The lysis of Trypanosoma cruzi epimastigotes by eosinophils and neutrophils

THE LYSIS OF TRYFANOSOMA CRUZZ EPIMASTIGOTES BY EOSINOPHILS AND NEUTROPHILS ANGEL F. LOPEZ,* MARLENEM. BUNN MORENO and COLIN J. SANDERSON* Institute de Biofisica and Institute de Microbiologica, Centro de Ci&ncias da Satide, Universidade Federal de Rio de Janeiro, Cidade Universitaria ZC-32, Rio de Janeiro 20,000, Brasil (Received 26 Jammry 1978) Abstract-Lcimxz A. F., BUNN MORENOM. M. and SANDERSON C. J. 1978. The lysis of Trypatwsoma cruziepimastigotes by wsinophils and neutrophils. International Journalfor Parasitology 8: 485-489. Antibody-dependent cell-mediated cytotoxicity of T. cruziepimastigotes has been studied by means of an isotopic assay for parasite lysis, in which the &ease of labelled RNA is measured. It is shown that rat wsinophils and neutrophils have approximately equal activity against the parasite in the presence of antibody. Antibody enhances the activity of neutrophils about S-fold, whereas eosinophils have no detectable activity in the absence of antibody. Attention is drawn to the tendency of eosinophils to be inactivated by in vitro manipulation, especially adherence techniques used for removing macrophages and neutrophils. INDEX KEY WORDS: Trypanosomn cruzi; cytotoxicity;

INTRODUCTION THE

IMMUNOLOGICALeffector

mechanisms

that

eosinophih

were capable

neutrophils;

T, cruzi. This report extends show that purified eosinophils

which can

play a role in the pathogenesis of diseases caused by protozoa ar4 largely unknown. This is particularly true of American T~panosomiasis (Chagas’ Disease) caused by Trypanosoma cruzi, where not only is little known about effector mechanisms, but also many results appear contradictory. For example, some authors have described intracellular growth of T. cruzi in macrophages (Behbehani, 1971; Nogueira & Cohn, 1976) while others, its destruction (Dvorak & Schmunis, 1972). The development of an isotopic assay for the lysis of T. cruzi in which the release of labelled RNA is measured, has provided the basis for a study of cell-mediated reactions in vitro (Sanderson, Bunn Moreno & Lbpez, 1978). These studies demonstrated antibody-de~ndent cell-mediated cytotoxocity by rat spleen cells, confirming independent studies with mouse spleen based on morphological criteria of parasite death (Abrahamsohn & Dias de Silva, 1977). It was shown that this activity in rat spleen did not reside in fractions enriched for lymphoid K cells, but appeared in high density fractions rich in granulocytes (Sanderson, Lttpez Bc Bunn Moreno, 1977). These preliminary experiments indicated

eosinophils;

of killing

*Permanent address and address for communications: Division of Surgical Sciences, Clinical Research Centre, Watford Rd., Harrow, Middlesex HA1 3UJ, U.K. 485

RNA release.

these studies and we and neutrophils have

high activity. ~TERlALS

AND METHODS

Cytotoxicity against T. cruzi was carried out by assaying the release of SH-uridine incorporated into RNA as previously described (Sanderson et al., 1977; Sanderson et al., in press). Briefly, the parasites (Y strain) were grown in modified Warren’s medium containing 10 @i/ml of SH-uridine, after 5-7 days the parasites were washed to remove uninco~~t~ isotope. Assays were carried out in small plastic tubes containing 105 parasites and different ratios of effector cells in RPM1 1640 containing 5% foetal calf serum and 10 mhi-HEPES buffer. After 4 h incubation at 37’C the tubes were centrifuged and an aliquot of the supernatant removed and suspended in water miscible scintillation fluid. The percentage isotope &ease was calculated in relation to the total number of counts in 1Ogparasites. The antiserum was pooled from four rats 14 days after injecting lOa blood forms of T. cruzi and was added before the incubation period at a dilution of 1 : 100. Effector cells were obtained by peritoneal lavage from AM-2 rats (kindly supplied by Professor S. Torres, Institute Biomedico, Universidade Federal Fhuninense, Niteroi). Normal rats served as a source of eosinoph~s and neutrophils were induced by injecting 5 ml of 3.5% dextran (MW 4-40 x 103 15 h before. Carbon&iron (approx iO0 mg) was added-to 5 ml of cell suspensi& and incubated for 15 min at 37°C. The non-adherent cells were recovered with the aid of a magnet (Sanderson, Clark & Taylor, 1975). Nylon wool was used as described by Julius, Simpson & Herzenberg, (1973), except

ANGEL F. LOPEZ, MARLENEM.

