the number of organisms per medium power field after 72 hours at 370. Normal lymphocytes from control rabbit C-4 also appeared to have no effect on free-swimming trypomastigotes. Cell-mediated immunity was further assayed when the degree of blast transformation
Experimental Chagas' Disease
407
TABLE 3 BLAST
TRANSFORMATION OF SENSITIZED BLOOD LYMPHOCYTES AFTER PRESENCE OF T. cruzi
Rabbit* CIRb-D AlRb-F5 (Group II)
Control RbC-4
72 HOURS INCUBATION AT 370 IN THE
Initial
Final number of
lymphocyte/
trypomastigotes/ MPF
lymphocytes/MPF
Percentage of lymphoblasts
1:1 2:1 3:1 1 :1 2:1 3:1 1:1 2:1 3:1
5 4 6 5 7 4 7 5 6
49 84 190 27 45 93 8 20 33
16 21 24 12 12 13 6 8 6
trypomastigote
Final number of
* CIRb-D = chronically infected rabbit D; AIRb-F5 = actively immunized rabbit, immunized with the lysosome-rich F5 fraction of T. cruzi; control RbC-4 = normal rabbit.
of sensitized blood lymphocytes (Zoschke and Bach, 1970) in the presence of viable forms of T. cruzi, was studied. Different ratios of lymphocytes to previously washed, free-swimming trypomastigotes were used. Results of these experiments are also summarized in Table 3. There was a marked increase in the lymphocyte to trypomastigote ratio, as well as an increase in the percentage of lymphoblasts after 72 hours' incubation at 37°. Blood lymphocytes collected from chronically infected rabbit D showed more blast transformation than blood lymphocytes obtained from the rabbit immunized with the lysosome-rich F5 fraction. The difference in response between these two rabbits is probably related to the continuous stimulus provided by the chronic infection of rabbit D with T. cruzi. The results obtained from these experiments contrast with those obtained when normal, non-sensitized lymphocytes derived from control rabbit C-4 were used. PASSIVE TRANSFER
Passive transfer of cell-mediated immunity to T. cruzi was achieved in two normal rabbits. One rabbit (T-1) received viable blood mononuclear cells derived from chronically infected rabbits (Landsteiner and Chase, 1942). The other animal, rabbit T-2, received disrupted blood mononuclear cells from the same pool of sensitized rabbits (Lawrence et al., 1963) (for details see Materials and Methods section). Care was taken to ensure that there was no demonstrable parasitaemia present when the blood mononuclear cells were collected. Inhibition of mononuclear cell migration in vitro was obtained after six injections of these preparations. The results of the experiment are given in Table 4. Cell-mediated immunity in recipient rabbits T- 1 and T-2 was assessed using the initial homogenate of T. cruzi (TH) and also the soluble cytosol F7 fraction. There was a marked difference in reactivity to the antigens in the migration inhibition test (see Fig. 2). The antigens in the initial homogenate elicited from 48-4 to 400 per cent inhibition of blood mononuclear cell migration, whereas inhibition of migration in the presence of cytosol F7 was not significantly different from inhibition obtained when blood mononuclear cells of control rabbits C-1, C-2 and C-3 were used (Table 4). Skin tests were also performed on rabbits T-1 and T-2, and control rabbit C-3 (Table 4). The antigens in the initial homogenate elicited hyperaemic, immediate-type response B
A. R. L. Teixeira and C. A. Santos-Buch
408
TABLE 4 PASSIVE TRANSFER OF CELL-MEDIATED IMMUNITY TO T. cruzi
Skin response Percentage inhibition of monocyte migration*
T. cruzi antigens TH F7
Delayed-type§
Immediate-type§
T-lt
T-2t
C-1, C-2, C-31
T-l
T-2
C-3
T-l
T-2
C-3
48-4+1-5 18-8+1*7
40 0+3 8 5-0+3-1
14-7+3-4 6 0+3 6
++ +
++ +
Negative Negative
00 0
00 0
Negative Negative
* Percentage inhibition = [100- (the average diameter of mononuclear cell migration exposed to antigen)/(the average diameter of mononuclear cell migration in the absence of antigen)] x 100. The results were recorded after 20 hours. t T-l Rb received viable blood mononuclear cells and T-2 Rb received disrupted blood mononuclear cells, both aliquots from infected rabbits at a time of negative parasitaemia. t Controls: C-1, C-2 and C-3 rabbits (see text). § + = Area of redness of 1-1-5 cm. + + = Area of redness over 2-5 cm. + + = Intermediate values. 0 = Area of induration of 1-1-5 cm. 000 = Area of induration measuring over 2-5 cm. 00 = Intermediate values.
FIG. 2. This plate shows the migration of blood mononuclear cells obtained from a rabbit which was the recipient of T. cruzi-sensitized lymphocytes (see text). Note the normal mononuclear cell migration in the top wells in contrast with slight inhibition of migration in the presence of the protozoan cytosol F7 fraction (middle wells), and marked inhibition of migration in the presence of the initial homogenate (TH) of T. cruzi forms (bottom wells).
in both rabbit T-1 and rabbit T-2, and also an induration after 36 hours. No skin reaction was given by control rabbit C-3. These results support the suggestion that the particles in the initial homogenate rather than the soluble substances in the cytosol (F7 fraction) were principally involved in eliciting cell-mediated immunity in rabbits.
