Trypanosoma lewisi: Influence of sera and peritoneal exudate cells

EXPERIMENTAL

P.4RhSITOLoGY

Trypanosoma

31, 370-377 (1972)

lewisi:

Influence of Sera Exudate Cells

and

Peritoneal

Curtis L. Patton’ Department

of Microbiology (Submitted

and Public Health, Michigan State University, East Lansing, Michigan

for publication

6 October, 1971)

PATTON, CURTIS L. 1972. Trypanosoma lewisi: Influence of sera and peritoneal exudate cells. Experimental Parasitology 31, 376-377. The administration of dexamethasane’ to rats depresses their ability to control infections with T. lewisi. Though sera from rats hyperimmune to T. lewisi protect normal rats, dexamethasone-treated rats are protected from trypanosomiasis only if they are given serum from hyperimmune rats and peritoneal exudate cells. Under this treatment, trypanosomes given intraperitoneally are detained in the peritoneal cavity where they are agglutinated, lysed, and phagocytized. INDEX DESCRIPTORS : Trypanosoma lewisi, Immunity passive ; Phagocytosis; Dexamethasone ; corticosteroid ; 9-a-Fluoro-16-a-methyl prednisolone ; Prednisolone.

Depression of the reticuloendothelial system favors increased levels of parasitemia in rats infected with Trypanosoma lewisi. The elevated levels of parasitemia observed in rats that have been blockaded and splenectomized (Taliaferro et al., 1931; Taliaferro, 1938), X-irradiated (Naiman, 1944; Templeis and Lysenko, 1965) or treated with corticosteroids (Sherman and Ruble, 1967; Patton and Clark, 1968) do not appear to result from an increased rate of reproduction of T. lewisi, but from an extension of t,he period of reproduction in treated rat(s. The means by which the rat controls T. lewisi infections and especially the role of phagocytes in this control has been a matter of controversy. Laveran and Mesnil (1901) observed that T. lewisi was phagocytized in the peritoneal cavities of rats which had been actively and passively 1Present address: Yale University School of Medicine, Department of Microbiology, New Haven, Connecticut 06510. ?Dexamethasone (9-a-fluoro-16-a-methyl prednisolone) is a synthetic corticosteroid supplied by Schering.

immunized. They, as did DelanoE (1911, 1912) and Brown (1915)) considered phagocytosis an essential mechanism in relieving the host of infection with this parasite. However, other workers stressed instead the action of lytic antibodies as the major trypanocidal action (MacNeal, 1904; Manteufel, 1909; Taliaferro, 1924). Lange and Lysenko (1960) reported that immune serum enhanced the phagocytosis of T. kwisi by exudate cells in vitro. The purpose of this investigation was to test the importance of phagocytes and antibodies in the control of T. lewisi infections. Dexamethasone reduces the absolute number of phagocytes and it inhibits antibody formation in rats (Nicol and Druce, 1961). Dexamet’hasone-treated rats provided an in vivo system to study how immune sera in conjunction with peritoneal exudate cells protect the rat against T. lewisi infections, MATERIALS

0 1972 by Academic Press, Inc.

METHODS

Rats used in this study were white albino males (average weight 74 g) from Spartan Research Animals Company, Williamston, 370

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AKD

T. k?WiSi: INFLUENCE OF SERA AND PERITONEAL EXUDATE Mich. The trypanosomes used in these experiments were obtained from rats 5 days after infection. The organisms were separated from the blood by repeated centrifugation at 1OOOgfor 10 min, washed twice in Hanks’ balanced salt solution (HBSS), and resuspended in it. The trypanosomes and all other substances injected into the rats in this study were given intraperitoneally tip). Hyperimmune sera were obtained from 12 rats which had recovered from infections with T. lewisi and had been challenged twice-45 and 59 days after the original infection. Blood samples were collected aseptically by cardiac puncture 10 days after the second challenge, and the sera were pooled. Acute sera were obtained from rats infected for 5 days, and normal sera were obtained from rats which had no history of trypanosomiasis. Peritoneal exudate cells were harvested from normal rats, acutely infected rats, and hyperimmune rats by the following procedure. The rats were injected ip with 5 ml of a 1% glycogen solution in sterile physiologic saline and 48 hr later with 20 ml HBSS containing 100 USP units of heparin. The animals were then anesthetized with ether, the abdominal skin was retracted, and an incision of approximately 1 cm was made through the abdominal wall. The rats were held over a sterile beaker in order to catch the fluid and cells which flowed through the incision. After centrifugation for 5 min at lOOOg, the cells were washed and resuspended in HBSS. Peritoneal exudate cells and the trypanosomes were counted in the Improved Neubauer hemacytometer. Disposable blood-diluting pipettes (supplied by Becton and Dickson and Co.) containing physiologic saline were used to make dilutions. The pipettes containing the trypanosomes were refrigerated for at least 2 hr at 4 C to reduce the motility of the organisms prior to counting them.

