Analysis of the thiol status of peripheral blood leukocytes in rheumatoid arthritis patients

Article (PDF Available)inJournal of Leukocyte Biology 81(4):934-41 · May 2007with11 Reads
DOI: 10.1189/jlb.0806533 · Source: PubMed
Abstract
Although the exact etiology of rheumatoid arthritis (RA) remains unknown, there is increasing evidence that reactive oxygen species and a pro-oxidant/antioxidant imbalance are an important part of the pathogenesis of joint tissue injury. Flow cytometry was used to evaluate the thiol status [surface-thiols and intracellular glutathione (iGSH)] of leukocytes from RA patients and controls. Levels of surface-thiols and iGSH of leukocytes from RA patients were significantly lower than of leukocytes from controls. CD53, a glycoprotein of the tetraspanin superfamily, which coprecipitates with the GSH recycling enzyme gamma-glutamyl transpeptidase, was elevated significantly on leukocytes from RA patients compared with leukocytes from controls. Surface-thiols and GSH play important roles in redox buffering of cells, providing protection from oxidative stress. The chronic inflammation of RA has been associated with oxidative stress, which is shown to cause a decline in the levels of cellular antioxidant sulfhydryls (R-SH). As antioxidant-protective levels also decline with age, the problem is compounded in older RA patients, who did have fewer R-SH. Chronic stress can also have an effect on telomere lengths, determining cell senescence and longevity. Although telomeres shorten with increasing age, our flow cytometry studies indicate that accelerated shortening in telomere lengths occurs with increasing age of RA patients, suggesting premature cellular aging. The paradox is that lymphocytes from RA patients are believed to resist apoptosis, and we suggest that the elevated expression of CD53, which results from the increased oxidative stress, may protect against apoptosis.
Analysis of the thiol status of peripheral blood leukocytes in
rheumatoid arthritis patients
Joan H. Pedersen-Lane,* Robert B. Zurier,
and David A. Lawrence*
,1
*Wadsworth Center, New York State Department of Health, Albany, New York, USA; and
Department of Medicine,
Division of Rheumatology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
Abstract: Although the exact etiology of rheuma-
toid arthritis (RA) remains unknown, there is in-
creasing evidence that reactive oxygen species and
a pro-oxidant/antioxidant imbalance are an impor-
tant part of the pathogenesis of joint tissue injury.
Flow cytometry was used to evaluate the thiol sta-
tus [surface-thiols and intracellular glutathione
(iGSH)] of leukocytes from RA patients and con-
trols. Levels of surface-thiols and iGSH of leuko-
cytes from RA patients were significantly lower
than of leukocytes from controls. CD53, a glyco-
protein of the tetraspanin superfamily, which co-
precipitates with the GSH recycling enzyme -glu-
tamyl transpeptidase, was elevated significantly on
leukocytes from RA patients compared with leuko-
cytes from controls. Surface-thiols and GSH play
important roles in redox buffering of cells, provid-
ing protection from oxidative stress. The chronic
inflammation of RA has been associated with oxi-
dative stress, which is shown to cause a decline in
the levels of cellular antioxidant sulfhydryls (R-
SH). As antioxidant-protective levels also decline
with age, the problem is compounded in older RA
patients, who did have fewer R-SH. Chronic stress
can also have an effect on telomere lengths, deter-
mining cell senescence and longevity. Although
telomeres shorten with increasing age, our flow
cytometry studies indicate that accelerated short-
ening in telomere lengths occurs with increasing
age of RA patients, suggesting premature cellular
aging. The paradox is that lymphocytes from RA
patients are believed to resist apoptosis, and we
suggest that the elevated expression of CD53,
which results from the increased oxidative stress,
may protect against apoptosis. J. Leukoc. Biol. 81:
934 –941; 2007.
Key Words: glutathione CD53 flow cytometry telomeres
INTRODUCTION
Rheumatoid arthritis (RA) is a chronic, systemic, inflamma-
tory, autoimmune syndrome, which produces degradation of
articular cartilage and bone erosion. The long-term outcomes of
this progressive disease are significant morbidity, loss of func-
tional capacity, and increased mortality [1, 2]. RA affects
1–2% of the general population worldwide [3], and the occur-
rence in women is three times greater than in men. Although
the onset of RA can occur at any age, the incidence increases
with age. The exact etiology of RA remains unknown.