DISCUSSION Rabbits chronically infected with T. cruzi and rabbits immunized with homogenates and subcellular antigens of T. cruzi showed both an immediate-type and also a delayedtype skin response to injections of these subcellular antigens. The particulate subcellular fractions showed a delayed type of skin reaction, principally characterized by induration, which persisted over 72 hours. On the other hand, the soluble cytosol F7 fraction showed an immediate type of skin reaction, principally characterized by a hyperaemic wheal. The initial homogenate elicited both an immediate and a delayed type of skin reaction.
Experimental Chagas' Disease 409 Further support for the suggestion that the particulate fractions derived from homogenates of T. cruzi are principally responsible for calling forth cell-mediated immunity in rabbits, came from the results of inhibition of blood mononuclear cell migration tests. The particulate lysosome-rich F5 fraction and the particulate ribosome-rich F6 fraction of T. cruzi homogenates elicited the highest degree of inhibition. Moreover, sensitized lymphocytes from rabbits immunized with the lysosome-rich fraction transformed to lymphoblasts and multipled in the presence of free-swimming trypomastigotes of T. cruzi. The low protein content of the lysosome-rich F5 fraction and of the ribosome-rich F6 fraction may be responsible for their failure to elicit a strong humoral antibody response in rabbits (Teixeira and Santos-Buch, 1974). Both these fractions were capable, however, of eliciting a marked cell-mediated immune response. Possibly only small quantities of antigenic principle in these particulate subcellular fractions of T. cruzi are necessary to adequately sensitize lymphocytes in rabbits. Which ofthe many membranebound substances in these fractions is responsible for the elicitation of cell-mediated immunity to T. cruzi is not known. Antigens capable of eliciting cell-mediated immunity are also present in the cytosol F7 fraction. The results of the experiments reported here suggest that the cytoplasmic antigens capable of eliciting cell-mediated immunity are extruded by active exocytosis of the T. cruzi organisms, since lymphoblast transformation was induced in the presence of previously washed, free-swimming trypomastigotes. However, intracellular antigens of T. cruzi may also be released by the action of sensitized cells in vivo. Transference of cellmediated immunity was achieved when viable lymphocytes, or the material from ruptured lymphocytes, derived from chronically infected rabbits was injected intramuscularly into normal rabbits. Furthermore, when the lymphocytes derived from the recipient rabbits, obtained after transference of cell-mediated immunity, were tested with both the initial homogenate and the cytosol F7 fraction of T. cruzi, marked inhibition of mononuclear cell migration was detected with the particle-rich homogenate. In contrast, little if any inhibition was elicited with the soluble substances in the cytosol of T. cruzi in the same assay system. In general, the skin response of the rabbits with transferred cellmediated immunity paralleled the results obtained in vitro. Delayed hypersensitivity might be responsible for the myocarditis of chronic Chagas' disease since this lesion is characterized by diffuse lymphocytic infiltrates and myocardial cell destruction in the absence of encysted parasites in situ. (Torres, 1929; Chagas, 1934). In the experiments reported here, the small particle or membrane fractions of T. cruzi elicited strong delayed hypersensitivity reactions in rabbits. We have also demonstrated that sensitized lymphocytes both from rabbits chronically infected with T. cruzi and from rabbits immunized with the particulate F5 fraction of T. cruzi showed cytotoxicity to normal, non-parasitized heart cells (Teixeira and Santos-Buch, 1974).
ACKNOWLEDGMENTS We are grateful to Mr Jose Avello, Mr Emilio Campo, Ms L. Yolanda Ortega and Ms Doris Heitmeyer for their contributions to this investigation. The research was partly supported by U.S. Public Health Grant number 5-ROI-HL12521. Dr Teixeira is a recipient of a Cornell-Bahia Program Fellowship from the Rockefeller Foundation and the Commonwealth Fund.
410
A. R. L. Teixeira and C. A. Santos-Buch REFERENCES
BLOOM, B. R. and BENNET, B. (1966). 'Mechanism of a reaction in vitro associated with delayed hypersensitivity.' Science, 153, 80. BLOOM, B. R. and GLADE, P. R. (1971). In Vitro Methods on Cell-Mediated Immunity, p. 289. Academic Press, New York. BOWUM, A. (1968). 'Isolation of monocytes and granulocytes from human blood.' Scand. J. clin. Lab. Invest., supplement 97. CHAGAS, C. (1934). 'Estado actual da Trypanosomiase Americana.' Rev. Biol. e Hyg., 5, 58. DAVID, J. R., AL-ASKARI, S., LAWRENCE, H. S. and THOMAS, L. (1964). 'Delayed hypersensitivity in vitro. I. The inhibition of cell migration by antigen.' J. Immunol., 93, 264. LANDSTEINER, K. and CHASE, M. W. (1942). 'Experiments on transfer of cutaneous sensitivity to simple compounds.' Proc. Soc. exp. Biol. (N. T.), 49, 688. LAWRENCE, H. S., AL-AsKARI, S., DAVID,J., FRANKLIN. E. C. and ZWEIMAN, B. (1 963). 'Transfer of immunological information in humans with dialysates of leucocyte extracts.' Trans. Ass. Amer. Phycns, 76, 84.
