Mar 15, 2019 - Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor ne- crosis factor (TNF). Using various ...
Vol. 267,No. 8,Issue of March 15,pp. 5317-5323, 1992 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytotoxic Activityof Tumor NecrosisFactor Is Mediated byEarly Damage of Mitochondrial Functions EVIDENCE FOR THE INVOLVEMENT OF MITOCHONDRIAL RADICAL GENERATION* (Received for publication, June 27, 1991)
Klaus Schulze-OsthoffS, Annette C. Bakkerg, Bart Vanhaesebroeckll, Rudi Beyaertll, Willem A. Jacob%, and Walter Fiersll From the Laboratow of Molecular BWloPv. State University, Gent, Belgiumand the §Department of Medicine, University of Antwerp, Wilrijk, B&iium - “ I
Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitorsof mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex I11 (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNFmediated injury. In contrast, when the electronflow to ubiquinone was blocked, either at complex I (NADHubiquinone oxidoreductase) with amytalor at complex I1 (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitorssignificantlyinterferedwith TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an earlydegeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive
* This research project was supported by grants from the Fonds voor Geneeskundig Wetenschappelijk Onderzoek, the Nationaal Stimuleringsprogramma voor Fundamenteel Onderzoek inzake Biowetenschappen, the Algemene Spaar en Lijfrentekas, and theSportvereniging tegen Kanker. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $’ Recipient of a fellowship from the Deutsches Krebsforschungszentrum. ll Research assistants with the Nationaal Fonds voor Wetenschappelijk Onderzoek. 11 To whom correspondence should be sent. Tel.: 32-91-64-51-31; Fax: 32-91-64-53-48.
evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is furthersupported by the protectiveeffect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristicalterations of the mitochondrial ultrastructure.
Tumor necrosis factor (TNF),’ primarily produced by activated macrophages, was originally described based on its ability to cause necrosis of Meth A sarcomas in vivo and selectively kill transformed and neoplastic cell lines in vitro (1).During the last few years, TNF has been shown to exert a wide range of biological activities mostly related to inflammatory and immunomodulatory functions. Beside its antitumor and anti-malignantcell effects, TNF has been reported to influence mitogenesis, differentiation, and immunoregulation of various cell types, which suggests an important role of this cytokine under several physiological and pathological conditions (reviewed in Refs. 2 and 3). Although recently some mechanisms of TNF-mediated cell regulatory activities have become better understood, the biochemical basis of the cytotoxic action against tumor cell lines is still largely unknown. TNF binds to its target cell via high affinity surface receptors. Two different receptor types have been cloned recently, but their primary structure does not reveal any significant consensus sequences pointing to the involvement of one of the classical signaling pathways (4-6). However, studies mainly based on inhibitors have indicated that, probably depending on the cell type, multiple intracellular pathways maybeinvolved in TNF-mediated killing. Among the reported effects, major steps seem to include Gprotein-coupled activation of phospholipases (7, 8), generation of reactive oxygen radicals (9, lo), and DNA damage (11, 12). Furthermore, it has been shown that inhibitors of proteases (13, 14) and lysosomal enzymes ( E ) , glucocorticoids (8, 16), and antioxidants (9, 10, 17) may confer some protection against TNF cytotoxicity. Certainly, the cytotoxic action of TNF does not depend on RNA or protein synthesis, and its activity is even considerably enhanced by transcription or translation inhibitors (18). In agreement with this observation, evidence has been obtained that cells may counteract The abbreviations used are: TNF, tumor necrosis factor; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone;DDC, diethyldithiocarbamate; DNP, 2,4-dinitrophenol; HQNO, 2-heptyl-4hydroxyquinoline N-oxide; TMPD, tetramethyl-p-phenylenediamine; TTFA, thenoyltrifluoroacetone; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
5317
5318
TNF Cytotoxicity Mediated by Early Damage of Mitochondrial Functions
TNF action by synthesis of rescue factors (19). Wong et al. (20) reported that, at least in some cell types, one of the proteins that can provide protection against TNF is manganous superoxide dismutase. Overexpression of this mitochondrial enzyme confers increased resistance against TNF. These findings further imply that superoxide radicals or other reactive oxygen species might participate in the TNF-initiated cytotoxic pathway. In the present study we show that specific mitochondrial electron transport inhibitors can either potentiate or block TNF cytotoxic activity. The effects observed with the inhibitors implicate oxygen radicals which are produced by the respiratory chain as akey cause of TNF-induced cytotoxicity. This conclusion is confirmed by further experiments showing that early events of TNF cytotoxicity are afunctional damage of selective components of the mitochondrial electron transport chain and structural alterations in mitochondrial morphology. MATERIALS AND METHODS
Cell Cultures L929, a murine fibrosarcoma cell line, was grown in Dulbecco's modifiedEagle'smedium with 10% heat-inactivated newborn calf serum, 100 units of penicillin/ml and 0.1 mgof streptomycin/ml. WEHI 164 clone 13, a murine fibrosarcoma cell line which is highly TNF-sensitive (obtained from Dr. T. Espevik, University of Trondheim, Norway), was cultured in RPMI 1640 supplemented with 10% fetal calf serum and antibiotics. The cell lines were repeatedly found to be mycoplasma-free as judged by a DNA-fluorochrome assay.
