Journal of Immunological Methods, 14 ( 1977 ) 243--251 © Elsevier/North-Holland Biomedical Press

A RAPID QUANTITATIVE

ASSAY FOR LYMPHOTOXIN

243

*

MARK E. SMITtt **, ROBERTA LAUDICO and BEN W. PAPERMASTER Department o f Human Biological Chemistry and Genetics, Division o f Biochemistry, and The Graduate School o f Biomedical Sciences, The University o f Texas Medical Branch, Galveston, Texas; and The Shriners Burns Institute, Galveslon, Texas

(Received 6 June 1976, accepted 30 September)

A rapid quantitative assay for lymphotoxin was developed with the use of a mouse cultured lymphoid cell line, L1210, as the target cell. The assay produces results which are substantially in agreement with assays used in other laboratories, and has the additional advantage of gaining four days over assays employing fibroblasts as targets. After incubation with lymphotoxin containing samples, target cells were labelled with [3Hlthymidine and harvested with a Multiple A u t o m a t e d Sample Harvester (MASH). The MASH allows multiple replicates to be obtained from which the calculation of an Is0 (50% inhibition) point and lymphotoxin specific activities can be performed with high statistical reliability by means of probit transformation and analysis.

INTRODUCTION Recent studies on the purification of lymphotoxin have emphasized the need for a rapid, quantitative test for determining specific activities of purll i e d f r a c t i o n s ( K o l b a n d G r a n g e r , 1 9 6 8 ; R u s s e l l e t al., 1 9 7 2 ; G r a n g e r e t al., 1 9 7 3 ; A m i n o e t al., 1 9 7 4 ; B o u l o s e t al., 1 9 7 4 ; T r i v e r s e t al., 1 9 7 6 ) . W e h a v e recently devised such a test based on the use of the Multiple Automated S a m p l e H a r v e s t e r ( M A S H ) ( T h u r m a n e t al., 1 9 7 3 ) a n d a c u l t u r e d m o u s e l y m p h o m a t a r g e t cell, t h e L 1 2 1 0 c u l t u r e d l y m p h o m a cell l i n e ( M o o r e e t al., 1 9 6 6 ) . T h e u p t a k e o f [ 3 H ] t h y m i d i n e in t h e L 1 2 1 0 l y m p h o m a t a r g e t cell h a s shown a direct dose-dependent response to active lymphotoxin fractions, from which a 50% inhibitory concentration and cytotoxic specific activities can be determined. MATERIALS AND METHODS Lymphokine-containing fractions of culture supernatants from the human c u l t u r e d l y m p h o i d cell l i n e R P M I 1 7 8 8 w e r e u s e d in t h e s e s t u d i e s ( P a p e r *-~F-h-es~-studies were supported in part by NIH grant CA 16964-01 and the Shriners Burns Institute. ** Mark E. Smith is the recipient (1975- 1977) of a James W. McLaughlin Predoctoral Fellowship for studies in immunology.