486

BUNN MOREN~

that columns were incubated for 15 min at 37°C. FicollHypaque was used as described by Bbyum (1968). The ratio of effector cells is based on the total cells determined in a Coulter counter. Differential counts were made after staining smears with MayyCrundwel Giemsa. Viability was assessed by trypan blue exclusion. Only data from experiments with 90% viability or higher are included. Results were analysed statistically as described (Sanderson et al., 1978) and only values significant at the 5% level are plotted, or calculated as specific release. Per cent isotope release was calculated from the supernatant counts (Sanderson et al., 1977). Per cent specific release was calculated from % isotope release : test value - control value % specific release = x 100. maximum - control value Where ‘test value’ = % release in the presence of antibody, ‘control value’ = ‘A release in the absence of antibody or absence of effector cells, ‘maximum’ = 100, as all the label is released when the parasites are totally lysed (Sanderson et. al., 1978).

and

COLIN

J.

SANDEKSON

I.J.P. \O,.. 8. 197s

lower cytotoxic activity than eosinophils, but as is discussed below, the lower activity in this experiment is more likely due to inhibition by the activated macrophages. The macrophages in the normal peritoneal washings appear to have little inhibitory activity. A number of experiments were carried out to purify eosinophils from normal peritoneal exudates. making use of the fact that they are relatively nonadherent cells compared to macrophages and neutrophils. Although carbonyl iron treatment and passage through nylon wool gave consistently good preparations of viable eosinophils, the cytotoxic activity of these cells varied considerably. Figure 2 P

L

N

F

P.1

0

9

. 0 0

0 n 0

12 2 1 7

15 4 1 3

62 94 98 90

7 r 0 il

25 -

RESULTS Normal AM-2 rats have relatively large numbers of resident eosinophils in the peritoneal cavity. Injecting dextran induces a large infiltration of neutrophils and macrophages without increasing the number of eosinophils. Thus the eosinophils become a small proportion of the exudate.

5- 40

B

4 zo-

0

1

0 0

‘2 10

P

L

N

E

M

17 11

21 4

7 80

52 3

3 2

8 Ratio

6 ItellS

-e

-30

2 ”

-20

z 2 in

- 10

s

*

2 w 15B u. L .s ‘3 lo%

- 50

1

60

10 -

20 -

-

0

0-’ I

I

4

2 Ratio

Icells

I 1 -parasites1

FIG. 2. Lysis of antibody coated T. cruzi by exudates from normal rats (0) and after treatment with carbonyliron ( 0). Cells recovered from the pellet of FkollHypaque and passed through nylon wool, non-adhered cells (M) and adherent cells (a). Differential count as in Table 1.

4

: ParaSiteS)

FIG.

1. Lysis of antibody coated T. cvuzi by unpurified exudates from normal rats (0) and rats injected with dextran ( 0). The arrow shows the central level of isotope release, which represents zero specific release. Differential cell count represented as in Table 1. Figure 1 shows typical data obtained with normal and dextran induced peritoneal exudates. The normal peritoneal washings containing about 50% eosinophils and give high levels of cytotoxicity against T. cruzi. The induced exudate, containing 80% neutrophils and only a few eosinophils, gives approx 4-fold lower isotope release. It was thought at first that this indicated that neutrophils had a

shows one of several experiments where the cytotoxic activity was decreased considerably. Cells passed through nylon wool gave approximately half the cytotoxic activity, although the proportion of eosinophils was increased from 62% to 94%. Cells recovered from the nylon wool, although 98 % eosinophils, had virtually no cytotoxic activity. Also, cells treated with carbonyl-iron and containing 90% eosinophils showed virtually no activity. Table 1 summarises data from seven experiments. It is clear from these results that the granulocytes are responsible for most, and probably all the cytotoxic activity, and there is no suggestion of cell interactions giving enhanced cytotoxicity. There is

I.J.P.

