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 spe

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: karim.echtay@balamand.edu.lb

(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 expres-sion of the mitochondrial protein uncoupling protein 2 (UCP2), and pre-treatment 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

mitochon-drial membrane Mammals express five UCP

homo-logues, UCP1–UCP5 UCP2 and UCP3 have 59% and

57% identity, respectively, with UCP1, and 73%

iden-tity 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

acti-vated by long-chain fatty acids and inhibited by purine

nucleotides [4] UCP2 is widely expressed in many

tis-sues with high levels detected in the spleen, thymus,

pancreatic b-cells, heart, lung, white and brown adi-pose 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 regu-late 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.

thermogenesis, protection against obesity, regulation of

the ATP⁄ ADP ratio, export of fatty acids, and

media-tion of insulin secremedia-tion (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

oxi-dative damage [10,11] This is mainly based on the

activation of mitochondrial proton conductance

medi-ated through UCPs by reactive oxygen species (ROS)

or by-products of lipid peroxidation [12,13], resulting

in a negative feedback loop that decreases ROS

pro-duction 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

perito-neal macrophages of UCP2) ⁄ ) mice are resistant to

infection by the intracellular parasite Toxoplasma

gon-dii through a mechanism proposed to involve higher

production of intracellular ROS [14] On the other

hand, studies in cells overexpressing UCP2 have

rein-forced 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]

More-over, cardiomyocytes transfected with a

UCP2-express-ing adenovirus were able to regulate ROS production

and protect against doxorubicin-mediated

cardiotoxic-ity [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

immunomodula-tors with diverse biological effects Their

immunologi-cal 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

adminis-tration into different hosts was associated with serious

toxicity Therefore, attempts have been made to

gener-ate 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

central nervous system, and the lack of induction of inflammatory responses [22]

Despite a long-standing interest in the field of mura-myl peptides, the impact of these molecules at the mitochondrial level has not yet been examined Recently the effect of these derivatives on mitochon-drial bioenergetics has been studied [23] MDP induced

in vivo a significant decrease in respiratory control ratio (RCR) in isolated mouse liver and spleen mito-chondria versus non-toxic analogues such as MB The decrease in RCR in mitochondria of MDP-treated mice is attributed to an increase in mitochondrial pro-ton 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 nitro-gen 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 func-tion in mitochondria isolated from tissues or cultured cells and to thoroughly localize the sites of impairment

of oxidative phosphorylation In this study, the activi-ties of the respiratory chain complexes are examined as the oxygen consumption rates after addition of various substrates and inhibitors The mitochondrial respira-tory function is conventionally separated into different states State 2 is the oxygen consumption rate of sub-strate (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 produc-tion State 4, the non-phosphorylation state, is a mea-sure of oxygen consumption in the presence of oligomycin (ATP synthase inhibitor) This state repre-sents the mitochondrial basal proton leak activity State 3⁄ state 4, termed the RCR, is used as an indica-tor to evaluate mitochondrial efficiency since it reflects the coupling between oxidative phosphorylation and the mitochondrial electron transport chain activity

Figure 1A shows the time-dependent inhibition of

succinate-linked RCR in mitochondria extracted from

MDP-treated (100 lgÆmL)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

MDP-treated 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

macro-phage-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

LPS-treated 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

mito-chondrial respirations of peritoneal macrophages after

2 h of treatment

Effect of MDP on cell viability

The viability of peritoneal macrophages under

condi-tions 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

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

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 lgÆmL)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 lgÆmL)1) Data are

normal-ized to state 3 rates of unstimulated mitochondria (black bars).

(C) RCRs of mitochondria isolated from cells treated for 2 h with MDP

(100 lgÆmL)1), murabutide (MB, 100 lgÆmL)1) or LPS (1 lgÆmL)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.

*

0 0.2 0.4 0.6 0.8 1 1.2

Contr

ol

MDP

C

A

*

*

0 20 40 60

10 30 50

Time (h) B

*

State 2 State 3 State 4 FCCP 0

0.4 0.8 1.2 1.6

–1 ·mg

–1 )

was noted between stimulated and MDP-treated

samples in the percentage of apoptotic or necrotic cells

(Fig 2C)

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

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 lgÆmL)1) on total cellular superoxide anion production by murine perito-neal macrophages As shown in Fig 3, total superox-ide production was unchanged after 30 min but was significantly elevated at 60 and 120 min (P < 0.05) in MDP-treated cells Interestingly, the O2 level decreased after 2 h of stimulation, returning almost to the resting level after 4 h On the other hand, stimula-tion with MB failed to induce superoxide producstimula-tion (Fig 3), even after 6 h of treatment, whereas stimula-tion with LPS only induced significant enhancement of superoxide production after a period of 6 h of stimula-tion (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

Table 1 Effects of MB and LPS on respiration rates in murine

peri-toneal macrophage mitochondria in vitro Mitochondria were

iso-lated from murine peritoneal macrophages after 2 h of treatment

with MB (100 lgÆmL)1) or LPS (1 lgÆmL)1) Data are presented as

the percentage of unstimulated cells Data are means ± standard

error of the mean of three independent experiments each

per-formed in triplicate.