371

RESULTS

Experiment 1: The Effects of Exudate Cells and Sera on T. lewisi in Vitro In vitro studies were carried out in 50-ml glass, round-bottomed centrifuge tubes which contained HBSS with trypanosomes, peritoneal exudate cells, and sera (Table I). The suspensions were incubated at 37 C. A drop of the contents from each tube was examined under a phase-contrast microscope three times during 3 hr. At the end of 3 hr, smears were made, dried, and stained with Wright’s stain. The trypanosomes were agglutinated when they were put into hyperimmune serum, but they were not agglutinated in normal or acute serum. At the end of 3 hr, the agglutinated trypanosomes were less motile and some appeared to be lysed (Table I). In all tubes that contained exudate cells from any of the sources used, the flagellates adhered to the surfaces of these cells and some were phagocytized (Fig. 1, A-C ; Table I). Although no quantitation, such as phagocytic index, was attempted, the presence of serum from all sources but especially serum from hyperimmune rats appeared to favor phagocytosis. Experiment 9: Passive Immunization Dexamethasone-Treated Rats

of

Rats were injected with 0.5 mg of dexamethasone daily beginning 3 days prior to infection with 5 x lo* trypanosomes. Rats were injected twice with sera and/or peritoneal exudate cells. The first injection was given at the time of infection and the second one 24 hr later. Sixty rats were allocated to 10 groups of 6 and also separated into two sections. All the rats in Section A of the experiment received dexamethasone. Rats in Section B did not receive the drug. Rats in Section A and B were treated as described in Table II and Fig. 2.

372

PATTON

TABLE --

In Vitro Added

Tube no.

1 2 3 4 5 (i 7 8 9 10 11

12 13 11 15 16

Hanks’ balanced salt solution

0.6 0.3 0.3 0.3 0.3 0.3 0.3

” hyperimmune rat

SerUll from acutely infected rat

Serum from normal i-at

System per tube

I

of Experiment ..~

I

(cc)

5 x 10’ Exudate cells from hyperimmllne rats

Results 5 x 10’ Exudate cells from acutely infected rats

Exudate cells from nonimmune rats

0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3

0.3 0.3 0.3

0. 3

0.3 0.3 0.3

0.3 0.3 0.3 0.3 0.3 0.3

0.3

As shown in Table II, rats which were given dexamethasone developed high and fatal parasitemias, except when administered both hyperimmune serum and exudate cells from normal rats or from hyperimmune rats. Rats not treated with dexamethasone were protected by hyperimmune serum with or without exudate cells. Though infections were established in untreated rats which were given a combination of normal serum and exudate cells from hyperimmune rats, the peak parasitemias were lower than those of rats which had been given serum and/or exudate cells from normal rats (Table II and Fig. 2). Experiment 8: Retention of T. lewisi in the Peritoneal Cavities of Rats Given Exudate Cells and Hyperimmune Serum Ten normal rats and 10 rats which had been given three daily injections of 0.5 mg dexamethasone were injected with 3 X lOlo trypanosomes in 1 ml HBSS. Four rats in each group were injected with 1 ml of hyperimmune serum along with 1 ml of HBSS which contained 6 x lOlo exudate cells from

