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Departments of
* Medicine,
Microbiology and Immunology, and
Pathology, State University of New York, College of Medicine, Syracuse, NY 13210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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
(pHi) elevation, followed by increased ROI production.
Baseline m, ROI production, and pHi 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-
1,
and IFN-
led to transient 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 pHi with no impact on
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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Disruption of the mitochondrial transmembrane potential
(m) has been proposed as the point of no
return in apoptotic signaling (8, 9, 10, 11). The
m is dependent upon the electron transport
chain transferring electrons from NADH to molecular oxygen and proton
transport mediated by the
F0F1-ATPase complex
(12). The energy stored in the electrochemical gradient is
used by F0F1-ATPase to
convert ADP to ATP during oxidative phosphorylation. We have previously
shown that elevation m, i.e., mitochondrial
hyperpolarization, occurs in the early phase of Fas-induced apoptosis
of Jurkat human leukemia T cells and normal human PBL
(13). Mitochondrial hyperpolarization precedes
phosphatidylserine externalization and disruption of
m in Fas (13)- and
H2O2-induced apoptosis
(14). These observations were confirmed and extended to
p53 (15), TNF-
(16), and
staurosporin-induced apoptosis (17). Elevation of
m is independent from activation of
caspases and represents an early event in apoptosis (13, 15). Recently, deviations in key mitochondrial checkpoints have
been associated with abnormal apoptosis of SLE T cells:
m and mitochondrial reactive oxygen
intermediate (ROI) production were elevated, whereas glutathione (GSH)
levels were diminished compared with healthy or rheumatoid arthritis
(RA) controls. Low GSH was consistent with increased ROI production due
to use of reducing equivalents. The m
elevation was correlated with ATP depletion and predisposition of lupus
T cells to necrosis, which in turn may significantly contribute to
inflammation in patients with SLE (18).
With m hyperpolarization and extrusion of
H+ ions from the mitochondrial matrix, the
cytochromes within the electron transport chain become more reduced,
which favors generation of ROI (19). ROIs mediate
signaling initiated via the CD3/CD28 receptors (20, 21).
Moreover, T cell activation via CD3/CD28 receptors induces
mitochondrial hyperpolarization and ROI production in normal PBL
(18). Abnormal T cell signaling has been related to an
imbalance of cytokine production in SLE (22). ROIs
influence activity of transcription factors AP-1 and NF-
B (23, 24), and, further downstream, may lead to the skewed expression
of IL-2, TNF, and IL-10 (25, 26). Increased spontaneous
apoptosis of lymphocytes has been linked to increased IL-10 production,
release of Fas ligand, and overexpression of Fas receptor in SLE
(27). Because increased ROI levels confer sensitivity to
H2O2, NO, TNF, and
Fas-induced cell death (13, 28), elevated baseline
m and ROI production may have key roles in
altered activation and death of lupus T cells. In the present study, we
show that mitochondrial hyperpolarization and increased ROI production
are associated with intracellular alkalinization in lupus patients.
IL-10, which is produced at elevated levels by lupus PBL, has a
differential impact on m, ROI production,
and intracellular pH (pHi) in lymphocytes of
lupus patients with respect to healthy and RA controls. IL-10 induced
mitochondrial hyperpolarization of control PBL without influencing
m of lupus PBL. Moreover, IL-10 enhanced
ROI production and cell death in lupus PBL without affecting ROI levels
or survival of control PBL. Ab-mediated IL-10 blockade or stimulation
with IL-12 normalized ROI production and pHi with
no impact on m of lupus PBL. The results
suggest that mitochondrial hyperpolarization and increased ROI
production play crucial roles in altered IL-10 responsiveness in
SLE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fifteen patients with SLE were investigated. All patients
satisfied the criteria for a definitive diagnosis (29). A
total of 13 females (age 39.3 ± 5.3 years; range 18–63) and 2
males (age 44.8 ± 10.2 years; range 25–55) was studied. As
controls, 10 age- and sex-matched healthy subjects and 10 patients with
RA (8 females, age 51.3 ± 6.7 years; 2 males, age 54.0 ±
0.0 years) (30) were studied. RA patients were treated
with methotrexate, sulfasalazine, cyclosporin A, leflunomide, or
etanercept. Of the 15 SLE patients, 11 were receiving prednisone (5–50
mg/day) and 13 were treated with immunosuppressive drugs, including
hydroxychloroquine (200–400 mg/day) and azathioprine (50 mg/day) or
methotrexate (7.5 mg/wk). Disease activity was assessed by the SLE
disease activity index (SLEDAI) score (31). Twelve
patients had a SLEDAI 
10 and were considered relatively
inactive. The remaining 3 patients with SLEDAI >10 were considered
active. The study has been approved by the Institutional Review Board
for the Protection of Human Subjects.