Percentage unstimulated cells

State 2 State 3 State 4 FCCP rate

MB (100 lgÆmL)1) 100 ± 0 112 ± 14.2 107 ± 10.8 95.68 ± 4.6

LPS (1 lgÆmL)1) 100 ± 1.5 102 ± 12.3 98 ± 7.6 98.27 ± 8.2

0

20

40

60

80

PI – /Ann

–

PI – /Ann

+

PI – /Ann

+

PI + /Ann +

C

10 0 10 1 10 2 10 3 10 4

Annex-FITC

A

10 0 10 1 10 2 10 3 10 4

Annex-FITC

B

Fig 2 The percentage of viable, dead and apoptotic cells in

trea-ted and untreatrea-ted 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

indepen-dent experiments.

0 1

6

1

2

2

2

3

3

4

4

4

5

Time (h)

*

*

*

Fig 3 Effect of MDP and MB on O 

2 and NO2=NO3 production

by murine peritoneal macrophages Macrophages (10 6 well)1) were stimulated with 100 lg of MDP (closed symbols) or MB (open sym-bols) per millilitre for various time intervals, and O2 and NO2=NO3 were measured as described in Experimental procedures Results for O2 (circle) and total NO (square) production were expressed as fold increase of unstimulated cells Data are means ± SEM of five independent experiments each performed in duplicate *P < 0.05.

100 lgÆmL)1 MDP for 2 h (Fig 3) (unstimulated cells

2.48 nmol NO⁄ 106cells ± 0.29; MDP treated cells

16.99 nmol NO⁄ 106cells ± 0.31; P < 0.05) However,

stimulation with MB (100 lgÆmL)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)

Macrophage activation by MDP leads to

overexpression of UCP2

Stimulation of peritoneal macrophages by MDP

increased cellular ROS and reactive nitrogen species

(RNS) production The increased production of

reac-tive species was apparent after 2 h of stimulation

Since UCP2 is described as a regulator of ROS

pro-duction, the expression of UCP2 in macrophages

stim-ulated or not with immunomodulators was then

investigated Results shown in Fig 4A clearly

demon-strate that stimulation of macrophages with MDP

(100 lgÆmL)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

signif-icant 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)

Free radical generation contributes to UCP2

upregulation

To determine if MDP-induced UCP2 upregulation

cor-related with free radical generation, cells stimulated

with MDP were pretreated with an antioxidant

(vita-min E) Figure 5A shows that both O2 and total NO

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

mac-rophage 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

inhib-itors of UCP1 [4] Also for UCP2 a purine nucleotide

binding domain has been predicted from the translated

mRNA sequence [4], and any effect of GDP on respi-ration (proton permeability) has broadly been equated with the involvement of the relevant UCP (here UCP2)

in the process Therefore, the effect of GDP on mito-chondrial 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 lgÆmL)1) after 2 h of incubation in peritoneal macrophages occurs partially through UCP2

1

3

2 4

0

5

* A

US MDP MB LPS

*

*

1 h 1

3

2 h 2

4

6 h 0

5

B

US MDP MB LSP

UCP2 GAPDH

UCP2 GAPDH

US 1 h 2 h 6 h

Fig 4 Immunodetection of UCP2 in murine peritoneal

macrophag-es Total cell lysates were prepared from unstimulated (US) and MDP (100 lgÆmL)1), MB (100 lgÆmL)1) or LPS (1 lgÆmL)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 per-formed 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.

The results obtained in this study demonstrate the

abil-ity of toxic MDP to potently induce impairment in

mitochondrial bioenergetics in murine peritoneal

mac-rophages The effect of MDP was observed in vitro at

a concentration of 100 lgÆmL)1 and after an

incuba-tion period of 1–2 h In contrast, the nontoxic

mura-myl 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 lgÆmL)1concentration were

iden-tical 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 toxicologi-cal 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

1

3

2

2

4

4 6 8

10 5

**

**

*

*

US MDP MDP + Vit E

Vit E

A

UCP2 GAPDH

1

3

2

4

0

*

US MDP Vit E MDP + Vit E

*

US MDP V it E MDP + V

it E B

**

Fig 5 Effect of vitamin E on UCP2 expression Macrophages

(10 6 well)1) were pretreated with vitamin E (100 l M ) for 10 min

and then stimulated with MDP (100 lg) for 2 h, and O2 and

NO2=NO3 were measured as described in Experimental

proce-dures Results for O 

2 (open bars) and total NO (black bars) produc-tion were expressed as fold increase of unstimulated cells Data

are means ± range of two independent experiments each

per-formed in duplicate (B) UCP2 western blot analysis Conditions are

as described in the legend to Fig 4 *P < 0.05 versus

unstimu-lated; **P < 0.05 versus MDP stimulation.