TNF and Reagents TNF Preparation-Recombinant murine TNF was produced in Escherichia coli and purified to at least 99% homogeneity (21). The preparation had a specific activity of 3.7 X lo7 IU/mg protein and contained less than 4 ng of endotoxin/mg of protein. TNF activity was determined as described by Ostrove and Gifford (22) using an international standard TNF preparation (code No. 88/532, obtained from the Institute for Biological Standards andControl, Potters Bar, United Kingdom) as a reference. Reagents-The following agents, used without further purification, were purchased from Sigma Chemical Co. (Deisenhofen, Federal Republic of Germany): actinomycin D, adenosine diphosphate, amytal (amobarbital), butylated hydroxyanisole, butylated hydroxytoluene, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), diethyldithiocarbamate (DDC), 2,4-dinitrophenol (DNP), 2heptyl-4-hydroxyquinolineN-oxide (HQNO), oligomycin, o-phenanthroline, rotenone, sodium nitroprusside, tetramethyl-p-phenylenediamine (TMPD), and thenoyltrifluoroacetone (TTFA). Antimycin A, succinate, and malate were from Serva (Heidelberg, Federal Republic of Germany) and desferrioxamine (Desferal) from Ciba Geigy (Basel, Switzerland). Stock solutions of the reagents were routinely prepared in medium, dimethyl sulfoxide, or ethanol as appropriate. For use in cytotoxicity assays, stock solutions were diluted in culture medium such that thefinal concentration of the organic solvent never exceeded 0.4%. Control experiments demonstrated that this concentration did not affect TNF cytotoxicity.
respiration medium consisting of 0.25 M sucrose, 0.1% bovine serum albumin, 10 mM MgC12, 10 mM K+-Hepes, 5 mM KH2P04,pH 7.2, with or without 1 mM ADP, and kept on ice.Cell viability was determined by trypan blue exclusion. Oxygen consumption was measured with a Clark electrode fitted in a 3.3-ml thermojacketed sample chamber (37 "C) under constant stirring (25). Briefly, cells were injected ina 200-plvolume and permeabilized by the addition of digitonin (final concentration 0.005%) in order to permit free entry of mitochondrial inhibitors and substrates. The latter were added in the following final concentrations: malate, 5 mM; rotenone, 100 nM; succinate, 5 mM; antimycin A, 50 nM; ascorbate, 1mM; TMPD, 0.4 mM; KCN, 1mM; oligomycin, 1 pM; DNP, 1 pM. Oxygen concentration was calibrated with airsaturated buffer, assuming 390 ng atoms/ml of O2 (25). Rates of oxygen consumption are expressed as nanogram atoms of oxygen/ min/1O7 cells. Electron Microscopy L929 cells were seeded on thermanox coverslips (diameter 1 cm; GIBCO, Bio-Cult, Paisley, United Kingdom) a t a density of 100,000 cells. 24 h later 10,000 IU/ml TNF were added. After various incubation times ranging from 1 to 17 h, cells were processed for electron microscopy. The growth medium was removed by two changes with serum-free medium and replaced by 100 mM cacodylate buffer, pH 7.4, containing 2% glutaraldehyde and 4% formaldehyde. After fixation for 15 min a t room temperature, coverslips were rinsed three times in cacodylate buffer for 5 min. Postfixation was carried out for 1h with potassium ferricyanide in 1%Os04. After a washing step in cacodylate buffer, specimens were stained en bloc in 2% aqueous uranyl acetate for 2 h and then washed with water. Dehydrations were performed with increasing concentrations (70-90-100%) of acetone. Cells were embedded overnight in a mixture of epoxy resin (LX112, Ladd) and acetone and polymerized at 60 "C for 60 h. After removal of the coverslips, sections (60-80nM)were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined in a Jeol 1200 EX microscope at 60 kV. RESULTS
Mitochondrial Respiratory Chain Inhibitors Strongly Modulate TNF Cytotoxicity-After analysis of the effects of various respiratory chain inhibitors, TNF cytotoxicity was found to be either increased or decreased, depending on the action site of the inhibitors. For reference, a simplified scheme of the mitochondrial respiratory chain is shown in Fig. 1. Inhibitors of complex 111, which inhibit the electron flow from ubiquinone to cytochrome cl, strongly potentiated TNF-mediated cytolysis of L929 cells. Indeed, both antimycin A and HQNO gave a dose-dependent enhancement of TNF cytotoxicity (Table I; Fig. 2a). At the concentrations used, neither inhibitor significantly influenced cell viability on its own. However, myxothiazol, which inhibits electron