CELLULAR

IMMUNOLOGY

Standardization

15, 208-221 (1975)

of a Sensitive and Rapid Assay for Lymphotoxin’

PATRICIA J. EIFEL, SHARYN Departnzents

of Medical

M. WALKER,

AND ZOLTAN J. LUCAS

Microbiology and Surgery, Stanford Medicine, Stanford, California 94305 Received

April

University

School

of

26, 1974

Actinomycin-treated mouse L cells and human HeLa cells are sensitive indicators of lymphotoxin activity in supernatant fluids of mitogen-stimulated lymphocytes. Without actinomycin, various strains of these cells are lo-200 times less sensitive. The concentration of actinomycin used amplifies the toxic effect of LT but is not itself cytotoxic. Actinomycin-treated indicator cells permit detection of LT activity where toxicity is not often found, as in supernatants of mixed lymphocyte cultures and of cell-mediated cytotoxicity reactions. This assay makes available to any investigator a sensitive indicator of LT activity.

Supernatant culture fluids of activated lymphocytes are toxic to other cells in culture (l-5). This activity, lymphotoxin (LT) (1)) has been detected in culture media from mitogen- or antigen-activated lymphocytes from human, guinea pig, mouse, and rat (2, 6-9). The function of LT is unknown, but because it kills indicator cells, it is purported to mediate lymphocyte cytotoxicity (10). Several observations hinder general acceptance of this hypothesis. LT has not been detected in supernatants of specific cell-mediated cytotoxic (CMC) reactions (11, 12)) and some investigators have been unable to detect it after mitogen treating mouse or guinea pig lymphocytes (13). Another argument against a soluble toxin is that CMC reactions often show exquisite specificity, without detectable lysis of innocent bystander cells (11, 14). However, because there is a wide range in cell susceptibility to LT, the indicator cells may have been relatively insensitive to LT. The most labile cell is the mouse L cell line 929 (IO). Other cells (bovine kidney, Chang liver cells) are at least 100 times less sensitive (15). Mouse L cell subtrains, however, may also vary greatly in susceptibility to LT (16)) making some inappropriate as indicator cells. In the present study, six mouse L cell substrains and two HeLa strains obtained from various laboratory and commercial sources were studied for their susceptibility to LT. They vary more than 200-fold when assayed as growing or mitomycin-inhibited cells. All were made highly sensitive by the addition of nonlethal amounts of actinomycin D. The synergistic action of actinomycin with LT in killing previously insensitive indicator cells now makes available to any investigator a rapid, sensitive assay for LT. We report here its applications to the detection of LT in supernatant fluids of mixed lymphocyte cultures (MLC) and target cultures undergoing cell-mediated lysis. 1 Supportedin part by NIH Grant AM 12458 and the Beta Sigma Phi Kidney Research Fund. 208 Copyright All rights

