Schistosoma mansoni: A diamidinophenylindole probe for in vitro death of schistosomula

EXPERIMENTALPARASITOLOGY

Schistosoma

57, 125131(1984)

mansoni:

A Diamidinophenylindole Death of Schistosomula

P. W. G. VAN DER LINDEN

Probe for in Vitro

AND A. M. DEELDER~

Laboratory of Parasitology, Medical Faculty, Wassenaarseweg 62, 2333 AL Leiden,

(Accepted for publication

University of Leiden, The Netherlands

16 September 1983)

VAN DER LINDEN, P. W. G., AND DEELDER, A. M. 1984. Schistosoma mansoni: A diamidinophenylindole probe for in vitro death of schistosomula. Experimental Parasitology 57, 12.5- 13 1. The following fluorochromes were studied as probes for discrimination between living and dead Schistosoma mansoni schistosomula: ethidium bromide (EB), propidium iodide (PI), diamidinophenylindole (DAPI), and carboxyfluoresceine diacetate (CFDA). While schistosomula stained with EB, PI, or C-FDA showed leakage of fluorochrome into the medium, this was not the case with DAPI. Dead schistosomula, which were stained with DAPI, showed an intense blue fluorescence, while living schistosomula were not stained even after prolonged incubation. In addition, the low DAPI concentration (1 &ml) in the medium proved not to be toxic to the schistosomula, nor did it cause any background fluorescence. These properties make DAPI an ideal probe: the viability of S. masoni schistosomula in cytotoxicity tests can be continuously monitored in tissue culture trays, using an inverted microscope with simultaneous transmitted light and incident fluorescent light illumination. INDEX DESCRIPTORS: Schistosoma mansoni; Trematode; Blood fluke; Schistosomule; Viability test; Diamidinophenylindole (DAPI); Fluorochrome; Staining, in vitro. INTRODUCTION

In studies on the mechanisms of immunity of schistosomiasis in humans and experimental animals, in vitro cytotoxicity tests are widely used. Such tests involve the combination of schistosomula and components of the immune system of the host. To assess the resultant damage to the schistosomula, several assays have been described, most of which are not entirely satisfactory. A review of such assays has recently been given by Butterworth et al. (1982). In short, assays may be based on (a) immobility or a swollen, granular appearance of the schistosomula in light-microscopical examination; (b) staining of the schistosomula, using eosin, methylene blue or toluidine blue and subsequent light-microscopical examination; and, (c) release of a radioactive label (‘%r). Although all of i To whom reprint requests should be addressed.

these techniques have their own disadvantages, there does not seem to exist a more reliable assay for damage, enabling continuous monitoring of the cytotoxicity process i.e., without a sampling procedure. In cytotoxicity tests in which separate cells are used, e.g., human peripheral lymphocytes in an HLA-DR typing test, fluorochromes are nowadays commonly used as an alternative to radioactive probes for discrimination between dead and living cells. Some regularly applied fluorescent probes are ethidium bromide (EB), propidium iodide (PI), and diamidinophenylindole (DAPI), which are known to label dead cells, whereas carboxyfluoresceine diacetate (C-FDA) is a good label for living cells (Ededin 1970; Garcia et al. 1971; Kapuscinsky and Skoczylas 1978; Lin et al. 1977; Rotman and Papermaster 1966; Stoehr and Vogt-Schaden 1979). In a recent study, the use of DAPI, EB, and PI was compared in experiments with human peripheral lym-

125 0014-4894/84 $3.00 Copyright 0 1984 by Academic Press, Inc. AII rights of reproduction in any form reserved.

