Modified procedure for labelling target cells in a europium release assay of natural killer cell activity

Journal oflmmunological Methods, 161 (1993) 135-137

135

© 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00

JIM 06686

Short communication

Modified procedure for labelling target cells in a europium release assay of natural killer cell activity * R o b e r t a Pacifici, Simonetta Di Carlo, Antonella Bacosi, Ilaria Altieri, Simona Pichini and Piergiorgio Zuccaro Clinical Biochemistry Department, Istituto Superiore di SanitY, Rome, Italy (Received 9 June 1992, revised received 12 January 1993, accepted 20 January 1993)

Lanthanide europium chelated to diethylenetriaminopentaacetate (EuDTPA) can be used to label target cells such as tumor cells and lymphocytes (Blomberg et al., 1986a,b; Granberg et al., 1988). This procedure has permitted the development of new non-radioactive methods for the detection of target cell cytolysis by natural killer (NK) cells (Blomberg et al., 1986a,b), cytotoxic T lymphocytes (CTL) (Granberg et al., 1988) or complement-mediated cytolysis (Cui et al., 1992). However, we had no success with this method because of a lack of comparability between human NK cell activity simultaneously measured by a classical 51Cr release assay (Seaman et al., 1981) and EuDTPA release assay (Blomberg et al., 1986a). Furthermore, cell division and cell viability were significantly impaired by the suggested concentrations of EuC13. In this paper, we present a modified non-cytotoxic method for target cell labelling with EuDTPA while cells are growing in culture medium. Key words: Europium labeling; Time-resolved fluorescence; Natural killer cell activity

The mycoplasma free human erythroleukaemic cell line, K562, was used as a source of target cells. Approximately 1.5 x 105 target cells/ml were grown in RPMI 1640 medium (Gibco, Paisley, England) containing penicillin (100 U/ml), streptomycin (100 mg/ml) and 10% fetal bovine serum (Flow Laboratories, Irvine, England) (complete medium), supplemented with a labelling mixture containing 25 mM Hepes buffer (Flow Laboratories), 30 mM NaC1, 0.5 mM KC1, 0.3 mM MgCI2, 20 /xg/ml dextran sulphate, 5 /zM

Correspondence to: R. Pacifici, Clinical Biochemistry Department, Istituto Superiore di Sanit?~, V.le Regina Elena 299, Rome, Italy. Tel.: 06-4990; Fax: 06-4461961. * This study was supported by a grant from the Ministry of Health - Istituto Superiore di Sanit~ - AIDS Project 1992.

DTPA (neutralized in NaOH) and 1 /~M EuCI 3. Half of the culture medium was changed every second day to ensure good growth and labelling. After 4 days of incubation, target cells labelled with this modified method, were washed three times with cold RPMI 1640 medium and resuspended in complete medium. Various concentrations of peripheral blood mononuclear cells from healthy volunteers were incubated with 5 x 10 4 K562 cells, labelled with SlCr or EuDTPA using both labelling procedures for 2-4 h in a 5% CO 2 atmosphere in microtiter plate wells at 37°C. Specific marker release from the NK sensitive K562 target cells using the 2 and 4 h cytotoxicity assay is shown in Fig. 1. The cytotoxicity values for the EuDTPA assay were higher than the corresponding mean values of the modified Eu-

136 100 -

2-HOUR-ASSAY

80-

60-

4.0-

2o,t

o I

6:1

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100 -

I

I

12:1

25:1

I

I

50:1 100:1 E.T. ratio

/+-HOUR-ASSAY

80-

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60-

4.0-

20-

I

i

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6:1

12:1

25:1

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E.T. ratio

Fig. 1. Percent specific marker release from target cells (K562) at different E : T ratios. NK activity of the same donors was determined by the EuDTPA assay (o), the modified EuDTPA assay (e) and 51Cr assay (zx). Incubation time: 2 h (top) and 4 h (bottom). The means±SD of 20 donors are plotted.

DTPA release assay and 51Cr release assay, both run simultaneously, The effect of several EuCI 3 concentrations on the growth and viability of the target cells was determined by incubating 1 × 104 K562 target cells in 10 ml of complete medium supplemented with 30 mM NaC1, 0.5 mM KC1, 0.3 mM MgCI 2, 2 0 / z g / m l dextran sulphate, 5 / z M DTPA and 1, 10 and 20 ~M EuC13, respectively. The different staining of viable target cells was evaluated by methylene blue incorporation and the growth of target cells was examined by optical microscopy. The best experimental results were obtained using a concentration of 1 /zM EuCI 3 (Table I). The labelling of target ceils with a concentration of 1 /zM EuCI 3 and 5 / z M DTPA in culture medium increased in a time-dependent fashion. After 1 day of growth in EuDTPA supplemented culture medium, the maximum release of fluorescence was only 10,000 + 4000 cps. However, after 4 days of growth it was 103,400 + 35,000 cps. The spontaneous release observed was not of the same order of magnitude for the different labelling conditions. After 24 h, it was significantly higher in the EuDTPA release assay (73.5%) than in the 51Cr release assay (15.4%), or the modified EuDTPA release assay (16.8%). In our study both cell division and viability were significantly modified by the presence of EuC13 at 10 and 20 ~M concentrations as suggested by Blomberg's method. Conversely, cellular physiology was apparently not compromised by a EuCI 3 concentration of 1 izM.

