Free Radical Biology & Medicine 42 (2007) 1625–1631www.elsevier.com/locate/freeradbiomed

Serial Review: Reactive Oxygen Species and Immune ResponsesSerial Review Editor: Matthew Grisham

Nitric oxide, mitochondrial hyperpolarization, and T cell activation☆

Gyorgy Nagy a,b, Agnes Koncz a, David Fernandez a, Andras Perl a,⁎

a Section of Rheumatology, Department of Medicine, Department of Microbiology, and Department of Immunology, State University of New York,Upstate Medical University, College of Medicine, Syracuse, NY 13210, USA

b Department of Rheumatology, Faculty of Medicine, Semmelweis University, Budapest, Hungary

Received 8 January 2007; revised 15 January 2007; accepted 8 February 2007Available online 3 March 2007

Abstract

T lymphocyte activation is associated with nitric oxide (NO) production, which plays an essential role in multiple T cell functions. NO acts as amessenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulatesmitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many celltypes, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both activation and apoptosis ofT lymphocytes, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cellsignaling pathways. Overproduction of NO in systemic lupus erythematosus induces mitochondrial biogenesis and alters Ca2+ signaling. Thus,whereas NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to thepathogenesis of autoimmunity.© 2007 Elsevier Inc. All rights reserved.

Keywords: Nitric oxide; Apoptosis; Mitochondrial hyperpolarization; Mitochondrial biogenesis; Calcium; Free radicals

Contents

TCR activation, Ca2+ signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627Mitochondrial Ca2+ and mitochondrial transmembrane potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627Role of nitric oxide in T cell activation, mitochondrial biogenesis, and hyperpolarization . . . . . . . . . . . . . . . . . . . . . . . . 1627Mitochondrial oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1628Mitochondrial hyperpolarization and increased NO production in systemic lupus erythematosus . . . . . . . . . . . . . . . . . . . . 1629Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629

Abbreviations: ETC, electron transport chain; ITAM, IgR family tyrosine-based activation motif; LAT, linker for activation; IP3, inositol-1,4,5-triphosphate; ROI,reactive oxygen intermediate; SOD, superoxide dismutase;Δψm, mitochondrial transmembrane potential; MHP, mitochondrial hyperpolarization; NOS, NO synthase;nNOS, neuronal NOS; eNOS, endothelial; iNOS, inducible NOS; RAPA, rapamycin; SLE, systemic lupus erythematosus; TCR, T cell antigen receptor; ZAP-70, ζ-associated protein-70.☆ This article is part of a series of reviews on “Reactive oxygen species and immune responses.” The full list of papers may be found on the home page of the journal.⁎ Corresponding author.E-mail address: perla@upstate.edu (A. Perl).

0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2007.02.026

1626 G. Nagy et al. / Free Radical Biology & Medicine 42 (2007) 1625–1631

The mitochondrion, the site of oxidative phosphorylation,has long been identified as a source of energy and cell survival[1,2]. The electrochemical gradient across the inner mitochon-drial membrane is maintained by the electron transport chain(ETC) and the mitochondrial membrane potential (Δψm) (neg-ative inside and positive outside) [3]. The Δψm is subject toregulation by an oxidation–reduction equilibrium of ROI,pyridine nucleotides (NADH/NAD + NADPH/NADP), andGSH levels [4]. Controlled levels of ROI modulate variousaspects of cellular function and are necessary for signal trans-duction pathways, including those mediating T cell activationand apoptosis [5] (Fig. 1). Elevation of Δψm or mitochondrialhyperpolarization (MHP) was discovered in our laboratory [6].MHP in T cells is facilitated by depletion of GSH and NADPHtriggered by overexpression of the PPP enzyme transaldolase[6,7]. Transient MHP is an early event preceding caspase acti-vation, phosphatidylserine externalization, and disruption ofΔψm in Fas- [6] and H2O2-induced apoptosis of Jurkat humanleukemia T cells and normal human peripheral blood lympho-cytes (PBL) [8]. These observations were widely confirmed andextended to other apoptosis pathways [9–14]. Transient MHP isalso triggered by activation of T cells by Con A [6] and CD3/CD28 costimulation [15] via ROI- and Ca2+-dependent pro-duction of nitric oxide (NO) [16]. Thus, MHP represents an

Fig. 1. Schematic diagram of T cell activation, NO production, and mitochondrial hyand at the immunological synapse of T cells. Localized NO production has been linksynapse. NO decomposes superoxide via ONOO− formation, thereby limiting the sinteraction of superoxide with NO will result in the elimination of superoxide-derivedby providing a NO-dependent mechanism to reduce both superoxide and H2O2. NO csuch as monocytes and dendritic cells, can affect T cell activation. NO regulatesmitochondrial hyperpolarization and mitochondrial biogenesis. Mitochondrial hypenecrosis. In turn, necrotic materials released from Tcells activate monocytes and dend

