lymphocyte in vitro cytotoxicity: characterization of human lymphotoxin

Lymphocyte in Vitro Cytotoxicity: Characterization of Human LymphotoxinAuthor(s): William P. Kolb and Gale A. GrangerSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 61, No. 4 (Dec. 15, 1968), pp. 1250-1255Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/58764 .

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LYMPHOCYTE IN VITRO CYTOTOXICITY: CHARACTElRIZATION OF HUMAN LYMPHOTOXIN

BY WILLIAM P. KOLB* AND GALE A. GRANGERt

DEPARTMENT OF MOLECULAR AND CELL BIOLOGY, UNIVERSITY OF CALIFORNIA (IRVINE)

Communicated by Edward A. Steinhaus, October 9, 1968

The central role of lymphoid cells in tumor immunity and allograft rejection has been well documented.' 2 However, the mechanism of how cell destruction occurs in these systems is still unclear. Development of in vitro systems has greatly facilitated the examination of immune reactions at the cellular and sub- cellular levels.3-8 Contact between immune cells and cellular antigen has been shown to be a very important initial step in all these reactions.9 It has been postulated that this initial step is promoted by antibody associated with the membrane of immune cells that have active sites directed toward target-cell anti- gens. Several authors have suggested that this close contact directly causes cell destruction as a result of lymphocyte-target-cell membrane interaction.10'-13 However, it has been reported that lymphoid-cell-target-cell membrane interactions are insufficient to account for target-cell damage and, moreover, only a viable lymphoid cell possesses the capacity to cause destruction of the target cell.14 15 Our studies suggest that membrane interactions can lead to additional steps: (a) lymphocyte activation and (b) release of a toxic cell-free factor termed lymphotoxin (LT).15 17 Once released, LT is nonspecifically toxic to cells and causes in vitro target-cell damage that is morphologically similar to lymphocyte- directed cytolysis.'6 Subsequent studies have revealed that lymphocytes obtained from many animal species, including maii, cain be induced to release LT in vitro (in the absence of target cells)."7 Substances which induce LT release in vitro are chemically unrelated,'8 cause lymphocyte transformation,'9 and induce nonimmune lymphocytes to cause target-cell destruction.6 7t 16 While the mechanism(s) of how these substances induce lymphocyte activation and LT release is unknown, they may interact with the lymphoid-cell membrane. The present report describes some of the physical and chemical characteristics of LT released in vitro by phytohemagglutinin-activated human lymphocytes.

Materials and Methods.-Tissue culture reagents, cell lines, and agglutinating agents: Tissue culture materials and cell lines used in these studies have been described previously."5 The cell culture medium used was Eagle's minimal essential medium (MEM) containing 10% fetal bovine serum. Phytohemagglutinin-P (PHA) was purchased from Difco Laboratories, Detroit, Michigan.

Human lymphotoxin (HLT) production: Suspensions of nonimmune small lymphocytes were obtained from human adenoid tissue as previously described.'7 The cell suspensions routinely consisted of 95%0 small lymphocytes which were 90-95% viable. Three hundred ug of PHA was added to a suspension of lymphocytes (300 X 106 cells) in 100 ml of MEM, and the suspensions were incubated at 37?C under an atmosphere of 5% CO2 and 95% air. After 72-96 hr of incubation, the medium was collected and pooled. Cells and cell debris were removed by centrifugation at 1500 X g for 30 min, and the supernatant fluid was passed through a Millipore filter (0.45-A pore size) and stored at -18OC until used.

Control medium: Three hundred million small lymphocytes were suspended in 10 ml of sterile distilled water, and after 10 min of swelling at 4?C, 300 Hg of PHA was added

1250



VOL. 6t, 1968 MICROBIOLOGY: KOLB AND GRANGER 1251

and the cells were homogenized in a Potter-Elvehjem grinder. The cell homogenate was added to 90 ml of MEM, incuibated for 72-96 hr at 37?C, cleared of cell debris, and stored as described for HLT medium.

Cytotoxicity assay: All in vitro assays were conducted using L-cell monolayers estab- lished in tube cultures (200,000 L cells in 2 ml of MEM), as previously described.'5 Each tube was microscopically examined before experimental use, and nonuniform monolayers were discarded. After this selection, the MEM was discarded and replaced with test medium. Following experimental incubation, target-cell viability was monitored by a C14-amino acid incorporation technique that has also been previously described.'5 A C14 protein hydrolysate with a specific activity of 1 mc/mg (obtained from Schwartz Bio- Research) was used in all experiments.

