Journal of Immunological Methods, 145 (1991) 199-203 © 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50
199
JIM06139
Rapid colorimetric assay for the quantification of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) Mitsuharu Ohno and Tsutomu Abe Life Science Research Laboratories, Asahi Chemical Industry Co., Ltd., Shizuoka, Japan (Received 14 January 1991, revised received 20 May 1991, accepted 19 August 1991)
The methods in general use for quantifying leukemia inhibitory factor (LIF) activity involve measurements of suppression of proliferation or stimulation of phagocytic activity of mouse myelocytic leukemia (M1) ceils. We have developed a novel assay for L1F activity which consists of measuring MTT reduction by M1 cells. The ability of M1 cells to reduce MT-I" correlates with the level of LIF used for stimulation. The MTT assay of LIF is much quicker than previously used quantitation methods and it is just as sensitive. The MTT assay can also be used to quantify IL-6. Key words: Colorimetric assay; Leukemia inhibitory factor (LIF); Differentiation inducing factor (DIF); Tetrazolium salt; MTT; lnterleukin 6
Introduction
The human macrophage differentiation inducing factor (DIF), which is capable of inducing differentiation of murine leukemia cells (M1) into macrophage-like cells, was discovered in the culture medium of stimulated human monocytic leukemia THP-1 cells and has been purified (Abe et al., 1989). DIF is glycoprotein of approximately 51,000 molecular weight and carbohydrate accounts for over half the mass of DIF. Comparison of the amino-acid sequences of leukemia in-
Correspondence to: M. Ohno, Medical Science Laboratory, Asahi Chemical Industry Co., Ltd., 2-1 Samejima, Fuji-shi, Shizuoka 416, Japan. Abbreviations: DIF, macrophage differentiation inducing factor from THP-1 cells; IL-6, interleukin 6; LIF, leukemia inhibitory factor; MTT, 3(4,5-dimethyl-thiazoyl-2-yl)2,5-diphenyltetrazolium bromide; M1, murine monocytic myeloid leukemia cell line; SDS, sodium dodecyl sulfate; THP-1, human monocytic myeloid leukemia cell line.
hibitory factor (LIF) (Gough et al., 1988) and DIF revealed that these proteins are identical. Other researchers have shown that factors identical to LIF have diverse functions such as the maintenance of embryonic stem cells (ES ceils) (Williams et al., 1988, Smith and Moreau, 1988), inhibition of lipoprotein lipase activity (MLPLI) (Mori et al., 1989) and the stimulation of both ciliary neurotrophic ceils (CNDF) (Yamamori et al., 1989) and hepatocytes (HSF-III) (Baumann et al., 1989). Because LIF is very efficient at inducing M1 cell differentiation to macrophage-like ceils, the assays commonly used to quantify LIF exploit its ability to promote phagocytosis or suppress colony formation. MTT (a tetrazolium salt, 3(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide) is a dye which is reduced by mitochondrial dehydrogenases to a purple formazan. The amount of MTT-derived formazan dye was found to be directly related to the number of viable cells and it has been shown that MTT reduction is correlated
200 with both lymphocyte cell growth stimulation or inhibition (Mosmann, 1983; Green et al., 1984). The MTT system has a number of advantages; it is rapid and precise and requires no washing steps or radioactive isotopes. In this study, we have shown that LIF accelerates the reduction of MTT in M1 cells and that the MTT assay is a highly sensitive, accurate method for the measurement of LIF.