0 of

1975 by AcademicPress, reproductionin any form

A SENSITIVE

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MATERIALS

ASSAY

AND

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LYMPHOTOXIN

209

METHODS

indicator target cells. Mouse L cell lines, designated Ll, L2, L4, L5, L7, and L8, were obtained from the following sources: Ll, strain 929, provided by G. Granger, Irvine, CA in 1969, was the substrain 2 used in previous work (5) ; L2, from Grand Island Biological Co., Grand Island, NY in 1969; L4, from G. Granger in 1973 (Granger’s strain a) (16) ; L5, from G. Granger in 1973 (Granger’s strain 8) (16) ; L7, from Microbiological Associates, Baltimore, MD in 1972; L8, from American Type Culture Collection Cell Repository, Rockville, MD (line CCL 1). HeLa cells, designated HeLa 1 and HeLa 2, were obtained from T. Merigan, Stanford, CA in 1971 and American Type Culture Collection Cell Repository (line CCL 2) in 1973, respectively. Two other lines chronologically tested, L3 and L6, are not reported because their sensitivities to LT were similar to Lines 1 and 7, respectively, and their origins were not defined sufficiently to consider them different “substrains.” Cells are maintained in monolayer culture or stored in liquid nitrogen. Frozen cells are initially cultured on plasma clots to attain logarithmic growth in 3 days. A plasma clot is prepared by spreading 0.5 ml chicken plasma (Grand Island) with 0.15 ml human fibrinogen (Merck, Sharp and Dohme, West Point, PA) (18 mg/ml medium) on a loo-mm petri dish (No. 3033, Falcon Plastics, Oxnard, CA). One quarter milliliter bovine thrombin (5000 units/ml 0.14 M NaCl) (Parke, Davis and Co., Detroit, MI) is dropped onto the plasma to induce clotting and 1 x lo6 viable cells are added to the clot in 10 ml maintenance medium containing 20% fetal calf serum (FCS). The usual maintenance medium is minimum essential medium (MEM) in Earle’s base (Grand Island), l-fold supplemented with nonessential amino acids, glutamine (2 pmoles/ml) , penicillin (50 units/ml), streptomycin (50 pg/ml), and mycostatin (20 units/ml) with 5% FCS. Monolayer cultures are trypsinized (0.25% trypsin in Earle’s buffer) and transferred to spinner culture (MEM without calcium) 2 or more days before their use as indicator cells. Incubation is at 37°C with CO2 adjusted to maintain the pH at 7.4. Sources of lymphotoxin. LT activity was assayed in supernatants from three sources : ( 1) human spleen cells grown with phytohemagglutinin-P (Difco Labs., Detroit, MI) in serum-free medium for 7 days (5) ; (2) human blood lymphocytes activated by mitogens, antigens, or allogeneic lymphocytes ; and (3) cultured Brown Norway (BN) rat fibroblasts undergoing lymphocyte-mediated cytolysis by Lewis (Le) lymphocytes sensitized 7 days previously with a BN skin graft (17). LT was partially purified from the splenic supernatant after ZO-fold concentrafollowed by elution from polyacrylamide gel tion by membrane ultrafiltration, electrophoresis (PAGE) columns (5,s). Human lymphocytes are obtained from defibrinated blood by an isopaque-ficoll gradient centrifugation procedure ( 18). Macrophages are removed by sequential adsorption to plastic (17). Lymphocytes serving as responder cells in MLC are not adsorbed. A 1 X lo6 -quantity of cells is cultured in 1 ml RPM1 1640 with 5% pooled, heat-inactivated human AB serum and antibiotics in 10 X 75-mm glass tubes. Various mitogens or antigens are added in the following amounts: PHA-P, 5 pi/ml ; concanavalin A (Con A) (Calbiochem, La Jolla, CA), 25 pg/ml ; poke2 The term “substrain” is in the context that cultures obtained from different sowces behave differently in their susceptibility to LT. The source and LT susceptibility are the only characteristics used to differentiate the substrains.

210

EIFEL,

WALKER

AND

LUCAS

weed mitogen (PWM) (Grand Island), 10 PI/ml; and purified protein derivative (PPD) (Connaught Medical Research Labs., Toronto, Canada), 100 units/ml. Supernatant fluids are collected at 3 days for nntogen and 4 days for antigenstimulated cultures. MLC are set up as one-way cultures by treating stimulator cells with mitomycin C, using 1 X 106 responder and 0.25 x 106 stimulator cells. After the fourth day supernatants are collected and transformation of lymphocytes is assessed by a [SH] thymidine pulse ( 19). Control fluids are prepared as described above except that mitogen or antigen is added at the time of supernatant collection. Rat lymphocytes (5 x lo6 spleen cells of Le or BN animals) are cultured in 16 X lO@mm tubes with PHA-P (1 rl/ml) for 3 days in 1 ml RPMI 1640 with 5% FCS and antibiotics. Supernatants from immune CMC reactions are obtained by incubating 7.5 X lo6 lymph node cells from sensitized Le rats on syngeneic or allogeneic fibroblasts in monolayers. Supernatant fluids are centrifuged free of lymphocytes and cellular debris, sterile filtered, and stored at -76°C for no more than 2 days. Assay for LT. Indicator L or HeLa cells are attached in 0.3 ml maintenance medium in l&mm wells of a 24-hole plastic plate (FB 16-24-TC, Linbro Chemical Co., New Haven, CT). Attachment is for 20 hr at an initial concentration of 2 X lo4 cells per well for L8 cells or 40 hr at 1 X lo4 cells per well for all other lines. Different attachment times are used because highest LT sensitivity is detected after 40-hr attachment for all cells except L8. Beyond 40 hr, sensitivity does not increase (see Results, Table 3). To facilitate uniform cell adherence, the plates are precoated with gelatin (Difco Labs) (50 mg/liter distilled water) by adding 0.3 ml to each well, flicking the plate, and drying at room temperature. After attachment each well is aspirated and up to 0.1 ml lympohcyte culture supernatant or 0.05 ml PAGE-fractionated LT are added in MEM-5% FCS to make a total volume of 0.3 ml. Actinomycin D (Calbiochem) is added simultaneously at a final concentration of 1 rg/ml. Actinomycin stock is prepared in distilled water at 200 pg/ml and stored at 4°C for no longer than 2 mo (20). After 24-hr incubation the number of viable indicator cells is determined by “jRb exchange (5, 17, 21). Briefly, the well fluid is aspirated and the monolayer washed with Earle’s balanced salt solution (EBSS ) with the potassium concentration reduced to 0.5 mM and containing 1% FCS, 50 units penicillin/ml, and 50 pg streptomycin/ml (modified EBSS). The fluid is aspirated, 0.3 ml modified EBSS with [*sRb] Cl (2 rCi/ml) (l-10 mCi/mg Rb, Amersham-Searle, Great Britain) added to each well, and the plates incubated at 37°C for 2 hr to attain isotopic equilibrium. To remove extracellular 8”Rb, the fluid is rapidly aspirated and the wells washed with 0.3 ml ice-cold phosphate-buffered saline (PBS) (0.14 M NaCl, 0.01 M sodium phosphate buffer, pH 7.4), aspirated, and washed again with 1 ml PBS which is removed by inverting the plate. All fluids are added by directing the fluid against the side of the well to prevent detachment of the monolayer. NO more than three wells are aspirated at one time to avoid drying out the cells. Intracellular *“Rb is released by lysing the cells with 0.2 ml saponin (0.02% in water), and the lysate is quantitatively transferred to vials containing 1.5 ml Bray’s solution (22). Radioactivity is determined in a Nuclear Chicago Mark II liquid scintillation spectrometer with a counting efficiency of 90% for 86Rb. All samples are performed in duplicate.

