Enzyme release assay of human NK cell activity using β-galactosidase-expressing K562 target cell line

Journal oflmmunological Methods, 164 (1993) 131-135

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

JIM06813

Short communication

Enzyme release assay of human NK cell activity using fi-galactosidase-expressing K562 target cell line Hitoshi Ohmori, Hidenori Ikeda, Takahiro Tanigawa, Toshiyuki Takai and Masaki Hikida Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700, Japan (Received 22 March 1993, revised received 8 June 1993, accepted 9 June 1993)

In the present report, we established a K562 cell line useful for an enzyme release assay of human natural killer (NK) activity. Human myelogenous leukemia cell line, K562, was transfected with a plasmid carrying Escherichia coli/3-galactosidase (/3-gal) gene. A colony that permanently expresses the enzyme activity was isolated, and designated K562/Zneo. Incubation of K562/Zneo cells (1 × 104) with nonadherent human peripheral blood lymphocytes (PBL) resulted in the release of fl-gal activity depending on the incubation time and the number of effector cells. Released fl-gal activity was assayed sensitively by using 4-methylumbelliferyl-fl-D-galactoside, a fluorescent substrate. The cytolytic activity of PBL was augmented significantly when the cells were preincubated with interleukin-2 for 20 h. This enzyme release assay showed a comparable sensitivity to that of 5tCr release assay. Thus, K562/Zneo cell line is thought to be useful for the nonradioactive assay of human NK and lyrnphokine-activated killer activities. Key words: Natural killer cell; Lymphokine-activated killer cell;/3-Galactosidase; K562 cell

Introduction

Natural killer (NK) cells play a vital role in vivo in preventing the development and metastasis of tumors (Herberman and Ortaldo, 1981; Grimm et al., 1982). The assay of these effector cells has been carried out by using target cells that were labeled with radioactive sodium [51Cr]chromate. K562, a human myelogenous leukemia cell line, is one of the most widely used

Correspondence to: H. Ohmori, Department of Biotechnology, Faculty of Engineering, Okayama University, TsushimaNaka, Okayama 700, Japan. Fax: 81-86-253-7399. Abbreviations: E l T ratio, effector to target ratio; /3-gal, /3-galactosidase; 4-MU, 4-methylumbelliferone; 4-MUG, 4methylumbelliferyl-fl-D-galactoside; PBL, peripheral blood lymphocytes.

target cell lines (Ortaldo and Longo, 1988). Although this is a very sensitive assay procedure, the use of radioactive chromate is hazardous, and one must prepare radiolabeled cells in every experiment. An enzyme release assay using lactate dehydrogenase (LDH) as an indicator enzyme has been reported (Korzeniewski and Callewaert, 1983). Since LDH is present both in target and effector cells, it appears more appropriate to select an enzyme that is exclusively expressed in the target cells. Recently, we have established a murine myeloma cell line, P3/NS1-Ag4-1, that permanently expresses Escherichia coli /3-galactosidase (fl-gal), and found that this cell line is useful for assaying H-2d-specific cytotoxic T cell activity by the release of fl-gal as an indicator enzyme (Ohmori et aL, 1992). The use of/3-gal is advantageous because this enzyme activity is very

132 stable under the assay conditions, and can be measured sensitively using 4-methylumbelliferyl/3-D-galactoside (4-MUG), a fluorescent substrate (Ishikawa and Kato, 1978). In the present report, we describe the establishment of a K562 cell line that permanently expresses /3-gal and its usefulness as the target cells for the measurement of human natural cytotoxicity.