flow via the Rieske iron sulfur center to cytochrome c1 (26),had no potentiating effect and was even marginally protective. Most striking was the observation that inhibitors of complex I, which interfere with the electron flow from NADHlinked substrates and NADH dehydrogenase to the ubiquinone pool, were protective against TNF, in contrast to the effect of complex I11 inhibitors. Amytal strongly decreased TNF Cytotoxicity Assay TNF susceptibility (Table I; Fig.2b), and also rotenone, which Cells were seeded in 96-microwellplates at 3 X lo' cells in 100 pl of medium. Twelve to 16 h later 50 p1 of a drug solution was given. inhibits the same step, was protective, albeit to a lesser extent T N F with or without actinomycin D (1pg/ml) was added 2 h later in (TableI).Furthermore, when the electron transport from succinate dehydrogenase wasblockedby the addition of a 50-p1 volumeand a concentrationrange of 2-5000 IU/ml. After 1824 h of further incubation, cell viability was routinely determined via TTFA, TNF cytotoxicity was also reduced (Table I). Finally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide stain- the combined inhibition of both potential electron entry sites ing (23).Similar results were obtained with crystal violet staining of to ubiquinone with amytal plus TTFA resulted in an almost the attached cells (24). complete inhibition of TNF-induced cell lysis (Table I), which was still observed at a high TNF concentration of 10,000 IU/ Measurement of Oxygen Consumption ml (not shown). On the other hand, inhibition of complex IV L929 cells were harvested by trypsinization a t the indicated times after TNFincubation and combined with the decanted medium. After (cytochrome c oxidase) with azide or cyanide gave ambiguous centrifugation and two followingwashing steps incalcium-free phos- results. Azidewas found to be slightly protective, whereas phate-buffered saline, cells were resuspended at 1 X lo' cells/ml in cyanide had marginal synergistic effects on TNF cytotoxicity.
5319
TNF Cytotoxicity Mediated by Early Damage of Mitochondrial Functions 0 2
COMPLEX
FIG. 1. Simplifiedscheme of the respiratorychainshowing sites of substrateentry,inhibitoraction, and potentialsites of superoxide anionformation. cyt, cytochrome; UQ, ubiquinone; Fe-S, iron sulfur center.
Ascorbate
?
NADH
TMPD (5Fe-S)
COMPLEX Ill Rotenone’Wvt-al
I
UO-cyt.b+cyt.cl
COMPLEX II
k-
COMPLEX IV
I [ c y t . c ] s F l1 K(11
co2 Ha0
Azide
n TABLE I Influence of mitochondrial and other inhibitors on TNF cytotoxicity i n L929 cells Influence of mitochondrial inhibitors, iron chelators, andantioxidants on TNF cytotoxicity in L929 cells in the presence and absence of actinomycin D (Act D). Survival values in the presence of TNF are expressed as percentage of the ODvalue at the same drug concentration without TNF. Only data obtained with drug concentrations resulting in cell survival higher than 65% in the absence of TNF are shown.SOD, superoxide dismutase. ND, not determined. Drug
None I. Mitochondrial chain inhibitors 1. Complex I Amytal Rotenone 2. Complex I1 TTFA TTFA + amytal 3. Complex 111 Antimycin A HQNO Myxothiazol 4. Complex IV Azide KCN 11. Uncouplers Dinitrophenol FCCP III.F1-ATPase Oligomycin IV. Iron chelators Desferrioxamine o-Phenanthroline V. SOD inhibitors Nitroprusside DDC VI. Antioxidants BHA BHT
DNg concentration
-Act D, % rel. +Act D, % rel. survival survival 555 IU/ml
61 IU/ml
6 0.7 IU/ml IU/ml
10.8 67.2 20.2 75.2
82.0 54.2
98.3 87.6
93.6 99.7
70.2 94.2
92.6 98.6
1.8 1.4 22.1
16.2 28.6 78.3
4.7 10.4 24.6
17.9 34.8 75.8
15.2 8.5
85.3 68.3
23.6 16.8
67.3 69.0
10.4 13.8
69.4 72.3
ND
ND
17.8
65.6
400 p M 25 p M
87.6 46.8
99.3 87.6
250pM
68.4 99.8
50 p M 50 p M 1 pM 500 p M 500 p M 1 mM 10 p M
TNF(IU/ml)
TNF(IU/mll
FIG. 2. Dose-dependent effects of antimycin A (a)and amytal ( b ) on TNF-induced cytotoxicity. Both inhibitors were tested with differentTNF concentrationsstarting with 5000 IU/ml, followed by serial 1:3 dilutions. Survival percentageis plotted against increasing TNF concentration. For each concentration of the substances, survival is defined as the percentage of the cell-staining value obtained in cultures treated with drug alone, but without TNF. The insets show the effect of amytal and antimycinA on survival in the absence of TNF. Symbols: a, antimycin A; 0, without drug; A, 12.5 pM; 0 , 2 5 p ~ ; 50 pM; b, amytal; 0,without drug;A, 50 pM; 0, 200 pM;
., ., 400 pM.