Cell culture, T cell activation, and cytokine treatments
PBMCs were isolated from heparinized venous blood on
Ficoll-Hypaque gradient. PBL were separated after the removal of
monocytes by adherence to autologous serum-coated petri dishes
(32). PBL were resuspended at 106
cells/ml in RPMI 1640 medium, supplemented with 10% FCS, 2 mM
L-glutamine, 100 IU/ml penicillin, and 100 µg/ml
gentamicin in 12-well plates at 37°C in a humidified atmosphere with
5% CO2. Cross-linking of the CD3 Ag was
performed by addition of PBL to plates precoated with 1 µg/ml/well
OKT3 mAb (CRL 8001 from American Type Culture Collection, Manassas, VA)
for 1 h at 37°C. CD28 costimulation was performed by addition of
500 ng/ml mAb CD28.2 (BD PharMingen, San Diego, CA). Proliferative
responses were assessed by [3H]TdR
incorporation assay (32). Following 72-h incubation and
subsequent pulsing of 105 PBL with 0.4 µCi
[3H]TdR per microtiter plate well,
[3H]TdR incorporation by CD3/CD28-costimulated
cells (12,129 ± 978 cpm) was enhanced with respect to
unstimulated control PBL (90 ± 24 cpm; p <
0.0001). In comparison with control donors, CD3/CD28-induced
[3H]TdR incorporation was diminished in lupus
PBL (7,888 ± 1,016; p < 0.0001). Cells were also
cultured in the presence or absence of human recombinant cytokines IL-2
(50–500 U/ml), IL-3 (10–100 ng/ml), IL-4 (10–100 ng/ml), IL-6 (5–50
ng/ml), IL-7 (5–50 ng/ml), IL-10 (10–100 ng/ml), IL-12 (5–50 ng/ml),
IL-15 (50–500 ng/ml), TNF- (20 ng/ml),
TGF-1 (5–50 ng/ml), and IFN- (500 U/ml).
All cytokines were obtained from PeproTech (Rocky Hill, NJ). Polyclonal
goat anti-human IL-10-neutralizing Ab was obtained from R&D Systems
(Minneapolis, MN).
Cell viability assays
Apoptosis was monitored by observing cell shrinkage and
nuclear fragmentation, and quantified by flow cytometry after
concurrent staining with fluorescein-conjugated annexin V (annexin
V-FITC; FL-1; R&D Systems) and propidium iodide (PI; FL-2), as
described earlier (13, 28, 33, 34). Staining with
PE-conjugated annexin V (annexin V-PE; R&D Systems) was used to monitor
phosphatidylserine externalization (FL-2) in parallel with measurement
of ROI levels and m (see below). Apoptosis
rates were expressed as percentage of annexin V-positive/PI-negative
cells. Necrosis was assessed by observing cellular and nuclear swelling
(3). Swollen nuclei of necrotic cells were observed by
staining with PI (50 µg/ml). Necrotic cells were enumerated by direct
PI staining using flow cytometry and fluorescence microscopy
(18). Necrosis rates were expressed as percentage of
PI-positive population within annexin-positive cells. As described
earlier (28, 35), live or apoptotic cells did not stain
directly with PI and required permeabilization with 0.1% Triton X-100.