LING, N. R. (1968). Lymphocyte Stimulation. NorthHolland, Amsterdam. Pizzi, T. and RuBio, M. (1955). 'Aspectos celulares de la immunidad en la enfermedad de Chagas.' Bol. chil. Parasit., 10, 4. SEAH, S. (1970). 'Delayed hypersensitivity in T. cruzi infection.' Nature (Lond.), 225, 1256. TALIAFERRO, W. H., and Pizzi, T. (1955). 'Connective tissue reactions in normal and immunized mice to a reticulotropic strain of T. cruzi.' J. infect. Dis., 96, 199. TEIxEIRA, A. R. L. and SANTOS-BUCH, C. A. (1974). 'The immunology of experimental Chagas' disease. I. Preparation of Trypanosoma cruzi antigens and humoral antibody response to these antigens.' J. Immunol., 113, 859. TORRES, C. M. (1929). 'Patogenia de la miocarditis cronica en la enfermedad de Chagas. 5. Reunidn Sociedad Argentina Patologia Regional Norte, 3, 902. ZOSCHKE, D. C. and BACH, F. H. (1970) 'Specificity of antigen recognition by human lymphocytes in vitro.' Science, 170, 1404.
The Immunology of Experimental Chagas' Disease II. DELAYED HYPERSENSITIVITY TO TRYPANOSOMA CRUZI ANTIGENS A. R. L. TEIXEIRA*
AND
C. A. SANTOS-BUCH
Department of Pathology, Cornell University Medical College, New York, New York, U.S.A.
(Received 9th March 1974; acceptedfor publication 6th June 1974)
Summary. Homogenates of suspensions of the trypomastigote and amastigote forms of the Ernestina strain of Trypanosoma cruzi, derived from tissue cultures, yielded two subcellular fractions which elicited strong delayed hypersensitivity reactions in rabbits. The 100,000 g x 90 minute fraction of T. cruzi homogenates gave rise to marked cell-mediated immunity. The 30,000 g x 35 minute fraction of these homogenates was also capable of eliciting a marked cell-mediated immune response. Cell-mediated immunity was assayed by experiments which established passive transfer, inhibition of blood mononuclear cell migration and blast transformation by T. cruzi-sensitized lymphocytes. Sensitized lymphocytes did not have observable effects on trypomastigotes of T. cruzi. The results of the experiments described here strongly suggest that constituents of intracytoplasmic particles of trypomastigotes and amastigotes of T. cruzi are involved in eliciting cell-mediated immunity in rabbits. INTRODUCTION In a previous report (Teixeira and Santos-Buch, 1974), a method for the partition of homogenates of trypomastigote and amastigote forms of Trypanosome cruzi into subcellular fractions was described. The soluble substances present in the cytosol fraction of T. cruzi homogenates appeared to possess antigenic properties principally related to the humoral antibody response in rabbits (Teixeira and Santos-Buch, 1974). Cell-mediated immune responses in Chagas' disease have not been fully explored (Seah, 1970). Studies have been based on morphological lesions formed in various tissues, which followed the systemic injection of viable T. cruzi forms (Pizzi and Rubio, 1955; Taliaferro and Pizzi, 1955). The findings reported in this paper indicate that cytoplasmic particles, derived from homogenates of T. cruzi elicited delayed hypersensitivity in rabbits. These particulate fractions also reacted strongly with lymphocytes derived from rabbits chronically infected with T. cruzi. Further, passive transfer of cellular immunity to normal rabbits was accomplished with lymphocytes collected from the peripheral blood of these chronically infected rabbits. * Present address: Departamento de Patologia, Faculdade de Ciencias da Saude da Universidade de Brasilia, 70000Brasilia-D.F., Brasil. Correspondence: Dr C. A. Santos-Buch, Department of Pathology, New York Hospital, Cornell Medical Center, 525 East 68th Street, New York 10021, U.S.A.
401
402
A. R. L. Teixeira and C. A. Santos-Buch
MATERIALS AND METHODS Strain of T. cruzi grown in tissue culture The Ernestina strain of T. cruzi utilized in these experiments was maintained in foetal rabbit heart and skeletal muscle primary cell line cultures as described previously by Teixeira and Santos-Buch (1974).
