Paul E. Phillips

Mitochondrial hyperpolarization and ATP depletion in patients with systemic lupus erythematosus

OBJECTIVE Peripheral blood lymphocytes (PBLs) from systemic lupus erythematosus (SLE) patients exhibit increased spontaneous and diminished activation-induced apoptosis. We tested the hypothesis that key biochemical checkpoints, the mitochondrial transmembrane potential (deltapsim) and production of reactive oxygen intermediates (ROIs), mediate the imbalance of apoptosis in SLE. METHODS We assessed the deltapsim with potentiometric dyes, measured ROI production with oxidation-sensitive fluorochromes, and monitored cell death by annexin V and propidium iodide staining of lymphocytes, using flow cytometry. Intracellular glutathione levels were measured by high-performance liquid chromatography, while ATP and ADP levels were assessed by the luciferin-luciferase assay. RESULTS Both deltapsim and ROI production were elevated in the 25 SLE patients compared with the 25 healthy subjects and the 10 rheumatoid arthritis patients. Intracellular glutathione contents were diminished, suggesting increased utilization of reducing equivalents in SLE. H2O2, a precursor of ROIs, increased deltapsim and caused apoptosis in normal PBLs. In contrast, H2O2-induced apoptosis and deltapsim elevation were diminished, particularly in T cells, and the rate of necrotic cell death was increased in patients with SLE. The intracellular ATP content and the ATP:ADP ratio were reduced and correlated with the deltapsim elevation in lupus. CD3:CD28 costimulation led to transient elevation of the deltapsim, followed by ATP depletion, and sensitization of normal PBLs to H2O2-induced necrosis. Depletion of ATP by oligomycin, an inhibitor of F0F1-ATPase, had similar effects. CONCLUSION T cell activation and apoptosis are mediated by deltapsim elevation and increased ROI production. Mitochondrial hyperpolarization and the resultant ATP depletion sensitize T cells for necrosis, which may significantly contribute to inflammation in patients with SLE.

N‐acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: A randomized, double‐blind, placebo‐controlled trial

OBJECTIVE Systemic lupus erythematosus (SLE) patients exhibit T cell dysfunction, which can be regulated through mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione (GSH). This randomized, double-blind, placebo-controlled study was undertaken to examine the safety, tolerance, and efficacy of the GSH precursor N-acetylcysteine (NAC). METHODS A total of 36 SLE patients received either daily placebo or 1.2 gm, 2.4 gm, or 4.8 gm of NAC. Disease activity was evaluated monthly by the British Isles Lupus Assessment Group (BILAG) index, the SLE Disease Activity Index (SLEDAI), and the Fatigue Assessment Scale (FAS) before, during, and after a 3-month treatment period. Mitochondrial transmembrane potential and mTOR were assessed by flow cytometry. Forty-two healthy subjects matched to patients for age, sex, and ethnicity were studied as controls. RESULTS NAC up to 2.4 gm/day was tolerated by all patients, while 33% of those receiving 4.8 gm/day had reversible nausea. Placebo or NAC 1.2 gm/day did not influence disease activity. Considered together, 2.4 gm and 4.8 gm NAC reduced the SLEDAI score after 1 month (P = 0.0007), 2 months (P = 0.0009), 3 months (P = 0.0030), and 4 months (P = 0.0046); the BILAG score after 1 month (P = 0.029) and 3 months (P = 0.009); and the FAS score after 2 months (P = 0.0006) and 3 months (P = 0.005). NAC increased Δψm (P = 0.0001) in all T cells, profoundly reduced mTOR activity (P = 0.0009), enhanced apoptosis (P = 0.0004), reversed expansion of CD4-CD8- T cells (mean ± SEM 1.35 ± 0.12-fold change; P = 0.008), stimulated FoxP3 expression in CD4+CD25+ T cells (P = 0.045), and reduced anti-DNA production (P = 0.049). CONCLUSION This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes.