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211

LYMPHOTOXIN

Indicator cells, in logarithmic phase of growth or growth inhibited (mitomycin treated), are used for comparison with the actinomycin-treated cell assay. Growing cells are as described above except without actinomycin. After 20-hr attachment growth-inhibited cells are prepared by incubation for 30 min at 37°C with mitomycin C (Calbiochem) (20 pg/ml), after which the cells are washed with MEM5% FCS. They are used as indicators 20 hr later. This method results in no cell division over the 24-hr assay period as determined by *ORb uptake. Protein synthesis. This was measured by the amount of [ 3H]leucine (Schwartz/ Mann, Orangeburg, NY) (55 C’/1 mmole) incorporated in 45 min by adding 10 &I [ 3H]leucine/ml leucine-free MEM (Grand Island) to the l&mm culture wells washed once with EBSS. After incubation the supernatant is removed, monolayer washed with EBSS, cells detached with 0.25 N NaOH, and protein precipitated with 2 ml cold trichloroacetic acid (10% in distilled water). The content of each well is transferred quantitatively to test tubes and boiled for 10 min. The precipitate is collected on filter paper, dried, and radioactivity determined by liquid scintillation spectrometry. The counting efficiency for 3H is 47%. Cell-mediated cytotoxicity assay. Sensitized lymphocytes are obtained from the local draining lymph nodes of Lewis rats grafted 7 days earlier with skin from Brown Norway animals. This is a major histocompatibility mismatch (23). Lymph node cells from nonimmune Le animals serve as control. Syngeneic and allogeneic fibroblast targets (25 x lo”), obtained from explants of Le and BN skin, are attached in 0.3 ml MEM-5% FCS in wells of the plate used in the LT assay. Prior to attachment, CMC target cells are treated with mitomycin to prevent cell multiplication. After 20 hr, lymphocytes (7.5 X 10e) are added in 0.25 ml fresh MEM-5% FCS. Target cell killing is determined at various times thereafter by the 8FRb quantitation of residual target cell number (17,21) :

% cel’s ki11ed =

86Rb cpm (target + immune cells) ’ - s6Rb cpm (target + nonimmune cells)

1

x 100.

RESULTS Relationship between indicator cell death and LT concentration. Cytotoxicity of LT (PAGE fraction) on L8 cell in three different metabolic states is depicted in Fig. 1. The fraction of cells killed is proportional to the logarithm of the amount LT added to the 0.3-ml assay volume. This relationship is maintained over a lOO-fold range in LT volume (0.01-10 ~1) for actinomycin-inhibited cells. The minimum amount LT detected with growing or mitomycin-treated cells is 1 ~1, at least 10 times more than with actinomycin. Although data are presented only for PAGE LT, the same stoichiometric relationship obtains for nonpurified supernatants of mitogen- or antigen-activated cultures (8) or for supernatants from MLC or CMC reactions. Each point on the graph is the average of duplicate determinations with a standard deviation of less than 3 %. Several laboratories have adopted the convention of defining unit cytotoxic activity as the volume or amount necessary to kill 50% of the indicator cells (LD,,) under precisely defined experimental conditions (5, 9). Thus, from Fig. 1, the LD5,, volume for the same standard LT is 0.7, 3.1, 7, or 9 ~1 for actinomycininhibited, 4%hr growing, mitomycin-inhibited, or 24-hr growing L8 cells, respectively. These yield cytotoxic titers of 1428, 322, 142, and 111 LT units per ml of the standard