various time points before and after TNF addition, in order to determine whether their effects occurred a t a n early step 25 p M 12.2 70.3 23.6 73.4 of T N F action (Fig. 3). Addition of antimycin A or amytal from 2 h before up to 4 h after start of the TNF treatment 41.2 ND ND 92.8 100 p M did not affect the inhibition or protection, respectively. But 1 mM 27.8 96.3 40.2 88.4 after 4 h, the potentiating effect of antimycin A and the 1mM 46.3 4.6 8.3 45.6 protection by amytal gradually declined,although effects were 2 mM 65.1 6.8 ND ND still detectable 10h after T N F addition. ATP SynTNF-mediated Cytotoxicity Is Not Dependent on 200 p M 78.6 98.6 78.3 98.2 thesis-Protonophorous uncouplers of the mitochondrialelec200 UM 70.3 92.3 69.6 94.7 tron flow, such as dinitrophenol orFCCP, were found toexert no effect on T N F cytotoxicity (Table I). This result indicates All the inhibitors were tested in the presence and absence that the phenomena observed with mitochondrial chain inof actinomycin D. Inhibition of transcription, however, did hibitors were not due toa blockade of ATP synthesis. Morenot lead to qualitative differences in drug effects on TNF- over, oligomycin, whichdirectlyblocks ATPsynthesis by mediated killing, although a much lower dose of T N F was inhibiting the F1-ATPase subunit, did influence not the activnecessary for a cytotoxic effect (Table I). Furthermore, in ity of T N F on L929 or WEHI 164 clone 13 cells. WEHI 164 clone 13cells, which were studied as an additional Antioxidants and Iron Chelators Protect against TNF Cycell line, similar effects of the mitochondrial inhibitors were totoxicity-The modulation of T N F cytotoxicity by respiraobserved (not shown). tory chain inhibitors strongly points to an involvement of Time Kinetics of the Influence of Mitochondrial Inhibitors oxidative events in the cytotoxic action of TNF. We therefore on TNF Cytotoxicity-Antimycin A or amytal were added at analyzed the effects of antioxidants and iron chelators on
5320
TNF Cytotoxicity Mediated by Early Damage of Mitochondrial Functions a
Diaitrin
timdh)
FIG. 3. Effects of antimycin A and amytal added at various time points before and after the start of TNF treatment (time 0 h). Antimycin A (50 PM) was used with a TNF concentration of