20

0

40 60 80 100

*

**

US

+ GDP

B

A

40

0

80 120 160 200

*

**

Fig 6 Effect of GDP on respiration rates of mitochondria extracted from murine peritoneal macrophages Cells were treated for 2 h with 100 lgÆmL)1 of MDP and oxygen consumption of extracted mitochondria was analysed in the presence or absence of 1 m M 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.

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

mito-chondrial respiration rates within the time period

stud-ied 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

pro-ton 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

mem-brane protein such as UCP adenine nucleotide

translo-case or others which can induce a proton leak and

thus increase the inefficiency of oxidative

phosphoryla-tion 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

Toxo-plasma gondii or Listeria monocytogenes infection than

Ucp2+⁄ + mice [14,15] Third, the development of

unstable atherosclerotic plaques is greater in the

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

elec-trons 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

macro-phages after stimulation with MDP and are involved

in cellular signalling Also, nitric oxide (NO)

produc-tion after stimulaproduc-tion 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, O2 and

NO combine to form the potent oxidant peroxynitrite

(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 phe-nomenon 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 regula-tion of intracellular redox state and macrophage-medi-ated 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 O2 genera-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 dismu-tases and peroxidases and by reaction with low molecu-lar weight reductants and sulfhydryl-containing protein reductants The mechanisms for removal of mitochon-drial ROS are thus well described (reviewed in [9]) Additionally, regulated expression of UCP2 would vide a mechanism for adjusting mitochondrial ROS pro-duction 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, oxida-tive stress has been induced and there is a need to counteract the toxic effects of inflammation and over-stimulation 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 feed-back regulatory cycle Finally, these data suggest the interesting possibility that UCP2 may serve as an anti-oxidant, guarding against an excess of oxygen free rad-icals Further studies on signal transduction cascades that participate in the positive⁄ negative regulation of UCP2 expression would contribute to designing possi-ble drugs that control bacterial infections

Experimental procedures

Animals

Animals were housed under standard conditions (12 h

approved by the Institutional Animal Care and Use

Com-mittee 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

intraperito-neally 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

Analysis of murine peritoneal macrophages

After 2 h of adherence, cells were washed twice with cold

detached by trypsinization, rewashed twice with cold

PE-Cy7-conjugated rat mouse CD11b monoclonal

monoclonal immunoglobulin for 30 min at room

resus-pended in 500 lL cell fix solution (containing formaldehyde

and 1% sodium azide) and subjected to flow cytometry

anal-ysis Data from the experiments were analysed using

Isolation of mitochondria

Mitochondria from murine peritoneal macrophages were

pre-pared as described previously [12], with all steps carried out

homogenizer in isolation medium consisting of 250 mm

homogenate was centrifuged at 1047 g for 3 min The super-natant 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 cal-culated assuming that the concentration of oxygen in the

incubated in standard assay medium (500 lL) containing

supplemented with 0.3% defatted BSA and 2 lm rotenone

succi-nate as substrate State 3 respiration was measured in the presence of 200 lm ADP and state 4 respiration by adding

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

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

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 deter-mination of its stable decomposition products nitrate and nitrite using Griess’s reaction [39] Nitrate was detected

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

The supernatants were collected after the appropriate

until analysis A mixture at 1 : 1 of 0.1%

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

difluo-ride) membranes (GE Healthcare, Chalfont St Giles, UK)

that were probed with either an anti-UCP2 antibody or a

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

tempera-ture The films were scanned using the Gel Documentation

System (Biorad) and quantification of the proteins was

achieved using quantity one software (Biorad,

Marnes-la-Coquette, France)

Viability test

AnnexinV-FITC Apoptosis Detection Kit II was used to

determine the percentage of viable, apoptotic and dead cells

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

400 lL of 1· binding buffer was added to each tube The

sus-pension 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

Statistical analysis

All results are shown as the mean of data from at least

three independent experiments The statistical significance

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 Zak-hem for technical assistance This work is supported

by grants from the University of Balamand Research Council

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