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Autism Research and Treatment
Volume 2012, Article ID 986519, 10 pages
doi:10.1155/2012/986519
Research Article
Intracellular and Extracellular Redox Status and Free Radical
Generation in Primary Immune Cells from Children with Autism
Shannon Rose, Stepan Melnyk, Timothy A. Trusty, Oleksandra Pavliv,
Lisa Seidel, Jingyun Li, Todd Nick, and S. Jill James
Department of Pediatrics, Arkansas Children’s Hospital Research Institute, University of Arkansas for Medical Sciences,
Little Rock, AR 72202, USA
Correspondence should be addressed to S. Jill James, jamesjill@uams.edu
Received 30 July 2011; Revised 12 August 2011; Accepted 12 September 2011
Academic Editor: Antonio M. Persico
Copyright © 2012 Shannon Rose et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The modulation of the redox microenvironment is an important regulator of immune cell activation and proliferation. To
investigate immune cell redox status in autism we quantified the intracellular glutathione redox couple (GSH/GSSG) in resting
peripheral blood mononuclear cells (PBMCs), activated monocytes and CD4 T cells and the extracellular cysteine/cystine redox
couple in the plasma from 43 children with autism and 41 age-matched control children. Resting PBMCs and activated monocytes
from children with autism exhibited significantly higher oxidized glutathione (GSSG) and percent oxidized glutathione equivalents
and decreased glutathione redox status (GSH/GSSG). In activated CD4 T cells from children with autism, the percent oxidized
glutathione equivalents were similarly increased, and GSH and GSH/GSSG were decreased. In the plasma, both glutathione
and cysteine redox ratios were decreased in autistic compared to control children. Consistent with decreased intracellular and
extracellular redox status, generation of free radicals was significantly elevated in lymphocytes from the autistic children. These
data indicate primary immune cells from autistic children have a more oxidized intracellular and extracellular microenvironment
and a deficit in glutathione-mediated redox/antioxidant capacity compared to control children. These results suggest that the loss
of glutathione redox homeostasis and chronic oxidative stress may contribute to immune dysregulation in autism.
1. Introduction
Autism is a behaviorally defined neurodevelopmental disor-
der that usually presents in early childhood and is charac-
terized by significant impairments in social interaction and
communication and by abnormal repetitive hyper-focused
behaviors. The prevalence of autism spectrum disorders has
increased more than 10-fold in the last two decades, now
affecting one in 110 US children, yet the etiology of these
disorders remains elusive [1]. Glutathione depletion and oxi-
dative stress have been implicated in the pathology of numer-
ous neurobehavioral disorders including schizophrenia [2],
bipolar disorder [3], and Alzheimer’s disease [4]. Accumu-
lating evidence suggests that redox imbalance and oxidative
stress may also contribute to autism pathophysiology. Mul-
tiple biomarkers of oxidative stress have been identified in
blood samples from children with autism [5–12]. Our group
has reported a decrease in concentrations of glutathione
(GSH) and several of its metabolic precursors, an increase
in oxidized glutathione disulfide (GSSG), and a decrease in
glutathione redox ratio (GSH/GSSG) in case-control evalua-
tions of plasma and lymphoblastoid cell lines derived from
children with autism [13–16]. Recently, several interactive
polymorphisms in enzymes regulating glutathione synthesis
were found to be more prevalent in children with autism
suggesting that the glutathione deficit and predisposition to
oxidative stress may be genetically based in some children
[17].
Oxidative stress occurs when cellular antioxidant defense
mechanisms fail to counterbalance endogenous ROS produc-
tion and/or exogenous prooxidant environmental exposures.
Glutathione (γ-L-glutamyl-L-cysteinylglycine) is a tripeptide
that functions as the major intracellular antioxidant and
redox buffer against macromolecular oxidative damage. The
glutathione thiol/disulfide redox couple (GSH/GSSG) is the
predominant mechanism for maintaining the intracellular
2 Autism Research and Treatment
microenvironment in a highly reduced state that is essential
for antioxidant/detoxification capacity, redox enzyme regula-
tion, cell cycle progression, and transcription of antioxidant
response elements (ARE) [18–23]. Subtle variation in the
relative concentrations of reduced and oxidized glutathione
provides a dynamic redox signaling mechanism that regu-
lates these vital cellular processes [24–27]. For example, in
both CNS precursor cells and na¨
ıve immune cells, intracel-
lular glutathione redox status is the primary determinant
modulating the cellular decision to undergo cell cycle arrest,
differentiation, or proliferation [27]. A reducing intracellular
environment is required for proliferation, while a more
oxidized microenvironment favors cell cycle arrest and dif-
ferentiation. A chronic deficit in the GSH/GSSG redox ratio
is considered to be a reliable indicator of oxidative stress and
increased vulnerability to oxidative damage from prooxidant
environmental exposures [28,29].