MEM concentrate: In order to test low-strength solutions for cytotoxicity, they were reconstituted to physiologic salt and MEM nutritional levels by the addition of a 4 X MEM concentrate. This concentrate contained Hank's balanced salt solution, MEM amino acids, MEM vitamins, 4.2% sodium bicarbonate, glutamine, streptomycin-peni- cillin-mycostatin, and fetal bovine serum.

pH studies: The pH of a se-ries of test tubes containing HLT or control medium was adjusted to various levels by a(lding either 1 M HCI or 1 M NaOH. After 24 hr of incubation at 37?C, the medium was dialyzed 18 hr against 3 X 10- M Tris-HCl, pH 7.6, 1 X 10-4 M MgCl2 (TM buffer) at 4?C. The dialysate was collected, reconstituted to MEM, and sterilized by filtration.

Phenol extraction: Test and control medium was extracted twice with an equal volume of phenol at 56?C for 30 min. The aqueous phase was washed six times with ethyl ether and dialyzed 12 hr at 4?C against TM buff er, pH 5.0. The dialysates were reconstituted to MEM and filter-sterilized.

CsCl equilibrium density gradient centrifugation: Preformed linear density gradients of HLT and control medium ranging from 8 to 43% CsCl were prepared in 5-ml cellulose nitrate tubes. They were centrifuged in a SW50L rotor in a Beckman L-2 ultracentrifuge at 44,000 rpm for 38 hr at 4?C. The tubes were punctured from the bottom and 20 drop fractions collected. The refractive index of each fraction was measured and the density obtained from a standard curve. The volume of each fraction was adjusted to 1.5 ml with distilled water, and each sample was dialyzed 15 hr at 4?C against TM buffer, pH 7.2. The dialysates were collected, supplemented with the MEM concentrate, and filtered.

Gel filtration: Sephadex G-150 slurry was poured into a Siliclad (Clay-Adams) coated- glass column 1 cm in diameter to a final height of 30 cm. The gel was equilibrated for 48 hr with 2 X 10-3 M Tris-HCl, pH 8.6, 1 X 10-4 MgCl2, 0.02 M NaCl buffer. A 2.5-ml sample of HLT or control media containing 5 X 10-3 units of E. coli alkaline phosphatase (80,000 mol wt, Worthington Biochemical Corp.) and 0.05 ml of blue dextran solution (2 X 106 mol wt) was applied to the column. The flow rate was adjusted to 12 ml/hr and 1.6-ml fractions were collected. Each fraction was assayed for alkaline phosphatase activity20 and the blue dextran peak was measured by adsorption at 610 m, in a Gilford model 2000 spectrophotometer. Each fraction was supplemented with the MEM concentrate and filter-sterilized.

Results.-Heat stability: Aliquots of HLT and control medium were placed in a 100?C water bath and after various intervals samples were removed and cooled to 4?C. After samples were collected, they were filtered to remove precipitates. Toxicity of the various filtrates was tested on L-cell monolayers and after 36 hours of incubation, target-cell viability was measured. The results shown in Figure 1 indicate that medium toxicity was lost after two minutes of heating. The decrease of growth in cultures treated with medium heated for greater than ten minutes is probably due to the removal of serum proteins from the MEM because of precipitation.



1252 MICROBIOLOGY: KOLB AND GRANGER PROC. N. A. S.

CPM 14C

11000 Control medium

9000

7 00 0 Human Lymphotoxin

5000

3000

1000 11 * * I 1 2 5 10 15 30

Minutes at 1000C

FIG. 1.-Control and HLT media were heated at 1000C for the various time periods indicated. L-cell monolayers were used to assay for medium cytotoxicity and after 36 hr of incubation, they were labeled with 0.5 ,ic/ml C'4-amino acids for 20 min.

C P M l4c

8000

Control medium

6000

a 4 000 \

2000 Human \ Lymphotoxin

2 5 p6 8 11

FIG. 2.-Control and HLT media were incubated at various pH's for 24 hr and then assayed for cytotoxicity as described in the text (cells were labeled with 0.25 ,uc/ml C14-amino acids for 30 min).

pH stability: Control and HLT media were incubated at various pH's, as described in MIaterials and Methods. After this treatment, the media were assayed for toxicity with duplicate tubes for each pH value tested. Cell viability was measured after 36 hours of incubation, and the duplicate values were averaged and are presented in Figure 2.

Enzyme sensitivity: Samples of HLT and control medium were incubated with various enzymes for 12 hours at 37?C. After this enzyme treatment, the media were tested for toxicity, and cell viability was measured after 48 hours. The results of this experiment are shown in Figure 3.

