SUMMARY

Activation, proliferation, and cell death pathway selection of T lymphocytes depend on reactive oxygen intermediate (ROI) production and ATP synthesis, which are tightly regulated via the mitochondrial transmembrane potential (Δψ_m). Mitochondrial hyperpolarization (MHP) and ATP depletion represent early and reversible steps in T-cell activation and apoptosis. By contrast, T lymphocytes of systemic lupus erythematosus (SLE) patients exhibit persistent MHP, cytoplasmic alkalinization, increased ROI production, and ATP depletion that mediate enhanced spontaneous and diminished activation-induced apoptosis and sensitize lupus T cells to necrosis.

Necrotic, but not apoptotic, cell lysates activate dendritic cells and may account for increased interferon-a production and inflammation in lupus patients. Persistent MHP is associated with increased mitochondrial mass and increased mitochondrial and cytoplasmic Ca²⁺ content in T cells of SLE patients. Nitric oxide (NO) has recently been recognized as a key signal of MHP and mitochondrial biogenesis in human T lymphocytes. NO-induced mitochondrial biogenesis in normal T cells regenerated the Ca²⁺ signaling profile of lupus T cells. Mitochondria constitute major Ca²⁺ stores, and NO-dependent mitochondrial biogenesis may account for altered Ca²⁺ handling by lupus T cells. MHP is proposed as a key mechanism of T-cell dysfunction and target of pharmacologic intervention in SLE.

INTRODUCTION

SLE is a chronic inflammatory disease characterized by T- and B-cell dysfunction and production of antinuclear antibodies. Potentially autoreactive T and B lymphocytes during development¹ and after completion of an immune response are removed by apoptosis.¹ Abnormal T-cell activation and cell death underlie the pathology of SLE.^3,4 Paradoxically, lupus T cells exhibit both enhanced spontaneous apoptosis¹ and defective activation-induced cell death.^6–10 Increased spontaneous apoptosis of PBL has been linked to chronic lymphopenia¹ and compartmentalized release of nuclear autoantigens in patients with SLE.¹ By contrast, defective CD3-mediated cell death may be responsible for persistence of autoreactive cells.¹

Both cell proliferation and apoptosis are energy-dependent processes. Energy in the form of ATP is provided through glycolysis and oxidative phosphorylation.¹ The synthesis of ATP is driven by an electrochemical gradient across the inner mitochondrial membrane maintained by an electron transport chain and the membrane potential (Δψ_m, negative inside and positive outside). A small fraction of electrons react directly with oxygen and form ROI. Disruption of Δψ_m has been proposed as the point of no return in apoptotic signaling.^13–15 Mitochondrial membrane permeability is subject to regulation by an oxidation-reduction equilibrium of ROI, pyridine nucleotides (NADH/NAD + NADPH/NADP), and GSH levels.¹

Regeneration of GSH by glutathione reductase from its oxidized form GSSG depends on NADPH produced by the pentose phosphate pathway (PPP).^17–19 Metabolic fluxes between glycolysis and the PPP are particularly relevant for balancing cellular requirements for energy and ROI production.¹ Although ROI have been considered toxic by-products of aerobic existence, evidence is now accumulating that controlled levels of ROI modulate various aspects of cellular function and are necessary for signal-transduction pathways (including those mediating T-cell activation and apoptosis).¹

Elevation of Δψ_m or MHP was discovered in our laboratory as an early event preceding caspase activation, phosphatidylserine (PS) externalization, and disruption of Δψ_m in Fas-(19) and H₂O₂-induced apoptosis of Jurkat human leukemia T cells and normal human PBL,¹ as well as in HIV-1-induced apoptosis.¹ These observations were confirmed and extended to p53;¹ tumor necrosis factor a;¹ and staurosporin,¹ camptothecin,¹ and NO-induced apoptosis.¹ MHP is also triggered by activation of the CD3/CD28 complex¹ via ROI- and Ca²⁺-dependent production of NO¹ or stimulation with Con A¹ or galectin 1,¹ IL-10, IL-3, IFN-?, and TGFβ.¹ Thus, elevation of Δψ_m or MHP represents an early but reversible switch not exclusively associated with apoptosis.