In the extracellular plasma compartment, the cysteine/
cystine (thiol/disulfide) redox couple independently provides
the ambient redox environment for circulating immune cells.
The ambient extracellular cysteine/cystine redox potential
has been shown to be more oxidized than the intracellular
GSH/GSSG redox potential and is independently regulated
[30]. Dynamic shifts in the plasma cysteine/cystine redox
potential alter the redox status of cysteine moieties in cell
surface proteins to induce conformational changes in protein
structure that can reversibly alter function [31,32]. For ex-
ample, under oxidizing extracellular conditions, redox-sensi-
tive cysteine residues in the catalytic core of protein tyrosine
phosphatases become oxidized and reversibly inactivate
enzyme activity depending on the ambient cysteine/cystine
redox potential [31,33,34]. Extracellular cysteine/cystine
redox status is emerging as an important new signal trans-
duction mechanism that can induce posttranslational alter-
ations in downstream redox-sensitive proteins including a
variety of enzymes, transcription factors, receptors, adhesion
molecules, and membrane signaling proteins resulting in the
dynamic modulation of their activity and function [32,35,
36].
Recent studies have revealed numerous immunologic
abnormalities among children with autism including alter-
ations in immune cell proportions [37–40] and shifts in
helper T-cell subpopulations after mitogenic stimulation
[41,42]. Peripheral blood mononuclear cells (PBMCs) from
individuals with autism have been shown to produce higher
levels of proinflammatory cytokines and abnormal levels of
regulatory cytokines compared to control PBMCs at baseline
and upon mitogenic stimulation [43–46]. Taken together,
the immunological studies suggest a role for a dysregulated
immune system in autism that potentially could be related to
a deficit in glutathione-mediated antioxidant capacity and an
oxidized microenvironment in immune cells. To investigate
this possibility, we examined whether primary immune cells
(PBMCs) from children with autism exhibit decreased in-
tracellular glutathione redox capacity compared to PBMCs
from age-matched control children and whether a more
oxidized intracellular and extracellular microenvironment
is associated with increased production of oxidizing intra-
cellular free radicals. Because immune cells from children
with autism have been shown to have abnormal responses
to stimulation, we also elected to challenge the PBMCs with
immune activators known to promote oxidative stress and
measure the resulting intracellular glutathione redox status
in activated isolated monocytes and T cells.
2. Subjects and Methods
2.1. Participants. This investigation was conducted on a
subset of children from the autism IMAGE (Integrated
Metabolic and Genomic Endeavor) study at Arkansas Chil-
dren’s Hospital Research Institute (ACHRI) that has recruit-
ed over 162 case and control families to date. The IMAGE
cohort for this study consisted of 43 children diagnosed
with autistic disorder and 41 unaffected control children
(16 of which were unaffected siblings). The autism case
families were recruited locally after referral to the University
of Arkansas for Medical Sciences (UAMS), Dennis Devel-
opmental Center and diagnosed by trained developmental
pediatricians. Children aged 3 to 10 with a diagnosis of
autistic disorder as defined by the Diagnostic and Statistical
Manual of Mental Disorders, Fourth Edition (DSM-IV 299.0),
the Autism Diagnostic Observation Schedule (ADOS),
and/or the Childhood Autism Rating Scales (CARS >30)
were enrolled. Children diagnosed with other conditions on
the autism spectrum or rare genetic diseases associated with
symptoms of autism were excluded from the study. Children
with chronic seizure disorders, recent infection, and high-
dose vitamin or mineral supplements exceeding the RDA
were also excluded because these conditions are potential
confounders that could affect redox status. Unaffected sib-
lings and unrelated, neurotypical children aged 3 to 10 with
no medical history of behavioral or neurologic abnormalities
by parent report made up the comparison group. The
protocol was approved by the Institutional Review Board at
UAMS, and all parents signed informed consent.
2.2. Materials. Culture flasks, plates, and pipettes were ob-
tained from Corning Life Sciences (Lowell, Mass, USA).