0.3 0.3

2.5 x 10’ TWPanosomes/ mmr

0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3

Agglutination

Phagocy to&

+ -

-

+ + + -

+ + + + + + + + +

.-

hyperimmune rats. Two rats in each group were injected with 1 ml of hyperimmune serum and 1 ml of HBSS; two others with a 2-ml suspension of exudate cells, and the remaining two rats with 2 ml of HBSS. The tail blood of each rat was examined for the presence of trypanosomes 30 min and 1 hr after injection. The rats were then killed and the peritoneal cavities were washed with HBSS. The contents of t’he wash were examined for the presence of trypanosomes as well as the occurrence of phagocytosis by macrophages. As shown in Table III, serum from hyperimmune rats alone or in combination with exudate cells prevented the appearance of trypanosomes after an hour in the peripheral blood stream of normal rats. Only when dexamethasonetreated rats were given a combination of hyperimmune serum and exudate cells were trypanosomes not found in their blood. Agglutinated trypanosomes and phagocytes containing trypanosomes were recovered from the peritoneal cavities of all rats administered both exudate cells and hyperimmune serum (Fig. 1D). When dexa-

T. lewisi:

INFLUENCE OF mm.4 AND PERITONEAL EXUDATE

FIG. 1. Exudate cells in various stages of phagocytizing Trypanosoma lewisi. A-C: in vitro. A. Cells from nonimmune rats which have engulfed trypanosomes in normal serum. B. Trypanosomes in hyperimmune serum adhering to an exudate cell from a rat with an acute infection. C. Exudate cells from hyperimmune rats in serum from hyperimmune rats showing vacuoles containing trypanosomes. D-E: in viva. D. Peritoneal washing from dexamethasone-treated rats given exudate cells and serum from hyperimmune rats. Note adhering and partially engulfed trypanosomes. E. Peritoneal cavity washing from an untreated rat given HBSS showing a macrophage in the process of engulfing a trypanosome. F. Peritoneal cavity washing from an untreated rat given hyperimmune serum. The cell shows two vacuoles which contain trypanosomes.

373

374

PATTON

-Deaamethosone

o----o

somes some of which were associated with peritoneal exudate cells (Fig. 1E). As shown in Table III, both exudate cells and antiserum were required to prevent the appearance of trypanosomes in the tail blood of rats treated with dexamethasone. In the intact controls the same effect was obtained by injecting antiserum alone.

No dexomethosona

DISCUSSION

c

1X1O2o h

E

1XlO2O

tf

2

, , 4

, , , 8 6 Days

ILL--

2

4

6

8

postlnfectlon

FIG. 2. Parasitemia curves from Experiment 2. Rats were infected with T. Eewisi and inoculated with the following solutions: (a) HBSS, (b) HBSS suspension of exudate rells from normal rats, (c) HBSS suspension of cells from hyperimmune rats, (d) normal serum, (e) serum from hpperimmune rats, (f) exudate cells and serum from normal rats, (g) exudate cells from normal rats and serum from hyperimmune rats, (11) exudate cells and serum from hyperimmune rats, (i) exudate cells from hyperimmune rats and normal serum. D = dead.

methasone-treated rats were administered singly exudate cells, serum, or only HBSS,, trypanosomes were found infrequently in the peritoneal cavities. When rats had not been given dexamethasone, but were given hyperimmune serum, the peritoneal cavities contained agglutinated as well as lysed trypanosomes. Some of the parasites were in combination with peritoneal exudate cells (Fig. 1F). The peritoneal cavities of normal rats which had been given HBSS contained few trypano-