150 IU/ml (m); amytal (400WM)was given with 1500 IU/ml TNF (0). Assays were stopped after 18 h of TNF treatment.
TIME
b
TNF activity. Iron ions have been shown to exert a strong influence on the toxicity of reactive oxygen species, as Fe(I1) can catalytically dismute superoxide radicals (0;) and hydrogen peroxide to theextremely toxic hydroxyl radical (reviewed in Refs. 27 and 28). We found indeed that the antioxidants butylated hydroxytoluene and butylated hydroxyanisole strongly protected against the cytotoxic effects of TNF (Table I). The importance of an involvement of reactive oxygen species was further supported by the finding that both desferrioxamine, a specific iron chelator, and o-phenanthroline, a chelator of heavy metal ions, inhibited TNF cytotoxicity (Table I). On the other hand, inhibitors of superoxide dismutases, which counteract oxidative damage of superoxide radicals, rendered the cells more susceptible to TNF. Nitroprusside, which inhibits all classes of superoxide dismutases, including the mitochondria-located manganese superoxide dismutase (29), had a marked potentiating effect, whereas DDC, which blocks activity of Cu/Zn superoxide dismutase (30), was less synergistic (Table I). TNF Causes an Early Damage of Mitochondrial Electron Transport-We next analyzed by polarographic measurements of oxygen consumption whether TNF affects mitochondrial electron flow. L929 cells were treated with digitonin, which selectively permeabilizes the plasma and outer mitochondrial membranes, while leaving the inner mitochondrial membrane intact. Three different substrates wereused to study the characteristics of oxygen consumption: malate, which generates intramitochondrial NADH; succinate, which provides electrons at the ubiquinone level; and ascorbate plus TMPD, which reduces the respiratory chain at the cytochrome c level. Traces from typical measurements are shown in Fig. 4a. We found that L929 cells, treated with 250 IU/ml TNF, became markedly inhibited in their ability to oxidize a NADH-linked substrate. Already after 3 h, oxidation of malate had decreased to 61% of its original activity; after 6 h of TNF incubation, less than 31% of activity remained detectable (Fig. 4b). This rapid loss of electron transport activity preceded cell lysis by at least 3-6 h. By blocking the electron flow through complex I with rotenone and by adding succinate as reducing substrate, the electron flow through complexes 11, 111, and IV (succinoxidase) was analyzed. Inhibition of the activity of succinoxidase was similarly observed, although it was somewhat delayed as compared with the rapidly decreased NADH-linked oxidation. After injection of antimycin A followed by TMPD plus ascor-
1
0
3
6
12
18
24
1
time(h)
FIG. 4. a, oxygen consumption in digitonin-permeabilized cells after different incubation times with 250 IU/ml TNF. The initial electron donor in the respiration medium was malate; after addition of mitochondrial respiratory chain inhibitors, succinate and ascorbate/TMPD wereinjected as further electron donors as described under “Materials and Methods.” Rates of oxygen consumption are expressed in nanogram atoms/min/107 cells. b, time kinetics of TNFinduced inhibition of mitochondrial electron transport in relation to cell death (250 IU/ml TNF). Values of oxygen consumption are taken from measurements in a. Oxygen consumption in the presence of malate (m), succinate (A), and TMPD/ascorbate (0).Cell viability was determined by trypan blue exclusion.
bate as electron donor, the activity of cytochrome c oxidase (complexIV)was measured. The activity of this enzyme complex wasfound to be relatively resistant to TNF, asmore than 50%of its original activity was still detectable 12 h after TNF addition. The observed decrease in the different mitochondrial enzyme activities could be due either to a direct effect on mitochondrial electron flow, to a block in ADP/Pi translocation, or to an inhibition of the F1-ATPase complex. After addition of oligomycin, inhibition of the mitochondrial electron consumption was observed (data not shown). This indicates that TNF-treated mitochondria were still energy-coupled, since oligomycin would not have exerted any effects on uncoupled mitochondria. Dose Dependence and Time Course of Electron Transport Inhibition in Uncoupled Mitochondria-In order to establish a dose dependence of TNF-induced inhibition of mitochondrial electron transport, oxygen consumption was measured in the presence of the protonophorous uncoupler dinitrophenol, which induces maximum electron transfer activity. Also under these conditions, TNF-induced inhibition of mitochondrial electron transfer was clearly dose-dependent (Fig. 5a).
TNF Cytotoxicity Mediated
10
loo
1000
1000
TNF(IU/ml)
1
2
3
4 tirnefh)
by Early Damage
5
of Mitochondrial Functions
5321
6
FIG. 5. Dose dependence (a)and time course ( b ) of TNFinduced inhibition of electron transport in uncoupled mitoinrespiration chondria. Cells were permeabilizedwithdigitonin medium without ADP and phosphate. Respirationwas uncoupled by the addition of 1 p~ dinitrophenol. Cell viability was determined by trypan blue exclusion. Dose dependence of T N F on electronflow was determined 6 h after addition of T N F in a titration series from 50 to 10,000 IU/ml T N F ( a ) . The time course of T N F effect on oxygen consumption was determined using a T N F concentration of 10,000 IU/ml T N F (b).