212

EIFEL,

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AND

LUCAS

FIG. 1. Relationship between the concentration of LT and cytotoxicity. PAGE-purified LT is added to cultures of L8 cells treated in three ways : with actinomycin (ACT), mitomycin (MIT), or untreated [logarithmically growing (LG)]. After 24 or 48 hr as indicated, cell number is measured by 86Rb exchange (Materials and Methods). The standard LT preparation is diluted lo- to IOO-fold to permit workable volumes (10-100 ~1) to be added to the 0.3-ml total reaction volume. Results are always expressed as volumes of the original LT preparation.

LT, respectively. The titers show actinomycin-inhibited cells to be about 10 times more sensitive to LT than growing or mitomycin-inhibited cells. Actinomycin has a more marked effect on other substrains (Table 1). Each LDbO volume in this paper is the result of titration of the one PAGE LT preparation over at least three concentrations, giving killing between 10 and 90%. LT sensitivity of various L and HeLa cell substrains. Six mouse L-929 cell cultures and two HeLa cell cultures from different sources were titered for LT sensitivity using the same standard LT. Volumes representing LDEO are shown in Table 1. Three general findings are readily apparent. First, there is a wide variation in LT susceptibility of the different cultures in exponential growth. At 48 hr, there is over a 150-fold difference between the least sensitive (L7) and most sensitive (L4) L cell substrains. Second, inhibition of growth by mitomycin does not markedly affect the sensitivity of the indicator cell to LT in any of the substrains. There is a modest increase in sensitivity with lines L2, L4, and L5. Use of nongrowing indicator cells has the advantage that any decrease in cell number relative to control cultures may be directly interpreted as cell death rather than growth inhibition. Finally, actinomycin potentiates the action of LT in all tested lines, and makes all but one of the substrains equally sensitive. The LDsO volumes for substrains Ll, L2, L4, L5, and L8 are between 0.5 and 1 ~1. The LD50 volume is 8 ~1 for substrain L7, which is also the most insensitive line without actinomycin. This volume is IO-fold greater than the others, but still about 40 times less than the 300~~1 volume required for nonactinomycin-treated cultures. HeLa cells from two different sources also become 30-100 fold more susceptible to LT with actinomycin.

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LYMPHOTOXIK

Table 1 also indicates the ranges of values for the LDSO volumes determined by titration experiments over a 6-mo period. They show remarkably small variation. For example, titrations of the same PAGE LT on LB over 20 times maintained the same exponential linearity shown in Fig. 1; the day-to-day variation in the volume giving 50% killing was no more than 3-fold (Table 1). This shows both the stability of the LT preparation and the indicator cell line and the reproducibility of the assay. Kinetics of killing of actinonzycin-treated cells by LT. Killing is linear with time as shown in Fig. 2. The onset of cytotoxicity by both 1 and 100 units LT, when added simultaneously with actinomycin, extrapolates to the same point on the time axis, about 4 hr. Accepting a 20% difierence from the control cell number as the minimum detectable cytotoxicity, the effects of 1 unit LT (standard preparation) can be detected in 14 hr ; IOO-fold more LT (100 units) is detectable in 6 hr. Incubation times longer than 24 hr were not used because after 30 hr, actinomycintreated cells begin to die and detach from the monolayer. However, as shown by the bottom line in Fig. 2, actinomycin without LT causes no cell loss for 24 hr. Efect of indicator cell nzmber on LT cytofosicity. The number of actinomycintreated indicator cells has relatively little effect on the sensitivity of the assay. This

TABLE

1

EFFECT OF ACTINOMYCIN ON THE SENSITIVITY TO LT OF VARIOUS I, AND HELA CELLS OBTAINED FROM DIFFERENT SOURCES Volume

Cell strain Actinomycin” (24 hr)

LT (~1) killing

50% of the cells (LDs~)o

Mitomycinr (24 hr)

Growingd (24 hr)