The observation that T. lewisi is phagocytized in vitro by peritoneal exudate cells in the presence of normal serum and that phagocytosis of the trypanosomes is enhanced by the addition of immune serum is in accord with the findings of Lange and Lysenko (1960). The evidence here shows that phagocytes have an active role in the control of trypanosomiasis. Very likely such cells in the normal host actively influence the whole course of infection. Dexamethasone, an anti-inflammatory steroid 20-35 times as potent as cortisone, inhibits the formation of cells and their function in phagocytosis and antibody formation in rats (Sic01 and Druce, 1961). The hyperimmune serum used in the experiments described here agglutinated and lysed trypanosomes in vitro and protected normal rats from infection with T. lewisi, but it did not protect dexamethasone-treated rats. Treated rats (Fig. 2, Table II) were protected only when they were given peritoneal exudate cells and hyperimmune serum. These rats were not only protected from fatal infections, but also from patent infections (Table II, Fig. 2). The results indicate that phagocytes assume an active and very likely essential role in the control of T. lewisi infections. Further evidence for the role of phagocytes in these infections is seen in Experiment 3 (Fig. 1, Table III). Trypanosomes were retained, agglutinated, lysed, and phagocytized in the peritoneal cavities of dexamethasone-treated rats if the rats were given hyperimmune serum and exudate cells. When either exudate cells or hyperim-

T. lewisi:

INFLUENCE

OF SERA AND PERITONEAL

TABLE Average

Parasitemias

375

II

of Rats Given Dexamethasone,

Sera, and Exudate

Trypanosomes

Cells

per mm3 X 102

Days after infection

Administered 1 HBSS HBSS and dexamethasone Normal exudate cells Normal exudate cells and dexamethasone Normal serum Normal serum and dexamethasone Hyperimmune serum Hyperimmune serum and dexamethasone Normal exudate cells plus normal serum Normal exudate cells plus normal serum and dexamethasone Normal exudate cells and hyperimmune serum Normal exudate cells plus hyperimmune serum and dexamethasone exudate Hyperimmune cells and HBSS exudate Hyperimmune cells plus HBSS and dexamethasone exudate Hyperimmune cells and normal serum exudate Hyperimmune cells plus normal serum and dexamethasone

EXUDATE

4

5

2

3

5.83 5.83 1.67

217 350 108

2630 9430 883

3930 13,000 3600

4300 I3 5100

2.5 4.17

225 102

7230 2370

9630 3180

10,600 3670

540 <2.5

7130 <2.5

9670 <2.5

3.33 2.5

368 120

7400 933

12,100 3230

D 3930

4480

3.33

290

5960

8800

10,600

D

8.3 <2.5

6

7

4900

4670

5200

4400

D 3930

9900 <2.5

D <2.5

<2.5

4500

<2.5

<2.5

~2.5

<2.5

<2.5

<2.5

<2.5

<2.5

<2.5

<2.5

<2.5

<2.5

<2.5

<2.5

2.5

91

882

2670

4.17

380

8530

10,000

720

2230

2370

11,700

14,500

8.3

1.67

88.3

243

4130

4130

4150

2400

2333

D

D

a D=dead.

TABLE Effects

III

of Dexamethasone, Exudate Cells, and Serum on the Occurrence in Tail Blood 30 and 60 Min After Inoculation

of Trypanosomes

Dexamethasone

Control

Treatment

Exudate cells and sera from hyperimmune rats Exudate cells from hyperimmune rats Sera from hyperimmune rats Hanks’ balanced salt solution

30 min

60 min

30 mm

60 min

Negative Positive Negative Positive

Negative Positive Positive Positive

Negative Negative Negative Positive

Negative Positive Negative Positive

376

PATTON

mune serum were given alone to dexamethasone-treated rats, the trypanosomes escaped from the peritoneal cavity and were found in the tail blood within an hour. Normal rats which were given hyperimmune serum alone also retained trypanosomes in the peritoneal cavities where they agglutinated, lysed, and were phagocytosed by peritoneal cells normally present in the host or by cells which had been injected. The finding that the trypanosomes were retained in the peritoneal cavities of rats passively immunized is in agreement with results reported by Cox (1964)) but differs in that he did not observe agglutination, lysis, or phagocytosis of trypanosomes recovered in peritoneal washings. The different observations may be due to experimental design. Cox (1964) used actively immune animals or he passively immunized his animals subcutaneously 1 day prior to infecting them. In the present study trypanosomes, hyperimmune serum, and peritoneal exudate cells were put into contact with one another in the peritoneal cavities of the rats. Exudate cells from hyperimmune rats did not protect dexamethasone-treated rats (Tables II and III) ) but they did appear to give some protection to normal rats. Among a heterogeneous population of exudate cells are cells which contain antibodies (Nossal and Makela, 1961). These cells might have reduced the intensity of infections in this group by providing immediately available specific antibodies or cells which more rapidly responded in the production of specific antibodies. Some of these results offer a basis for cxplaining in part the early higher levels of parasitemia in rats treated with dexamethasone (Patton and Clark, 1968) compared with rats not given the drug. Cells of the reticuloendothelial system are essential for the effective removal of microorganisms as well as necessary for antibody formation. Dexamethasone-treated rats are deficient in

