Trypanosoma gambiense: Phagocytosis in vitro

EXPERIMENTAL

36, 106-113

PARASITOLOGY

Trypanosoma TAN

Department

( 1974)

gambiense:

TAKAYANAGI

of Medical

AND

Phagocytosis YOSHISADA

in Vitro

NAKATAKE

Zoology, Medical School, Nagoya Mizuho-ku, Nagoya, Japan

City

University,

AND GLORIA

L.

ENRIQUEZ

1

Department of ProtozookJgy, the Research Institute Osaka University, Yamada kami, Suita-city, (Submitted

for publication

April

for Microbial Diseases, Osaka, Japan

19, 1973)

TAKAYANAGI, T., NAKATAKE, Y., AND ENHIQUEZ, G. L. 1974. Typanosoma gambiense: Phagocytosis in vitro. Experimental Parasitology 36, 106-113. When Trypanosomu gambiense was exposed in vitro to rat macrophages, the addition of homologous antiserum resulted in immediate adherence of the parasites to the surface of macrophages. The antigenic components of the parasite were fractionated by Sephadex G-200, DEAE-cellulose, and block electrophoresis. Each fraction was used for immunizing rats, and the antisera obtained were each treated for agglutinating antibodies and for their effect on in vitro phagocytosis by rat macrophages. Only the antiserum capable of agglutination could enhance phagocytosis. A strong possibility that the agglutination antigen is mostly responsible for phagocytosis, hence protection, is indicated. Scanning electron micrographs showed that adherence to the macrophage is mostly by the anterior part of the parasite. INDEX DESCRIPTORS: Tqpanosoma gambiense; Rat; Macrophage; Tissue culture; Sephadex; Chromatography; Column; ElectroPhagocytosis; Scanning microscope; phoresis; Antigens; Antibodies; Agglutinins; Immunity; Mice; Filtration; Gel; Immunization.

ported that immune serum enhanced the phagocytosis of T. lewisi by exudate cells in vitro and that a phagocytic enhancing antibody may be of significance in the rapid clearing of parasites injected into the circulating blood of immune rats. Patton (1972) reported that serum from rats hyperimmune to T. bwisi protects normal rat and that synthetic corticosteroid-treated rats are protected from trypanosomiasis only if they are given serum from hyperimmune rats and peritoneal exudate cells. With this treatment, trypanosomes given

are reports of the interaction beThere tween phagocytes and trypanosomes. Laveran and Mesnil (1901) observed that Typanosoma lewisi was phagocytized in the peritoneal cavities of rats which had been actively and passively immunized. Delano& (1911; 1912) and Brown (1915) considered phagocytosis an essential mechanism in relieving the host of infection with T. lewisi. Lange and Lysenko (1960) re1 Present address: Department of Zoology, versity of the Philippines, Diliman, Quezon D-505, Philippines.

UniCity,

106 Ox-wright 0 1974 by Academic Press, All rights of reproduction in any form

PHACOCYTOSIS OF T. gUfflbie?lSe

intraperitoneally are detained in the peritoneal cavity where they are agglutinated, phagocytized, and lysed. This report deals with the interaction between phagocytes and T. gambiense, and the fractionations of the antigens concerned with immune phagocytosis. MATERIALS AND METHODS

Parazites The Wellcome strain, antigenically 0 type T. gambiense, maintained in female mice (dd strain), 18-20 g by weight and serially transferred at 3-day intervals, was used throughout the experiment. Collection

and Separation of Organisms

For this study, Wistar male rats (lOO150 g) were experimentally infected intraperitoneally (ip) with 5 x lo7 parasites from a mouse infected 3 days earlier. The blood was collected by heart puncture 3 days after infection. Parasites free of host blood cell components were obtained by the method ‘of Lanham and Godfrey (1970). The parasites were washed 3 times with 1% glucose phosphate buffer ( GPB ), p = 0.271, pH 7.5, and centrifuged at SOOg for 8 min. After final centrifugation, some parasites were resuspended in GPB in suitable numbers for the phagocytic test. The remaining parasites were homogenized in a sonicator (20 kc) for 5 min at 4 C. After centrifugation at 15,000g for 60 min, the supernatant fluid was stored at -25 C until used as a source of antigens for further fractionation. Harvesting

