Central role of nitric oxide in the pathogenesis of rheumatoid arthritis and sysemic lupus erythematosus

Nitric oxide (NO) has been shown to regulate T cell functions under physiological conditions, but overproduction of NO may contribute to T lymphocyte dysfunction. NO-dependent tissue injury has been implicated in a variety of rheumatic diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Several studies reported increased endogenous NO synthesis in both SLE and RA, and recent evidence suggests that NO contributes to T cell dysfunction in both autoimmune diseases. The depletion of intracellular glutathione may be a key factor predisposing patients with SLE to mitochondrial dysfunction, characterized by mitochondrial hyperpolarization, ATP depletion and predisposition to death by necrosis. Thus, changes in glutathione metabolism may influence the effect of increased NO production in the pathogenesis of autoimmunity.

NOS (iNOS), and endothelial NOS (eNOS) [5]. NO also serves as a potent immuno regulatory factor, and infl uences the cytoplasmic redox balance through the generation of peroxynitrite (ONOO -) following its reaction with superoxide (O 2 -) [6]. In addition, NO regu lates signal transduction by regulating Ca 2+ signaling, by regulating the structure of the immuno logical synapse, or through the modifi cation of intra cellular proteins, such as by inter actions with heme groups ( Figure 1). Here we summarize the eff ects of NO on T lymphocyte functions in both systemic lupus erythe matosus (SLE) and rheumatoid arthritis (RA).
NO regulates mitochondrial membrane potential in human T cells [7], and may both stimulate and inhibit apop tosis [8]. It was shown to inhibit cytochrome c oxidase, leading to cell death through ATP depletion ( Figure 1). In addition, NO was shown to regulate mitochondrial biogenesis in U937 and HeLa cells and adipocytes through the cGMP-dependent peroxisome proliferator-activating receptor λ coactivator 1α [9]. According to our earlier work, NO regulates mito chondrial biogenesis in human lymphocytes as well [10]. Nitrosylation of sulfhydryl groups represents an important cGMP-independent, NO-dependent post-translational modifi cation. Several important signal transduction proteins are potential targets of S-nitrosylation, such as caspases and c-Jun-N-terminal kinase (JNK) [11,12].

The role of nitric oxide in T cell activation and diff erentiation
NO regulates T lymphocyte function in several ways: T cell activation is associated with NO production and mitochondrial hyperpolarization (MHP) [13]. According to our previous data, eNOS and nNOS are expressed in human peripheral blood lymphocytes and both are upregulated several times following T cell activation [13]. TCR stimulation induces Ca 2+ infl ux and, through inositol-1,4,5-triphosphate (IP 3 ), the release of Ca 2+ from intracellular stores. Th e IP 3 inhibitor 2-APB (2-aminoethoxydiphenyl borane) decreases T-cellactivation-induced Ca 2+ and NO production, and NO

