Down-regulation of tumor necrosis factor receptors by blockade of mitochondrial respiration.

We have studied the effect of blockade of mitochondrial respiration on the binding of human I-TNFα to L929 cell receptors. Specific TNFα binding was decreased to about 20-40% of controls by blocking mitochondrial respiration. This effect was dose- and time-related and was observed independently of the level at which the respiration was blocked (respiratory chain, proton backflow, ATPase, anaerobiosis). This blockade had no effect on the half-life of the specific TNFα binding, the internalization or degradation of TNFα-receptor complexes, or the number of TNFα-binding sites. Scatchard analysis of TNFα binding data indicated a 2-4-fold decrease in the affinity of these binding sites. These effects did not appear to be related to the protein kinase C activity or to reactive oxygen radicals, since they were not antagonized by pretreatment of cells with oxygen radical scavengers, deferoxamine, or inhibitors of protein kinase C. Decrease in TNFα binding capacity correlated significantly with cellular ATP content (r = 0.94; p < 0.01) and with the cytocidal activity of TNFα against L929 cells. These findings suggest that blockade of mitochondrial respiration down-regulates the binding of TNFα to cells, most likely by changing the affinity of receptors for this cytokine. This down-regulation may increase the resistance of cells to TNFα cytotoxicity.

We have studied the effect of blockade of mitochondrial respiration on the binding of human 125 I-TNF␣ to L929 cell receptors. Specific TNF␣ binding was decreased to about 20 -40% of controls by blocking mitochondrial respiration. This effect was dose-and timerelated and was observed independently of the level at which the respiration was blocked (respiratory chain, proton backflow, ATPase, anaerobiosis). This blockade had no effect on the half-life of the specific TNF␣ binding, the internalization or degradation of TNF␣-receptor complexes, or the number of TNF␣-binding sites. Scatchard analysis of TNF␣ binding data indicated a 2-4-fold decrease in the affinity of these binding sites. These effects did not appear to be related to the protein kinase C activity or to reactive oxygen radicals, since they were not antagonized by pretreatment of cells with oxygen radical scavengers, deferoxamine, or inhibitors of protein kinase C. Decrease in TNF␣ binding capacity correlated significantly with cellular ATP content (r ‫؍‬ 0.94; p < 0.01) and with the cytocidal activity of TNF␣ against L929 cells. These findings suggest that blockade of mitochondrial respiration down-regulates the binding of TNF␣ to cells, most likely by changing the affinity of receptors for this cytokine. This down-regulation may increase the resistance of cells to TNF␣ cytotoxicity.
Tumor necrosis factor ␣ (TNF␣) is a polypeptide cytokine with a wide range of biological activities (1)(2)(3)(4), including tumor cytotoxicity (5). TNF␣ initiates its biological effects by its binding to high affinity receptors (6). The expression of these receptors is necessary to determine responsiveness of target cells (7). It has also shown that some intracellular events, such as protein kinase C activity or intracellular levels of cAMP may regulate the binding of TNF␣ to these specific receptors (8 -14). Two TNF␣ receptors have been recognized: a type I receptor of 55-60 kDa and a type II receptor of 75-80 kDa. The cytocidal effect of TNF␣ is believed to be transduced by the type I receptor (15), although the type II receptor may also contribute to this effect (16,17). Unlike the species-specific type II receptor, the type I receptor in the murine L929 cell will bind heterologous (i.e. human) TNF␣ (18) and may initiate apoptotic cell death. In this paper, we show that mitochondrial function modulates the binding of TNF␣ to cell receptors, an event that might influence the effects of this cytokine on cell cytotoxicity. Mitochondrial dysfunction has been suggested to be an early event of TNF␣ cytotoxicity in tumor cells (19) EXPERIMENTAL PROCEDURES Materials-Recombinant human TNF␣ was purchased from Genzyme Co. (Cambridge, MA). RPMI 1640 medium was from Biochrom (Berlin, Germany), and 125 I-Bolton-Hunter-labeled human TNF␣ (specific activity, 48.8 Ci/g) was from DuPont NEN. SDS, rotenone, thenoyltrifluoroacetone (TTFA), 1 antimycin A, potassium cyanide (KCN), malonic acid, 2,4-dinitrophenol, oligomycin, actinomycin D, cycloheximide, sodium orthovanadate, okadaic acid, staurosporine, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) were purchased from Sigma. Fetal calf serum (FCS) was from Sera-lab (Sussex, United Kingdom), and phosphate buffered saline was from SCN Biomedicals, Inc. (Costa Mesa, CA). Plastic cell culture flasks and dishes were from Nunc (Roskilde, Denmark) and Falcon Division of Becton Dickinson Co. (Oxnard, CA).