I, 1 I, 2 I. 4 L5 I, 7 1, 8

0.5 1 0.5 1 8 0.7

(0.3-0.9)’ (0.6-1.8)e (0.2-0.8)’ (0.6-1.8)’ (5-14)’ (0.4-1.3)e

HeLa 1 HeLa 2

3 9

(2.5, 3.5)h (8, 10)”

100 15 2 50 >300h 9

(60-180)f,” (9-21)’ (1.2-3.6)’ (30-90)’ (4.2, 14)h

350 (200, 500)" 350 (200, 500)”

100 2.5 5 100 >300' 10

(60-l 80)’ (15, 35)h (3-9)j (60-150)’ (6-18)i

(48 hr) 4 10 2 9 >300' 3

(3.6-7)’ (6, 14)h (1.2, 2.8)” (4.2, 14)” (2-S)'

-

a PA gel-fractionated LT is added to cells and after 24- or 48-hr incubation as indicated, the voIume LT killing 500/, of the cells is determined by plotting percent cells killed against volume LT added (Fig. 1). The numbers in parentheses indicate the range of LDN values of all experiments done. b Actinomycin concentration is 1.0 rg/ml for all L cells, 0.1 pg/ml for HeLa 1 and 0.025 pg/ml for HeLa 2. c Cells are inhibited from multiplying by prior treatment with mitomycin, 20 pg/ml. d Cells are in logarithmic growth. en, the number of titrations done on different days, is 10-20. J n = 3-5. g To permit titration in the 0.3-ml total reaction volume, the standard LT preparation was concentrated lo-fold whenever the test volume would have been greater than 0.1 ml. Results are always expressed as volumes of the original standard LT preparation. hn=2.

214

EIFEL, 100

WALKER I

AND I

LUCAS I

I

90 -

80

1

100 UNITS

/

P

FIG. 2. 2 X 10’ L measured the onset

70

-

60

-

Kinetics of LT cytotoxicity on actinomycin-treated cells. After 20-hr attachment of cells (L8), actinomycin (1 rg/ml) is added with and without LT. LT cytotoxicity is at various times by =Rb exchange (Materials and Methods). Dashed lines extrapolate of killing to 4 hr.

is shown in Table 2 and Fig. 3. Figure 3 shows that the amount of LT required to kill 50% of the cells does not significantly change over a 100-fold range in indicator cell number (5-450 x 103). The slopes of the curves describing the stoichiometric relationship between cells killed and LT concentration are the same for all three target cell concentrations. TABLE EFFECT OF NUMBER ACTINOMYCIN-TREATED Cell numbera (X 103)

5 15 45 150 450

2 INDICATOR CELLS ON LT CYTOTOXICITY

LDso (~1) B Reaction

volume

0.3 ml (16-mm well)

1 ml (16-mm well)

1 ml (35mm well)

1.15 0.83

3.8 3.3

-

0.66 0.54 -

2.2 1.8 -

2.7 2.6 2.1

Number cells killed at LDso

2,500 7,500 22,500 75,000 225,000

5 Number Ll cells in assay wells at time LT and actinomycin added. The 16-mm and 35mm assay wells were used to accommodate cell number and for comparison of effect of different size wells on cytotoxicity. b Volume LT which kills 50% of the cells after 24-hr incubation. Cell number is determined by *6Rb exchange.

A SENSITIVE

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VOLUME

FOR

21.5

LYMPHOTOXIN

LT Cl)

FIG. 3. Titration of LT on various numbers of cells. Increasing amounts of LT are added to 5, 45, or 450 X 10’ attached cells in l-ml total culture volume containing actinomycin. The highest number of cells is assayed in 35-mm wells; the others in 16-mm assay wells. After 24 hr, cell death is determined by “Rb exchange.

Table 2 shows that titration of LT in either 0.3- or l.O-ml total reaction volume yields different LDsO volumes. The l-ml reaction volume requires about three times the amount LT to kill 50% of the cells than does the 0.3-ml volume. This indicates that it is the concentration, rather than the absolute amount of LT, which determines the fraction of cells killed. These results, obtained on Ll, hold for the other L cells tested. Other variables exavnined in attaining a reproducible assay with actinonzycin. A factor of importance in obtaining reproducible results is the length of attachment of indicator cells to the assay wells before addition of actinomycin and LT. As shown in Table 3, attachment times of about 40 hr are required for optimal sensitivity for four L cell substrains (Ll, L2, L4, and L7). If Ll cells are attached for only 20 hr, the LDSO volume is 1.2 ~1, four times greater than the final sensitivity attained at TABLE EFFECT