RPMI 1640, penicillin/streptomycin, Dulbecco’s phosphate-
buffered saline (PBS), fetal bovine serum (FBS), and glu-
tamine were purchased from Life Technologies (Carlsbad,
Calif, USA). Carboxy-H2DCFDA (6-carboxy-2,7-dichlo-
rodihydrofluorescein diacetate, diacetoxymethyl ester) was
obtained from Molecular Probes (Carlsbad, Calif, USA).
Human Monocyte Isolation Kit II and Human CD4 T
cell Isolation Kit II were purchased from Miltenyi Biotec
(Bergisch-Gladbach, Germany). Histopaque-1077 and all
other chemicals were obtained from Sigma-Aldrich (St.
Louis, Mo, USA).
2.3. Isolation of PBMCs and Stimulation of Monocytes and
CD4 T Cells. Fasting blood samples (≤20 mL) were collected
before 9:00 AM into EDTA-Vacutainer tubes and immedi-
ately chilled on ice before centrifuging at 1300 ×gfor10min
at 4◦C. Aliquots of plasma were stored at −80◦C in cryostat
tubes until extraction and HPLC quantification. PBMCs
were isolated by centrifugation over Histopaque-1077. Red
Autism Research and Treatment 3
blood cells were lysed using a brief (15 s) incubation with
1 mL ice-cold water. Approximately, 30 ×106PBMCs were
resuspended in RPMI 1640 medium (supplemented with
10% FBS, 1% penicillin/streptomycin, and 2 mM glutamine)
at a density of 106cells/mL. Note that because we were unable
to obtain 20 mL blood volume from every child, it was not
possible to isolate and analyze monocytes and CD4 T cells
for all participants. For monocyte stimulation, PBMCs were
treated with 0.1 μg/mL lipopolysaccharide (LPS); for T-cell
stimulation, PBMCs were treated with 10 ng/mL phorbol 12-
myristate 13-acetate (PMA) and 1 μg/mL ionomycin. Cells
were placed in a humidified 5% CO2incubator at 37◦C
for 4 hr. Stimulated monocytes and CD4 T cells were then
isolated by negative selection using magnetic cell labeling as
described by the manufacturer (Miltenyi Biotec, Bergisch-
Gladbach, Germany). Using flow cytometry, we determined
that ≥75% of isolated monocytes are positive for CD14 and
that ≥87% of isolated CD4 T cells are positive for CD4. For
HPLC quantification of GSH and GSSG, approximately 2 ×
106unstimulated (resting) PBMCs, stimulated monocytes,
or stimulated CD4 T cells were pelleted, snap frozen on dry
ice,andstoredat−80◦C.
2.4. Cell Extraction and HPLC Quantification of Intracellular
Glutathione and Plasma Cysteine Redox Status. The storage
interval at −80◦C before extraction was consistently between
1-2 weeks after blood draw and cell isolation to minimize
potential metabolite interconversion. The methodological
details for intracellular and extracellular GSH extraction
and HPLC elution and electrochemical detection have been
described previously [15,16], and metabolite detection does
not require derivatization. Although most GSSG is present
as a mixed disulfide with other thiols including cysteine,
our measurements detect only the free GSSG in plasma.
Glutathione and cysteine concentrations were calculated
from peak areas of standard calibration curves using HPLC
software. Intracellular results are expressed as nanomoles
per milligram of protein using the BCA Protein Assay Kit
(Pierce, Rockford, Ill, USA), and plasma results are expressed
as micromoles per liter.
2.5. Measurement of Intracellular Free Radicals. Carboxy-
H2DCFDA (DCF) is a membrane-permeable ROS/RNS-sen-
sitive probe that remains nonfluorescent until oxidized by
intracellular free radicals. The intensity of DCF fluorescence
is directly proportional to the level of free radical oxidation.
Approximately, 106PBMCs were resuspended in 1 mL RPMI
1640 medium supplemented with 10% FBS, 1% penicil-
lin/streptomycin, and 2 mM glutamine and stained in the
dark for 20 min with 1 μMDCFat37
◦C. Stained cells were
washed and resuspended in PBS and analyzed immediately
onaPartecCyFlowflowcytometer(G
¨
orlitz, Germany) using
488 nm excitation wavelength with 530/30 nm (FL1) emis-
sion filter. For each analysis, the fluorescence properties of
10000 cells were collected, and the data were analyzed using
the FCS Express software (De Novo Software, Los Angeles,
Calif, USA). Intracellular free radical levels are expressed as
median fluorescence intensity (MFI) of subject sample DCF
Tab le 1: Demographics of study population.
