Immunology Letters, 34 (1992) 249-256 0165 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved IMLET 01876

Augmentation of monocyte-mediated cytocidal activity by a low dose tumour necrosis factor measured by the kinetic colorimetric microplate assay Maciej Siedlar, Wojciech U r a c z a n d M a r e k Z e m b a l a Department of Clinical Immunology, Institute of Paediatrics, Copernicus Medical School, Cracow, Poland (Received 28 August 1992; accepted 22 September 1992)

1.

Summary

This paper describes a simple kinetic colorimetric assay for the quantitation of human peripheral blood monocyte-mediated cytotoxic activity against tumour cells. Isolated effector monocytes were cultured overnight with an increasing number of target cells in 96-well microplates. Cytotoxic activity of monocytes was determined by modified nitroblue tetrazolium (MTT) dye assay using standard ELISA reader offering possible automation. The test was performed with three different effector/target cell ratios using a fixed number of monocytes. This allowed the expression of cytotoxic activity of monocytes in cytotoxic activity units. The assay was found to be a simple method to demonstrate that low doses of TNF (1 U/ ml) enhanced monocyte-mediated cytotoxicity.

Key words: Monocyte; Human macrophage; TNF; Cytotoxicity test; Colorimetry method; Tetrazolium salt Correspondence to: Maciej Siedlar, M.D., Department of Clinical Immunology, Institute of Paediatrics, Copernicus Medical School, Wielicka 265, 30-663 Cracow, Poland. Abbreviations: E, effector cells; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; FACS, Becton-Dickinson Immunocytometry Systems; FCS, foetal calf serum; IFNy, interferon ~,; MTT, (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PBMC, human peripheral blood mononuclear cells; T, target cell; TNF, tumour necrosis factor (TNF~).

2.

Introduction

Human peripheral blood monocytes have significant cytolytic and cytostatic activity against tumour cells [l-3] and are regarded as important cells involved in tumour regression in vivo [4,5]. Cells of monocyte/macrophage lineage can be activated for tumour cytotoxicity by interferon 7 (IFNJ, tumour necrosis factor (TNF~), bacterial toxins, etc. [6,7]. The increased cytotoxic and/or cytostatic activity of monocytes was observed in some cancer patients [8-10]. A variety of assay systems have been used to measure the cytotoxic effector function of human monocytes. These include cytostasis and long term cytolysis. Monocyte-mediated cytotoxicity is quantitated by using target cells tagged with radioactive, enzymatic or colorimetric dye or a fluorescent label [11,12]. The present paper describes a simple microcolorimetric assay for the quantitation of target cell cytostasis/lysis that is performed in the microplates using a small number of effector monocytes. The MTT reduced to formazan by remaining viable target cells is used for the determination of cytotoxicity. By keeping the number of effector monocytes constant and performing the test at three different effector/target cell ratios, it was possible to express cytostatic/cytotoxic activity in units. The use of a standard ELISA microplate reader offers the possibility of automation. The test was applied to evaluate the augmenting effect of low dose TNF~ on cytotoxic activity of freshly 249

isolated human monocyte.

3.3.

3.

Isolated monocytes (1 x 105 cells/well) were incubated overnight at 37°C in RPMI/2% FCS with 5 × 103, 1 x 104 and 2 x 104 target cells, i.e., 20:1, 10:1 and 5:1 ratio. At the end of culture, 20 #1 of MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide, Sigma) dye in saline (5 mg/ml) was added to each well and the cells were incubated for an additional 4 h. The medium was removed and the microplates were put overnight into a deep freezer (-70°C). Formazan was solubilized by the addition of isopropyl alcohol (Fluka) containing 0.04 N HC1. The A was measured with a microplate ELISA reader (Multiscan Plus, Labsystems) using two different wavelengths: 570 and 630 n m ( . 4 5 7 0 / 6 3 0 ) , and the result of the second measurement was subtracted from the result of the first one. The percentage of monocyte-mediated cytotoxicity was calculated using the following formula:

3.1.