Cell strain

OF INDICATOR

LDso volumea derived 20 hr

Ll I, 2 L4 L7 L8

3

CELL ATTACHMENT ACTINOMYCIN-TREATED

1.0 (0.7-2.2) 3.3 (3.0-3.8) 0.4 (0.37-0.46) 20 (15-25) 0.7 (0.6-0.8)

TIME

ON THE LT SENSITIVITY CELLS”

from titration 40 hr

on cells attached

OF

for

60 hr

0.3 (0.28-0.35) 0.7 (0.6-0.8) 0.2 (0.18-0.23)

0.3 (0.28-0.35)

8.0 (7.0-9.0) 0.7 (0.6-0.8)

8.0 (7.0-9.0) -

-

a Different numbers of L cells are added to 16-mm assay wells so that after 20, 40, wells contain approximately the same number of cells (4 X 104). At that time the and unattached cells are removed ; LT in varying amounts and actinomycin in fresh added. Residual cell number is determined by *eRb exchange 24 hr later. b Value reported is the mean of three determinations done at different times; parentheses are the ranges of LDao values.

or 60 hr the initial fluid medium are numbers

in

216

EIFEL,

WALKER

AND

TABLE EFFECT

LUCAS

4

OF ACTINOMYCIN DOSE ON CELL NUMBER, SENSITIVITY IN L8 AND HELA

PROTEIN CELLS

[“HI-

Actinomycin

@Rb *

dosek/ml)

(cpm)

SYNTHESIS,

AND LT

L&a

C”HI-

Leucine

Leucine

(wm)

bm)

(rl)

s’Rb (wd L cell (L8)

HeLa

(HeLa

0 (24 hr) 0.5 (24 hr)

20,162 11,998

1.0 (24 hr)

11,382

2.0 (24 hr)

11,053

35,000 69,200 5,731 5,063 4,639

0 0 0.1 0.3

45,000 89,456 50,724 2,254

75,000 150,936 20,901 -

0

(“zero”

time)

11,651

-

3.4 3.4 0.48 0.44 0.42

10.4 1.2 0.8 0.8

(9.2, 11.5) (1.1,

1.4)

(0.7, 0.9) (0.7, 0.9)

1) (“zero” time) (24 hr) (24 hr) (24 hr)

-

1.7

1.7 0.41 -

>300 3.6 (2.8, 4.5) -

a sBRb uptake, [SH]leucine incorporation, and LT susceptibility studies were performed in duplicate on different sets of cells. The data are the mean of two experiments done at different times; LDSo volumes in parentheses are means of each experiment. b @Rb counts are directly proportional to the number of viable cells remaining in cluture (21).

40 or 60 hr. L8 cells have the same sensitivity (0.7 ~1 * 0.1) after either 20- or 40-hr attachment. It is not known why certain L cell substrains require different periods of attachment before attaining peak sensitivity. However, it is not the time required to regenerate receptors for LT because the L cells are maintained in suspension culture to avoid any such effect by trypsin. In addition, results identical to those in Table 3 are obtained on trypsinized cells. The same results obtain when the assays are performed in 2.5-100/o FCS (data not presented).

I 1

I

I

I

1

I

I

I

2

3

4

5

6

7

8

ACTINOMVCIN

Ilrglmll

FIG. 4. Effect of actinomycin dose on sensitivity of L cells to LT. 2 X 10’ L8 cells are attached for 20 hr before various amounts of actinomycin are added to control and LT-containing fluids. After 24 hr, viability is determined by “Rb exchange. Control viability is compared to the number of cells immediately prior to actinomycin addition.

A SENSITIVE AND RAPID AssAy

217

FOR LYMPHOTOXIN

L8 was chosen as the indicator cell for routine use because it can be obtained from a cell repository which maintains cell lines under well-defined conditions. However, some experiments (Fig. 3, Tables 2 and 5) were initially done on Ll because it was our initial standard L cell line. Efect uf actinomycin on cell number, protein synthesis, and LT sensitivity. Treatment of L8 cells with 0.5-2.0 pg actinomycin per ml for 24 hr results in complete inhibition of growth with no detectable cell loss (Table 4). At 1 pg actinomycin per ml, [3H]leucine incorporation is decreased to 13% of the control; LT sensitivity is maximal. A different optimr.1 concentration of actinomycin was obtained for HeLa cells ; again, it is possible to find a concentration that has a pronounced effect on protein synthesis and LT susceptibility which does not affect cell survival for 24 hr. Figure 4 relates the dose of actinomycin to indicator cell susceptibility to LT. There is no change in sensitivity with actinomycin concentrations between 1.0 and 7.5 pg per ml. Similar results are obtained on all L cell lines. One microgram per TABLE

