Agnes Koncz

T Cell Activation-Induced Mitochondrial Hyperpolarization Is Mediated by Ca2+- and Redox-Dependent Production of Nitric Oxide

Activation, proliferation, or programmed cell death of T lymphocytes is regulated by the mitochondrial transmembrane potential (Δψm) through controlling ATP synthesis, production of reactive oxygen intermediates (ROI), and release of cell death-inducing factors. Elevation of Δψm or mitochondrial hyperpolarization is an early and reversible event associated with both T cell activation and apoptosis. In the present study, T cell activation signals leading to mitochondrial hyperpolarization were investigated. CD3/CD28 costimulation of human PBL elevated cytoplasmic and mitochondrial Ca2+ levels, ROI production, and NO production, and elicited mitochondrial hyperpolarization. Although T cell activation-induced Ca2+ release, ROI levels, and NO production were diminished by inositol 1,4,5-triphosphate receptor antagonist 2-aminoethoxydiphenyl borane, superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, spin trap 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide, and NO chelator carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, mitochondrial hyperpolarization was selectively inhibited by carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (−85.0 ± 10.0%; p = 0.008) and, to a lesser extent, by 2-aminoethoxydiphenyl borane. Moreover, NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate diethylenetriamine elicited NO and ROI production, Ca2+ release, transient ATP depletion, and robust mitochondrial hyperpolarization (3.5 ± 0.8-fold; p = 0.002). Western blot analysis revealed expression of Ca-dependent endothelial NO synthase and neuronal NO synthase isoforms and absence of Ca-independent inducible NO synthase in PBL. CD3/CD28 costimulation or H2O2 elicited severalfold elevations of endothelial NO synthase and neuronal NO synthase expression, as compared with β-actin. H2O2 also led to moderate mitochondrial hyperpolarization; however, Ca2+ influx by ionomycin or Ca2+ release from intracellular stores by thapsigargin alone failed to induce NO synthase expression, NO production, or Δψm elevation. The results suggest that T cell activation-induced mitochondrial hyperpolarization is mediated by ROI- and Ca2+-dependent NO production.

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.

Regulation of CD4 Expression via Recycling by HRES-1/RAB4 Controls Susceptibility to HIV Infection

A novel 2986-base transcript encoded by the antisense strand of the HRES-1 human endogenous retrovirus was isolated from peripheral blood lymphocytes. This transcript codes for a 218-amino acid protein, termed HRES-1/Rab4, based on homology to the Rab4 family of small GTPases. Antibody 13407 raised against recombinant HRES-1/Rab4 detected a native protein of identical molecular weight in human T cells. HRES-1 nucleotides 2151-1606, located upstream of HRES-1/Rab4 exon 1, have promoter activity when oriented in the direction of HRES-1/Rab4 transcription. The human immunodeficiency virus, type 1 (HIV-1), tat gene stimulates transcriptional activity of the HRES-1/Rab4 promoter via trans-activation of the HRES-1 long terminal repeat. Transfection of HIV-1 tat into HeLa cells or infection of H9 and Jurkat cells by HIV-1 increased HRES-1/Rab4 protein levels. Overexpression of HRES-1/Rab4 in Jurkat cells abrogated HIV infection, gag p24 production, and apoptosis, whereas dominant-negative HRES-1/Rab4S27N had the opposite effects. HRES-1/Rab4 inhibited surface expression of CD4 and targeted it for lysosomal degradation. HRES-1/Rab4S27N enhanced surface expression, recycling, and total cellular CD4 content. Infection by HIV elicited a coordinate down-regulation of CD4 and up-regulation of HRES-1/Rab4 in PBL. Moreover, overexpression of HRES-1/Rab4 reduced CD4 expression on peripheral blood CD4+ T cells. Stimulation by HIV-1 of HRES-1/Rab4 expression and its regulation of CD4 recycling reveal novel coordinate interactions between an infectious retrovirus and the human genome.

Nitric oxide production of T lymphocytes is increased in rheumatoid arthritis

Experimental and clinical evidence for T cell involvement in the pathology of rheumatoid arthritis (RA) is compelling, and points to a local dysregulation of T cell function in the inflamed joint. Nitric oxide (NO) has been shown to regulate T cell function under physiological conditions, but overproduction of NO may contribute to lymphocyte dysfunction characteristic of RA. Several investigations in patients with RA have documented evidence of increased NO synthesis, but these studies have focused largely on macrophage-derived NO and its impact on innate immune and inflammatory responses. In this study, we set out to explore the contribution that T cells make to NO production. We find that T cells from RA patients produce >2.5 times more NO than healthy donor T cells (p<0.001). Although NO is an important physiological mediator of mitochondrial biogenesis, mitochondrial mass is similar in RA and control T cells. In contrast, increased NO production is associated with increased cytoplasmic Ca(2+) concentrations in RA T cells (p<0.001). In vitro treatment of human peripheral blood lymphocytes, or Jurkat cells with TNF increases NO production (p=0.006 and p=0.001, respectively), whilst infliximab treatment in RA patients decreases T cell derived NO production within 6 weeks of the first infusion (p=0.005). Together, these data indicate that TNF induced NO production in T lymphocytes may contribute to perturbations of immune homeostasis in RA.