early and reversible switch not exclusively associated withapoptosis. In contrast, as recently revealed in our laboratory, Tcells of SLE patients show persistent MHP and ATP depletion,which cause abnormal T cell death, shifting susceptibility fromapoptosis to necrosis [15]. ATP depletion in lupus T cells wasrecently confirmed by Krishnan et al. [17]. Increased necrosisrates could play a central role in enhanced inflammatoryresponses in SLE [18]. Necrotic cell death stimulates macro-phages [19] and dendritic cells [20] and leads to inflammation[15,18,21] and development of murine SLE [22]. We proposedthat increased release of necrotic materials from T cells wouldactivate macrophages and dendritic cells (DCs) and enhancetheir capacity to produce NO and IFN-α in SLE [18]. Indeed,DCs exposed to necrotic, but not apoptotic, cells induce lupus-like disease in MRL mice and accelerate the disease of MRL/lprmice [22]. These data suggest that principal mitochondrialfunctions are involved in T cell activation. NO is produced by Tcells during activation and it regulates T cell signal transduction[1,16,18]. NO affects both crucial functions of the mitochon-drion, the generation of energy and the control of cell death,through modulating production of reactive oxygen intermedi-ates and of ATP. This review will focus on the central role ofNO in mitochondrial control of activation and cell death path-way selection in human T lymphocytes.

perpolarization. NO is produced in the cytosol, in the mitochondrial membrane,ed to targeting of eNOS to the outer mitochondrial membrane and to the T cellteady-state concentrations of this oxygen-derived free radical. Furthermore, theH2O2. Indeed, these types of interactions would likely extend the life of the Tcellan freely diffuse through biological membranes and NO produced by other cells,many steps of T cell activation; production of cytokines, such as IL2; and

rpolarization is associated with depletion of ATP, which predisposes T cells toritic cells. Altered Tcell cytokine production also influences activation of B cells.

1627G. Nagy et al. / Free Radical Biology & Medicine 42 (2007) 1625–1631

TCR activation, Ca2+ signaling

Engagement of the T cell antigen receptor (TCR) leads toactivation of multiple protein tyrosine and serine kinases, result-ing in phosphorylation of intracellular substrates. The subsequenthydrolysis of phospholipids and elevation of cytoplasmic Ca2+

ultimately lead to clonal expansion of antigen-specific T cells[23]. An increase in cytoplasmic Ca2+ is essential for and is anearly marker of T cell activation. The TCR is associated with amultimeric receptor module, comprising the CD3 γδϵ and TCRζ chains. The cytoplasmic domains of CD3 and ζ chains containa common motif termed the IgR family tyrosine-based activa-tion motif (ITAM) [24,25]. Each ζ homodimer possesses threeITAMs, whereas other CD3 chains each contain one ITAM.

The most proximal biochemical event following T cell acti-vation is the activation of protein tyrosine kinases. The inter-action of the T-cell-specific CD4 or CD8 with P56lck, a memberof the Src family of PTKs, leads to an increase in P56lck kinaseautophosphorylation, leading to phosphorylation of ITAM.Overexpression of an activated form of P56lck enhances TCR-mediated activation. A signaling mutant of the Jurkat T cell line,JaCam1, that fails to demonstrate an elevation of the intracellularCa2+ upon TCR signaling has been shown to lack P56lck protein[26]. Phosphorylation of ITAMs is required for Src homology 2domain-mediated binding by ζ-associated protein (ZAP-70).ZAP-70 is activated through phosphorylation by P56lck. Acti-vated ZAP-70 phosphorylates LAT adaptor protein, followedby the direct binding of LAT to phospholipase C-γ1.

Activation of the TCR by monoclonal antibodies, mitogeniclectins, or a specific antigen on the surface of appropriateantigen-presenting cells elicits substantial, sustained increasesin IP3 and in the concentration of cytoplasmic free Ca2+ withinT lymphocytes. Phospholipase C-γ1 mediates IP3-dependentCa2+ signaling in T cells, similar to B cells [27,28]. Ca2+ servesas an intracellular messenger regulating different signalingpathways that lead to activation of protein kinase C (PKC),calmodulin, and calmodulin-dependent protein kinases, as wellas calcineurin and translocation of the nuclear factor of acti-vated T cells into the nucleus [29].

Activation of T cells through the TCR initiates a rapidincrease in cytoplasmic and mitochondrial Ca2+ levels (within5–10 min), followed by a plateau phase, lasting at least 48 h[27]. Inhibition of the endoplasmic reticulum Ca2+ ATPase bythapsigargin results in emptying of the intracellular Ca2+ storesand capacitative Ca2+ influx from extracellular space.

Mitochondrial Ca2+ and mitochondrial transmembranepotential

Isolated mitochondria increase their rate of ATP productionseveralfold in response to increasing concentrations of ADP andPi with unlimited supply of substrate and oxygen [30]. Inaddition to ADP, mitochondrial Ca2+ is another important regu-lator of mitochondrial ATP production. Exposure of isolatedmitochondria to artificially created Ca2+ pulses, like those seenin the cytosol of the cells, led to the discovery of mitochondrialCa2+ uptake. Mitochondria take up Ca2+ primarily through a

uniporter, whose molecular nature is still controversial. Thismight act like a channel, opening with increased probabilityonce the local Ca2+ rises [31]. The major targets of the mito-chondrial Ca2+ import pathway are the dehydrogenases of thecitric acid cycle, as the rate-limiting enzymes are all upregulatedby Ca2+-dependent processes. In addition intramitochondrialCa2+ activates the ETC, adenine nucleotide translocator, andF1F0-ATPase [32]; thus oxidative phosphorylation is rapidlystimulated by pulses of Ca2+. The continuous charging ofmitochondria with Ca2+ will sustain the activation state of themitochondrial dehydrogenases and may be important in sus-taining mitochondrial energy production (Fig. 1). MitochondrialCa2+ uptake is primarily driven by the electrochemical potentialgradient and a relatively low intramitochondrial Ca2+ concen-tration. Typically, mitochondrial Ca2+ uptake follows thecytosolic signal. Because reequilibration of mitochondrial Ca2+

is relatively slow, mitochondria effectively integrate cytosolicCa2+ signals over time [33].

