Blut

Blut (1990) 60:97-102

© Springer-Verlag 1990

A quantitative colorimetric method to evaluate the functional state of human polymorphonuclear leukocytes* Sadik O e z L2, Erich Platzer 2, and Karl Welte 1 i Laboratory of Cytokine Biology, Memorial Sioan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA 2 Medizinische Klinik III der Universit~itErlangen-NOrnberg, Krankenhausstrasse 12, D-8520 Erlangen, Federal Republic of Germany Received May 8, 1989/Accepted September 1, 1989

Summary. The colorimetric assay previously described by Mosmann [11] for the measurement of cell viability and proliferation has been modified for the assessment of the functional state of human polymorphnuclear cells (PMNs). The ability of PMNs to reduce the tetrazolium salt MTT to formazan reflects directly the degree of stimulation induced by various agents. The underlying mechanism of MTT-reduction to formazan seems to be similar to that of nitroblue tetrazolium (NBT)-reduction. In contrast to the NBT-reduction assay, the formazan produced from MTT can easily be measured by an ELISA reader. Parallel experiments revealed a qualitative correlation between the concentration of formazan produced from MTT and the concentration of cytochrome C reduced by PMNs. Although oxidative burst may not be the actual lyric mechanism in cellular cytotoxicity of PMN, we also observed an association between MTT-reduction capacity and the cytotoxic activity of PMNs from normal donors in antibody dependent cellular cytotoxicity. Our results indicate that the MTT-reduction assay can be employed to estimate the functional state of polymorphnuclear granulocytes.

Key words. Neutrophils Colorimetric assay

-

Oxidative burst

-

Introduction

The stimulation of human polymorphnuclear leukocytes (PMNs) by opsonized bacteria [2] or soluble substances such as N-formylated oligopeptides [1], phorbol 12-myristate 13-acetate (PMA) [13], gra* Supported in part by grant no. CA33484 from the NIH Offprint requests to: S. Oez (Erlangen)

nulocyte-macrophage colony stimulating factor (GM-CSF) [18, 19], and tumor necrosis factor alpha (TNFa) [4, 17] results directly or indirectly in a process referred to as oxidative burst, marked by a rapid oxygen uptake, O2-, H202 production, and hexose monophosphate shunt activity. Each of these steps was utilized by an appropriate method to measure the oxidative burst activity of PMNs. Nitroblue tetrazolium reduction assay [15] is one of the tests with which it is possible to assess the oxidative burst ability of PMNs. This test is used in the diagnosis of e.g. chronic granulomatous disease, characterized by a loss or microbicidal capacity of the PMNs due to the lack of oxidative burst generation [12]. Three-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a tetrazolium salt like nitroblue tetrazolium (NBT). We adopted the method reported by Mosmann [11] for the measurement of cellular survival and growth to obtain information about oxidative burst and functional capacity of PMNs. Consequently, we compared the reduction of MTT with the 02- generation measured in cytochrome C reduction assay, and the cytotoxic capacity in antibody dependent cellular cytotoxicity (ADCC) by standard 51Cr release assay. The latter was reported to correlate closely with the respiratory burst activity of PMNs [3, 7]. Materials and methods Reagents and cytokines. Three-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), phorbol 12-myristate 13-acetate (PMA), cytochrome C and superoxide dismutase (SOD) from bovine liver were purchased from Sigma (St. Louis, Mo.). Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and recombinant human granulocyte colony stimulating factor (rhG-CSF) were provided by Dr.L. Souza (Amgen, Thousand Oaks, Calif.). Recombinant human tumor necrosis factor alpha (rhTNFc0 was a gift of Dr. H. M. Shepard (Genentech, San Francisco, Calif.).

98

S. Oez et al.: Leukocyte function assay

°7o specific lysis = experimental cpm - spontaneous cpm maximal cpm - spontaneous cpm

