Báo cáo khoa học: Muramyl-dipeptide-induced mitochondrial proton leak in macrophages is associated with upregulation of uncoupling protein 2 and the production of reactive oxygen and reactive nitrogen species docx

Muramyl-dipeptide-induced mitochondrial proton leak in
macrophages is associated with upregulation of
uncoupling protein 2 and the production of reactive
oxygen and reactive nitrogen species
Takla G. El-Khoury, Georges M. Bahr and Karim S. Echtay
Faculty of Medicine and Medical Sciences and Faculty of Sciences, University of Balamand, Tripoli, Lebanon

Keywords
mitochondria; muramylpeptides; nitric oxide;
respiratory control ratio; superoxide anion;
UCP2
Correspondence
K. S. Echtay, Faculty of Medicine and
Medical Sciences, University of Balamand,
PO Box 100, Tripoli, Lebanon
Fax: +961 6 930279
Tel: +961 3 714125
E-mail:
(Received 5 May 2011, revised 13 June
2011, accepted 28 June 2011)
doi:10.1111/j.1742-4658.2011.08226.x

The synthetic immunomodulator muramyl dipeptide (MDP) has been
shown to induce, in vivo, mitochondrial proton leak. In the present work,
we extended these findings to the cellular level and confirmed the effects of
MDP in vitro on murine macrophages. The macrophage system was then
used to analyse the mechanism of the MDP-induced mitochondrial proton
leak. Our results demonstrate that the cellular levels of superoxide anion
and nitric oxide were significantly elevated in response to MDP. Moreover,
isolated mitochondria from cells treated with MDP presented a significant
decrease in respiratory control ratio, an effect that was absent following

treatment with a non-toxic analogue such as murabutide. Stimulation of
cells with MDP, but not with murabutide, rapidly upregulates the expression of the mitochondrial protein uncoupling protein 2 (UCP2), and pretreatment with vitamin E attenuates upregulation of UCP2. These findings
suggest that the MDP-induced reactive species upregulate UCP2 expression
in order to counteract the effects of MDP on mitochondrial respiratory
efficiency.

Introduction
Uncoupling proteins (UCPs) are members of the anion
carrier family molecules present in the inner mitochondrial membrane. Mammals express five UCP homologues, UCP1–UCP5. UCP2 and UCP3 have 59% and
57% identity, respectively, with UCP1, and 73% identity with each other [1], whereas UCP4 and UCP5 (also
referred to as brain mitochondrial carrier protein 1,
BMCP1) have much lower sequence identity with
UCP1 [2,3]. UCP1 is the best characterized of these
proteins, mediating non-shivering thermogenesis in
brown adipose tissue by catalysing proton leak activated by long-chain fatty acids and inhibited by purine
nucleotides [4]. UCP2 is widely expressed in many tissues with high levels detected in the spleen, thymus,

pancreatic b-cells, heart, lung, white and brown adipose tissue, stomach, testis and macrophages, whereas
low levels have been reported in the brain, kidney,
liver and muscle [5]. UCP3 is expressed predominantly
in skeletal muscles and brown adipose tissues [6,7], at
hundred-fold lower concentration than UCP1 in brown
adipose tissue [8]. UCP4 and UCP5 are only present in
the brain [2,3]. Due to their homology to UCP1 and
their distribution in several mammalian tissues, it has
been initially postulated that these proteins can regulate mitochondrial oxidative phosphorylation through
uncoupling activity. However, the physiological function
of UCPs other than UCP1 has remained controversial.
Suggested functions include mild uncoupling, adaptive

Abbreviations
FCCP, fluorocarbonyl cyanide phenylhydrazone; LPS, lipopolysaccharide; MB, murabutide; MDP, muramyl dipeptide; PI, propidium iodide;
RCR, respiratory control ratio; ROS, reactive oxygen species; RNS, reactive nitrogen species; UCP, uncoupling protein.

