Comparative evaluation of the isotopes 86Rb and 51Cr in a humoral cytotoxicity system

Journal o f lmmunological Methods 6 (1974) 23-29

© North-Holland Publishing Company

COMPARATIVE EVALUATION 8 6 R b a n d SICr I N A H U M O R A L

OF THE ISOTOPES CYTOTOXICITY SYSTEM

Ph. CHOLLET, J.M. BIDET, R. PLAGNE and B. SAUVEZIE With technical collaboration of Mrs Gilberte BARGOIN* and Miss Christine DELOSSEDAT** * INSERM, Unite 71, ** Centre de Lutte Contre le Cancer, B.P. 392, Centre de Transfusion Sanguine and Centre Hospitalier Universitaire, 63000 Clermont-Ferrand, France

Received 21 January 1974, accepted 10 May 1974 A comparative study of 86Rb and s 1Cr in a humoral cytotoxic system has been performed. Lymphocytes obtained from normal donors were separated in a gradient centrifugation system and labelled in vitro with isotope. In each tube, 106 lymphocytes were exposed to the action of antilymphocyte serum, and the activity remaining in the cells was determined and compared with that in controls. The fraction of marker not released is much larger with chromium than with rubidium; 86Rb release is very fast, and nearly complete after 30 min, as shown by comparison with physical lysis techniques. The ratios control/experiment are usually 2:1 with chromium and 4:1 or more with rubidium, despite higher spontaneous release of the latter due to passive diffusion through the cell wall. It appears that 86 Rb gives a more consistent and more sensitive method than s ~Cr.

1. Introduction Lymphocytotoxic methods provide useful assays for screening alloantibodies against leucocytes. These combine specifically with lymphocyte antigens and, if complement is present, the cell membrane is damaged. Several techniques are used to demonstrate this phenomenon: Trypan Blue Dye Exclusion (Terasaki and McClelland, 1964; Walford et al., 1964), use of fluorogenic substrates (Rotman and Papermaster, 1966; Marcelli-Barge et al., 1973), release of s l Cr by labelled cells (Sanderson, 1964). The results obtained by these methods are similar, and they have equal sensitivity (Mayer and Tongio, 1971). Attempts are now being made to find more sensitive techniques in order to display new antigens on the cell surface of lymphocytes, and we report here a comparative evaluation of 86 Rb with s 1 Cr in a system using in vitro lymphocyte membrane lysis by polyspecific antibody. Lymphocytes obtained from normal donors, and purified by a centrifugation gradient technique, were 23

24

Ph. Chollet et aL, Comparative evaluation of 86Rb and Sl Cr

divided and labelled either with s6 Rb or S~Cr. The cytotoxicity test was then performed using antilymphocyte serum (ALS) as polyspecific antibody source.

2. Materials and methods ALS was obtained from a commercial source (IMMOSSAR*, CHOAY Laboratories). One hundred ml of blood was drawn in a sterile flask containing 1 ml of heparin. 2.1. L y m p h o c y t e preparation

Lymphocyte separation was performed by centrifugation in a mixture of sodium metrizoate (9.6%) and Ficoll* (5.6%) having a final specific gravity of 1.077 (LYMPHOPREP*). A 100 ml of blood was spun at 400 g for 20 rain. Leucocyte-rich plasma and buffy coat were aspirated and resuspended. Four to five ml of this suspension were added to 3 ml of the above mixture. After centrifugation at 400 g for 30 rain, the supernatant was aspirated. The lymphocyte layer was resuspended in 2 0 - 3 0 ml of phosphate-buffered saline (PBS), pH 7.2. The cells were washed three times with PBS, and the lymphocytes resuspended in the following buffer (I): 0.15 M NaCI; 0.5 mM KC1; 1 mM MgC12 ; 1 mM sodium phosphate buffer, pH 7.4; 2.5 mM CaCI2 ; 0.1% glucose; 1% calf serum, decomplemented and filtered by Millipore filtration (0.22 ~). This buffer (I) has a low concentration of KC1, in order to enhance the entry of 86 Rb, which is competitive with that of potassium (Walker and Lucas, 1972). The same buffer (I) was used to adjust the cellular suspension to contain 10,000 lymphocytes per mm 3 . 2. 2. Labelling with 8 6Rb