The process of mitochondrial respiration establishes a largepotential gradient across the inner mitochondrial membrane, theΔψm, generally estimated to be on the order of 150–200 mV,negative inside [1]. The biochemical mechanism of oxidativephosphorylation involves two main steps: the transduction ofchemical redox potentials into an electrochemical H+ gradientacross the inner mitochondrial membrane and the ATP synthesisby the H+-driven molecular rotor of F0F1-ATPase. Whereas theprincipal function of the mitochondrial potential is clearly todrive ATP synthesis, it also provides the major mechanism forhandling Ca2+.

The mitochondrial membrane potential lies at the heart of allthe major bioenergetic functions of the mitochondrion, from thesynthesis of ATP to the accumulation of Ca2+. The collapse ofΔψm as a response to pathological states such as anoxia or as aresponse to disordered mitochondrial respiration will limitmitochondrial Ca2+ uptake and may contribute to cellularpathophysiology [32,34]. Mitochondrial Ca2+ import is an elec-trogenic process, as the movement of Ca2+ is not countered byany other ion exchange and therefore acts like an inwardcurrent, tending to depolarize the mitochondrial membranethrough increasing the permeability of the voltage-dependentanion channel [35]. This is observed as a small and transientdepolarization of the mitochondrial membrane in response inthe early phase of the Ca2+ flux. Although the activation ofdehydrogenases stimulates mitochondrial respiration, leading toMHP and increased ATP production, after the above-mentionedinitial depolarization in many cell types [1], Ca2+ influx by ion-omycin or Ca2+ release from intracellular stores by thapsigarginalone failed to induce Δψm elevation in lymphocytes [16].

Role of nitric oxide in T cell activation, mitochondrialbiogenesis, and hyperpolarization

Nitric oxide is a diffusible, multifunctional, transcellularmessenger that has been implicated in numerous physiologicaland pathological conditions [36]. NO is synthesized from L-arginine by NO synthases (NOS). Three distinct cytoplasmicisoforms of NOS are known, including neuronal NOS (nNOS),

1628 G. Nagy et al. / Free Radical Biology & Medicine 42 (2007) 1625–1631

inducible NOS (iNOS), and endothelial NOS (eNOS). nNOSand eNOS are expressed constitutively and generate NO forconstitutive cell-signaling purposes, whereas iNOS releases NOin larger quantities during inflammatory or immunologicaldefense reactions and is involved in host tissue damage [37].The physiological role of NO in the maintenance of vasculartone, in synaptic transmission, and in cellular defense is firmlyestablished. Recent evidence indicates that NO regulatesmitochondrial biogenesis. NO competes with molecular oxygen(O2) and reversibly inhibits cytochrome c oxidase, suggestingthat this competitive interaction has a physiological role in thecontrol of mitochondrial respiration [36].

Crosslinking of the TCR has been associated with elevationof the cytosolic Ca2+, ROI, and NO production and mitochond-rial hyperpolarization [16]. The NO chelator carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide profoundlyinhibited TCR crosslink-induced mitochondrial hyperpolariza-tion, cytoplasmic and mitochondrial Ca2+ response, ROIproduction, and NO levels, suggesting that TCR activation-induced MHP is mediated by NO [16]. In addition, NO signalwas required for CD3/CD28-induced Ca2+ flux, suggesting anessential role for NO in T cell activation. Western blot analysisrevealed expression of eNOS and nNOS and absence of iNOS inhuman PBL. eNOS and nNOS protein levels were stimulated upto 15-fold by CD3/CD28 costimulation [16,38]. Ibiza andcolleagues recently showed that within minutes of binding toantigen, T cells produce NO via eNOS [39]. NO promotedmitochondrial hyperpolarization, ATP depletion, and relativeresistance to apoptotic stimuli in astrocytes, lymphocytes, andJurkat cells [13,40]. Although pretreatment of normal T cellswith NO resulted in elevation of mitochondrial and cytoplasmicCa2+ levels, Ca2+ by itself was insufficient to induce NOsynthesis or mitochondrial hyperpolarization in lymphocytes[16,39].

NO has been recognized as a key signal for mitochondrialbiogenesis. This operates through the cGMP-dependentperoxisome proliferator-activating receptor γ coactivator-1α,a master regulator of mitochondrial biogenesis [41]. NO wasshown to induce mitochondrial biogenesis in brown adipo-cytes, U937 and HeLa cells, and human lymphocytes [42,43].Studies of T lymphocytes lacking eNOS will be important toestablish an absolute requirement of this NOS isoform in T cellactivation.