Abstract
Nitric oxide (NO) has been shown to regulate T cell functions under physiological conditions, but overproduction of NO may contribute to T lymphocyte dysfunction. NO-dependent tissue injury has been implicated in a variety of rheumatic diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Several studies reported increased endogenous NO synthesis in both SLE and RA, and recent evidence suggests that NO contributes to T cell dysfunction in both autoimmune diseases. The depletion of intracellular glutathione may be a key factor predisposing patients with SLE to mitochondrial dysfunction, characterized by mitochondrial hyperpolarization, ATP depletion and predisposition to death by necrosis. Thus, changes in glutathione metabolism may infl uence the eff ect of increased NO production in the pathogenesis of autoimmunity. treatment of T lymphocytes leads to an increase in mitochondrial and cytoplasmic Ca 2+ levels. In contrast, th e NO che lator C-PTIO (carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide) powerfully inhibits the T-cell-activation-induced Ca 2+ response, NO production and MHP, indicating that T cell receptor (TCR)activation-induced MHP is mediated by NO [13].
A central event in the antigen-specifi c interaction of T cells with antigen-presenting cells is the formation of the immunological synapse, in which the TCR complex and the adhesion receptor LFA-1 (leukocyte functionassociated antigen 1) are organized in central and peripheral supramolecular activation clusters. eNOS was shown to translocate with the Golgi apparatus to the immune synapse of T helper cells engaged with antigenpresenting cells [14] ( Figure 1). Overexpression of eNOS was associated with increased phosphorylation of the CD3ζ chain, ZAP-70, and extracellular signal-regulated kinases, and increased IFN-γ synthesis, but reduced produc tion of IL-2. Th ese data indicate that eNOS-derived NO selectively potentiates T cell receptor signaling to antigen at the immunological synapse [14].
Following activation, CD4 T cells proliferate and diff erentiate into two main subsets of primary eff ector Figure 1. Schematic diagram of T cell activation, nitric oxide production, and mitochondrial hyperpolarization. Nitric oxide (NO) is produced in the cytosol, the mitochondrial membrane, and at the immunological synapse of T cells. Localized NO production has been linked to targeting of endothelial NO synthase (eNOS) to the outer mitochondrial membrane and to the T-cell synapse. NO regulates many steps of T cell activation, the production of cytokines, such as IL-2, and mitochondrial hyperpolarization and mitochondrial biogenesis. NO regulates mammalian target of rapamycin (mTOR) activity. NO dependent mTOR activation induces the loss of TCRζ in lupus T cells through HRES-1/Rab4. Mitochondrial hyperpolarization is associated with depletion of ATP, which predisposes T cells to necrosis. In turn, necrotic materials released from T cells activate monocytes and dendritic cells. Solid arrows indicate processes upregulated by NO, while broken lines indicate processes down-regulated by NO. APC, antigen-presenting cell; DAG, diacylglycerol; IP 3 , inositol-1,4,5-triphosphate; LAT, linker for activation of T cells; MHC, major histocompatibility complex; PIP2, phosphatidylinositol 4,5-biphosphate; PLC, phospholipase C.

NO
T cell NO cells, T helper 1 (Th 1) and Th 2 cells, characterized by their specifi c cytokine expression patterns [15]. Th e Th 1/ Th 2 balance is considered to be essential in chronic infl ammatory diseases. NO selectively enhances Th 1 cell proliferation [16] and represents an additional signal for the induction of T cell subset response. According to our data, the NO precursor NOC-18 elicited IFN-γ production, whereas the NO synthase inhibitors N G -monomethyl-L-arginine and nitronidazole both inhibited its production, suggesting a role for NO in regulating IFN-γ synthesis [17]. NO preferentially promotes IFN-γ syn thesis and type Th 1 cell diff erentiation by selective induction of IL-12Rβ2 via cGMP. Together, these data indicate that NO has a crucial role in the regulation of Th 1/Th 2 polarization.

Nitric oxide regulates T lymphocyte activation in systemic lupus erythematosus
Considerable evidence supports that NO production is increased in SLE; for example, serum nitrite and nitrate levels were recently reported to correlate with disease activity and damage in SLE [18]. According to our previous work, NO plays a crucial role in T cell dysregulation in SLE [19][20][21]. Activation-induced rapid Ca 2+ signals are higher in T cells from patients with SLE [22]; in contrast, the sustained Ca 2+ signal is decreased in these lupus T cells. Interestingly, the mitochondrial membrane potential is permanently high in lupus T c ells [23][24][25]. Lupus and normal T cells produce comparable amounts of NO, but monocytes from lupus patients generate signifi cantly more NO than normal monocytes. As it is a diff usible gas, NO produced by neighboring cells may aff ect T cell functions. Accordingly, NO produced by mono cytes contributes to lymphocyte mitochondrial dysfunction in SLE [10]. Peripheral blood lymphocytes from SLE patients contain enlarged mitochondria, and as there are microdomains between mitochondria and the endoplasmic reticulum and because mitochondria may also serve as Ca 2+ stores, this increased mitochondrial mass may alter Ca 2+ signaling in SLE [10,26]. Although NO production was found to be increased in both lupus [10] and RA [27], MHP was confi ned to lupus T cells [10,13,28,29]. Th is diff erence may be attributed to the depletion of intracellular glutathione (GSH) in SLE but not in RA or healthy controls [28]. Indeed, low GSH predisposes to MHP in human T cells, as originally described by Banki and colleagues [30]. Increased exposure to IFN may contribute to the increased NO production of lupus monocytes [31].