1.5 ml of a cell suspension containing 15 × 10 6 cells were incubated in an Erlenmeyer flask with 100oCi of 86Rb (rubidium chloride, specific activity 1-2 /aCi//lg), in 0.1 ml of the same buffer (I). Incubation was performed with shaking at 37°C in a water bath for 90 rain (Walker and Lucas, !972). The suspension was then diluted to 10 ml with buffer lI. This buffer had the same composition as buffer I, except that it contained 5 mM KC1, this higher physiological extracellular potassium concentration tending to reduce effiux of 86Rb (Walker and Lucas, 1972). The labelled lymphocytes were washed twice with 10 ml of buffer II, cooled in melting ice, then spun down at 400 g for 8 min and resuspended in 1.5 ml of buffer II. A small quantity of this suspension was used to determine the number of cells, and to verify their viability by Trypan Blue Dye Exclusion.

Ph. Chollet et al., Comparative evaluation o f 86Rb and SlCr

25

2. 3. Labelling with s 1 Cr

1.5 ml of the same cell suspension were incubated in the same manner with I00/ICi of 5 a Cr (sodium chromate, specific activity 5 0 - 2 0 0 ~Ci//ag Cr) in 0.1 ml of buffer I. Incubation was performed at 37°C with shaking in a water bath, for 45 rain. The suspension was then diluted to 10 ml with buffer II. The labelled cells were washed three times with 10 ml of buffer If, cooled in melting ice, and spun down at 4 0 0 g for 8 min and resuspended in 1,5 ml of buffer II. The number of remaining cells and their viability were determined in the same way. 2.4. C y t o t o x i c i t y test with A L S

This test was performed in the same manner with both isotopes. Each tube contained 106 viable labelled lymphocytes in 100/11 of buffer II. For each isotope the following were prepared: 6 tubes with 100 IA of pure ALS and 100/A of complement (a frozen stock of pooled rabbit serum was used; several dilutions were tested and the dilution used always gave 100% cell lysis, as judged by Trypan Blue Dye Exclusion); 8 control tubes with 100/~1 of human serum AB, and 100/11 of complement. The tubes were incubated at 37°C for 30 min (86Rb) or for 1 or 2 hr (StCr). After incubation further centrifugation at 400g for 8 min to separate cells and supernatant was carried out. The cells and supernatant were then counted in a two channel gamma counter (Nuclear Chicago). At the beginning of the experiment, a control tube was spun down and counted immediately, so as to make a control 'time 0'. Results were expressed as percent of total activity remaining in the cells at the end of the experiment. In order to make valid comparisons, percentages were not calculated on the basis of the zero time sample being zero percent lysis or the sample obtained by physical methods being I00% lysis, but on the actual experimental values. Total lysis samples were prepared by two methods: repeated freezing and thawing in liquid nitrogen; and hypotonic shock with distilled water, so as to induce a maximal release of the isotope. The final cellular viability was tested by Trypan Blue Dye Exclusion.

3. Results We confirmed the fact that incorporation of a 6Rb is significantly higher when using buffer I, which contained a low concentration of KC1 (Walker and Lucas, 1972). To be of use, each experiment needed a Trypan Blue Dye Exclusion test to be carried out at the beginning and end of the procedure, for both control and ALS-

26

Ph. Chollet et al., Comparative evaluation of 86Rb and 51Cr

Table 1 Cytotoxicity with ALS: Comparison of 86Rb and 5 ZCr Percent cpm remaining in cells at the end of experiment Rubidium