Binding Assays-Binding assays were performed in confluent cultures in 35-mm plastic plates containing approximately 1 ϫ 10 6 L929 cells, as described by Tsujimoto et al. (20). The effect of inhibitors of mitochondrial respiratory chain on the binding of TNF␣ to specific cell receptors was evaluated by the same assay after incubation of L929 cells with these agents for 1, 2, or 3 h at 37°C. NADH-dehydrogenase complex (complex I) was inhibited with rotenone; succinate dehydrogenase (complex II) was inhibited with TTFA and malonic acid; b-c 1 complex (complex III) was inhibited with antimycin A; cytochrome oxidase complex (cytochrome a-a 3 ; complex IV) was inhibited with KCN and NaF; and ATPase was inhibited with oligomycin. As uncoupling agent, 2,4-dinitrophenol was used, and as phosphatase inhibitors, sodium orthovanadate and okadaic acid were employed. Anaerobiosis was induced by incubating cells in a nitrogen atmosphere, after replacing oxygen by nitrogen and sealing culture flasks tightly. All of these assays were carried out in quadruplicate. At the time of the TNF binding studies, no evidence of cytotoxicity was detected. Calculation of the dissociation constant (K d ) and the number of binding sites was done by Scatchard plot analysis of the binding (21), as described by Linge and Green (22), under control conditions and after incubation of cells with either 10 g/ml antimycin A or 0.24 M rotenone for 2 h at 37°C. These assays were performed in duplicate.
Internalization of cell-bound TNF␣ was evaluated by the procedure described by Costlow and Hample (23) and modified by Tsujimoto et al. (20). Degradation of internalized TNF␣ was measured following the indication of Tsujimoto et al. (20).
Cytotoxicity-Fifty microliters of solution containing the inhibitory drug were added to L929 cells cultured in 35-mm plates at 1 ϫ 10 6 cells in 2 ml RPMI 1640 medium. At the time indicated, 25 ng/ml human TNF␣ and 1 g/ml actinomycin D were added to the culture medium. Cytotoxicity was measured using the index of lactate dehydrogenase enzyme leakage from damaged cells and expressed as a percentage of total cellular activity, as described by Decker and Lohmann-Matthes (24). Intracellular ATP Concentration-ATP content was measured using a modified bioluminiscence assay as described by Wulff and Döppen (25). Cellular ATP levels were expressed in nmol/10 6 cells.
Statistical Analysis-All results are expressed as mean Ϯ S.D. unless otherwise mentioned. Student's t test was used to evaluate the difference in means between groups, accepting p Ͻ 0.05 as level of significance (26). Pearson's correlation coefficient was used for correlation analysis between variables that demonstrated statistically significant changes after treatment of cells.

Effect of Blocking the Mitochondrial Respiratory Chain on
Specific TNF␣ Binding to Cell Receptors-Complex I of the respiratory chain accepts electrons from NADH and transfers them to ubiquinone (27) (Fig. 1). The blockade of this transfer of electrons through complex I by incubating cells with 0.24 M and 30 M rotenone for 1-3 h led to a marked decrease in the binding of TNF␣ to specific type I cell receptors. This effect increased in proportion to the incubation time, there being only 19 Ϯ 7% of the control binding after 3 h of incubation with 30 M rotenone (Table I).