Partition of T. cruzi forms into subcellular fractions Trypomastigotes and amastigotes of T. cruzi harvested from rabbit heart and skeletal muscle cultures were isolated and homogenized as described by Teixeira and SantosBuch (1974). The initial homogenates of parasites were partitioned into seven subcellular fractions (Fl to F7) (Teixeira and Santos-Buch, 1974). The speed of centrifugation for the different fractions are shown in Table 2. Previous partial characterization had shown that the F5, 30,000 g x 35 minute, fraction was rich in lysosomal bodies, whereas the F6, 100,000 g x 90 minute, fraction was rich in ribosomes. The supernatant of the F6 pellet, the cytosol F7 fraction, contained about 50 per cent of the protein of the initial homogenate (Teixeira and Santos-Buch, 1974). Immunization of rabbits with homogenates and subcellular fractions of T. cruzi Two groups of eight, 6- to 12-month-old New Zealand white rabbits were each immunized with T. cruzi homogenates (TH) and subcellular antigens derived from differential centrifugation. The initial dose consisted of 0-1 ml of antigen injected intravenously and this was followed by two consecutive, weekly, subcutaneous injections of 1 ml of the antigen homogenized in 1 ml of complete and incomplete Freund's adjuvants, respectively. Subsequent courses of 0-2 ml of the antigen suspensions intravenously were spaced 1 week apart. Each rabbit of the first group (group I) received injections of antigen in the following concentrations: TH, 220 ug/ml; F1, 460 pg/ml; F2, 280 jug/ml; F3, 240 ug/ml; F4, 180 ig/ml; F5, 40 ug/ml; F6, 90 ug/ml; F7, 120 ug/ml. Each rabbit of the second group (group II) received higher dosage of antigen with the following concentrations: TH, 320 pg/ml; Fl, 780 ug/ml; F2, 670 ug/ml; F3, 460 ug/ml; F4, 250 ug/ml; F5, 70 pg/ml; F6, 115 ug/ml; and F7, 180 pg/ml. Controls consisted of rabbit C-1, immunized with homogenates of cultured allogeneic heart cells; rabbit C-2, immunized with culture medium with 5 per cent (v/v) horse serum, and normal rabbit C-3 and C-4 (Teixeira and Santos-Buch, 1974).
Infection of rabbits with T. cruzi Eight, 1-month-old, New Zealand white rabbits were injected intraperitoneally with trypomastigotes of T. cruzi derived from tissue culture. The inoculum was calculated by multiplying the body weight in grams x I05 trypomastigotes. Wet preparations showed parasites in the peripheral blood between days 21 and 30 following inoculation. NNN media inoculated with 0-5 to 1 ml of peripheral blood from these rabbits were positive by day 15 and they became repeatedly negative after the 3rd month following inoculation. Skin response Each rabbit immunized with its subcellular fraction of T. cruzi was skin tested with specific antigen 1 week after the 5th and 1 week after the 10th immunizing dose. The chronically infected rabbits were skin tested with each fraction during the period of
403 Experimental Chagas' Disease demonstrable negative blood cultures. 01 ml of antigen was injected intradermally. The protein concentration of antigens was the same used for immunization. Control injections consisted of 0 1 ml of the solution used for suspension of the antigens. Readings were taken after 6, 12, 24, 48, and 72 hours. A positive, immediate type of reaction was considered an area of redness measuring at least 1 cm in diameter which developed within 24 hours, and the degree of reaction was assayed as follows: one plus (+) was equivalent to an area of redness of 1-1 5 cm, and three plus ( + + + ) was an area of redness of over 2*5 cm. An intermediate reading was considered as (+ +). A delayed-type reaction was considered an area of induration persisting for 72 hours after antigen injection, and a similar scale of one zero (0) to three zeros (000) was used to assay the degree of induration.
Collection of blood mononuclear cells Seven to 10 days after the last injection of antigen, the rabbits were bled from a marginal ear vein. The first collection of blood was obtained after the twelfth immunizing dosage, and later collections were made at other times, following further courses of antigen
injection. Peripheral mononuclear cells were isolated from the blood of eight rabbits with T. cruzi infection in a similar fashion, after negative blood culture tests had been obtained.
Assay of inhibition of monocyte migration A modification of the capillary tube method for studying the inhibition of migration of blood monocytes was used. This method was reviewed recently by Bloom and Glade (1971). Monocytes and lymphocytes were isolated from 35-40 ml of fresh heparinized rabbit venous blood (sodium heparin, Upjohn, 10 i.u./ml) by the Ficoll-Hypaque (Pharmacia, Uppsala, Sweden; Winthrop Laboratories, New York) density gradient centrifugation technique of Boyum (1968). For convenience, the Hypaque concentration was used at full strength (50 per cent w/v) before it was mixed with Ficoll at 9 per cent (w/v) in proportions of 2*4 volumes of Ficoll: 1 volume of Hypaque. The blood was layered on top of the Ficoll-Hypaque mixture (3 parts of blood: 2 parts Ficoll-Hypaque), and centrifuged at 1000 g for 50 minutes at 200. The layer of mononuclear cells at the interface was collected with a sterile Pasteur pipette. This modification of the method gave good yields of mononuclear cells. The suspension of cells revealed an average differential count of 87-90 per cent lymphocytes and 10-13 per cent monocytes. More than 90 per cent of the cells were viable as judged by trypan blue exclusion. The mononuclear cells were washed once with phosphate-buffered saline+0 003 M ethylenediaminotetraacetate (PBS-EDTA) and three times in Hanks's buffered saline solution (HBSS). The lymphocyte and monocyte pellet was then suspended in 50 p1 Hanks's minimal essential medium (MEM) (Flow Laboratories, Maryland, U.S.A.) to a concentration of approximately 60 x 106 cells/ml and were sucked into capillary tubes attached to a rubber nipple of an Eppendorff microlitre pipette (Sargent-Welch Scientific, Springfield, New Jersey). The capillary tips were anchored in a drop of sterile silicone high vacuum grease (DowCorning, Midland, Michigan) in the wells of a microchamber which were sealed with glass coverslips. The culture medium used in the well was MEM with 10 per cent horse serum (HS). All the antigens were made up to a concentration of 150 jg/ml, except for F5 and F6 which were made up to 100 ug/ml concentration. All tests were run in triplicate. The largest diameters of the migration areas were measured, after 20 hours incubation at
A. R. L. Teixeira and C. A. Santos-Buch 370, with a magnifying comparator with a scale in mm (ACE Scientific Supply, Linden, New Jersey). The migration in the presence and absence of antigen was then compared.