244 master et al., 1976). The target cells, L 1 2 1 0 , a DBA/2 mouse l y m p h o m a cell line, were grown in culture medium RPMI 1640 supplemented with 10% fetal calf serum (FCS) (Moore et al., 1966}. L 1210 cells were alternately passaged in vivo in DBA/2 mice, and propagated in vitro to insure the identity of the cell line. The mouse fibroblast t u m o r cell line, L959 (obtained from Dr. Sam Barranco, University of Texas Medical Branch), was propagated and used as a target cell as described by Kramer and Granger (1972). Control materials for l y m p h o t o x i n included u n f r a c t i o n a t e d sterile RPMI 1640 culture medi um containing 2% humm~ AB serum and RPM1 1640 culture medium fractionated in a similar m anner to l y m p h o t o x i n media (McDaniel et al., 1976; Papermaster et al., 1976). L F10, a cyt ol yt i c detergent (National Laboratories, Sterling Drug, Montvale, N.J.), was used at 5% c o n c e n t r a t i o n in RPMI 1640, 10% FCS as a positive control for 1 0 0 ~ cell death. Viability of target cell culture before assay was determined by T r y p a n Blue exclusion (0.1% T r y p a n Blue in Hanks balanced salt solution prepared in our laboratory). Only cells with viability greater than 95% were used in the assay. Protein concentrations of all samples were determined by the m e t h o d of BShlen et al. (1973) using fluorescamine (Roche Laboratories, N u tley , N.J.). L 1 2 1 0 cultured cells were distributed into 0.3 ml wells of microculture trays (Microtest II, Falcon, Oxnard, California) in 100 pl volumes containing 10 s cells in RPMI 1640, 10% FCS. Replicate 2-fold serial dilutions of each test fraction were made in RPMI 1640, 10% FCS. Control materials were diluted similarly. Samples were added (100 pl per well) to the target cells usually in four replicate rows containing identical dilutions. Target cells and test fractions in a total volume of 200 pl were incubated in a humidified incubator at 37°C in 5% CO2 for 20 h in covered trays. At the end of this time period, 4 pC [3H]thymidine (Sehwarz-Mann, Orangeburg, New York, 40 Ci/mM) in 40 pl of RPMI 1640, 10% FCS were added to each well with a Hamilton repeating syringe (Fisher Scientific Co., Houston, Texas). The target cells were allowed to label for 4 h at 37°C in 5% CO2, and were then harvested in the MAStt o n t o filter paper (Thurman et al., 1973). The filter paper discs were dried, placed in 5 ml glass scintillation vials with 3.0 ml of scintillation fluid, and c o u n t e d in a Packard Scintillation Counter. When assays were carried out with L959 fibroblasts as target cells, test fractions were added to m onol ayer s of 10 s cells growing in microculture trays. The L 9 5 9 fibroblasts were pulsed with ['~H]thymidine, as above, and harvested in a balanced salt solution containing 0.5% EDTA in the MASH apparatus as described above. Mean counts were det er m i ned for cont rol cells incubated in cell culture medium only (12---24 wells). C y t o t o x i c activity or inhibition of [3H]thymidine uptake was d e t e r m i n e d for cells treated with l y m p h o t o x i n by comparison to [3H]thymidine uptake of cells treated with control medium. Control counts for 100% e y t o t o x i c effect were obtained by comparison with wells containing L F 1 0 detergent.

245

% Cytotoxicity = (1 -- Mean cpm experimental ) Mean cpm control × 100 Percent cytotoxicity values were transformed to probits of the percent of mean control counts as described elsewhere (Papermaster et al., 1974). The assay results were then plotted as probit percent c y t o t o x i c i t y versus log protein in the test solution added per well. This type of transformation regularly yielded data points which followed dilution and on which linear regression analysis could readily be performed. The a m o u n t of protein necessary to produce 50% inhibition of [~H]thymidine uptake was determined from the linear regression plots which thus become dose response plots (see fig. 1), and was termed an Is0 dose (50% inhibitory concentration). Specific cytotoxic activities for lymphotoxin (Is0 units) (mg protein -z) were calculated from the Iso values.

795 90 6-

.80 -70

5. . . . . . . . . . .

-....

..~

SO

5 -30 4-

-20

-10 -5 3-

LOG P l O T [ I N (ng)

Fig. 1. P e r c e n t e y t o t o x i c i t y t r a n s f o r m e d to p r o b i t values is p l o t t e d against log sample p r o t e i n per well. Linear regression analysis is p e r f o r m e d on the data and a least squares fitted line is o b t a i n e d by c o m p u t e r or e l e c t r o n i c calculator. The p r o j e c t i o n o f the 50% e y t o t o x i c i t y p o i n t on the regression line is s h o w n by the h o r i z o n t a l d o t t e d line m a d e o n t o the abscissa and the Is0 value of ng p r o t e i n for 50% c y t o t o x i c i t y is d e t e r m i n e d (vertical d o t t e d lines). F i f t y p e r c e n t c y t o t o x i c i t y , c o r r e s p o n d i n g to 50% i n h i b i t i o n o f [ ' ~ H ] t h y m i d i n c u p t a k e , is t e r m e d a unit of l y m p h o t o x i n activity. The a m o u n t o f p r o t e i n responsible for one unit o f activity, Is(), is d e t e r m i n e d for each fraction assayed and c o n v e r t e d to (units) (mg p r o t e i n - I ) , as s h o w n in table 1. ) = a g a r o s e e l e c t r o p h o r e s i s ; f J = a l b u m i n - c o n t a i n i n g fraction from gel filtration o f S e p h a d e x G-200, see table 1); • = crude cell free media.