VOL.

Granulocyte lysis of T. cruzi

8. 1978 TABLEI

____-.--

F-“‘-

Experiment Treatment* Cell types? 1 { None 11 12 14 62 2 a 5 6 7

Fe None Pellet Pellet-NW Pellet-Fe

3 17 1 12 1 3 0 4 0 0 3

Pellet Pellet Pellet

2 16 2

3 1 2

0 15 9 4 0 I2 0 0

“/o SpeclfiC isotope release ___.42.7 5&7 20.6 19.0 14.8 14.5 18.5 33.2 21-2 23.4

1 0 2 0 0 17 0

80 62 87 94 97 83 83 82 96

0 1 0

487

neutrophiis and good cytotoxic activity was passed through nylon wool. A small proportion passed through the column giving a preparation of 92% neutrophils plus 6% eosinophils, with enhanced cytotoxic activity. Cells recovered from the column also showed good activity. The difference between these preparations suggests an inhibitory effect of the macrophages. P

L

N

00

2

0

95

3

0

mo

4

6

1 _..--._

87

2

40 -l

E

Cl

*The treatment given to the cells recovered from the peritoneum. Pellet = cell recovered from pellet of Ficoll-Hypaque. = carbonyl-iron treated. Fe NW = nylon wool passaged, and the non-adherent

recovered. ?Cell types determined by differential count. Figures rounded to nearest whole number, values under 0.5% shown as zero. P = macrophages. L = lymphoid cells. N =: neutrophils. E = eosinophils. M = mast cells. fspecific isotopic release at a ratio of 4 effector cells to

each parasite. Incubation period 4 h at 37°C.

OJ

a

q I

8

I

4

I

2

I

1

Ratio(cells : parasites)

considerable variation between experiments, although the eosinophils showed high viability and normal staining. These results emphasise the difficulties of working with eosinophils, but in contrast neutrophils appear to be more resistant to manipulation. For example (Fig. 3), an exudate with 93%

P

5

0 0

0oJ

L

5 1 0 2 8 ~~10

,

I

4

2 Ratio

fceiis

N

E

93 92 79

1 6 3

&I 0 0 0 1 I

FIG. 4. Lysis of T. cruzi by eosinophils (0, a) from normal rats, and neutrophils (0, 0) from dextran injected rats. Filled symbols show results obtained with antibody and empty symbols show results without antibody. Jn both cases the granulocytes were obtained from the pellet of a Ficoll-Hypaque separation. Figure 4 shows the results of exudates purified by Ficoll-Hypaque, and shows that whereas eosinophil cytotoxicity is entirely antibody dependent, the killing by neutrophils has a significant antibodyindependent component. Dextran induced peritoneal exudate cells were separated on FicoilHypaque, the pellet recovered and the technique repeated. The recovered cells contained 95 % neutrophils plus 3% eosinophils. Cytotoxic activity was 8-fold higher with antibody than in the absence of antibody (a ratio of 1 : 1 with antibody giving slightly more isotope release than a ratio of 8 : 1 in the control). The cells washed from a normal rE.t were centrifuged once through Ficoll-Hypaque and tested with a minimum of manipulation. Cytotoxic activity of this eosinophil enriched preparation in the presence of antibody is similar to that with the neutrophils, but in the absence of antibody isotope release was not significantly different from medium controls (no effector cells).

: parasitesi DISCUSSION

Ro. 3. Lysis of T. cruzi by exudates induced with dextran ( 0) and after passage through nylon wool, non-adherent cells (0) and adherent cells (H). Differential counts

-.. _ as rn iable 1.

