Mitochondrial hyperpolarization and ATP depletion in patients with systemic lupus erythematosus

Abstract

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.

Figure 1

Fluorescence microscopy (top panels) and flow cytometry (bottom panels) of the mitochondrial transmembrane potential (ΔΨm) in peripheral blood lymphocytes (PBLs) from a healthy control subject and a patient with systemic lupus erythematosus (SLE). Cells were cultured for 16 hours in vitro and stained with annexin V–phycoerythrin (PE) (red) and with 3,3′-dihexyloxacarbocyanine iodide (DiOC₆) (green) and visualized by fluorescence microscopy (original magnification × 200). The ΔΨm (green fluorescence; fluorescence channel 1 [FL-1]) was increased in nonapoptotic cells of SLE PBLs, and the frequency of apoptotic cells (red fluorescence; FL-2) was increased in SLE PBLs. Numbers in the upper right corner of the dot-plots show the percentage of annexin V–positive cells; x mean = mean channel fluorescence of DiOC₆ (FL-1) in annexin V–negative cells; numbers at the top of the histograms show the mean channel fluorescence of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1; FL-2) gated on live cells, based on forward/side scatter analysis.

Figure 2

Prominence of mitochondrial hyperpolarization in T cells from SLE patients. A, PBLs were cultured in vitro for 16 hours, and the ΔΨm was measured by DiOC₆ fluorescence (FL-1). T cells were detected by staining with Cy5-conjugated anti-CD3 monoclonal antibody (FL-3 in the dot-plots). Annexin V–positive cells were electronically gated out. Histograms showing the ΔΨm (DiOC₆ fluorescence; FL-1) of CD3-positive cells (open) are overlaid on those showing the ΔΨm of CD3-negative cells (shaded). Numbers at the top of the histograms show the ΔΨm ratio of CD3-positive to CD3-negative cells. B, The ΔΨm ratio of CD3-positive to CD3-negative cells in 25 patients with SLE and 25 healthy donors. See Figure 1 for definitions.

Figure 3

Analysis of cell death in SLE patients and healthy controls in response to stimulation with H₂O₂. PBLs from 15 SLE patients and 15 controls were cultured in the presence and absence of 50 μM H₂O₂ for 16 hours and analyzed by fluorescence microscopy and flow cytometry. A, Fluorescence micrographs of H₂O₂-stimulated PBLs from representative control and SLE patient donors. Cells were stained with annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI). After 16 hours' incubation in the absence of H₂O₂ (no treatment), the frequency of annexin V–FITC–staining apoptotic cells was increased in SLE PBLs. After 16 hours of treatment with H₂O₂, the frequency of apoptotic cells was lower in SLE PBLs, while that of necrotic cells with swollen nuclei directly staining with PI was elevated (original magnification × 400). B, Flow cytometric analysis of apoptotic (annexin V–positive and PI-negative; lower right quadrant) and necrotic (annexin V–positive and PI-positive; upper right quadrant) cells after stimulation with 50 μM H₂O₂ for 16 hours. PBLs were from different donors than those used in A. C, Apoptosis rates were determined in 15 healthy donors and 15 SLE patients, based on the percentage of annexin V–phycoerythrin (PE)–positive/PI-negative cells. D, After H₂O₂ treatment, necrosis was prominent in SLE patients compared with healthy controls. Data points correspond to the percentage of PI-positive cells within the annexin V–positive population induced by H₂O₂ treatment. See Figure 1 for other definitions.

Figure 4

Effect of H₂O₂ treatment on the production of reactive oxygen intermediates (ROIs) in SLE patients. A, ROI levels were monitored in live cells by flow cytometry after staining with annexin V–phycoerythrin (PE; FL-2) and dihydrorhodamine (DHR; FL-1) (dot-plots). Numbers in the upper right corner of the dot-plots show the percentage of annexin V–positive cells; numbers at the top of the first row of histograms show the mean channel fluorescence of DHR (FL-1) in annexin V–negative cells. Histograms of H₂O₂-treated cells (shaded) are overlaid on those of untreated control cells (open). ROI production was also monitored by hydroethidine (HE) staining. Numbers at the top of the second row of histograms show the mean channel fluorescence of HE-positive cells (FL-2) gated on annexin V–FITC-negative cells (FL-1). B, Correlation between H₂O₂-induced ROI production and apoptosis, as determined by Pearson's correlation coefficient. Increases in apoptosis and ROI production were estimated by annexin V positivity (Δannexin V) and DHR fluorescence (ΔDHR), respectively. C, H₂O₂-induced ROI production in SLE patients and controls. Bars show the mean ± SEM of H₂O₂-induced changes in DHR and HE fluorescence in PBLs from SLE patients and controls. See Figure 1 for other definitions.