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T. G. El-Khoury et al.

thermogenesis, protection against obesity, regulation of
the ATP ⁄ ADP ratio, export of fatty acids, and mediation of insulin secretion (reviewed in [9]).
The hypothesis that has good experimental support
is the function of UCP2 to attenuate mitochondrial
production of free radicals and to protect against oxidative damage [10,11]. This is mainly based on the
activation of mitochondrial proton conductance mediated through UCPs by reactive oxygen species (ROS)
or by-products of lipid peroxidation [12,13], resulting
in a negative feedback loop that decreases ROS production by lowering both the proton-motive force and
local oxygen consumption. UCP2 was shown to play a
regulatory role in macrophage-mediated immune
and ⁄ or inflammatory responses [14,15]. Infected peritoneal macrophages of UCP2) ⁄ ) mice are resistant to
infection by the intracellular parasite Toxoplasma gondii through a mechanism proposed to involve higher
production of intracellular ROS [14]. On the other
hand, studies in cells overexpressing UCP2 have reinforced the belief that UCP2 plays a role in limiting
intracellular ROS production, as has been shown in
the murine macrophage cell line Raw-264 [16]. Moreover, cardiomyocytes transfected with a UCP2-expressing adenovirus were able to regulate ROS production
and protect against doxorubicin-mediated cardiotoxicity [17]. Therefore, by acting as a modulator of ROS
production, particularly in monocytes ⁄ macrophages,
UCP2 may impact the outcome of an innate response.

However, whether UCP2 functions to attenuate ROS
production by simply catalysing mild uncoupling
remains to be tested.
Muramyl peptides are a family of immunomodulators with diverse biological effects. Their immunological activities include adjuvanticity, enhancement of
non-specific resistance to viral and bacterial infections,
potentiation of anti-tumour activity of macrophages,
manipulation of cytokine release and restoration
of haematopoiesis [18–20]. The parent molecule of this
family is muramyl dipeptide (MDP), which has been
reported as the minimal adjuvant-active structure of
bacterial peptidoglycan [21]. However, MDP administration into different hosts was associated with serious
toxicity. Therefore, attempts have been made to generate analogues with desirable properties and reduced
toxicities. One of these derivatives is murabutide (MB),
a hydrophilic derivative of MDP that has eventually
reached a clinical stage of development [20,22]. It has
been tested in vivo comparing its pharmacological,
inflammatory and toxic effects with those of the parent
molecule MDP. The results reported establish the
safety of MB, the absence of undesirable effects on the

UCP2 modulates MDP-induced mitochondrial inefficiency

central nervous system, and the lack of induction of
inflammatory responses [22].
Despite a long-standing interest in the field of muramyl peptides, the impact of these molecules at the
mitochondrial level has not yet been examined.
Recently the effect of these derivatives on mitochondrial bioenergetics has been studied [23]. MDP induced
in vivo a significant decrease in respiratory control
ratio (RCR) in isolated mouse liver and spleen mitochondria versus non-toxic analogues such as MB. The
decrease in RCR in mitochondria of MDP-treated

mice is attributed to an increase in mitochondrial proton leak (i.e. mitochondrial uncoupling). In the present
study we use the immunomodulators to reveal the
mechanism of action of toxic MDPs on mitochondrial
respiration by correlating the uncoupling effect
induced by these molecules with the level and function
of UCP2 and free radical production in macrophages.
We find that MDP induces reactive oxygen and nitrogen species production and upregulates UCP2 protein
level, whereas MB does not. We further show that the
activity of UCP2 is consistent with the level of free
radicals.

Results
In vitro effect of muramyl peptides and
lipopolysaccharide on respiratory mitochondrial
activity of murine peritoneal macrophages
Measurement of oxygen consumption represents a
potent technique to characterize the respiratory function in mitochondria isolated from tissues or cultured
cells and to thoroughly localize the sites of impairment
of oxidative phosphorylation. In this study, the activities of the respiratory chain complexes are examined as
the oxygen consumption rates after addition of various
substrates and inhibitors. The mitochondrial respiratory function is conventionally separated into different
states. State 2 is the oxygen consumption rate of substrate (succinate) oxidation. State 3 is defined as the
phosphorylation state and is dependent on the oxygen
consumption in the presence of ADP, thus reflecting
the mitochondrial respiration coupled to ATP production. State 4, the non-phosphorylation state, is a measure of oxygen consumption in the presence of
oligomycin (ATP synthase inhibitor). This state represents the mitochondrial basal proton leak activity.
State 3 ⁄ state 4, termed the RCR, is used as an indicator to evaluate mitochondrial efficiency since it reflects
the coupling between oxidative phosphorylation and
the mitochondrial electron transport chain activity.

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T. G. El-Khoury et al.

