INFECTION AND IMMUNrrY, Aug. 1975, p. 225-232 Copyright 01975 American Society for Microbiology
Vol. 12, No. 2 Printed in U.SA.
Sensitive Assay for Detection of Toxin-Induced Damage to the Cytoplasmic Membrane of Human Diploid Fibroblasts MONICA THELESTAM* AND ROLAND MOLLBY Department of Bacteriology, Karolinska Institutet, S-104 01 Stockholm 60, Sweden Received for publication 13 March 1975
A sensitive assay was developed for detection and quantitation of subtle permeability changes in the cytoplasmic membrane of human diploid fibroblasts. Release of the non-metabolizable amino acid [1-14C]alphaaminoisobutyric acid (AIB; molecular weight 103) from the cytoplasm of prelabeled cells was used as an indicator of toxin-induced membrane damage. An optimal procedure for labeling these cells was designed after varying the conditions with regard to pH, temperature, concentration of AIB, composition of medium, and incubation time. Toxin-induced release of AIB was compared with release of a previously described nucleotide label, [3H ]uridine. Melittin from bee venom and the polyene antibiotics filipin and amphotericin B in low concentrations induced a strikingly greater release of AIB than of nucleotide label. The sensitivity of this assay was furthermore demonstrated by treatment with the following bacterial cytolysins: phospholipase C and theta-toxin from Clostridium perfringens, alpha-, beta-, delta-, and gamma-toxins from Staphylococcus aureus, and streptolysin S from Streptococcus pyogenes. In spite of their different modes of action, all these membrane-active toxins at low concentrations induced a significant release of AIIB label. For an equal release of nucleotide label, several times higher concentrations were required.
Release of intracellular substances has been used by several investigators as a criterion of damage to the cytoplasmic membrane of cells (13, 18, 26, 35, 43). To detect minor permeability changes and assess kinetic aspects of cell lysis, the ions K+ and Rb+ have been used as cytoplasmic markers (6, 8, 16, 20, 27). The sequence of reactions leading to hemolysis induced by different agents has been studied by comparison of release of K+ or Rb+ and hemoglobin (8, 23, 25). Adenosine 5'-triphosphate and ["IC]nicotinamide have also been used as low-molecular-weight cytoplasmic markers (22, 29). To investigate the effects of bacterial cytolysins on human diploid embryonic lung fibroblasts, we used 3H-labeled nucleotide as a
low-molecular-weight cytoplasmic marker (31, 39, 40). It became desirable to use a more sensitive assay for detecting subtle changes in membrane permeability. In our system, measurement of release of potassium was not suitable due to a very rapid spontaneous leakage of this ion (M. Thelestam, unpublished data). 42K or "IRb are not ideal for routine cytotoxicity screening purposes due to their gamma emission and short half-lives. A good marker should be readily accumulated
by the cell, should not be incorporated into macromolecules, and should be treated as nonforeign material in the cell. We hypothesized that non-metabolizable amino acids might offer such qualities (4, 7) and evaluated the use of [1- 14CJalpha-aminoisobutyric acid (AIB; molecular weight 103) as a low-molecular-weight cytoplasmic marker. Optimal labeling for a leakage assay should result in a high cytoplasmic radioactivity paired with a low spontaneous release during the test. In this report we describe suitable conditions for the use of AIB as a low-molecular-weight cytoplasmic marker. Furthermore, the sensitivity of this assay is demonstrated with a number of bacterial cytolysins and other cytolytic agents of nonbacterial origin. MATERIALS AND METHODS Chemicals. Eagle medium and Hanks balanced salt solution (BSS) were obtained from the National Bacteriological Laboratory, Stockholm, Sweden. [5'H]uridine (specific activity, 24 Ci/mmol), [1-4C IAIB (specific activity, 12 mCi/mmol), and Aquasol TM Universal Cocktail were from New England Nuclear Chemicals GmbH, Frankfurt, West Germany. Sodium pyruvate, sodium borate, sodium chloride, potassium chloride, boric acid, and tris(hydroxymethyl)aminomethane (Tris) were purchased from E. 225
226
THELESTAM AND MOLLBY
Merck, Darmstadt, West Germany, dimethyl sulfoxide from Mallinckrodt, and BioGel from BioRad Laboratories, Richmond, Calif. Unless otherwise stated, all chemicals were of analytical grade. Toxic substances. Triton X-100 (technical grade) was purchased from Rohm and Haas Co., Philadelphia, Pa. Purified melittin, free from phospholipase activity, was a kind gift from W. Vogt and P. G. Lankisch, Max-Planck Institute, Gottingen, Germany. Filipin complex (U-5956, ref. 8393-DEC-11-18, crystalline complex, 66% pure) was a kind gift from G. B. Whitfield, The Upjohn Co., Kalamazoo, Mich. Amphotericin B (batch 22-380-39568-001) was generously supplied by the Squibb Institute, Princeton, N.J. Filipin and amphotericin B were dissolved in dimethyl sulfoxide to 0.1 M stock solutions. Phospholipase C from Clostridium perfringens was highly purified according to Mollby and Wadstrom (32). Highly purified theta-toxin from the same organism was obtained by similar methods (36; R. M3llby, unpublished data). Streptolysin S from Streptococcus pyogenes, prepared according to Bernheimer (1), was kindly supplied by C. J. Smyth. Crude enterotoxin, containing 4 mean effective doses per mg of protein, was obtained by concentrating (39 times) the culture supernatant after cultivation of Escherichia coli (porcine strain 853/67) under controlled conditions in a fermentor (R. Mollby, 0. Sioderlind, and T. Wadstriom, unpublished data). Highly purified alpha-, beta-, delta-, and gamma-toxins from Staphylococcus aureus were obtained as recently described (40). Cultivation of cells. The basic methods have been described in detail by Thelestam et al. (40). A line of human diploid embryonic lung fibroblasts [Lu(S)], isolated and kindly supplied by Goran Wadell (National Bacteriological Laboratory, Stockholm), was used. For cytotoxicity testing, cells were cultivated in wells (culture area, 7 cm2) in disposable polystyrene trays (FB-6-TC, Linbro Chemicals Co., Inc., New Haven, Conn.). Seeding density was 90,000 cells per culture in 3 ml of Eagle medium (9) supplemented with 10% calf serum, 5 mM glutamine, 1 mM sodium pyruvate, penicillin (100 IU/ml), and streptomycin (100 Ag/ml). Cultures were incubated at 37 C in a humid atmosphere containing 5% C02-95% air and used for testing purposes after 6 to 8 days. Labeling of cells. Confluent monolayers were incubated for 30 min in 1 ml of Hanks BSS. The medium was changed to 1 ml of AIB (1 ACi/ml) in Hanks BSS prewarmed to 37 C. A 1-h labeling period was followed by a nonradioactive chase in 1 ml of fresh medium for 30 min. This marker will be referred to as the AIB label. For comparison, parallel cultures were nucleotide labeled as earlier described (40). Confluent monolayers were incubated with 1 ml of [aHlluridine (1 MCi/ml) in Eagle medium for 2 h. A 2-h nonradioactive chase in 1 ml of fresh medium followed. This marker will be referred to as the nucleotide label. All incubations were performed at 37 C and pH 7.5. The cells were washed three times with Hanks BSS before the test. Maximal and spontaneous release. The maximal release of cytoplasmic radioactivity was determined
INF-ECT. IMMUN. after cell membrane rupture with a 0.06 M borate buffer (pH 7.8) as described by Thelestam et al. (40). Radioactivity in 0.1-ml samples, diluted in 10 ml of Aquasol, was measured by counting in a Nuclear Chicago liquid scintillator. The absolute values for the maximal releases of AIB and nucleotide labels were usually 100,000 to 200,000 counts/min per culture (0.6 x 106 to 0.7 x 10' cells), depending on cell density and slight variations in labeling conditions. The spontaneous release of AIB label during standard test conditions, i.e., incubation at 37 C for 30 min in Tris-buffered saline, pH 7.0 (TBS), varied between 15 and 30%. For the nucleotide label the spontaneous release was 3 to 7%. Size of the AIB label. The molecular size of the AIB label in cell lysates free of nuclei was estimated, in comparison with plain AIB, by gel chromatography on a BioGel P-2 column. The column was 2.5 cm by 24.5 cm, the flow rate was 2.7 ml/h x cm2, and 4.5-ml fractions were collected. TBS was used for equilibration of the gel and for elution. Radioactivity was determined in a 0.1-ml sample of each fraction. Toxin treatment of cells. Labeled cultures were incubated with test substances in TBS. Unless otherwise stated, incubation was performed at 37 C for 30 min at pH 7.0. After this, the incubation medium was sucked off and centrifuged (1,000 x g, 10 min, 4 C). Radioactivity was measured in 0.1 ml of the supernatant. Released radioactivity was expressed as percentage of maximal release, i.e., (toxin-induced release spontaneous release)/(maximal release - spontaneous release) x 100. In this formula, a relatively low spontaneous release is necessary (38). Therefore, when the spontaneous release occasionally exceeded 30%, the experiment was discarded. All tests were performed in duplicate or triplicate. Hemolytic and phospholipase C assays. Alphatoxin was assayed on rabbit erythrocytes, beta- and theta-toxins were assayed on sheep erythrocytes, and delta- and gamma-toxins and streptolysin S were assayed on human erythrocytes as previously described (37, 40). Phospholipase C activity was determined by a titrimetric method on egg yolk suspension as substrate (32).