Material and Methods

Monocyte isolation and activation

Human peripheral blood mononuclear cells (PBMC) were isolated from EDTA blood on Ficoll-Isopaque (Pharmacia, Sweden) gradient centrifugation. PBMC were depleted of T cells by tosetting with neuraminidase-treated sheep erythrocytes followed by Ficoll-Isopaque gradient. Monocyte-enriched cells were resuspended at 1 x 106/ml in RPMI 1640 medium (Gibco) with 10% foetal calf serum (FCS) and incubated for 1 h at 37°C in 5% CO2 atmosphere on plastic Petri dishes. After removal of non-adherent cells by vigorous shaking and three washes with medium, adherent cells were harvested by rubbing off with a rubber policeman [13]. The adherent cell population contained > 90% monocytes as judged by FACS analysis (FACScanV, Becton-Dickinson) using Leu-M3 monoclonal antibody (BectonDickinson). Adherent cells (effector cells, E) were suspended at 0.5 × 106/ml in RPMI 1640 with 2% FCS. Isolated monocytes were incubated with medium alone or medium containing human recombinant TNF~ (gift from Professor W. Stec, L6d~, Poland) for various durations and different doses as indicated in Results. 3.2.

Target cells

The following established cell lines were used as a target: mouse fibroblast cell line L929 and melanoma B16. Cell lines were placed in tissue culture flasks (28 cm 2, Falcon) and were grown to confluency in RPMI 1640 medium supplemented with 100 units/ml penicillin (Polfa, Poland), 100 /~g/ml streptomycin (Polfa, Poland), 2 mM glutamine (Gibco) and 10% FCS at 37°C in 5% CO2 atmosphere. For the in vitro analysis, L929 cells were trypsinized and replated at varying concentrations in RPMI 1640 medium containing 2% FCS. The B16 cells were suspended at 0.1 x 106/ ml in RPMI 1640 with 2% FCS.

250

Cytotoxic assay

% cytotoxicity = { T - [ ( E x T ) - E ] / T} x 100% where: E = formazan production by monocytes alone, T = formazan production by target cells alone, and E x T = formazan production by effector and target cell cocultures. The plot of the results of MTT assay against effector/target cell ratios, at least at three different E/T ratios, permitted linearization (see Fig. 3) and allowed the transformation of the results into quantitative activity units on a continuous scale. The results obtained were fitted by quadratic regression analysis. The cytotoxic activity at 1:1 E/T ratio was estimated and expressed as units (see Appendix). 3.4.

Statistical analysis

Curve fitting, correlation coefficients and graphs were performed using SigmaPlot ver.5 software (license 280211) from J A N D E L Scientific G m b H (Schimmelbusstr. 25, 4006 Erkrath, Germany).

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Fig. 1. MTT cleavage by monocytes (open triangles) and L929 ceils (filled triangles). Dot line shows that MTT reduction by l x l05 monocytes did not interfere with the signal from as few as 5000 living L929 target cells. Cells were incubated in 0.2 ml medium with 2% FCS overnight at 37°C in a CO2 incubator. MTT were added for an additional 4 h. Plates were placed overnight at -70°C and then developed with acidified isopropanol and read on a Multiscan Plus ELISA reader using a test wavelength of 570 nm, and a reference wavelength of 630 nm. The results show that the absorbance of L929 is directly proportional to the number of cells. 4.

4.1.

Results

Colorimetric M T T assay can be used to measure cytotoxicity

T h e c o l o r i m e t r i c a s s a y b a s e d o n the t e t r a z o l i u m salt M T T m e a s u r e s o n l y l i v i n g a n d p r o l i f e r a t i n g cells. As s h o w n in Fig. 1, f i b r o b l a s t i c cell line L929 r e d u c e d M T T to f o r m a z a n w h i c h was l i n e a r l y p r o p o r t i o n a l to the n u m b e r o f cells a d d ed. I n c o n t r a s t , there was n o s i g n i f i c a n t f o r m a z a n production by human monocytes. MTT reduction b y 1 × 105 m o n o c y t e s d i d n o t i n t e r f e r e w i t h the signal f r o m as few as 5000 living t a r g e t cells (see d o t line in Fig. 1). W h e n a d i f f e r e n t n u m b e r o f effector m o n o c y t e s were c u l t u r e d w i t h the s a m e n u m b e r o f t a r g e t cells, the c y t o t o x i c a c t i v i t y was d e p e n d e n t o n the E / T r a t i o (Fig. 2). I n the n e x t series o f experim e n t s the n u m b e r o f effector m o n o c y t e was k e p t c o n s t a n t while a d i f f e r e n t n u m b e r o f t a r g e t cells were a d d e d . Fig. 3 s h o w s essentially the s a m e resuits, h o w e v e r the b a c k g r o u n d c y t o t o x i c effect was lower. It was c o n c l u d e d t h a t the a s s a y m i g h t be used for d e t e r m i n a t i o n o f m o n o c y t e c y t o t o x i c a c t i v i t y in vitro.