404
Interaction between T. cruzi and peripheral blood lymphocytes in vitro Fresh mononuclear cells were obtained from rabbit peripheral blood as described above. After the first washing in PBS-EDTA and three more washes in HBSS, the cells were resuspended in MEM with 10 per cent (v/v) horse serum and transferred into a culture plastic vessel in order to allow monocytes to stick onto its surface. The supernatant population of lymphocytes was collected after 4 hours incubation at 36-370. These lymphocytes were transferred into sterile 5-ml plastic tubes and adjusted to concentrations of 1, 2, and 3 x 105 cells/ml in MEM. Trypomastigotes (1 x 105 parasites/ml) were added to the tubes to make 1: 1, 1: 2, and 1: 3 parasite to lymphocyte ratios. One millilitre of each of the lymphocyte/living parasite mixtures was transferred to wells of a microchamber and sealed with coverslips. The tubes and the microchambers were incubated at 36-370. The wells of the microchamber were inspected periodically with a microscope. At the end of 72 hours incubation, the tubes were centrifuged, and the cells were washed with PBS and resuspended to the original volume. A sample of the suspension was smeared on a slide and stained by the Giemsa-May-Grunwald method. Lymphocytes were quantitated by counting the number of cells in three medium power fields of the smear and blast transformation was determined by standard morphological criteria (Ling, 1968).
Transfer of cell-mediated immunity from chronically infected rabbits to normal rabbits Mononuclear cells were isolated from T. cruzi-infected rabbits with negative blood cultures. Two male, 8-10-month-old, New Zealand white rabbits were used as recipients. The first rabbit received six intramuscular injections of approximately 3 x 107 intact lymphocytes (Landsteiner and Chase, 1942). The second rabbit received six intramuscular injections of the homogenate derived from equal numbers of disrupted cells following repeated freezing and thawing (Lawrence, Al-Askari, David, Franklin and Zweiman, 1963). RESULTS SKIN RESPONSE TO ANTIGENS OF
T. cruzi
Skin reactivity of the actively immunized and chronically infected rabbits infected with the antigens derived from the initial homogenate of T. cruzi was tested. The results of the experiment are given in Table 1. Both immediate- and delayed-type skin reactions occurred in the specifically immunized rabbits after the intradermal injection of homologous antigen. The initial homogenate contained antigens capable of eliciting a strong immediate-type reaction and also marked delayed-type skin response. The cytosol F7 fraction elicited an immediate- rather than a delayed-type response. In contrast, the particulate antigens gave rise to a greater degree of induration and delayed-type of skin reaction. Also, delayed skin reactivity was more marked in chronically infected rabbits E, F and G when the particulate fractions F4, F5 and F6 were used, than when the cytosol F7 fraction was used.
Experimental Chagas' Disease
405
TABLE 1
SKIN RESPONSE
Chronically infected rabbits
Actively immunized rabbits*
T. cruzi antigen TH Fl F2 F3 F4 F5 F6 F7
Group II
Group I Immediate
Immediate
typet
Delayed type:
+++ ++ ++ + + + + +++
000 00 00 0 0 0 0 0
+++ +++ ++ ++ ++ + + +++
Immediate
Delayed
typet
Rabbit
typet
Delayed type:
A B C D E F G H
+++ ++ ++ ++ + + + +++
000 00 00 0 00 00 00 0
typel 000 000 00 00 00 00 0 0
* Each specific T. cruzi antigen was injected into a correspondingly immunized rabbit intradermally. Immediate-type reaction was recorded after 24 hours and delayed-type reaction was recorded after 48 hours. t Development of hyperaemic wheal within 24 hours of injection. + = Area of redness of 1-1 5 cm. + + + = Area of redness of over 2-5 cm. + + = Intermediate values. $ Induration of skin persisting for 72 hours of injection. 0 = Area of induration of 1-1 5 cm. 000 = Induration measuring over 2-5 cm. 00 = Intermediate values. CELL-MEDIATED IMMUNITY
Inhibition of blood mononuclear cell migration (David, Al-Askari, Lawrence and Thomas, 1964; Bloom and Bennet, 1966) was used to assess cell-mediated immunity in rabbits actively immunized with subcellular fractions of T. cruzi. This test was also used to determine the responsiveness of blood mononuclear cells derived from a chronically infected rabbit (Rabbit E). The results of these experiments are given in Table 2. The TABLE 2
CELL-MEDIATED
MIGRATION* (BLOOD MONUCLEAR CELLS
IMMUNE RESPONSE: INHIBITION OF MONOCYTE