Rat Macrophuges

Wistar male rats, weighing 100-150 g, were used. Production of peritoneal exudates was induced in rats by the injection ip of 5 ml sterile paraffin liquid. After 4 days, the rats were killed rapidly with ether. A small incision was made on the lower left abdomen and the peritoneal cavity rinsed with 30 ml Hanks’ solution containing 100 I.U. of heparin. The cellular

107

suspension was collected with a sterile pipette. In most instances, all exudates employed in this study were free of significant numbers of red blood cells. Appropriate volumes of lthe exudate were dispersed into sterile 50 ml centrifuge tubes. The cells were sedimented at 28Og for 5 min in a refrigerated centrifuge. The cell-free supernatant fluid was carefully removed with a sterile fine tipped pipette, and the residual fluid was allowed to drain. The cells were gently resuspen,ded in Hanks’ solution, and then counted in a hemocytometer. Methylene blue was used to check the number of intact cells, and the concentration of cells was adjusted to the desired number. Three further washes were carried out when cells from immunized animals were used. The entire procedure was done with the cells either refrigerated or kept on ice. Tissue Culture of Rat Peritoneal Exudate Cells were cultured in culture tubes containing 8 x 30 mm coverslips. One milliliter ‘of cell suspension (2 X 106/ml) in complete medium, consisting of TCM 199, 20% calf serum, 50 units/ml penicillin, and 50 ,ag/ml streptomycin, was dispensed into each culture tube. The tubes were gassed with a 5% COzair mixture, closed with rubber stoppers, and incubated at 37 C for 60 min. Each culture tube was then vigorously shaken and the medium withdrawn. The cell layer was next washed with 2 ml of complete medium with agitation. The wash fluid was removed and the cells overlayed with 0.8 ml complete medium, gassed, and incubated for a few hours until used for the phagocytic test. The agitation and washing procedure removed more than 95% of the lymphocytes, and in most preparations it was difficult to find any cells other than macrophages on the coverslip. Before adding the parasite suspension, the culture medium was first discarded, and 0.25 ml suspension containing 4 x lo7 parasites/ml was added. After 1 min, 0.25

108

TAKAYANAGI,

NAKATAKE

ml serum was added. After mixing thoroughly, the culture tubes were incubated at 37 C for 5 min. Fractionation

of Antigens

(I ) Gel filtration. Sephadex G-200 (Pharmacia, Uppsala, Sweden) was used. A column, 3.0 x 100 cm, was prepared according to the method ‘of Flodin and Killander ( 1962) and equilibrated in a solution of 0.1 M tris-hydroxymethylaminomethane (Tris-HCl), pH 8.0, in 0.15 M NaCl. Five milliliters of the antigen preparation were applied to the column. Elution was carried out at room temperature with the Tris-HCl buffer. Five milliliter fractions per tube were collected, and the optical density was read at 280 nm with the aid ,of Toyo’s Uvicon 540 recording spectrophotometer. Individual effluent fractions were dialyzed against 0.02 M phosphate buffered saline solution ( pH 7.5). (2) DEAE-cellulose column chromatography. Chromatographic separation of fractions of 10 ml of the antigen preparation was carried ,out on columns containing 40 g of DEAE-cellulose (Whatman DE52). An initial buffer (pH 8.0) of 0.02 M phosphate was used to equilibrate the column, and elutions were made by gradually increasing the phosphate concentration to 0.2 M while maintaining a constant pH. (3) Block electrophoresk. Samples were dialyzed overnight against veronal buffer, p = 0.05, pH 8.6, at 4 C. Denaturated protein, if present, was removed by centrifugation. The superna,tant fluid (0.3 ml) was applied to a 2.5 x 7.0 x 0.1 cm agar gel block (1.5% wt/vol). A direct current of 5 mA/plate for 5 hr at 4 C was applied. Millimeter-wi.de fractions were cut and extracted twice by freezing an’d thawing in dry ice-acetone.