RESULTS Labeling conditions. To obtain optimal labeling conditions, the following variables were studied: concentration of AIB, type of labeling medium, temperature, pH, and time for labeling, as well as nonradioactive preincubations and chases. A concentration of 1 ACi/ml was found to be optimal for labeling. The cytoplasmic activity obtained with this concentration was dependent on the composition of the labeling medium (Table 1). The presence of other amino acids in Eagle medium depressed the uptake of AIB. The uptake was stimulated by glucose in phosphate-buffered saline (PBS) and by Hanks BSS. Prewarming of the AIB solution to 37 C before
VOL. 12, 1975
ASSAY FOR DETECTING TOXIN-INDUCED DAMAGE
TABLE 1. Cellular uptake of AIB in different mediaa Labeling medium con-
Cytoplasmic activity
taining 1 uCi of AIB/ml
(counts/min per 10' cells)
TABLE 2. Cellular uptake of AIB in relation to the starting temperature of the AIB solutiona
application to cells increased the uptake of AIB significantly (Table 2). The shock of a sudden decrease in temperature evidently caused a disturbance of the amino acid transport system. The uptake of AIB was highly sensitive to changes in pH with an optimum at 7.5 (Fig. 1). No further increase in cytoplasmic activity was evident after a labeling period of 1 h. The intracellular radioactivity was further increased by a 30-min preincubation in Hanks BSS before labeling. A 30- to 60-min nonradioactive chase in Eagle medium lowered the spontaneous release during the subsequent test, whereas a similar chase in Hanks BSS caused an extremely high spontaneous release. Spontaneous release. The spontaneous release of AIB label during standard test conditions, i.e., 30 min at 37 C (pH 7.0), was studied in different solutions (Table 3). Roughly similar results were obtained in Tris-buffered potassium chloride (TBP), TBS, PBS, and these solutions supplemented with 0.1% glucose. The spontaneous release of AIB label from confluent monolayers was also followed with incubation time (Fig. 2). The release increased rapidly, reaching more than 50% in 1 h. Release in TBP was lower than in TBS, especially during incubations longer than 30 min. A standard procedure for labeling and testing with AIB was designed on basis of these results, as described in Materials and Methods. Molecular size of the AIB label. To confirm that AIB was not bound to cytoplasmic substances under the conditions used, lysates from
cells labeled with AIB were chromatographed on BioGel P-2. The labeled cytoplasmic material was eluted as a single peak coinciding with plain AIB that had not been in contact with cells. Induced release of AIB and nucleotide labels. Parallel cultures were labeled with AIB and [3H ]uridine and treated with cytolytic agents (Fig. 3). The nonionic detergent Triton X-100 induced the release of equal amounts of nucleotide and AIB labels. Low concentrations
Cytoplasmic activity
Starting temp (C)
(counts/min per 10' cells)
+4 +20 +37
90,302 237,188 275,800
8,583 ......... TBS .... PBS + 0.1% glucose ............. 131,683 Hanks BSS ....... ...... 290,950 Eagle medium ............. 48,667 a Confluent cultures were labeled with AIB for 1 h at 37 C as indicated. Cytoplasmic radioactivity was estimated after cell membrane rupture with a borate buffer as described in the text.
227
a Confluent cultures were labeled with AIB in Hanks BSS (1 AiCi/ml) for 1 h at 37 C. The labeling medium had the starting temperatures indicated. Cytoplasmic radioactivity was estimated after cell membrane rupture with a borate buffer as described in the text. I
3.105 2X105 u
a. i.io5
6.0
8.0
7.0 pH
FIG. 1. Effect of pH on the uptake of AIB. Confluent cultures were labeled with AIB as described in the text. NaHCO was added to Hanks BSS in amounts sufficient to give the pH values indicated, after equilibration with an atmosphere containing 5% CO2. Maximal cytoplasmic radioactivity was estimated after cell membrane rupture and expressed as counts per minute per 10J cells. TABLE 3. Spontaneous release of AIB label in different solutionsa
Solution
release) of maximal release (%Spontaneous
.......... TBP ... .......... TBS ... TBS + 0.1% glucose ............. PBS ............. PBS + 0.1% glucose ............. ........ Hanks BSS ..... ...... Eagle medium .......
12.1 19.8 15.3 18.0 12.8 31.4 52.0
aConfluent cultures were labeled with AIB in Hanks BSS as described in the text. Spontaneous release during 30 min at 37 C in the solutions indicated was measured and expressed as percentage of the maximal release determined in parallel cultures.
of melittin, a cationic polypeptide with surfaceactive properties (34), induced the release of significantly greater amounts of AIB than nu-
cleotide label. Similar results were obtained with the polyene antibiotics filipin and am-
228
INFECT. IMMUN.
THELESTAM AND MOLLBY
hemolytic unit (HU)/ml, remarkably more AIB label was liberated. Phospholipase C induced the release of only slightly more AIB than 100 nucleotide label (Fig. 4B). Treatment with streptolysin S at concentrations below 2.5 HU/ml caused the release of about 75% of AIB /g E label without any nucleotide release (Fig. 4C). Considerably more, in terms of HU, was needed E 50s I of streptolysin S than of theta-toxin for release c of the nucleotide label. E. coli enterotoxin appeared to be without effect on membrane permeability since neither nucleotide nor AIB label was released after treatment with a crude " of this toxin (Fig. 4D). preparation f i is 30 45 120 60 Treatment with staphylococcal alpha-, beta-, Incubation time (min) FIG. 2. Spontaneous release in relation to incuba- and gamma-toxins induced a concentrationtion time. The spontaneous release of A1B label in dependent release of the AIB label, whereas the TBS and TBP was followed for 2 h and e.xpressed as nucleotide label was retained at the concentrapercentage of the initial cytoplasmic raodioactivity. tions indicated in Fig. 5. By contrast, after exposure to delta-toxin, both nucleotide and Symbols: O, Release in TBS; *, release ijn TBP. AIB labels were released. One-hundredth of a hemolytic unit caused the release of more than 50% of the AIB label. However, for an equal 100 release of nucleotide label, 0.1 HU was required. At higher concentrations the release curves coincided. a The time courses for the release of AIB and nucleotide labels induced by amphotericin B 50 and melittin are shown in Fig. 6. A release of more than 50% of the AIB label was caused in 5 min by both amphotericin B and melittin. The L. corresponding amount of nucleotide label was detected only 1 to 2 h later at the concentrations used. These time curves illustrate how effectively the release of AIB label may demonstrate Ampho- Hypotonic Filipin Triton X-100 Melittin NaC tericin B rapidly occurring changes in membrane 16.1mM 100pM 10pM 1.5 yM 0,2mM permeability.