246 In order to determine whether our assay was capable of measuring quantitative differences in l y m p h o t o x i n specific activity, l y m p h o t o x i n containing 1788 culture supernatants were subjected to partial purification and assayed. After centrifugation at 700 g for 15 min, the supernatant was concentrated and diafiltered over an Amicon PM-10 ultrafiltration membrane (Amicon Corporation, Lexington, Massachusetts), lyophilized, resuspended in 0.01 M phosphate, 0.15 M NaC1, pH 8.0, and subjected to gel filtration on Sephadex G-200 (Pharmacia, Piscataway, N.J.) (McDaniel et al., 1976). The retained peak in the molecular weight range 60,000--90,000 (serum albumin) was pooled, dialyzed against 0.01 M phosphate buffer, lyophilized, and electrophoresed in 0.1 M borate buffer, ptI 8.3 (Cann, 1968) at 20 ma and 15 volts/ cm for 4 h at room temperature on a 4 × 8 × 0.3 cm preparative 0.6% agarose slab gel (Sigma Chemical Co., St. Louis, Mo.). A strip was cut parallel to the line of migration, stained, and, using the stained section as a guide, protein fractions were cut and eluted into 0.01 M phosphate, 0.15 M NaC1, pH 8.0, for 18 h. Samples were then harvested by centrifugation in 15 ml centrifuge tubes at 20,000 g for 30 min, and tested for l y m p h o t o x i n activity. RESULTS The relative or specific cytotoxic activity in our assay of a lymphotoxin containing sample is determined as follows: From the dose response curve in fig. 1, the protein concentration corresponding to 50% inhibition of [3H]thymidine uptake is determined graphically. This is performed by projecting the point on the regression line corresponding to 50% inhibition onto the abscissa or log ng protein axis (fig. 1). This value is termed an Is,, dose, or the a m o u n t of test sample protein added to the wells responsible for 50% inhibition of thymidine uptake. The a m o u n t of test sample protein at the 50% c y t o t o x i c i t y point is arbitrarily defined as a unit of cytotoxicity, and has the dimension: (ng protein) (unit-~). Inversion of this value thus yields specific activity with dimensions: (units) (ng protein-t), which is converted to: (units) (rag protein-') for convenience (table 1). Examples of assay results with different activity are shown in fig. 1, and represent three different degrees of purification. They were obtained from the successive steps in l y m p h o t o x i n isolation, described above in Methods, and are presented in order to illustrate the quantitative features of the method. Table 1 shows the specific activities attained by purification. Each fraction of higher specific activity requires successively less protein to achieve a 50% inhibitory dose (fig. 1). These Iso values are determined from the regression analysis performed on each sample assay result, since the linearity of the transformation allows accurate determination of the Is0. Thus, specific activities are determined from the regression line graphically or by calculation, and an adequate description of the variation involved is obtained from 95% confidence intervals about each Is~) point which are extracted from a standard analysis of variance calculation (Snedecor and Cochran, 1967).