5

ASSAYOF LT IN SUPERNATANTSOF~TIMULATEDLYMPHOCYTES Source

No. of experiments

Units LT* L cell

Supernatant” Cells

Agent

M.I.c

___ Actinomycin

Growing

1 23-47 lo-28 12-24 6-12

o-7 105-330 33-85 30-70 2.5-40

0 10-33 3-9 3-8 3-4

4-5 7-12

13-17 20-26

l-2 2-3

3-20 990-3500

0 100-350

O-10 100-120

0 10-12

0 23-30

0 2-3

(X%“) Human blood

10 6 3 3 2

1 1 1 1 1

None PHA PWM Con A PPD

1.25 1.25

AXBm B. ~Cm

MLC 2 2

Rat spleen

Cell-mediated Rat lymph

cytolysis node

2 2

10 10

None PHA

3 3

10 10

None PH,4

1 8-10

2

25

CMC L target BN target

-

a Human and rat lymphocytes are cultured in 1 ml medium for 72 hr with mitogen or 96 hr with antigen or in an MLC at which time supernatant fluids are assayed for LT and stimulation assessed by a [sH]thymidine pulse. Rat lymph node cells from a Le rat sensitized by a BN skin graft for 7 days are incubated on Le and BN fibroblasts for 48 hr at which time the allogeneic target is 75’% killed and the syngeneic target is undamaged as assessed by a @Rb pulse (17). Cell-free supernatant fluids are assayed for LT. Values where expressed as a range indicate extremes within all experiments done. *Units LT/ml determined by a 24-hr assay on actinomycin-treated L cells (Ll) or a 48-hr assay on nontreated, growing L cells (Ll). c Mitogenic index; exDerimental cpm pH]thymidine divided by control cpm.

218

EIFEL,

0

4

WALKER

d

12

AND

16

20

LUCAS

24

28

32

LT (UNITS1

FIG. 5. Correlation between immune lymphocyte-mediated cytotoxicity and LT activity. Supernatants from CMC reactions are obtained by incubating 7.5 X IO’ lymphocytes from skin-graftsensitized rats on allogeneic (0, l ) and syngeneic targets in 0.3 ml medium. After various times killing is determined by %Rb (17), and supernatants are titered for LT activity on actinomycin-treated L cells. LT is not found in CMC reactions on syngeneic targets (Table 5).

milliliter ity.

was chosen since it was the minimal concentration giving maximal sensitiv-

Detection

of

LT

in supernatants of variously

activated

lymphocytes.

Cultures

of

human lymphocytes activated by various mitogens (PHA, PWM, Con A), antigen (PPD), and allogeneic lymphocytes contain cytotoxins when assayed with actinomycin-treated L cells (Ll) as indicator cells (Table 5). Control cultures contain little or no toxin. The small amounts of toxin in controls may originate from FCS or antibiotic activation of the cells. Alternatively, it may represent the basal level of LT in blood lymphocytes or LT synthesized by a few naturally occurring activated cells. There is a general correlation between degree of blastogenesis as measured by the mitogenic index and the amount LT detected. PHA-stimulated cells produced the most LT; Con A and PWM induced l/4-1/3 as much, and antigen and MLC, about l/10-1/5. However, discrepancies appeared at both extremes; some cultures with low mitogenic indices had large amounts of LT; others with high indices, low LT titers. These observations are being examined in greater detail. Previous experiments utilizing growin g cell indicators permitted detection of toxin only in mitogen-stimulated, unconcentrated supernatants (5). Cultures of PHA-stimulated rat spleen cells also produce toxin detected easily with actinomycin-treated cells, but less readily with growing L cells. Detection of LT in supernatants of target cultures undergoing cell-mediated cytolysis. Lymphocytes from skin-grafted rats destroy the appropriate allogeneic fibroblast monolayers but not syngeneic monolayers in in vitro assays of CMC. In

two experiments, each performed in duplicate, supernatants of CMC assays, demonstrating total lysis of the allogeneic monolayer, contain 23-30 units I>T (Table 5). In contrast, aliquots of the :ame lymphocytes on syngeneic fibroblasts showed no cytotoxicity and produced no toxin.