Case children
n=43
Control children
n=41
Age; mean (SD) 5.42 (1.98) 6.16 (2.29)
Male; n(%) 36 (84) 20 (49)
White; n(%) 38 (88.4) 31 (75.6)
Asian; n(%) 2 (4.65) 0 (0)
African American; n(%) 2 (4.65) 8 (19.5)
Hispanic; n(%) 1 (2.3) 2 (4.9)
OTC multivitamin use; n(%) 17 (39.5) 8 (19.5)
fluorescence normalized to DCF fluorescence of a standard
PBMC preparation. As an internal control, the standard
PBMC preparation was isolated from a 100 mL blood sample
from an unaffected healthy adult volunteer, aliquoted and
frozen at −180◦C in 90% FBS/10% DMSO. An aliquot of
the standard PBMC preparation was stained and analyzed
with each subject sample. Evaluation of oxidizing free radical
production was possible only in those case and unrelated
control samples for which sufficient (∼20 mL) blood volume
was obtained.
2.6. Statistical Analysis. Within the control group, 16 of the
41 unaffected control children were case siblings. There were
27 additional case children without a sibling and 25 addi-
tional unrelated control children comprising the total case-
control cohort of 84 children. To down-weight the impact of
outliers, three metabolites observations were curtailed at the
extremes of the distributions for PBMC GSH, PBMC GSSG,
and Monocytes GSH/GSSG (see footnote in Table 2). The
sibling data are correlated resulting in a combined sample
of correlated and uncorrelated data; thus, the assumption of
all data being independent is not satisfied for the standard
two-sample t-test. To make use of all data from dependent
and independent observations, we used the corrected Z-test
proposed by Looney and Jones [47]. This statistical approach
provides adequate control of Type 1 errors and has more
power than a standard Student’s t-test. Because the DCF data
compared cases and unrelated controls (without siblings)
the standard Student’s t-test was used with significance set
at 0.05. Nonparametric intercorrelations (Spearman correla-
tion coefficients) between age and gender and the 7 outcome
variables, GSH, GSSG, GSH/GSSG, % oxidized glutathione,
cysteine, cystine, and cysteine/cystine were determined with
the significance level set at 0.05. Data was analyzed using SAS
9.2 software (SAS Institute Inc, Cary, NC, USA).
3. Results
3.1. Demographics of Study Population. Ta b l e 1 lists the
demographics of the study population. The only major
differences between cases and controls are that the control
group was composed of a greater proportion of females and
African Americans, whereas the case group had a greater pro-
portion of Asian subjects. Over-the-counter multivitamin
supplement use was higher among cases (39.5%) compared
4 Autism Research and Treatment
Tab le 2: Intracellular glutathione redox status in resting PBMCs and activated monocytes and CD4 T cells.
Metabolite Case children Control children Corrected Z-test
nMean ±SD nMean ±SD Difference
(95% CI) Pvalue
Resting PBMCs
GSH (nmol/mg protein) 43 25.45 ±8.16 41 23.35 ±6.38 2.09 (−1.09, 5.29) 0.19
GSSG (nmol/mg protein) 43 0.90 ±0.3410.66 ±0.23 0.24 (0.13, 0.35) <0.001
GSH/GSSG 43 29.58 ±9.04 41 37.58 ±10.89 −7.99 (−12.51, −3.48) <0.001
Oxidized GSH (%) 43 0.07 ±0.02 41 0.05 ±0.01 0.02 (0.0075, 0.024) <0.001
Activated monocytes
GSH (nmol/mg protein) 18 7.73 ±3.16 20 8.55 ±2.5−0.82 (−2.02, 0.38) 0.18
GSSG (nmol/mg protein) 18 0.62 ±0.24 20 0.47 ±0.17 0.14 (0.03, 0.25) 0.01
GSH/GSSG 18 13.31 ±7.26 20 19.30 ±6.35 −5.98 (−9.99, −1.97) 0.003
Oxidized GSH (%) 18 0.14 ±0.05 20 0.10 ±0.03 0.04 (0.02, 0.07) <0.001
Activated CD4 T cells
GSH (nmol/mg protein) 18 6.82 ±3.01910.16 ±3.74 −3.33 (−5.24, −1.42) <0.001
GSSG (nmol/mg protein) 18 0.68 ±0.29 19 0.63 ±0.24 0.05 (−0.11, 0.22) 0.51
GSH/GSSG 18 10.47 ±4.19 19 17.49 ±6.95 −7.02 (−10.17, −3.87) <0.001
Oxidized GSH (%) 18 0.17 ±0.05 19 0.11 ±0.05 0.05 (0.03, 0.08) <0.001
GSH: glutathione; GSSG: oxidized glutathione disulfide; oxidized GSH: (%)2GSSG/(GSH+2GSSG); curtailment: PBMC GSH >45 set =45 (n=1); PBMC
GSSG >1.75 set =1.75 (n=1); Monocytes GSH/GSSG >35 set =35 (n=1).
to controls (19.5%); however, the glutathione redox status
was statistically unaffected by vitamin use (data not shown).