With ??_m hyperpolarization and extrusion of H+ from the mitochondrial matrix, the cytochromes within the electron transport chain become more reduced (which favors generation of ROI).¹ Therefore, MHP is a likely cause of increased ROI production and may be ultimately responsible for increased susceptibility to apoptosis following T-cell activation.¹ MHP and ATP depletion play key roles in abnormal T-cell death (which shift susceptibility from apoptosis to necrosis) in patients with SLE.¹ In turn, increased necrosis rates could play a central role in enhanced inflammatory responses in SLE. Δψ_m and ROI levels (as well as cytoplamic pH) are elevated in patients with SLE in comparison to healthy or RA controls.^7,8 Baseline MHP and ROI levels correlated with diminished GSH levels, suggesting increased utilization of reducing equivalents in patients with SLE.

It is presently unclear whether de novo synthesis of GSH or its regeneration from the oxidized form (GSSG) is deficient in lupus patients. GSH is required for interleukin-2-dependent T-cell proliferation¹ as well as CD2- and CD3-mediated T-cell activation.¹ Thus, low GSH content may also inhibit CD3-induced H₂O₂ production. Moreover, GSH deficiency predisposes cells for ROI-induced cell death.^18,21,33 Diminished H₂O₂-induced apoptosis of cells with low baseline GSH levels indicates a severe dysfunction of redox signaling in patients with SLE.

Increased ROI production may lead to skewed expression of redox-sensitive surface receptors and lymphokines. As examples, ROIs regulate gene transcription and release of TNFa and interleukin-10,¹ both of which are elevated in sera^35,36 and freshly isolated PBL of SLE patients.^37,38 Elevated serum levels of type I interferon (IFN) raised the possibility of a viral etiology in lupus.¹ Recently, overexpression of genes regulated by IFN were noted by microarray analysis of lupus PBL.^40,41 Interestingly, expression of type I IFN is enhanced by ROI.^42–45 Microarray studies also indicated overexpression of cytokines, cytokine receptors,¹ bcl-2, superoxide dismutase,¹ apoptosis mediators TNFR6 (Fas/CD95), TRAIL,¹ TRAIL DR3, and TRAIL DR4,¹ all consistent with oxidative stress in SLE. Interestingly, NADH dehydrogenase/complex-I of the electron transport chain was found to be down-regulated in SLE.¹

Expression of TCR? chain is sensitive to oxidative stress¹ and thus increased ROI levels may explain, at least in part, low TCR? chain expression in lupus T cells.¹ Cell surface expression of the Fas receptor^51–53 and Fas ligand is also redox sensitive.¹ Thus, higher ROI levels may lead to increased IL-10 production, release of FasL, and overexpression of the FasR in SLE.^55–57 Mitochondrial ROI production and ??_m are early checkpoints in Fas-¹ and H₂O₂-induced apoptosis.¹ Increased ROI levels confer sensitivity to H₂O₂-, NO-, TNFa-, or Fas-induced cell death.¹ Enhanced NO production may also contribute to increased spontaneous apoptosis.^58,59 Therefore, elevated baseline ROI and ??_m levels may have key roles in abnormal T-cell activation and apoptosis in patients with SLE. Indeed, MHP could represent a novel target of pharmacological intervention in patients with SLE.^60–62

REDOX CONTROL OF T-CELL ACTIVATION AND APOPTOSIS SIGNAL PROCESSING

ROI modulate T-cell activation, cytokine production, and proliferation at multiple levels.¹ The antigen-binding αβ or γδTCR is associated with a multimeric receptor module comprised of the CD3γδe and TCRζ chains. The cytoplasmic domains of CD3 and δ chains contain a common motif termed the immunoglobulin receptor family tyrosine-based activation motif (ITAM), which is crucial for coupling of intracellular tyrosine kinases.¹ Expression of TCRζ chain is suppressed by ROI.¹ Binding of p56^lck to CD4 or CD8 attracts this kinase to the TCRζ-CD3 complex, leading to phosphorylation of ITAM. Phosphorylation of both tyrosines of each ITAM is required for SH-2-mediated binding by zeta-associated protein-70 (ZAP-70) or the related SYK. ZAP-70 is activated through phosphorylation by p56^lck. Activated ZAP-70 and SYK target the two key adaptor proteins LAT and SLP-76.¹