T cell activation involves considerable reordering of cellsurface and cytoplasmic components into an immunologicalsynapse [44]. Synapse formation is dependent on TCR-mediated signals that, in concert with costimulatory signals,cause the cellular polarization of the T cell cytoskeleton,membrane receptors, and selected cytoplasmic signalingeffectors toward the T cell–antigen presenting cell (APC)interface. Although not required for TCR signal initiation,synapse formation has been associated with the induction of Tcell proliferation, cytokine production, and lytic granule release.Quantitative subcellular analysis of NO production in T cellAPC conjugates showed that local NO production was twofoldhigher at cell–cell contacts than in the cytoplasm [39]. Thelocalized production of NO by eNOS at the immunological

synapse is a newly recognized checkpoint of T cell activationwhich leads to increased phosphorylation of CD3 ζ chain, ZAP-70, and extracellular signal-regulated kinases and increasedIFN-γ synthesis, but reduced production of IL-2 [39] (Fig. 1).

In addition to the NO originating from the cytoplasm, now itis clear that the mitochondrion has its own NO production. NOproduction by the co-oxidation of L-arginine and NADPH by O2

has been observed in mitochondrial membranes isolated from aseries of mammalian organs. The responsible enzyme has beennamed initially mitochondrial nitric oxide synthase (mtNOS),referring to its intracellular localization, in contrast to nNOS,eNOS, and iNOS, which were defined according to the cell typeof origin [45]. The mitochondrial NOS isoenzyme is aconstitutive protein of the mitochondrial outer membrane thatgenerates NO in a Ca2+-dependent reaction. mtNOS activityshows very strong and exponential dependence on mitochon-drial membrane potential in the physiological potential range(150–200 mV), which observation suggests that mtNOS isregulated by Δψm [46]. However, a distinct mtNOS gene hasnot yet been cloned and its very existence is controversial.Recent findings suggest that mtNOS may correspond to eNOSselectively targeted to the outer mitochondrial membrane [47].

Mitochondrial oxidative stress

In most cell types, mitochondria seem to represent one of themajor sources of generation of ROI, such as superoxide anion(O2

−) [1] (Fig. 1). Formation of the superoxide anion in themitochondrion occurs at the level of complex I and complex IIIof the ETC [48]. Although they are well known for theirdestructive effect on biomolecules, ROI are more and moreaccepted as necessary constituents in signaling pathways andmodulators of responses in physiological and pathologicalconditions [49]. In T cells, it has been reported that the radicalscavenger N-acetylcysteine inhibited the activation of NF-κBby phorbol 12-myristate 13-acetate, tumor necrosis factor-α,and interleukin-1, strongly supporting the idea that oxygenradicals are implicated in physiological activation processes[50,51]. ROI species trigger several proximal and distalsignaling pathways in T lymphocytes, affect the activities oftranscription factors, and lead to expression of specific genes(Fig. 1). In Jurkat T cells ROI induce increases in proteintyrosine phosphorylation and activity of p56lck, ZAP-70, andPKC as well as elevations in intracellular Ca2+ levels [52]. ROIare known to mediate the activation of NF-κB, but chronicexposure to ROI inhibits NF-κB phosphorylation andactivation.

As was mentioned above, increased intramitochondrial Ca2+

activates the ETC; thereafter it stimulates mitochondrialrespiration and ROI production [1]. Superoxide is effectivelyconverted to hydrogen peroxide (H2O2) by the mitochondrialsuperoxide dismutase (SOD) (Fig. 1). H2O2 has no unpairedelectrons and, by itself, is not a ROI. H2O2 could leave themitochondrion and act as a second messenger that can mediategene transcription and cell proliferation. Induction of apoptosisor necrosis by H2O2 requires transformation into an ROI, e.g.,OH−, through the Fenton reaction [53].

1629G. Nagy et al. / Free Radical Biology & Medicine 42 (2007) 1625–1631

The role of NO in apoptosis has been widely investigated,and both pro- and antiapoptotic actions have been reported [54–56]. Fas-induced apoptosis was found to be associated with anearly, concentration-dependent NO production in Jurkat cells[57]. NO can induce apoptosis in a variety of cell lines,including macrophages, thymocytes, T cells, and myeloid cells.Low concentrations of NO may specifically inhibit cytochromec oxidase, leading to ATP depletion [58]. A site for NO mo-ldulation of the apoptotic process that has received littleattention is the controlled release of cytochrome c from themitochondria [59]. The release of cytochrome c from mito-chondria leads to activation of caspases 9 and 3; thus theproapoptotic effect of NO may be connected to its effect onmitochondrial respiration.

NO reacts rapidly with O2− to produce peroxynitrite

(ONOO−), which may act as an oxidant itself, or it mayisomerize to nitrate. Peroxynitrite reacts with protein andnonprotein thiols, tyrosine residues, unsaturated fatty acids, andDNA, and it was reported to cause Ca2+ efflux from livermitochondria [60–62]. Addition of peroxynitrite to themitochondria also causes opening of the permeability transitionpore and opening of this pore contributes to the aforementionedloss of cytochrome c from the mitochondrion. Furthermoreperoxynitrite inhibits, or damages, mitochondrial complexesand SOD (Fig. 1). ROS may cause lipid peroxidation anddamage to cell membranes and to DNA, so that mitochondriarepresent not only a major source of ROS generation, but also amajor target of ROS-induced damage.

Mitochondrial hyperpolarization and increased NOproduction in systemic lupus erythematosus

SLE is a complex autoimmune disease characterized byproduction of antinuclear autoantibodies, clinical involvementin multiple organ systems, and increased NO production inmonocytes [43]. Lupus T cells exhibit persistent MHP orelevation of the mitochondrial transmembrane potential,increased ROI levels, and ATP depletion [15]. T lymphocytesof patients with SLE showed increased spontaneous and de-creased activation-induced apoptosis [63]. Upon T cell activa-tion, cell death preferentially occurred via necrosis rather thanapoptosis in patients with SLE. ATP levels and ATP/ADP ratioswere profoundly diminished in lupus PBL, which accounts forpredisposition to necrosis [64].