vs
T. cruzi
ANTIGENS)
T. cruzi antigen used in testt
Actively immunized rabbits of groups I and 11:
Chronically infected
T. cruzi antigen
Control rabbits C-1, C-2 and C-4§
TH (initial homogenate) F 1 (350 g x 10 minutes) F2 (700 g x 10 minutes) F3 (5000 g x 10 minutes) F4 (15,000 gx25 minutes) F5 (30,000 gx35 minutes) F6 (100,000 g x 90 minutes) F7 (cytosol)
47-8+ 9-8 52 8+ 6-9 48-2 +4-7 36-5+8-1 415+5 3 518+ 7-311 42-4+ 9 1l¶
56-7 + 9 4 46-3+ 16 53-7+ 1P6 49-1 +4-3 56-4+5-9 90-6+ 12-1** 76-9 + 3.9** 48-4+2-3**
TH
14 7+ 3*4
F5
9 7+3 1
F7
6-0+3-6
33-2+2±0¶
rabbit E
* Percentage inhibition = [100- (the average diameter of mononuclear cell migration exposed to antigen)/(the average diameter of mononuclear cell migration in the absence of antigen)] x 100. The results were recorded after 20 hours. t Blood mononuclear cells were exposed to 150 yg/ml of antigens TH, Fl, F2, F3, F4 and F7. For F5 and F6, 100 pg/ml were used. T The antigen used in the test was the antigen used to immunize the rabbit. % Controls = C-1, C-2 and C-4 rabbits (see text). ¶ F5 vs F7, t11 = 6-0609, 0 005>P>0 001; F6 vs F7, tj1 = 2-0172, 0 1 >P>0-05. ** F5 vs F7, t11 = 5-8708, 0-005>P>0-00l; F6 vs F7, t11 = 10-4790, 0-001 >P.
406
A. R. L. Teixeira and C. A. Santos-Buch blood mononuclear cells derived from rabbits immunized with the F 1 fraction and the lysosome-rich F5 fraction showed the highest degree of inhibition of migration. The degree of migration of the cells derived from rabbits immunized with the lysosome-rich F5 fraction (51 8+ 7-3 per cent) contrasted sharply with the degree of migration of those cells derived from rabbits immunized with the cytosol F7 fraction (33-2+2-0 per cent), when these sensitized blood mononuclear cells were tested with their specific antigens. This would suggest that the particulate component of the lysosome-rich F5 fraction was involved in eliciting a cell-mediated immune response. Further evidence in support of this suggestion was provided by the reactivity of the blood mononuclear cells of chronically infected rabbit E when exposed to subcellular antigens F5, F6, and F7 (Table 2). This experiment showed that the degree of inhibition elicited by the antigens present in the lysosome-rich F5 fraction and also the antigens present in the ribosome-rich F6 fraction (Fig. 1) was marked, as compared to the degree
FIG. 1. This plate shows the migration of blood mononuclear cells, obtained from a chronically infected rabbit (CIRb-E) in the absence of T. cruzi antigen (top wells), in the presence of lyosome-rich F5 fraction (middle wells) and in the presence of ribosome-rich F6 fraction (bottom wells) derived from homogenates -of T. cruzi. Note the marked inhibition of mononuclear cell migration in the presence of T. cruzi antigens (see text).
of inhibition observed when the cytosol F7 fraction was used. These differences were statistically significant. All these results contrast sharply with those obtained from control rabbits C- 1, C-2 and C-4 where the degree of inhibition obtained ranged from 14 7 + 3 4 per cent to 6-0+3 6 per cent fcr TH, F5 and cytosol. Do sensitized blood lymphocytes have a cytotoxic effect upon trypomastigotes of T. cruzi? Results of experiments that shed light on this question are given in Table 3. Peripheral blood lymphocytes from chronically infected rabbit D, and from a rabbit immunized with the lysosome-rich F5 fraction, which had shown a marked degree of inhibition of monocyte migration on survey tests, were used. Incubation of these sensitized lymphocytes with previously washed trypomastigotes showed that there was no reduction in the number of organisms per medium power field after 72 hours at 370. Normal lymphocytes from control rabbit C-4 also appeared to have no effect on free-swimming trypomastigotes. Cell-mediated immunity was further assayed when the degree of blast transformation
Experimental Chagas' Disease
407
TABLE 3 BLAST
TRANSFORMATION OF SENSITIZED BLOOD LYMPHOCYTES AFTER PRESENCE OF T. cruzi
Rabbit* CIRb-D AlRb-F5 (Group II)
Control RbC-4
72 HOURS INCUBATION AT 370 IN THE
Initial
Final number of
lymphocyte/
trypomastigotes/ MPF
lymphocytes/MPF
Percentage of lymphoblasts
1:1 2:1 3:1 1 :1 2:1 3:1 1:1 2:1 3:1
5 4 6 5 7 4 7 5 6
49 84 190 27 45 93 8 20 33
16 21 24 12 12 13 6 8 6
trypomastigote
Final number of
* CIRb-D = chronically infected rabbit D; AIRb-F5 = actively immunized rabbit, immunized with the lysosome-rich F5 fraction of T. cruzi; control RbC-4 = normal rabbit.