Materials and methods
Cell culture Clone T22 (Tomida et al., 1984) from the murine myeloid leukemic cell line M1 was kindly provided by Dr. Hozumi (Saitama Cancer Research Center, Japan). M1 cells were routinely maintained in Eagle's minimal essential medium (MEM: Flow Laboratories) supplemented with 5% (v/v) heat-inactivated horse serum (Flow), twice the normal concentration of amino acids (Flow), and vitamins (Flow), 50 I U / m l of penicillin G (Sigma Chemical) and 50/xg/ml of streptomycin (Sigma). Human monocytic leukemia cell THP-1 was maintained in RPMI-1640 medium supplemented with twice the normal concentration of amino acids and vitamins, and 10% heat-inactivated FCS (Flow) and antibiotics. Preparation of LIF LIF was prepared as described previously with minor modifications (Abe et al., 1989). THP-1 cells (6 × 105 cells/ml) were stimulated and cultured in serum-free RPMI-1640 medium supplemented with twice the normal concentrations of amino acids and vitamins, 100 ng/ml (w/v) Mezerein (Sigma), 1 /zg/ml retinoic acid (Sigma). After incubation for 3 or 4 days at 37°C, the conditioned medium was centrifuged and filtered through a ZetaPrep 15 (QAE) chromatography column (AMF), and was then concentrated at 4°C using a polysulphone hollow-fibre type filter for ultrafiltration with a molecular weight exclusion limit of 10,000 (Asahi Chem. Inc., Japan). The concentrated activity was applied directly to a lentil lectin-Sepharose 4B column (Pharmacia) and then eluted with P B S ( - ) containing 0.5 M
a-methylmannose (Sigma). The pooled activity was further purified by ion exchange chromatography using a Mono Q and Mono S column (Pharmacia). Final gel chromatography was performed using a Superose 12 column (1 × 30 cm, Pharmacia) equilibrated with Tris-HCl buffer, pH 7.4 containing 0.14 M NaC1 and 0.01% (w/v) PEG 6000. The pooled active material obtained was used in the present experiments.
Reagents Recombinant human interleukin l a and 1/3 (1000 units/ml each), recombinant human interleukin 6 (2 x 104 units/ml), recombinant human granulocyte colony stimulating factor (5000 CFU/ml) and recombinant human macrophage colony stimulating factor (5000 CFU/ml) were purchased from Genzyme. MTT was purchased from Sigma. MTT reduction assay for LIF Serial two-fold dilutions of the various LIF preparations were prepared in a 96-well flat-bottomed microtitre plate (Falcon) by diluting samples with a multichannel pipette using complete medium composed of MEM, 10% heat-inactivated fetal calf serum (FCS: Flow) and twice the normal concentration of amino acids and vitamins. M1 cells were harvested by centrifugation and were suspended at 4 x 105 cells/ml in complete medium. Fifty-/xl aliquots of the M1 cell suspension were transferred in duplicate to the 96-well microtitre plate containing diluted sample (total volume, 100 /~1). Plates were then incubated for a suitable period at 37°C in a humidified 5% CO2/95% air mixture. The MTT solution was freshly prepared at 5 mg/ml in phosphate-buffered saline (PBS) and filtered through a 0.2-p~m pore size filter. An aliquot of 20 tzl of MTT stock solution was added to each well using a multichannel pipette and the plate was incubated for 4 h at 37°C in a humidified 5% CO2/95% air mixture. To each well 100 ~1 of acidified isopropanol (0.04 N HC1 in isopropanol) (Mosmann, 1983) or acidified SDS solution (10% SDS, 0.01 N HC1) (Tada et al., 1986) were added in order to solubilize the formazan. The formazan solubilized by isopropanol was measured after 15 min. Complete solubilization of the dye
201
by SDS solution was achieved by incubation at 37°C for over 6 h. The optical density of each well was measured with a microplate spectrophotometer equipped with a 540- or 570-nm filter.
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Results
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Measurement of accelerated of M T T reduction Because the amount of reduced M T T was linearly related to the number of viable cells, growth factors or growth inhibitory factors for lymphocytes were quantified using the MT-F colorimetric assay (Mosmann, 1983; Green et al., 1984). Using murine myelocytic leukemia M1 cells the amount of reduced M T T was also dependent on the number of viable cells, but the reaction was accelerated in M1 cells stimulated by LIF. The amount of dye reduced by 2 x 104 M1 cells in a 96-well microtitre plate well was less than half the amount of dye reduced by LIF-stimulated cells after 24 h (0.25 OD units vs 0.65 OD units). However, after 48 h, almost the same amount of M T T formazan was reduced by unstimulated and LIF-stimulated ceils. The activation of M1 cells by LIF was observed after 12 h of LIF exposure (Fig. 1). The conversion of M T T to formazan was maximal 0.5
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0.1 zJ i
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24 Culture period
36
48
(hour)
Fig. 1. The accelerated reduction of M T T to formazan following the addition of LIF. Wells of microtitre plate were seeded with M1 cells in 100 ~1 of complete m e d i u m containing 125 u n i t s / m l of LIF. 20 /zl of M T T solution (5 m g / m l ) were added to each well after the indicated period and the cells were then cultured for an additional 4 h. A570 was measured after complete solubilization of M T T formazan by the SDS solution. Each point and vertical line show the m e a n and standard deviation of 4 replicates.