'"',-
a
x
E
0
i
FIG. 3. Effects of various cytolytic agernts. Release of AIB and nucleotide labels from culture,s incubated at 37 C for 30 min with various cytolyti[c agents at concentrations chosen so as to give apj 75% release of the AIB label. White bars, hatched bars, nucleotide label.
pAIBlatbely 'l
photericin B, which are known to ireact with membrane cholesterol (19). Howlever, amphotericin B caused the release of strikingly more AIB than nucleotide label, w]hereas the difference between AIB and nucleotiide release induced by filipin was less conspicuc)us. Hypotonic swelling of the cells also causesI a greater release of AIB than of nucleotide Ilabel. The effects of these agents at different close levels will be reported separately (Thele stam and M6llby, unpublished data). Clostridial theta-toxin induced a 4concentration-dependent release of both AIB a nd nucleotide labels (Fig. 4A). At concentratio gns below 1
DISCUSSION AIB was initially considered for use as a cytoplasmic marker for the following reasons: (i) it is known to be actively accumulated by different kinds of cells (5, 7, 14, 28); (ii) it is not metabolized in the cell (3); and (iii) it has a low molecular weight, 103. Furthermore, "4Clabeled AIB is commercially available. The subsequent study of optimal labeling conditions with human diploid fibroblasts indicated that uptake of AIB by these cells was dependent on the same factors as described for other cell types (21). The uptake was competitively inhibited by the presence of other amino acids in Eagle medium in accordance with Christensen and Liang (5) (Table 1). Foster and Pardee (14) used PBS + glucose for incorporation of AIB into 3T3 cells. In the present system, glucose improved uptake of AIB as compared
VOL. 12, 1975
ASSAY FOR DETECTING TOXIN-INIDUCED DAMAGE I
~~~I
I
I
229
I
A
B
100
100 Go
50 L0
E x
1.0 HU/m
0.5
0
E
2
1.5
4
U/mi
oa
D
0 c
100
E5c
100
50
0.25
2.5 HU/ml
25
Concentration
2.5 mg/ml
5.0
FIG. 4. Effects of various bacterial toxins. Release of AIB and nucleotide labels from cultures incubated at 37 C for 30 min with (A) theta-toxin, (B) phospholipase C, (C) streptolysin S, and (D) enterotoxin at the concentrations indicated. Symbols: 0, AIB label; 0, nucleotide label.
A
B 100
100
to W
a# 50
50
I x
a
.
4
E
-8
16
25
10
50
-a E
1-
x a
E
D
C
E 100
9
0
a
100
c u
so.
50
I
4
8
I
.
a
0.5
16
I
,
1.0
Concentration (HU/ml)
FIG. 5. Effects of staphylococcal toxins. Release of AIB and nucleotide labels from cultures incubated at 37 C for 30 min with (A) alpha-toxin, (B) beta-toxin, (C) gamma-toxin, and (D) delta-toxin at the concentrations indicated. Symbols as in Fig. 4.
with TBS -nly. However, Hanks BSS proved to be even more suitable for efficient labeling of our cells. The uptake of AIB was highly sensitive to changes in temperature and pH, as reported for
the Ehrlich cell (5). A ratio of equilibrium distribution between intra- and extracellular AIB
was
reached within 1 to 2 h with several
types of cells (7, 11, 28). Also, in our cell system 1 h
was
sufficient for maximal labeling. Prein-
230
THELESTAM AND MOLLBY
INFECT. IMMUN.
B
100
*
I I
I
E
I
x
a
I 0
E so;
50
0-
E
0
30
60
90
30
60
90
Incubation time (min)
FIG. 6. Effects of melittin and amphotericin B in relation to incubation time. Release of AIB and nucleotide labels from cultures treated with (A) melittin (1.5 MM) and (B) amphotericin B (100 MM) for the periods of time indicated. Symbols as in Fig. 4.
cubation in a solution free from amino acids in TBP than in TBS. The flux of AIB through (Hanks BSS) before labeling increased the sub- cell membranes is Na+ dependent (33), which sequent uptake of AIB, possibly due to deple- may explain the differences found in TBP and TBS. The stimulation of AIB efflux with intion of the endogenous amino acid pool (14). Gel chromatography of AIB-labeled cell ly- creasing intracellular concentrations of Na+ sates on BioGel P-2 indicated that this amino (10, 41) could be an important factor in release acid was not metabolized in the present cell of AIB from only slightly damaged cells. In such system. The effective diameter of AIB as a cases the measured release of AIB may repremarker should thus be 0.6 to 0.8 nm (17). This sent a stimulated efflux in addition to the actual leakage due to changed permeability of the agrees with the fact that the sensitivity of the test with AIB as a marker was comparable with membrane. This may involve the interesting that found when release of K+ was measured possibility of obtaining an increased sensitivity (Thelestam, unpublished data). The high sensi- for agents acting as Na+ ionophores. Additional tivity should enable detection and quantitation information may thus be achieved regarding the of subtle permeability changes during early mode of action of cytolytic toxins by performing stages of cytolysis. The diffusion rate of the parallel tests in solutions without Na+ ions. marker used in leakage tests is an important When investigating effects of bacterial toxins factor for adequate reflection of permeability on the fibroblast membrane, we have used the changes (30). Thus, membrane alterations may nonionic detergent Triton X-100, the surface be detected early simply because the small AIB active polypeptide melittin, and the polyene molecules diffuse very rapidly out of the cell antibiotics filipin and amphotericinx B as referafter perturbation of the cell membrane. ence substances. The present study indicates AIB is actively transported out of the cell by that release of AIB may be used to detect lower the same system mediating its uptake (4). The concentrations of these agents more rapidly active efflux explains the relatively high sponta- than is possible when measuring release of nuneous release observed in the present cell syscleotide label (Fig. 3 and 6). The fact that a tem (Table 3 and Fig. 2). The high spontaneous 'sublytic concentration of Triton X-100 caused release is a disadvantage with this assay and the release of the same amount of nucleotide as makes it unsuitable for long-term studies of of AIB label agrees with our earlier observamembrane damage. The same limitation has tions regarding the size of the "functional been reported for K+ and Rb+ (22, 23, 29). A holes" produced by this detergent (39). Effects somewhat lower spontarneous release was con- of these reference substances as indicated by sistently found when the solution contained K+ release of four different cytoplasmic markers (TBP) instead of Na+ ions (TBS). However, in will be reported elsewhere in more detail (Thethis study the incubations with toxins were lestam and M6llby, unpublished data). performed in TBS because preliminary experiRecently we described the effects of crude and ments indicated that the sensitivity was lower highly purified preparations of staphylococcal
VOL. 12, 1975
ASSAY FOR DETECTING TOXIN-INDUCED DAMAGE
toxins on the fibroblast membrane, using release of nucleotide label as criterion of membrane damage (40). Highly purified alpha-, beta-, and gamma-toxins appeared to be without effect on these cells. However, the present study showed that these toxins caused a release of the AIB label, indicating a changed permeability of the fibroblast plasma membrane (Fig. 5). The fact that this change did not result in any morphological effects may be interpreted as partly due to the high repair capacity in the membrane of contact-inbibited fibroblasts (42). These hemolytic toxins induce more drastic effects on liposomes and erythrocytes (2, 15), which may be explained in part by the lack of such repair mechanisms. The agents used in this investigation were hemolytic, with the exception of the E. coli enterotoxin. This toxin has recently been shown to bind to membrane receptors, causing an activation of the adenylate cyclase in human intestinal cells (24). Such an activation results in an active efflux of potassium chloride and bicarbonate ions as shown with Vibrio cholerae enterotoxin (12). However, the E. coli enterotoxin did not cause any release of AIB label from the fibroblasts used in this study. In conclusion, suitable conditions have been presented for the use of AIB as a cytoplasmic marker in a standardized leakage test. Release of the AIB label is a sensitive indicator of alterations in cell membrane permeability as evidenced by the dose response curves and time course relationships presented. This assay may be applied in the study of alterations in membrane permeability caused by various cytolytic toxins and enzymes, membrane-active antibiotics, immune lysis, and industrial chemicals such as detergents and heavy metals. ACKNOWLEDGMENTS We are greatly indebted to C. J. Smyth and T. Wadstrom for stimulating discussions and generous support. The skillful technical assistance of G. Blomquist, M. Kjellgren, and L. Norenius is gratefully acknowledged. This work was supported by the Swedish Medical Research Council (grant no. 16X-2562) and Karolinska institutets fonder. R.M. had a research position at the Swedish Medical Research Council (no. 40P-4576).