The formation and scavenging activity of free radicals in
biological systems have been linked closely to a number of
pathological conditions. In healthy individuals, reactive oxygen
species (ROS) and associated oxidative stresses are kept in
check by a combination of antioxidant activities [4, 5]. Human
cells have developed a formidable antioxidant defense against
oxidant reactions. In particular, they possess enzymatic and
nonenzymatic antioxidant molecules, including thiols [mainly
glutathione (GSH)], for defense. One key chemical barrier
against stress-induced damage is the redox equilibrium of
sulfhydryl (SH)/disulfides, by which low molecular weight thi-
ols can be oxidized reversibly to disulfides and/or protein
mixed disulfides in response to an oxidative stress [68].
There is increasing evidence that ROS and the resulting pro-
oxidant/antioxidant imbalance play a major role in RA, as well
as in other disease states [9 –11]. It has been shown that
lymphocytes, which are highly sensitive to thiol modification,
are impaired in many of their immune functions when exposed
to oxidative stress or SH modifiers such as those found in the
cellular microenvironment [12].
-Glutamyl transpeptidase (GGT) is a cell-surface enzyme
in the recycling pathway of GSH, part of the antioxidant
defense mechanism. Through GGT cleavage of -glutamyl from
GSH, the cysteinylglycine dipeptide is released, thus eventu-
ally increasing the supply of cysteine available to the cell [13].
Studies indicate that the metabolism of extracellular GSH by
GGT is also important in T cell signaling and in the activation
of transcription factors [14]. CD53, a glycoprotein of the tet-
raspanin superfamily, has been reported to coprecipitate with
GGT activity [15]. The functional significance of this associa-
tion is still not fully understood; the association may be im-
portant in regulating local intracellular redox potential by
playing a role in the binding or recycling of the end products
of the GGT reaction. It has been proposed that tetraspanins act
as molecular facilitators, grouping specific cell-surface pro-
1
Correspondence: Biggs Laboratory, Wadsworth Center, New York State
Department of Health, Empire State Plaza, Albany, NY 12201-0509, USA.
E-mail: lawrenced@wadsworth.org
Received August 28, 2006; revised October 14, 2006; accepted November
8, 2006.
doi: 10.1189/jlb.0806533
934 Journal of Leukocyte Biology Volume 81, April 2007 0741-5400/07/0081-934 © Society for Leukocyte Biology
teins together, thereby increasing the formation of stable and
functioning signaling complexes [16]. Overexpression of CD53
in stably transfected cells resulted in elevated levels of GSH
and reduced levels of peroxides [17]. Increases in mRNA
transcripts and protein expression of CD53 have been associ-
ated with increased resistance to H
2
O
2
, UVB, -irradiation,
and apoptosis [18]. The ligation of CD53, with an associated
reduction in caspase activation, triggers a survival response
and reduces the number of cells that enter apoptosis [19]. It
has been known for some time that resistance to apoptotic cell
death through low-level, proapoptotic or high-level, antiapo-
ptotic stimuli can initiate and perpetuate autoimmune dis-
eases, including RA [20].
Several studies have demonstrated links between chronic
oxidative stress, as occurs with chronic inflammation, and
shortened telomeres, which are determinants of cell senes-
cence and accelerated aging [21, 22]. Telomeres are unique
DNA-protein complexes consisting of G-rich hexanucleotide
repeats, located at the termini of eukaryotic chromosomes.
These complexes play an important role in maintaining chro-
mosomal integrity. In the absence of compensatory mecha-
nisms, they are not fully replicated with each cell division, as
a result of limitations of DNA polymerase to complete repli-
cation to the ends of a linear chromosome, and they, therefore,
shorten with each replication. When the telomeres have short-
ened sufficiently, the cell is arrested in senescence [23]. In a
study using normal human endothelial cells, mild, chronic
oxidative stress, induced by interference with GSH-dependent,
antioxidant defenses, accelerated telomere erosion and the
onset of replicative senescence. Aging is closely associated
with an oxidative shift in the thiol/disulfide redox state of the
intracellular GSH (iGSH) and cysteine pools, with a decline in
cellular thiol concentration [24].