3.2. Decreased Intracellular Glutathione Redox Status in
Autism. Table 2 presents the relative intracellular concen-
trations of GSH, GSSG, the glutathione redox ratio, and
the percentage of oxidized glutathione equivalents in resting
(unstim-ulated) PBMCs and in isolated stimulated mon-
ocytes and CD4 T cells from children with autism and
age-matched control children. The percent oxidized gluta-
thione is expressed in absolute glutathione equivalents as
2GSSG/(GSH+2GSSG). Relative to controls, the intracellular
concentration of GSSG and the percent oxidized glutathione
were significantly increased (∼40%), and the GSH/GSSG
ratio decreased (∼21%) in PBMCs from children with
autism (P<0.001). After stimulation with LPS, mono-
cytes from children with autism also exhibited significantly
decreased GSH/GSSG (∼31%, P=0.003), increased GSSG
concentration (∼32%, P=0.01), and 40% higher percent
oxidized glutathione (P<0.001). In mitogen-stimulated
CD4 T cells from children with autism, the intracellular GSH
concentration was ∼33% lower, the GSH/GSSG was ∼40%
lower (P<0.001), and the percent oxidized glutathione
was ∼55% higher than in stimulated CD4 T cells from
control children (<0.001). As expected, activation with LPS
and PMA both resulted in decreased intracellular GSH
levels and GSH/GSSG in isolated monocytes and CD4 T
cells compared to resting (unstimulated) PBMCs. Upon
stimulation, there was a greater decrease in intracellular
GSH and GSH/GSSG in both CD4 T cells and monocytes
from children with autism compared to control children.
Neither age nor gender was significantly correlated with any
of the outcome measures. The protein content per 106cells
did not differ between case and control children (data not
shown).
3.3. Decreased Extracellular Glutathione and Cysteine Redox
Status in Autism. Ta b l e 3 presents the relative concentrations
of GSH, GSSG, GSH/GSSG, % oxidized GSH, cysteine,
cystine, and the cysteine/cystine redox ratio in the extracel-
lular plasma compartment. Children with autism exhibited
a significantly decreased extracellular concentration of GSH
(∼21%) and GSH/GSSG (∼54%) and increased concentra-
tion of GSSG and the percent oxidized glutathione (52%
and 82%, resp., P<0.001). Figures 1(a) and 1(b) com-
pare GSH/GSSG and % oxidized glutathione equivalents,
respectively, in plasma, T cells, and monocytes from case and
control children and graphically demonstrates the consistent
decrease in both extracellular and intracellular glutathione
redox status among the case children.
The dynamic balance between the reduced and oxidized
forms of glutathione can also be expressed as the redox
potential or reducing power of the GSH/GSSG redox couple
(Eh) and can be calculated from the Nernst equation, Eh=
E0+RT/nF ln[disulfide]/([thiol 1] ∗[thiol 2]), where E0
is the standard potential for the glutathione redox couple
(−264 mV), Ris the gas constant (8.314 J/◦Kmol), Tis the
absolute temperature of analytical measurement (25◦C=
298◦K), nis 2 for the number of electrons transferred,
and Fis Faraday’s constant (96,485 coulomb/mol) [48].
The calculated Ehvalue for the GSH pool in the children
with autism is −116 mV, which is 12 mV more oxidized
than in the control children, with an Ehvalue of −128 mV
(Table 3).
Autism Research and Treatment 5
Tab le 3: Extracellular (plasma) glutathione and cysteine redox status.