f r o m 3 - (4,5 - d i m e t h y l t h i a z o l - 2 - y l ) - 2 , 5 - d i p h e n y l t e t r a z o l i u m b r o m i d e ( M T T ) was l a r g e l y d e p e n d e n t o n t h e s t i m u l a t o r y s t a t e a n d t h e e x p o s i t i o n time. W h i l e P M N s in R P M I 1640 m e d i u m c o n t a i n i n g 10% fetal c a l f s e r u m ( F C S ) d i s p l a y e d a n a v e r a g e O D o f 0.09 + 0.030 in t h e a b s e n c e o f a n y s t i m u l a n t , t w o t o e i g h t f o l d h i g h e r O D s were a c h i e v e d f r o m P M N s s t i m u l a t e d w i t h 10 n g / m l P M A , 1,000 u / m l r h T N F a o r 1,000 u / m l r h G M - C S F . A l t h o u g h a f t e r 15 m i n a n i n c r e a s e d M T T - r e d u c t i o n was a l r e a d y o b s e r v e d in t h e s e e x p e r i m e n t s , we d e c i d e d in f a v o r o f 60 m i n i n c u b a t i o n t i m e , since at t h a t t i m e a n a p p r o p r i a t e d i f f e r e n c e b e t w e e n t h e activity of stimulated and unstimulated PMNs could be measured. On the other hand, one hour preincubation of PMNs with rhTNFa or rhGM-CSF prior to M T T e x p o s i t i o n a d d i t i o n a l l y a c c e l e r a t e d t h e red u c t i o n rate, a n d y i e l d e d s i g n i f i c a n t l y h i g h e r O D s t h a n w i t h o u t p r e i n c u b a t i o n (Fig. 2). H o w e v e r , a t 1,000 u / m l r h T N F a t h e r a n g e o f O D s was s i m i l a r for both time periods. I n o r d e r t o e v a l u a t e w h e t h e r M T T - r e d u c t i o n is a s s o c i a t e d w i t h o t h e r f u n c t i o n s o f P M N s , we s i m u l taneously tested the release of superoxide anion (O2-) o f s t i m u l a t e d P M N s in a s u p e r o x i d e d i s m u t a se ( S O D ) i n h i b i t a b l e c y t o c h r o m e C assay. A l t h o u g h e a c h a s s a y was f o u n d t o b e s u f f i c i e n t f o r q u a n t i t a t i n g t h e effect o f P M A a n d r h T N F a o n P M N s , t h e s t i m u l a t o r y effect o f r h G M - C S F c o u l d b e d e t e c t e d in M T T a s s a y o n l y (Fig. 3). F u r t h e r m o r e , a c o m p l e te s u p r e s s i o n o f c y t o c h r o m e C r e d u c t i o n , b u t n o t t h e r e d u c t i o n o f M T T , c o u l d b e o b s e r v e d in t h e presence o f 100 ~tg/ml S O D (Fig. 3 a vs. F i g . 3 c). A n o t h e r f u n c t i o n o f P M N s t h a t we c o m p a r e d t o M T T - r e d u c t i o n was t h e c y t o t o x i c i t y a g a i n s t H L - 6 0 cell line in A D C C assay. A s i l l u s t r a t e d in Fig. 4, t h e results f r o m M T T - r e d u c t i o n a n d A D C C assays revealed the same relative potencies of stimulating a g e n t s a t v a r i o u s e f f e c t o r - t a r g e t r a t i o s ( E : T). T h e m e a n p e r c e n t a g e s p e c i f i c lysis o f t a r g e t cell (10 4 c e l l s / w e l l ) were, a f t e r s t i m u l a t i n g w i t h 1 n g / m l P M A , 6 8 % ; w i t h 1,000 u / m l r h T N F a , 4 9 % a n d w i t h 1,000 u / m l r h G M - C S F , 3 8 % . T h e m e a n O D s o b t a i n e d in M T T - r e d u c t i o n a s s a y p e r f o r m e d s i m u l t a n e o u s l y f r o m t h e s a m e n u m b e r o f e f f e c t o r cells (2 x 105/well) were 0.784, 0.321 a n d 0.152 in c a s e o f s t i m u l a t i o n w i t h P M A , rhTNFGt o r r h G M - C S F at t h e s a m e c o n c e n t r a t i o n s , respectively, r h G - C S F t e s t e d in all t h r e e a s s a y s was f o u n d to be r a t h e r m o d e r a t e in r e g u l a t i n g P M N f u n c t i o n s .

Results

Discussion

A s s h o w n in F i g . 1, t h e q u a n t i t y o f f o r m a z a n p r o d u c e d b y h u m a n p o l y m o r p h n u c l e a r cells ( P M N s )

Various investigators have shown that the method o f M T T - r e d u c t i o n first d e s c r i b e d b y M o s m a n n [11]