0.2
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8
DIF activity
i
i
i
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32
63
125
250
(unit/ml)
Fig. 2. Relationship between M T T reduction and level of LIF added to M1 cells. LIF was added at a concentration of 250 u n i t s / m l and then serially diluted (2-fold steps) in microtitre plates. M T F solution was added at 24 h and the SDS solution was added at 28 h. Each point and vertical line shows the mean and standard deviation of triplicate samples.
after 24 h and then levelled off up to 36 h of exposure. Production of formazan declined after 36 h.
Calibration curve for LIF LIF containing a small amount of lipopolysaccharide ( ~ 1 f g / u n i t LIF) was purified from THP-1 cells and used in these experiments. The level of LIF activity was determined by the ability of LIF to stimulate phagocytic activity of M1 cells. The half maximal value of the LIF-stimulating ability was 50 units/ml (Abe et al., 1989). M1 cells (2 x 104) were cultured for 24 h in 100 /1.1 complete medium containing LIF (0-250 units/ml). After 24 h, 20 /.1.1 of M T T solution were added to the culture medium. The amount of M T T reduced by LIF-stimulated cells was proportional to the amount of LIF added to the cells (Fig. 2). A linear relationship between the amount of LIF added and the absorbance of the solubilized dye existed over a LIF range from 10 to 100 units/ml. Use of the M T T assay to quantify other inducers M1 cells were shown to be induced to differentiate by other factors, such as interleukin 1 (IL-1) (Tamatani et al., 1987), interleukin 6 (IL-6) (Miyaura et al., 1988), granulocyte colony stimulating factor (G-CSF) (Tomida et al., 1986), lipopolysaccharide (LPS) (Weiss and Sachs, 1978),
202 0.4
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0.2
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/
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,"
/
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01
1.0
10
100
Concentration of factors (ng/ml) Fig. 3. E f f e c t s o f ] [ , - 6 o n the a c c e l e r a t i o n o f M T T
reduction.
Purified LIF (e) and IL-6 (o) were diluted to final concentration of 6.0 and 100 ng/ml, respectively, and then serially diluted (2-fold steps) in 96-well microtitre plates. MTT solution was added at 24 h and the acidified SDS solution was added at 28 h. Each point and vertical line shows the mean and standard deviation of triplicate samples. Solubilized MTT formazan was measured at A540.
and dexamethasone (Lotem and Sachs, 1975). Acceleration of MTI" reduction by M1 cells was induced by IL-6 (Fig. 3), but not by I L - l a , IL-1/3, G-CSF, or macrophage colony stimulating factor (M-CSF). IL-6 and L I F induced the MTT-reducing capability of M1 cells. The half maximal values were approximately 20 u n i t s / m l (0.33 n g / m l ) of LIF, and approximately 125 u n i t s / m l (12.5 n g / m l ) of IL-6. L I F was more effective on a molar basis than IL-6 at stimulating M T T reduction by M1 cells. Both I L - l a and IL-1/3 exhibited a much weaker MTT-reducing capability. Interestingly, T N F - a is known to be capable of enhancing the differentiation of M1 cells in combination with L I F or IL-1, but a similar enhancing effect by T N F - a could not be demonstrated using the M T T assay (data not shown).