Metabolite Case children Control children Corrected Z-test
nMean ±SD nMean ±SD Difference
(95% CI) Pvalue
Plasma
GSH (μM) 38 1.58 ±0.23 41 1.99 ±0.22 −0.41 (−0.50, −0.31) <0.001
GSSG (μM) 38 0.20 ±0.06 41 0.13 ±0.04 0.07 (0.05, 0.09) <0.001
GSH/GSSG 38 8.24 ±2.20 41 17.14 ±5.54 −8.73 (−10.52, −6.94) <0.001
Oxidized GSH (%) 38 0.20 ±0.05 41 0.11 ±0.03 0.09 (0.07, 0.10) <0.001
Ehfor GSH −116 mV −128 mV
Cysteine (μM) 41 21.7±4.88 41 21.43 ±4.08 0.13 (−1.88, 2.14) 0.90
Cystine (μM) 41 29.2±10.641 19.26 ±4.89.73 (6.25, 13.2) <0.001
Cysteine/Cystine 41 0.79 ±0.18 41 1.14 ±0.18 −0.33 (−0.41, −0.26) <0.001
Ehfor Cysteine −106 mV −111 mV
GSH: glutathione; GSSG: oxidized glutathione disulfide; Eh: steady-state redox potential; Ehfor GSH: −264 mV +(30 mV) ∗log([GSSG]/[GSH]2); Ehfor
cysteine: −250 mV + (30 mV) ∗log([CySSCy]/[Cys]2).
0
5
10
15
20
25
30
GSH/GSSG
Control
Case
Plasma Activated monocytes Activated T cells
∗
∗
∗
(a)
Control
Case
Plasma Activated monocytes Activated T cells
0
0.05
0.1
0.15
0.2
0.25
0.3
Oxidized GSH (%)
∗
∗
∗
(b)
Figure 1: Intracellular and extracellular glutathione redox imbalance in autism. (a) presents the GSH/GSSG in plasma, isolated activated
monocytes, and CD4 T cells from case and control children; (b) presents the % oxidized glutathione equivalents. Both extracellular and
intracellular glutathione redox status are consistently significantly decreased among the case children (∗P<0.01).
The concentration of cystine, the oxidized form of
cysteine, was significantly elevated (∼52%), while the cys-
teine/cystine redox ratio was significantly decreased (∼31%)
in plasma from children with autism (P<0.001). The Eh
value for the cysteine pool can also be calculated from the
Nernst equation (see above) where the E0for cysteine is equal
to −250 mV [30]. The calculated Ehvalue for the cysteine
pool in children with autism is −106 mV, or 5 mV more
oxidized than the control Ehvalue of −111 mV (Table 3).
3.4. Elevated Free Radical Production in Autism. The level of
intracellular free radicals was measured in available resting
PBMCs from children with autism (n=15) and unaffected
control children (n=16) using DCF, an ROS/RNS-sensitive
fluorescent probe. Monocytes and lymphocytes were gated
based on light scatter properties (size and density) and ana-
lyzed separately. Figure 2 presents the median fluorescence
intensity (MFI) of lymphocytes from children with autism
and unaffected control children (normalized to MFI of the
standard PBMC preparation). Gated lymphocytes from chil-
dren with autism exhibited a significantly higher mean level
of intracellular free radicals compared to lymphocytes from
control children (P<0.05). No differences in free radical
production were observed in gated monocytes from case and
control children. Intracellular free radical production was
not correlated with age or gender in this cohort.
4. Discussion
Oxidative stress is generally defined as an imbalance between
oxidant production and endogenous antioxidant defense
mechanisms and can be clinically defined in humans by a
decrease in the redox status of GSH/GSSG and cysteine/cys-
tine thiol/disulfide redox couples [49].Therelativeequi-
librium between reduced and oxidized sulfhydryl groups
defines the ambient redox state. Low glutathione redox status
6 Autism Research and Treatment
has been associated with the pathophysiology of several neu-
robehavioral disorders including schizophrenia [2,50], bipo-
lar disorder [3], alcoholism [51], HIV [52], and Alzheimer’s
disease [53]. This is the first study to evaluate intracellular
glutathione-mediated antioxidant/redox capacity in primary
cells from children with autism as well as the extracellular
plasma cysteine/cystine redox status. Because these two redox
systems are compartmentalized and independently regulat-
ed, evaluation of both redox couples provides a complete
picture of the primary immune cell microenvironment in
children with autism. Supporting and extending our previ-
ous findings of decreased plasma and lymphoblastoid cell
GSH/GSSG, we now report that both primary immune cell
GSH/GSSG and plasma cysteine/cystine redox couples are
similarly compromised resulting in a more oxidized immune
cell microenvironment in children with autism compared to
control children.