Ubiquinone passes these electrons to the b-c 1 complex (complex III), which transfers them to cytochrome c (27). The function of complex III can be blocked with antimycin A (Fig. 1). Therefore, we incubated L929 cells either with 10 g/ml or 26 g/ml antimycin A for 1 to 3 h. Our study showed that the blockade of this complex decreased TNF␣ binding to about 40 and 30%, respectively, of the control after 3 h of incubation (Table I).
Cytochrome c is involved in carrying electrons from the b-c 1 complex to the cytochrome oxidase complex (cytochrome a-a 3 , or complex IV). This complex finally transfers these electrons to oxygen (27). KCN binds to this complex and thereby blocks the transport of electrons. Treatment of cells with either 250 M or 500 M KCN for 3 h reduced TNF␣ binding to 47 and 37%, respectively, of controls (Table I). Likewise, 5 mM NaF, another inhibitor of cytochrome c oxidase, decreased specific binding of TNF␣ to 18% of the control level after 3 h of incubation ( Fig. 2A).
Transport of electrons through the respiratory chain is coupled to the pumping of protons from the mitochondrial matrix to the intermembrane space. ATPase synthesizes ATP when protons flow back from the intermembrane space into the matrix (27). Oligomycin inhibits ATPase activity, and lipophilic weak acids uncouple electron transport from ATP synthesis. Incubation of L929 cells for 3 h with either 10 g/ml oligomycin or 2 M 2,4-dinitrophenol, an uncoupling agent, decreased the binding of TNF␣ to 27 and 57%, respectively, of control values (Table I and Fig. 2A).
FADH 2 remains as a part of the succinate dehydrogenase complex (complex II), which passes its electrons directly to ubiquinone ( Fig. 1). Addition of TTFA (250 M) to the cells led to a gradual decrease in TNF␣ binding from 81% after 1 h of incubation to 63 and 29% after 2 and 3 h of incubation, respectively (Table I). Likewise, incubation of cells with malonic acid, a competitive inhibitor of complex II, induced a dose-related inhibition of the TNF␣ binding ( Fig. 2A).
In the absence of oxygen, respiratory enzyme complexes are in their reduced state, being unable to generate ATP. Incubation of L929 cells in anaerobic conditions for 3 h reduced the TNF␣ binding to 45% of the control ( Fig. 2A).

Effect of Inhibitors of the Respiratory Chain on Number, Affinity, Internalization, Degradation and Half-life of the TNF␣ Cell
Receptors-Scatchard analysis of the binding data showed that the dissociation constant of TNF␣ receptors in L929 cells was 5.7 ϫ 10 Ϫ10 M and that the average number of specific binding sites was about 3372/cell. These results concurred with those reported by Tsujimoto et al. (20). Scatchard analysis after treatment of L929 cells for 2 h with either 10 g/ml antimycin A or 0.24 M rotenone, two powerful inhibitors of TNF␣ binding, indicated a 2-4-fold decrease in the receptor affinity (rotenone-treated: K d ϭ 11.7 ϫ 10 Ϫ10 M; antimycin A-treated: K d ϭ 21.9 ϫ 10 Ϫ10 M). In rotenone-treated cells, the number of specific binding sites did not differ significantly (3308 binding sites) from those of control cells. However, this number decreased slightly, to 82% of the controls, in cells

TABLE I
Effect of antimycin A, olygomycin, potassium cyanide, rotenone, and TTFA on 125 I-TNF binding to L929 cells Approximately 1 ϫ 10 6 L929 cells were cultured for the indicated times at 37°C with 10 g/ml and 26 g/ml antimycin A, 10 g/ml olygomycin, 250 M potassium cyanide, 0.24 M and 30 M rotenone, or 250 M and 500 M TTFA. No evidence of cytotoxicity of these inhibitors was detected during the period in which these experiments were carried out. After removal of the culture medium, cells were incubated with 400 pM human 125 I-TNF␣ for 18 h at 4°C in 2 ml RPMI-1640 medium containing 10% FCS. To measure nonspecific binding, a 100-fold excess of unlabeled human TNF␣ was added together with human 125 I-TNF␣ during 18 h of exposure at 4°C. Cells were washed four times with ice-cold RPMI-1640 medium and were solubilized in 0.1% SDS, and radioactivity was measured in a ␥ counter. Cell binding is expressed as percentage of radioactivity of control cultures incubated without inhibitors of the respiratory chain but otherwise processed in the same way. Data represent mean values from a single experiment, which was conducted in quadruplicate. r, correlation coefficient of Pearson. treated with antimycin A (2752 binding sites) (Fig. 3). Neither 10 g/ml antimycin A nor 250 M KCN, the less effective blocker of TNF␣ binding, modified the rate of internalization of human 125 I-TNF␣. Intracellular radioactivity resistant to acid elution rose early after incubation of cells at 37°C, but this rise was followed by a slow decline. No significant differences were seen in the kinetics of intracellular TNF␣ between control cells and cells treated with either antimycin A or KCN (Fig. 4A). Analysis of 125 I radioactivity in the TCA-soluble fraction of culture medium showed a significant increase after 2 h of incubation and continued rising gradually until 6 h (Fig. 4A). The kinetics of this increase were identical in control cells and after blocking the mitochondrial respiratory chain with antimycin A or KCN.