Phenol extraction: Control and HLT media were phenol-extracted as outlined in Materials and Methods. In addition, HLT and control media were treated in an identical manner except that the phenol extraction step was omitted, i.e., test media were heated at 560C for one hour, centrifuged, dialyzed, supplemented, and filtered. The different samples were placed on target-cell monolayers and incubated for 48 hours before they were assayed for viability. The

Eg Control go Human medium Lymphotoxin

100

-680 r-60 0

C.)

0 40 0.

20 0\O0

Untreated R Nase DNase Trypsin control 15ug/mi 5ug/ml 10Oug,m,

FIG. 3.-Control and HLT media were incubated for 12 hr at 37?C with the enzymes indicated and then assayed for cytotoxicity as described in Fig. 1 (cells were labeled with 0.30 mc/mI C'4-amino acids for 30 min).



VOL. 61, 1968 MICROBIOLOGY: KOLJB AND GRA-NGER 1253

TABLE 1. The effect of phenol exctraclion on JILT cytotoxicity. Target-cell viability (epni (2.4-anino acid

TreaktyeijA Saiiiple inicorporationt) Phetiol extraction I:LT 4331

Control medium 4589 Controls HLT 349

Control medium 5057

amount of C14-amino acid incorporation into cellular protein is presented in Table 1.

Buoyant density studies: Cesium chloride equilibrium density gradient centrifugation was performed using HLT and control media as outlined in Materials and Methods. In initial experiments, different density ranges were tested and a 8-43 per cent gradient was chosen. The fractions were assayed for cytotoxicity in the usual manner and the indicator monolayers were labeled with C'4-amino acids after 36 hours of incubation. From the results presented in Figure 4, it is apparent that HLT activity was preseiit in the same density range as the cytochrome c protein marker.

Column chromatography: Sephadex column chromatography was employed to determine the number of components responsible for HLT toxicity and to ap- proximate molecular weights. The fractions collected were assayed for cytotoxicity on L-cell monolayers, which were labeled after 48 hours of incubation with C'4-amino acids. The results of this experiment are illustrated in Figure 5, where target-cell viability is plotted versus fraction number (Vle/Vt equals elu- tion volume/total volume). Two marker peaks are shown, i.e., the excluded blue dextran was located in fraction 7, while the 80,000 mol wt alkaline phospha-

