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

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.

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

thermogenesis, protection against obesity, regulation of the ATP ⁄ ADP ratio, export of fatty acids, and media- tion of insulin secretion (reviewed in [9]).

role

the uncoupling

correlating

Despite a long-standing interest in the ﬁeld of mura- myl peptides, the impact of these molecules at the level has not yet been examined. mitochondrial 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 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

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 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.

cytokine

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.

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, release and restoration manipulation of 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

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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 changes were state 4 respiration. No signiﬁcant 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

0 State 2

State 4

State 3

FCCP

1.2 C

viable

1

0.8

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

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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.

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

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

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

100 ± 1.5 102 ± 12.3

112 ± 14.2 107 ± 10.8 95.68 ± 4.6 98.27 ± 8.2 98 ± 7.6

MB (100 lgÆmL)1) 100 ± 0 LPS (1 lgÆmL)1)

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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 level MDP-treated 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 production (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).

2 0 1

1 0 1

The effect of muranyl peptides on the total NO (nitrite and nitrate) production of murine peritoneal macrophages was determined by Griess assay. The the culture supernatant was NO concentration of with signiﬁcantly

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Fig. 2. The percentage of viable, dead and apoptotic cells in trea- ted 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 indepen- dent experiments.

2 and NO(cid:2)

Fig. 3. Effect of MDP and MB on O(cid:3)(cid:2) 3 production 2 by murine peritoneal macrophages. Macrophages (106 well)1) were stimulated with 100 lg of MDP (closed symbols) or MB (open sym- bols) per millilitre for various time intervals, and O(cid:3)(cid:2) 2 =NO(cid:2) 3 were measured as described in Experimental procedures. Results for O(cid:3)(cid:2) (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.

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

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5 A

DP

MB

LSP

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100 lgÆmL)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 lgÆmL)1) failed to generate stimulation with LPS only NO (Fig. 3), whereas induced a high and signiﬁcant level of NO after 48 h of treatment (data not shown).

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Macrophage activation by MDP leads to overexpression of UCP2

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2 h

6 h

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

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 MDP-induced UCP2

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 O(cid:2) 2 and total NO signiﬁcantly decreased in MDP-treated cells. Figure 5B that vitamin E signiﬁcantly clearly demonstrates reduced upregulation, thus showing that free radicals contribute to UCP2 upregulation.

Evidence for the involvement of UCP2 in the mitochondrial impairment caused by MDP

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.

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

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10

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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 O(cid:3)(cid:2) and 2 NO(cid:2) 3 were measured as described in Experimental proce- dures. Results for O(cid:3)(cid:2) (open bars) and total NO (black bars) produc- 2 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.

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 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 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)1 concentration 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

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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 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.

(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.

is greater

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 in the Ucp2) ⁄ ) mouse model of atherosclerosis [26]. Fourth, transgenic mice overexpressing UCP2 show a reduced inﬂammatory response following LPS treatment [27]. from ob ⁄ ob mice were Moreover, macrophages 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 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, O(cid:3)(cid:2) and 2 NO combine to form the potent oxidant peroxynitrite

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 O(cid:2) 2 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 pro- 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.

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Experimental procedures

Animals

sucrose, 5 mm Tris ⁄ HCl (pH 7.4) and 2 mm EGTA. The 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

cycle,

Chemicals and reagents

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

Macrophage harvesting and cultivation

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).

Assay for superoxide anion generation

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 air-saturated incubation medium was 406 nmolÆmL)1 [12]. Mitochondria (3 mgÆmL)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 (cid:2)C). Respiration was initiated with 2 mm succi- nate as substrate. State 3 respiration was measured in the presence of 200 lm ADP and state 4 respiration by adding 1 lgÆmL)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.

inhibitable ferricytochrome

Analysis of murine peritoneal macrophages

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 seeded in 75 cm2 ﬂasks to a ﬁnal concentration of 5 · 105 cellsÆcm)2. Non-adherent cells were washed with NaCl ⁄ Pi.

to remove non-adherent cells;

) as readout for NO

) and NO3

Isolation of mitochondria

Measurement of NO2 production

Superoxide anion release was determined by superoxide c. dismutase reduction of Brieﬂy, macrophages (1 · 106 well)1) were covered with 450 lL of Kreeb’s ringer phosphate buffer (123 mmolÆL)1 NaCl, 1.23 mmolÆL)1 MgCl2, 4.9 mmolÆL)1 KCl and 16.7 mmolÆL)1 Na phosphate buffer, pH 7.4), containing 5 mmolÆL)1 glucose, 0.5 mmolÆL)1 CaCl2 and 2 mmolÆL)1 NaN3 and supplemented with 80 lmolÆL)1 cytochrome c (Sigma). After 10 min incubation at 37 (cid:2)C (5% CO2), cells were treated with MDP (100 lgÆmL)1), MB (100 lgÆmL)1) or LPS (1 lgÆmL)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 lgÆmL)1 superoxide dismutase (Sigma) were also made in order to provide correction for the O(cid:2) independent 2 reduction of cytochrome c. The results were expressed as nanomoles of superoxide anion per million cells. After 2 h of adherence, cells were washed twice with cold then they were NaCl ⁄ Pi detached by trypsinization, rewashed twice with cold NaCl ⁄ Pi and ﬁnally resuspended at a ﬁnal concentration of 106 cellsÆ100 lL)1 in cold NaCl ⁄ Pi. Cells were labelled with PE-Cy7-conjugated rat anti-mouse CD11b monoclonal anti- body or its isotype control PE-Cy7-conjugated rat IgG2b, j monoclonal immunoglobulin for 30 min at room tempera- ture (25 (cid:2)C). Cells were washed once with NaCl ⁄ Pi, 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 cell- quest software. The collected events per sample were 10 000.

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NO production was evaluated by spectrophotometric deter- mination of its stable decomposition products nitrate and nitrite using Griess’s reaction [39]. Nitrate was detected Mitochondria from murine peritoneal macrophages were pre- pared as described previously [12], with all steps carried out at 4 (cid:2)C. Cells were homogenized using a glass Dounce homogenizer in isolation medium consisting of 250 mm

T. G. El-Khoury et al.

UCP2 modulates MDP-induced mitochondrial inefﬁciency

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.

References

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.

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 con- centration of 1 · 106 cellsÆmL)1 in DMEM phenol red free. the appropriate The supernatants were collected after incubation period with MDP (100 lgÆmL)1) or MB (100 lgÆmL)1) or LPS (1 lgÆmL)1) and stored at )20 (cid:2)C until analysis. A mixture at 1 : 1 of 0.1% naphthylenedi- amine 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.

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