Molecular mimicry and immunomodulation by the HRES-1 endogenous retrovirus in SLE

Genetic and environmental factors are believed to influence development of systemic lupus erythematosus (SLE). Endogenous retroviruses (ERV) correspond to the integrated proviral form of infectious retroviruses, which are trapped within the genome due to mutations. ERV represent a key molecular link between the host genome and infectious viral particles. ERV-encoded proteins are recognized by antiviral immune responses and become targets of autoreactivity. Alternatively, ERV protein may influence cellular processes and the life cycle of infectious viruses. As examples, the HRES-1 human ERV encodes a 28-kDa nuclear autoantigen and a 24-kDa small GTP-ase, termed HRES-1/Rab4. HRES-1/p28 is a nuclear autoantigen recognized by cross-reactive antiviral antibodies, while HRES-1/Rab4 regulates surface expression of CD4 and the transferrin receptor (TFR) through endosome recycling. Expression of HRES-1/Rab4 is induced by the tat gene of HIV-1, which in turn down-regulates expression of CD4 and susceptibility to re-infection by HIV-1. CD4 and the TFR play essential roles in formation of the immunological synapse (IS) during normal T-cell activation by a cognate MHC class II peptide complex. The key intracellular transducer of T-cell activation, Lck, is brought to the IS via binding to CD4. T-cell receptorζ (TCRζ) chain binds to the TFR. Abnormal T-cell responses in SLE have been associated with reduced lck and TCRζ chain levels. HRES-1 is centrally located on chromosome 1 at q42 relative to lupus-linked microsatellite markers and polymorphic HRES-1 alleles have been linked to the development of SLE. 1q42 is one of the three most common fragile sites in the human genome, and is inducible by DNA demethylation, a known mechanism of retroviral gene activation. Molecular mimicry and immunomodulation by a ERV, such as HRES-1, may contribute to self-reactivity and abnormal T and B-cell functions in SLE.

Natural autoantibodies reactive with glycosaminoglycans in rheumatoid arthritis.

IntroductionAlthough natural autoantibodies make up the majority of circulating immunoglobulins and are also present in high numbers in therapeutically used intravenous immunoglobulin preparations, they have received little attention and their precise role remains largely unknown. An increasing awareness of the importance of posttranslational autoantigen modifications and glycobiology led us to explore carbohydrate-reactive natural autoantibodies in patients with rheumatoid arthritis. This study examined systematic antibodies reactive to glycosaminoglycans (GAGs), the carbohydrate components of proteoglycans that are released in large amounts from degrading cartilage.MethodsTo measure antibodies reactive to six different types of GAGs, a specialised ELISA was used in which the carbohydrates were covalently linked to the plastic surface through a 2 nm spacer. Sera from rheumatoid arthritis patients (n = 66), umbilical cord serum samples (n = 11) and adult controls (n = 54) were studied. In order to explore cross-reactivity with microbial antigens, bacterial peptidoglycans and fungal polysaccharides were used. Sera and synovial fluid samples were also tested using a GlycoChip carbohydrate array to characterise individual carbohydrate recognition patterns. We followed a multistep statistical screening strategy for screening GAG-reactive antibodies as predictive disease markers.ResultsWhile anti-GAG antibodies were absent in the umbilical cord sera, they were readily detectable in adult controls and were significantly elevated in patients with rheumatoid arthritis (p < 0.001). Anti-GAG antibodies showed significant cross-reactivity among different types of GAGs. They also reacted with bacterial peptidoglycans and fungal polysaccharides. Interestingly, anti-chondroitin sulphate C IgM antibody levels showed inverse correlation both with the Disease Activity Score (DAS) 28 scores and C-reactive protein (CRP) levels in rheumatoid arthritis.ConclusionThe highly abundant and cross-reactive, GAG-specific natural autoantibodies in serum may serve as novel disease-state markers in patients with rheumatoid arthritis.

Nitric oxide mediates T cell cytokine production and signal transduction in histidine decarboxylase knockout mice