6 h afteraddition of 500IU/ml TNF, theoxygen consumption was inhibited for more than 60%; at thistime, cell death, as determined by trypan blue exclusion, was detectable in less than 20% of the cells. The amplified system of TNF-induced decrease in oxygen consumption in the presence of an uncoupler was further used to investigate the time course of mitochondrial electron transport inhibition (Fig. 56). Applying a high concentration of 10,000 IU/ml TNF, a marked decrease in electron transport could already be observed 1h after TNF addition, reaching a plateau in about 5-6 h. Consistent with the results shown in Fig. 4b, the TNF-induced inhibition largely preceded the onset of cell death as defined by trypan blue exclusion. Ultrastructural Changes Caused by TNF"L929 cells were grown in the presence of TNF for varying times ranging from 1up to 17 h. After each hour, cells were fixed and analyzed by electron microscopy. Cells grown in the presence of TNF revealed an early degeneration of their mitochondrial ultrastructure, which became evident about 2 h after TNFaddition. The most prominent and initial change was the appearance of onion-like structures inside the mitochondrial matrix, which could generally not be recognized in untreated cells (Fig. 6, A and B ) . The cristae became more electron-dense and rounded up, and appeared to protrude into themitochondrial matrix. With prolonged TNF incubation, the stacking of cristae became increasingly pronounced, resulting in the gradual formation of multilamellar bodies inside the mitochondrion (Fig. 6, C and D).However, extensive swelling of mitochondria was not observed before 9 h afterTNF addition. In the first 5 h, other cellular compartments still retained a generally normal ultrastructure. In some TNF-treated cells, a slight margination of nuclear chromatin could be observed in the nucleus. Sometimes, the endoplasmic reticulum also displayed alterations similar to those observed in the mitochondria (Fig. 6C). The exposure of L929 cells to a combination of TNF and antimycin Aseemed to potentiate the degenerating process of the mitochondria (Fig. 7A). The cells exhibited more swollen mitochondria with an increased number of disorientated cristae. Manv cristae were amarentlv detached from the inner membrane. In comparison with TNF-treated cells, onion-like 1 1
!
I
FIG. 6. Electron micrographs demonstrating the effects of TNF on the mitochondrial ultrastructure. L929 cells were treated with 10,000 IU/ml TNF for 3 h. A , untreated L929 cell displaying a normal ultrastructure of mitochondria (bar = 500 nm). B, representative overview of a TNF-treated cell showing that most of the mitochondria became degenerated.The arrows a-c indicate the apparent evolution of the degradation process (bar = 500 nm). C, clustering and vesiculation of mitochondrial cristae with the appearance of onion-like structures inside the mitochondrial matrix (arrow a). Note also the degradation of the endoplasmic reticulum (arrow b) (bar = 350 nm). D, high magnification of a mitochondrion from TNFtreated L929 cells showing a multilamellarvesicle of clustered cristae inside the mitochondrial matrix (bar = 100 nm).
FIG. 7. Effects of antimycin A and amytal on the mitochondrial ultrastructure of TNF-treated L929 cells (bar = 500 nm). A , mitochondria of L929 cells treated with TNF and antimycin A (60 p ~ reveal ) an apparent loss in the number of cristae, with many remaining cristae being disorientated and detached. B, L929 cells treated with TNF and amytal (400 p M ) exhibit many mitochondria with a normal ultrastructure and transverse orientation of the cristae.
structures apparently were not increased by the combined treatment with antimycin A and TNF. On the other hand, the exposure of L929 cells to a combination of amytal and TNF seemed to diminish the deleterious TNF effects on the mitochondrial ultrastructure (Fig. 7 B ) . A smaller number of
5322
TNF Cytotoxicity Mediated by Early Damageof Mitochondrial Functions
mitochondria seemed to be affected. The cristae of most mitochondria exhibited the normal transverse orientation. DISCUSSION
Several cellular pathways have been suggested leading to cell death, including perturbation of ion homeostasis, activation of proteases and phospholipases, generation of reactive oxygen species,or degradation of DNA(31). Despite a growing number of investigations dealing mainly with immunomodulatory activities of TNF, itsmechanism of cytotoxicity is still largely unknown. TNF has been described to initiate two forms of cell killing, although both forms presumably cannot be strictly separated for many cell types (32,33). The necrotic form of cell death is mainly characterized by swelling of the cytoplasm and organelles followedby lysis of the plasma membrane. In apoptotic or so-called programmed cell death, which is exemplified by growth factor-deprived thymocytes and embryonic cells, the cytoplasm becomes condensed and the nucleus compact. Characteristic patterns of 183-base pair DNA fragments can be observed