60

A

RCR inhibition (%)

50

*

40

*

30

20

10

0

2
Time (h)

1

6

4

1.6

B

1.2
Respiration rate
(nmol O·min–1·mg–1)

Figure 1A shows the time-dependent inhibition of
succinate-linked RCR in mitochondria extracted from
MDP-treated (100 lgỈmL)1) macrophages. A maximum
decrease in RCR (about 42% compared with untreated
cells) was noted after 2 h of treatment and the value
returned to its basal level after 4 h. Figure 1B shows
that the decrease in RCR in mitochondria of MDPtreated macrophages was attributed to an increase in
state 4 respiration. No significant changes were
observed in state 2, state 3 and fluorocarbonyl cyanide
phenylhydrazone (FCCP) rates between untreated and
MDP-treated cells. The conditions at which MDP
exerted its maximum effects on mitochondria were
applied to examine the impact of the other derivatives.

Figure 1C and Table 1 summarize the effect of MB
(non-toxic muramyl peptide) and lipopolysaccharide
(LPS) on the mitochondrial bioenergetics of macrophage-treated cells. The results demonstrate clearly the
inability of MB and LPS to induce any impairment in
mitochondrial function after 2 h of treatment. RCR
and states 2, 3, 4 and FCCP rates of MB- and LPStreated cells were the same as those of unstimulated
cells. These results demonstrate clearly the ability of
only toxic muramyl peptides (such as MDP) to impair
mitochondrial function whereas non-toxic muramyl
peptides (such as MB) and LPS have no effect on mitochondrial respirations of peritoneal macrophages after
2 h of treatment.

0.8
*
0.4

Effect of MDP on cell viability
0

Fig. 1. Effects of muramyl peptides and LPS on respiration rates
and RCR in murine peritoneal macrophage mitochondria in vitro.
(A) Oxygen consumption was measured in the presence of
100 lgỈmL)1 of MDP after 1, 2, 4 and 6 h of incubation. The
decrease in RCR is presented as a percentage of inhibition.
(B) Mitochondrial respiratory states were measured in mitochondria
after 2 h of treatment with MDP (100 lgỈmL)1). Data are normalized to state 3 rates of unstimulated mitochondria (black bars).
(C) RCRs of mitochondria isolated from cells treated for 2 h with MDP
(100 lgỈmL)1), murabutide (MB, 100 lgỈmL)1) or LPS (1 lgỈmL)1).
Data are normalized to the values of unstimulated cells (black
bar, taken as 1). Data are means ± SEM of three independent

experiments each performed in triplicate. *P < 0.05.

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State 2

State 3

State 4

FCCP

1.2

C

1

0.8
RCR

The viability of peritoneal macrophages under conditions of maximum impairment of mitochondrial activity
of MDP-treated cells was examined. The proportions
of viable (Annexin V-FITCneg ⁄ propidium iodide
(PIneg)), early apoptotic (Annexin V-FITCpos ⁄ PIneg)
and late apoptotic ⁄ necrotic (Annexin V-FITCpos ⁄
PIpos) cells were identified (Fig. 2A–C). The mean

*

0.6

0.4

0.2

0

l
ro

nt

Co

DP

M

B

M

S

LP

percentage of viable cells in unstimulated and in
MDP-treated cells was 69.05% and 65.45% respectively (P > 0.05). Moreover, no significant difference

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T. G. El-Khoury et al.

UCP2 modulates MDP-induced mitochondrial inefficiency

Table 1. Effects of MB and LPS on respiration rates in murine peritoneal macrophage mitochondria in vitro. Mitochondria were isolated from murine peritoneal macrophages after 2 h of treatment
with MB (100 lgỈmL)1) or LPS (1 lgỈmL)1). Data are presented as
the percentage of unstimulated cells. Data are means ± standard
error of the mean of three independent experiments each performed in triplicate.
Percentage unstimulated cells
State 3

State 4

FCCP rate

B

100

101

PI
102

103

A

104

MB (100 lgỈmL)1) 100 ± 0
112 ± 14.2 107 ± 10.8 95.68 ± 4.6
LPS (1 lgỈmL)1)
100 ± 1.5 102 ± 12.3 98 ± 7.6 98.27 ± 8.2

100

101 102 103
Annex-FITC

104 100

101 102 103
Annex-FITC

In order to investigate the mechanism of action of
MDP on the mitochondrial bioenergetics system and
since mitochondria are an important source of ROS
production and especially of superoxide anion, we
investigated the effect of MDP (100 lgỈmL)1) on total
cellular superoxide anion production by murine peritoneal macrophages. As shown in Fig. 3, total superoxide production was unchanged after 30 min but was
significantly elevated at 60 and 120 min (P < 0.05) in
MDP-treated cells. Interestingly, the OÀ level
2
decreased after 2 h of stimulation, returning almost to
the resting level after 4 h. On the other hand, stimulation with MB failed to induce superoxide production
(Fig. 3), even after 6 h of treatment, whereas stimulation with LPS only induced significant enhancement of

superoxide production after a period of 6 h of stimulation (data not shown).
The effect of muranyl peptides on the total NO
(nitrite and nitrate) production of murine peritoneal
macrophages was determined by Griess assay. The
NO concentration of the culture supernatant was
significantly
increased
after
stimulation
with