Histamine is a key regulator of the immune system. Several lines of evidence suggest the role of histamine in T cell activation and accelerated Th1 immune response is a hallmark of histidine decarboxylase knockout (HDC-KO) mice, with a complete lack of endogenously produced histamine. According to our previous work, T lymphocytes produce NO upon activation, and NO is necessary for effective T cell activation. To study the role of histamine in T cell activation, we investigated cytokine production and T cell signal transduction in HDC-KO and wild-type (WT) mice. In the absence of histamine, an elevated IFN-γ mRNA and protein levels of splenocytes (p < 0.001; p = 0.001, respectively) were associated with a markedly increased (2.5-fold, p = 0.0009) NO production, compared with WT animals. Furthermore, histamine treatment decreased the NO production of splenocytes from both WT and HDC-KO mice (p = 0.001; p = 0.0004, respectively). NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate-diethylenetriamine elicited IFN-γ production (p = 0.0002), whereas NO synthase inhibitors NG-monomethyl-l-arginine and nitronidazole both inhibited IFN-γ production (p = 0.002 and p = 0.01, respectively), suggesting the role of NO in regulating IFN-γ synthesis. Cytoplasmic Ca2+ concentration of unstimulated T cells was increased in the HDC-KO mice (p = 0.02), whereas T cell activation-induced δ Ca2+-signal was similar in both HDC-KO and WT animals. Our present data indicate that, in addition to its direct effects on T lymphocyte function, histamine regulates cytokine production and T cell signal transduction through regulating NO production.

The effect of balneotherapy on antioxidant, inflammatory, and metabolic indices in patients with cardiovascular risk factors (hypertension and obesity)-A randomised, controlled, follow-up study

INTRODUCTION The primary objective of our study was to explore the changes of antioxidant, inflammatory, and metabolic parameters in obese and hypertension people patients during balneotherapy and to evaluate the safety of balneotherapy in these participants. METHODS Following randomisation, 22 obese and 20 hypertensive patients underwent balneotherapy with thermal water of 38°C temperature, in 15 sessions of 30 minutes. An additional 22 obese and 20 hypertensive patients served as controls. Antioxidant, inflammatory, and metabolic parameters were determined at baseline, as well as post-treatment and at the end of follow-up (at 15 weeks). RESULTS As regards changes observed in hypertensive patients subjected to balneotherapy, differences could be detected between baseline and post-treatment albumin and haemoglobin A(1c) levels only; however, these were no longer significant after 3 months. Although the difference between transferrin levels determined at the end of balneotherapy and 3 months later was significant, it remained within the physiological range, as well as it was accompanied by normal serum iron level and therefore, it was considered irrelevant. C-reactive protein levels of balneotherapy patients decreased significantly after treatment. In obese patients, haemoglobin A(1c) level decreased after balneotherapy, but this difference was not observed either after 3 months. Similarly, both transferrin and C-reactive protein levels changed from baseline, but not between groups. CONCLUSIONS This study contributes important information regarding the safety of balneotherapy in hypertensive and obese diabetics by showing no alterations of antioxidant, inflammatory, or metabolic indices. The findings of this study confirm that balneotherapy is not contraindicated for hypertensive or obese patients.

Histidine deficiency does not protect against aggrecan-induced arthritis

Increased numbers of mast cells are found in the synovial tissues and fluids of patients with rheumatoid arthritis, and at sites of cartilage erosion. Mast cell activation has been reported for a significant proportion of rheumatoid specimens. Because the mast cell contains potent mediators, including histamine, its potential contributions to the processes of inflammation and matrix degradation have recently become evident; thereafter, we investigated the potential protective effect of histamine deficiency against aggrecan-induced arthritis. To study the role of histamine in rheumatoid arthritis we investigated cartilage proteoglycan (aggrecan)-induced arthritis in histidine decarboxylase knockout (HDC-KO) mice, with complete lack of endogenously produced histamine. Aggrecan-induced arthritis was similar in HDC-KO and wild-type (WT) mice. Arthritis was even more severe in HDC-KO mice than in the WT animals 10 weeks following the immunization with aggrecan (arthritis score 1.2 ± 1.5 and 0 ± 0, respectively; P = 0.01). At later time points, arthritis scores were similar in both HDC-KO and WT mice. Since T-lymphocyte dysfunction has an important role in the pathogenesis of rheumatoid arthritis, next we investigated T-cell signal transduction and cytokine production in HDC-KO and WT mice. In the absence of histamine, elevated INFγ mRNA and protein levels of splenocytes (P < 0.001 and P = 0.001, respectively) were associated with a markedly increased (2.5-fold, P = 0.0009) nitric oxide (NO) production, compared with WT animals. Furthermore, histamine treatment decreased the NO production of splenocytes from both WT and HDC-KO mice (P = 0.001 and P = 0.0004, respectively). NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate-diethylenetriamine (NOC-18) elicited IFNγ production (P = 0.0002), suggesting the role of NO in regulating IFNγ synthesis. The cytoplasmic Ca2+ concentration of unstimulated T cells and the T-cell activation-induced Ca2+ signal were increased in T cells from the HDC-KO mice (P = 0.02 and P = 0.04, respectively), while the T-cell activation-induced CD3 internalization was similar in both HDC-KO and WT animals. Our present data indicate that histamine deficiency does not protect against aggrecan-induced arthritis. The Th1 cytokine pattern and increased NO production may both contribute to the sensitivity of HDC-KO mice to aggrecan-induced arthritis. Furthermore, our data indicate that histamine, in addition to its direct effects on T-lymphocyte function, regulates cytokine production and T-cell signal transduction through regulating NO production.