When using hydroethidine (HE; FL-2) for ROI measurement, cells were
costained with annexin V-FITC (FL-1; R&D Systems). Thus, annexin V-PE
or annexin V-FITC was matched with emission spectra of potentiometric
and oxidation-sensitive fluorescent probes. Specific combinations are
described in each figure legend. Staining with annexin V alone or in
combination with dihydrorhodamine 123 (DHR), HE,
3,3'-dihexyloxacarbocyanine iodide (DiOC6),
chloromethyl-X-rhosamine (CMXRos), or tetramethylrhodamine methyl
esther (TMRM) (see below) was conducted in 10 mM HEPES, pH 7.4, 140 mM
NaCl, and 2.5 mM CaCl2.
Flow cytometric analysis of ROI production and
m
Production of ROI was assessed fluorometrically
using oxidation-sensitive fluorescent probes
5,6-carboxy-2',7'-dichlorofluorescein-diacetate (DCFH-DA), DHR,
and HE (Molecular Probes, Eugene, OR), as described earlier
(28). Following apoptosis assay, cells were washed three
times in 5 mM HEPES-buffered saline, pH 7.4; incubated in
HEPES-buffered saline with 0.1 µM DHR for 2 min, 1 µM DCFH-DA for
15 min, or 1 µM HE for 15 min; and samples were analyzed using a
FACStarPlus flow cytometer (BD Biosciences, Palo
Alto, CA) equipped with tunable argon ion laser delivering 200 mW power
at 488 nm. Fluorescence emission from
5,6-carboxy-2',7'-dichlorofluorescein (DCF; green) or DHR (green) was
detected at a wavelength of 530 ± 30 nm. Fluorescence emission
from oxidized HE, ethidium (red), was detected at a wavelength of 605
nm. Dead cells and debris were excluded from the analysis by electronic
gating of forward and side scatter measurements. Although R123, the
fluorescent product of DHR oxidation, binds selectively to the inner
mitochondrial membrane, ethidium and DCF remain in the cytosol of
living cells. DHR and HE were more sensitive than DCF for measurement
of ROI production. m was estimated by
staining with 20 nm DiOC6 (Molecular Probes), a
cationic lipophilic dye (8, 36, 37), for 15 min at 37°C
in the dark before flow cytometry (excitation, 488 nm; emission, 525 nm
recorded in FL-1). Fluorescence of DiOC6 is
oxidation independent and correlates with m
(37). The m was also
quantitated using a potential-dependent J-aggregate-forming lipophilic
cation,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine
iodide (JC-1) (38). JC-1 selectively incorporates into
mitochondria, where it forms monomers (fluorescence in green, 527 nm)
or aggregates, at high transmembrane potentials (fluorescence in red,
590 nm) (38, 39). Cells were incubated with 0.5 µM JC-1
for 15 min at 37°C before flow cytometry.
m changes were also confirmed by staining
with 1 µM CMXRos (excitation, 579 nm; emission, 599 nm recorded in
FL-2) and 1 µM TMRM (excitation, 543 nm; emission, 567 nm recorded in
FL-2; all from Molecular Probes). Cotreatment with a protonophore, 5
µM carbonyl cyanide m-chlorophenylhydrazone
(Sigma-Aldrich, St. Louis, MO) for 15 min at 37°C resulted in
decreased DHR, DiOC6, CMXRos, TMRM, and JC-1
fluorescence, and served as a positive control for disruption of
m (13). Each measurement was
conducted on 10,000 cells.