of mediated exodus of amino acids from the Ehrlich ascites tumor cell. J. Biol. Chem. 243:5428-5438. 5. Christensen, H. N., and M. Liang. 1966. On the nature of the "non-saturable" migration of amino acids into Ehrlich cells and into rat jejunum. Biochim. Biophys. Acta 112:524-531. 6. De Kruijff, B., W. J. Gerritsen, A. Oerlemans, R. A. Demel, and L. L. M. van Deenen. 1974. Polyene antibiotic sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. I. Specificity of the membrane permeability changes induced by the polyene antibiotics. Biochim. Biophys. Acta
339:30-43. 7. Dickson, J. A. 1970. The uptake of non-metabolizable amino acids as an index of cell viability in vitro. Exp. Cell Res. 61:235-245. 8. Duncan, J. L. 1974. Characteristics of streptolysin 0 hemolysis: kinetics of hemoglobin and 'lrubidium release. Infect. Immun. 9:1022-1027. 9. Eagle, H. 1959. Amino acid metabolism in mammalian cell cultures. Science 130:432-437. 10. Eddy, A. A. 1968. The effects of varying the cellular and extracellular concentrations of sodium and potassium ions on the uptake of glycine by mouse ascites tumor cells in the presence and absence of sodium cyanide. Biochem. J. 108:489-498. 11. Elsas, L. J., and L. E. Rosenberg. 1967. Inhibition of amino acid transport in rat kidney cortex by puromycin. Proc. Natl. Acad. Sci. U.S.A. 57:371-378. 12. Finkelstein, R. A. 1973. Cholera. CRC Crit. Rev. Microbiol. 2:553-623. 13. Forbes, I. J. 1963. Studies of cytotoxicity using P32. Aust. J. Exp. Biol. 41:255-264. 14. Foster, D. O., and A. B. Pardee. 1969. Transport of amino acids by confluent and non-confluent 3T3 and polyoma virus-transformed 3T3 cells growing on glass cover slip. J. Biol. Chem. 244:2675-2681. 15. Freer, J. H., J. P. Arbuthnott, and A. W. Bernheimer. 1968. Interaction of staphylococcal alpha-toxin with artificial and natural membranes. J. Bacteriol. 95:1153-1168. 16. Gale, E. F. 1974. The release of potassium ions from Candida albicans in the presence of polyene antibiotics. J. Gen. Microbiol. 80:451-465. 17. Giese, A. C. 1973. Cell physiology, p. 291, 4th ed. W. B. Saunders Co., Philadelphia. 18. Green, H., R. A. Fleischer, P. Barrow, and B. Goldberg. 1959. The cytotoxic action of immune gamma globulin and complement on Krebs ascites tumor cells. II. Chemical studies. J. Exp. Med. 109:511-521. 19. Hamilton-Miller, J. M. T. 1974. Fungal sterols and the mode of action of the polyene antibiotics. Adv. Appl.
Microbiol. 18:109-134. 20. Hammond, S. M., P. A. Lambert, and B. N. Kliger. 1974. The mode of action of polyene antibiotics; induced potassium leakage in Candida albicans. J. Gen. Micro-
biol. 81:325-330.
21. Heinz, E. 1972. Transport of amino acids by animal cells, p. 455-501. In L. E. Hokin (ed.), Metabolic pathways, vol. 6, Metabolic transport. Academic Press Inc., New
York.
LITERATURE CITED 1. Bernheimer, A. W. 1949. Formation of a bacterial toxin
(Streptolysin S) by resting cells. J. Exp. Med. 90:373-392. 2. Bernheimer, A. W. 1974. Interactions between membranes and cytolytic bacterial toxins. Biochim. Biophys. Acta 344:27-50. 3. Christensen, H. N., A. J. Aspen, and E. G. Rice. 1956. Metabolism in the rat of three amino acids lacking alpha-hydrogen. J. Biol. Chem. 220:287-294. 4. Christensen, H. N., and M. E. Handlogten. 1968. Modes
231
22. Henney, C. S. 1973. Studies on the mechanism of lymphocyte mediated cytolysis. II. The use of various target ceil markers to study cytolytic events. J. Immunol. 110:73-84. 23. Hingson, D. J., R. K. Massengill, and M. M. Mayer. 1969. The kinetics of release of serubidium and hemoglobin from erythrocytes damaged by antibody and complement. Immunochemistry 6:295-307. 24. Kantor, H. S., P. Tao, and C. Wisdom. 1974. Action of Escherichia coli enterotoxin: adenylate cyclase behavior of intestinal epithelial cells in culture. Infect. Immun. 9:1003-1010.
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25. MacGregor, R. D., II, and C. A. Tobias. 1972. Molecular sieving of red cell membranes during gradual osmotic hemolysis. J. Membr. Biol. 10:345-356. 26. Madoff, M. A., M. S. Artenstein, and L. Weinstein. 1963. Studies on the biologic activity of purified staphylococcal alpha-toxin. II. The effect of alpha-toxin on Ehrlich ascites carcinoma cells. Yale J. Biol. Med. 35:382-389. 27. Madoff, M. A., L. Z. Cooper, and L. Weinstein. 1964. Hemolysis of rabbit erythrocytes by purified staphylococcal alpha-toxin. III. Potassium release. J. Bacteriol. 87:145-149. 28. Mahoney, M. J., and L. E. Rosenberg. 1970. Uptake of alpha-aminoisobutyric acid by cultured human fibroblasts. Biochim. Biophys. Acta 219:500-502. 29. Martz, E., S. J. Burakoff, and B. Benacerraf. 1974. Interruption of the sequential release of small and large molecules from tumor cells by low temperature during cytolysis mediated by immune T-cells or complement. Proc. Natl. Acad. Sci. U.S.A. 71:177-181. 30. Miller, R. G., and M. Dunkley. 1974. Quantitative analysis of the 1Cr release cytotoxicity assay for cytotoxic lymphocytes. Cell. Immunol. 14:284-302. 31. Mollby, R., M. Thelestam, and T. Wadstrom. 1974. Effect of Clostridium perfringens phospholipase C (alpha-toxin) on the human diploid fibroblast membrane. J. Membr. Biol. 16:313-330. 32. Mollby, R., and T. Wadstrom. 1973. Purification of phospholipase C (alpha-toxin) from Clostridium perfringens. Biochim. Biophys. Acta 321:569-584. 33. Schultz, S. G., and P. F. Curran. 1970. Coupled transport of sodium and organic solutes. Physiol. Rev. 50:637718. 34. Sessa, G., J. H. Freer, G. Colacicco, and G. Weissmann. 1969. Interaction of a lytic polypeptide, melittin, with lipid membrane systems. J. Biol. Chem.
INFECT. IMMUN. 244:3575-3582. 35. Siegel, I., and S. Cohen. 1964. Action of staphylococcal toxin on human platelets. J. Infect. Dis. 114:488-502. 36. Smyth, C. J. 1975. The identification and purification of multiple forms of theta-haemolysin (theta-toxin) of Clostridium perfringens type A. J. Gen. Microbiol. 87:219-238. 37. Strandberg, K., R. Mollby, and T. Wadstrom. 1974. Histamine release from mast cells by highly purified phospholipase C (alpha-toxin) and theta-toxin from Clostridium perfringens. Toxicon 12:199-208. 38. Stulting, R. D., and G. Berke. 1973. The use of 51Cr release as a measure of lymphocyte-mediated cytolysis in vitro. Cell. Immunol. 9:474-476. 39. Thelestam, M., and R. Mollby. 1975. Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts. Infect. Immun. 11:640-648. 40. Thelestam, M., R. Mollby, and T. Wadstrom. 1973. Effects of staphylococcal alpha-, beta-, delta-, and gamma-hemolysins on human diploid fibroblasts and HeLa cells: evaluation of a new quantitative assay for measuring cell damage. Infect. Immun. 8:938-946. 41. Vidaver, C. A., and S. L. Shepherd. 1968. Transport of glycine by hemolyzed and restored pigeon red blood cells, Symmetry properties, trans effects of sodium ion and glycine and their description by a single rate equation. J. Biol. Chem. 243:6140-6150. 42. Warren, L., and M. C. Glick. 1968. Membranes of animal cells. II. The metabolism and turnover of the surface membrane. J. Cell Biol. 37:729-746. 43. Wigzell, H. 1965. Quantitative titrations of mouse H-2 antibodies using Cr5l-labeled target cells. Transplantation 3:423-431.