Persistent MHP has been associated with increased mito-chondrial mass of T lymphocytes in patients with SLE [43].Mitochondria can take up, store, and release Ca2+; thereafterincreased mitochondrial mass may account for altered Ca2

handling [2]. Although SLE T cells and normal T cells producecomparable amounts of NO, lupus monocytes were found toproduce a significantly higher amount of NO than normalmonocytes [43,65]. In comparison to control monocytes, lupusmonocytes increased Δψm and Ca2+ of normal T cells aftercoculture [43], suggesting that NO produced by monocytes isresponsible for mitochondrial dysfunction and predispositionto necrosis by lupus T cells. Release of necrotic materials fromT cells activates monocytes and dendritic cells, which may

operate as a positive feedback loop sustaining a proinflamma-tory state in SLE [2,18].

Baseline cytoplasmic Ca2+ levels are increased in SLE Tcells. Whereas the early phase of Ca2+ signal was enhanced,sustained elevation of CD3/CD28-induced Ca2+ signaling wasmarkedly reduced in lupus T cells [43]. Rapamycin (RAPA), amacrolide immunosuppressant, has been shown to interferewith T cell activation events in the course of spontaneousdisease progression in the MRL/lpr mouse model of lupus. ARAPA-sensitive pathway has been shown to regulate mito-chondrial membrane potential in irradiated human breast cancer(MCF-7) cells [66]. RAPA significantly reduced or preventedmany pathologic features of lupus normally seen in the MRL/lpr mouse [67]. RAPA treatment of patients with SLE nor-malized cytosolic and mitochondrial Ca2+ levels and T cellactivation-induced rapid Ca2+ fluxing, without influencingMHP [68], indicating that increased Ca2+ flux is downstreamor independent of MHP in the pathogenesis of T cell dysfunc-tion in SLE. Interestingly, the mammalian target of rapamycin(mTOR) may be activated by enhanced production of NO[69]. The effectiveness of RAPA in murine and human SLEsuggests that mTOR is a potential sensor [70] and downstreameffector of MHP and activation of mTOR may precede T celldysfunction and autoimmunity in SLE. These new data sug-gest that the clinically beneficial role of RAPA may be relatedto its selective effect on Ca2+ flux in SLE.

Acknowledgments

This work was supported by grants RO1 AI 48079, RO1DK49221, and R21 AI 061066 from the National Institutes ofHealth, F 61030 from OTKA, the Alliance for Lupus Research,and the Central New York Community Foundation.

References

[1] Duchen, M. R. Mitochondria in health and disease: perspectives on a newmitochondrial biology. Mol. Aspects Med. 25:365–451; 2004.

[2] Nagy, G.; Koncz, A.; Perl, A. T- and B-cell abnormalities in systemic lupuserythematosus. Crit. Rev. Immunol. 25:123–140; 2005.

[3] Skulachev, V. P. Mitochondrial physiology and pathology: concepts ofprogrammed death of organelles, cells and organisms. Mol. Aspects Med.20:139–140; 1999.

[4] Constantini, P.; Chernyak, B. V.; Petronilli, B.; Bernardi, P. Modulation ofthe mitochondrial permeability transition pore by pyridine nucleotides anddithiol oxidation at two separate sites. J. Biol. Chem. 271:6746–6751;1996.

[5] Perl, A.; Gergely Jr., P.; Puskas, F.; Banki, K. Metabolic switches of T-cellactivation and apoptosis. Antioxid. Redox Signaling 4:427–443; 2002.

[6] Banki, K.; Hutter, E.; Gonchoroff, N.; Perl, A. Elevation of mitochondrialtransmembrane potential and reactive oxygen intermediate levels are earlyevents and occur independently from activation of caspases in Fassignaling. J. Immunol. 162:1466–1479; 1999.

[7] Banki, K.; Hutter, E.; Colombo, E.; Gonchoroff, N. J.; Perl, A. Glutathionelevels and sensitivity to apoptosis are regulated by changes in transaldolaseexpression. J. Biol. Chem. 271:32994–33001; 1996.

[8] Puskas, F.; Gergely, P.; Banki, K.; Perl, A. Stimulation of the pentosephosphate pathway and glutathione levels by dehydroascorbate, theoxidized form of vitamin C. FASEB J. 14:1352–1361; 2000.

[9] Li, P.-F.; Dietz, R.; von Harsdorf, R. p53 regulates mitochondrialmembrane potential through reactive oxygen species and induces

1630 G. Nagy et al. / Free Radical Biology & Medicine 42 (2007) 1625–1631

cytochrome c-independent apoptosis blocked by bcl-2. EMBO J. 18:6027–6036; 1999.

[10] Gottlieb, E.; Vander Heiden, M. G.; Thompson, C. B. Bcl-XL prevents theinitial decrease in mitochondrial membrane potential and subsequentreactive oxygen species production during tumor necrosis factor alpha-induced apoptosis. Mol. Cell. Biol. 20:5680–5689; 2000.