Materials and methods

Cell line K562, a human myelogenous leukemia cell line was obtained from Riken Cell Bank, Tsukuba, Japan. Materials A plasmid, pRoZtk, that allows the expression in mammalian cells of E. coli /3-gal gene under the control of Rous sarcoma virus long terminal repeat was presented by Prof. H. Kondoh (Nagoya University, Nagoya, Japan). pSV2neo was obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan). Other materials were purchased from the following sources: 4MUG and 4-methylumbelliferone (4-MU) from Sigma (St. Louis, MO); RPMI 1640 medium from Nissui Seiyaku (Tokyo, Japan); fetal calf serum (FCS) from Gibco (Grand Island, NY); sodium [51Cr]chromate from New England Nuclear (Boston, MA). The culture medium for K562 cells was RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U / m l penicillin G, 50 /zg/ml streptomycin and 1 x 10 -5 M 2-mercaptoethanol. Transfection and establishment of stable transformant K562 cells (5 x 106) were suspended in 500/zl of serum-free RPMI 1640 medium, and plated into two wells in a 24 well culture plate (Nunc, Roskilde, Denmark) at 250 /xl/well. Then each well received 250 /xl of serum-free RPMI 1640 medium containing 10% (v/v) lipofectin reagent (Bethesda Research Laboratories, Bethesda, MD), 8 /xg of pRoZtk that was linearized by ScaI and 4/zg of pSV2neo that was linearized by EcoRI. After incubation of the plate at 37°C for

11 h in an atmosphere of 5% C O 2 and 95% air, the cells were washed, resuspended in 10% FCScontaining RPMI 1640 medium at 1 x10 ~ cells/ml, and plated into 96 well multidish culture plates (Nunc) at 0.1 ml/well. They were cultured for 14-18 days. On day 2, each well was supplemented with the same volume of the culture medium containing 800/zg/ml G418 (Sigma) for selecting neomycin-resistant colonies. A half of the culture medium was replaced by the G418-containing fresh medium every 5 days. In total, 156 G418-resistant colonies were found. Growing colonies were split and examined for their expression of/3-gal activity by in situ staining using 5-bromo-4-chloro-3-indolyl-/3-D-galactoside (Sigma), a chromogenic substrate as described by Sanes et al. (1986). About 30% of G418-resistant colonies were found to express /3-gal activity at various frequencies. A colony expressing the highest level of/3-gal was selected. A stable transformant was isolated from this colony after two cycles of limiting dilution, and designated K562/Zneo. K562/Zneo cells were maintained in the culture medium without altering the level of /3-gal activity for at least 50 days when they were recultured from frozen stock. Usually, the cells were used at late logarithmic phase to avoid the increase of spontaneous /3-gal release.

Enzyme release assay of NK cell activity Human peripheral blood lymphocytes (PBL) were separated from heparinized blood of healthy volunteers by density gradient centrifugation on 1.077 Histopaque (Sigma) and depleted of monocytes by adherence on plastic dishes for 2 h. In some experiments, these PBL were used as effector cells after they were preincubated for 20 h at 2 × 106/mi with 300 U / m l human recombinant IL-2 (a kind gift from Takeda Chemical Industry, Osaka, Japan). K562/Zneo cells (1 X 104) were mixed with varying numbers of effector cells (1-4 × 105) in 0.2 ml of RPMI 1640 medium containing 2% heat-inactivated FCS in a 96 V-bottomed well plate (Nunc). The plates were centrifuged for 2 min at 800 rpm and incubated at 37 °C for 2-8 h under 5% CO 2 and 95% air. For measuring released /3-gal activity, 0.1 ml of the supernatant

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from each well was incubated for 30 min at 37°C with 0.3 ml of 10 mM sodium phosphate buffer (pH 7.0) containing 0.1 M NaC1, 1 mM MgC12, 0.1% bovine serum albumin and 10 ~g of 4-MUG. The reactions were terminated by addition of 2 ml of 0.1 M glycine buffer (pH 10.3). The amount of 4-MU released from 4-MUG was determined fluorometrically with excitation at 360 nm and emission at 450 nm. Relative fluorescent intensities were read using Fluororead fluorescence spectrophotometer (Ajinomoto, Tokyo, Japan). This apparatus allows the measurement of 10 -9 M 4-MU. The fluorescent intensity of 10 -7 M 4-MU was usually defined as 10.

Percent enzyme release was defined by the following equation R-S % enzyme release =

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× 100

where R and S are observed enzyme release and spontaneous enzyme release, respectively. Total enzyme activity of the used target cells (T) was determined after disrupting the cells with 0.0425% Triton X-100. This concentration of the detergent had no effects on /3-gal activity. Usually, assays were carried out in triplicate, and data were presented as the mean + standard deviation (SD). Typical data from several repeated experiments were presented.