Discussion H u m a n leukemia inhibitory factor (LIF) is capable of inducing the differentiation of M1 cells. To date, the most popular assay for the quantitation of L I F activity has been the m e a s u r e m e n t of stimulation of phagocytic ability or growth inhibition of M1 cells. For example Tomida et al. (1984) and Abe et al. (1989) measured the phagocytic activities of M1 by microscopic counting
after 2 days, whereas Hilton et al. (1988) assayed colony formation of M1 cells after 7 days. However, such assays are complicated and tedious. In contrast, the MTI" colorimetric assay for LIF using microtitre plates is rapid (1 or 2 days) and can easily handle multiple samples. In this assay, the increase in M T T reduction by M1 cells was dependent on the amount of LIF added (5-200 units/ml), but the amount of M T T reduction decreased at high levels of added LIF ( > 500 units/ml). This p h e n o m e n o n was probably due to M1 cell damage. Thus, optimal results using this assay will be achieved by serially diluting the LIF-containing samples. The lower limit of the conventional phagocytic assay after two days of culturing is 10 u n i t s / m l of L I F and the M T T reduction assay has the same lower limit of sensitivity. However, the M T T reduction assay only takes one day and is hence much quicker than the conventional assay. M T T reduction is catalysed by mitochondrial dehydrogenases (Mosmann, 1983). However, the mechanism of LIF stimulation of dehydrogenase activity has not been fully elucidated. U p o n differentiation into macrophage-like cells, human promyelocytic leukemia cells (HL-60) acquire the ability to reduce nitroblue tetrazolium (NBT). M1 cells have very little NBT-reducing capability after differentiation. We have found that other human leukemic cell lines, such as THP-1, HL-60, U937 and KG-1 are capable of reducing M T T even without L I F stimulation. Furthermore, no LIF-induced acceleration of M T T reduction was observed in these cells. Thus, M1 cells appear to be the most suitable for m e a s u r e m e n t of LIF levels. Other cytokines such as IL-1, IL-6, G-CSF, and M-CSF were also capable of inducing the differentiation of M1 cells into macrophage-like cells. However, only IL-6 was capable of inducing M T T reduction in M1 cells. The specific activity of L I F for the induction of M T T activity was much higher than that of IL-6. Although typical macrophage functions stimulated by LIF (the induction of phagocytic activity or the expression of lysozyme esterase) are not observable until after two days of culture with LIF, the M T T reduction response was observed within 12 h of LIF addition.
203
Both LIF and IL-6 play a role in the early stages of M1 cell differentiation. Although there is no significant homology between the amino-acid sequences of LIF and IL-6, they have been shown to have similar functions. IL-6 regulates the production of acute phase proteins in human hepatoma (HepG2) cells (Gauldie et al., 1987). Hepatocyte stimulating factor III (HSF-III) which is considered to be identical to LIF has been shown to possess the ability to induce the same set of acute phase proteins as the proteins induced by IL-6 (Baumann et al., 1989). IL-6 induced neuronal differentiation of rat pheochromocytoma PC12 ceils (Satoh et al., 1988) and LIF is known to be identical to cholinergic neuronal differentiation factor (CNDF) which acts on sympathetic neurons (Yamamori et al., 1989). Thus, both LIF and IL-6 play a role in the differentiation of many tissue types.
References Abe, T., Murakami, T., Sato, T. Kajiki, M., Ohno, M. and Kodaira, R. (1989) Macrophage differentiation inducing factor from human monocytic cells is equivalent to murine leukemia inhibitory factor. J. Biol. Chem. 264, 8941. Baumann, H. and Wong, G.G. (1989) Hepatocyte-stimulating factor III shares structural and functional identity with leukemia-inhibitory factor. J. Immunol. 143, 1163. Gauldie, J., Richards, C., Harnish, D., Lansdorp, P. and Baumann, H. (1987) Interferon b2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc. Natl. Acad. Sci. U.S.A. 84, 7251. Gough, N.M., Gearing, D.P., King, J.A., Willson, T.A., Hilton, D.J., Nicola, N.A. and Metcalf, D. (1988) Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia inhibitory factor. Proc. Natl. Acad. Sci. U.S.A. 85, 2623. Green, L.M., Reade, J.N. and Ware, C.F. (1984) Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. J. Immunol. Methods 70, 257. Hilton, D.J., Nicola, N.A. and Metcalf, D. (1988) Purification of a murine leukemia inhibitory factor from Krebs ascites cells. Anal. Biochem. 173, 359. Lotem, J. and Sachs, L. (1975) Induction of specific changes in the surface membrane of myeloid leukemic cells by steroid hormones. Int. J. Cancer, 15, 731.