AND

ENRIQUEZ

were immunized ip with 2 x 10” killed trypanosomes 3 times weekly to obtain standard test antisera. Rats were also immunized ip with 1 ml of trypanosomal fractions obtained by gel filtration, DEAEcellulose column chromatography, and block electrophoresis to examine the PI activity. After 10 days, the animals were bled from the heart. The sera from each group of animals were inactivated at 56 C for 30 min and stored at -25 C until used. Standard test antisera against killed parasites had a titer of 1: I28 in agglutination tests. Agglutination

Test

Five-tenths milliliters of twofold diluted serum was mixed with an equal volume of GPB containing 1 x lo8 trypanosomes. After 10 min, the agglutination titer was determined as the last dilution of antiserum in which agglutination occurred. Observations were made under the microscope. Staining When necessary, covershps were fixed in methanol for 10 min and stained in Giemsa. Nitrogen Determination of the AntigewAntibody Complex One milliliter of fractionated antigen solution was mixed with the antiserum in optimal ratio. After gentle agitation and addition of NaN3 to a final concentration of O.l%, the mixture was incubated at 37 C for 1 hr and then allowed to stand for a week at 4 C in a refrigerator. After centrifugation at 15,000g for 60 min, the sediment was washed several times with chilled phosphate buffered saline, pH 7.5, at 4 C. Nitrogen was measured in a Microkjeldahl apparatus (Kabat and Mayer 1961) .

Immunization Trypanosomes collected from rats were killed by repeated freezing and thawing in dry ice-acetone. Rats, rabbits, and mice

Pre paratkms

for Scanning

Microscopy

For fixation of the material, the coverslip containing the specimens was immersed

PHAGOCYTOSIS OF

T. gambiense

109

into 1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. After 1 hr, the material was washed 3 times with 7.5% sucrosecacodylate buffer, and then was doublefixed in 1% 0~0~ in 0.2 M sucrose-cacodylate buffer for 1 hr. Dehydration was done with acetone. All procedures were carried out in ice. After ‘dehydration, the material was air dried. The scanning microscope used was JSMdOA, JEOL. RESULTS

Macrophages mixed with the antiserum attracted the parasites almost instantaneously to their surface. Since the adherence of the parasite is considered to be the first step to engulfment, the phenomenon is taken as the phagocytic index, % (PI). Figure 1 shows the change in PI with time with varying concentrations of the standard test rat antiserum. A close relationship between the PI effect on the macrophage and the agglutinin titer of the antiserum is shown. The highest concentrations (l/8) shows a PI of approximately 90% 5 min after adding serum to the parasite PI

123456 MINUTE

FIG. 1. Phagocytic index of macrophage in relation to time in minutes and dilutions of standard test rat antiserum. It had a titer of 1: 128 in agglutination test. X-X, l/8; *-*, l/32; O-0, l/128; 0-0, control (normal serum).

16

32

64

128

DILUTION

FIG. 2. Phagocytic index of macrophages in the presence of standard test antiserum after incubation for 5 min. O-O, rat; X-X, mouse; O-O, rabbit.

suspension and the macrophage culture. The phagocytic activity is induced by antisera from mouse and rabbit, but not as much as the antiserum from rat (Fig, 2). In both cases, the PI is relate’d to the agglutinin titer. Figure 3 shows the O.D. measurement of elutions after Sephadex G-290 fractionation. For phagocytic test, rats were immunized against each fraction, and the antisera were obtained 10 days later. To estimate the amount of antigenic substances, each fraction was reacted with the standard test rat antiserum. Nitrogen determination was carried out from the antigen-antibody complex. Figure 3 also shows that the antigenic substances were distributed in all fractions although, understandably, a large amount was found in the fraction that gave the highest O.D. reading. Agglutination test was done on all antisera, but only that which corresponded to the first peak showed agglutinating ability. The PI of the first peak is shown in Fig. 4. All other fractions were unable to enhance phagocytosis. The same steps were ‘done with the fractions obtained by use of DEAE-cellulose

110

TAKAYANAGI, OJ. 280~1~

1

lJG N/ML

AT

320

NAKATAKE

0 0 0 0

AND ENRIQUEZ

9~8~ , 0.021 ri 16

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0,OSM

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0.m

0

0

0

,

"G NhL

. 1200 , 1000

1300

800 .