51Cr release assay K 5 6 2 / Z n e o (5 × 106) cells were labeled with 50 mCi of sodium [SaCr]chromate in 0.2 ml of the culture medium at 37°C for 45 min as described by Grabstein and Chen (1980). After washed extensively with the same medium, the radiolabeled cells (1 x 104) were mixed with varying numbers of effector cells in RPMI 1640 medium containing 2% FCS. 5~Cr release assay of NK cell activity was carried out under the same experimental conditions as those in the enzyme release assay.

Results and discussion

We established a stable transformant of K562 cell line that permanently expresses E. coli fl-gal

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Fig. 1. A: stability of/3-gal activity in the NK assay medium. The cell-free extracts equivalent to 1 × 104 K562/Zneo cells were incubated at 37°C in 0.2 ml of RPMI 1640 medium containing 2% FCS. At the time indicated, a 0.1 ml aliquot was assayed for its fl-gal activity as described in the materials and methods section. B: total fl-gal activity and the spontaneous release of the enzyme in K562/Zneo cells during incubation in the assay medium. K562/Zneo cells (1 × 104) were incubated in 0.2 ml of the assay medium for 2-8 h. At the indicated time, 0.1 ml of the supernatant was collected for the assay of spontaneous fl-gal release (e). Total fl-gal activity ( © ) was measured after disrupting the cells with 0.0425% Triton X-100. /3-Gal activity was expressed as a relative fluorescent intensity of liberated 4-MU, where the intensity of 10-7M 4-MU was defined as 10. Data were presented as the mean of duplicate assays.

gene, and designated it K 5 6 2 / Z n e o . As shown in Fig. 1A, it was possible to assay the enzyme activity sensitively using a fluorescent substrate, 4-MUG with cell-free extracts derived from as few as 1 x 104 ceils. When the cell-free extracts of K 5 6 2 / Z n e o cells were incubated at 37°C in the medium used for the assay of NK cell activity, no significant inactivation of the enzyme occurred during incubation for up to 8 h (Fig. 1A). We also confirmed that the incubation for 16-18 h did not result in a significant loss of the released enzyme activity (data not shown). Fig. 1B shows that neither the total level nor the spontaneous release of the enzyme activity was significantly altered when K 5 6 2 / Z n e o cells were cultured at 37°C in the assay medium. Percent spontaneous release of fl-gal was always kept below 10% of the total if the fresh cells harvested at logarithmic phase were used. The ceils at stationary phase sometimes gave a higher spontaneous enzyme-release. These results suggest that /3-gal expressed in K 5 6 2 / Z n e o ceils has excellent properties as an indicator enzyme for the enzyme release assay of NK cell activity. Thus, we tested whether or not

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time and E~ T ratio. K562/Zneo cells (1 x 104) were incubated in triplicate with nonadherent human PBL at E / T ratio of 10-40 for 2 (©), 4 (1), 6 ( A) or 8 ( • ) h. Percent/3-gal release was expressed as the mean +_SD from triplicate wells.

K 5 6 2 / Z n e o cells could be used as target cells of h u m a n N K cells. I n c u b a t i o n of 1 x 104 K 5 6 2 / Z n e o cells with varying n u m b e r s of a d h e r e n t cell-depleted h u m a n P B L for 2 - 8 h resulted in the release of /3-gal activity f r o m the target cells d e p e n d i n g on effector to target ( E / T ) ratio and incubation time as illustrated in Fig. 2. T h e experimental variation a m o n g triplicate assays was usually less than 10% of the m e a n value. As d e m o n s t r a t e d in Fig. 3 A , the cytolytic activity of h u m a n P B L against K 5 6 2 / Z n e o was markedly a u g m e n t e d w h e n the P B L were preinc u b a t e d with h u m a n r e c o m b i n a n t IL-2 for 20 h. This increased cytolysis is, at least in part, t h o u g h t

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Fig. 3. Use of K562/Zneo cells for the measurement of LAK activity. A: K562/Zneo cells (1×104) were incubated in triplicate for 4 h with varying numbers of PBL that had been cultured with (o) or without (©) 300 U/ml IL-2 for 20 h. B: the same experiment was repeated using target cells that had been maintained in the culture for a month after experiment A. Percent enzyme release at each point was expressed as the mean +_SD from triplicate wells.