In light of the “inflamm-aging” model of longevity [25], we
determined if the chronic inflammation of RA contributes to a
faster decline of aging-associated biomarkers. Our analysis of
the thiol status (surface-thiols and iGSH) of peripheral blood
leukocytes (PBL) from RA patients revealed significant differ-
ences compared with the cellular thiols status of leukocytes
from a normal, non-RA control group. The decline of cellular
thiols of leukocytes from RA patients inversely affected the
cell-surface levels of CD53. In addition, the mean length of
telomeres (adjusted for age) was shorter in RA patients than in
non-RA control subjects, an indication that chronic stresses
accelerate cellular aging.
MATERIALS AND METHODS
Specimen collection
EDTA venous blood samples were obtained with informed consent from
healthy volunteers and from RA patients who fulfilled the 1987 criteria for RA
by the American Rheumatism Association and were in functional Class I, II,
or III (Table 1), according to the revised criteria of the American College of
Rheumatology [26]. Patients with an active disease duration of at least 6
months, as manifest by at least three joints that were swollen and six joints that
were tender at the time of the blood donation, were accepted for the study
(Table 2). In addition, RA patients had an erythrocyte sedimentation rate
28, a CRP 1.4, or morning stiffness of at least 45 min in duration. All
standard therapy for RA including nonsteroidal anti-inflammatory drugs, dis-
ease-modifying antirheumatic drugs (DMARDs), and combinations of
DMARDs were allowed as long as doses were stable for 2 months prior to
participation in the study. Specimens were randomized blindly for the different
analyses, and not all specimens collected were used for all analyses; no
analyses were excluded. The New York State Department of Health Institu-
tional Review Board (IRB; Protocol #04-025), University of Massachusetts
Medical School IRB, and the New England IRB approved participation in this
study.
Flow cytometric analysis
For analysis of surface-thiols, all steps were done using ice-cold HBSS (Sigma
Chemical Co., St. Louis, MO, USA). Whole blood (100 l) was washed to
remove any serum thiols and resuspended in 200 L, 25 M AlexaFluor
488-C
5
maleimide (Invitrogen, Carlsbad, CA, USA), for 15 min on ice in the
dark. The blood was then diluted to 4 mL with HBSS, washed twice, resus-
pended in 100 l cold HBSS, and stained with one or more of the following
mAb: CD3-PerCP, CD45-allophycocyanin (APC), CD4-PE, CD8-PE, CD19-
PE, CD16/56-PE, and CD14-PE (BD Biosciences, San Jose, CA, USA). The
suspension was incubated for 30 min on ice, lysed with a 1 solution of
Pharmlyse (BD Biosciences), washed twice, and resuspended in 300 l HBSS.
The specimens were analyzed with a FACSCalibur flow cytometer (BD Bio-
sciences) using CellQuest software (BD Biosciences).
For determination of iGSH levels, PBMC were isolated on a Histopaque-
1077 (Sigma Chemical Co.) gradient, washed twice, and resuspended in PBS
(Sigma Chemical Co.) at 10
7
cells/ml. Aliquots (100 l) were stained with one
or more of the following mAb: CD3-FITC, CD4-PE, CD8-PE, CD19-PE,
CD16/56-PE, and CD14-PE (BD Biosciences). The specimens were incubated
30 min on ice, washed thrice, and resuspended in 100 l PBS. Cells were fixed
with 1% paraformaldehyde (Polyscience, Inc., Warrington, PA, USA) for 10
min on ice, washed once, and resuspended in 5 mM N-ethylmaleimide (NEM;
Invitrogen)/Cytofix/Cytoperm solution (BD Biosciences). The cells were incu-
bated overnight at 4°C, washed twice with 1 PermWash solution (BD
Biosciences), and resuspended in 100 l1 PermWash containing 2 g
AlexaFluor 488-conjugated 8.1-GSH mAb {anti-GSH adduct with NEM (anti-
GS-NEM); refs. [27, 28]}. After 30 min incubation on ice, the suspension was
washed twice with PermWash and resuspended in 300 l PBS. The specimens
were analyzed on a FACSCalibur flow cytometer using CellQuest software.