[11] Scarlett, J. L.; Sheard, P. W.; Hughes, G.; Ledgerwood, E. C.; Ku, H.-H.;Murphy, M. P. Changes in mitochondrial membrane potential duringstaurosporin-induced apoptosis in Jurkat cells. FEBS Lett. 475:267–272;2000.

[12] Sanchez-Alcazar, J. A.; Ault, J. G.; Khodjakov, A.; Schneider, E. Increasedmitochondrial cytochrome c levels and mitochondrial hyperpolarizationprecede camptothecin-induced apoptosis in Jurkat cells. Cell Death Differ.7:1090–1100; 2000.

[13] Almeida, A.; Almeida, J.; Bolanos, J. P.; Moncada, S. Different responsesof astrocytes and neurons to nitric oxide: the role of glycolyticallygenerated ATP in astrocyte protection. Proc. Natl. Acad. Sci. USA 98:15294–15299; 2001.

[14] Norman, J. P.; Perry, S. W.; Kasischke, K. A.; Volsky, D. J.; Gelbard, H. A.HIV-1 trans activator of transcription protein elicits mitochondrialhyperpolarization and respiratory deficit, with dysregulation of complexIVand nicotinamide adenine dinucleotide homeostasis in cortical neurons.J. Immunol. 178:869–876; 2007.

[15] Gergely, P. J.; Grossman, C.; Niland, B.; Puskas, F.; Neupane, H.; Allam,F.; Banki, K.; Phillips, P. E.; Perl, A. Mitochondrial hyperpolarization andATP depletion in patients with systemic lupus erythematosus. ArthritisRheum. 46:175–190; 2002.

[16] Nagy, G.; Koncz, A.; Perl, A. T cell activation-induced mitochondrialhyperpolarization is mediated by Ca2+- and redox-dependent production ofnitric oxide. J. Immunol. 171:5188–5197; 2003.

[17] Krishnan, S.; Kiang, J. G.; Fisher, C. U.; Nambiar, M. P.; Nguyen, H. T.;Kyttaris, V. C.; Chowdhury, B.; Rus, V.; Tsokos, G. C. Increasedcaspase-3 expression and activity contribute to reduced CD3{zeta} ex-pression in systemic lupus erythematosus T cells. J. Immunol. 175:3417–3423; 2005.

[18] Perl, A.; Gergely Jr., P.; Nagy, G.; Koncz, A.; Banki, K. Mitochondrialhyperpolarization: a checkpoint of T cell life, death, and autoimmunity.Trends Immunol. 25:360–367; 2004.

[19] Cohen, J. J.; Duke, R. C.; Fadok, V. A.; Sellins, K. A. Apoptosis andprogrammed cell death in immunity. Annu. Rev. Immunol. 10:267–293;1992.

[20] Sauter, B.; Albert, M. L.; Francisco, L.; Larsson, M.; Somersan, S.;Bhardwaj, N. Consequences of cell death: exposure to necrotic tumorcells, but not primary tissue cells or apoptotic cells, induces the matu-ration of immunostimulatory dendritic cells. J. Exp. Med. 191:423–434;2000.

[21] Eisner, M. D.; Amory, J.; Mullaney, B.; Tierney Jr., L.; Browner, W. S.Necrotizing lymphadenitis associated with systemic lupus erythematosus.Semin. Arthritis Rheum. 26:477–482; 1996.

[22] Ma, L.; Chan, K. W.; Trendell-Smith, N. J.; Wu, A.; Tian, L.; Lam, A. C.;Chan, A. K.; Lo, C. K.; Chik, S.; Ko, K. H.; To, C. K.; Kam, S. K.; Li, X.S.; Yang, C. H.; Leung, S. Y.; Ng, M. H.; Stott, D. I.; MacPherson, G. G.;Huang, F. P. Systemic autoimmune disease induced by dendritic cells thathave captured necrotic but not apoptotic cells in susceptible mouse strains.Eur. J. Immunol. 35:3364–3375; 2005.

[23] Lanzaveccia, A.; Lezzi, G.; Viola, A. From TcR engagement to T cellactivation: a kinetic view of T cell behavior. Cell 96:1–4; 1999.

[24] Germain, R. N. The T cell receptor for antigen: signaling and liganddiscrimination. J. Biol. Chem. 276:35223–35226; 2001.

[25] Abraham, N.; Miceli, M. C.; Parnes, J. R.; Veillette, A. Enhancement ofT-cell responsiveness by the lymphocyte-specific tyrosine protein kinasep56lck. Nature 350:62–66; 1991.

[26] Samelson, L. E.; Klauser, R. D. Tyrosine kinases and tyrosine basedactivation motifs. J. Biol. Chem. 267:24913–24916; 1992.

[27] Imboden, J. B.; Weiss, A. The T-cell antigen receptor regulates sus-tained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influxand ongoing intracellular Ca2+ mobilization. Biochem. J. 247:695–700;1987.

[28] Lewis, R. S. Calcium signaling mechanism in T lymphocytes. Annu. Rev.Immunol. 19:497–521; 2001.

[29] Premack, B. A.; Gardner, P. Signal transduction by T cell receptors:mobilization of Ca and regulation of Ca-dependent effector molecules.Am. J. Physiol. 263:19–40; 1992.

[30] Chance, B. The nature of electron transfer and energy coupling reactions.FEBS Lett. 23:3–20; 1972.