Figure 5

H₂O₂-induced changes in ΔΨm in SLE patients and healthy controls. PBLs from 15 control donors and 15 SLE patients were cultured in media for 16 hours in the presence and absence of 50 μM H₂O₂. A, The ΔΨm was monitored 20 minutes and 16 hours after exposure to H₂O₂ by concurrent staining with annexin V–phycoerythrin (PE) (FL-2) and DiOC₆ (FL-1) (dot-plots and first row of histograms) or staining with JC-1 alone (second row of histograms). Numbers in the upper right corner of the dot-plots show the percentage of annexin V–positive cells; numbers at the top of the first row of histograms show the mean channel fluorescence of DiOC₆ (FL-1) of annexin V–negative cells. Histograms of H₂O₂-treated cells (shaded) are overlaid on those of untreated control cells (open). The FL-2 fluorescence of JC-1 aggregates is shown in the second row of histograms. Numbers at the top of the second row of histograms show the mean channel fluorescence of JC-1–positive cells (FL-2). B, H₂O₂-induced changes in ΔΨm in SLE and control donors. Bars show the mean ± SEM of changes in ΔΨm measured by DiOC₆ and JC-1 fluorescence after 20 minutes of H₂O₂ treatment. See Figure 1 for other definitions.

Figure 6

Cell surface phenotyping of T cells from SLE patients and controls during H₂O₂-induced apoptosis. A, Representative dot-plots of PBLs costained with annexin V–phycoerythrin (PE; FL-2) and Cy5-conjugated anti-CD3 monoclonal antibody (FL-3), after treatment with H₂O₂ for 16 hours. Numbers in the upper right corner show the percentage of CD3-positive cells within the annexin V–positive compartment. B, After treatment with H₂O₂, the percentage of CD3-positive cells within the annexin V–positive compartment was diminished in patients with SLE. See Figure 1 for other definitions.

Figure 7

A, Intracellular ATP and ADP content in PBLs from 24 SLE patients, 10 rheumatoid arthritis (RA) patients, and 17 healthy donors. Cells were cultured for 16 hours in vitro and assayed for ATP and ADP content. Values are the mean ± SEM. B, In parallel, cell aliquots were stained with DiOC₆ and JC-1, and the ΔΨm was measured by flow cytometry. The correlation between intracellular ATP levels and the ΔΨm in patients with SLE was calculated using Pearson's correlation coefficient. See Figure 1 for other definitions.

Figure 8

A, Effect of CD3/CD28 stimulation and oligomycin treatment on intracellular ATP content and the ΔΨm (DiOC₆ fluorescence) in normal PBLs. PBLs from 4 healthy donors were pretreated with CD3/CD28 antibodies or 2.5 μM oligomycin. Values are the mean ± SEM percentage of control cells incubated in parallel (n = 4 independent experiments). B, Effect of pretreatment with CD3/CD28 (1 hour) or oligomycin (2.5 μM for 30 minutes) on H₂O₂-induced elevation of the ΔΨm in PBLs from 4 healthy donors. Values are the percentage elevation of the ΔΨm elicited by exposure to H₂O₂ (50 μM for 20 minutes) in CD3/CD28- or oligomycin-pretreated cells compared with control cells. C, Effect of pretreatment with CD3/CD28 (1 hour) or oligomycin (2.5 μM for 30 minutes) on H₂O₂-induced necrosis of PBLs from healthy donors. Untreated (control), oligomycin-pretreated, and CD3/CD28-pretreated PBLs were exposed to 50 μM H₂O₂ for 16 hours, and necrosis was assessed by the ratio of propidium iodide–positive/annexin V–positive cells. Values are the mean ± SEM of 6 independent experiments. See Figure 1 for definitions.