For CD53 surface level analysis, whole blood (100 l) was stained with one
or more of the following mAb: CD3-PerCP, CD45-APC, CD4-PE, CD8-PE,
CD19-PE, CD56/16-PE, CD14, CD53-FITC, or mouse IgG
1
-FITC (BD Bio
-
sciences). The specimens were incubated with the mAb 15 min at room
temperature, lysed with a 1 solution of FACSLyse (BD Biosciences), and run
on a FACSCalibur flow cytometer using CellQuest software.
Telomere length analysis
Previously isolated and frozen PBMC were thawed, and PBMC (110
6
) were
washed and centrifuged (10 min; 200 g). The cell pellets were resuspended in
hybridization buffer containing 70% deionized formamide (Sigma Chemical
Co.), 10 mM Tris, pH 7.0, 10% FCS, and 0.3 g/ml telomere-specific,
FITC-conjugated peptide nucleic acid probe (FITC-OO-CCCTAACCCTAAC-
CCTAA-COOH; Applied Biosystems, Foster City, CA, USA). Samples were
TABLE 1. American College of Rheumatology Revised Criteria
for Classification of Functional Status in RA
a
Class I Completely able to perform usual activities of daily
living (self-care, vocational, and avocational)
Class II Able to perform usual self-care and vocational
activities but limited in avocational activities
Class III Able to perform usual self-care activities but limited in
vocational and avocational activities
Class IV Limited in ability to perform usual self-care,
vocational, and avocational activities
a
Usual self-care activities include dressing, feeding, bathing, grooming, and
toileting. Avocational (recreational and/or leisure) and vocational (work,
school, homemaking) activities are patient-desired and age- and gender-
specific.
Pedersen-Lane et al. Cellular thiol changes associated with arthritis 935
heat-denatured at 82°C for 10 min, followed by hybridization in the dark for
16 h at room temperature. The cells were washed (PBS, Sigma Chemical Co.),
centrifuged (10 min; 200 g) twice, and then resuspended in PBS with 10%
FBS, RNase (10 g/ml; Sigma Chemical Co.), and propidium iodide for2hat
room temperature. Cells were analyzed on a FACSCalibur (Becton Dickinson,
San Jose, CA, USA) flow cytometer. The telomere fluorescence signal is defined
as the mean fluorescence signal in G
0
/G
1
cells after subtraction of the back
-
ground fluorescence signal [fluorescence in situ hybridization (FISH) proce-
dure without probe]; results are expressed in molecular equivalents of soluble
fluorochrome units. Human T cell lines (Jurkat and CEM-CCRF) were used as
controls for short and long telomeres, respectively. Telomere length was
calculated [29] from the molecules of an equivalent, soluble fluorochrome units
standard curve (Quantum Beads, Bangs Labs, Fischers, IN, USA) using the
following equation: telomere length (kb) [Flchannel #–Flchannel # (blank)]
0.019 0.02604/slope.
Statistical analysis
Statistical analysis was performed using Sigma Plot 9.0 software. In all tests,
comparisons with associated P values less than 0.05 were considered signif-
icant. Age and gender correlations in each study group were quantified by the
Pearson correlation test.
RESULTS
RA patients have decreased levels of
surface-thiols on PBL
Earlier studies (e.g., ref. [30]) have shown that there are at least
15 different cell-surface proteins, which may contain free SH
groups, depending on the redox status of the cells. To measure
the overall level of cell surface R-SH, we took advantage of the
readily available AlexaFluor-maleimide reagents. The nucleo-
philic maleimide covalently couples to an available SH moiety.
Maleimide is effective in its ability to react with thiols and is
considered to be highly specific for thiol groups. The Alexa
dyes that are coupled to maleimide do not interfere with the
maleimide-thiol interaction and as they are charged molecules,
do not enter the cell, thus enabling a FACS-detectable, overall
cell surface-thiol determination.