NO regulates mammalian target of rapamycin activity and TCRζ expression in SLE
Th e mammalian target of rapamycin (mTOR) is a serine/ threonine protein kinase and a sensor of the mitochon drial transmembrane potential that regulates protein synthesis, cell growth, cell proliferation and survival [32]. Th e activity of mTOR is increased in lupus T cells [29] (Table 1); furthermore, NO regulates mTOR activity, which leads to enhanced expression of HRES-1/ Rab4, a small GTPase that regulates recycling of surface receptors through early endosomes [29,33]. HRES-1/ Rab4 over expression inversely correlates with TCRζ protein levels. TCR/CD3 expression is regulated by TCRζ, and dimin ished ζ chain expression disrupts TCR transport and function [34]. Th e TCR ζ chain is defi cient in lupus T cells [35,36], although this defi ciency has been shown to be independent of SLE disease activity [3 7,38]. Sequencing o f genomic DNA and TCRζ transcripts showed mutations in the coding region of TCRζ from lupus T cells [39]. Th ere is a direct interaction between HRES-1/Rab4, CD4 and TCRζ. Rapamycin treatment of lupus patients reversed the TCRζ defi ciency of lupus T cells, and normalized T-cell-activation-induced calcium fl uxing [29]. Th ese data suggest that NO-dependent mTOR activation induces the loss of TCRζ in lupus T cells through HRES-1/Rab4. Several earlier fi ndings indicate that decreased TCRζ chain expression may also be independent of NO in SLE [40][41][42][43][44].

Consequences of increased nitric oxide production in rheumatoid arthritis
Several studies in patients with RA have documented evidence for increased endogenous NO synthesis, suggest ing that overproduction of NO may be important in the pathogenesis of RA. Th e infl amed joint in RA is the predominant source of NO [45,46]. Several investigators found correlations between serum nitrite concentration and RA disease activity or radiological progression while others did not fi nd such correlations [47,48]. NOS polymorphism has been observed in RA [49]. iNOS is regulated at the transcriptional level, while eNOS and nNOS are regulated by intracellular Ca 2+ . Several diff er ent cell types are capable of generating NO in the infl amed synovium, including osteoblasts, osteoclasts, macro phages, fi broblasts, neutrophils and endothelial cells [50][51][52]. NOS inhibition was reported to decrease disease activity in experimental RA [53].
We have shown recently that T cells from RA patients produce more than 2.5 times more NO than healthy donor T cells (P < 0.001) [27]. Although NO is an importa nt physiologic al mediator of mitochondrial biogenesis, mitochondrial mass is similar in both RA and control T cells ( Table 1). By contrast, increased NO production is associated with increased cytoplasmic Ca 2+ concentrations in RA T cells (P < 0.001). Furthermore, in vitro treat ment of human peripheral blood lymphocytes or Jurkat cells with TNF increases NO production (P = 0.006 and P = 0.001, respectively), whilst infl iximab treatment of RA patients decreases T-cell-derived NO production within 6 weeks of the fi rst infusion (P = 0.005) [27]. Increased NO production of monocytes is associated with increased mitochondrial biogenesis in lupus T cells, while increased NO production of T cells is not associated with increased mitochondrial mass in RA. Monocytes express iNOS, while lymphocytes express both eNOS and nNOS. Although NO is generated more rapidly via the eNOS or nNOS than the iNOS pathway, iNOS can generate much larger quantities of NO than eNOS and nNOS. Th us, the lower amount of NO generated by T cells compared to monocytes may explain the diff erences in T lymphocyte mitochondrial biogenesis that we observed for lupus and RA T cells.
iNOS knockout mice are resistant to IL-1-induced bone resorption, suggesting that NO plays a central role in the pathogenesis of bone erosions in RA [51,54]. TNF blockade decreases iNOS expression in human peripheral blood mononuclear cells [55]. Tripterygium wilfordii Hook F (TWHF) was also reported to be eff ective in the treatment of experimental arthritis [56]. Th e specifi c inhibition of iNOS by TWHF is probably responsible for the anti-infl ammatory eff ects of this medicinal plant. NO treatment may lead to necrosis rather than apoptosis by decreasing intracellular ATP levels. Th e release of intracellular antigens through necrosis may accelerate autoimmune reactions leading to chronic infl ammation [57,58].