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By properly selecting the duration and geometric conditions of the assay (confluent layer of target cells and sensitized lymphocytes relative to the assay well) (17), varying degrees of target cell lysis are obtained. Thus, 35% and 50% CMCinduced cytotoxicity was seen at 12 hr, 6070 and 87% at 24 hr, and 96% and 100% at 48 hr (Fig. 5). Amounts of LT found in supernatants of these CMC reactions are directly correlated with CMC cytotoxicity. Only the actinomycin-treated cell was sensitive enough to detect the small amounts in unconcentrated supernatants. Although growing L cells may detect low levels of toxin in supernatant fluids, the large volumes required would preclude titration. In addition, the large volumes of “spent” culture supernatant can introduce nonspecific cultural artifacts. DISCUSSION This study shows that mouse L cells and human HeLa cells from various sources are more sensitive to LT when treated with an inhibitor of RNA synthesis, actinomycin D. With actinomycin, toxicity of LT-containing supernatants is increased IO200 times depending on the cell substrain. Although the actinomycin assay is routinely carried out to 24 hr, significant LT lysis occurs as early as 6 hr (Fig. 2). In contrast, most LT assays on L cells yield little killing at 24 hr, requiring at least 48 hr incubation (1, 3, 5, 7). The actinomycin assay is not only rapid, but highly reproducible, with only a 2- to 3-fold variation in the volume of a standard preparation of LT giving 50% killing (Ll, L2, and L8, 20 experiments, Table 1). It is not known how actinomycin increases the sensitivity of L cells to LT. Rosenau et al. (24) postulated that the drug acts by inhibiting repair of membrane damage. Perhaps central to dissecting the mechanism is the observation that actinomycin renders five of the six L cell substrains to near equal sensitivity (Table 1). One substrain, L7, not made as responsive as the others, is also the least responsive cell Iine tested without actinomycin. Increased sensitivity of L cells to LT has been reported with other metabolic inhibitors, 2,4-dinitrophenol, puromycin, and cycloheximide (25) ; however, the increased sensitivity is only 3-fold or less. The demonstration that approximately the same fraction of cells is killed at one concentration of LT despite a 100-fold difference in the initial number of target cells suggests that an L cell culture is composed of cells of varying LT susceptibility. The basis of variability to LT susceptibility is not known but it is remarkably stable since LD5,, volumes have not changed in over 6 mo. It would appear that actinomycin increases the sensitivity of all susceptible cells, but it cannot make a population of refractive cells, such as found in L7, sensitive to LT. Experiments are in progress to determine if a totally insensitive L cell line could be selected by culturing cells in differing amounts LT and subculturing the residual, viable cells. While the effect of actinomycin is not known, it nonetheless permits a sensitive assay for LT. Since the assay end-point is cell death, a phenomenon inducible by other cell constituents (lysosomal enzymes), it is necessary to demonstrate that cell death in actinomycin-treated L cells measures the same substance(s) that induces lysis in growing L cells. Human LT purified by polyacrylamide gel electrophoresis has an Rj of 0.33-0.42 by either method of assay (5, 8). Antibody made to this partially purified LT neutralizes the toxin regardless of the assay used (8). And, finally, concentration of supernatants from mitogen-stimulated or MLC or

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CMC reaction mixtures 20- to lOO-fold give parallel results on growing L cells as unconcentrated supernatants on actinomycin-treated cells (Table 5). The actinomycin assay enables demonstration of significant amounts LT iI1 supernatants of targets undergoing immune lysis (Fig. 5). Although from these data one cannot conclude that LT mediates CMC, its presence is clearly correlated with the degree of immune lysis. LT may be simply a product of activated lymphocytes, having little to do with the actual mechanism of immune lysis. However, other studies with antibody to human LT suggest that LT mediates CMC (26, 27). More definitive work is in progress. Of greater practical significance at the present is that the assay of LT in unconcentrated supernatant fluids may be a preferable alternative to [ 3H] thymidine incorporation as a means of assessing delayed hypersensitivity, cell-mediated immunity, or immunocompetence. LT is consistently detected in supernatant fluids from variously activated lymphocytes at cell concentrations as low as 1 x lo6 per ml (Table 5). The preliminary data presented here show only a general correlation between LT synthesis and mitogenesis; cultures having the highest mitogenic index usually, but not invariably, have the highest LT titer. Studies continue to show disparities between mitogenesis and elaboration of lymphokines (28, 29). Although the latter authors feel that the exceptions are not sufficient to prevent a direct correlation to be made (29), there is little evidence that thymidine incorporation and lymphokine release (as MIF or LT) are directly related and equal indicators of in viva cellular immune competence, The standardized actinomycin assay for LT presented in this paper is a simple, easily quantitated test that could prove to be a reliable indicator of immunocompetent cells. REFERENCES 1. 2. 3. 4. 5. 6. 7.

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