Vol. 12, No. 2 Printed in U.SA.
Sensitive Assay for Detection of Toxin-Induced Damage to the Cytoplasmic Membrane of Human Diploid Fibroblasts MONICA THELESTAM* AND ROLAND MOLLBY Department of Bacteriology, Karolinska Institutet, S-104 01 Stockholm 60, Sweden Received for publication 13 March 1975
A sensitive assay was developed for detection and quantitation of subtle permeability changes in the cytoplasmic membrane of human diploid fibroblasts. Release of the non-metabolizable amino acid [1-14C]alphaaminoisobutyric acid (AIB; molecular weight 103) from the cytoplasm of prelabeled cells was used as an indicator of toxin-induced membrane damage. An optimal procedure for labeling these cells was designed after varying the conditions with regard to pH, temperature, concentration of AIB, composition of medium, and incubation time. Toxin-induced release of AIB was compared with release of a previously described nucleotide label, [3H ]uridine. Melittin from bee venom and the polyene antibiotics filipin and amphotericin B in low concentrations induced a strikingly greater release of AIB than of nucleotide label. The sensitivity of this assay was furthermore demonstrated by treatment with the following bacterial cytolysins: phospholipase C and theta-toxin from Clostridium perfringens, alpha-, beta-, delta-, and gamma-toxins from Staphylococcus aureus, and streptolysin S from Streptococcus pyogenes. In spite of their different modes of action, all these membrane-active toxins at low concentrations induced a significant release of AIIB label. For an equal release of nucleotide label, several times higher concentrations were required.
Release of intracellular substances has been used by several investigators as a criterion of damage to the cytoplasmic membrane of cells (13, 18, 26, 35, 43). To detect minor permeability changes and assess kinetic aspects of cell lysis, the ions K+ and Rb+ have been used as cytoplasmic markers (6, 8, 16, 20, 27). The sequence of reactions leading to hemolysis induced by different agents has been studied by comparison of release of K+ or Rb+ and hemoglobin (8, 23, 25). Adenosine 5'-triphosphate and ["IC]nicotinamide have also been used as low-molecular-weight cytoplasmic markers (22, 29). To investigate the effects of bacterial cytolysins on human diploid embryonic lung fibroblasts, we used 3H-labeled nucleotide as a
low-molecular-weight cytoplasmic marker (31, 39, 40). It became desirable to use a more sensitive assay for detecting subtle changes in membrane permeability. In our system, measurement of release of potassium was not suitable due to a very rapid spontaneous leakage of this ion (M. Thelestam, unpublished data). 42K or "IRb are not ideal for routine cytotoxicity screening purposes due to their gamma emission and short half-lives. A good marker should be readily accumulated
by the cell, should not be incorporated into macromolecules, and should be treated as nonforeign material in the cell. We hypothesized that non-metabolizable amino acids might offer such qualities (4, 7) and evaluated the use of [1- 14CJalpha-aminoisobutyric acid (AIB; molecular weight 103) as a low-molecular-weight cytoplasmic marker. Optimal labeling for a leakage assay should result in a high cytoplasmic radioactivity paired with a low spontaneous release during the test. In this report we describe suitable conditions for the use of AIB as a low-molecular-weight cytoplasmic marker. Furthermore, the sensitivity of this assay is demonstrated with a number of bacterial cytolysins and other cytolytic agents of nonbacterial origin. MATERIALS AND METHODS Chemicals. Eagle medium and Hanks balanced salt solution (BSS) were obtained from the National Bacteriological Laboratory, Stockholm, Sweden. [5'H]uridine (specific activity, 24 Ci/mmol), [1-4C IAIB (specific activity, 12 mCi/mmol), and Aquasol TM Universal Cocktail were from New England Nuclear Chemicals GmbH, Frankfurt, West Germany. Sodium pyruvate, sodium borate, sodium chloride, potassium chloride, boric acid, and tris(hydroxymethyl)aminomethane (Tris) were purchased from E. 225
226
THELESTAM AND MOLLBY
Merck, Darmstadt, West Germany, dimethyl sulfoxide from Mallinckrodt, and BioGel from BioRad Laboratories, Richmond, Calif. Unless otherwise stated, all chemicals were of analytical grade. Toxic substances. Triton X-100 (technical grade) was purchased from Rohm and Haas Co., Philadelphia, Pa. Purified melittin, free from phospholipase activity, was a kind gift from W. Vogt and P. G. Lankisch, Max-Planck Institute, Gottingen, Germany. Filipin complex (U-5956, ref. 8393-DEC-11-18, crystalline complex, 66% pure) was a kind gift from G. B. Whitfield, The Upjohn Co., Kalamazoo, Mich. Amphotericin B (batch 22-380-39568-001) was generously supplied by the Squibb Institute, Princeton, N.J. Filipin and amphotericin B were dissolved in dimethyl sulfoxide to 0.1 M stock solutions. Phospholipase C from Clostridium perfringens was highly purified according to Mollby and Wadstrom (32). Highly purified theta-toxin from the same organism was obtained by similar methods (36; R. M3llby, unpublished data). Streptolysin S from Streptococcus pyogenes, prepared according to Bernheimer (1), was kindly supplied by C. J. Smyth. Crude enterotoxin, containing 4 mean effective doses per mg of protein, was obtained by concentrating (39 times) the culture supernatant after cultivation of Escherichia coli (porcine strain 853/67) under controlled conditions in a fermentor (R. Mollby, 0. Sioderlind, and T. Wadstriom, unpublished data). Highly purified alpha-, beta-, delta-, and gamma-toxins from Staphylococcus aureus were obtained as recently described (40). Cultivation of cells. The basic methods have been described in detail by Thelestam et al. (40). A line of human diploid embryonic lung fibroblasts [Lu(S)], isolated and kindly supplied by Goran Wadell (National Bacteriological Laboratory, Stockholm), was used. For cytotoxicity testing, cells were cultivated in wells (culture area, 7 cm2) in disposable polystyrene trays (FB-6-TC, Linbro Chemicals Co., Inc., New Haven, Conn.). Seeding density was 90,000 cells per culture in 3 ml of Eagle medium (9) supplemented with 10% calf serum, 5 mM glutamine, 1 mM sodium pyruvate, penicillin (100 IU/ml), and streptomycin (100 Ag/ml). Cultures were incubated at 37 C in a humid atmosphere containing 5% C02-95% air and used for testing purposes after 6 to 8 days. Labeling of cells. Confluent monolayers were incubated for 30 min in 1 ml of Hanks BSS. The medium was changed to 1 ml of AIB (1 ACi/ml) in Hanks BSS prewarmed to 37 C. A 1-h labeling period was followed by a nonradioactive chase in 1 ml of fresh medium for 30 min. This marker will be referred to as the AIB label. For comparison, parallel cultures were nucleotide labeled as earlier described (40). Confluent monolayers were incubated with 1 ml of [aHlluridine (1 MCi/ml) in Eagle medium for 2 h. A 2-h nonradioactive chase in 1 ml of fresh medium followed. This marker will be referred to as the nucleotide label. All incubations were performed at 37 C and pH 7.5. The cells were washed three times with Hanks BSS before the test. Maximal and spontaneous release. The maximal release of cytoplasmic radioactivity was determined
INF-ECT. IMMUN. after cell membrane rupture with a 0.06 M borate buffer (pH 7.8) as described by Thelestam et al. (40). Radioactivity in 0.1-ml samples, diluted in 10 ml of Aquasol, was measured by counting in a Nuclear Chicago liquid scintillator. The absolute values for the maximal releases of AIB and nucleotide labels were usually 100,000 to 200,000 counts/min per culture (0.6 x 106 to 0.7 x 10' cells), depending on cell density and slight variations in labeling conditions. The spontaneous release of AIB label during standard test conditions, i.e., incubation at 37 C for 30 min in Tris-buffered saline, pH 7.0 (TBS), varied between 15 and 30%. For the nucleotide label the spontaneous release was 3 to 7%. Size of the AIB label. The molecular size of the AIB label in cell lysates free of nuclei was estimated, in comparison with plain AIB, by gel chromatography on a BioGel P-2 column. The column was 2.5 cm by 24.5 cm, the flow rate was 2.7 ml/h x cm2, and 4.5-ml fractions were collected. TBS was used for equilibration of the gel and for elution. Radioactivity was determined in a 0.1-ml sample of each fraction. Toxin treatment of cells. Labeled cultures were incubated with test substances in TBS. Unless otherwise stated, incubation was performed at 37 C for 30 min at pH 7.0. After this, the incubation medium was sucked off and centrifuged (1,000 x g, 10 min, 4 C). Radioactivity was measured in 0.1 ml of the supernatant. Released radioactivity was expressed as percentage of maximal release, i.e., (toxin-induced release spontaneous release)/(maximal release - spontaneous release) x 100. In this formula, a relatively low spontaneous release is necessary (38). Therefore, when the spontaneous release occasionally exceeded 30%, the experiment was discarded. All tests were performed in duplicate or triplicate. Hemolytic and phospholipase C assays. Alphatoxin was assayed on rabbit erythrocytes, beta- and theta-toxins were assayed on sheep erythrocytes, and delta- and gamma-toxins and streptolysin S were assayed on human erythrocytes as previously described (37, 40). Phospholipase C activity was determined by a titrimetric method on egg yolk suspension as substrate (32).