resulting from the activation of a nuclear endonuclease during the apoptotic process (34). We have also observed some typical DNA degradation patterns in L929 cells, but these arose as a rather late event in the cytotoxic action of TNF.' L929 and other fibroblast cell lines seem to be more likely killed by necrotic processes. One of the first consequences inflicted by TNF in L929 cells includes morphological alterations of the mitochondria. We observed that TNF-treated cells reveal a degenerative clumping of mitochondrial cristae, which resulted in the formation of multilamellar vesicles inside the mitochondrial matrix. In addition, Matthews et al. (35,36) have shown that besides mitochondrial degeneration, TNF-treated cells respond with an increased rate of glycolysis and decreased rate of ATP synthesis. Conceivably, the increase in glycolysis compensates for the damage of mitochondrial electron transport as observed in this study. Functional inhibition of the respiratory chain has been demonstrated previously as aconsequence of the cytotoxic action of macrophages on tumor cells (25, 37, 38). Moreover, Lancasteret al. (39) recently reported that TNF induces mitochondrial electron transfer inhibition in a number of transformed cell lines which may be a causative event in the cytotoxicity of TNF. We have detailed these studies in highly TNF-susceptible L929 cells, and we show that functional damage of the electron flow occurs as anearly consequence of TNF action. The ability of mitochondria to oxidize succinate and NADH-linked substrates was seriously affected by TNF treatment, preceding cell death by at least 3-6 h. The activity of complexIV, however, was fairly resistant to TNF,with over 50% activity remaining even 12 h after incubation with 250 IU/ml TNF. Analysis of the electron flow in permeabilized cells with uncoupled mitochondria and high TNF concentrations allowed the detection of a TNF effect on the respiratory chain already after 60 min, without any evidence for compromised cell viability at this time point. In the TNFcytotoxicity assay, we observed that inhibition of mitochondrial electron transportper se or depletion of ATP synthesis were not detrimental to the viability of L929 and WEHI 164 clone 13 cells. Moreover, we show that inhibition of mitochondrial electron transport at specific sites can differentially interfere with TNF-mediated cytotoxicity. Inhibition of the electron transport at complex I with amytal or rotenone or at complex I1with TTFA markedly protected the cells against TNF. On the other hand, we found that electron K. Schulze-Osthoff,A. C. Bakker,B. Vanhaesebroeck,R. Beyaert, W. A. Jacob, and W. Fiers, unpublished results.
transport inhibition behind the ubiquinone region with antimycin A potentiated TNF cytotoxicity. These results suggest that oxidative events generated in the mitochondrion, not inhibition of energy-coupled processes, are crucial in TNFinduced cytotoxicity. It has long been recognized that mitochondria are an important source of reactive oxygen species under certain conditions. According to Boveris et al. (40) about 1-2% of the oxygen consumed in state 4 (resting respiration) is utilized by the formation of reactive oxygen species. In the presence of various drugs (i.e. mitochondrial inhibitors, quinoid compounds), mitochondrial generation of reactive oxygen radicals can increase severalfold. TWO sites of the mitochondrial respiratory chain have been identified as sources responsible for the formation of reactive oxygen species. Oneis dependent on the autooxidation of the flavin mononucleotide from the NADH-dehydrogenase (complex I), whereas the other, probably the most relevant one, depends on autooxidation of the unstable ubisemiquinone (complex 111),which is an intermediate of the Q-cycle reaction (41, 42). Mitochondrial substrates and inhibitors have been described as effective modulators of reactive oxygen species. The production of oxygen radicals at the ubiquinone site supported by NADH-linked substrates isdiminished by complex I and complex I1 inhibitors (43-45). On the other hand, formation of reactive oxygen species increases several times in thepresence of antimycin A, proposing ubisemiquinone as the main reductant site of oxygen (46). The opposite effects observed with specific mitochondrial inhibitors in this study, uiz. enhancement of TNF cytotoxicity with antimycin A and inhibition with amytal and TTFA, strongly indicate that TNF activates radical production in mitochondria mainly at the ubiquinone site. The first oxygen reduction product generated in mitochondria under both physiological and pathological conditions appears to be the superoxide radical, which can subsequently be converted to hydrogen peroxide (47). Dismutation of 0; and HzOzcan result in theproduction of the more deleterious hydroxyl radical and singlet oxygen. Conversion of superoxide and hydrogen peroxide to these harmful oxygen intermediates is catalyzed by transition metals such as iron ions in the Fenton reaction and