Persistent Mitochondrial Hyperpolarization, Increased Reactive Oxygen Intermediate Production, and Cytoplasmic Alkalinization Characterize Altered IL-10 Signaling in Patients with Systemic Lupus Erythematosus

Abnormal death signaling in lymphocytes of systemic lupus erythematosus (SLE) patients has been associated with elevation of the mitochondrial transmembrane potential (Δψm) and increased production of reactive oxygen intermediates (ROI). The resultant ATP depletion sensitizes T cells for necrosis that may significantly contribute to inflammation in patients with SLE. In the present study, the role of mitochondrial signal processing in T cell activation was investigated. CD3/CD28 costimulation of PBL elicited transient mitochondrial hyperpolarization and intracellular pH (pHi) elevation, followed by increased ROI production. Baseline Δψm, ROI production, and pHi were elevated, while T cell activation-induced changes were blunted in 15 patients with SLE in comparison with 10 healthy donors and 10 rheumatoid arthritis patients. Similar to CD3/CD28 costimulation, treatment of control PBL with IL-3, IL-10, TGF-β1, and IFN-γ led to transient Δψm elevation. IL-10 had diametrically opposing effects on mitochondrial signaling in lupus and control donors. Unlike healthy or rheumatoid arthritis PBL, cells of lupus patients were resistant to IL-10-induced mitochondrial hyperpolarization. By contrast, IL-10 enhanced ROI production and cell death in lupus PBL without affecting ROI levels and survival of control PBL. Ab-mediated IL-10 blockade or stimulation with antagonistic lymphokine IL-12 normalized baseline and CD3/CD28-induced changes in ROI production and pHi with no impact on Δψm of lupus PBL. The results suggest that mitochondrial hyperpolarization, increased ROI production, and cytoplasmic alkalinization play crucial roles in altered IL-10 responsiveness in SLE.

Activation of mammalian target of rapamycin controls the loss of TCRzeta in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation.

Persistent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation and death pathway selection in systemic lupus erythematosus. Treatment with rapamycin, which effectively controls disease activity, normalizes CD3/CD28-induced calcium fluxing but fails to influence MHP, suggesting that altered calcium fluxing is downstream or independent of mitochondrial dysfunction. In this article, we show that activity of the mammalian target of rapamycin (mTOR), which is a sensor of the mitochondrial transmembrane potential, is increased in lupus T cells. Activation of mTOR was inducible by NO, a key trigger of MHP, which in turn enhanced the expression of HRES-1/Rab4, a small GTPase that regulates recycling of surface receptors through early endosomes. Expression of HRES-1/Rab4 was increased in CD4+ lupus T cells, and in accordance with its dominant impact on the endocytic recycling of CD4, it was inversely correlated with diminished CD4 expression. HRES-1/Rab4 overexpression was also inversely correlated with diminished TCRζ protein levels. Pull-down studies revealed a direct interaction of HRES-1/Rab4 with CD4 and TCRζ. Importantly, the deficiency of the TCRζ chain and of Lck and the compensatory up-regulation of FcεRIγ and Syk, which mediate enhanced calcium fluxing in lupus T cells, were reversed in patients treated with rapamcyin in vivo. Knockdown of HRES-1/Rab4 by small interfering RNA and inhibitors of lysosomal function augmented TCRζ protein levels in vitro. The results suggest that activation of mTOR causes the loss of TCRζ in lupus T cells through HRES-1/Rab4-dependent lysosomal degradation.

Nitric Oxide-Dependent Mitochondrial Biogenesis Generates Ca2+ Signaling Profile of Lupus T Cells