of sensitized blood lymphocytes (Zoschke and Bach, 1970) in the presence of viable forms of T. cruzi, was studied. Different ratios of lymphocytes to previously washed, free-swimming trypomastigotes were used. Results of these experiments are also summarized in Table 3. There was a marked increase in the lymphocyte to trypomastigote ratio, as well as an increase in the percentage of lymphoblasts after 72 hours' incubation at 37°. Blood lymphocytes collected from chronically infected rabbit D showed more blast transformation than blood lymphocytes obtained from the rabbit immunized with the lysosome-rich F5 fraction. The difference in response between these two rabbits is probably related to the continuous stimulus provided by the chronic infection of rabbit D with T. cruzi. The results obtained from these experiments contrast with those obtained when normal, non-sensitized lymphocytes derived from control rabbit C-4 were used. PASSIVE TRANSFER
Passive transfer of cell-mediated immunity to T. cruzi was achieved in two normal rabbits. One rabbit (T-1) received viable blood mononuclear cells derived from chronically infected rabbits (Landsteiner and Chase, 1942). The other animal, rabbit T-2, received disrupted blood mononuclear cells from the same pool of sensitized rabbits (Lawrence et al., 1963) (for details see Materials and Methods section). Care was taken to ensure that there was no demonstrable parasitaemia present when the blood mononuclear cells were collected. Inhibition of mononuclear cell migration in vitro was obtained after six injections of these preparations. The results of the experiment are given in Table 4. Cell-mediated immunity in recipient rabbits T- 1 and T-2 was assessed using the initial homogenate of T. cruzi (TH) and also the soluble cytosol F7 fraction. There was a marked difference in reactivity to the antigens in the migration inhibition test (see Fig. 2). The antigens in the initial homogenate elicited from 48-4 to 400 per cent inhibition of blood mononuclear cell migration, whereas inhibition of migration in the presence of cytosol F7 was not significantly different from inhibition obtained when blood mononuclear cells of control rabbits C-1, C-2 and C-3 were used (Table 4). Skin tests were also performed on rabbits T-1 and T-2, and control rabbit C-3 (Table 4). The antigens in the initial homogenate elicited hyperaemic, immediate-type response B
A. R. L. Teixeira and C. A. Santos-Buch
408
TABLE 4 PASSIVE TRANSFER OF CELL-MEDIATED IMMUNITY TO T. cruzi
Skin response Percentage inhibition of monocyte migration*
T. cruzi antigens TH F7
Delayed-type§
Immediate-type§
T-lt
T-2t
C-1, C-2, C-31
T-l
T-2
C-3
T-l
T-2
C-3
48-4+1-5 18-8+1*7
40 0+3 8 5-0+3-1
14-7+3-4 6 0+3 6
++ +
++ +
Negative Negative
00 0
00 0
Negative Negative
* Percentage inhibition = [100- (the average diameter of mononuclear cell migration exposed to antigen)/(the average diameter of mononuclear cell migration in the absence of antigen)] x 100. The results were recorded after 20 hours. t T-l Rb received viable blood mononuclear cells and T-2 Rb received disrupted blood mononuclear cells, both aliquots from infected rabbits at a time of negative parasitaemia. t Controls: C-1, C-2 and C-3 rabbits (see text). § + = Area of redness of 1-1-5 cm. + + = Area of redness over 2-5 cm. + + = Intermediate values. 0 = Area of induration of 1-1-5 cm. 000 = Area of induration measuring over 2-5 cm. 00 = Intermediate values.
FIG. 2. This plate shows the migration of blood mononuclear cells obtained from a rabbit which was the recipient of T. cruzi-sensitized lymphocytes (see text). Note the normal mononuclear cell migration in the top wells in contrast with slight inhibition of migration in the presence of the protozoan cytosol F7 fraction (middle wells), and marked inhibition of migration in the presence of the initial homogenate (TH) of T. cruzi forms (bottom wells).
in both rabbit T-1 and rabbit T-2, and also an induration after 36 hours. No skin reaction was given by control rabbit C-3. These results support the suggestion that the particles in the initial homogenate rather than the soluble substances in the cytosol (F7 fraction) were principally involved in eliciting cell-mediated immunity in rabbits.
DISCUSSION Rabbits chronically infected with T. cruzi and rabbits immunized with homogenates and subcellular antigens of T. cruzi showed both an immediate-type and also a delayedtype skin response to injections of these subcellular antigens. The particulate subcellular fractions showed a delayed type of skin reaction, principally characterized by induration, which persisted over 72 hours. On the other hand, the soluble cytosol F7 fraction showed an immediate type of skin reaction, principally characterized by a hyperaemic wheal. The initial homogenate elicited both an immediate and a delayed type of skin reaction.