Polymorphonuclear leukocytes. Polymorphonuclear granulocytes (PMNs) from peripheral blood were separated by discontinuous density gradient centrifugation on Percoll (Pharmacia, Uppsala, Sweden). In brief: Heparinized blood from healthy donors was diluted 1:1 with RPMI 1640 medium and centrifuged at 2800 rpm for 20 rain at room temperature on two layers of Percoll (1,077 and 1,086 g/ml). PMNs forming an interphase on density 1,086 g/ml were collected and pelleted. After lysing contaminating erythrocytes with distilled water for 30 s, PMNs were washed twice and resuspended in RPMI 1640 medium containing 10% fetal calf serum (FCS) for the assay. The purity of neutrophilic granulocytes by this single-step separation was > 96% (with 1 % - 4 % eosinophil contamination)and the viability > 99% estimated by May-Griinwald-Giemsa staining and trypan blue exclusion. MTT-reduction assay. MTT dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg/ml was sterilized by filtering and kept at 4 °C. Fifty microliter of this solution was then added to each well of a 96 well flat bottom microplate containing PMNs (2.10 [18] per well) in 150 ~tl RPMI 1640 10% FCS and various stimulants at different concentrations in triplicates. The plates were centrifuged at 1,000 rpm for 1 min and incubated for 1 h (or up to 3 h for time kinetic studies) at 37 °C and 5°70 CO2. The plates were then centrifuged for 5 min at 2,500 rpm, and the supernatants were removed by rapid inversion. Formazan produced by PMNs in each well was dissolved in 100 gl 2-propanol without acidification. Optical densities (ODs) were measured by an ELISA reader (Bio-tek, model EL 309) at dual wave lengths (560 nm and 630 nm). Cytochrome C assay. This test procedure [14] was modified slightly and performed in 96 well flat bottom microplates for comparison with MTT-reduction assay. Briefly, after a short centrifugation at 1,000 rpm, PMNs were incubated at 37 °C for 1 h in phenol red-free RPMI 1640 containing 2% FCS, cytochrome C (1 mg/ml final concentration), and stimulants at varying concentrations. Control samples were wells containing superoxide dismutase (SOD) at a final concentration of 100 ~tg/ml, in comparison to which the reduction of cytochrome C was measured. The reaction was stopped by centrifuging the plates for 5 min at 2,500 rpm and transferring 100 gl supernatant from each well to measuring plates. The absorbance of the samples was then determined immediately using the ELISA reader at 550 nm. Cytotoxic assay. HL-60, a human promyelocytic leukemia cell line which is cultured continuously in RPMI 1640 medium supplemented with 10% FCS, L-glutamine, and antibiotics, was used as target cell in an antibody dependent cellular cytotoxicity (ADCC) assay. Serum from a rabbit hyperimmunized with HL-60 was utilized as antibody source. Target cells were labelled 1 h prior to test with antibody and 5aCrNaO4 (E. I. du Pont de Nemours & Co, Boston, Mass.) and coincubated with freshly isolated PMNs for 4 h at 37°C and 5°70 CO2. Supernatant from each well was harvested by using SCS frames and transferring tubes (Skatron, Sterling, Va.). Maximal release was obtained by lysing the target cells in 2% SDS. The percent specific lysis from average cpms of triplicates measured was calculated as follows:

S. Oez et al.: Leukocyte function assay

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Fig. 1. Time kinetics of MTT-reduction by polymorphnuclear leukocytes stimulated with PMA, rhTNFct or r h G M - C S E PMNs were preincubated in four separate plates in the absence or presence of stimulant for varying periods of time. The reduction was terminated as described in Materials and methods. O - - O P M A (1 ng/ml); O--I# rhTNFct (1,000 u/ml); A ,_~ rhGM-CSF (1,000 u/ml); ~ medium

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Fig. 3 A - D . Comparison of polymorphnuclear leukocytes activity in superoxide dismutase inhibitable cytochrome C and MTTreduction assays. PMNs were stimulated with rhTNF~, rhGM-CSF, rhG-CSF (100 u/ml [52~ or 1,000 u/ml [ ] ), or PMA (0.1 ng/ml ESI or 1 ng/ml [ ] ) in the presence or absence of 100 gg/ml SOD. Reduced cytochrome C or MTT were measured after 1 h incubation at 550 nm, or 560 nm and 630 rim, respectively. MTT (A) and cytochrome C-reduction (C) in presence of SOD; MTT (B) and cytochrome C-reduction (D) in the absence of SOD

constitutes a very useful technique for measuring cell viability, proliferation and activation [6, 8, 16]. Although the different quantity of formazan production by untreated versus PMA-stimulated EL-4 cells was also previously demonstrated [5], an association between MTT-reduction and other functional activities of cells has not yet been reported. Because of the chemical resemblance, the mechanism of MTT-reduction is presumably similar to that of nitroblue tetrazolium (NBT). It seems that the enzymes reducing tetrazolium salts are various NADPH-oxidases located both in mitochondria [11] and plasma membrane [9]. One of the membrane-bound NADPH-oxidases has been recently shown to be linked with the oxidative burst activity

of human PMNs [9]. Preactivation of this enzyme in vivo may account for the varying base line activity and heterogenous response of PMNs to stimulants observed in vitro. We ascertained that PMNs can reduce both MTT and NBT equally well. Still we preferred MTT because formazan derived from this substance displays a better solubility than that derived from NBT (cf. Protocol for the quantitative NBT assay [10]). By virtue of comparative experiments with the cytochrome C reduction assay, we were able to demonstrate that the activity state of stimulated neutrophils can be quantified in MTT assay. Although the same mechanism is not responsible for reducing these two substances, a close association with oxi-