Recent evidence supports the notion that subtle fluctu-
ations in ambient redox status may provide an important
regulatory mechanism that can dynamically modulate im-
mune cell function. Activation and proliferation of T cells
require a reducing intracellular microenvironment, whereas
a more oxidized environment promotes cell cycle arrest and
blunted responsiveness to immune stimulation [54–57]. For
example, a mechanism involving extracellular redox mod-
ulation by regulatory T cells (Tregs) was recently elucidated
by Yan et al. [35]. Tregs were shown to inhibit the release of
cysteine into the immune synapse between dendritic cells
and na¨
ıve T cells, which effectively reduces GSH levels in T
cells by eliminating the rate-limiting amino acid for GSH
synthesis. A high ratio of reduced to oxidized glutathione is
required for cell cycle progression from G1 to S phase and
induction of the T-cell proliferative response [55]. Thus, the
more oxidized GSH/GSSG redox state of the intracellular
glutathione pool in PBMCs and in activated CD4 T cells
observed in children with autism (Table 2) would suggest
a hyporesponsive phenotype that is less conducive to T-cell
activation and proliferation. Consistent with this hypothesis,
several recent studies have documented abnormalities in
the adaptive immune response in children with autism
[44,58].
A glutathione deficit in T cells has been shown to nega-
tively affect the adaptive immune response and T-cell pro-
liferation by reducing IL-2 receptor turnover and IL-2-
dependent DNA synthesis [59,60]. In monocytes, an oxi-
dized intracellular environment has been shown to alter the
cytokine profile and skew the Th1 and Th2 balance [61,62].
Studies in mice have demonstrated that the intracellular GSH
content of antigen presenting cells (APCs) reversibly alters
the Th1 and Th2 cytokine response pattern [61]. Specifically,
a GSH deficit reduced Th1-associated IFN-γproduction and
exaggerated Th2-associated IL-4 production. Restoration of
GSH restored the Th1 cytokine response and normalized
the Th2 response. Consistent with these observations, two
independent studies have reported that helper T-cell sub-
populations in PBMCs from children with autism are shifted
towards T helper 2 (Th2) dominance [41,42]. Further, a
decrease in T-cell IL-2 receptor expression has been reported
∗
Intracellular free radicals
(normalized to standard)
Control Case
1.5
1
0.5
0
Figure 2: Intracellular Free Radicals are Elevated in Lymphocytes
from Children with Autism. Intracellular free radicals were mea-
sured in freshly isolated PBMC from children with autism and
unaffected control children using 1uM DCF. Presented is median
fluorescent intensity (MFI) of the gated lymphocyte population
from subject samples normalized to MFI of a standard PBMC
preparation also treated with 1 uM DCF and analyzed with each
subject sample. Lymphocytes from children with autism exhibited
a significantly higher mean level of intracellular free radicals than
controls (P=0.04). Control median (95% CI) =0.576 (0.551–
0.640); case median (95% CI) =0.689 (0.561–1.086).
to be associated with decreased proliferative response after
mitogen stimulation in children with autism [58].
The more oxidized GSH/GSSG redox status in plasma
and primary immune cells in children with autism (Figure 1)
may offer a mechanistic explanation for the abnormal adap-
tive immune response previously reported in these children.
When intracellular oxidative stress exceeds glutathione redox
capacity, cells export GSSG into the plasma as a mechanism
to restore internal redox homeostasis [49,63]. The elevated
GSSG concentrations in PBMCs (Table 2) suggest that
the GSSG export mechanism and intracellular antioxidant
capacity were not sufficient to maintain intracellular redox
homeostasis and that redox imbalance was chronic in these
children. The association between a more oxidized immune
cell microenvironment and an abnormal adaptive immune
response warrants continued investigation especially in light
of the potential reversibility of immune dysfunction with
targeted treatment to restore redox homeostasis [15].
The calculated Ehvalues for the extracellular GSH and
cysteine pools (Table 3) in our control population differ
somewhat from previously published values. In adults, the
plasma glutathione Ehis more reduced at around −137 mV,
and the plasma cysteine redox couple is more oxidized at
−80 mV [30,48]. These discrepancies may reflect method-
ological differences in sample preparation in that our electro-
chemical detection does not require derivatization for detec-
tion. It is also possible that children (age 3–10 years) may
have less reducing capacity than previously reported in adults
(age 25–35 years) [48]. Nonetheless, our calculated Ehvalues
are consistent with previous reports that plasma cysteine Eh
(−111 mV) is more oxidized than that of GSH (−128 mv).