Miyaura, C., Onozaki, K., Akiyama, U., Taniyama, T., Hirano, T., Kishimoto, T. and Suda, T. (1988) Recombinant human interleukin 6 (B-cell stimulatory factor 2) is a potent inducer of differentiation of mouse myeloid leukemia cells (M1). 234, 17. Mori, M., Yamaguchi, K. and Abe, K. (1989) Purification of a lipoprotein lipase-inhibiting protein produced by a melanoma cell line associated with cancer cachexia. Biochem. Biophys. Res. Commun. 160, 1085. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55. Saiki, R.K., Scharf, S. Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Enzymatic amplification of /3-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350. Satoh, T., Nakamura, S., Taga, T., Matsuda, T., Hirano, T., Kishimoto, T. and Kaziro, Y. (1988) Induction of neuronal differentiation in PC12 cells by B-cell stimulatory factor 2/interleukin 6. Mol. Cell. Biol. 8, 3546. Smith, A.G. and Moreau, J. (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688. Tada, H., Shiho, O., Kuroshima, K., Koyama, M. and Tsukamoto, K. (1986) An improved colorimetric assay for interleukin 2. J. Immunol. Methods 93, 157. Tamatani, T., Urawa, H., Hashimoto, T. and Onozaki, K. (1987) Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells. Biochem. Biophys. Res. Communun. 136, 94. Tomida, M., Yamamoto-Yamaguchi, Y. and Hozumi, M. (1984) Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells. J. Biol. Chem. 259, 10978. Tomida, M., Yamamoto-Yamaguchi, Y., Hozumi, M., Okabe, T. and Takaku, F. (1986) Induction by recombinant human granulocyte colony-stimulating factor of differentiation of mouse myeloid leukemic M1 cells. FEBS Lett., 207, 271. Weiss, B. and Sachs, L. (1978) Indirect induction of differentiation in myeloid leukemic cells by lipid A. Proc. Natl. Acad. Sci. U.S.A. 75, 1374. Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewate, C.L, Geraring, D.P., Wagner, E.F., Metcalf, D., Nicola, N.A. and Gough, N.M. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684. Yamamori, T., Fukada, K., Aebersold, R., Korshing, S., Fann, M. and Patterson, P.H. (1989) The cholinergic neuronal differentiation factor from heart cells is identical to leukemia inhibitory factor. Science 246, 1412.
199
JIM06139
Rapid colorimetric assay for the quantification of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) Mitsuharu Ohno and Tsutomu Abe Life Science Research Laboratories, Asahi Chemical Industry Co., Ltd., Shizuoka, Japan (Received 14 January 1991, revised received 20 May 1991, accepted 19 August 1991)
The methods in general use for quantifying leukemia inhibitory factor (LIF) activity involve measurements of suppression of proliferation or stimulation of phagocytic activity of mouse myelocytic leukemia (M1) ceils. We have developed a novel assay for L1F activity which consists of measuring MTT reduction by M1 cells. The ability of M1 cells to reduce MT-I" correlates with the level of LIF used for stimulation. The MTT assay of LIF is much quicker than previously used quantitation methods and it is just as sensitive. The MTT assay can also be used to quantify IL-6. Key words: Colorimetric assay; Leukemia inhibitory factor (LIF); Differentiation inducing factor (DIF); Tetrazolium salt; MTT; lnterleukin 6
Introduction
The human macrophage differentiation inducing factor (DIF), which is capable of inducing differentiation of murine leukemia cells (M1) into macrophage-like cells, was discovered in the culture medium of stimulated human monocytic leukemia THP-1 cells and has been purified (Abe et al., 1989). DIF is glycoprotein of approximately 51,000 molecular weight and carbohydrate accounts for over half the mass of DIF. Comparison of the amino-acid sequences of leukemia in-
Correspondence to: M. Ohno, Medical Science Laboratory, Asahi Chemical Industry Co., Ltd., 2-1 Samejima, Fuji-shi, Shizuoka 416, Japan. Abbreviations: DIF, macrophage differentiation inducing factor from THP-1 cells; IL-6, interleukin 6; LIF, leukemia inhibitory factor; MTT, 3(4,5-dimethyl-thiazoyl-2-yl)2,5-diphenyltetrazolium bromide; M1, murine monocytic myeloid leukemia cell line; SDS, sodium dodecyl sulfate; THP-1, human monocytic myeloid leukemia cell line.