Oxidative stress and TCRζ expression in RA T cellsthe possible role of NO
Reduced GSH levels may contribute to the hypo responsive ness of T cells from synovial fl uid of RA patients [59,60]. Th e expression of the TCR ζ chain protein is decreased in synovial fl uid T cells of RA patients, similar to lupus T cells, which may contribute to the abovementioned hyporesponsiveness of the synovial fl uid T cells [61]. TNF-α treatment decreases TCR ζ chain expression of T cells [62] in a GSH-precursor-sensitive way, showing the role of redox balance in the regulation of TCR ζ chain expression. TCRζ overexpression does not restore signaling in TNF-α-treated T cells [63]. Increased NO production may alter redox balance through generating peroxynitrite following its reaction with superoxide. In this way NO may contribute to the decreased TCR ζ chain expression of T lymphocytes from synovial fl uid [61]. Importantly, FcR gamma substitutes for the TCR ζ chain in SLE T cells [64], which may explain the enhanced T-cell-activation-induced Ca 2+ fl uxing. Th e potential role of NO in the regulation of FcR gamma expression clearly needs further investi gation.

Th17 and regulatory T cells
Recently, the Th 1/Th 2 paradigm has been updated follow ing the discovery of a third subset of Th cells, known as Th 17 cells. Th 17 cells have been identifi ed as cells induced by IL-6 and TGF-β and expanded by IL-23 [65]. Similarly to Th 1 and Th 2 subsets, Th 17 development relies on the action of a lineage-specifi c transcription factor. Th 17 cells have emerged as an independent subset because their diff erentiation was independent of the Th 1 and Th 2 promoting transcription factors T-bet, STAT1, STAT4 and STAT6. ROR-γt, RORα and STAT3 appear to be critical for the development of Th 17 cells. Th 17 cells produce IL-17 and are thought to clear extracellular pathogens that are not eff ectively handled by either Th 1 or Th 2 cells, and have also been strongly implicated in allergic diseases [66]. In addition to IL-17, Th 17 cells produce other proinfl ammatory cytokines such as IL-21 and IL-22. Increased levels of IL-17 have been observed in patients with RA. Indeed, IL-17 can directly and indirectly promote cartilage and bone destruction. IL-17defi cient mice develop attenuated collagen-induced arthritis. Th e role of NO in IL-6-and TGF-β-induced Th 17 cell diff erentiation has not been studied yet.
Regulatory T cells (Tregs) represent a subset of T cells involved in peripheral immune tolerance. Th ere are at least three major types of Tregs with overlapping functions: Th 3, Treg1, and CD4 + CD25 + Tregs. CD4 + CD25 + Tregs (naturally occurring cells or nTREGs) are the best characterized, principally because it is relatively easy to obtain large numbers of these cells. Tregs seem to have Table 1

Altered T cell function SLE RA
Mitochondrial hyperpolarization and biogenesis Higher [10] Normal [27] T lymphocyte NO production Normal [10] Increased [27] TCR-induced rapid and sustained Ca 2+ signal Rapid-increased, sustained-decreased [10] Normal [22] TCRζ expression Decreased [34] Decreased [61] mTOR activity Increased [29] Not known ATP level Decreased [28] Normal [28] Monocyte NO production Increased [10] Increased [46] an impaired regulatory function in RA. It was recently reported that NO, together with anti-CD3, induces the proliferation and sustained survival of mouse CD4 + CD25 -T cells, which became CD4 + CD25 + but remained Foxp3 -. Th is previously unrecognized population of Tregs (NO-Tregs) downregulated the proliferation and function of freshly purifi ed CD4 + CD25eff ector cells in vitro and suppressed colitis-and collagen-induced arthritis in mice in an IL-10-dependent manner [67]. Th e existence of human NO-Tregs has not been investigated yet. Although NO profoundly alters T cell activation and Th 1/Th 2 balance, the precise role of NO in Th 17 and Treg diff erentiation is not known.

Conclusion
Whilst NO plays a central role in many physiological processes, its increased production is pathological. NO mediates many diff erent cell functions at the site of synovial infl ammation, including cytokine production, signal transduction, mitochondrial functions and apoptosis (Table 1). Th e eff ects of NO depend on its concentration. Increased NO production plays an important role in the pathogenesis of both SLE and RA. Further studies are needed to determine the cellular and molecular mechanisms by which NO regulates immune cell functions. NOS inhibition may represent a novel therapeutic approach in the treatment of chronic autoimmune diseases.

Competing interests
The authors declare that they have no competing interests.