[GANN, 62, ; April, 1971]

Transcription

1 [GANN, 62, ; April, 1971] UDC : EVALUATION OF CELL DAMAGE IN IMMUNE REACTIONS BY RELEASE OF RADIOACTIVITY FROM 3H-URIDINE LABELED For quantitative evaluation of cell damage in immune reactions, release of radioactivity from target cells labeled with 3H- or 14Cthymidine for DNA,6-9,13) radioactive amino acids for protein,2) and 32P-phosphate11) or 51Cr-chromate1,3,8,12,14) for cell constituents has been determined. However, each of these methods has some disadvantages such as spontaneous or incomplete release of radioactivity, reutilization of released radioactivity by viable cells, and cytotoxicity or short life of the isotope. In the present paper we report that release of radioactivity from 3H-uridine labeled cells well reflects cell damage in immune reactions in vitro. Isotope-labeled Test Cells Mouse ascites leukemia cells, RADA1 (strain A, H-2a CELLS*1 Yoshiyuki HASHIMOTO and Hisayo SUDO (Tokyo Biochemical Research Institute*2) obtained from mice transplanted with syngeneic tumor cells. Leukemia cells were washed once with RPMI 1640 medium and suspended in RPMI 1640 medium containing 10% heat-inactivated calf or rat serum. One volume of a solution of 3H-uridine (uridine- [5-3H]) or 3H-thymidine (thymidine[methyl- 3H]) (New England Nuclear, Mass., U.S.A.) in physiological saline was added to 10 volumes of the cell suspension containing 106cells/ml for 30min, the cells were sedimented by centrifugation at 1000rpm for 10min and then washed 3 times with the medium containing 10% serum. Washed cells were resuspended in the medium for test. Immune Sera and Immune Lymphocytes C57BL/6 mice (H-2b) were immunized with RADA1 leukemia cells and their sera were obtained 10 days after the last immunization. Peritoneal fluid lymphocytes and spleen cells were obtained from the immune mice 7 days after the last immunization. The method for cell fractionation has been reported in a previous paper.5) Guinea pig serum used as the complement was obtained from a frozen stock of fresh serum which was nontoxic to mouse leukemia cells. Cytotoxicity Test with Immune Sera A mixture of 0.1ml of a test cell suspension RPMI 1640 medium, 0.1ml of serially diluted immune serum, and 0.1ml of 1:3 diluted min. The reaction was stopped by immersing the test tubes into an ice-water bath, 0.2ml of the medium was added to the mixture, and 0.4ml of the mixture was filtered through size, 24mm diameter). The filter was washed with 20ml of cold 5% trichloroacetic acid 10ml of toluene base scintillator (POP 5g and POPOP 0.3g in 1 L of toluene) was added. The radioactivity was determined in a liquid scintillation counter, Nuclear Chicago Mark I. Extent of target cell death was calculated from the following equation: Target cells died (%)=100-(test sample cpmbackground/serum control cpm-background) For visual counting of viable and killed cells, 0.1ml of Trypan Blue solution was added to 0.1ml of the reaction mixture, and stained

2 Y. HASHIMOTO AND H. SUDO H-2a leukemia cells H-2d leukemia cells and unstained cells were counted under a microscope in a Burker type hemocytometer. Cytotoxicity Test with Immune Lymphocytes A mixture of 0.25ml of a suspen- ml and 0.25ml of a suspension containing in a CO2-incubator. As a control, target cells alone were cultured in the same concentration of cells as the test samples. Medium used was RPMI 1640 supplemented with 15% heatinactivated rat serum, 100U/ml of penicillin, At specified intervals the tubes were taken out from the incubator and 0.4ml of the cell mixture was filtered through a Millipore membrane filter. Radioactivity of the sample was determined as described above. Extent of target cell death was calculated from the following equation: Target cells died (%)= 100-(test sample cpm-background/control Fig. 1. Titration with immune serum against target leukemia cells Visual counting of viable target cells was carried out under a phase-contrast microscope in a Burker type hemocytometer. Cytotoxicities of Immune Serum and Immune Lymphocytes Based on Isotope Releasing Method and Visual Counting Results of titration with C57BL/6 anti- RADA1 leukemia cell serum (H-2b anti- leukemia cells are shown in Table I and in Fig. 1. Percentages of dead leukemia cells calculated from radioactivity released from 3H-uridine-labeled leukemia cells well reflected the results obtained by visual counting. More than 80% of radioactivity was released from the target cells when 100% of the cells were dead according to visual counting. Initial radioactivity of 3H-uridine-labeled was 17,500-20,000 and 12,500-15,000cpm/ 106 cells, respectively. Table II and Fig. 2 illustrate cell damage by cell-mediated immune reaction, in which peritoneal lymphocytes obtained from the same immune mice as the serum donor were cultured with RADA1 leukemia cells. Percentages of dead target cells determined by the two methods were in good agreement. On the other hand, as shown in Fig. 3, release of radioactivity from 3H-thymidinelabeled leukemia cells was incomplete both in cytotoxic reactions with immune serum and immune lymphocytes. Reutilization of Released Radioactivity by Viable Leukemia Cells A suspension of 3H-uridine-labeled RADA1 leukemia cells was irradiated with ultraviolet ray for 30min 140 GANN