1200 0,s

600

1100

500 400

0,3

300 0.1

200 100

0

10

20

30

40

50

60

70

FRACTl0N

FIG. 5. O.D. measurement of fractions with DEAE-cellulose column chromatography ( O-O ) and nitrogen measurements of antigen-antibody complex ( O-O). Agglutination ‘titer is shown as AT.

25 35 45 55 65 75 85 95 FRACTION FIG. 3. O.D. measurements of elutions from Sephadex G-200 (0-O) and nitrogen measurements of complexes ( O-O). Agglutination titer of positive fraction after immunization is shown as AT.

column chromatography and block electrophoresis. Figure 5 depicts the O.D. measurement of fractions ‘obtained by DEAEcellulose fractionation and the respective amount of nitrogen. The fraction eluted with 0.02 M phosphate buffer, pH 8.0, pro-

duced antiserum with an agglutination titer of l/16. All other fractions gave negative results with the agglutination test. Figure 6 shows the PI of the same fraction as compared with all other fractions. Figure 7 shows the amount of nitrogen in each of the fractions obtained by block electrophoresis and the respective amount of nitrogen from the antigen-antibody complex. The agglutination titers of the positive fractions are also indicated. Figure 8 shows PI 100

PI

O-0

100 O-0

\

0

50 50 \

0

\

\

c 0

0 .-.-.-. 4

-’

7

2 8

16

32

DILUTION

FIG. 4. Phagocytic index of macrophages in the presence of antiserum against fraction No. 35 ( 0-O) and antiserum against other fraotions and normal control serum ( 0-O ).

0

.-.-.-.

4

8

16

DILUTION

FIG. 6. Phagocytic index of macrophages in the presence of antiserum against the DEAE-cellulose fraction in 0.02 M buffer (O-0) and antiserum against fraction in 0.05 M, 0.1 M, and 0.2 M buffer, and normal control serum ( O-O ).

PHAGOCYTOSIS

OF

111

T. gumbhse

800 AT

t

01:

I,

1

32

AT

I,.

I

I

I+) 3 2 1 0 1 2 3 4 5

I,

16

I,,

6 7 8 9 10 (-I

FRACTION

FIG. 7. Nitrogen measurements of fraotions from block electrophoresis ( 0-O) and Ag-Ab complex ( X-X ). Agglutination titers are shown as AT.

the PI of antisera to the fractions from block electrophoresis with an agglutination titer of l/32 and l/16. Macrophages from hyperimmune rats showed enhanced phagocytic ability only with the addition of the antiserum (Fig. 9). Almost all parasites adhered to the macrophage surface by means of their anterior end in the first step as shown by the scanning electron micrograph (Fig. 10). DIXXJSSION

Laboratory rats experimentally infected with T. gambknse show parasitemia for varying periods of time depending on the dose and then invariably die. However, immunophagocytosis and passive transfer of immunoglobulins have been found effective for protection. Ono and Inoki ( 1972) showed electron microscopically that the phagocytosed parasites were digested or lysed within the cell in vioo upon injection of the antiserum. Takayanagi and Em-iquez ( 1973) used purified immunoglobulins to protect the experimental host against T. gambknse infection. Accordingly, it might be suggested that the immunoglobulins facilitated phagocytosis in tiuo. The same possibility has been advanced by a number of reports on T. lewki (Laveran and Mesnil 1901; Delano6 1911, 1912; Brown 1915; Lange and Lysenko 1960; Patton 1972).

FIG. 8. Phagocytic index of macrophages in the presence of antiserum against zero fraction from block electrophoresis ( O-O), antiserum against -9 fraction (X-X), and control and antisera against other fractions ( O-O ) after incubation for 5 min.