Oxidative stress reduces phosphorylation and displacement of LAT from the cell membrane, causing T-cell hyporesponsiveness.¹ Phosphorylated LAT binds directly to phospholipase C-γ1, which controls hydrolysis of phosphatydilinositol-4,5-biphosphate (PIP2) to inositol-1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). Phosphorylation of inositol lipid second messengers is mediated by phosphatidylinositol 3′hydroxyl kinase (PI₃K). The stimulatory effect of the TCR alone on PI₃K activity is small. Concurrent triggering of the CD28 co-stimulatory molecule by its ligands CD80 or CD86 is required for optimal PI₃K activation. IP₃ binds to its receptors in the endoplasmic reticulum, opening Ca²⁺ channels that release Ca²⁺ to the cytosol.

Increased cytosolic Ca²⁺ concentration activates the phosphatase calcineurin, which dephosphorylates the transcription factor NFAT. Dephosphorylated NFAT can translocate to the nucleus, where it promotes transcription of IL-2 in concert with AP-1, NF?B, and Oct-1. Whereas activities of AP-1 and NF?B are increased by oxidative stress,¹ both thiol insufficiency and H₂O₂ treatment suppress calcineurin-mediated activation of NFAT.¹ Thus, expression of cytokines [i.e., IL-2 (with AP-1 and NFAT motif-containing promoter) and IL-4 (with AP-1-less NFAT enhancer)] can be selectively regulated by oxidative stress (depending on the relative expression level of transcription factors involved).¹

Programmed cell death (PCD) or apoptosis is a physiologic mechanism for elimination of autoreactive lymphocytes during development. Signaling through the Fas or structurally related TNF family of cell surface death receptors has emerged as a major pathway in elimination of unwanted cells under physiologic and disease conditions.¹ Fas and TNF receptors mediate cell death via cytoplasmic death domains (DD) shared by both receptors.¹ They trigger sequential activation of caspases, resulting in cleavage of cellular substrates and the characteristic morphologic and biochemical changes of apoptosis.¹

Disruption of the mitochondrial membrane potential (Δψ_m) has been proposed as the point of no return in apoptotic signaling that leads to caspase activation and disassembly of the cell.¹ Interestingly, MHP and ROI production precede disruption of Δψ_m, activation of caspases, and phosphatidylserine (PS) externalization in Fas-,¹ TNFa-¹ and H₂O₂-induced apoptosis of Jurkat human leukemia T cells and normal human peripheral blood lymphocytes.¹ Elevation of Δψ_m is independent of activation of caspases and represents an early event in apoptosis.¹

Pretreatment with caspase inhibitors completely abrogated Fas-induced PS externalization, indicating that activation of caspase-3, caspase-8, and related cysteine proteases were absolutely required for cell death.¹ ROI levels were partially inhibited in caspase inhibitor-treated Jurkat cells, suggesting that caspase-3 activation (perhaps through damage of mitochondrial membrane integrity) contributes to ROI production and serves as a positive feedback loop at later stages of the apoptotic process. Cleavage of cytosolic Bid by caspase-8 generates a p15 carboxyterminal fragment that translocates to mitochondria. This may increase mitochondrial membrane permeability and lead to secondary elevation of ROI levels in the Fas and TNF pathway.¹

MITOCHONDRIAL CHECKPOINTS OF CELL DEATH PATHWAY SELECTION: δψ_m, ATP SYNTHESIS, ROI AND NO PRODUCTION, CA²⁺ FLUXING, AND REDUCING CAPACITY PROVIDED BY GSH AND NADPH

MHP appears to be the earliest change associated with several apoptosis pathways.^10,20 Elevation of Δψ_m is also triggered by activation of the CD3-CD28 complex¹ or stimulation with Con A,¹ IL-10, IL-3, IFN-γ, or TGFβ.¹ Therefore, MHP represents an early but reversible switch not exclusively associated with apoptosis. MHP is a likely cause of increased ROI production¹ and may be ultimately responsible for increased susceptibility to apoptosis following T-cell activation.¹