104

*

*

80

4
60

Fold increase of superoxide

% peritoneal macrophages

8

5

C

40

20

0

–

n

– /An
PI

+

n

– /An
PI

+

n

– /An
PI

6

3
4

*

2

2
1

Fold increase of total NO

State 2

Time course effect of MDP on ROS and reactive
nitrogen species production by murine peritoneal
macrophages

+

n

+ /An
PI

Fig. 2. The percentage of viable, dead and apoptotic cells in treated and untreated cells is shown in (C). Data (A,B) represent one of
three separate experiments with similar results. The percentage of
decrease in cell viability (C) is the mean ± SEM of three independent experiments.

was noted between stimulated and MDP-treated
samples in the percentage of apoptotic or necrotic cells
(Fig. 2C).

0

0
0

1

2

Time (h)

3

4

5

Fig. 3. Effect of MDP and MB on OÀ and NOÀ =NOÀ production
2
2
3
by murine peritoneal macrophages. Macrophages (106 well)1) were
stimulated with 100 lg of MDP (closed symbols) or MB (open symbols) per millilitre for various time intervals, and OÀ and NOÀ =NOÀ
2
2
3

were measured as described in Experimental procedures. Results
for OÀ (circle) and total NO (square) production were expressed as
2
fold increase of unstimulated cells. Data are means ± SEM of five
independent experiments each performed in duplicate. *P < 0.05.

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T. G. El-Khoury et al.

Free radical generation contributes to UCP2
upregulation
To determine if MDP-induced UCP2 upregulation correlated with free radical generation, cells stimulated
with MDP were pretreated with an antioxidant (vitamin E). Figure 5A shows that both OÀ and total NO
2
significantly decreased in MDP-treated cells. Figure 5B
clearly demonstrates that vitamin E significantly
reduced the MDP-induced UCP2 upregulation,
thus showing that free radicals contribute to UCP2
upregulation.
Evidence for the involvement of UCP2 in the
mitochondrial impairment caused by MDP
The results obtained suggested a role of UCP2 in macrophage activation by MDP. The question raised at
this stage is whether UCP2 is responsible for the
increase in mitochondrial proton permeability (state 4)

induced in macrophages after stimulation with MDP.
Purine nucleotides (such as GDP) are recognized inhibitors of UCP1 [4]. Also for UCP2 a purine nucleotide
binding domain has been predicted from the translated
3058

US
M
D
M P
B
LS
P

GAPDH

3
2
1

MDP

MB

LPS

5

6h

US

2h

0

B

UCP2

*

US
1h

Stimulation of peritoneal macrophages by MDP
increased cellular ROS and reactive nitrogen species
(RNS) production. The increased production of reactive species was apparent after 2 h of stimulation.
Since UCP2 is described as a regulator of ROS production, the expression of UCP2 in macrophages stimulated or not with immunomodulators was then
investigated. Results shown in Fig. 4A clearly demonstrate that stimulation of macrophages with MDP
(100 lgỈmL)1) for 2 h results in significant increase in
UCP2 expression (3.6-fold, P < 0.05). On the other
hand, analysis of the kinetics of induction of UCP2
protein in MDP-treated macrophages revealed a significant increase starting 1 h after stimulation (2.2-fold,
P < 0.05), a peak level after 2 h (3.6-fold, P < 0.05)
and a return to baseline level after 6 h of treatment
(Fig. 4B).

5
4

Relative expression of
UCP2/GAPDH

Macrophage activation by MDP leads to
overexpression of UCP2

A

UCP2
Relative expression of
UCP2/GAPDH

100 lgỈmL)1 MDP for 2 h (Fig. 3) (unstimulated cells
2.48 nmol NO ⁄ 106 cells ± 0.29; MDP treated cells
16.99 nmol NO ⁄ 106 cells ± 0.31; P < 0.05). However,
stimulation with MB (100 lgỈmL)1) failed to generate
NO (Fig. 3), whereas stimulation with LPS only
induced a high and significant level of NO after 48 h
of treatment (data not shown).