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phocytes (Tanke el al. 1982). DAPI proved to be the most suitable label, as it showed no cytotoxic effects. Although the applicability of such fluorescent probes has now been generally accepted for cytotoxicity tests involving separate cells, they have, to our knowledge, not yet been applied to systems with multicellular organisms. In the present study we have investigated the use of such probes for cytotoxicity tests with Schistosoma mansoni schistosomula. MATERIALS

AND METHODS

Schistosomula were mechanically transformed from Schistosoma mansoni cercariae (Puerto Rico strain) by a modified version (Van der Linden et al. 1982) of the technique originally described by Ramalho-Pinto ef al. (1974). Three hours after transformation, the schistosomula, which were incubated in RPMI-1640 medium supplemented with 100 IU Penicillin/ml, 0.1 mg Streptomycin/ml, 1 mg NaHC03/ml, 20 ng glycylL-histidyl-L-lysine acetate/ml and 10% heat-inactivated foetal calf serum, were used in assays. Peritoneal exudate cells were recovered from outbred Swiss mice, 9 weeks after infection with 120 S. mansoni cercariae. The cells were recovered in Hanks’ balanced salt solution (pH 7.2) and layered in SO-ml tissue culture flasks (Costar, Cambridge, England, UK) for 1 hr at 37 C to separate the macrophages from the other cells. After washing and centrifuging the suspension twice in the Hanks’ (10 min, 4OOg), the pellet was resuspended in the supplemented RPMI- 1640 medium in a final concentration of 5 x 10s cells/ml medium. The resultant cell suspension contained three to seven times more eosinophils than the original suspension. Serum from S. mansoni-infected mice was recovered by cardiac puncture from Swiss mice, 14-16 weeks after infection with 120 cercariae each. Pooled, it was added to the supplemented RPMI- 1640 medium in a final dilution of 1:5. DAPI was dissolved in phosphate-buffered saline (PBS; 0.05 M, pH 7.2) to make a stock of 100 pg DAPI/ ml PBS. In assays, stock solution was added to a final concentration of 1 pg DAPI/ml medium. Apart from DAPI, stock solutions of the fluorochromes ethidium bromide (EB) and propidium iodide (PI) (both in a concentration of 100 kg/ml in PBS containing 5% EDTA) and C-FDA (1: 1500 solution in PBS of a 10 mg/ ml solution in acetone) were prepared and used in the test suspensions at final concentrations of 1 pg/ml medium for EB and PI and of 0.3 t&ml medium for CFDA. EDTA and acetone were, at a final concentration of 0.05 and 0.003%, respectively, found to be not

AND DEELDER toxic to the schistosomula under the test conditions used. All assays were carried out in 24-well tissue cluster plates (Costar). Two incubation procedures were followed. 1. Incubation of a suspension of schistosomula with each of the four fluorochromes. Due to the mechanical transformation procedure, suspensions of schistosomula contained 3-5% dead schistosomula, as determined by methylene blue staining and by morphological criteria. For ah fluorochromes except DAPI, it was necessary to subsequently wash the schistosomula and transfer them to medium without fluorochrome to avoid fluorescence of the background. 2. Incubation of a suspension of living schistosomula with peritoneal exudate cells and infected mouse serum (1000 cells for each schistosomulum and a final dilution of 1:30) in DAPI-containing medium. Assay plates were inspected on a Leitz Diavert inverted microscope (Leitz, Wetzlar, F.G.R.), which was equipped with transmitted light and a Ploemopak illuminator for incident fluorescent light. The filter combinations used for the different fluorochromes have been described by Tanke et al. (1982). For experiments with DAPI, ultraviolet excitation and the filters UGl, CBS 400, LP 420, and SP 490 were used. RESULTS

In an initial set of experiments, the fluorochromes C-FDA, EB, PI, and DAPI were tested for their ability to differentiate between dead and living Schistosoma mansoni schistosomula. The only fluorochrome staining living schistosomula, C-FDA, also showed staining of the preacetabular glands in dead schistosomula. In addition, the contrast between stained, living schistosomula and the background strongly diminished in a few hours, probably due to continuous leakage of the C-FDA into the medium. With two of the fluorochromes which stained dead schistosomula, EB and PI, an intense red fluorescence of dead schistosomula was observed, while living schistosomula showed no fluorescence at fluorochrome concentrations of lower than 1 kg fluorochrome/ml medium. At higher EB or PI concentrations living schistosomula also showed fluorescence, although only at the level of the tegument. This staining of the tegument showed a very diffuse pattern, without any staining of nuclei. After a few