hibitory factor (LIF) (Gough et al., 1988) and DIF revealed that these proteins are identical. Other researchers have shown that factors identical to LIF have diverse functions such as the maintenance of embryonic stem cells (ES ceils) (Williams et al., 1988, Smith and Moreau, 1988), inhibition of lipoprotein lipase activity (MLPLI) (Mori et al., 1989) and the stimulation of both ciliary neurotrophic ceils (CNDF) (Yamamori et al., 1989) and hepatocytes (HSF-III) (Baumann et al., 1989). Because LIF is very efficient at inducing M1 cell differentiation to macrophage-like ceils, the assays commonly used to quantify LIF exploit its ability to promote phagocytosis or suppress colony formation. MTT (a tetrazolium salt, 3(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide) is a dye which is reduced by mitochondrial dehydrogenases to a purple formazan. The amount of MTT-derived formazan dye was found to be directly related to the number of viable cells and it has been shown that MTT reduction is correlated
200 with both lymphocyte cell growth stimulation or inhibition (Mosmann, 1983; Green et al., 1984). The MTT system has a number of advantages; it is rapid and precise and requires no washing steps or radioactive isotopes. In this study, we have shown that LIF accelerates the reduction of MTT in M1 cells and that the MTT assay is a highly sensitive, accurate method for the measurement of LIF.
Materials and methods
Cell culture Clone T22 (Tomida et al., 1984) from the murine myeloid leukemic cell line M1 was kindly provided by Dr. Hozumi (Saitama Cancer Research Center, Japan). M1 cells were routinely maintained in Eagle's minimal essential medium (MEM: Flow Laboratories) supplemented with 5% (v/v) heat-inactivated horse serum (Flow), twice the normal concentration of amino acids (Flow), and vitamins (Flow), 50 I U / m l of penicillin G (Sigma Chemical) and 50/xg/ml of streptomycin (Sigma). Human monocytic leukemia cell THP-1 was maintained in RPMI-1640 medium supplemented with twice the normal concentration of amino acids and vitamins, and 10% heat-inactivated FCS (Flow) and antibiotics. Preparation of LIF LIF was prepared as described previously with minor modifications (Abe et al., 1989). THP-1 cells (6 × 105 cells/ml) were stimulated and cultured in serum-free RPMI-1640 medium supplemented with twice the normal concentrations of amino acids and vitamins, 100 ng/ml (w/v) Mezerein (Sigma), 1 /zg/ml retinoic acid (Sigma). After incubation for 3 or 4 days at 37°C, the conditioned medium was centrifuged and filtered through a ZetaPrep 15 (QAE) chromatography column (AMF), and was then concentrated at 4°C using a polysulphone hollow-fibre type filter for ultrafiltration with a molecular weight exclusion limit of 10,000 (Asahi Chem. Inc., Japan). The concentrated activity was applied directly to a lentil lectin-Sepharose 4B column (Pharmacia) and then eluted with P B S ( - ) containing 0.5 M
a-methylmannose (Sigma). The pooled activity was further purified by ion exchange chromatography using a Mono Q and Mono S column (Pharmacia). Final gel chromatography was performed using a Superose 12 column (1 × 30 cm, Pharmacia) equilibrated with Tris-HCl buffer, pH 7.4 containing 0.14 M NaC1 and 0.01% (w/v) PEG 6000. The pooled active material obtained was used in the present experiments.
Reagents Recombinant human interleukin l a and 1/3 (1000 units/ml each), recombinant human interleukin 6 (2 x 104 units/ml), recombinant human granulocyte colony stimulating factor (5000 CFU/ml) and recombinant human macrophage colony stimulating factor (5000 CFU/ml) were purchased from Genzyme. MTT was purchased from Sigma. MTT reduction assay for LIF Serial two-fold dilutions of the various LIF preparations were prepared in a 96-well flat-bottomed microtitre plate (Falcon) by diluting samples with a multichannel pipette using complete medium composed of MEM, 10% heat-inactivated fetal calf serum (FCS: Flow) and twice the normal concentration of amino acids and vitamins. M1 cells were harvested by centrifugation and were suspended at 4 x 105 cells/ml in complete medium. Fifty-/xl aliquots of the M1 cell suspension were transferred in duplicate to the 96-well microtitre plate containing diluted sample (total volume, 100 /~1). Plates were then incubated for a suitable period at 37°C in a humidified 5% CO2/95% air mixture. The MTT solution was freshly prepared at 5 mg/ml in phosphate-buffered saline (PBS) and filtered through a 0.2-p~m pore size filter. An aliquot of 20 tzl of MTT stock solution was added to each well using a multichannel pipette and the plate was incubated for 4 h at 37°C in a humidified 5% CO2/95% air mixture. To each well 100 ~1 of acidified isopropanol (0.04 N HC1 in isopropanol) (Mosmann, 1983) or acidified SDS solution (10% SDS, 0.01 N HC1) (Tada et al., 1986) were added in order to solubilize the formazan. The formazan solubilized by isopropanol was measured after 15 min. Complete solubilization of the dye
201
by SDS solution was achieved by incubation at 37°C for over 6 h. The optical density of each well was measured with a microplate spectrophotometer equipped with a 540- or 570-nm filter.