phagocytes which are available and effect phagocytosis in normal rats (Nicol and Druce, 1961). Phagocytes in normal rats may reduce the number of trypanosomes which initiate parasitemias and in addition augment the protection given by humoral antibodies which arise during infection. REFERENCES BROWN, W. H. 1915. Concerning changes in the biological properties of Trypanosoma Eewisi produced by experimental means, nith especial reference to virulence. Journal of Experimental Medicine 21,345-364. Cox, H. W. 1964. Immune response of rats and mice to trypanosome infections. Journal of Parasitology 50, 15-22. DELANO& P. 1911. Sur la rCceptivitk de la souris au Trypanosoma lewki. Comptes Rendus Societe Biologie 70, 649-651. DELANO& P. 1912. L’importance de l& phagocytose dans I’immunit6 de la souris a 1’Cgard de quelques flagellCs. Anna/s de Z’lnstitute Pasteur

26,172-203. LANGE. D. E., AND LYSEKKO, M. G. 1960. In vitro phagoc)-tosis of Trypanosomn lewisi by rat ex10, udative cells. Experimental Parasitology

3942. LAVERAN, A., AND MESKIL, F. 1901. Recherches morphologiques et expRrimentales sur lr trq-panosome des rats (Trypanosoma lewisi, Kent). Annals de l’lnstitule Pasteur 15,673-714. MACNEAL. W. J. 1904. The life-history of Trypanosoma lewisi and Trypanosoma brucei. Journal of Infectious Disenses 1,517-543. MANTEuFEL. P. 1909. Stud& uber die Trypanosomiasis der Ratten mit Berucksichtigung der unternaturlichen Vrrhaltnissen Ubertragung und der Immunitat. ArbeifergeslLnrlheits-bibliothek Berlin 33,46-83. ?S.~IX~X, D. N. 1944. Effect of S-irradiation of rats upon their resistance to Trypanosoma lewisi. Journal of Parasitology 30,209-228. NICOL. T.. .4x11 DR~J~E,. C. 1961. Effrct of Drxamethasone on the phagocvtic activity of the reticuloendothelial system. Nature, London

190,91-92. NOSSAL, G. J. V.. AXD MAKELA. 0. 1961. Autoradiographic studies on the immune response: the kinetics of plasma erll proliferation. Jo,lmnZ of Experimental Medicine 115,209-233. PATTOS. C. I,.. AXD CLARK, D. T. 1968. Trypanosoma lewisi infections in normal rats and in rats treated Ivith dexamethasone. Journal of Protozoology 15,31-35.

T. kWi&

INFLUENCE OF SERA AND PERITONEAL EXUDATE

I. W., AND RUBLE, J. A. 1967. Virulent Trypanosoma lewisi infections in cortisonetreated rats. Journal of Parasitologg 53, 253-

SHERMAN,

262.

W. H. 1924. A reaction product in infections with Trypanosoma lewisi which inhibits the reproduction of trypanosomes. Journal of Experimental Medicine 39,171-199. TALIAFERRO, W. H. 1938. The effects of splenectomy and blockade on the passive transfer of anti-

TALIAFERRO,

377

bodies against Trypanosoma lewisi. Journal of Infectious Diseases 62,98-111. TALIAFERRO, W. II., CANNON, P. R., AND GOODJJJE, SARA. 1931. The resistance of rats to infection lewisi as affected by splenwith Trypanosoma ectomy. American Journal of Hygiene 14, lC. H., AND LYSENKO, M. G. 1965. Effect lewisi infecof X-irradiation on Trypanosoma tion in the rat. Experimental Parasitology 16,

TEMPELIS,

174-181.