Blocking protein synthesis with 0.1 mM cycloheximide revealed a half-life of TNF␣-receptors of about 2 h in control cells. This result is in agreement with those reported by others (11,14). Treatment of cells with 10 g/ml antimycin A did not modify this half-life (Fig. 4B).
To explore the role played by reactive oxygen radicals formed throughout the mitochondrial respiratory chain, we analyzed the effect of scavengers of these radicals and iron chelators on antimycin A-induced inhibition of TNF␣ binding capacity. Panel A, human 125 I-TNF␣ was allowed to bind to L929 cells for 2 h at 4°C. Then cells were washed four times with ice-cold RPMI 1640 medium containing 10% FCS and further incubated at 37°C in the absence (control) or presence of either 10 g/ml antimycin A or 250 M KCN. After the indicated times, cells were washed once with ice-cold phosphate-buffered saline and incubated for 5 min at 4°C with 2 ml 0.05 M glycine-HCl buffer (pH 3.0) containing 0.15 NaCl. After removal of this buffer, cells were washed twice with RPMI 1640 medium containing 10% FCS and solubilized in 0.1% SDS. 125 I-radioactivity found in the trichloracetic acid-soluble fraction of culture media collected before addition of the glycine buffer and in solubilized cells gave a measure of degradation and internalization, respectively, of TNF␣receptor complex. Diagram is representative of two separate experiments. Panel B, L929 cells were incubated with or without 10 g/ml antimycin A in the presence or absence of 0.1 mM cycloheximide at 37°C. At the indicated times TNF␣ binding capacity was determined as indicated in Table I and under "Experimental Procedures." Specific human 125 I-TNF␣ binding is given as percentage of untreated cells (11,300 Ϯ 635 cpm). Data represent the mean of triplicate samples. CHX, cycloheximide. Treatment of cells with 10 g/ml antimycin A decreased the specific binding of TNF␣ to 37 Ϯ 7% of control cells (Fig. 5). However, the addition of 10 g/ml superoxide dismutase, an enzyme that removes superoxide anion from the cells, as well as 100 mM mannitol, a scavenger of hydroxyl radicals, or 100 M deferoxamine, a specific iron chelator, did not antagonize this effect of antimycin A (Fig. 5). We also studied the effect of the protein kinase C inhibitors H-7 and staurosporine on the decreased binding of TNF␣ to L929 cells induced by antimycin A (10 g/ml). As Fig. 5 shows, neither H-7 (50 M) nor staurosporine (0.5 nM) had any significant effect on the specific binding of TNF␣ to these cells, and neither of them antagonized the antimycin A-induced reduction of TNF␣ binding activity. Likewise, the addition of orthovanadate (1 g/ml) or okadaic acid (20 ng/ml), two phosphatase inhibitors, did not reverse the effect of antimycin A on TNF␣ binding to the cells (data not shown).