* GAPDH

4
3

*
2
1
0

1h

2h

6h

Fig. 4. Immunodetection of UCP2 in murine peritoneal macrophages. Total cell lysates were prepared from unstimulated (US) and
MDP (100 lgỈmL)1), MB (100 lgỈmL)1) or LPS (1 lgỈmL)1) treated
macrophages, and 50 lg of total cell lysate proteins were loaded
onto an SDS ⁄ 12% PAGE gel (A). (B) Time course effect of MDP on
UCP2 expression in macrophages. Western blot analysis was performed as described under Experimental procedures. Inserts in (A)
and (B) show western immunoblot analysis. Data are relative to the
value for unstimulated cells (black bars, taken as 1). Each result
shown is the mean ± SEM of three independent experiments.
*P < 0.05. GAPDH, glyceraldehydes-3-phosphate dehydrogenase.

mRNA sequence [4], and any effect of GDP on respiration (proton permeability) has broadly been equated
with the involvement of the relevant UCP (here UCP2)
in the process. Therefore, the effect of GDP on mitochondrial respiration in macrophages was analysed.
Figure 6A shows that GDP added to mitochondria
extracted from the cells treated with MDP for 2 h
induced a significant decrease in state 4 (14.94%).
Consequently, the RCR value increased significantly
by 15.15% in GDP-treated mitochondria (Fig. 6B).
These results clearly suggest that the mitochondrial
inefficiency caused by MDP (100 lgỈmL)1) after 2 h of
incubation in peritoneal macrophages occurs partially
through UCP2.

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T. G. El-Khoury et al.

UCP2 modulates MDP-induced mitochondrial inefficiency

10
8

3

6

**

2

1

4

**

120

80

40

US

*

4

UCP2
GAPDH

3

MDP + Vit E

M
+ V DP
it E

Vit E

DP
Vi
tE

MDP

M

US

0

B 100

**

80

**
RCR (%)

Relative expression of
UCP2/GAPDH

**

0

0

B

2

*

160

State 4 (%)

*

4

*

A 200

Fold increase of total NO

Fold increase of superoxide

A 5

2

1

0

**

60

*

40

20

US

MDP

Vit E

MDP + Vit E

Fig. 5. Effect of vitamin E on UCP2 expression. Macrophages
(106 well)1) were pretreated with vitamin E (100 lM) for 10 min
and then stimulated with MDP (100 lg) for 2 h, and OÀ and
2
NOÀ =NOÀ were measured as described in Experimental proce2
3
dures. Results for OÀ (open bars) and total NO (black bars) produc2
tion were expressed as fold increase of unstimulated cells. Data
are means ± range of two independent experiments each performed in duplicate. (B) UCP2 western blot analysis. Conditions are
as described in the legend to Fig. 4. *P < 0.05 versus unstimulated; **P < 0.05 versus MDP stimulation.

0

US

DP

M

DP
M DP
+G

Fig. 6. Effect of GDP on respiration rates of mitochondria extracted

from murine peritoneal macrophages. Cells were treated for 2 h
with 100 lgỈmL)1 of MDP and oxygen consumption of extracted
mitochondria was analysed in the presence or absence of 1 mM of
GDP. Respiration states (A) and RCR (B) of treated cells are
presented as a percentage of unstimulated samples. Data are
means ± SEM of three independent experiments each performed
in duplicate. *P < 0.05 versus control. **P < 0.05 versus MDP
treated.

Discussion
The results obtained in this study demonstrate the ability of toxic MDP to potently induce impairment in
mitochondrial bioenergetics in murine peritoneal macrophages. The effect of MDP was observed in vitro at
a concentration of 100 lgỈmL)1 and after an incubation period of 1–2 h. In contrast, the nontoxic muramyl dipeptide derivative MB was not able to provoke
any defect in macrophage mitochondria since the RCR
and the respiration rate values obtained after 2 h of
treatment and at 100 lgỈmL)1 concentration were identical to those of the unstimulated cells. This view is

consistent with a previous report showing that MDP,
but not a safe analogue such as MB, is capable of
inducing mitochondrial proton leak in the spleen and
liver of injected mice. Moreover, it is of importance to
note that the maximum in vivo effect of MDP and
some of its derivatives on mitochondrial respiration
was observed 2 h after administration, a time peak
which has been reported for several of the toxicological effects of MDP in vivo [24]. The results obtained in
this study and in the previous report [23] shed light on
mitochondria as a new target affected by MDP and