RESULTS Labeling conditions. To obtain optimal labeling conditions, the following variables were studied: concentration of AIB, type of labeling medium, temperature, pH, and time for labeling, as well as nonradioactive preincubations and chases. A concentration of 1 ACi/ml was found to be optimal for labeling. The cytoplasmic activity obtained with this concentration was dependent on the composition of the labeling medium (Table 1). The presence of other amino acids in Eagle medium depressed the uptake of AIB. The uptake was stimulated by glucose in phosphate-buffered saline (PBS) and by Hanks BSS. Prewarming of the AIB solution to 37 C before
VOL. 12, 1975
ASSAY FOR DETECTING TOXIN-INDUCED DAMAGE
TABLE 1. Cellular uptake of AIB in different mediaa Labeling medium con-
Cytoplasmic activity
taining 1 uCi of AIB/ml
(counts/min per 10' cells)
TABLE 2. Cellular uptake of AIB in relation to the starting temperature of the AIB solutiona
application to cells increased the uptake of AIB significantly (Table 2). The shock of a sudden decrease in temperature evidently caused a disturbance of the amino acid transport system. The uptake of AIB was highly sensitive to changes in pH with an optimum at 7.5 (Fig. 1). No further increase in cytoplasmic activity was evident after a labeling period of 1 h. The intracellular radioactivity was further increased by a 30-min preincubation in Hanks BSS before labeling. A 30- to 60-min nonradioactive chase in Eagle medium lowered the spontaneous release during the subsequent test, whereas a similar chase in Hanks BSS caused an extremely high spontaneous release. Spontaneous release. The spontaneous release of AIB label during standard test conditions, i.e., 30 min at 37 C (pH 7.0), was studied in different solutions (Table 3). Roughly similar results were obtained in Tris-buffered potassium chloride (TBP), TBS, PBS, and these solutions supplemented with 0.1% glucose. The spontaneous release of AIB label from confluent monolayers was also followed with incubation time (Fig. 2). The release increased rapidly, reaching more than 50% in 1 h. Release in TBP was lower than in TBS, especially during incubations longer than 30 min. A standard procedure for labeling and testing with AIB was designed on basis of these results, as described in Materials and Methods. Molecular size of the AIB label. To confirm that AIB was not bound to cytoplasmic substances under the conditions used, lysates from
cells labeled with AIB were chromatographed on BioGel P-2. The labeled cytoplasmic material was eluted as a single peak coinciding with plain AIB that had not been in contact with cells. Induced release of AIB and nucleotide labels. Parallel cultures were labeled with AIB and [3H ]uridine and treated with cytolytic agents (Fig. 3). The nonionic detergent Triton X-100 induced the release of equal amounts of nucleotide and AIB labels. Low concentrations
Cytoplasmic activity
Starting temp (C)
(counts/min per 10' cells)
+4 +20 +37
90,302 237,188 275,800
8,583 ......... TBS .... PBS + 0.1% glucose ............. 131,683 Hanks BSS ....... ...... 290,950 Eagle medium ............. 48,667 a Confluent cultures were labeled with AIB for 1 h at 37 C as indicated. Cytoplasmic radioactivity was estimated after cell membrane rupture with a borate buffer as described in the text.
227
a Confluent cultures were labeled with AIB in Hanks BSS (1 AiCi/ml) for 1 h at 37 C. The labeling medium had the starting temperatures indicated. Cytoplasmic radioactivity was estimated after cell membrane rupture with a borate buffer as described in the text. I
3.105 2X105 u
a. i.io5
6.0
8.0
7.0 pH
FIG. 1. Effect of pH on the uptake of AIB. Confluent cultures were labeled with AIB as described in the text. NaHCO was added to Hanks BSS in amounts sufficient to give the pH values indicated, after equilibration with an atmosphere containing 5% CO2. Maximal cytoplasmic radioactivity was estimated after cell membrane rupture and expressed as counts per minute per 10J cells. TABLE 3. Spontaneous release of AIB label in different solutionsa
Solution
release) of maximal release (%Spontaneous
.......... TBP ... .......... TBS ... TBS + 0.1% glucose ............. PBS ............. PBS + 0.1% glucose ............. ........ Hanks BSS ..... ...... Eagle medium .......
12.1 19.8 15.3 18.0 12.8 31.4 52.0
aConfluent cultures were labeled with AIB in Hanks BSS as described in the text. Spontaneous release during 30 min at 37 C in the solutions indicated was measured and expressed as percentage of the maximal release determined in parallel cultures.
of melittin, a cationic polypeptide with surfaceactive properties (34), induced the release of significantly greater amounts of AIB than nu-
cleotide label. Similar results were obtained with the polyene antibiotics filipin and am-
228
INFECT. IMMUN.
THELESTAM AND MOLLBY
hemolytic unit (HU)/ml, remarkably more AIB label was liberated. Phospholipase C induced the release of only slightly more AIB than 100 nucleotide label (Fig. 4B). Treatment with streptolysin S at concentrations below 2.5 HU/ml caused the release of about 75% of AIB /g E label without any nucleotide release (Fig. 4C). Considerably more, in terms of HU, was needed E 50s I of streptolysin S than of theta-toxin for release c of the nucleotide label. E. coli enterotoxin appeared to be without effect on membrane permeability since neither nucleotide nor AIB label was released after treatment with a crude " of this toxin (Fig. 4D). preparation f i is 30 45 120 60 Treatment with staphylococcal alpha-, beta-, Incubation time (min) FIG. 2. Spontaneous release in relation to incuba- and gamma-toxins induced a concentrationtion time. The spontaneous release of A1B label in dependent release of the AIB label, whereas the TBS and TBP was followed for 2 h and e.xpressed as nucleotide label was retained at the concentrapercentage of the initial cytoplasmic raodioactivity. tions indicated in Fig. 5. By contrast, after exposure to delta-toxin, both nucleotide and Symbols: O, Release in TBS; *, release ijn TBP. AIB labels were released. One-hundredth of a hemolytic unit caused the release of more than 50% of the AIB label. However, for an equal 100 release of nucleotide label, 0.1 HU was required. At higher concentrations the release curves coincided. a The time courses for the release of AIB and nucleotide labels induced by amphotericin B 50 and melittin are shown in Fig. 6. A release of more than 50% of the AIB label was caused in 5 min by both amphotericin B and melittin. The L. corresponding amount of nucleotide label was detected only 1 to 2 h later at the concentrations used. These time curves illustrate how effectively the release of AIB label may demonstrate Ampho- Hypotonic Filipin Triton X-100 Melittin NaC tericin B rapidly occurring changes in membrane 16.1mM 100pM 10pM 1.5 yM 0,2mM permeability.
