cytochrome-c-t and Lupus-Erythematosus--Systemic

Mitochondria, main producers of reactive-oxygen species (ROS), were studied to examine their role in the pathogenesis of systemic lupus erythematosus (SLE). PBMCs and mitochondria were isolated from SLE patients and healthy volunteers for various parameters. Mitochondrial ROS, swelling, hyperpolarization and levels of cytochrome c, caspase3 in the cells were assessed by flow cytometry. ROS was significantly increased in SLE patients (SLE vs controls: 1.83 ± 1.03 vs 1.10 ± 0.35; p < 0.0001). Depolarized state of mitochondria was greater in patients (SLE vs controls: 7.10 ± 5.50% vs 2.5 ± 1.8%; p < 0.05). Mitochondria swelling was found to be significantly altered in patients (SLE vs controls: 112.65 ± 36.56 vs 60.49 ± 20.69; p < 0.001). Expression of cytochrome c and caspase 3 (SLE vs controls: 1.37 ± 0.37% vs 1.01 ± 0.03%; 1.57 ± 0.46% vs 1.06 ± 0.07%; p < 0.05) respectively was found to be significantly increased in SLE. Further, the enzymatic activity of mitochondrial complex was assessed in isolated mitochondria. A significant decrease in activity of Complex I (SLE vs controls: 11.79 ± 3.18 vs 15.10 ± 6.38 nmol NADH oxidized/min/mg protein, p < 0.05); Complex IV (SLE vs control: 9.41 ± 5.16 vs 13.56 ± 5.92 nmol cytochrome c oxidized/min/mg protein, p < 0.05) and Complex V (SLE vs controls: 4.85 ± 1.39 vs 6.17 ± 2.02 nmol ATP hydrolyzed/min/mg protein, p < 0.05) was found in SLE patients in comparison to healthy controls. However, Complex II did not show significant variation in either group (SLE vs controls: 42.2 ± 28.6 vs 61.71 ± 42.3 nmol succinate oxidized/min/mg protein; ns). The decrease in enzyme activities of mitochondrial Complexes I, IV and V on one hand and ROS, hyperpolarization and apoptosis on the other points toward a possible role of mitochondria in the pathogenesis of lupus.

Topics: Adult; Apoptosis; Case-Control Studies; Caspase 3; Cytochromes c; Electron Transport Chain Complex Proteins; Female; Humans; Leukocytes, Mononuclear; Lupus Erythematosus, Systemic; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; Mitochondrial Swelling; Reactive Oxygen Species

Cystamine, a disulphide metabolite, has been demonstrated to ameliorate various lupus-associated tissue damages by animal models. However, effects of cystamine on apoptosis of cardiac tissue, a main cardiac damage attributing to lupus, are less obvious. Therefore, we aimed to investigate whether or not cystamine possesses anti-apoptotic effects with emphasis on LV tissue of lupus-prone mice NZB/W-F1. Cystamine treatment was performed by daily intraperitoneal administration. Morphology and apoptotic status of ventricular tissues in the treated mice were assessed by microscopy and TUNEL assay, respectively. Levels of apoptotic biomarkers were determined using immunoblot. Our results revealed that cystamine significantly attenuated the apoptosis of LV tissues in NZB/W-F1 mice, whereas the morphology of the tissues was slightly altered. In addition, cystamine reduced level of Fas and inhibited activation of caspase-8. Cystamine also increased level of Bcl-2 and phosphorylation of Bad, and decreased level of Bad and truncated Bid (tBid). Moreover, level of cytosolic cytochrome c and Apaf-1, and activation of caspase-9 and caspase-3 were suppressed in response to cystamine treatment. In Balb/c mice, as normal control mice, changes in cell morphology and levels of the tested apoptotic components were found insignificant in the LV tissues. These findings indicate that cystamine treatment attenuates apoptosis of LV tissues of NZB/W-F1 mice through suppressing both intrinsic and extrinsic apoptotic pathways. Therefore, cystamine is considered beneficial to alleviating lupus-associated cardiac damages.

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-Associated Death Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Caspase 8; Caspase 9; Cystamine; Cytochromes c; fas Receptor; Heart Ventricles; Immunoblotting; In Situ Nick-End Labeling; Lupus Erythematosus, Systemic; Male; Mice; Mice, Inbred BALB C; Mice, Inbred NZB; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

Autoreactive T cell activation is a consistent feature of murine lupus; however, the mechanism of such activation remains unclear. We hypothesized that naive CD4+ T cells in lupus have a lower threshold of activation through their TCR-CD3 complex that renders them more susceptible to stimulation with self-Ags. To test this hypothesis, we compared proliferation, IL-2 production, and single cell calcium signaling of naive CD4+ T cells isolated from Fas-intact MRL/+(Fas-lpr) mice with H-2k-matched B10.BR and CBA/CaJ controls, following anti-CD3 stimulation in the presence or absence of anti-CD28. We also assessed the responsiveness of naive CD4+ T cells isolated from Fas-intact MRL and control mice bearing a rearranged TCR specific for amino acids 88-104 of pigeon cytochrome c to cognate and low affinity peptide Ags presented by bone marrow-matured dendritic cells. TCR transgenic and wild-type CD4+ T cells from MRL mice displayed a lower threshold of activation than control cells, a response that was class II MHC dependent. The rise in intracellular calcium in MRL vs controls was enhanced and prolonged following anti-CD3 triggering, suggestive of proximal defects in TCR-engendered signaling as the mechanism for the observed hyperactivity. These findings were observed as early as 1-2 mo postweaning and, based on analysis of F1 T cells, appeared to be dominantly expressed. This genetically altered threshold for activation of MRL T cells, a consequence of a proximal defect in CD3-mediated signal transduction, may contribute to the abrogation of T cell tolerance to self-Ags in lupus.

Topics: Animals; Antigen Presentation; Autoimmunity; Calcium Signaling; CD4-Positive T-Lymphocytes; Cell Proliferation; Columbidae; Cytochromes c; Dendritic Cells; fas Receptor; Genes, Dominant; Interleukin-2; Lupus Erythematosus, Systemic; Lymphocyte Activation; Mice; Mice, Inbred MRL lpr; Mice, Inbred Strains; Mice, Knockout; Mice, Transgenic; Phenotype; Receptor-CD3 Complex, Antigen, T-Cell; Receptors, Antigen, T-Cell; Signal Transduction