MHP in T lymphocytes is associated with a dramatic increase, more than sixfold, of NO production lasting 24 hours after CD3-CD28 co-stimulation. Molecular ordering of T-cell activation-induced NO production revealed critical roles for ROI production and cytoplasmic and mitochondrial Ca²⁺ influx.¹ CD3-CD28 co-stimulation-induced ROI production (similar to H₂O₂) enhances expression of NOS isoforms eNOS and nNOS, which require elevated Ca²⁺ levels for enzymatic activity. These results suggest that T-cell activation-induced ROI and Ca²⁺ signals contribute to NO production, with the latter representing a final and dominant step in MHP.

Proteins of the Bcl-2 family are localized to membranes of distinct organelles, including mitochondria.¹ Both the proapoptotic (Bax, Bad) and antiapoptotic (Bcl-2, Bcl-X_L) members of the family can form ion-conducting channels in lipid membranes.¹ Bax can create a channel in the outer mitochondrial membrane, thus releasing cytochrome c and other caspase-activating moieties into the cytosol. Bcl-2 and Bcl-X_L inhibit this process through dimerization with Bad or Bax. Bcl-2 expression appears to be unaltered in lupus PBL.¹

The mitochondrion is the site of ATP synthesis via oxidative phosphorylation. The synthesis of ATP is driven by an electrochemical gradient across the inner mitochondrial membrane maintained by an electron transport chain and the membrane potential. Activity of caspases require ATP to the extent that depletion of ATP by inhibition of F₀F₁-ATPase with oligomycin¹ or exhaustion of intracellular ATP stores by prior apoptosis signals, Fas stimulation,¹ or H₂O₂ pretreatment leads to necrosis.¹ Thus, intracellular ATP concentration is a key switch in the cell’s decision to die via apoptosis or necrosis.¹

MHP, INCREASED MITOCHONDRIAL MASS, ROI PRODUCTION, CYTOPLASMIC ALKALINIZATION, AND ATP DEPLETION IN LUPUS T CELLS

Coordinate MHP and ATP depletion play key roles in abnormal T-cell death in lupus patients.¹ Δψ_m and ROI levels as well as cytoplamic pH are elevated in patients with SLE in comparison to healthy or rheumatoid arthritis controls.^7,8 Baseline MHP and ROI levels correlated with diminished GSH levels, suggesting increased utilization of reducing equivalents in patients with SLE. It is presently unclear whether synthesis of GSH or its regeneration from its oxidized form is deficient in lupus patients. GSH is also required for interleukin-2-dependent T-cell proliferation¹ as well as CD2- and CD3-mediated T-cell activation.¹ Thus, low GSH content may also inhibit CD3-induced H₂O₂ production. Nevertheless, GSH deficiency predisposes for ROI-induced cell death.^18,21 Diminished H₂O₂-induced apoptosis of cells with low baseline GSH levels indicate a severe dysfunction of redox signaling in patients with SLE.¹

Increased ROI production may lead to skewed expression of redox-sensitive surface receptors and lymphokines in SLE (Table 25.1). As examples, ROIs regulate gene transcription and release of TNFa and interleukin-10,¹ both of which are elevated in sera¹ and freshly isolated PBL of SLE patients.¹ Expression of TCR? chain is sensitive to oxidative stress,¹ and thus increased ROI levels may in part explain low TCR? chain expression in lupus T cells.¹ Cell surface expression of the Fas receptor¹ and ligand is also redox sensitive.¹ Increased ROI levels may be related to increased IL-10 production, release of FasL, and overexpression of the FasR in SLE.¹ Elevated NO production may also contribute to increased spontaneous apoptosis.¹ Increased ROI levels confer sensitivity to H₂O₂, NO, TNFa, and Fas-induced cell death.¹ Therefore, persistent MHP [causing increased ROI production (a trigger of apoptosis) and depletion of ATP (required for AICD)] may be responsible for the paradox of increased spontaneos apoptosis and diminished AICD in SLE.

TABLE 25.1 REDOX SIGNALING ABNORMALITIES IN T CELLS OF PATIENTS WITH SLE