[31] Rizzuto, R.; Bernardi, P.; Pozzan, T. Mitochondria as all-round players ofthe calcium game. J. Physiol. 15:37–47; 2000.

[32] Duchen, M. R. Mitochondria and calcium: from cell signalling to celldeath. J. Physiol. 529:57–68; 2000.

[33] Hajnoczky, G.; Robb-Gaspers, L. D.; Seitz, M. B.; Thomas, A. P.Decoding of cytosolic Ca2+ oscillations in the mitochondria. Cell 82:415–424; 1995.

[34] Perl, A.; Qian, Y.; Chohan, K. R.; Shirley, C. R.; Amidon, W.; Banerjee, S.;Middleton, F. A.; Conkrite, K. L.; Barcza, M.; Gonchoroff, N.; Suarez,S. S.; Banki, K. Transaldolase is essential for maintenance of themitochondrial transmembrane potential and fertility of spermatozoa. Proc.Natl. Acad. Sci. USA 103:14813–14818; 2006.

[35] Bathori, G.; Csordas, G.; Garcia-Perez, C.; Davies, E.; Hajnoczky, G. Ca2+-dependent control of the permeability properties of the mitochondrialouter membrane and voltage-dependent anion-selective channel (VDAC).J. Biol. Chem. 281:17347–17358; 2006.

[36] Bredt, D. S. Endogenous nitric oxide synthesis: biological functions andpathophysiology. Free Radic. Res. 31:577–596; 1999.

[37] Song, H.; Xia, S. L.; Liao, C.; Li, Y. L.; Wang, Y. F.; Li, T. P.; Zhao, M. J.Genes encoding Pir51, Beclin 1, RbAp48 and aldolase b are up or down-regulated in human primary hepatocellular carcinoma. World J. Gastro-enterol. 10:509–513; 2004.

[38] Reiling, N.; Kroncke, R.; Ulmer, A. J.; Gerdes, J.; Flad, H. D.; Hauschildt,S. Nitric oxide synthase: expression of the endothelial, Ca2+/calmodulin-dependent isoform in human B and T lymphocytes. Eur. J. Immunol. 3:511–516; 1996.

[39] Ibiza, S.; Victor, V. M.; Bosca, I.; Ortega, A.; Urzainqui, A.; O'Connor,J. E.; Sanchez-Madrid, F.; Esplugues, J. V.; Serrador, J. M. Endothelialnitric oxide synthase regulates T cell receptor signaling at theimmunological synapse. Immunity 24:753–765; 2006.

[40] Beltran, B.; Mathur, A.; Duchen, M. R.; Erusalimsky, J. D.; Moncada, S.The effect of nitric oxide on cell respiration: a key to understanding its role incell survival, or death. Proc. Natl. Acad. Sci. USA 26:14602–14607; 2000.

[41] Nisoli, E.; Carruba, M. O. Nitric oxide and mitochondrial biogenesis.J. Cell Sci. 119:2855–2862; 2006.

[42] Nisoli, E.; Clementi, E.; Paolucci, C. Mitochondrial biogenesis inmammals: the role of endogenous nitric oxide. Science 299:896–899;2003.

[43] Nagy, G.; Barcza, M.; Gonchoroff, N.; Philips, P. E.; Perl, A. Nitric oxide-dependent mitochondrial biogenesis generates Ca2+ signaling profile oflupus T cells. J. Immunol. 173:3576–3583; 2004.

[44] Grakoui, A.; Bromley, S. K.; Sumen, C.; Davis, M. M.; Shaw, A. S.; Allen,P. M.; Dustin, M. L. The immunological synapse: a molecular machinecontrolling T cell activation. Science 285:221–227; 1999.

[45] Bates, T. E.; Loesch, A.; Burnstock, G.; Clark, J. B. Mitochondrial nitricoxide synthase: a ubiquitous regulator of oxidative phosphorylation?Biochem. Biophys. Res. Commun. 218:40–44; 1996.

[46] Valdez, L. B.; Zaobornyj, T.; Boveris, A. Mitochondrial metabolic statesand membrane potential modulate mtNOS activity. Biochim. Biophys.Acta 1757:166–172; 2006.

[47] Gao, S.; Chen, J.; Brodsky, S. V.; Huang, H.; Adler, S.; Lee, J. H.;Dhadwal, N.; Cohen-Gould, L.; Gross, S. S.; Goligorsky, M. S. Docking ofendothelial nitric oxide synthase (eNOS) to the mitochondrial outermembrane: a pentabasic amino acid sequence in the autoinhibitory domainof eNOS targets a proteinase K-cleavable peptide on the cytoplasmic faceof mitochondria. J. Biol. Chem. 279:15968–15974; 2004.

[48] Moncada, S.; Erusalimsky, J. D. Does nitric oxide modulate mitochondrialenergy generation and apoptosis?Nat. Rev. Mol. Cell. Biol. 3:214–220; 2002.

[49] Cemerski, S.; Cantagrel, A.; Van Meerwijk, J. P.; Romagnoli, P. Reactiveoxygen species differentially affect T cell receptor-signaling pathways.J. Biol. Chem. 277:19585–19593; 2002.

1631G. Nagy et al. / Free Radical Biology & Medicine 42 (2007) 1625–1631

[50] Schreck, R.; Rieber, P.; Baeuerle, P. A. Reactive oxygen intermediates asapparently widely used messengers in the activation of the NF-kappa Btranscription factor and HIV-1. EMBO J. 10:2247–2258; 1991.