Abnormal T cell activation and cell death underlie the pathology of systemic lupus erythematosus. Although mitochondrial hyperpolarization (MHP) represents an early and reversible checkpoint of T cell activation and apoptosis, lupus T cells exhibit persistent MHP. NO has recently been recognized as a key signal of mitochondrial biogenesis and mediator of MHP in human T lymphocytes. In this study, we show that persistent MHP was associated with increased mitochondrial mass (+47.7 ± 2.8%; p = 0.00017) and increased mitochondrial (+21.8 ± 4.1%; p = 0.016) and cytoplasmic Ca2+ content in T cells from 19 systemic lupus erythematosus patients with respect to 11 control donors (+38.0 ± 6.4%; p = 0.0023). Electron microscopy revealed that lupus lymphocytes contained 8.76 ± 1.0 mitochondria, while control donors contained 3.18 ± 0.28 mitochondria per cell (p = 0.0009). Increased mitochondrial mass in T cells was associated with 2.08 ± 0.09-fold enhanced NO production by lupus monocytes (p = 0.0023). Activation of T cells through the TCR initiates a biphasic elevation in cytosolic free Ca2+ concentration, a rapid initial peak observed within minutes, and a plateau phase lasting up to 48 h. In response to CD3/CD28 costimulation, rapid Ca2+ fluxing was enhanced while the plateau phase was diminished in lupus T cells. NO-induced mitochondrial biogenesis in normal T cells enhanced the rapid phase and reduced the plateau of Ca2+ influx upon CD3/CD28 costimulation, thus mimicking 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.

Comparative analysis of antibody and cell-mediated autoimmunity to transaldolase and myelin basic protein in patients with multiple sclerosis.

Antibody and T cell-mediated immune responses to oligodendroglial autoantigens transaldolase (TAL) and myelin basic protein (MBP) were examined in patients with multiple sclerosis (MS). Immunohistochemical studies of postmortem brain sections revealed decreased staining by MBP- and TAL-specific antibodies in MS plaques, indicating a concurrent loss of these antigens from demyelination sites. By Western blot high titer antibodies to human recombinant TAL were found in 29/94 sera and 16/23 cerebrospinal fluid samples from MS patients. Antibodies to MBP were undetectable in sera or cerebrospinal fluid of these MS patients. Proliferative responses to human recombinant TAL (stimulation index [SI] = 2.47+/-0.3) were significantly increased in comparison to MBP in 25 patients with MS (SI = 1.37+/-0.1; P < 0.01). After a 7-d stimulation of PBL, utilization of any of 24 different T cell receptor Vbeta gene segments in response to MBP was increased less than twofold in the two control donors and six MS patients investigated. In response to TAL-H, while skewing of individual Vbeta genes was also less than twofold in healthy controls, usage of specific Vbeta gene segments was differentially increased ranging from 2.5 to 65.9-fold in patients with MS. The results suggest that TAL may be a more potent immunogen than MBP in MS.

ANGIOGENIC FACTOR FROM SYNOVIAL FLUID RESEMBLING THAT FROM TUMOURS

Synovial fluids from 2 of 2 patients with osteoarthritis, 2 of 8 with rheumatoid arthritis, and 2 of 2 with ankylosing spondylitis contained a low-molecular-weight angiogenesis factor apparently identical with that derived from tumours. The amount of factor present was highest in patients with osteoarthritis, 2 patients with psoriatic arthropathy gave negative results. It is suggested that angiogenesis factor is involved, directly or indirectly, in the production of synovial exudate.

Mechanistic Target of Rapamycin Activation Triggers IL-4 Production and Necrotic Death of Double-Negative T Cells in Patients with Systemic Lupus Erythematosus

The mechanistic target of rapamycin (mTOR) is recognized as a sensor of mitochondrial dysfunction and effector of T cell lineage development; however, its role in autoimmunity, including systemic lupus erythematosus, remains unclear. In this study, we prospectively evaluated mitochondrial dysfunction and mTOR activation in PBLs relative to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) during 274 visits of 59 patients and 54 matched healthy subjects. Partial least square–discriminant analysis identified 15 of 212 parameters that accounted for 70.2% of the total variance and discriminated lupus and control samples (p < 0.0005); increased mitochondrial mass of CD3+/CD4−/CD8− double-negative (DN) T cells (p = 1.1 × 10−22) and FOXP3 depletion in CD4+/CD25+ T cells were top contributors (p = 6.7 × 10−7). Prominent necrosis and mTOR activation were noted in DN T cells during 15 visits characterized by flares (SLEDAI increase ≥ 4) relative to 61 visits of remission (SLEDAI decrease ≥ 4). mTOR activation in DN T cells was also noted at preflare visits of SLE patients relative to those with stable disease or healthy controls. DN lupus T cells showed increased production of IL-4, which correlated with depletion of CD25+/CD19+ B cells. Rapamycin treatment in vivo blocked the IL-4 production and necrosis of DN T cells, increased the expression of FOXP3 in CD25+/CD4+ T cells, and expanded CD25+/CD19+ B cells. These results identify mTOR activation to be a trigger of IL-4 production and necrotic death of DN T cells in patients with SLE.