TABLE I EFFECT OF SEVERAL EuCI 3 CONCENTRATIONS ON THE GROWTH AND VIABILITY OF TARGET CELLS Numbers represent means ± SD of ten determinations. Incubation

Cell number ( × 104 )

Cell viability (%)

time (h)

Eucl 3 (/zM)

EuCI 3 (#M)

0 2 4 10 24 48

0

1

10

20

0

1

10

20

1.0±0.1 1.3±0.1 1.5±0.1 2.2±0.2 4.0±0.2 7.8±0.3

1.0±0.1 1.3±0.1 1.4±0.1 2.3±0.2 3.8±0.2 8.0±0.3

1.0±0.1 1.0±0.1 1.0±0.1 2.0±0.2 3.0±0.2 4.8±0.2

1.0±0.1 1.0±0.1 1.0±0.1 1.9±0.2 2.5±0.2 3.0±0.2

100±2 100±3 100±2 100±2 97±1 90±2

1~±2 1~±2 100±3 100±2 90±2 87±1

100±2 91±3 88±1 79±3 60±1 51±2

1~±3 89±2 78±4 65±2 48±2 24±1

137 A c o n c e n t r a t i o n o f 1 / z M E u C I 3 was sufficient to label t a r g e t cells if u s e d to s u p p l e m e n t c u l t u r e m e d i u m d u r i n g t h e 4 days o f c e l l u l a r growth, a n d t h e E u D T P A i n c o r p o r a t i o n by t a r g e t cells p e r m i t t e d m e a s u r e m e n t s o f N K cell activity by t h e E u D T P A r e l e a s e assay. T h i s m o d i f i e d m e t h o d d i d n o t a l t e r c e l l u l a r physiology b u t d i d i n c r e a s e t h e analysis time. W i t h this l a b e l l i n g p r o c e d u r e v a l u e s for b o t h s p o n t a n e o u s r e l e a s e a n d h u m a n N K cell activity w e r e similar to t h o s e o b t a i n e d with t h e c o m m o n l y u s e d 5~Cr r e l e a s e assay. T h e n e g a t i v e effect o f E u C I 3 o n t h e physiology o f t h e cells was also d e m o n s t r a t e d for t h e n o n a d h e r e n t t u m o r cell line M O L T - 4 f r o m a c u t e l y m p h o b l a s t i c l e u k a e m i a a n d for t h e a d h e r e n t t u m o r cells such as A L A B d e r i v e d f r o m b r e a s t c a n c e r a n d T-24 f r o m b l a d d e r c a r c i n o m a . T h i s effect was c o n c e n t r a t i o n - d e p e n d e n t in all cases. W h e t h e r o r n o t t h e labelling p r o c e d u r e p r o p o s e d in this study is also a d e q u a t e for a d h e r e n t cell lines will r e q u i r e f u r t h e r study.

References Blomberg, K., Granberg, C., Hemmila, I. and Lovgren, T. (1986a) Europium-labelled target cells in an assay of natural killer cell activity. I. A novel non-radioactive method based on time-resolved fluorescence. J. Immunol. Methods 86, 225. Blomberg, K., Granberg, C., Hemmila, I. and Lovgren, T. (1986b) Europium-labelled target cells in an assay of natural killer cell activity. II. Significance and specificity of the method. J. Immunol. Methods 92, 117. Cui, J. and Bystryn, J.C. (1992) An improved europium release assay for complement-mediated cytolysis. J. Immunol. Methods 147, 13-19. Granberg, C., Blomberg, K., Hemmila, I. and Lovgren, T. (1988) Determination of cytotoxic T lymphocyte activity by time-resolved fluorometry using europium-labelled concanavalin A-stimulated cells as targets. J. Immunol. Methods 114, 191. Seaman, W.E., Gindhart, T.D., Blackman, M.A., Dalai, B., Talal, N. and Werh, Z.(1981) Natural killing of tumor cells by human peripheral blood cells: suppression of killing in vitro by tumor promoting phorbol diesters. J. Clin. Invest. 67, 1324-1333.