Experimental Chagas' Disease 409 Further support for the suggestion that the particulate fractions derived from homogenates of T. cruzi are principally responsible for calling forth cell-mediated immunity in rabbits, came from the results of inhibition of blood mononuclear cell migration tests. The particulate lysosome-rich F5 fraction and the particulate ribosome-rich F6 fraction of T. cruzi homogenates elicited the highest degree of inhibition. Moreover, sensitized lymphocytes from rabbits immunized with the lysosome-rich fraction transformed to lymphoblasts and multipled in the presence of free-swimming trypomastigotes of T. cruzi. The low protein content of the lysosome-rich F5 fraction and of the ribosome-rich F6 fraction may be responsible for their failure to elicit a strong humoral antibody response in rabbits (Teixeira and Santos-Buch, 1974). Both these fractions were capable, however, of eliciting a marked cell-mediated immune response. Possibly only small quantities of antigenic principle in these particulate subcellular fractions of T. cruzi are necessary to adequately sensitize lymphocytes in rabbits. Which ofthe many membranebound substances in these fractions is responsible for the elicitation of cell-mediated immunity to T. cruzi is not known. Antigens capable of eliciting cell-mediated immunity are also present in the cytosol F7 fraction. The results of the experiments reported here suggest that the cytoplasmic antigens capable of eliciting cell-mediated immunity are extruded by active exocytosis of the T. cruzi organisms, since lymphoblast transformation was induced in the presence of previously washed, free-swimming trypomastigotes. However, intracellular antigens of T. cruzi may also be released by the action of sensitized cells in vivo. Transference of cellmediated immunity was achieved when viable lymphocytes, or the material from ruptured lymphocytes, derived from chronically infected rabbits was injected intramuscularly into normal rabbits. Furthermore, when the lymphocytes derived from the recipient rabbits, obtained after transference of cell-mediated immunity, were tested with both the initial homogenate and the cytosol F7 fraction of T. cruzi, marked inhibition of mononuclear cell migration was detected with the particle-rich homogenate. In contrast, little if any inhibition was elicited with the soluble substances in the cytosol of T. cruzi in the same assay system. In general, the skin response of the rabbits with transferred cellmediated immunity paralleled the results obtained in vitro. Delayed hypersensitivity might be responsible for the myocarditis of chronic Chagas' disease since this lesion is characterized by diffuse lymphocytic infiltrates and myocardial cell destruction in the absence of encysted parasites in situ. (Torres, 1929; Chagas, 1934). In the experiments reported here, the small particle or membrane fractions of T. cruzi elicited strong delayed hypersensitivity reactions in rabbits. We have also demonstrated that sensitized lymphocytes both from rabbits chronically infected with T. cruzi and from rabbits immunized with the particulate F5 fraction of T. cruzi showed cytotoxicity to normal, non-parasitized heart cells (Teixeira and Santos-Buch, 1974).
ACKNOWLEDGMENTS We are grateful to Mr Jose Avello, Mr Emilio Campo, Ms L. Yolanda Ortega and Ms Doris Heitmeyer for their contributions to this investigation. The research was partly supported by U.S. Public Health Grant number 5-ROI-HL12521. Dr Teixeira is a recipient of a Cornell-Bahia Program Fellowship from the Rockefeller Foundation and the Commonwealth Fund.
410
A. R. L. Teixeira and C. A. Santos-Buch REFERENCES
BLOOM, B. R. and BENNET, B. (1966). 'Mechanism of a reaction in vitro associated with delayed hypersensitivity.' Science, 153, 80. BLOOM, B. R. and GLADE, P. R. (1971). In Vitro Methods on Cell-Mediated Immunity, p. 289. Academic Press, New York. BOWUM, A. (1968). 'Isolation of monocytes and granulocytes from human blood.' Scand. J. clin. Lab. Invest., supplement 97. CHAGAS, C. (1934). 'Estado actual da Trypanosomiase Americana.' Rev. Biol. e Hyg., 5, 58. DAVID, J. R., AL-ASKARI, S., LAWRENCE, H. S. and THOMAS, L. (1964). 'Delayed hypersensitivity in vitro. I. The inhibition of cell migration by antigen.' J. Immunol., 93, 264. LANDSTEINER, K. and CHASE, M. W. (1942). 'Experiments on transfer of cutaneous sensitivity to simple compounds.' Proc. Soc. exp. Biol. (N. T.), 49, 688. LAWRENCE, H. S., AL-AsKARI, S., DAVID,J., FRANKLIN. E. C. and ZWEIMAN, B. (1 963). 'Transfer of immunological information in humans with dialysates of leucocyte extracts.' Trans. Ass. Amer. Phycns, 76, 84.
LING, N. R. (1968). Lymphocyte Stimulation. NorthHolland, Amsterdam. Pizzi, T. and RuBio, M. (1955). 'Aspectos celulares de la immunidad en la enfermedad de Chagas.' Bol. chil. Parasit., 10, 4. SEAH, S. (1970). 'Delayed hypersensitivity in T. cruzi infection.' Nature (Lond.), 225, 1256. TALIAFERRO, W. H., and Pizzi, T. (1955). 'Connective tissue reactions in normal and immunized mice to a reticulotropic strain of T. cruzi.' J. infect. Dis., 96, 199. TEIxEIRA, A. R. L. and SANTOS-BUCH, C. A. (1974). 'The immunology of experimental Chagas' disease. I. Preparation of Trypanosoma cruzi antigens and humoral antibody response to these antigens.' J. Immunol., 113, 859. TORRES, C. M. (1929). 'Patogenia de la miocarditis cronica en la enfermedad de Chagas. 5. Reunidn Sociedad Argentina Patologia Regional Norte, 3, 902. ZOSCHKE, D. C. and BACH, F. H. (1970) 'Specificity of antigen recognition by human lymphocytes in vitro.' Science, 170, 1404.