247 TABLE 1 Purification of human lymphotoxin to illustrate quantitation of the assay. Specific activity (units)(mg protein -l )

Is0 + 95% Confidence limits (lOgl0 ng protein) a) Concentrated 1788 Supernatant

5.18 + 0.69

7.0

b) Gel filtration, G-200 Sephadex, Peak 3

4.11 + 0.29

77.0

c) Preparative Agarose gel electrophoresis-Fraction B

3.04 + 1.8

912.0

(a) Starting material. (b) The Sephadex fraction is the retained peak containing serum albumin or a molecular weight range of 60,000--90,000. (c) Preparative agarose electrophoresis was performed utilizing the gel filtration fraction in an agarose slab in 0.1 M borate buffer, pH 8.3, for 4 h a( room temperature. The fraction shown was the most active for lymphotoxin activity. Confidence limits per Is0 values were computed by analysis of variance (see text). Is 0 refers to the amount of sample protein added which produces a 50% reduction in [3H]thymidine uptake. Is0 values are listed in logl0 protein. After conversion to antilog values, inversion yields specific activity in (units)(ng protein -1 ), which is then converted to (units)(mg protein -1 ) for convenience.

T a b l e 2 s h o w s t h a t t h e u p t a k e o f [ ~ H ] t h y m i d i n e is a g o o d m e a s u r e o f v i a b i l i t y o f L 1 2 1 0 c e l ls w h e n c o r r e l a t e d w i t h T r y p a n B l u e d y e e x c l u s i o n . F i g . 2 c o m p a r e s t h e r e s u l t s o b t a i n e d w i t h L 9 5 9 m o u s e f i b r o b l a s t l i n e as a t a r g e t in a 1 2 0 - h a s s a y ( G r a n g e r e t al., 1 9 7 3 ) , a n d t h e L 1 2 1 0 m o u s e l y m p h o m a l i n e in a 2 4 - h a s s a y . A c o n c e n t r a t e d s u p e r n a t a n t f r o m h u m a n l y m p h o b l a s t 1 7 8 8 ( P a p e r m a s t e r e t al., 1 9 7 6 ) w a s u s e d t o d e t e r m i n e t h e s i m i l a r i t y in r e s p o n s e o f t h e t w o cell t y p e s u n d e r t h e d i f f e r i n g c o n d i t i o n s . S t a t i s t i cal a n a l y s i s c o m p a r i n g t h e r e s p o n s e o f t h e t w o t a r g e t cell t y p e s w a s p e r -

TABLE 2 Comparison of assay techniques. Trypan Blue exclusion counts were taken directly from wells containing cells incubated in the same tray as [3H]thymidine labelled cells. Cells were labelled with [3H]thymidine (40 Ci/mM in RPMI 1640, 10% FCS) by a 4 h pulse after 20 h of incubation with test solutions, harvested in the MASH, and counted (see text). Counts were normally transformed as in figs. 1 and 2. Correlation between Trypan Blue, viability and thymidine incorporation was high (r = 0.9852). Dilution

1/1 1/4 1/16

Concentrated 1788 supernatant

Control media

-

-

[ 3H ]thymidine incorporation

Trypan Blue exclusion % Viability

% Viability

cpm

30 49 99 99

22 55 100 100

6,960 17,800 32,700 32,200

~ 236 + 3248 + 2122 + 2040

248 A. L1210 24hr.

B. L959 120hr.

I

o

5 ..........

o

.'Z_ 0

4.0

4.5

5.0

Log Protein (ng/

5.5

4:o

41s

s:o

sls

Log Protein (hi)

Fig. 2. ' A ' shows probil percent cytotoxicity w, rsus log protein using L1210 mouse lymphoma as target cells incubated for 24 h. 'B' shows the same dose response using the standard assay method with L959 mouse fibrot)lasts as target cells and a 120-h incubation. The slopes or regression lines for each type of assay were compared by students t test, and the analysis indicated a high enough d~,gree of correspondence t)etween the assay (P > 0.50) to consider them homogeneous.

