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. 2002 Jul 15;169(2):1092-101.
doi: 10.4049/jimmunol.169.2.1092.

Persistent mitochondrial hyperpolarization, increased reactive oxygen intermediate production, and cytoplasmic alkalinization characterize altered IL-10 signaling in patients with systemic lupus erythematosus

Peter Gergely Jr  1 , Brian Niland, Nick Gonchoroff, Rudolf Pullmann Jr, Paul E Phillips, Andras Perl
Affiliations

Persistent mitochondrial hyperpolarization, increased reactive oxygen intermediate production, and cytoplasmic alkalinization characterize altered IL-10 signaling in patients with systemic lupus erythematosus

Peter Gergely Jr et al. J Immunol. .

Abstract

Abnormal death signaling in lymphocytes of systemic lupus erythematosus (SLE) patients has been associated with elevation of the mitochondrial transmembrane potential (Delta psi(m)) 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 (pH(i)) elevation, followed by increased ROI production. Baseline Delta psi(m), ROI production, and pH(i) 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-beta(1), and IFN-gamma led to transient Delta psi(m) 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 pH(i) with no impact on Delta psi(m) 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.

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Figures

FIGURE 1
FIGURE 1
A, Measurement of pHi based on the FL3/FL2 ratio of SNARF-1 fluorescence. A standard calibration curve was recorded for each experiment by SNARF-1 staining of PBL incubated in high K+ buffers of pH values preset between 7.0 and 7.55. Histograms from control donor and a representative lupus patient show SNARF-1 fluorescence recorded without (open curves) and with CD3/CD28 costimulation for 1 h (shaded curves). Values over histograms indicate pHi based of standard calibration. B, Effect of CD3/CD28 costimulation on the ΔΨm, ROI production, ATP content, and pHi of normal PBL. The ΔΨm was measured by DiOC6 and JC-1 fluorescence, while ROI production was assessed by DHR and HE fluorescence of annexin V-negative cells. Data represent mean of three independent experiments. DiOC6, JC-1, DHR, and HE fluorescence as well as ATP levels are shown along the left y-axis relative to baseline (0 h) values normalized at 100.0%. Absolute pHi values are represented along the right y-axis.
FIGURE 2
FIGURE 2
A, Increased ROI production, mitochondrial hyperpolarization, and cytoplasmic alkalinization in PBL of lupus patients with respect to healthy and RA controls. Before measurements, PBL from 10 healthy controls, 15 patients with SLE, and 10 patients with RA were cultured in vitro for 16 h. The Δψm was measured by DiOC6 and JC-1 fluorescence, while ROI production was assessed by DHR and HE fluorescence of annexin V-negative cells and displayed on the left y-axis with respect to mean of controls normalized at 100.0%; pHi values are represented along the right y-axis. B, Effect of CD3/CD28 costimulation on ROI levels (DHR and HE fluorescence), Δψm (DiOC6 and JC-1 fluorescence), and pHi. ROI levels were assessed 6 h, while Δψm and pHi were determined 1 h after CD3/CD28 costimulation. Columns and error bars represent mean ± SEM.
FIGURE 3
FIGURE 3
Effect of IL-3 (10 ng/ml), IL-10 (10 ng/ml), TGF-β1 (5 ng/ml), and IFN-γ (500 U/ml) on Δψm (DiOC6 and JC-1 fluorescence) and ROI productions (DHR and HE fluorescence) of control PBL. Significant elevation of Δψm was noted after 1-h exposure to each cytokine (p < 0.01). Relative fluorescence values are shown along the y-axis with respect to baseline (0 h) values normalized at 100.0%. Data represent mean ± SEM of four experiments.
FIGURE 4
FIGURE 4
A, Correlation of IL-10 production and SLEDAI in patients with SLE. IL-10 production was measured in supernatant of PBL cultured in vitro for 5 days. Correlation between IL-10 levels and SLEDAI was calculated using Pearson's correlation coefficient. B, Effect of IL-10 on cell survival in control and lupus PBL. After 16-h treatment with 10 ng/ml IL-10, cell survival was analyzed by flow cytometry following staining with annexin V-FITC and PI. Dot plots (row 1) show percentage of apoptotic (annexin V-positive and PI-negative, lower right quadrants) and necrotic (annexin V-positive and PI-positive, upper right quadrants) cells. Histograms in row 2 indicate the percentage of annexin V-positive population, representing total cell death (M1). ROI production was monitored in parallel by HE and annexin V-FITC staining. Values over histograms (row 3) show mean channel HE fluorescence (FL2) gated on annexin V-FITC-negative (FL1) cells.
FIGURE 5
FIGURE 5
Effect of treatment with IL-10 (10 ng/ml), Ab to IL-10 (25 μg/ml anti-IL-10), and IL-12 (10 ng/ml) on Δψm (DiOC6 and JC-1 fluorescence), ROI production (DHR and HE fluorescence), and pHi in PBL from healthy controls and patients with SLE or RA. Values of p indicate significant effects by IL-10; *, p values < 0.05 for effects by anti-IL-10 and IL-12.
FIGURE 6
FIGURE 6
Effect of IL-10 (10 ng/ml), anti-IL-10 (25 μg/ml), and IL-12 (10 ng/ml) on CD3/CD28 induced changes in Δψm (DiOC6 and JC-1 fluorescence, A), ROI production (DHR and HE fluorescence, A), and pHi (B). PBL from 10 patients with SLE were pretreated for 16 h with IL-10 (10 ng/ml), anti-IL-10 (25 μg/ml Ab), or IL-12 (10 ng/ml), then washed and stimulated with CD3 and CD28 mAbs. The Δψm and pHi were assessed 1 h, while ROI production was determined 6 h after CD3/CD28 costimulation. Values of p reflect significant enhancement of CD3/CD28-induced ROI production and elevation of pHi following pretreatment with anti-IL-10 and IL-12.
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