Polymorphic genotypes of the HRES-1 human endogenous retrovirus locus correlate with systemic lupus erythematosus and autoreactivity

Abstract Antinuclear autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Autoantibodies to HRES-1/p28, a 28 000 Mr nuclear protein, commonly occur in patients with SLE. HRES-1 is a single-copy endogenous retroviral element mapped to human Chromosome 1 at q42. A polymorphic Hin dIII site defines two different allelic forms of the genomic locus. The HRES-1/1 probe [5.5 kilobases (kb)] anneals to three polymorphic fragments and three genotypes can be differentiated: I, 5.5 kb fragment only; II, 3.7 kb and 1.8 kb fragments only; and III, all three polymorphic fragments. By cloning of the HRES-1 locus from homozygous type I and type II human DNA samples, the polymorphic Hin dIII site was identified as a G to C transition at position 653 of the long terminal repeat region. Family studies showed that Hin dIII genotypes of the HRES-1 locus are inherited in a Mendelian pattern. The relative frequency of genotype I with respect to genotype III was 3.1-fold lower in patients with SLE (14 : 40=0.35) in comparison to 100 ethnically matched control donors (47 : 43=1.09;P=0.0084). Frequency of genotype I vs genotype II alleles was lower in SLE (68/52) than in normal donors (137/63;P=0.033), suggesting that a genotype I allele of the HRES-1 locus may be protective against SLE. Western blot seroreactivity with recombinant HRES-1/p28 was noted in 4/14 (29%) of genotype I patients and 13/19 (68%) of genotype III patients (P<0.025). These data raise the possibility that the HRES-1 element or a gene in linkage disequilibrium with this genomic locus may influence autoimmunity in SLE.

Haplotypes of the HRES-1 Endogenous Retrovirus Are Associated With Development and Disease Manifestations of Systemic Lupus Erythematosus

OBJECTIVE Endogenous retroviral sequences represent a link between viral and genetic factors that may influence the development of systemic lupus erythematosus (SLE). The HRES-1 human endogenous retroviral sequence is centrally located at the 1q42 chromosomal region relative to microsatellites previously associated with SLE. We therefore undertook the present study to determine the haplotypes of the HRES-1 locus and their linkage to SLE. METHODS One hundred six patients with SLE, 82 unrelated healthy Caucasian individuals, and 70 healthy members of 34 lupus families were examined. HRES-1 was amplified by polymerase chain reaction (PCR) and analyzed by sequencing and restriction enzyme mapping. Microsatellites were analyzed by PCR. Haplotype construction and transmission disequilibrium testing (TDT) were performed in lupus families. RESULTS Based on 4 single-nucleotide polymorphisms (SNPs) within a 935-base interval, we detected 6 HRES-1 haplotypes that were differentially segregated in unrelated Caucasian patients and control subjects (chi(2) = 16.86, P = 0.0048) and were in linkage disequilibrium (LD) with the D1S225 microsatellite (P = 0.0002). The microsatellites D1S225, D1S235, and D1S2785 (but not D1S229) were linked to SLE by TDT. Interestingly, LD between HRES-1 SNPs at bases 653 and 1259 was reduced in patients with SLE (P = 0.048). The HRES-1 653C/1259C-harboring alleles were associated with the presence of renal disease (P = 0.0021) and with the absence of lung disease (P = 0.0323), while the 956A allele was associated with the antiphospholipid syndrome in patients with SLE (P = 0.0036). CONCLUSION The HRES-1 locus represents a recombination hot spot at the 1q42 chromosomal region that influences the development and disease manifestations of SLE.