f o r m e d a n d s h o w e d a s i g n i f i c a n t h o m o g e n e i t y (P > 0 . 5 0 ) u s i n g t h e s t u d e n t l d i s t r i b u t i o n . T h e o n l y s i g n i f i c a n t d i f f e r e n c e o b s e r v e d b e t w e e n t h e t w o cell types was the increased variance of [~Iflthymidine uptake for each sample d i l u t i o n t h a t c a u s e d a g r e a t e r o v e r a l l v a r i a t i o n in t h e a s s a y r e s u l t s in t h e L959 fibrobt~sts. DISCUSSION T h e t e c h n i q u e d e s c r i b e d in t h i s p a p e r r e p r e s e n t s a s i g n i f i c a n t r e d u c t i o n in time required for the quantitation of lymphotoxin activity. When necessary, i n d i v i d u a l f r a c t i o n s o r p o o l e d f r a c t i o n s cml b e a s s a y e d w i t h i n 24 h w i t h g r e a t statistical accuracy. T h e r e is a h i g h l y s i g n i f i c a n t c o r r e l a t i o n ( P > 0 . 5 ) b e t w e e n use o f t h e L 1 2 1 0 a n d t h e c o m m o n l y u s e d L 9 5 9 f i b r o b l a s t cell line as t a r g e t cell t y p e s in t h e l y m p h o t o x i n a s s a y . S t a t i s t i c a l a n a l y s i s r e v e a l s t h e cytotoxie r e s p o n s e o f e a c h cell l i n e t o be i n d i s t i n g u i s h a b h , in t h e s p e c i f i e d a s s a y c o n d i t i o n s . W e feel c o n f i d e n t , t h e r e f o r e , in u s i n g c u l t u r e d L 1 2 1 0 l y m p h o m a as a target (:ell

249

line. The ease of propagating the suspension cell line L 1 2 1 0 , as com pared to the L959 fibroblast (which grows as a m onol ayer), makes it a good choice overall for a rapid, quantitative assay. I m p r o v e m e n t in our technique is also based on the use of the MASH, which has been used in the fibroblast assay by Knudsen et al. (1974). The speed and repeatability of assay harvest is greatly increased, as well as the n u m b e r of samples which can be done in a given time. The availability of ['~H]thymidine in high specific activities (40--60 Ci/ raM) provides an inexpensive and highly sensitive source to measure the t h y m i d i n e uptake. Control cell counts can average as high as 400,000 cpm per 10 s cells and c o m m o n l y range between 1 0 0 , 0 0 0 - - 4 0 0 , 0 0 0 cpm per l 0 s cells in our experiments with a coefficient of variation usually in the range of 8 - 1 0 % of the mean. Thus the variation normally found with isotope uptake in cells is greatly reduced because of the high counts in control cells and the large n u m b e r of replicate wells harvested in the MASH. The high counts in control cells also make the assay more sensitive with reduction in counts often measurable over a 3--4 log range. The large n u m b e r of replicates for l y m p h o t o x i n sample dilutions allows a substantial i m p r o v e m e n t in the ability to process the data. The sigmoid nature of the dose response curve can be linearized by transforming percent c y t o t o x i c i t y values to probits of per c e nt c y t o t o x i c i t y . Once a linear transfo rmatio n is attained, a regression analysis enables specific activity to be d e t e r m i n e d with an accurate estimate of its variance. Even greater precision can be obtained by iterative pr obi t analysis as discussed elsewhere (Papermaster et al., 1974). There are at present several c y t o t o x i c assays used in different laboratories. Both direct cell counts of fibroblast monolayers (Granger et al., 1973) and exclusion of vital stains, such as T r y p a n Blue (Sullivan et al., 1972) or fluorochromasia ( R o t m a n and Papermaster, 1966) have been used for determination of cell viability. This requires either visual counting of stained cells or an electronic or laser-activated cell counting apparatus (Hulett et al., 1973). I n c o r p o r a t i o n of labelled amino acids into protein has been used as anot her measure of cell viability (Granger et al., 1973). S~Cr prelabelled cells have been incubated with toxic fractions and the leakage of S~Cr labelled target cell protein into supernatant measured to d et ect cell lysis (Todd, 1975). The ability to incorporate [~H]thymidine into DNA has also been used to determine cell viability (Henney, 1973). A more recent assay has m o n i t o r e d the internal/external K * equilibrium of target cells by measuring S~Rb (an ion which co mp etes with potassium in active transport) in cells after pulse labelling (Walker and Lucas, 1972). All these assays a t t e m p t to m o n i t o r by different parameters a specific signal of cell damage, w he t he r at the cell m e m b r a n e or internally, leading to reduction o f DNA synthesis or cell death. In all cases, cellular response to l y m p h o t o x i n is c om pl ex and there is a great deal of variation within any given assay. In the assay described here, we have a t t e m p t e d to minimize as