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reveal a new approach by which muramyl peptides
could exert their toxic effect. Furthermore, LPS, which
constitutes a chemically different immunomodulator
from muramyl dipeptides but exerts a high toxic effect
in vivo, does not show any significant effect on mitochondrial respiration rates within the time period studied. It has been demonstrated previously that LPS
requires a period of 16 h to induce a significant impact
on rat mitochondrial respiration in vivo [25]. Therefore,
the mechanism of action of LPS is completely different
from MDP in inducing mitochondrial proton leak.
MDP decreases mitochondrial RCR by increasing state
4 respiration (non-phosphorylation state), without
affecting state 2 (succinate-link respiration) or state 3
(phosphorylation state). This increase in the basal proton leak activity of mitochondria (i.e. state 4) from
MDP-treated cells could be the result of activation or
an induction of expression of a mitochondrial membrane protein such as UCP adenine nucleotide translocase or others which can induce a proton leak and
thus increase the inefficiency of oxidative phosphorylation. In this regard, the effect on state 4 is similar to
an uncoupling effect.
UCP2 acts as a mild uncoupler, controlling both
ATP synthesis and the production of ROS (reviewed
in [9]). Several lines of evidence emphasize a role for
UCP2 in immunity. First, UCP2 is expressed in
immune cells such as phagocytes and lymphocytes [15].
Second, Ucp2) ⁄ ) mice are more resistant to a Toxoplasma gondii or Listeria monocytogenes infection than

Ucp2+ ⁄ + mice [14,15]. Third, the development of
unstable atherosclerotic plaques is greater in the
Ucp2) ⁄ ) mouse model of atherosclerosis [26]. Fourth,
transgenic mice overexpressing UCP2 show a reduced
inflammatory response following LPS treatment [27].
Moreover, macrophages from ob ⁄ ob mice were
reported to express lower UCP2 and higher ROS levels
than lean mice [28]. These findings agree with the
hypothesis [29] that an increase in the mitochondrial
membrane potential would slow the transport of electrons through the respiratory chain, increasing the time
of interaction between these electrons and molecular
oxygen and facilitating the formation of ROS.
Activation of innate immune cells by MDP is known
to be crucial for stimulating host antimicrobial defence
reactions [30]. ROS are rapidly produced from macrophages after stimulation with MDP and are involved
in cellular signalling. Also, nitric oxide (NO) production after stimulation plays a pivotal role in numerous
and diverse biological functions, in particular as a
principal mediator of the microbicidal and tumoricidal
actions of macrophages [31]. Furthermore, OÀ and
2
NO combine to form the potent oxidant peroxynitrite
3060

(ONOO)) which mediates bactericidal activity [32].
Thus, both ROS and NO are important mediators of
cellular immune response. It is well established that
mitochondria are the main source of ROS. Moreover,
mitochondrial ROS production is particularly sensitive
to membrane potential and to mild uncoupling [33].
However, the role of mild uncoupling in the regulation

of the response to MDP has not been elucidated.
Thus, we aimed in the present study (a) to demonstrate
the involvement of mitochondria in MDP-induced
ROS signalling and (b) to identify the mitochondrial
protein UCP2 as a physiological brake on this phenomenon. As anticipated, both ROS and RNS were
markedly higher in MDP-treated macrophages than in
unstimulated cells and the overexpression of UCP2
protein correlated with the production of both reactive
species. However, cells stimulated with MB did not
present any modification in the level of detectable
ROS or UCP2 expression. This finding indicated that
UCP2 is a constitutive modulator of reactive species
production, suggesting a role for UCP2 in the regulation of intracellular redox state and macrophage-mediated immunity.
As stated earlier, mitochondria are the major source
of ROS production and the primary ROS generated is
superoxide anion as a consequence of monoelectronic
reduction of O2. Moreover, the main sites of OÀ genera2
tion at the level of the mitochondrial electron transport
chain are complexes I and III [34]. The ROS generated
in mitochondria are removed by local superoxide dismutases and peroxidases and by reaction with low molecular weight reductants and sulfhydryl-containing protein
reductants. The mechanisms for removal of mitochondrial ROS are thus well described (reviewed in [9]).
Additionally, regulated expression of UCP2 would provide a mechanism for adjusting mitochondrial ROS production in cell types such as macrophages by lowering
membrane potential and thereby limit ROS production.
Taken together, our data support a model of UCP2
regulation consisting of a late phase response to MDP.
At this stage, 1 to 2 h after MDP stimulation, oxidative stress has been induced and there is a need to
counteract the toxic effects of inflammation and overstimulation of immune cells. Upregulation of UCP2
expression may be seen as a response to reduce the
production of ROS in immune cells in a negative feedback regulatory cycle. Finally, these data suggest the
interesting possibility that UCP2 may serve as an antioxidant, guarding against an excess of oxygen free radicals. Further studies on signal transduction cascades

that participate in the positive ⁄ negative regulation of
UCP2 expression would contribute to designing possible drugs that control bacterial infections.