ATP measurement
Intracellular ATP levels were determined using the luciferin-luciferase method (40). A total of 5 x 106 PBL that had been cultured in vitro for 16 h was collected by centrifugation and washed in PBS. The pellet was resuspended in 50 µl of PBS and mixed with equal volumes of 2.5% trichloroacetic acid. Such extracts were stored at -20°C. The total protein content of each sample was determined using the Lowry assay (41). ATP content of PBL from SLE patients and control donors was assayed in parallel. The bioluminescence assay was performed in an AutoLumat LB953 automated luminometer (Berthold, Wildbad, Germany) using an ATP determination kit (Molecular Probes), according to the manufacturer’s instructions. ATP standard curves were established in each experiment and were linear in the 5–5000 nM range. To eliminate the impact of nonspecific inhibitors in the cellular extracts, standard amounts of ATP were added to the reaction mixtures as controls, and ATP levels were remeasured (42). The sample volume added to the reaction mixtures was less than 2% of the total assay volume. ATP/ADP ratio and ADP levels were assessed with the ApoGlow kit (Lumitech, Nottingham, U.K.).
Intracellular pH measurements
The pHi measurements were conducted with flow cytometry using the pH-sensitive dye carboxy seminaphthorhodafluor-1 (SNARF-1)-acetoxymethyl ester acetate (SNARF-1; Molecular Probes), as described earlier (43). SNARF-1 enters cells passively as a nonpolar ester. It is then hydrolyzed by intracellular esterases into a polar compound unable to leave membrane-intact cells. The emission spectrum of SNARF-1 undergoes a pH-dependent wavelength shift. To assess pHi, PBL were centrifuged at 1000 rpm for 10 min and resuspended in PBS. SNARF-1 was added to the cells at 5 µg/ml, and the samples were then incubated for 30 min at 37°C. Fresh 0.5 mg/ml stock solutions of SNARF-1 were made in DMSO for each experiment. After incubation, cells were washed in PBS and analyzed by flow cytometry. The dye was excited with 200 mW of the 488 nm argon laser, and fluorescence was collected in two wavelengths (FL2, 580 nm, and FL3, 650 nm) in the pulse-processing mode. The pHi was calculated from the FL3/FL2 ratio. A standard calibration curve was generated for each experiment by staining the cells in high K+ buffers of varying pH values (120 mM KCl, 30 mM NaCl, 0.5 mM MgSO4, 1 mM CaCl2, 1 mM NaHPO4, 5 mM glucose, and 10 mM HEPES) and in the presence of 5 µg/ml nigericin (Sigma-Aldrich; diluted from a stock solution of 500 µg/ml in ethanol) to equilibrate the intracellular/extracellular pH.
IL-10 production
IL-10 levels were measured in supernatants of PBL after 5-day culture in vitro using an ELISA kit (Endogen, Woburn, MA).
Statistics
Results were analyzed by Student’s t test or Mann-Whitney rank sum test for nonparametric data. Correlation was measured using Pearson’s correlation coefficient. Changes were considered significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m, ROI production, and
pHi in peripheral blood T cells
PBL of patients with SLE exhibit increased spontaneous
(5) and diminished activation-induced apoptosis
(7). Previous studies suggested that an increase of
m or mitochondrial hyperpolarization was an
early event in apoptosis and T cell activation (13, 18).