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Fig. 4. Comparison of cytolysis of K562/Zneo cells between /3-gal release assay and 51Cr release assay. Untreated (©) or IL-2-activated (e) PBL were incubated in triplicate at indicated E / T ratios with 1 × 10 4 unlabeled or 51Cr-labeled K562/Zneo cells for 4 h. (A) /3-gal release and (B) 51Cr release were measured simultaneously in parallel experiments. The spontaneous release of/3-gal and of 51Cr were 8% and 15%, respectively. Each point represents the mean + SD from triplicate wells.

to be due to lymphokine-activated killer ( L A K ) activity as r e p o r t e d by several authors (Rosenberg and Lotze, 1986; G r i m m and Rosenberg, 1984). Thus, it is likely that K 5 6 2 / Z n e o is also useful for the m e a s u r e m e n t of L A K activity. In Fig. 3B, the same experiment was r e p e a t e d using target cells that had b e e n maintained in the culture for a m o n t h after carrying out the experim e n t of Fig. 3A. Similar data were obtained in this experiment, suggesting the stability of K 5 6 2 / Z n e o as target cells. S p o n t a n e o u s /3-gal release did not significantly increase during this culture period (data not shown). Next, we c o m p a r e d the sensitivity of t h e / 3 - g a l release assay and that of 5~Cr release assay as shown in Fig. 4. In the case of 51Cr release assay, K 5 6 2 / Z n e o cells labeled with sodium [SlCr]chrom a t e were used as target cells. Either u n t r e a t e d or IL-2-activated P B L were examined for their cytolytic activity against K 5 6 2 / Z n e o cells. Percent release of SlCr was always approximately two-fold higher than that of /3-gal. However, s p o n t a n e o u s release of the enzyme was usually a half of those observed in 51Cr release assay (see legend in Fig. 4), thus suggesting that this enzyme release assay exhibits sufficient sensitivity. Similar observations were m a d e i n / 3 - g a l release assay of cytotoxic T lymphocyte activity as r e p o r t e d previously ( O h m o r i et al., 1992). Since /3-gal is a m a c r o m o l e c u l e , it might be released from the

135

cells only after larger pores are formed in the plasma membrane. Therefore, the enzyme release assay is assumed to reflect more extensive disruption of the target cells. In conclusion, it was confirmed that K 5 6 2 / Zneo is an excellent target cell line useful for the enzyme release assay of human N K / L A K activity. This procedure is economical, non-hazardous and convenient in that one does not have to prepare labeled target cells prior to every experiment.

References Grabstein, K. and Chen, Y.U. (1980) In: B.B. Mishell and S.M. Shiigi (Eds.), Selected Methods in Cellular Immunology. W.H. Freeman, San Francisco, CA, p. 124. Grimm, E.A. and Rosenberg, S.A. (1984) The human lymphokine-activated killer phenomenon. Lymphokines 9, 279.

Grimm, E.A., Mazumder, A., Zhang, H.Z. and Rosenberg, S.A. (1982) Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J. Exp. Med. 155, 1823. Herberman, R.B. and Ortaldo, J.R. (1981) Natural killer cells: their role in defences against diseases. Science 214, 24. Ishikawa, E. and Kato, K. (1978) Ultrasensitive enzyme immunoassay. Scand. J. Immunol. 8 (suppl. 7), 7. Korzeniewski, C. and Callewaert, D.M. (1983) An enzyme release assay for natural cytotoxicity. J. Immunol. Methods 64, 313. Ohmori, H., Takai, T., Tanigawa, T. and Honma, Y. (1992) Establishment of an enzyme release assay for cytotoxic T lymphocyte activity. J. Immunol. Methods 147, 119. Ortaldo, J.R. and Longo, D.L. (1988) Human natural lymphocyte effector cells: definition, analysis of activity, and clinical effectiveness. J. Natl. Cancer Inst. 80, 999. Rosenberg, S.A. and Lotze, M.T. (1986) Cancer immunotherapy using interleukin 2 and interleukin 2-activated lymphocytes. Annu. Rev. Immunol. 4, 681. Sanes, J.R., Rubenstein, J.L.R. and Nicolas, J.-F. (1986) Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 5, 3133.