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Fig. 2. The plot of the results of MTT assay against effector/ target cell ratio (dashed line). Ten thousand living target L929 per well were incubated overnight with an increasing number of monocytes. E/T ratio and number of effector and target cells per well are also shown. The mean (+ SE from four experiments) percentage of monocyte-mediated cytotoxicity (open bars) was calculated using the following formula: % cytotoxicity = {T - [(E x T) - E] / T} x 100%; where: E = formazan production by monocytes alone, T = formazan production by target cells alone, and E x T = formazan production by effector and target cell cocultures. The second order line plotted (thin solid line) is the best fit line calculation using all points (dot lines show 95% confidence limits).

4.2.

Simultaneous measurements o f monocyte cytotoxicity at different E / T ratios allows the determination o f cytotoxic activity

M e d i u m d a s h c u r v e s s h o w n in Fig. 2 a n d Fig. 3 were o b t a i n e d b y p l o t t i n g E / T r a t i o a g a i n s t cytotoxicity. T h e s e c o n d o r d e r line (Y = a X 2 + b X + c, t h i n solid line) p l o t t e d was the best fit line c a l c u l a t i o n u s i n g all p o i n t s ( d o t lines s h o w 9 5 % c o n f i d e n c e limits, R 2 = 0.88 a n d R 2 = 0.96, re-

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Fig. 3. The plot of the results of MTT assay against effector/ target cell ratio (dashed line). One hundred-thousand monocytes per well were incubated overnight with an increasing number of living target L929. E/T ratio and number of effector and target cells per well are also shown. The mean (4- SE from four experiments) percentage of monocyte-mediated cytotoxicity (open bars) was calculated as in Fig. 2. The second order line plotted (thin solid line) is the best fit line calculation using all points (dot lines show 95% confidence limits). 251

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Fig. 4. The percentage of cytotoxicity and the cytotoxic activity in units of monocytes against L929 (open bars) and BI6 (filled bars) target cells in three consecutive experiments. One hundred thousand monocytes per well were incubated overnight with an increasing number of living target L929 or B16 (E/T ratio: 1:0.2, l:0.1 and 1:0.05, respectively). The percentage of monocyte-mediated cytotoxicity varied between experiments and E/T ratio. Estimated cytotoxic activity in units against tumour B16 target cells was unequivocally higher than against fibroblast L929 cells. spectively). Linearization of these curves allowed the transformation of cytotoxicity values into quantitative activity units. Apparent linearity of cytotoxic results of the M T T assay was observed at 1:0.2, 1:0.1 and 1:0.05 E/T ratios (Fig. 3). Therefore, cytotoxic results obtained at 1:0.2, 1:0.1 and 1:0.05 E/T ratios were used to estimate cytotoxic activity at 1:1 E/T ratio by quadratic regression procedure (see Appendix). In three consecutive experiments, monocyte-dependent cytotoxicity against L929 and B16 targets was tested in parallel (see Fig. 4). The percentage of monocyte-mediated cytotoxicity varied significantly and depended on origin of target cells, E/T ratio, and source of monocytes between experiments. Transformation of the results into activity units showed that in every experiment, monocytes were stronger effectors against B 16 than L929 target cells. In subsequent experiments, isolated monocytes were cultured in medium alone for 3, 18 and 42 h before B16 melanoma target cells were added. Freshly isolated monocytes (3 h) expressed the strongest cytotoxicity (% cytotoxicity) against B16 target cells (Fig. 5). Overnight cultured monocytes were less cytotoxic, while two day cultured monocytes showed the weakest cytotoxicity. However, the calculated cytotoxic activity in units 252

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Fig. 5. The percentage of cytotoxicityand the cytotoxic activity in units of monocytes cultured in vitro for different periods of time before putting them into the test. Following the indicated time of incubation in medium alone, 1 x 105 monocytes per well were then incubated overnight with an increasing number of living target BI6 melanoma cells. The percentage of cytotoxicity decreased with the duration of culture of monocytes in medium while the cytotoxic activity in units remained constant. according to the formula shown in the Appendix, was the same. It was concluded that multiple kinetic measurements of monocytes and target cells at different E/T cell ratios allow the determination of monocyte cytotoxic activity in" vitro.

4.3.