Chromium

Incubation 30 Min ALS 12.5 10.9 13.7 12.7 13.9 13.6 12.9 12 10.6 10.8 6.8 7.8 14.8 10.9 11.7 6.4

Control 56.6 57 47.8 63.8 49.4 40.9 50.7 41.2 57.6 40.5 39 40.3 37.8 45.8 56.9 51.8

Experiment number 11 14 15 17 18 19 20 24 25 29 30 32 33 34 35 36

Incubation 1 hr

Incubation 2 hr

ALS

Control

ALS

-

38.6 33.5 34.7 34 38.5 33.6 33.7 40.2 35.6 32.9 35.8 30.6 34.9 40.4 43

69.9 74 87.1 77.2 79.7 80.1 41.7 59.8 70.8 55.9 50 64.5 52.6 79.4 82.9

35.9 -+ 4,1

66.3 -+13.8

Control

31.7 31.8 32.6 32.3 33.2 33.2 31.5

69.7 62 69.5 82.4 75.5 76.9 74.8

-

-

32.4 -+ 1.8

73 -+ 6.5

Mean -+ S.D. 11.7 + 2,9

47.7 -+ 9.1

t r e a t e d t u b e s . W h e n the p r o p o r t i o n o f cells taking up dye was m o r e t h a n 1 0 - 1 5 % b e f o r e labelling, or in the c o n t r o l t u b e s at the end o f t h e p r o c e d u r e , the e x p e r i m e n t was discarded. Usually t u b e s c o n t a i n i n g ALS, w h e n t e s t e d for dye exclusion at the e n d o f the e x p e r i m e n t , s h o w e d 100% dye u p t a k e w i t h r u b i d i u m as well as w i t h c h r o m i u m . A n t i l y m p h o c y t e s e r u m always gave the same result, being p o l y s p e c i f i c in vitro at the c o n c e n t r a t i o n used. C o m p a r a t i v e results o b t a i n e d w i t h r u b i d i u m and c h r o m i u m are listed in table 1 and p l o t t e d in fig. 1, T h e y s h o w t h a t the c y t o t o x i c e f f e c t o f ALS can be d e m o n s t r a t e d w i t h b o t h isotopes. Total lysis b y the t w o d i f f e r e n t m e t h o d s gave equivalent results listed in table 2. The p e r c e n t o f tracer n o t released on t o t a l lysis was less t h a n 10% w i t h r u b i d i u m , b u t varied b e t w e e n 20 and 30% w i t h c h r o m i u m .

Ph. Chollet et al., Comparative evaluation of 86Rb and SlCr

,o!

27

8 6 Rb

60-

50-

"

40.

=

J.

I

30-

20.

10. ÷

0

r

10

i

90

+

~

30

40

r

50

~

60

70

i

f

80

90

r

100

51Cr

Fig. 1. Percent radioactivity remaining in cells labelled with ~ 6 Rb or s l Cr after ALS (+) in human AB serum (,),

Table 2 Total tysis controls - eight samples using two different methods of lysis. Rubidium 1

Chromium 2

9.6 10.1 6.4 7.9 7.8 8.7 10.5 8.3

1 10.2 7.9 5.4 5.2 7.4 5.4 8.6 9.6

2 25.9 28.2 27 24.7 25.7 27.4 24.8 26

30.3 28.7 32.8 28.1 22.5 26.3 -

Mean -+ S.D. 8.7 +- 1.4

7.5 +- 2.0

+-

26.2 1.2

28.1 ± 3.5

Figures are percent ¢pm remaining in cells at the end of experiment: 1 - repeated freezing and thawing in liquid nitrogen; 2 - hypotonic shock against distilled water.