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/
o
~- 0.3
Results
£ o
Measurement of accelerated of M T T reduction Because the amount of reduced M T T was linearly related to the number of viable cells, growth factors or growth inhibitory factors for lymphocytes were quantified using the MT-F colorimetric assay (Mosmann, 1983; Green et al., 1984). Using murine myelocytic leukemia M1 cells the amount of reduced M T T was also dependent on the number of viable cells, but the reaction was accelerated in M1 cells stimulated by LIF. The amount of dye reduced by 2 x 104 M1 cells in a 96-well microtitre plate well was less than half the amount of dye reduced by LIF-stimulated cells after 24 h (0.25 OD units vs 0.65 OD units). However, after 48 h, almost the same amount of M T T formazan was reduced by unstimulated and LIF-stimulated ceils. The activation of M1 cells by LIF was observed after 12 h of LIF exposure (Fig. 1). The conversion of M T T to formazan was maximal 0.5
E
0.4
o
0.3
\
°~ 0.2
\\ \
o
0.1 zJ i
12
24 Culture period
36
48
(hour)
Fig. 1. The accelerated reduction of M T T to formazan following the addition of LIF. Wells of microtitre plate were seeded with M1 cells in 100 ~1 of complete m e d i u m containing 125 u n i t s / m l of LIF. 20 /zl of M T T solution (5 m g / m l ) were added to each well after the indicated period and the cells were then cultured for an additional 4 h. A570 was measured after complete solubilization of M T T formazan by the SDS solution. Each point and vertical line show the m e a n and standard deviation of 4 replicates.
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1
2
4
8
DIF activity
i
i
i
i
;
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63
125
250
(unit/ml)
Fig. 2. Relationship between M T T reduction and level of LIF added to M1 cells. LIF was added at a concentration of 250 u n i t s / m l and then serially diluted (2-fold steps) in microtitre plates. M T F solution was added at 24 h and the SDS solution was added at 28 h. Each point and vertical line shows the mean and standard deviation of triplicate samples.
after 24 h and then levelled off up to 36 h of exposure. Production of formazan declined after 36 h.
Calibration curve for LIF LIF containing a small amount of lipopolysaccharide ( ~ 1 f g / u n i t LIF) was purified from THP-1 cells and used in these experiments. The level of LIF activity was determined by the ability of LIF to stimulate phagocytic activity of M1 cells. The half maximal value of the LIF-stimulating ability was 50 units/ml (Abe et al., 1989). M1 cells (2 x 104) were cultured for 24 h in 100 /1.1 complete medium containing LIF (0-250 units/ml). After 24 h, 20 /.1.1 of M T T solution were added to the culture medium. The amount of M T T reduced by LIF-stimulated cells was proportional to the amount of LIF added to the cells (Fig. 2). A linear relationship between the amount of LIF added and the absorbance of the solubilized dye existed over a LIF range from 10 to 100 units/ml. Use of the M T T assay to quantify other inducers M1 cells were shown to be induced to differentiate by other factors, such as interleukin 1 (IL-1) (Tamatani et al., 1987), interleukin 6 (IL-6) (Miyaura et al., 1988), granulocyte colony stimulating factor (G-CSF) (Tomida et al., 1986), lipopolysaccharide (LPS) (Weiss and Sachs, 1978),
202 0.4
/
t,-4~4
°
c 0.3
0.2
/
/
c
,"
/
o 0.1
01
1.0
10
100
Concentration of factors (ng/ml) Fig. 3. E f f e c t s o f ] [ , - 6 o n the a c c e l e r a t i o n o f M T T
reduction.