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T. G. El-Khoury et al.

Experimental procedures
Animals
Experiments were done on Balb ⁄ C mice weighing 30–40 g.
Animals were housed under standard conditions (12 h
light ⁄ dark cycle, 22 ± 2 °C). All experiments were
approved by the Institutional Animal Care and Use Committee of the University of Balamand and complied with
the principles of laboratory animal care.

Chemicals and reagents
Muramyl peptides (MDP and MB) used in this work were
kindly provided by ISTAC-SA (Lille, France) and were
synthesized as described previously [35]. LPS, derived
from Escherichia coli (0127:B8), was purchased from Sigma
(Steinheim, Germany).

Macrophage harvesting and cultivation
Macrophages were obtained from mice peritoneum following
the method described in [36]. BALB ⁄ c mice were intraperitoneally injected with 3% thioglycollate (Difco, Lawrence, KS,
USA) broth. Four days later, the animals were killed by neck
dislocation, and the peritoneal exudates were collected and
centrifuged at 400 g. The cell sediment was resuspended in
Dulbecco’s modified Eagle’s medium (DMEM) phenol red

free, supplemented with 10% fetal bovine serum. Cells were
seeded in 75 cm2 flasks to a final concentration of 5 · 105
cellsỈcm)2. Non-adherent cells were washed with NaCl ⁄ Pi.

Analysis of murine peritoneal macrophages
After 2 h of adherence, cells were washed twice with cold
NaCl ⁄ Pi to remove non-adherent cells; then they were
detached by trypsinization, rewashed twice with cold
NaCl ⁄ Pi and finally resuspended at a final concentration of
106 cellsỈ100 lL)1 in cold NaCl ⁄ Pi. Cells were labelled with
PE-Cy7-conjugated rat anti-mouse CD11b monoclonal antibody or its isotype control PE-Cy7-conjugated rat IgG2b, j
monoclonal immunoglobulin for 30 min at room temperature (25 °C). Cells were washed once with NaCl ⁄ Pi, resuspended in 500 lL cell fix solution (containing formaldehyde
and 1% sodium azide) and subjected to flow cytometry analysis. Data from the experiments were analysed using cellquest software. The collected events per sample were 10 000.

Isolation of mitochondria
Mitochondria from murine peritoneal macrophages were prepared as described previously [12], with all steps carried out
at 4 °C. Cells were homogenized using a glass Dounce
homogenizer in isolation medium consisting of 250 mm

UCP2 modulates MDP-induced mitochondrial inefficiency

sucrose, 5 mm Tris ⁄ HCl (pH 7.4) and 2 mm EGTA. The
homogenate was centrifuged at 1047 g for 3 min. The supernatant was centrifuged at 11 360 g for 11 min. Mitochondrial
pellets were resuspended in the isolation medium and protein
concentration was determined by the Biuret method [37]. All
results are expressed per milligram mitochondrial protein.

Measurement of oxygen consumption
Measurements of oxygen consumption were performed
using an oxygen electrode (Clark electrode; Rank Brothers

Ltd, Cambridge, UK). Oxygen consumption rates were calculated assuming that the concentration of oxygen in the
air-saturated incubation medium was 406 nmolỈmL)1 [12].
Mitochondria (3 mgỈmL)1) isolated from culture cells were
incubated in standard assay medium (500 lL) containing
120 mm KCl, 5 mm KH2PO4, 3 mm HEPES, 1 mm EGTA
supplemented with 0.3% defatted BSA and 2 lm rotenone
(pH 7.2, 37 °C). Respiration was initiated with 2 mm succinate as substrate. State 3 respiration was measured in the
presence of 200 lm ADP and state 4 respiration by adding
1 lgỈmL)1 oligomycin. Electrode linearity was checked by
following the uncoupled respiration rate in the presence of
2 mm FCCP from 100% to 0% air saturation. RCRs were
calculated as state 3 divided by state 4 respiration rates.