Apoptosis has been generally characterized by cellular acidification
that is required for activity of caspases (44). However,
growth factor withdrawal-induced apoptosis of hemopoetic cells has
recently been associated with initial mitochondrial hyperpolarization
and cytoplasmic alkalinization (45). T cell activation by
mitogenic lectins or stimulation of CD3 or CD2 receptors also leads to
cytoplasmic alkalinization (46, 47). Therefore, changes in
pH may be relevant for signaling abnormalities in SLE. The
pHi was determined based on the FL3/FL2 ratio of
SNARF-1 fluorescence. A standard calibration curve was generated for
each experiment by staining the cells in high K+
buffers of pH values varying between 7.0 and 7.55 (Fig. 1
A). CD3/CD28 costimulation of
normal PBL was accompanied by an increase of pHi,
i.e., alkalinization, as early as 6 min and lasted up to 16 h
following T cell activation (Fig. 1B). By 72 h,
pHi returned to baseline. Induction of apoptosis
by serum withdrawal or treatment with 1 µg/ml Fas Ab or 50 µM
H2O2 caused a transient
elevation of pHi lasting for up to 3 h,
followed by a drop below baseline (not shown). These results were
consistent with intracellular acidification at later stages of cell
death in correlation with previous findings (44). In
parallel, m was assessed by the
potentiometric dyes, monitoring DiOC6
fluorescence in annexin V negative and red fluorescence of
JC-1 (FL-2), as described earlier (13, 14, 18). CD3/CD28
costimulation led to elevation of m,
beginning as early as 6 min and peaking 
30 min to 1 h after
treatment (Fig. 1B), as noted by both
DiOC6 and JC-1 measurements. CD3/CD28-induced
mitochondrial hyperpolarization was followed by depletion of
intracellular ATP 30–60 min later. ATP depletion lasted up to 4 h,
then returned to baseline levels (Fig. 1B). ROI production,
as monitored by DHR and HE fluorescence, increased gradually and became
maximal 72 h after CD3/CD28 costimulation (Fig. 1B).
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m, ROI production,
and pHi are elevated, while T cell activation-induced
changes are blunted in patients with SLE
Because pHi can affect processing of
activation and apoptosis signals, it was assessed in PBL of lupus
patients and healthy as well as RA controls. As shown in Figs. 1
A and 2A, baseline pHi was
increased in patients with SLE (7.236 ± 0.021) with respect to
healthy (7.151 ± 0.019; p = 0.013) or RA controls
(7.17 ± 0.02; p = 0.03). A pH calibration
curve was generated in each experiment (Fig. 1A). In
accordance with previous findings (18), elevation of
m and ROI production were noted in lupus
patients with respect to healthy and RA controls. By contrast,
CD3/CD28-induced mitochondrial hyperpolarization, ROI production, and
elevation of pHi were reduced in lupus patients
(pHi: +0.069 ± 0.019) as compared with
healthy controls (pHi: +0.155 ± 0.026;
p = 0.005). No significant differences were noted
between healthy and RA donors (pHi:
+0.133 ± 0.019) studied in parallel (Fig. 2B).
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m and ROI production
by IL-10 in normal and lupus lymphocyte PBL
Aberrant T cell activation has been associated with altered
lymphokine milieu in SLE (22). Moreover, cytokines also
influence susceptibility of T cells to apoptosis (48).
Therefore, we investigated the effect of IL-2 (50–500 U/ml), IL-3
(10–100 ng/ml), IL-4 (10–100 ng/ml), IL-6 (5–50 ng/ml), IL-7 (5–50
ng/ml), IL-10 (10–100 ng/ml), IL-12 (5–50 ng/ml), IL-15 (50–500
ng/ml), TNF- (20 ng/ml), TGF-1 (5–50
ng/ml), and IFN- (500 U/ml) on m. As
shown in Fig. 3, IL-3 (10 ng/ml), IL-10
(10 ng/ml), TGF-1 (5 ng/ml), and IFN- (500
U/ml) significantly augmented m after
incubation for 1–16 h in normal PBL. With regard to the lymphokines
causing mitochondrial hyperpolarization, increased production of IL-10
(26, 49), while diminished levels of IFN-
(50) and TGF-1 (51)
were noted in patients with SLE. Following 5-day incubation, IL-10
production, ROI levels, m, and pH were
determined in lupus and control PBL. Indeed, IL-10 production was
increased in PBL of lupus patients (934.7 ± 85.8 pg/ml) in
comparison with healthy controls (566.1 ± 58.9 pg/ml;
p = 0.014). IL-10 production by lupus PBL correlated
with SLEDAI (Fig. 4, p =
0.0006). As described earlier, m did not
correlate with disease activity or medications (18).