For assessment of the surface-thiol levels of RA patients,
whole blood specimens were treated as described in Materials
and Methods, and the measured levels were compared with the
levels assessed for a healthy control population (Fig. 1).
Overall, RA patients had significantly lower levels of surface-
thiols on most of their PBMC subpopulations as compared with
the control population. The relative quantity of the surface-
thiols in both groups followed a pattern seen consistently
among the analyses within this study, as well as in our previous
surface-thiol study [11]. The ordering seen, namely (CD19
)B
cells (CD8
) T cells (CD4
) T cells, appears to correlate
well with the differing sensitivities of the cells to radiation [31]
and thiol-reactive chemicals [32]. In addition, in RA patients,
there was a trend toward decreasing overall surface-thiol levels
with increasing age (Fig. 2). This trend was not seen in the
healthy, aging, control population; in fact, surface-thiols in-
creased on CD4
and CD8
lymphocytes with age (Fig. 3
).
The decrease with leukocytes from RA patients is consistent
with the idea that a decrease in oxidative stress management as
a function of age is compounded by chronic inflammatory
disease states, as in RA [33, 34]. There was no significant
correlation between surface-thiol levels and gender in the
patient group or the control group.
RA patients have decreased levels of
iGSH in PBMC
The GSH-8.1 antibody used in this test recognizes a GS-NEM
[27]. To stabilize the iGSH and prevent the loss of GS-NEM,
PBMC were fixed with cold 1% paraformaldehyde prior to
NEM treatment. Cells not treated with NEM prior to antibody
Fig. 1. Surface-thiol levels on leukocyte subsets. Patients with RA (n 93)
have significantly lower levels of surface-thiols on most leukocyte subpopula-
tions than do the non-RA healthy controls (n26). The rank order by relative
quantity of the surface-thiols in both groups was monocytes (Monos)
granulocytes (Grans) lymphocytes (CD19CD8CD4). The levels of thiols
on the surface of leukocytes are presented as units of mean fluorescence
intensity (MFI). The data are mean SD; *, significant difference (P0.001)
from the appropriate control subset.
TABLE 2. Baseline Characteristics of the RA Cohort
Demographics:
Race Gender Age
White (non-Hispanic) 107 Female 95 Range 28–85
Hispanic 3 Male 22 Mean 59.3 11
Black (non-Hispanic) 4
Asian 2
Other 1
Disease category:
Classification Joint count
I 15 Range 11–52
II 44 Mean 34 15
III 58
Lymphoid immunophenotyping:
Percentages
Range
Absolute
numbers
RangeMean
SD Mean SD
CD3 73 11 16–91 1440 583 182–2881
CD8 21 10 2–52 418 276 37–1655
CD4 52 11 5–76 1020 436 313–2870
NK 13 7 3–37 243 150 13–625
CD19 14 7 2–46 263 161 14–979
936 Journal of Leukocyte Biology Volume 81, April 2007 http://www.jleukbio.org
staining were used as a negative control and were not labeled.
It has been shown that the immune system works optimally if
lymphoid cells can maintain a delicately balanced level of
GSH [35]. Patients with RA had significantly lower levels of
iGSH, except for CD4
T lymphocytes, than did the controls
(Fig. 4). The observed subset-wise pattern of iGSH levels,
namely, monocytes (CD8
) T cells (CD4
) T cells
(CD19
) B cells, is similar to that reported previously [36].
Although this is a different pattern than what was seen for
surface-thiol levels, it may again be related to the sensitivities
of each subpopulation to stresses. GSH functions as an intra-
cellular reductant in oxidation-reduction processes. The prod-
uct of GSH oxidation is GSH disulfide (GSSG), which functions
as an intracellular quencher of ROS by behaving as a redox
buffer. Decreased levels of iGSH could therefore hinder the
ability of cells to maintain a normal redox balance. There was
a trend toward decreasing iGSH levels with age in RA patients
(Fig. 5), but a significant decline was seen only for B cells and
monocytes; no significant positive or negative correlation with
age was seen in a healthy, aging population (data not shown).