[51] Williams, M. S.; Kwon, J. T cell receptor stimulation, reactive oxygenspecies, and cell signaling. Free Radic. Biol. Med. 37:1144–1151; 2004.

[52] Hardwick, J. S.; Sefton, B. M. Activation of the Lck tyrosine proteinkinase by hydrogen peroxide requires the phosphorylation of Tyr-394.Proc. Natl. Acad. Sci. USA 92:4527–42531; 1995.

[53] Blokhina, O.; Virolainen, E.; Fagerstedt, K. V. Antioxidants, oxidativedamage and oxygen deprivation stress: a review. Ann. Bot. (London) 91:179–194; 2003.

[54] Kim, Y. M.; Bombeck, C. A.; Billiar, T. R. Nitric oxide as a bifunctionalregulator of apoptosis. Circ. Res. 19:253–256; 1999.

[55] Chung, H. T.; Pae, H. O.; Choi, B. M.; Billiar, T. R.; Kim, Y. M. Nitricoxide as a bioregulator of apoptosis. Biochem. Biophys. Res. Commun.282:1075–1079; 2001.

[56] Melino, G.; Catani, M. V.; Corazzari, M.; Guerrieri, P.; Bernassola, F.Nitric oxide can inhibit apoptosis or switch it into necrosis. Cell Mol.Life. Sci. 57:612–622; 2000.

[57] Beltran, B.; Quintero, M.; Gracia-Zaragoza, E.; O'Connor, E.; Esplugues,J. V.; Moncada, S. Inhibition of mitochondrial respiration by endogenousnitric oxide: a critical step in Fas signalling. Proc. Natl. Acad. Sci. USA13:8892–8897; 2002.

[58] Brown, C. G. Nitric oxide and mitochondrial respiration. Biochem.Biophys. Acta 1411:351–369; 1999.

[59] Brookes, P. S.; Salinas, E. P.; Darley-Usmar, K.; Eiserich, J. P.; Freeman,B. A.; Darley-Usmar, V. M.; Anderson, P. G. Concentration dependenteffects of nitric oxide on mitochondrial permeability transition andcytochrome c release. J. Biol. Chem. 275:20474–20479; 2000.

[60] Packer, M. A.; Murphy, M. P. Peroxynitrite causes calcium efflux frommitochondria which is prevented by cyclosporin A. FEBS Lett. 2-3:237–240; 1994.

[61] Weinberg, J. B.; Granger, D. L.; Pisetsky, D. S. Alterations to glutathioneand nicotinamide nucleotides during the mitochondrial permeability tran-

sition induced by peroxynitrite. Biochem. Pharmacol. 7:1047–1055;1996.

[62] Packer, M. A.; Murphy, M. P. Peroxynitrite formed by simultaneous nitricoxide and superoxide generation causes cyclosporin-A-sensitive mito-chondrial calcium efflux and depolarisation. Eur. J. Biochem. 1:231–239;1995.

[63] Caricchio, R.; Cohen, P. L. Spontaneous and induced apoptosis in systemiclupus erythematosus: multiple assays fail to reveal consistent abnormal-ities. Cell. Immunol. 1:54–60; 1999.

[64] Leist, M.; Single, B.; Castoldi, A. F.; Kuhnle, S.; Nicotera, P. Intracellularadenosine triphosphate (ATP) concentration: a switch in the decisionbetween apoptosis and necrosis. J. Exp. Med. 185:1481–1486; 1997.

[65] Amin, A. R.; Attur, M.; Vyas, P. Expression of nitric oxide synthase inhuman peripheral blood mononuclear cells and neutrophils. J. Inflamma-tion 4:190–205; 1995.

[66] Paglin, S.; Lee, N. Y.; Nakar, C.; Fitzgerald, M.; Plotkin, J.; Deuel, B.;Hackett, N.; McMahill, M.; Sphicas, E.; Lampen, N.; Yahalom, J.Rapamycin-sensitive pathway regulates mitochondrial membrane poten-tial, autophagy, and survival in irradiated MCF-7 cells. Cancer Res. 65:11061–11070; 2005.

[67] Warner, L. M.; Adams, L. M.; Sehgal, S. N. Rapamycin prolongs survivaland arrests pathophysiologic changes in murine systemic lupus erythema-tosus. Arthritis Rheum. 37:289–297; 1994.

[68] Fernandez, D.; Bonilla, E.; Mirza, N.; Niland, B.; Perl, A. Rapamycinreduces disease activity and normalizes T cell activation-induced calciumfluxing in patients with systemic lupus erythematosus. Arthritis Rheum.54:2983–2988; 2006.

[69] Ban, H.; Shigemitsu, K.; Yamatsuji, T.; Haisa, M.; Nakajo, T.; Takaoka,M.; Nobuhisa, T.; Gunduz, M.; Tanaka, N.; Naomoto, Y. Arginine andleucine regulate p70 S6 kinase and 4E-BP1 in intestinal epithelial cells.Int. J. Mol. Med. 13:537–543; 2004.

[70] Desai, B. N.; Myers, B. R.; Schreiber, S. L. FKBP12-rapamycin-associatedprotein associates with mitochondria and senses osmotic stress via mito-chondrial dysfunction. Proc. Natl. Acad. Sci. USA 99:4319–4324; 2002.