(Received for publication, April 28, 1995; and in revised form, June 23, 1995)
From the
We have studied the effect of blockade of mitochondrial
respiration on the binding of human 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 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 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, 9, 10, 11, 12, 13, 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
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) .
Figure 1:
Schema of the
mitochondrial respiratory chain, showing action sites of inhibitors. DFX, deferoxamine; DNP, 2,4-dinitrophenol; OH, hydroxyl radical; SOD, superoxide
dismutase; TTFA, thenoyltrifluoroacetone; UQ,
ubiquinone.
Ubiquinone passes these
electrons to the b-c 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
µg/ml or 26 µg/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 1).
Cytochrome c is involved in carrying
electrons from the b-c complex to the
cytochrome oxidase complex (cytochrome a-a
, 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 µM or 500 µM KCN for 3
h reduced TNF
binding to 47 and 37%, respectively, of controls (Table 1). 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).
Figure 2:
Effect of 2,4-dinitrophenol, NaF, malonic
acid, and anaerobiosis on TNF binding to L929 cells. Confluent
cultures of L929 cells were incubated with either 2 µM 2,4-dinitrophenol or 5 mM NaF or were incubated in a
nitrogenous atmosphere for 3 h at 37 °C (panelA). In panelB, confluent cells were
incubated with increasing concentrations of malonic acid for 3 h at 37
°C. After incubation, specific binding was measured as described
under ``Experimental Procedures'' and Table 1. Data are
expressed as percentage of radioactivity of control cultures and are
representative of two independent experiments that were carried out in
quadruplicate.
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 µg/ml oligomycin or
2 µM 2,4-dinitrophenol, an uncoupling agent, decreased the
binding of TNF to 27 and 57%, respectively, of control values ( Table 1and Fig. 2A).
FADH remains
as a part of the succinate dehydrogenase complex (complex II), which
passes its electrons directly to ubiquinone (Fig. 1). Addition
of TTFA (250 µM) 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 1). 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).
Figure 3:
Equilibrium saturation of TNF binding
to L929 cells and Scatchard analysis of the binding data. Cultures of 1
10
L929 cells were incubated with increasing
concentrations of human
I-TNF
in the absence (control) or presence of either 10 µg/ml antimycin A or
0.24 µM rotenone. Specific binding was calculated as
described in Fig. 2and under ``Experimental
Procedures.'' Scatchard plot analysis of the binding data at 4
°C is shown in the inset. Diagrams are
representative of experiments that were carried out twice. The data
represent the mean of duplicate samples.
Neither 10 µg/ml antimycin A nor 250 µM KCN, the
less effective blocker of TNF binding, modified the rate of
internalization of human
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
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.
Figure 4:
Effect of blockade of mitochondrial
respiration on internalization, degradation, and half-life of TNF
binding sites. PanelA, human
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 µg/ml antimycin
A or 250 µM 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.
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. PanelB, L929 cells were incubated with or without 10 µg/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 1and under ``Experimental
Procedures.'' Specific human
I-TNF
binding is
given as percentage of untreated cells (11,300 ± 635 cpm). Data
represent the mean of triplicate samples. CHX,
cycloheximide.
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 µg/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. Treatment of cells
with 10 µg/ml antimycin A decreased the specific binding of
TNF
to 37 ± 7% of control cells (Fig. 5). However,
the addition of 10 µg/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 µM 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
µg/ml). As Fig. 5shows, neither H-7 (50 µM)
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 µg/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).
Figure 5:
Effect of oxygen radical scavengers and
protein kinase C inhibitors on antimycin A-induced reduction of
TNF binding capacity to L929 cells. L929 cells were pretreated for
3 h with 10 µg/ml antimycin A (A) in the presence or
absence of one of the following agents: 0.5 nM staurosporine (St), 50 µM H-7 (H7), 10 µg/ml
superoxide dismutase (SD), 100 µM deferoxamine (DF), or 100 mM mannitol (M). Afterward,
specific TNF
binding was quantified as described in Fig. 2and ``Experimental Procedures.'' Specific
binding is expressed as percentage of radioactivity of untreated cells (C) cultured in RPMI 1640 medium without antimycin A but
otherwise processed in the same way (15,187 ± 695 cpm). Values
are means ± S.D. of triplicate
samples.
Figure 6:
Effect of blockade of the mitochondrial
respiration on intracellular content of ATP. PanelA,
L929 cells were incubated for 2 h at 37 °C with a series of
inhibitors of mitochondrial respiration and then solubilized. The ATP
content was determined as described under ``Experimental
Procedures.'' The data represent values from triplicate samples
and are expressed as mean ± S.D. C, controls; R, 0.24 µM rotenone; A1, 10 µg/ml
antimycin A; A2, 26 µg/ml antimycin A; CN, 250
µM potassium cyanide; O, 10 µg/ml oligomycin; T, 250 µM TTFA. PanelB, diagram represents correlation between intracellular ATP
content and specific TNF binding in cells pretreated with a number
of inhibitors of mitochondrial respiration. Data represent the mean of
triplicate samples.
Figure 7:
Resistance of L929 cells to TNF
cytotoxicity induced by inhibition of mitochondrial respiration. PanelA, L929 cells were pretreated for 2 h with 250
µM potassium cyanide, 10 µg/ml oligomycin, 0.24
µM rotenone, 10 µg/ml antimycin A, 2 µM 2,5-dinitrophenol (DNP), or in anaerobiosis and incubated
for the indicated time periods with 25 ng/ml human TNF
and 1
µg/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. PanelB, diagram
represents correlation between TNF
cytotoxicity and specific
TNF
binding to cells treated with 10 µg/ml antimycin A or 0.24
µM 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 Table 1and Table 2.
Cytotoxicity of TNF on L929 cells
pretreated with rotenone or antimycin A correlated closely with the
binding of TNF
to these cells (Fig. 7B).
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
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, 9, 10, 11, 12) , which
are involved in the down-regulation 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
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
10
M in control
cells, this constant increased between 2- and 4-fold in cells treated
with rotenone (K
= 11.7
10
M) or antimycin A (K
= 21.9
10
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, 35, 36, 37, 38, 39, 40, 41, 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 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, 9, 10, 11, 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, 47, 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, 51, 52, 53, 54, 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, down-regulation 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.