Because increased production of IL-10 may account, at least in part,
for mitochondrial dysfunction, effect of IL-10 in control and lupus PBL
was comparatively analyzed. Treatment with IL-10 increased
DiOC6 (+45.5 ± 7.4%; p =
0.008) and JC-1 fluorescence (+33.9 ± 6.4%; p =
0.0019), i.e., elicited mitochondrial hyperpolarization (Fig. 5), while it did not affect ROI
production (Fig. 5) or apoptosis of control PBL (Fig. 4). By contrast,
IL-10 enhanced ROI production, as measured by increased DHR (+28.8
± 5.5%; p = 0.0005) and HE fluorescence (+38.4
± 6.4%; p = 0.0002) without influencing
m of lupus PBL (Fig. 5). IL-10 induced
apoptosis and necrosis of lupus PBL (Fig. 4). Selective induction of
cell death by IL-10 in lupus vs control PBL was consistent with
previous findings (27). IL-10 increased
pHi in control (+0.156 ± 0.026;
p = 0.0011) and lupus cells as well (+0.069 ±
0.018; p = 0.02; Fig. 5).
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Because IL-10 levels were elevated in patients with SLE and IL-10
increased m of normal PBL (Fig. 3), we
investigated the effect of Ab-mediated blockade of IL-10 and
stimulation by the IL-10 antagonist lymphokine IL-12 on mitochondrial
dysfunction of lupus T cells. As shown in Fig. 5, pretreatment of lupus
PBL with 25 µg/ml neutralizing IL-10 Ab for 16 h significantly
diminished ROI production, as measured by DHR (-19.9 ± 6.9%;
p = 0.017) and HE fluorescence (-19.0 ± 4.0%;
p = 0.0011). Pretreatment with 10 ng/ml IL-12 also
reduced DHR (-15.4 ± 6.8%; p = 0.02) and HE
fluorescence (-24.2 ± 5.4%; p = 0.005) in lupus
cells. Neither Ab to IL-10 nor IL-12 affected ROI production by normal
or RA PBL (Fig. 5). Ab to IL-10 (p = 0.03), but
not IL-12 (p = 0.056), reduced
pHi of lupus PBL (Fig. 5). Baseline
m of normal or RA PBL or mitochondrial
hyperpolarization of lupus PBL was not influenced by IL-10 Ab or IL-12.
TGF-1 did not affect ROI production,
pHi, or mitochondrial hyperpolarization of lupus
PBL (data not shown).
The effect of IL-10, Ab to IL-10, IL-12, and
TGF-1 on CD3/CD28-induced mitochondrial
hyperpolarization, ROI production, and cytoplasmic alkalinization was
also investigated in lupus patients and healthy as well as RA controls.
Pretreatment for 16 h with Ab to IL-10 and IL-12 increased
CD3/CD28-induced ROI production in lupus PBL comparable with those in
normal and RA controls (Fig. 6A). Ab to IL-10 and IL-12
also enhanced CD3/CD28-induced cytoplasmic alkalinization in lupus PBL
(Fig. 6B). Neither Ab to IL-10 nor IL-12 normalized
CD3/CD28-induced m elevation in lupus PBL.
TGF-1 had no significant effect on
CD3/CD28-induced changes in m, ROI
production, or pHi (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m (negative inside and positive
outside) is of key importance for ATP synthesis and ROI production,
thus regulating cellular activation, survival, and death signals.
Although disruption of the mitochondrial membrane potential has been
proposed as the point of no return in apoptotic signaling
(8, 9, 10), recent data from this laboratory indicate that
elevation of m, i.e., mitochondrial
hyperpolarization, occurs in the early phase of Fas- and
H2O2-induced apoptosis of
Jurkat human leukemia T cells and normal human PBL (13, 14). Abnormal apoptosis of SLE T cells has been associated with
deviations in key mitochondrial checkpoints:
m and mitochondrial ROI production were
elevated, whereas GSH levels were diminished compared with healthy or
RA controls (18). Intracellular ATP content and ATP/ADP
ratio were reduced and correlated with m
elevation in lupus. Mitochondrial hyperpolarization can be induced by T
cell activation via CD3/CD28 costimulation or inhibition of
F0F1-ATPase by oligomycin.