Effect of Blockade of the Mitochondrial Respiratory Chain on Intracellular ATP Levels-As expected, incubation of L929 cells for 2 h with any inhibitor of cellular respiration resulted in a significant decrease in the intracellular ATP content (Fig. 6A). These levels correlated significantly with the binding of TNF␣ to cells treated for 2 h with the inhibitor (r ϭ 0.94; p Ͻ 0.01) (Fig. 6B). Fig. 7A. L929 cells are sensitive to the cytocidal activity of 25 ng/ml human TNF␣ in the presence of 1 g/ml actinomycin D. Cells treated with any inhibitor of the respiratory chain become more resistant to this effect of TNF␣. In most cases, resistance to cytolysis persisted 24 h after application of the cytokine. Blockade of complex I (0.24 M rotenone) reduced cytocidal activity of TNF␣ to 20% of controls after 9 h of incubation with TNF␣. Likewise, inhibition of the b-c 1 complex (10 g/ml antimycin A), blockade of ATPase (10 g/ml oligomycin) or treatment of cells with 2 M 2,4-dinitrophenol decreased cytotoxicity of TNF␣ to 34, 25, and 11%, respectively, of controls. In contrast, blockade of cytochrome c oxidase with 250 M KCN resulted in a moderate reduction in the cell death after 9 h of incubation with TNF␣ to 68% of control values. Treatment of cells with 25 ng/ml TNF␣ and 1 g/ml actinomycin D under aerobic conditions for 12 h induced the cytolysis of about 50% of the cells. However, only 10% of the cells died in the absence of oxygen. Anaerobiosis 4 h after the addition of TNF␣ did not significantly protect cells from TNF␣ (Table II). Likewise, resistance to cytolysis decreased when the respiratory chain was inhibited by other agents 1-4 h after the addition of TNF␣.

Effect of Inhibition of the Mitochondrial Respiratory Chain on TNF␣-induced Cytotoxicity-The effect of the pretreatment of cells for 2 h with various inhibitors of the respiratory chain on TNF␣-induced cytotoxicity is shown in
Cytotoxicity of TNF␣ on L929 cells pretreated with rotenone or antimycin A correlated closely with the binding of TNF␣ to these cells (Fig. 7B).

DISCUSSION
In this study, we show that treatment of L929 cells with a number of inhibitors of cellular respiration resulted in a significant decrease in the binding of human TNF␣ to cell surface receptors. Although there were some differences in the degree of this inhibition depending on the drug used for blocking the transport of electrons throughout the respiratory chain, this effect was observed independently of the level at which the inhibition was induced. Down-regulation of TNF␣ binding by blocking cellular respiration was intensified in proportion to the incubation time and to the concentration of inhibitor in the culture medium. After 3 h of inhibition, the binding of human 125 I-TNF␣ to cell receptors was only 18 -47% of the control. This effect on TNF␣ binding had biological implications, since it was followed by a decrease in the cytocidal activity of TNF␣ in L929 cells, and both effects, TNF␣ binding and TNF␣ cytotoxicity, were closely correlated. Thus, the interpretation of changes in TNF␣-mediated cytotoxicity induced by pretreatment of cells with inhibitors of the mitochondrial respiratory chain as well as by any other factor demands that this effect on TNF␣ binding be taken into account.
Several factors have been identified as modulators of TNF␣receptor function. These factors include protein kinase C and interleukin-1 (8 -12), which are involved in the down-regula-tion of TNF␣ binding. In contrast, cAMP, protein kinase A (13,14) and interferon-␥ up-regulate this binding (14,28,29). Now we demonstrate that there is a close relationship between cellular respiration and the binding of TNF␣ to its specific type I receptors.
The mechanisms by which the blockade of the mitochondrial respiratory chain led to a down-regulation of the TNF␣ binding capacity of L929 cells are not known. This effect cannot be attributed to a reduced cell viability as a result of these treatments, since no cell death was detected at the time these experiments were done. On the contrary, these treatments protect the cells from TNF-induced cytotoxicity. This effect could be also due to changes in the number of receptors or in their affinity for the ligand but might also reflect modifications in the kinetics of receptor-ligand complex internalization and degradation or an increase in the shedding of TNF␣ receptor into the culture medium.