3 CELL DAMAGE IN IMMUNE REACTIONS Table I. Titration with H-2b anti-h-2a Serum to H-2a Leukemia Cells S=serum control C'=complement control. Cf. Fig. 1. Table II. Cytotoxicity of H-2b anti-h-2a Immune Peritoneal Lymphocytes against H-2a Leukemia Cells Data indicated are average of 3 test samples. a) Net count-background (19cpm). Cf. Fig. 2. Table III. Reutilization of Radioactivity Released from Ultraviolet Irradiated Cells by Viable Leukemia Cells Data indicated are average of 2 test samples. *RADA1=3H-uridine-labeled RADA1 leukemia cells. at 20-cm distance from a 15-W lamp. The irradiated leukemia cells were incubated with or without untreated RADA1 leukemia cells, and the radioactivity of cultured cells was determined after 48hr. As shown in Table III, a large percentage of radioactivity released from the irradiated leukemia cells was reutilized by the untreated leukemia cells which had not been labeled. The principle of the present 3H-uridine method is that RNA of target cells labeled with 3H-uridine is released from the cells after their death, majority of the released RNA is washed out with trichloroacetic acid solution through a Millipore membrane filter, and radioactivity retained only on viable cells can be determined. Green et al.4) have reported that exposure of tumor cells to antibody plus complement resulted in a loss of about 3/4 of the cell RNA into the medium. A similar result was obtained in the present experiments; 80-90% of radio- 62(2)

4 Y. HASHIMOTO AND H. SUDO Fig. 2. Cytotoxicity of H-2b anti-h-2a immune peritoneal lymphocytes against H-2a leukemia cells Fig. 3. Cytotoxicity of H-2b anti-h-2a immune serum and immune spleen cells against H-2a leukemia cells Comparative results of release of radioactivity from leukemia cells. Ab; 1:64 diluted immune serum in Table I. s-spc; immune spleen cells from the same origin as the immune serum. activity was released from 3H-uridine-labeled leukemia cells when they were killed by immune serum plus complement. Radioactivity of target lukemia cells labeled with 3H-uridine was well preserved during the incubation period as shown in Table II, and even after 48hr the radioactivity was not changed, if the cells remained viable during the incubation. Increase of radioactivity in labeled leukemia cells seen in the first few hours may be due to incorporation of acid-soluble isotope retained on the cells into cytoplasmic constituents of the cells. The present 3H-uridine method for evaluation of cell damage is simple, sensitive, and quantitative, and the prepared specimens are stable for a long period. This method will be valuable not only for immunological experiments but also for other types of tests such as determination of cytotoxic activity of carcinostatic agents. However, it is not suitable for experiments in which cells grow progressively over a relatively long period of experimental time, because the released radioactivity will be utilized by the viable cells. (Received December 12, 1970) 142 GANN

5 CELL DAMAGE IN IMMUNE REACTIONS REFERENCES 1) Berke, G., Ax, W., Ginsberg, H., Feldman, M., Immunology, 16, 643 (1969). 2) Bickis, I. J., Quastel, J. H., Vas, S. I., Cancer Res., 19, 602 (1959). 3) Brunner, K. T., Mauel, J., Cerottini, J. C., Chapuis, B., Immunology, 14, 181 (1968). 4) Green, H., Fleischer, R. A., Barrow, P., Goldberg, B., J. Exptl. Med., 109, 511 (1959). 5) Hashimoto, Y., Sudo, H., GANN, 59, 7 (1968). 6) Holm, G., Perlmann, P., Nature, 207, 818 (1965). 7) Holm, G., Perlmann, P., Werner, B., ibid., 203, 841 (1964). 8) Holm, G., Perlmann, P., Immunology, 12, 525 (1967). 9) Klein, G., Perlmann, P., Nature, 199, 451 (1963). 10) Old, L. J., Boyse, E. A., Stockert, E., J. Natl. Cancer Inst., 31, 977 (1963). 11) Perlmann, P., Broberger, O., J. Exptl. Med., 117, 717 (1963). 12) Sanderson, A. R., Nature, 204, 250 (1964). 13) Vanio, T., Koskimies, O., Perlmann, P., Perlmann, H., Klein, G., Nature, 204, 453 (1964). 14) Wigzell, H., Transplantation, 3, 423 (1965). 62(2)