the (metal-catalyzed) Haber-Weiss reaction (27, 28). Therefore, chelation of iron by desferrioxamine or o-phenanthroline reduces susceptibility of L929 and WEHI 164 clone 13 cells toward TNF. That generation of radicals is probably a crucial event in TNF action is further supported by our finding that antioxidants and free radical scavengers protect against TNF-induced cytolysis (Table I; Refs. 8 and 13). As already noticed by Neale et al. (7), we confirmed that L929 cells kept under anaerobic conditions, or treatedwith succinylacetone in order to block heme synthesis and deplete cells from cytochromes, became less sensitive to TNF. Furthersupport for the importance of radicals arises from our observation of the characteristic degeneration pattern of mitochondrial ultrastructure. Similar typical clumping and vesiculation of mitochondrial cristae has been reported to appear after perfusion of rat hearts with a free radical-generating system (48). Normally, cells counteract the toxicity of reactive oxygen species by means of superoxide dismutases and other detoxifying enzymes. Indeed, TNF has been shown to induce synthesis of manganous superoxide dismutase, but not of other known antioxidant enzymes such as CufZn superoxide dismutase, glutathione peroxidase, or glutathione transferase (49). On the other hand, inhibition of superoxide dismutase or depletion of reduced glutathione (50) leads to an enhance-
Functions TNF Cytotoxicity Mediated by EarlyDamage of Mitochondrial ment of TNF cytotoxicity. Additionally, the rather selective specificity of TNF action on tumor cells may berelated to the long-standing observation that tumor cells generally contain low levels of enzymes dealing with oxygen radicals and hydrogen peroxide (51). Oxygen radicals escaping detoxifying enzymes are capable of inflicting various damage (27, 28). Attack by radicals can result in lipid peroxidation, protein insults, and DNA degradation. Although some 0; and H202 will diffuse from the mitochondrion to damage more distant cellular components, the reactions of the highly toxic short-lived hydroxyl radical are largely diffusion controlled. It is conceivable that the toxicity of all three oxygen species should be greatest at the inner mitochondrial membrane. It has been shown that mitochondria treated with peroxidants have an impaired ability to retain calcium (52). Elevation of cytoplasmic calcium from intracellular stores has been further reported to cause activation of proteases and phospholipases, both of which seem to be involved in TNF cytotoxicity (7,8,12,13).Furthermore, oxygen radicals on their own can directly impair the mitochondrial electron transport (53), whichwouldsuggest an autocatalytic inhibition of mitochondrial chain components. The TNF-induced inhibition of the mitochondrial electron transport, together with the effects observed for different mitochondrial inhibitors, favor the explanation that TNF damages the mitochondrial chain at complex 111, which consequently results in the increased production of oxygen radicals inside the mitochondrion. However, it remains unclear from our results in which way TNF or activated second messengers affect the mitochondrial electron transport. Further studies areunder way in order to unravel primary effects of TNF on components of the electron transport chain. Acknowledgments-We thank Dr. F. Van Roy, Dr. J. Grooten, and Dr. B. Meier for helpful discussions and comments, F. Van Houtte f0I technical assistance, and W. Drijvers for artistic contribution. REFERENCES 1. Carswell, W. A., Old, L. J., Kassel, R.L., Green, S., Fiore, N., and Williamson, B. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3666-3670 2. Beutler, B., and Cerami, A. (1989) Annu. Reu. Immunol. 7 , 625655 3. Grunfeld, C., and Feingold, K. R. (1991) Biotherapy 3 , 143-158 4. Loetscher, H., Pan, Y.-C. E., Lahm, H.-W., Gentz, R., Brockhaus, M., Tabuchi, H., and Lesslauer, W. (1990) Cell 61,351-359 5. Schall, T. J., Lewis, M., Koller, K. J., Lee, A., Rice, G. C., Wong, G. H. W., Gatanaga, T., Granger, G. A., Lentz, R., Raab, H., Kohr, W. J., and Goeddel, D. V. (1990) Cell 6 1 , 361-370 6. Smith, C. A., Davis, T., Anderson, D., Solam, L., Beckmann, M. P., Jerzy, R., Dower, S. K., Cosman, D., and Goodwin, R. G. (1990) Science 2 4 8 , 1019-1022 7. Neale, M. L, Fiera, R. A., and Matthews, N. (1988) Immunology 64,81-85 8. Suffys, P., Beyaert, R., VanRoy, F., and Fiers, W. (1987) Bwchem. Biophys. Res. Commun. 1 4 9 , 735-743 9. Zimmerman, R. J., Chan, A., and Leadon, S. A. (1989) Cancer Res. 4 9 , 1644-1648 10. Yamauchi, N., Kuriyama, H., Watanabe, N., Neda, H., Maeda, M., and Niitsu, Y. (1989) Cancer Res. 49, 1671-1675 11. Schmid, D. S., Hornung, R., McGrath, K.M., Paul, N., and Ruddle, N. H. (1987) Lymphokine Res. 6, 195-202 12. Dealtry, G. B., Naylor, M. S., Fiers, W., and Balkwill, F. R. (1987) Eur. J. Immunol. 17,689-693 13. Suffys, P., Beyaert, R, Van Roy, F., and Fiers, W. (1988) Eur. J . Biochem. 178, 257-265
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