Purified LIF (e) and IL-6 (o) were diluted to final concentration of 6.0 and 100 ng/ml, respectively, and then serially diluted (2-fold steps) in 96-well microtitre plates. MTT solution was added at 24 h and the acidified SDS solution was added at 28 h. Each point and vertical line shows the mean and standard deviation of triplicate samples. Solubilized MTT formazan was measured at A540.
and dexamethasone (Lotem and Sachs, 1975). Acceleration of MTI" reduction by M1 cells was induced by IL-6 (Fig. 3), but not by I L - l a , IL-1/3, G-CSF, or macrophage colony stimulating factor (M-CSF). IL-6 and L I F induced the MTT-reducing capability of M1 cells. The half maximal values were approximately 20 u n i t s / m l (0.33 n g / m l ) of LIF, and approximately 125 u n i t s / m l (12.5 n g / m l ) of IL-6. L I F was more effective on a molar basis than IL-6 at stimulating M T T reduction by M1 cells. Both I L - l a and IL-1/3 exhibited a much weaker MTT-reducing capability. Interestingly, T N F - a is known to be capable of enhancing the differentiation of M1 cells in combination with L I F or IL-1, but a similar enhancing effect by T N F - a could not be demonstrated using the M T T assay (data not shown).
Discussion H u m a n leukemia inhibitory factor (LIF) is capable of inducing the differentiation of M1 cells. To date, the most popular assay for the quantitation of L I F activity has been the m e a s u r e m e n t of stimulation of phagocytic ability or growth inhibition of M1 cells. For example Tomida et al. (1984) and Abe et al. (1989) measured the phagocytic activities of M1 by microscopic counting
after 2 days, whereas Hilton et al. (1988) assayed colony formation of M1 cells after 7 days. However, such assays are complicated and tedious. In contrast, the MTI" colorimetric assay for LIF using microtitre plates is rapid (1 or 2 days) and can easily handle multiple samples. In this assay, the increase in M T T reduction by M1 cells was dependent on the amount of LIF added (5-200 units/ml), but the amount of M T T reduction decreased at high levels of added LIF ( > 500 units/ml). This p h e n o m e n o n was probably due to M1 cell damage. Thus, optimal results using this assay will be achieved by serially diluting the LIF-containing samples. The lower limit of the conventional phagocytic assay after two days of culturing is 10 u n i t s / m l of L I F and the M T T reduction assay has the same lower limit of sensitivity. However, the M T T reduction assay only takes one day and is hence much quicker than the conventional assay. M T T reduction is catalysed by mitochondrial dehydrogenases (Mosmann, 1983). However, the mechanism of LIF stimulation of dehydrogenase activity has not been fully elucidated. U p o n differentiation into macrophage-like cells, human promyelocytic leukemia cells (HL-60) acquire the ability to reduce nitroblue tetrazolium (NBT). M1 cells have very little NBT-reducing capability after differentiation. We have found that other human leukemic cell lines, such as THP-1, HL-60, U937 and KG-1 are capable of reducing M T T even without L I F stimulation. Furthermore, no LIF-induced acceleration of M T T reduction was observed in these cells. Thus, M1 cells appear to be the most suitable for m e a s u r e m e n t of LIF levels. Other cytokines such as IL-1, IL-6, G-CSF, and M-CSF were also capable of inducing the differentiation of M1 cells into macrophage-like cells. However, only IL-6 was capable of inducing M T T reduction in M1 cells. The specific activity of L I F for the induction of M T T activity was much higher than that of IL-6. Although typical macrophage functions stimulated by LIF (the induction of phagocytic activity or the expression of lysozyme esterase) are not observable until after two days of culture with LIF, the M T T reduction response was observed within 12 h of LIF addition.
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Both LIF and IL-6 play a role in the early stages of M1 cell differentiation. Although there is no significant homology between the amino-acid sequences of LIF and IL-6, they have been shown to have similar functions. IL-6 regulates the production of acute phase proteins in human hepatoma (HepG2) cells (Gauldie et al., 1987). Hepatocyte stimulating factor III (HSF-III) which is considered to be identical to LIF has been shown to possess the ability to induce the same set of acute phase proteins as the proteins induced by IL-6 (Baumann et al., 1989). IL-6 induced neuronal differentiation of rat pheochromocytoma PC12 ceils (Satoh et al., 1988) and LIF is known to be identical to cholinergic neuronal differentiation factor (CNDF) which acts on sympathetic neurons (Yamamori et al., 1989). Thus, both LIF and IL-6 play a role in the differentiation of many tissue types.
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