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dative burst can be assumed in MTT-reduction. However, while 100 ~tg/ml of SOD could entirely suppress the reduction of cytochrome C, this substance could not influence the reduction of MTT. Moreover, we observed further differences between MTT and cytochrome reduction by the cells stimulated with rhGM-CSE The direct effect of rhGMCSF on PMNs could be demonstrated in MTT but not in cytochrome C reduction assay. This finding agrees with previous studies which have shown that rhGM-CSF merely prime for, but do not induce oxidative burst [18, 19]. Likewise, the comparison of MTT-reduction with ADCC assay has indicated that these two functions might be closely associated with each other; i.e. the more formazan produced from MTT by PMNs stimulated, the higher the percent specific lysis in ADCC. However, it must be stressed that the outcomes compared here were not from the same actions of PMNs, but the consequences of a process of stimulation, because MTT was reduced by the effectors, whereas 51Cr isotopes

Fig. 4 A, B. Cytotoxic activity and MTTreduction of polymorphnuclear leukocytes in response to various stimulants. MTT-reduction and cytotoxic activity of PMNs in ADCC were tested simultaneously in the presence of 1 ng/ml PMA, or rhTNF~, rhGM-CSF, rhG-CSF (1,000 u/ml each). Formazan produced during the first hour of incubation is given as ODs (A). Cytotoxicity against HL-60 cells (1 × 104 per well) labelled with antibody and 5]Cr at various effector: target ratios were expressed in percent specific lysis (B). C)--C) PMA (1 ng/ml); 0 - - 0 rhTNFc~ (1,000 u/ml); A. A rhGM-CSF (1,000 u/ml); ~ rhG-CSF (1,000 u/ml); E3--[] medium control

in ADCC were released from the target cells lysed. Yet MTT-reduction assay may be useful for the anticipation of cytotoxic ability of PMNs. In summary, these data demonstrate that the MTT-reduction assay as a simple and non-radioactive method allows to quantitate the response of human polymorphnuclear leukocytes to various stimulants and thus their activity state. Hence, this technique may be a useful tool to test the polymorphnuclear leukocyte in vitro. References 1. Boxer LA, Yoder M, Bonsib S, Schmidt M, Ho P, Jersild R, Baehner R (1979) Effect of a chemotactic factor, N-formylmethionyl peptide, on adherence, superoxide anion generation, phagocytosis and microtubule assembly of human polymorphnuclear leukocytes. J Lab Clin Med 93:506-514 2. Curnutte JY, Babior BM (1974) Biological defense mechanisms: The effect of bacteria and serum on superoxide production by granulocytes. J Clin Invest 53:1662-1672 3. Dallegri F, Frumento G, Minervini F, Muttini P, Patrone F (1982) Role of the oxidative metabolic burst in the anti-

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9.

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S. Oez et al.: Leukocyte function assay body-dependent cellular cytotoxicity metiated by neutrophil polymorphnuclears. Exp Hematol 10:859-866 Figari IS, Mori NA, Palladino MA, Jr (1987) Regulation of neutrophil migration and superoxide production by recombinant tumor necrosis factors alpha and beta: Comparison to recombinant interferon gamma and interleukin 1 alpha. Blood 7 0 : 9 7 9 - 9 8 4 Gerlier D, Thomasset N (1986) Use of MTT colorimetric assay to measure cell activation. J Immunol Methods 94: 57-63 Green LM, Reade JL, Ware CF (1984) Rapid colorimetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines. J Immunol Methods 7 0 : 2 5 7 - 2 6 8 Hafeman DG, Lucas ZJ (1979) Polymorphnuclear leukocyte mediated, antibody-dependent, cellular cytotoxicity against tumor cells: Dependence on oxygen and the respiratory burst. J Immunol 1 2 3 : 5 5 - 6 2 Heeg K, Reimann J, Kabelitz D, Hardt C, Wagner H (1985) A rapid colorimetric assay for the determination of IL-2 producing helper T cell frequencies. J Immunol Methods 77:237-246 Kakinuma K, Fukuhara Y, Kaneda M (1987) The respiratory burst oxidase of nentrophils. J Biol Chem 262: 12316-12322 Metcalf JA, Gallin JI, Nauseef WM, Root RK (1986) Laboratory manual of neutrophil function. Raven-Press, New York, pp 101-102 Mosmann T (1983) Rapid colorimetric assay for cellular

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