28

Ph. Chollet et al., Comparative evaluation o f 86Rb and Sl Cr

4. Discussion Chromium becomes bound to cellular proteins in a non-specific, although firm way, while rubidium is actively concentrated by the living cell membrane in the intracellular medium, in the same way as potassium (Kahn Jr., 1962; Bernstein and Israel, 1970; Gilbert, 1970). This active gradient is similar to that for K, established by the ' N a - K pump', a physiologic mechanism of some complexity involving an enzyme of the cell membrane, N a - K ATPase, which is inhibited by cardiotonic glycosides (Dunham and Gunn, 1972; Parker and Welt, 1972). In addition to this active mechanism, there is also a passive transfer by simple diffusion through the cell wall, in both directions. It was for this reason that the cells labelled with s 6 Rb were washed only twice; each new washing caused 86 Rb efflux, and decreased the lymphocyte labelling. This did not occur with s ~Cr. 86 Rb efflux was always present in control tubes after an incubation of thirty minutes. The lymphocyte labelling time is longer with 86 Rb, but rubidium release after the cytotoxic action of ALS is much quicker than chromium release by damaged cells (Green et al., 1959; Hingson et al., 1969; Hoffmann, 1969; Henney, 1973). The results in table 1 demonstrate some advantages of rubidium over chromium: The fraction of the marker not released on total cell lysis is much larger with chromium (20-30%) than with rubidium (about 10%). Rubidium release was fast and nearly complete; chromium release was slower and a considerable fraction remained bound to cellular elements (Sanderson, 1964). As a consequence, the difference between control tubes and ALS-containing tubes is much larger with rubidium than with chromium; the ratio of control to experimental values was usually 2:1 with chromium, but at least 4:1 with rubidium, reaching 5:1 or 6:1, despite the fact that spontaneous release of the marker in controls was higher with rubidium than with chromium. Cooling with ice during washing of labelled cells gave better results than washing at room temperature in agreement with Walker and Lucas (1972). Control tubes containing 10 6 labelled lymphocytes with 200 tal of minimum Eagle's medium, or 200/al of complement alone, or 100/al of calf serum and 100/al of complement, gave similar results to standard control tubes containing 100/al of human serum AB and 100/al of complement. From time to time chromium labelling was poor, with a low level in control tubes. These cells were not eliminated from the study, because rubidium and chromium tests were performed simultaneously and in a similar way, and the rubidium test in parallel gave a normal result. Such fluctuations do not modify the validity of the comparison. In our hands, the rubidium test is more consistent and more satisfactory than the chromium test.

Ph. Chollet et al., Comparative evaluation of S6Rb and StCr

29

References Bernstein, J.C. and Y. Israel, 1970, J. Pharmacol. Exptl. Ther. 174,323. Dunham, P.B. and R.B. Gunn, 1972, Arch. Intern. Med. 129,241. Gilbert, I.G.F., 1970, J. Membrane Biol. 2, 277. Green, H., P. Barrow and B. Goldberg, 1959, J. Exptl. Med. 110,699. Henney, C.S., 1973, J. Immunol. 110, 73. Hingson, D.J., R.K. Massengil and M.M. Mayer, 1969, Immunochemistry, 6,295. Hoffmann, L.G., 1969, lmmunochemistry 6,309. Kahn Jr., J.B., 1962, J. Pharmacol. Exptl. Therap. 136, 197. MarceUi-Barge, A., A. Benajam, J.C. Poirier, L. Beraud and J. Dausset, 30 Mai-2 Juin 1973, IXe Cong. Nat. Transf. Sang. Nancy. Mayer, S. and M.M. Tongio, 1971, Rev. Fr. Transf. 14, 147. Parker, J.C. and L.G. Welt, 1972, Arch. Intern. Med. 129,310. Relman, A.S., 1956, Yale J. Biol. Med. 29,248. Rotman, B. and B.W. Papermaster, 1966, Proc. Natl. Acad. Sci. U.S, 55,134. Sanderson, A.R., 1964, Nature 204,250. Terasaki, P.I. and J.D. McClelland, 1964, Nature 204,928. Walford, R.L., R. Gallagher and J.R. Sjaaraa, 1964, Science 144,868. Walker, S.M. and Z.J. Lucas, 1972, J. Immunol. 109, 1223.