Trypanosomes have been shown to have a number of antigenic components (Gray 1961; Seed 1963). In the present work, the antigenic component(s) responsible for agglutination (agglutination antigen) when

16

32

64

.I28

DILUTION

FIG. 9. Phagocytic index of the macrophage from normal (X-X ) and immunized (0-O) rat using standard test rat antiserum.

112

TAKAYANAGI,

NAKATAKE AND ENRIQUEZ

FIG. 10. Micrograph showing the parasite just attached to the macrophage. M, macrophage; ae, anterior end of the parasite; pe, posterior end; bu, beginning of the undulating membrane, x3000.

used for immunization consistently gave antibody that enhanced phagocytosis in uitro. The fractions obtained by different methods always gave identical results. The present work shows that agglutination quantitatively parallels phagocytic enhancement activity and that the antigenic component ( s ) and the antibody ( -ies ) involved are probably the same for both activities. In an earlier report, Takayanagi et al. (1973) showed that the soluble antigen (agglutinin) was present in the flagellum (undulating membrane) by ferritin-coupled antibody through immunoelectron microscopic observations. Scanning electron microscopic observations showed that the antigen ( s ) involved in immunophagocytosis was also present in the anterior part of the parasite. It is also possible that the surface of the anterior part of the parasite, included flagellum, contains mainly the agglutinin( s) which is also responsible for the process of phagocytosis. ACKNOWLEDGMENT We thank Professor Sigefusa able advice and criticism.

Sato for his valu-

REFERENCES BROWN, W. H. 1915. Concerning changes in the biological properties of Typanosomu lewisi produced by experimental means, with special reference to virulence. Journul of Experimental Medicine 21, 345-364. DELANO& P. 1911. Sur la receptivitk au Typanosoma lewisi. Comptes ciete Biobgie 70, 649-651.

de la souris Rendus So-

DELANO& P. 1912. L’importance de la phagocytose Bdans l’immunitk de la souris a l’egard de quelques flagelk. Annals de Z’Institute Pasteur 26, 172-203. FLOD~V, P., AND KILLANDER, J. 1962. Fractionation of human-serum proteins by gel filtration. Biochimica et Biophysics Acta 63, 403-410. GRAY, A. It. 1961. Soluble antigens of Typanosoma vivax and of other trypanosomes. Immunology 4, 253-261. KABAT, E. A., AND MAYER, M. M. 1961. “Experimental Immunochemistry,” p. 476. Charles C Thomas, Springfield, Ill. LANGE, D. E., AND LYSENKO, M. G. 1960. In vitro phagocytosis of Typanosoma lewisi by rat exudative cells. Experimental Pumsitology 10,

3942. LANHAM, S. M., AND GODFREY, D. G. 1970. Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Experimental Parasitology 28, 521-534.

PHAGOCYTOSIS

LAVERAN, A.,

AND MESNIL, F. 1901. Recherches morphologiques et exp&imentales sur le trypanosome des rats (Trypanosomu lewisi, Kent). Annals de Plnstitute Pasteur 15, 673714.

ONO, T., AND INOKI,

S. 197.2. Studies on the phagocytosis of Typanosoma gambiense by mouse ascites cell. Japanese Journfzl of Parasitology 21, 41 (in Japanese).

PATTON, C. L. 1972. Typanosoma

lewisi: In&ence of sera and peritoneal exudate cells. Experimental Parasitology 31, 370-377.

OF

T. gUd&??We SEED, J. Il. 1963. The characterization

113

of antigens isolated from Typanosoma rhodesiense. JOUTnal of Protozoology IO, 380-389. TAKAYANAGI, T., AND ENRIQUEZ, G. L. 1973. Effects of IgG and IgM immunoglobulins in Typanosoma gambiense. Journul of Parasitology 59, 644-647. TAKAYANAGI, T., ENRIQUEZ, G. L., KAMBARA, H., AND 0~~0, Y. 1973. An immunoelectron microscopy of the soluble antigen of Tlypanosoma gambiense. Southeast Asian Journal of Tropical Medicine and Public Health 4, 165167.