The resultant ATP depletion sensitizes T cells for necrosis, which may
significantly contribute to inflammation in patients with SLE. This was
consistent with a role for intracellular ATP concentration in the
decision of the cell to die via apoptosis or necrosis
(52). With m hyperpolarization
and extrusion of H+ ions from the mitochondrial
matrix, the cytochromes within the electron transport chain become more
reduced, which favors generation of ROI (19). Thus,
mitochondrial hyperpolarization is a likely cause of increased ROI
production, and may be ultimately responsible for increased spontaneous
cell death in patients with SLE. Mitochondrial hyperpolarization also
occurred during T cell activation, which indicated that this event
represents an early and reversible step in apoptosis. In parallel with
mitochondrial hyperpolarization, T cell activation through CD3/CD28
costimulation also elicited cytoplasmic alkalinization. In comparison
with healthy and RA controls, lymphocytes of lupus patients exhibited
cytoplasmic alkalinization, while CD3/CD28-induced changes in
m, ROI production, and
pHi were diminished in SLE. Because
caspase-mediated apoptosis requires a disruption of
m and cellular acidification, baseline
mitochondrial hyperpolarization and cytoplasmic alkalinization
represent an altered state of T cell activation rather than ongoing
apoptosis in patients with SLE.
The present data reveal that, similar to CD3/CD28 costimulation,
certain lymphokines, such as IL-3, IL-10,
TGF-1, and IFN-, can also elicit
a transient elevation of m. Among these
lymphokines, production of IL-10 was increased in patients with SLE and
correlated with disease activity. IL-10 elicited cell death of lupus
PBL, while it did not influence viability of normal PBL, in accordance
with previous observations (27). IL-10 had diametrically
opposing effects on m and ROI
production in lupus patients on the one hand and healthy as well as
RA controls in contrast. Although m was
increased by IL-10 in control and RA PBL, it remained unchanged after
IL-10 stimulation of lupus PBL. Maximal baseline hyperpolarization may
account for the inability of IL-10 or CD3/CD28 costimulation to further
augment m in SLE. In contrast, ROI
production was selectively increased by IL-10 in lupus PBL. Of note,
ROI were shown to increase expression of IL-10 mRNA (25)
and IL-10 secretion (53), suggesting that increased IL-10
levels and ROI production represent interacting components of a
positive feedback loop, amplifying cell death signaling in patients
with SLE (18). IL-10 blockade or stimulation with
antagonistic lymphokine IL-12 reduced ROI production and
pHi without affecting baseline
m in SLE. This suggested that increased
IL-10 production was not responsible for mitochondrial
hyperpolarization; however, it may have contributed to ROI production
and alkalinization. Moreover, IL-10 blockade or IL-12 normalized
CD3/CD28-induced ROI production and cytoplasmic alkalinization without
affecting m of lupus PBL. These results, in
agreement with previous studies (27, 54), indicate that
increased IL-10 production may partially interfere with
CD3/CD28-induced T cell activation in SLE. Alternatively,
TGF-1 had no significant effect on baseline or
CD3/CD28-induced changes in m, ROI
production, and pHi, arguing against its role in
aberrant T cell activation. Indeed, diminished production of
TGF-1 may be important in augmented Ig
production by lupus B cells (51).
The m reflects the energy stored in the
electrochemical gradient across the inner mitochondrial membrane,
which, in turn, is used by
F0F1-ATPase to convert ADP
to ATP during oxidative phosphorylation. Both T cell activation and
apoptosis require the energy provided by ATP (55).