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FLUOROCHROME

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FIGS. 1,2. Living (arrows) and dead Schistosoma mansoni schistosomula after incubation with diamidinophenylindole, as visualized by transmitted light microscopy (Fig. 1) and incident light fluorescence microscopy (Fig. 2).

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mansoni schistosomulum after incubation with diamiFIG. 3. Anterior part of a dead Schistosoma dinophenvlindole. as visualized bv combined transmitted light and incident fluorescence microscopy. Nuclei are brightly fluorescent. ’

hours incubation, the contrast between stained schistosomula and the background became low, probably due to staining of serum components and of RNA which had leaked into the medium. As, in contrast to the results with these three fluorochromes, DAPI-treated schistosomula showed no leakage of fluorochrome, the applicability of this fluorochrome was further investigated. DAPI stock solution was added to 3 hr transformed schistosomula in a supplemented RPMI-1640 medium to a final concentration of 1 p,g DAPI/ml medium. These suspensions contained 3-5% dead schistosomula, as determined both by morphological and staining (eosin) criteria. These dead schistosomula became strongly fluorescent within 2 min, showing an intense blue fluorescence of the nuclei. No leakage of fluorochrome was ever observed, as shown by the absence of background fluorescence in the suspension, even during incubation periods of up to 48 hr (Figs. l-3). In contrast, no fluorescence at all was seen in living,

moving schistosomula (Figs. l-2), even after a 48-hr incubation. Control experiments in suspensions with or without DAPI showed that the fluorochrome is, in the concentration used, not toxic to S. munsoni schistosomula. When schistosomula in a DAPI-containing medium were mechanically damaged, an immediate staining of the cells surrounding the damaged area occurred, followed by a more gradual staining of the dying organism. In a medium containing infected mouse serum and a mouse eosinophil-enriched peritoneal cell preparation, similar results were obtained. Although dead eosinophils and other cells were also stained by the DAPI, the fluorescence pattern of dead schistosomula was easily distinguishable from individual dead peritoneal exudate cells or cell clumps (Figs. 4-5). DISCUSSION

Fluorescent probes are now generally accepted as an advantageous alternative to ra-

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munsoni:

FLUOROCHROME

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FIGS. 4, 5. Dead Schisrosoma mansoni schistosomulum and both living and dead mouse peritoneal exudate cells after diamidinophenylindole incubation, as visualized by transmitted light microscopy (Fig. 4) and by a combination of transmitted light and incident fluorescent light.