Assay for superoxide anion generation
Superoxide anion release was determined by superoxide
dismutase inhibitable reduction of ferricytochrome c.
Briefly, macrophages (1 · 106 well)1) were covered with
450 lL of Kreeb’s ringer phosphate buffer (123 mmolỈL)1
NaCl, 1.23 mmolỈL)1 MgCl2, 4.9 mmolỈL)1 KCl and
16.7 mmolỈL)1 Na phosphate buffer, pH 7.4), containing
5 mmolỈL)1 glucose, 0.5 mmolỈL)1 CaCl2 and 2 mmolỈL)1
NaN3 and supplemented with 80 lmolỈL)1 cytochrome c
(Sigma). After 10 min incubation at 37 °C (5% CO2), cells
were treated with MDP (100 lgỈmL)1), MB (100 lgỈmL)1)
or LPS (1 lgỈmL)1). A 350 lL aliquot from each well was
aspirated at different time intervals and diluted 1 : 3 with
cold buffer. The reduced cytochrome c was measured by
analysing the difference in absorbency at 550–468 nm using
a micromolar extinction coefficient of 0.0245 [38]. All
assays were performed in duplicate. Controls containing

30 lgỈmL)1 superoxide dismutase (Sigma) were also made
in order to provide correction for the OÀ independent
2
reduction of cytochrome c. The results were expressed as
nanomoles of superoxide anion per million cells.
)
)
Measurement of NO2 and NO3 as readout for NO
production

NO production was evaluated by spectrophotometric determination of its stable decomposition products nitrate and
nitrite using Griess’s reaction [39]. Nitrate was detected

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UCP2 modulates MDP-induced mitochondrial inefficiency

T. G. El-Khoury et al.

after reduction to nitrite using a commercially available
preparation of nitrate reductase from Aspergillus (Sigma).
Macrophages were seeded in 24-well plates to a final concentration of 1 · 106 cellsỈmL)1 in DMEM phenol red free.
The supernatants were collected after the appropriate
incubation period with MDP (100 lgỈmL)1) or MB
(100 lgỈmL)1) or LPS (1 lgỈmL)1) and stored at )20 °C
until analysis. A mixture at 1 : 1 of 0.1% naphthylenediamine dihydrochloride and 1% sulfanilamide in 5% H3PO4
was added and incubated at room temperature for 10 min.

The absorbance was measured at 540 nm in a microplate
automated multiscan reader (Thermo, Runcorn, UK). The
results were expressed as nanomoles of NO per million cells.

Western blot analysis
About 50 lg of total cell lysate proteins were resolved by
SDS ⁄ PAGE and then transferred to poly(vinylidene difluoride) membranes (GE Healthcare, Chalfont St Giles, UK)
that were probed with either an anti-UCP2 antibody or a
mouse
anti-glyceraldehyde-3-phosphate
dehydrogenase
antibody used as a loading control. The immunoblots were
developed by enhanced chemiluminescence (GE Healthcare),
and the band intensity was recorded using high performance
chemiluminescence films (GE Healthcare) at room temperature. The films were scanned using the Gel Documentation
System (Biorad) and quantification of the proteins was
achieved using quantity one software (Biorad, Marnesla-Coquette, France).

Viability test
AnnexinV-FITC Apoptosis Detection Kit II was used to
determine the percentage of viable, apoptotic and dead cells
after treatment, or not, with MDP (100 lgỈmL)1) for 2 h.
Cells were washed twice with cold NaCl ⁄ Pi, detached by
trypsinization (1· trypsin), rewashed twice with cold NaCl ⁄ Pi
and finally resuspended at a final concentration of 106
cellsỈmL)1 in 1· binding buffer (10· binding buffer contains
0.1 m HEPES ⁄ NaOH (pH 7.4), 1.4 m NaCl, 25 mm CaCl2).
The solution (100 lL, 1 · 105 cells) was transferred to a 5 mL
culture tube containing 5 lL of FITC Annexin V and 5 lL
propidium iodide. The cells were gently mixed and incubated

for 15 min at room temperature (25 °C) in the dark. Finally,
400 lL of 1· binding buffer was added to each tube. The suspension was analysed by flow cytometry within 1 h using a
FACSCalibur (Becton Dickinson, Erembodegem, Belgium)
equipped with a 488-nm argon laser and a 635-nm red diode
laser. Data from the experiments were analysed using cellquest software. The collected events per sample were 10 000.

Statistical analysis
All results are shown as the mean of data from at least
three independent experiments. The statistical significance

3062

of the differences was calculated using Student’s t-test and
values of P < 0.05 were accepted as statistically significant.
Data were analysed using the spss 11.0 software.

Acknowledgements
We would like to thank Samer Bazzi and Michel Zakhem for technical assistance. This work is supported
by grants from the University of Balamand Research
Council.

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