Autism Research and Treatment 7
Mean intracellular free radical production was higher in
primary lymphocytes from children with autism relative to
lymphocytes from age-matched control children (Figure 2)
and was driven by a subset of 5 (33.3%) children whose
lymphocytes exhibited especially high levels of free radicals.
Mitochondria are the primary producers and targets of
intracellular free radicals, and mitochondrial dysfunction has
been postulated to be a contributing factor in the pathogen-
esis of autism and numerous other neurological disorders
[64–67]. In a lymphoblastoid cell model, we previously dem-
onstrated that the GSH/GSSG redox ratio in mitochondria
was significantly lower in autism compared to control cells
and was associated with a significantly lower mitochondrial
membrane potential after nitrosative stress [16]. It is well
established that mitochondria are highly concentrated in
presynaptic terminals and that loss of redox control can neg-
atively affect the efficiency of neurotransmission and synap-
tic plasticity [68,69]. Similarly, mitochondrial localization
and redox signaling at the immunological synapse between
lymphocytes and antigen presenting cells are required for
immune activation, and excessive ROS can interrupt these
signaling pathways [70–72]. A recent study of mitochondrial
defects in lymphocytes from children with autism found
decreased complex I activity and overreplication of and de-
letions in mitochondrial DNA compared to control lym-
phocytes [73]. Based on this evidence, it is plausible to hy-
pothesize that mitochondria may be the source of the
increased levels of lymphocyte free radicals observed in the
subset of autistic children presented in Figure 2. Consistent
with this hypothesis, a recent meta-analysis estimated that
mitochondria dysfunction may affect up to 30% of children
with autism [64]. Based on this evidence, further study of
mitochondrial function and redox status in lymphocytes
from children with autism is warranted.
Relevant to our observations, two recent papers have
revealed that an oxidized extracellular cysteine/cystine redox
status can initiate a redox signaling cascade that stimulates
intracellular mitochondrial ROS production as a mechanism
to initiate an inflammatory immune response [74,75]. The
signal transduction from the extracellular to intracellular
compartments occurs through oxidative modification of
redox-reactive cysteines on cell surface proteins. Exposed
cysteine sulfhydryl groups on proteins can be reversibly oxi-
dized to sulfenic acid or disulfide bonds resulting in altered
protein structure and function that initiate downstream
redox signaling cascades [33,76,77]. In an elegant series
of experiments, Imhoffand Hansen demonstrated that mi-
tochondrial ROS production was significantly increased in
cells incubated under extracellular oxidized cysteine/cystine
redox conditions [74]. The stimulated intracellular ROS
production resulted in the expression of Nrf-2, the transcrip-
tion factor responsible for initiation of the inflammatory
response. Treatment to block the availability of cell surface
cysteine thiol groups abrogated mitochondrial ROS produc-
tion and Nrf-2 expression. Go et al. confirmed and extended
these observations by demonstrating that treatment to main-
tain mitochondrial redox status abrogated ROS production
in the presence of oxidized extracellular cysteine/cystine
[75]. Although the precise mechanism for the oxidative
cysteine/cystine-dependent signaling for mitochondrial ROS
production is not yet clear; the authors provide evidence of
a possible link to changes in the redox state of cytoskeletal
proteins that could be functionally linked to the mitochon-
drial membrane. Other studies have demonstrated that an
oxidized plasma cysteine/cystine redox potential is associated
with proinflammatory conditions [78,79]andcanbemod-
ulated by diet [80,81]. These observations support the pos-
sibility that the oxidized plasma cysteine/cystine in children
with autism may be functionally related to the increase in
lymphocyte free radical production observed and contribute
to immune cell abnormalities in these children.
In summary, we show for the first time that both the
extracellular and intracellular immune cell compartments
are more oxidized in children with autism compared to age-
matched unaffected control children. Randomized clinical
trials will be needed to determine whether treatment to
normalize plasma and intracellular redox status will improve
immune cell function and possibly the health and behavior
in children with autism.
Conflict of Interests
The authors declare no conflict of interests.
Acknowledgments
The authors would like to express their gratitude to the
families in Arkansas affected by autism whose participa-
tion made this study possible. They also acknowledge the
invaluable help of the nurses and clinicians at the Dennis
Developmental Center for referral and evaluation. This
research was supported, in part, with funding from the
National Institute of Child Health and Development (RO1
HD051873; SJJ), the Department of Defense (AS073218P1;
SJJ), and by grants from the Arkansas Children’s Hospital
and Arkansas Biosciences Institute (SJJ).
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