The effect of TNFc~ on monocyte cytotoxic activity in vitro

Monocytes were preincubated with medium alone or medium containing 1, 100 and 10000 U/ ml of recombinant human T N F ~ for 3, 18 and 42 h and then washed. Subsequently, B 16 target cells were added at 1:0.2, 1:0.1 and 1:0.05 E/T ratios. Cytotoxicity was determined after overnight incubation and results were expressed as activity units as described above. Fig. 6 shows that 3 h incubation of monocytes with 1 U/ml of T N F caused almost a double increase of cytotoxic activity while the highest dose of T N F (10 000 U/ml) had no effect. The increased cytotoxic activity of monocytes was less profound after 18 and 42 h treatments with T N F . The 42 h treatment paradoxically decreased the monocyte cytotoxic activity. It suggested that short period low dose T N F priming of monocytes induced monocyte cytotoxic activity against B 16 target cells in vitro.

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Fig. 6. The effect of TNF~ priming of monocytes in vitro on the induction of cytotoxic activity against target B16 cells. One hundred thousand cells per well of unprimed and TNF (1, 100 and 10000 U/ml) primed monocytes (3, 18, and 42 h priming duration) were subsequently incubated with increasing numbers of living melanoma BI6 cells (E/T ratio 1:0.2, h0.1 and 1:0.05, respectively).The percentage of monocyte-mediatedcytotoxicity was obtained and the cytotoxic activity was calculated in units. Results are from a representative experiment of four independent experiments each done in triplicate. Standard deviations were consistently less than 10% of mean. Small dose (1 U/ml) short (3 h) TNFct priming caused significant increase of cytotoxic activity of monocytes against tumour cells in vitro. 5.

Discussion

Monocytes aad macrophages play an important role in restricting tumour growth [10]. Activated mononuclear phagocytes can kill tumour cells both in vitro and in vivo [8,14]. Monocytes mediate cytotoxicity through the production of cytotoxic effectors including T N F [15,16], nitrogen intermediates [17] etc. Therefore the T N F sensitive cell line L929 were included as a target. The reduction of M T T has been used to measure the cytotoxic effect of h u m a n monocytes against target cells. This colorimetric assay allows quantitation of the number of active and living cells at the end of the assay [11,12]. As shown on Fig. 1, the amount of formazan generated by the target cells was directly proportional to the cell number over a wide range. This paper showed that monocyte reduction of M T T to a formazan was almost independent of the cell number (in range tested) and very low in comparison to the target cells run in parallel. It offered

the possibility to use this assay to measure the interaction between monocyte and target cell. Similar results were obtained by van de Loosdrecht et al, using peripheral blood monocytes and U937 monocytic cell line as a target [12]. As shown in Fig. 2, monocytes expressed cytocidal activity against L929 targets. At high effector:target ratio, the M T T reduction by target cells seemed to be masked. It might be due to the bystander effect caused by the large number of cells in the test well (see Fig. 2). To avoid this problem, the number of effector monocytes was fixed while the number of target cells was changed. This caused relatively smaller changes in the cell number in test wells (see Fig. 3). The results obtained were essentially the same but in addition the background cytotoxic effect was lower. The M T T test used in this study was modified by including overnight freezing of microplates at the end of M T T reduction. This step caused cell disruption and facilitated formazan solubilization in acidified isopropanol resulting in a lowering of well to well variation among triplicates. An improved signal could also be obtained by dissolving formazan in D M S O with the addition of glycine instead of using acidified isopropanol [12]. Since the colorimetric assay was rapid and the use of microplates decreased the number of cells used, it was possible to perform cytotoxic assay at different E/T ratios routinely. Furthermore, colorimetric assay offers the advantage of precise quantitation by the use of an ELISA reader and allows compatibility with computer analysis programs and further automation. The basic concept for the evaluation of the assay was that results of the assay depended on cell to cell ratio, the activity of the effector monocytes and 'resistance' of target cells to growth inhibition and/or lysis in an assay. In an individual test, the 'resistance' of target cells is constant. Thus, the result of cytotoxic test depends on the cell number in the well and their activity. As shown in Fig. 3, the cytotoxic effect of monocytes was linearly dependent on target number at 1:0.2 to 1:0.05 E/T ratios. The existence of this linearity offered the advantage that the absorbance values might be transformed into quantitative units on a continuous scale. Cytotoxic results obtained at different E/T ratios might be regarded as points to con253

struct a dose-response curve. It allows expression of dose-response curves in standard units [18]. It was found that the second order line (Y = aX 2 + bX + c) plotted was the best fit line calculation using all points (R 2 = 0.96 on Fig. 3, P