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. 2004 Sep 15;173(6):3676-83.
doi: 10.4049/jimmunol.173.6.3676.

Nitric oxide-dependent mitochondrial biogenesis generates Ca2+ signaling profile of lupus T cells

Gyorgy Nagy  1 , Maureen Barcza, Nick Gonchoroff, Paul E Phillips, Andras Perl
Affiliations

Nitric oxide-dependent mitochondrial biogenesis generates Ca2+ signaling profile of lupus T cells

Gyorgy Nagy et al. J Immunol. .

Abstract

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.

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Figures

Figure 1
Figure 1
Effect of CD3/CD28 costimulation on NO production and Ca2+ signaling in T cells from 11 control and 19 lupus donors. NO production was monitored by DAF-FM fluorescence (A) while [Ca2+]c and [Ca2+]m levels were assessed by Fluo-3 (B) and Rhod-2 fluorescence (C), respectively, in CD3+/annexin V− cells. D, Detection of CD3+/annexin V− cell population with elevated Δψm, [Ca2+]c, and [Ca2+]m as well as and NO production. Following CD3/CD28 costimulation, Δψm was measured by TMRM fluorescence (FL-2); [Ca2+]c and [Ca2+]m levels were assessed by Fluo-3 (FL-1) and Rhod-2 fluorescence (FL-2), while NO production was monitored by DAF-FM fluorescence (FL-1) gated on CD3+/annexin V− cells. Values in dot plots indicate mean channel FL-1 and FL-2 fluorescence, respectively. Results are expressed as RF values with respect to those of unstimulated cells normalized at 1.0 (100%). Data present mean ± SE. Error bars are occasionally covered by data symbols. Values of p reflect differences between 19 lupus and 11 control donors.
Figure 2
Figure 2
Rapid Ca2+ fluxing in CD3/CD28 costimulated PBL from 19 lupus and 11 control donors. PBL were preloaded with Fluo-3 and stimulated with OKT3 and anti-CD28 mAbs while Fluo-3 fluorescence of CD3+/annexinV− T cells was continuously recorded by flow cytometry. Results are expressed as RF values with respect to those of unstimulated cells normalized at 1.0. Data present mean ± SE. Numbers over each time point indicate p values reflecting differences between lupus and control donors.
Figure 3
Figure 3
Assessment of Δψm (A) and mitochondrial mass (B) in lupus and control T cells. PBL from 11 healthy controls and 19 patients with SLE were stimulated with CD3/CD28 Abs for 4 and 24 h. Δψm was monitored by TMRM and mitochondrial mass was assessed by MTG fluorescence in CD3+/annexin V− cells. Results are expressed as RF values with respect to those of unstimulated cells normalized at 1.0. Data present mean ± SE. Values of p reflect differences between lupus and control donors.
Figure 4
Figure 4
Transmission electron microscopy of control and lupus PBL. Images represent analysis of 100 cells per donor from five healthy and five lupus subjects. Size scale is shown for each image. Green arrows show mitochondria of normal size, while red arrows indicate megamitochondria. Apoptotic cells (AC) with shrunken size and fragmented nuclei and necrotic cells (NC) with round-shaped swollen nuclei and cytoplasm lacking mitochondria are marked.
Figure 5
Figure 5
Assessment of NO production in control and lupus monocytes. NO production was monitored by DAF-FM fluorescence in CD14+/annexin V− cells. Data reflect mean ± SE of measurements in 10 healthy and 11 lupus donors.
Figure 6
Figure 6
Effect of NO pretreatment on CD3/CD28-induced Ca2+ fluxing. A, Dose-dependent induction by NO donor NOC-18 of MHP monitored by TMRM, elevation of [Ca2+]c monitored by Fluo-3, and mitochondrial mass assessed by MTG fluorescence. Values in dot plots (row 1) indicate mean channel FL-1 (Fluo-3) and FL-2 fluorescence (TMRM), respectively. Values over histograms (row 2) indicate mean channel of MTG fluorescence (FL-1). Histograms of NOC-18-treated cells (open curves) are overlayed on control cells (shaded curves). B, Monitoring of NO levels by DAF-FM fluorescence in cells exposed to 200 µM NOC-18 for 24 h and subsequent CD3/CD28 stimulation for 4 h. C, Effect of NO on rapid Ca2+ fluxing induced by CD3/CD28 costimulation. PBL were pretreated with 200 µM NOC-18 for 24 h, washed, preloaded with Fluo-3, exposed to CD3/CD28, and analyzed by flow cytometry. D, Effect of NO on CD3/CD28-induced MHP. After pretreatment with 200 µM NOC-18 for 24 h, PBL were stimulated with CD3/CD28 for 24 h and Δψm was assessed by TMRM fluorescence in CD3+/annexin V− cells. E, Effect of NO on sustained elevation of [Ca2+]c. After pretreatment with 200 µM NOC-18 for 24 h, PBL were stimulated with CD3/CD28 for 24 h and [Ca2+]c levels were assessed by Fluo-3 fluorescence in CD3+/annexin V− cells. Data in C–E represent mean ± SE of independent experiments using four healthy donors. Values of p reflect the effect of NOC-18 pretreatment.
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