Mitochondrial hyperpolarization also occurs during T cell activation,
which indicates that this event represents an early and reversible step
in apoptosis. In this study, we showed that stimulation of normal T
cells through the CD3 and CD28 receptors or incubation with IL-3,
IL-10, TGF-1, and IFN- caused a transient
elevation of m. Thus, repetitive T cell
activation in vivo could be responsible for prolonged mitochondrial
hyperpolarization. However, repeated T cell activation through CD3/CD28
costimulation, Fas stimulation, or IL-10 treatment at 1- to 6-day
intervals did not elicit persistent m
elevation, but led to a disruption of m,
followed by activation-induced cell death (data not shown), as
described earlier (18, 56). The lack of correlation
between IL-10 levels and m as well as the
failure of IL-10 blockade or IL-12 treatment to reduce
m argue against a role for IL-10 in
mitochondrial hyperpolarization in SLE. Incubation of lupus PBL up to 5
days in vitro failed to affect the extent of
m elevation with respect to control PBL.
Persistent mitochondrial hyperpolarization may originate from an
intramitochondrial block affecting reversal of commonly arising
activation signals. There are a number of potential mechanisms
underlying m elevation (11).
Lupus patients exhibit deficiency of protein kinase A (57, 58) that may favor intramitochondrial translocation of Bad, a
Bcl-2 family protein (59, 60). Other mitochondrial
membrane ion transporters,
OH-/Pi antiporter,
K+/H+ antiporter,
Na+/H+ exchanger,
H+/Pi and
H+/pyruvate symotransporters, and/or electrogenic
K+ uniporter (12), may also be
involved. Diminished ADP import through the voltage-dependent anion
channel of the outer membrane and/or the adenine nucleotide
translocator of the inner membrane could interfere with use of the
electrochemical gradients and result in elevation of
m. The latter mechanism is favored by a
relative increase of the ADP/ATP ratio in cytosol of lupus PBL
(18).
Mitochondrial hyperpolarization predisposes for increased ROI
production (19). Oxidative stress affects activity of
transcription factors AP-1 and NF-B (23, 24), and,
further downstream, may lead to the skewed expression of IL-2, TNF, and
IL-10 (25). Increased spontaneous apoptosis of lymphocytes
has been linked to increased IL-10 production, release of Fas ligand,
and overexpression of Fas receptor in SLE (27). Because
increased ROI levels confer sensitivity to
H2O2, NO, TNF, and
Fas-induced cell death (13, 28), elevated baseline
m, ROI production, and
pHi may have key roles in altered activation and
death of lupus T cells. Although mitochondrial hyperpolarization was
not affected, IL-10 Ab or IL-12 normalized ROI production and
intracellular alkalinization in lupus PBL. Therefore, while the
mechanism of mitochondrial hyperpolarization is further investigated,
the present study supports a role for IL-10 antagonists in correcting
signaling dysfunction and treatment of patients with SLE.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andras Perl, Department of Medicine, State University of New York, 750 East Adams Street, Syracuse, NY 13210. E-mail address: perla{at}upstate.edu
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; m, mitochondrial transmembrane potential; CMXRos, chloromethyl-X-rosamine; DCF, 5,6-carboxy-2',7'-dichlorofluorescein; DCFH-DA, DCF-diacetate; DHR, dihydrorhodamine 123; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; GSH, reduced glutathione; HE, hydroethidine; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide; pHi, intracellular pH; PI, propidium iodide; ROI, reactive oxygen intermediate; SLEDAI, SLE disease activity index; SNARF, seminaphthorhodafluor; TMRM, tetramethylrhodamine methyl ester.
Received for publication March 25, 2002. Accepted for publication May 6, 2002.
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S. Fiorucci, A. Mencarelli, E. Distrutti, M. Baldoni, P. del Soldato, and A. Morelli Nitric Oxide Regulates Immune Cell Bioenergetic: A Mechanism to Understand Immunomodulatory Functions of Nitric Oxide-Releasing Anti-Inflammatory Drugs J. Immunol., July 15, 2004; 173(2): 874 - 882. [Abstract] [Full Text] [PDF]
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