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diolabels in a wide variety of cytotoxicity perior to those stains which can only be tests involving individual cells (e.g., HLAused on samples taken from a schistosoDR typing). One of these fluorochromes, mula suspension in an experiment, like toDAPI, has the advantage of emitting a luidine blue or eosin. bright blue fluorescence, thus enabling a Other advantages of DAPI are that no rapossible combination with other fluorodioactive or toxic labels have to be used; that it can be used in a low concentration chromes, such as the red TRITC. DAPI also shows an excellent penetration of dead (1 pg/ml), which makes it a cheap reagent; cells and binding to DNA (Tanke ef al. that there is no background fluorescence of 1982). DAPI penetration is dependent on DAPI in the test suspension; and that the the damage of the cell membrane, and bright blue DAPI fluorescence easily surTanke et al. have shown by means of fluo- passes the autofluorescence of the plastic tray. rescence activated cell sorting that living lymphocytes are not stained at all. While Although our initial experiments with basic data on the use of DAPI have been schistosomula and mouse eosinophil-enobtained with individual cells, it seemed riched peritoneal exudate cells clearly likely that similar results could be obtained showed that fluorescent dead schistosowith multicellular organisms, although dif- mula and cells can easily be differentiated, of fusion of the fluorochrome and the fluores- a further analysis of the applicability DAPI in cytotoxicity systems using difcence through several cell layers might prove to be a problem. ferent effector cell types has to be carried In the present study we have tested the out. applicability of DAPI as a probe for damage REFERENCES to Schistosoma mansoni schistosomula. Artificially killed schistosomula showed a BUTTERWORTH, A. E., TAYLOR, D. W., VEITH, M. C., bright fluorescence, without leakage of the VADAS, M. A., DESSEIN, A., STURROCK, R. E, fluorochrome into the medium, in contrast AND WELLS, E. 1982. Studies on the mechanisms of to observations with the fluorochromes EB immunity in human schistosomiasis. Immunological Reviews 61, 5-39. and PI. Damaged schistosomula showed EDEDIN, M. 1970. A rapid quantitative fluorescence local fluorescence near the damaged reassay for cell damage by cytotoxic antibodies. The gion. Journal of Immunology 104, 1303-1306. As the fluorescence caused by DAPI is GARCIA, I?, RODRIGUEZ, J. L., AND VINAS, J. 1971. A new rapid and more sensitive microcytotoxicity extremely bright, suspensions of schistotests. The Journal of Immunological Methods 1, somula can easily be studied under test con303-307. ditions (i.e., in a tissue culture tray) on an KAPUSCINSKY, J., AND SKOCZYLAS, B. 1978. Fluoresinverted fluorescence microscope using sicent complexes of DNA and DAPI 4’-6-diamidinomultaneous illumination with transmitted 2-phenylindole. 2HCl or DC1 4’-6-dicarboxyamide(normal) light and incident fluorescence 2-phenylindole. Nucleic Acids Research 5, 37753799. (UV) excitation for DAPI fluorescence. In this way, living and dead schistosomula can LIN, M. S., COMINGS, D. E., AND ALFI, 0. S. 1977. Optical studies of the interaction of 4’-6-Diamidinoeasily be differentiated. Even after pro2-Phenylindole with DNA and metaphase chromolonged incubation, DAPI did not show any somes. Chromosoma 60, 15-25. toxic effect to schistosomula, as had al- RAMALHO-PINTO, F. J., GAZZINELLI, G., HOWELLS, R. E., MOTA-SANTOS, T. S., FIGUEIREDO, E. A., ready been proven for individual lymphoAND PELLEGRINO, J. 1974. Schistosoma mansoni: A cytes (Tanke et al. 1982). This permits the defined system for the step-wise transformation of use of DAPI for continuous monitoring of cercaria to schistosomule in vitro. Experimental damage to schistosomula in cytotoxicity Parasitology 36, 360-372. tests. As such, this fluorescent probe is su- ROTMAN, B., AND PAPERMASTER, B. W. 1966. Mem-

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brane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proceedings of the National Academy of Sciences (USA) 55, 134-141. STOEHR, M., AND VOGT-SCHADEN, M. 1979. A new dual staining technique for simultaneous flow cytometric DNA analysis of living and dead cells. In “Proceedings of the Fourth International Symposium on Flow Cytometry” (0. D. Laerum, T. Lindmd, and E. Thorud, eds.), pp. 96-99. Universitetsforlaget, Bergen.

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W. G., AND LANfluorochromes to ethidium bromide for automated read out of cytotoxicity tests. Journal of Immunological Methods 52, 91-96.

TANKE, H. J., VAN DER LINDEN, GERAK, J. 1982. Alternative

P.

VAN DER LINDEN, P. W. G., KOERTEN, H. K., AND DEELDER, A. M. 1982. Scanning electron micro-

scopical observations on antigen-antibody coat formation on mechanically transformed Schistosoma mansoni schistosomula. Zeitschrift fir Parasitenkunde 68, 73-80.