C PM 14c 9000

Control medium

7000

5000

Human Lymphotoxin

0. D. 3000

1.0

~~~~~~ ~~~~.8 Dextr-an / .Iaie .6

10 00 ekaie 4 phosphatase2

Fraction no. 51 10 15 20 ve/vt 0.25 0.50 0.75

FIG. 4.-Control, HILT medium, and a protein marker, cytochrome c, were subjected to CsCl equilibrium density gradient centrifugation. Fractions were collected and assayed for cytotoxicity as described in the text (cells were labeled with 0.25 ,uc/ml C'4-amino acids for 30 mIir).



1254 MICROBIOLOGY: KOLB AND GRANGER PRoc. N. A. S.

CPM 14C 11000 1.5

Control medium

9000 ' 9t)00 s \( / t 1.4

7000 ~0 Human

| Lymphotoxin 4. ~~~~ 1.31

4.~~~~~~. 4. C

5 0 00 Cytochrme3.4.

0

1000 1.1

0 5 10 FRACTION NUMBER

FIG. 5.-Control and HLT media were passed through a Sephadex G-150 column, and the experimental fractions were collected and assayed for cytotoxicity as described in Fig. 1 (cells were labeled with 0.25 mc/ml C14-amino acids for 20 min).

tase enzyme demonstrated peak activity in fraction 13. Both control medium and HLT possessed components of molecular weight greater than 150,000, which caused slight toxicity. However, monolayers that received control medium beyond tube 8 supported good cell growth, while HLT fractions showed a single peak of strong cytotoxicity with maximum activity in tubes 12 and 13.

Discussion.-It is apparent from the data presented that lymphotoxin released in vitro by phytohemagglutinin-activated human lymphocytes is proteinaceous in nature. This is supported by several observations: sensitivity of HLT activity to (1) heat, (2) extremes in pH, and (3) phenol extraction. Sensitivity of HLT to phenol extraction in conjunction with its insusceptibility to DNase and lRNase strongly suggests that HLT activity is not due to nucleic acid. Although its activity was insusceptible to trypsin digestion, the proteinaceous nature of HLT was demonstrated by CsCl equilibrium density gradient centrifugation. The buoyant density of HLT was shown to be 1.30 i 0.04, a value characteristic of a proteiin (the buoyant density of lipid being less than 1.0, that of protein equal to 1.33 i 0.1, DNA equal to 1.71 i 0.04, carbohydrates approximately 1.8, and RNTA equal to 2.0 i 0.04).21

Human LT medium subjected to gel filtration showed a single, symmetrical peak of cytotoxicity with a spread equivalent in width to the marker protein of 80,000 mol wt. This observation suggests that in vitro cytotoxicity is due to a single component. In addition, the molecular weight of HLT was estimated to be 80,000-90,000.

Human lyrmphotoxin does not appear to be related to the proteins present in normal serum. A primary consideration was to establish the relationship between HLT, immunoglobins, and/or complemenrt. The results indicate that HLT is not an immunoglobulin because of its low molecular weight and non-



VoL. 61, 1968 MICROBIOLOGY: KOLB AND GRANGER 1255

specific cytotoxicity.17 Furthermore, the heat-labile components of complement do not participate in cell cytolysis because HLT was not inactivated by heating at 56?C for one hour (see 'Fable 1). Moreover, HLT is not antigenically related to normal serum components because rabbit antiserum (1:20,000 precipitin titer) directed against whole human serum does not affect cytotoxicity, while serum obtained from rabbits immunized with HLT inhibited cytotoxicity.22

Although mouse lymphotoxin (MLT) also appears to be proteinaceous, it dif- fers from HLT in that it is remarkably stable to heating and to extremes in pH.22 In addition, the molecular weight of MLT has been estimated at 90,000-100,000,22 a value similar to that shown for HLT in this report. However, MLT is antigenically unrelated to HLT because rabbit and goat antisera directed against MLT block the in vitro cytolytic action of IVILT but not HLT.22

We have previously shown that a mechanism by which activated mouse lymphocytes mediate the in vitro destruction of target cells is by releasing lymphotoxin. Since human lymphocytes also release LT when activated, the mechanism by which these cells direct target-cell cytolysis may be similar.

Summary.-This communication describes some of the physical and chemical characteristics of human Iymphotoxin (HLT), a cytotoxic factor released by human lymphocytes in vitro after stimulation with phytohemagglutinin. Human LT was found to be a heat-sensitive, trypsin-resistant molecule exhibiting proper- ties characteristic of a protein having a molecular weight of approximately 85,000 and a pH stability having a broad optimum centering around neutrality. The differences and similarities between HLT and mouse lymphotoxin are briefly discussed.

* Predoctoral fellow supported by grant no. FJ-GM-37, 389-02 from the National Institutes of Health.

t Supported by grant no. AI 07839-02 from the National Institutes of Health and the Cancer Research Coordinating Committee.

1 Weaver, J., G. Algire, and IR. T. Prehn, J. Natl. Cancer Inst., 15, 1737 (1958). 2 Snell, G. D., H. J. Winn, and A. A. Kandutsch, J. Immunol., 87, 1 (1961). 3 Govaerts, A., J. Immunol., 85, 516 (1960). 4Koprowski, H., and M. V. Fernandes, J. Exptl. Med., 116, 467 (1962). I Rosenau, W., and H. D. Moon, J. Natl. Cancer Inst., 27, 471 (1961).

6 Holm, G., P. Perlman, and B. Werner, Nature, 203, 841 (1964). 7Moller, E., Science, 147, 873 (1965). 8 Wilson, D. B., J. Cellular Comp. Physiol., 62, 273 (1963). 9 Rosenau, W., in Cell Bound Antibodies, ed. B. Amos and H. Koprowski (Philadelphia:

Wistar Institute Press, 1963), p. 75. '0Moller, G., and E. Moller, Ann. N. Y. Acad. Sci., 129, 735 (1966). "Moller, G., V. Beckman, aind G. Lundgren, Nature, 212, 1203 (1966). 12 Hellstrom, K. E., I. Hellstrorn, and G. Haughton, Nature, 204, 661 (1964). 13 Hellstrom, K. E., I. Hellstr6m, and C. Bergheden, Nature, 208, 458 (1965). 14 Holm, G., Exptl. Cell Research, 48, 327 (1967). " Granger, G. A., and W. P. Kolb, J. Immunol., 101, 111 (1968). 16 Kolb, W. P., and G. A. Granger, Federation Proc., 27, 687 (1968). 17 Williams, T. W., and G. A. Granger, Nature, 219, 1076 (1968). 18 Granger, G. A., and T. W. Williams, Nature, 218, 1253 (1968). 19 Oppenheim, J. J., Federation Proc., 27, 21 (1968). 20 Garen, A., and C. Levinthal, Biochim. Biophys. Acta, 38, 470 (1960). 21 Hu, A. S. L., R. M. Bock, and H. 0. Halvorson, Anal. Biochem., 4, 489 (1962). 22 Kolb, W. P., and G. A. Grmnger, manuscript in preparation.



lymphocyte in vitro cytotoxicity: characterization of human lymphotoxin

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