'"',-
a
x
E
0
i
FIG. 3. Effects of various cytolytic agernts. Release of AIB and nucleotide labels from culture,s incubated at 37 C for 30 min with various cytolyti[c agents at concentrations chosen so as to give apj 75% release of the AIB label. White bars, hatched bars, nucleotide label.
pAIBlatbely 'l
photericin B, which are known to ireact with membrane cholesterol (19). Howlever, amphotericin B caused the release of strikingly more AIB than nucleotide label, w]hereas the difference between AIB and nucleotiide release induced by filipin was less conspicuc)us. Hypotonic swelling of the cells also causesI a greater release of AIB than of nucleotide Ilabel. The effects of these agents at different close levels will be reported separately (Thele stam and M6llby, unpublished data). Clostridial theta-toxin induced a 4concentration-dependent release of both AIB a nd nucleotide labels (Fig. 4A). At concentratio gns below 1
DISCUSSION AIB was initially considered for use as a cytoplasmic marker for the following reasons: (i) it is known to be actively accumulated by different kinds of cells (5, 7, 14, 28); (ii) it is not metabolized in the cell (3); and (iii) it has a low molecular weight, 103. Furthermore, "4Clabeled AIB is commercially available. The subsequent study of optimal labeling conditions with human diploid fibroblasts indicated that uptake of AIB by these cells was dependent on the same factors as described for other cell types (21). The uptake was competitively inhibited by the presence of other amino acids in Eagle medium in accordance with Christensen and Liang (5) (Table 1). Foster and Pardee (14) used PBS + glucose for incorporation of AIB into 3T3 cells. In the present system, glucose improved uptake of AIB as compared
VOL. 12, 1975
ASSAY FOR DETECTING TOXIN-INIDUCED DAMAGE I
~~~I
I
I
229
I
A
B
100
100 Go
50 L0
E x
1.0 HU/m
0.5
0
E
2
1.5
4
U/mi
oa
D
0 c
100
E5c
100
50
0.25
2.5 HU/ml
25
Concentration
2.5 mg/ml
5.0
FIG. 4. Effects of various bacterial toxins. Release of AIB and nucleotide labels from cultures incubated at 37 C for 30 min with (A) theta-toxin, (B) phospholipase C, (C) streptolysin S, and (D) enterotoxin at the concentrations indicated. Symbols: 0, AIB label; 0, nucleotide label.
A
B 100
100
to W
a# 50
50
I x
a
.
4
E
-8
16
25
10
50
-a E
1-
x a
E
D
C
E 100
9
0
a
100
c u
so.
50
I
4
8
I
.
a
0.5
16
I
,
1.0
Concentration (HU/ml)
FIG. 5. Effects of staphylococcal toxins. Release of AIB and nucleotide labels from cultures incubated at 37 C for 30 min with (A) alpha-toxin, (B) beta-toxin, (C) gamma-toxin, and (D) delta-toxin at the concentrations indicated. Symbols as in Fig. 4.
with TBS -nly. However, Hanks BSS proved to be even more suitable for efficient labeling of our cells. The uptake of AIB was highly sensitive to changes in temperature and pH, as reported for
the Ehrlich cell (5). A ratio of equilibrium distribution between intra- and extracellular AIB
was
reached within 1 to 2 h with several
types of cells (7, 11, 28). Also, in our cell system 1 h
was
sufficient for maximal labeling. Prein-
230
THELESTAM AND MOLLBY
INFECT. IMMUN.
B
100
*
I I
I
E
I
x
a
I 0
E so;
50
0-
E
0
30
60
90
30
60
90
Incubation time (min)
FIG. 6. Effects of melittin and amphotericin B in relation to incubation time. Release of AIB and nucleotide labels from cultures treated with (A) melittin (1.5 MM) and (B) amphotericin B (100 MM) for the periods of time indicated. Symbols as in Fig. 4.
cubation in a solution free from amino acids in TBP than in TBS. The flux of AIB through (Hanks BSS) before labeling increased the sub- cell membranes is Na+ dependent (33), which sequent uptake of AIB, possibly due to deple- may explain the differences found in TBP and TBS. The stimulation of AIB efflux with intion of the endogenous amino acid pool (14). Gel chromatography of AIB-labeled cell ly- creasing intracellular concentrations of Na+ sates on BioGel P-2 indicated that this amino (10, 41) could be an important factor in release acid was not metabolized in the present cell of AIB from only slightly damaged cells. In such system. The effective diameter of AIB as a cases the measured release of AIB may repremarker should thus be 0.6 to 0.8 nm (17). This sent a stimulated efflux in addition to the actual leakage due to changed permeability of the agrees with the fact that the sensitivity of the test with AIB as a marker was comparable with membrane. This may involve the interesting that found when release of K+ was measured possibility of obtaining an increased sensitivity (Thelestam, unpublished data). The high sensi- for agents acting as Na+ ionophores. Additional tivity should enable detection and quantitation information may thus be achieved regarding the of subtle permeability changes during early mode of action of cytolytic toxins by performing stages of cytolysis. The diffusion rate of the parallel tests in solutions without Na+ ions. marker used in leakage tests is an important When investigating effects of bacterial toxins factor for adequate reflection of permeability on the fibroblast membrane, we have used the changes (30). Thus, membrane alterations may nonionic detergent Triton X-100, the surface be detected early simply because the small AIB active polypeptide melittin, and the polyene molecules diffuse very rapidly out of the cell antibiotics filipin and amphotericinx B as referafter perturbation of the cell membrane. ence substances. The present study indicates AIB is actively transported out of the cell by that release of AIB may be used to detect lower the same system mediating its uptake (4). The concentrations of these agents more rapidly active efflux explains the relatively high sponta- than is possible when measuring release of nuneous release observed in the present cell syscleotide label (Fig. 3 and 6). The fact that a tem (Table 3 and Fig. 2). The high spontaneous 'sublytic concentration of Triton X-100 caused release is a disadvantage with this assay and the release of the same amount of nucleotide as makes it unsuitable for long-term studies of of AIB label agrees with our earlier observamembrane damage. The same limitation has tions regarding the size of the "functional been reported for K+ and Rb+ (22, 23, 29). A holes" produced by this detergent (39). Effects somewhat lower spontarneous release was con- of these reference substances as indicated by sistently found when the solution contained K+ release of four different cytoplasmic markers (TBP) instead of Na+ ions (TBS). However, in will be reported elsewhere in more detail (Thethis study the incubations with toxins were lestam and M6llby, unpublished data). performed in TBS because preliminary experiRecently we described the effects of crude and ments indicated that the sensitivity was lower highly purified preparations of staphylococcal
VOL. 12, 1975
ASSAY FOR DETECTING TOXIN-INDUCED DAMAGE
toxins on the fibroblast membrane, using release of nucleotide label as criterion of membrane damage (40). Highly purified alpha-, beta-, and gamma-toxins appeared to be without effect on these cells. However, the present study showed that these toxins caused a release of the AIB label, indicating a changed permeability of the fibroblast plasma membrane (Fig. 5). The fact that this change did not result in any morphological effects may be interpreted as partly due to the high repair capacity in the membrane of contact-inbibited fibroblasts (42). These hemolytic toxins induce more drastic effects on liposomes and erythrocytes (2, 15), which may be explained in part by the lack of such repair mechanisms. The agents used in this investigation were hemolytic, with the exception of the E. coli enterotoxin. This toxin has recently been shown to bind to membrane receptors, causing an activation of the adenylate cyclase in human intestinal cells (24). Such an activation results in an active efflux of potassium chloride and bicarbonate ions as shown with Vibrio cholerae enterotoxin (12). However, the E. coli enterotoxin did not cause any release of AIB label from the fibroblasts used in this study. In conclusion, suitable conditions have been presented for the use of AIB as a cytoplasmic marker in a standardized leakage test. Release of the AIB label is a sensitive indicator of alterations in cell membrane permeability as evidenced by the dose response curves and time course relationships presented. This assay may be applied in the study of alterations in membrane permeability caused by various cytolytic toxins and enzymes, membrane-active antibiotics, immune lysis, and industrial chemicals such as detergents and heavy metals. ACKNOWLEDGMENTS We are greatly indebted to C. J. Smyth and T. Wadstrom for stimulating discussions and generous support. The skillful technical assistance of G. Blomquist, M. Kjellgren, and L. Norenius is gratefully acknowledged. This work was supported by the Swedish Medical Research Council (grant no. 16X-2562) and Karolinska institutets fonder. R.M. had a research position at the Swedish Medical Research Council (no. 40P-4576).