250

much of this variaLion as is practically possible. The primary advantage of our assay is the ease of maintenance and handling the L1210 l y m p h o t o x i n target cells, which grow in suspension cultures, as opposed to the fibroblast or other adherent target cell types. The second is a 24 h turn about. The efficiency of handling a large number of sample replicates obtainable with the MASH is shown here and also by Knudsen et al. (1974). The assay described here employs [3H]thymidine, an inexpensive and readily available radiolabelled c o m p o u n d which is also more easily handled than SICr, 86Rb or 14C. The need for only a 24 h incubation results in a significant time saving in doing the assay and yields results by the following day. By the quantitative linearization of data, endpoints can be established with greater accuracy and definite Is0 estimates can be determined, as well as their confidence limits. ACKNOWLEDGEMENTS

The authors would like to acknowledge the excellent help in manuscript typing and figure preparation by Mrs. Stephanie Sordahl, the technical assistance of Mr. Christopher Skisak, and helpful discussions with Mr. John McEntire. REFERENCES Amino, N., E.S. Linn, T.J. Pysher, R. Mier, G.E. Moore and L.J. De Groot, 1974, d. Immunol. 113, 1334. B6hlen, P., S. Stein, W. Dairman and S. Udenfriend, 1973, Arch. Bioehem. Biophys. 115, 213. Boulos, G.N., W. Rosenau and M.L. Goldberg, 1974, J. Immunol. 112, 1347. Cann, J.R., 1968, in: Methods in immunology and immunochemistry, vol. 2, eds, C. Williams and M.W. Chase (Academic Press, New York) p. 73. Granger, G.A., E.C. Laserna, W.P. Kolb and F. Chapman, 1973, Proc. Natl. Acad. Sci. 70, 27. ttenney, C.S., 1973, J. Immunol. 110, 73. Hulett, H.R., W.A. Bonner, R.G. Sweet and L.A. Herzenberg, 1973, Clin. Chem. 19,813. Knudsen, R.C., A. Ahmed and K.W. Sell, 1974, J. Immunol. Methods 5, 55. Kolb, W.P. and G.A. Granger, 1968, Proe. Natl. Aead. Sci. 61, 1250. Kramer, J.d. and G.A. Granger, 1972, Cell. Immunol. 3, 144. McDaniel, M.C., R. Laudico and B.W. Papermaster, 1976, Clin. Immunol. Immunopathol. 5,91. Moore, G.E., A.A. Sandberg and K. Ulrich, 1966, J. Natl. Cane. Inst. 36,405. Papermaster, B.W., O.A. tloltermann, E. Klein, S. Parmett, D. Dobkin, R. Laudico and I. Djerassi, 1976, Clin. Immunol. Immunopathol. 5, 48. Papermaster, B.W., J.H. Pincus, P. Lincoln and R.A. Reisfeld, 1974, Transplantation 18, 541. Rotman, B. and B.W. Papermaster, 1966, Proe. Natl. Acad. Sei. 55, 134. Russell, S.W., W. Rosenau, M.L. Goldberg and G. Kunitomi, 1972, J. Immunol. 109, 784. Snedeeor,G.W. and W.G. Coehran, 1967, Statistical methods (Iowa State University Press, Ames). Sullivan, K.A., G. Berke and B. Amos, 1972, Transphmtation 13, 627.

251 Thurman, G.B., D.M. Strong, A. Ahmed, S.S. Green, K.W. Sell, R.J. Hartzman and F.H. Bach, 1973, Clin. Exp. Immunol. 15, 1. Todd, R.F., III, 1975, Cell. Immunol. 20, 257. Trivets, G., D. Braungart and E. Leonard, 1976, J. Immunol. 117, 130. Walker, S.M. and Z.J. Lucas, 1972, J. Immunol. 109, 1223.