In these experiments the activity of different cell preparations Pa3 been compared by diluting the cells to change the ratio to a fixed number of

3XX

Aluc;r L 1’. L(‘)rb/,

MARLENI: M. BUNP. MORF~O and C.OI.IN .I. SA~IIFHSO~

parasites. Although the determination of isotope release is relatively accurate (Sanderson et al., 1978) there are two factors which can influence the measurement of cytotoxic activity: firstly, interf-rence by other cell types, for example macrophages inhibit granulocyte lysis of erythrocytes (Sanderson & Thomas, 1978). Secondly, errors in cell counting and in the preparation of cell dilutions. Thus data might show differences of statistical significance which may not be biologically relevant. For this reason our interpretation has been restricted to relatively large differences in cytotoxic activity.

The results indicate that both eosinophils and neutrophils can cause substantial lysis of T. cruzi epimastigotes at low cell to parasite ratios. The possibility that macrophages might also be cytotoxic cannot be excluded by these experiments. Indeed, Nogmzria & Cohn (1976) demonstrated degeneration of epimastigotes within macrophages. This was independent of antibody. However, it would seem unlikely that macrophages would contribute significantly in the present 4-h assay system, because although macrophages caused damage to erythrocytes after phagocytosis, the cell contents of the erythocytes (measured by Yr release) were not released over a 20-h incubation period (Sanderson & Thomas, 1978). Eosinophils and neutrophils showed essentially similar cytotoxic activity, although eosinophils were the more difficult to work with and showed a variable tendency to be inactivated by in vitro manipulation, particularly cell adherence techniques. Eosinophils were dependent on antibody, with no significant lysis in its absence. Neutrophils, on the other hand, showed a significant antibody independent effect, but cytotoxicity was enhanced approx &fold by antibody. They could be recovered from nylon wool without significant loss of activity. The lytic activity of neutrophils against bacteria (Klebanoff, 1970) and erythrocytes (Simchowitz & Schur, 1976) is well known. On the other hand, less is known about eosinophils, probably in part due to the problems of obtaining normal eosinophils in pure form. In some systems they appear to have less phagocytic and bactericidal activity than neutrophils (Mickenberg, Root & Wolff, 1972; Butterworth, 1978). Eosinophils and not neutrophils are active against schistosomulae (Butterworth, David, Franks, Mahmoud, David, Sturrock & Houba, 1977). In contrast, these two cell types appear to have approximately equal activity against chick erythrocytes (Sanderson & Thomas, 1978) and in the lysis of T. cruzi. These differences may lie in the requirement for different enzymes to bring about the lysis of different targets. The biological role of this antibody dependent granulocyte lysis of T. cruzi remains to be tested. Although the role of humoral antibody in resistance to infection has been demonstrated (Kierszenbaum

lJ.1’. \OI

x. 197x

Rr Howard,

1976), K cells are inactive (Sanderson and macrophages and monocytes appear to have limited ability to destroy the parasites (Kress, Bloom, Wittner, Rowen & Tanowitz, 1975; Williams & Remington, 1977). Thus the possibility that granulocytes may be important in the pathogenesis of the disease deserves further study.

rt

al.,

1977)

A~l\no~~,/ed~en;erlt.s-This work was supported

by grants from the Brazilian National Research Council (CNP,), and the National Fund for Research in Brazil (FINEP). CJS was supported in part by a visiting professorship from the Federal University of Rio de Janeiro.

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I.J.P. VOL.8. 1978

Granulocyte

SANDERSON C. J., LOPEZ A. F. & BUNN MORENOM. M. 1977. Eosinophils and not lymphoid K cells kill Tryanosoma cruzi epimastigotes. Nature 268: 340-341. SANDERSON C. J., BUNN MORENOM. M. & LOPEZ A. F. 1978. Antibody-dependent cell-mediated cytotoxity of Trypanosoma cruzi: the release of tritium labelled RNA, DNA and protein. Parasito’ogy 76: 299-307. SANDERSON C. J & THOMASJ. A. 19X. A comparison of the cytotoxic activity of eosinophils and other cells by

lysis of T. cruzi Tr

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489

and time lapse microcinematography. 34: 771-780. SIMCHOWITZL. & SCHUR P. H. 1976. Lectin dependent neutrophil-mediated cytotoxicity-1. Characteristics. Immunology 31: 303-31 I. WILLIAMSD. M. & REMINGTONJ. S. 1977. Effect of human monocytes and macrophages on Trypanosoma cruzi. Immunology 32 : 19-23. Immunology