The binding of TNF␣ to specific cell receptors is usually followed by internalization of the receptor-TNF␣ complex into the cell and by degradation of TNF␣ by lysosomal hydrolases (30). However, our study detected no significant difference in the kinetics of internalization or degradation of human 125 I-TNF␣ between untreated cells and cells treated with either antimycin A or KCN (Fig. 4). Moreover, determination of the half-life of TNF␣ binding sites showed that it is not affected by blocking of the respiratory chain, suggesting that it is unlikely that increased degradation or shedding of these receptors can account for the down-regulation of the TNF␣ binding capacity of cells. On the other hand, Scatchard analysis of antimycin A or rotenone-treated L929 cells indicated that the number of TNF␣ receptors did not change significantly, ruling out the possibility that a change in TNF␣-receptor synthesis may account for this down-regulation. Likewise, this result led to doubts about an enhanced shedding of TNF␣ receptors as the cause of the reduced binding of TNF␣ to these cells. Our data indicate that this effect is better explained by a decrease in receptor affinity. Scatchard analysis showed that the affinity of TNF␣ binding sites was markedly decreased in cells treated with either antimycin A or rotenone. Thus, while TNF␣ binding sites had a dissociation constant of 5.7 ϫ 10 Ϫ10 M in control cells, this constant increased between 2-and 4-fold in cells treated with rotenone (K d ϭ 11.7 ϫ 10 Ϫ10 M) or antimycin A (K d ϭ 21.9 ϫ 10 Ϫ10 M).
Blockade of the respiratory chain has a number of consequences for cell metabolism and function. Mitochondria are the major source of reactive oxygen species (31)(32)(33), and, under certain conditions, including mitochondrial inhibition and TNF␣ treatment, generation of reactive oxygen radicals increases severalfold (19, 34 -42). Our results do not support the involvement of these radicals as mediators for this relationship between mitochondrial respiration and TNF␣ binding to the cells. Neither superoxide dismutase nor mannitol prevented FIG. 7. Resistance of L929 cells to TNF␣ cytotoxicity induced by inhibition of mitochondrial respiration. Panel A, L929 cells were pretreated for 2 h with 250 M potassium cyanide, 10 g/ml oligomycin, 0.24 M rotenone, 10 g/ml antimycin A, 2 M 2,5-dinitrophenol (DNP), or in anaerobiosis and incubated for the indicated time periods with 25 ng/ml human TNF␣ and 1 g/ml actinomycin D. Cytotoxicity was assessed as described under "Experimental Procedures." Data were collected from triplicate samples. Values are expressed as percent of TNF␣ cytotoxicity on control cells. Panel B, diagram represents correlation between TNF␣ cytotoxicity and specific TNF␣ binding to cells treated with 10 g/ml antimycin A or 0.24 M rotenone. Cells were pretreated with these inhibitors for 1, 2, or 3 h. After these pretreatment times, TNF binding and TNF cytotoxicity were measured as indicated in Tables I and II.   TABLE II Time course of the effect of the blockade of cellular respiration on TNF␣-induced cytotoxicity Confluent L929 cells were cultured at 1 ϫ 10 6 cells in 2 ml of RPMI 1640 medium and treated with 25 ng/ml TNF␣ and 1 g/ml actinomycin D. At the indicated times prior to or after TNF␣ treatment, cell respiration was inhibited with 0.24 M rotenone, 10 g/ml antimycin A, 10 g/ml oligomycin, 250 M potassium cyanide, or anaerobiosis. After 12 h of treatment with TNF␣/actinomycin D with or without inhibition of cell respiration, cytotoxicity was measured as indicated under "Experimental Procedures." Data are expressed as the percentage of cytotoxicity of control cultures incubated for identical times without inhibitors of cellular respiration but otherwise processed in the same way.