of mediated exodus of amino acids from the Ehrlich ascites tumor cell. J. Biol. Chem. 243:5428-5438. 5. Christensen, H. N., and M. Liang. 1966. On the nature of the "non-saturable" migration of amino acids into Ehrlich cells and into rat jejunum. Biochim. Biophys. Acta 112:524-531. 6. De Kruijff, B., W. J. Gerritsen, A. Oerlemans, R. A. Demel, and L. L. M. van Deenen. 1974. Polyene antibiotic sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. I. Specificity of the membrane permeability changes induced by the polyene antibiotics. Biochim. Biophys. Acta
339:30-43. 7. Dickson, J. A. 1970. The uptake of non-metabolizable amino acids as an index of cell viability in vitro. Exp. Cell Res. 61:235-245. 8. Duncan, J. L. 1974. Characteristics of streptolysin 0 hemolysis: kinetics of hemoglobin and 'lrubidium release. Infect. Immun. 9:1022-1027. 9. Eagle, H. 1959. Amino acid metabolism in mammalian cell cultures. Science 130:432-437. 10. Eddy, A. A. 1968. The effects of varying the cellular and extracellular concentrations of sodium and potassium ions on the uptake of glycine by mouse ascites tumor cells in the presence and absence of sodium cyanide. Biochem. J. 108:489-498. 11. Elsas, L. J., and L. E. Rosenberg. 1967. Inhibition of amino acid transport in rat kidney cortex by puromycin. Proc. Natl. Acad. Sci. U.S.A. 57:371-378. 12. Finkelstein, R. A. 1973. Cholera. CRC Crit. Rev. Microbiol. 2:553-623. 13. Forbes, I. J. 1963. Studies of cytotoxicity using P32. Aust. J. Exp. Biol. 41:255-264. 14. Foster, D. O., and A. B. Pardee. 1969. Transport of amino acids by confluent and non-confluent 3T3 and polyoma virus-transformed 3T3 cells growing on glass cover slip. J. Biol. Chem. 244:2675-2681. 15. Freer, J. H., J. P. Arbuthnott, and A. W. Bernheimer. 1968. Interaction of staphylococcal alpha-toxin with artificial and natural membranes. J. Bacteriol. 95:1153-1168. 16. Gale, E. F. 1974. The release of potassium ions from Candida albicans in the presence of polyene antibiotics. J. Gen. Microbiol. 80:451-465. 17. Giese, A. C. 1973. Cell physiology, p. 291, 4th ed. W. B. Saunders Co., Philadelphia. 18. Green, H., R. A. Fleischer, P. Barrow, and B. Goldberg. 1959. The cytotoxic action of immune gamma globulin and complement on Krebs ascites tumor cells. II. Chemical studies. J. Exp. Med. 109:511-521. 19. Hamilton-Miller, J. M. T. 1974. Fungal sterols and the mode of action of the polyene antibiotics. Adv. Appl.
Microbiol. 18:109-134. 20. Hammond, S. M., P. A. Lambert, and B. N. Kliger. 1974. The mode of action of polyene antibiotics; induced potassium leakage in Candida albicans. J. Gen. Micro-
biol. 81:325-330.
21. Heinz, E. 1972. Transport of amino acids by animal cells, p. 455-501. In L. E. Hokin (ed.), Metabolic pathways, vol. 6, Metabolic transport. Academic Press Inc., New
York.
LITERATURE CITED 1. Bernheimer, A. W. 1949. Formation of a bacterial toxin
(Streptolysin S) by resting cells. J. Exp. Med. 90:373-392. 2. Bernheimer, A. W. 1974. Interactions between membranes and cytolytic bacterial toxins. Biochim. Biophys. Acta 344:27-50. 3. Christensen, H. N., A. J. Aspen, and E. G. Rice. 1956. Metabolism in the rat of three amino acids lacking alpha-hydrogen. J. Biol. Chem. 220:287-294. 4. Christensen, H. N., and M. E. Handlogten. 1968. Modes
231
22. Henney, C. S. 1973. Studies on the mechanism of lymphocyte mediated cytolysis. II. The use of various target ceil markers to study cytolytic events. J. Immunol. 110:73-84. 23. Hingson, D. J., R. K. Massengill, and M. M. Mayer. 1969. The kinetics of release of serubidium and hemoglobin from erythrocytes damaged by antibody and complement. Immunochemistry 6:295-307. 24. Kantor, H. S., P. Tao, and C. Wisdom. 1974. Action of Escherichia coli enterotoxin: adenylate cyclase behavior of intestinal epithelial cells in culture. Infect. Immun. 9:1003-1010.
232
THELESTAM AND MOLLBY
25. MacGregor, R. D., II, and C. A. Tobias. 1972. Molecular sieving of red cell membranes during gradual osmotic hemolysis. J. Membr. Biol. 10:345-356. 26. Madoff, M. A., M. S. Artenstein, and L. Weinstein. 1963. Studies on the biologic activity of purified staphylococcal alpha-toxin. II. The effect of alpha-toxin on Ehrlich ascites carcinoma cells. Yale J. Biol. Med. 35:382-389. 27. Madoff, M. A., L. Z. Cooper, and L. Weinstein. 1964. Hemolysis of rabbit erythrocytes by purified staphylococcal alpha-toxin. III. Potassium release. J. Bacteriol. 87:145-149. 28. Mahoney, M. J., and L. E. Rosenberg. 1970. Uptake of alpha-aminoisobutyric acid by cultured human fibroblasts. Biochim. Biophys. Acta 219:500-502. 29. Martz, E., S. J. Burakoff, and B. Benacerraf. 1974. Interruption of the sequential release of small and large molecules from tumor cells by low temperature during cytolysis mediated by immune T-cells or complement. Proc. Natl. Acad. Sci. U.S.A. 71:177-181. 30. Miller, R. G., and M. Dunkley. 1974. Quantitative analysis of the 1Cr release cytotoxicity assay for cytotoxic lymphocytes. Cell. Immunol. 14:284-302. 31. Mollby, R., M. Thelestam, and T. Wadstrom. 1974. Effect of Clostridium perfringens phospholipase C (alpha-toxin) on the human diploid fibroblast membrane. J. Membr. Biol. 16:313-330. 32. Mollby, R., and T. Wadstrom. 1973. Purification of phospholipase C (alpha-toxin) from Clostridium perfringens. Biochim. Biophys. Acta 321:569-584. 33. Schultz, S. G., and P. F. Curran. 1970. Coupled transport of sodium and organic solutes. Physiol. Rev. 50:637718. 34. Sessa, G., J. H. Freer, G. Colacicco, and G. Weissmann. 1969. Interaction of a lytic polypeptide, melittin, with lipid membrane systems. J. Biol. Chem.
INFECT. IMMUN. 244:3575-3582. 35. Siegel, I., and S. Cohen. 1964. Action of staphylococcal toxin on human platelets. J. Infect. Dis. 114:488-502. 36. Smyth, C. J. 1975. The identification and purification of multiple forms of theta-haemolysin (theta-toxin) of Clostridium perfringens type A. J. Gen. Microbiol. 87:219-238. 37. Strandberg, K., R. Mollby, and T. Wadstrom. 1974. Histamine release from mast cells by highly purified phospholipase C (alpha-toxin) and theta-toxin from Clostridium perfringens. Toxicon 12:199-208. 38. Stulting, R. D., and G. Berke. 1973. The use of 51Cr release as a measure of lymphocyte-mediated cytolysis in vitro. Cell. Immunol. 9:474-476. 39. Thelestam, M., and R. Mollby. 1975. Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts. Infect. Immun. 11:640-648. 40. Thelestam, M., R. Mollby, and T. Wadstrom. 1973. Effects of staphylococcal alpha-, beta-, delta-, and gamma-hemolysins on human diploid fibroblasts and HeLa cells: evaluation of a new quantitative assay for measuring cell damage. Infect. Immun. 8:938-946. 41. Vidaver, C. A., and S. L. Shepherd. 1968. Transport of glycine by hemolyzed and restored pigeon red blood cells, Symmetry properties, trans effects of sodium ion and glycine and their description by a single rate equation. J. Biol. Chem. 243:6140-6150. 42. Warren, L., and M. C. Glick. 1968. Membranes of animal cells. II. The metabolism and turnover of the surface membrane. J. Cell Biol. 37:729-746. 43. Wigzell, H. 1965. Quantitative titrations of mouse H-2 antibodies using Cr5l-labeled target cells. Transplantation 3:423-431.