Hours prior to or after treatment with TNF␣/actinomycin D

Ϫ3
Ϫ2 the inhibitory effect of antimycin A on the TNF␣ binding capacity of cells (Fig. 6). Conversion of superoxide anion and hydrogen peroxide to hydroxyl radical can be catalyzed by iron ions. However, chelation of these ions by deferoxamine had no effect on TNF␣ binding either. Moreover, anaerobiosis, a condition under which no reactive oxygen radicals are generated, significantly decreased TNF binding to L929 cells ( Fig. 2A). It has been demonstrated that TNF␣ binding to the cell receptors can be significantly decreased by activation of protein kinase C (8 -12, 43). The mechanism of this effect is unclear (8,9,11,14). However, it has been shown that activation of protein kinase C enhances shedding of TNF␣ receptors into the medium (43)(44)(45). It has been suggested that the decrease in TNF␣ binding sites caused by phorbol myristate acetate is most likely mediated through enhanced phosphorylation and activation of a cellular protease, which cleaves the TNF␣-receptors (43). Protein kinase C has been shown to have similar down-regulating effects on other cell receptors (46 -48), and Dowing et al. (49) reported that phorbol myristate acetate increased shedding of CSF-1 receptors through protein kinase C-mediated activation of a protease. The results of our study appear to indicate that the effect of blockade of the mitochondrial respiratory chain on TNF␣ binding activity is not likely to be mediated by a protein kinase C-phosphorylated protein. Neither the inhibition of this enzyme with H-7 or staurosporine nor the blockade of cellular phosphatase with orthovanadate or okadaic acid antagonized the effect of antimycin A on TNF␣ binding activity (Fig. 5). Moreover, blocking the mitochondrial transfer of electrons decreased TNF␣ receptor affinity markedly, with no significant effect on the number of these receptors (Fig. 3). As we have previously commented, receptor shedding also seems unlikely since the half-life of TNF␣-receptors was not reduced.
In this study, we found that the binding capacity of L929 cells for TNF␣ was significantly correlated with the intracellular concentration of ATP. This finding suggests that the cellular ATP content may have a direct or indirect modulatory role in the TNF␣ binding activity of TNF␣ receptors. As far as we are aware, no data regarding cellular ATP levels and TNF␣ receptors have been published so far. A series of studies have found some relationships between other hormone receptors and ATP content in cells (50 -55). However, results from these studies are heterogeneous and frequently contradictory and thus fail to indicate a possible common mechanism of action. It is conceivable that the binding of TNF␣ to specific cell receptors is an energy-consuming process. However, further studies are required to explain this relationship.
This down-regulation could be simply a side effect of mitochondrial dysfunction, secondary to the loss of cellular energy and, therefore, lacking in teleologic meaning. However, we could speculate that this mechanism might protect cells from being killed by TNF␣. It has been shown that TNF␣ treatment leads to a dose-dependent inhibition of mitochondrial transport of electrons and to the formation of reactive oxygen species (19,(37)(38)(39)(40)42). Based on abundant evidence, it has been suggested that these reactive metabolites play a major role in the cytotoxicity induced by TNF␣ (19,37,38,42,56,57). Thus, downregulation of TNF␣ binding by blockade of the mitochondrial respiratory chain might reduce the biological effects of TNF␣ and, consequently, could diminish oxidative stress, hydroxyl radical generation, and cell death. Holtmann and Wallach (8), Wallach et al. (58) and Hahn et al. (59) showed that cells pretreated with TNF␣ for 1-12 h became more resistant to the cytocidal effect of TNF␣, and Aggarwal et al. (28) demonstrated that TNF␣ and TNF␤ inhibit the binding of TNF␣ to the cells. Other mechanisms of self-protection have been shown to be induced by TNF␣. This is the case for manganous superoxide dismutase, a mitochondrial enzyme involved in the scavenging of superoxide radicals. Wong et al. (56,57) and Hirose et al. (60) demonstrated that treatment of cells with TNF␣ induces the expression of mRNA for this enzyme, and these investigators proposed manganese superoxide dismutase as one of the protective proteins whose synthesis is induced by TNF␣.
We conclude that blockade of the mitochondrial respiratory chain down-regulates the binding of TNF␣ to L929 cells, most likely by decreasing the affinity of TNF␣ receptors for this cytokine. The mechanism of this down-regulation is not known, but it appears to be closely related to intracellular ATP levels. This down-regulation may increase the resistance of cells to TNF␣ cytotoxicity.