RA patients have increased levels of surface
CD53 on PBL
Exofacial plasma membranal expression of CD53 was analyzed
on each subpopulation of PBL isolated from RA patients and
was compared with the expression on PBL from the non-RA
control group. Consistently, RA patients had a significantly
higher expression of CD53 on the cell surface of PBL subpopu-
lations than did the control group (Fig. 6). The relative ex-
pression of CD53 followed the pattern monocytes B cells
T cells granulocytes, which has been seen in other studies
[37]. It is interesting that the pattern, monocytes (CD19
)B
cells (CD8
) T cells (CD4
) T cells granulocytes,
matches that seen for relative surface-thiol levels [36].
RA patients show shortened telomere lengths
in PBMC
Overall, RA patients had significantly shorter telomeres in
PBMC than did the non-RA control group (Fig. 7A). Telo-
meres in healthy individuals are relatively intact until the third
decade, when they begin to erode progressively, until finally
plateauing at a much shorter length in the sixth decade [38].
The mean telomere length (when adjusted for age) of PBMC
from RA patients was shorter than the mean length for the
non-RA control group (Fig. 7B). Patients with RA had a much
sharper decrease in the telomere length through the decades
than did the non-RA controls; in common with healthy indi-
viduals, the erosion appears to plateau in the sixth decade but
at a markedly shorter length.
DISCUSSION
The need for an intact redox balance in cells has led to the
evolution of several effective, intracellular, antioxidant defense
systems, which can sense elevated levels of oxidative stress
and swiftly reinstate a healthy redox environment [4, 5]. The
Fig. 3. Correlation of surface-thiols and age on leukocyte subsets from
healthy, control donors. Unlike the CD4
lymphocyte population from RA
patients, this subset showed a significant increase of surface-thiols with age.
The surface-thiols of CD8
lymphocytes also increase with age, whereas all of
the other subsets had no significant changes with age.
Fig. 2. Correlation of surface-thiols and age on leukocyte subsets from RA
patients. The surface-thiols of CD4
lymphocytes, NK cells, and monocytes
declined significantly with age.
Pedersen-Lane et al. Cellular thiol changes associated with arthritis 937
response of a cell to stress often involves changes in cell thiol
content. Thiols are first consumed in reactions that protect
the cell by removing deleterious compounds; the thiols are
then replaced through enzymatic reduction of disulfides or
de novo synthesis. It has been suggested that the pro-oxidant/
antioxidant imbalance seen in cells from patients with RA,
resulting from accumulation of ROS, is a result of acceleration
of some cellular reaction or an impaired antioxidant defense
system [9, 10].
In this study, we evaluated the thiol status (surface-thiols
and iGSH) of PBL from patients with RA. We found consis-
tently that RA patients had significantly lower levels of sur-
face-thiols and iGSH than did the control group. During the
maintenance of a balanced redox environment, an initial mild
stress causes an elevation in thiols and iGSH, which provide
protection against more severe forms of stress. Cellular GSH
levels are maintained by GSH reductase activity, which con-
verts GSSG back to GSH, and by an up-regulation of GSH
synthesis [5]. Severe and/or chronic oxidative stress, such as
that resulting from inflammation and tissue damage in RA,
leads to a decline in these defense mechanisms [35, 39].
Continuous exposure to even low levels of oxidants can even-
tually cause depletion of GSH, by depleting the substrate
required to replenish GSH. Decreased protection leads to DNA
damage, a rise in intracellular-free Ca
2
and iron, damage to
proteins, and lipid peroxidation, resulting in cell toxicity and
cell death [39, 40]. The status of GSH in a cell may reflect the
ability of a cell to protect itself against oxidative injury. Pa-
tients with RA exhibit significantly higher than normal plasma
levels of a variety of oxidant end products and of altered
plasma thiol patterns [41, 42], suggesting the presence of
increased oxidative stress. The significantly reduced levels of
surface-thiols and iGSH in PBMC of RA patients, which we
observed, may represent the constant efforts of the cells to
function in the presence of pro-oxidant compounds in the
cellular microenvironment [43, 44]. It is interesting that a
significant increase in erythrocyte GSH level in RA patients
has been reported, suggesting an additional protective re-
sponse against continuous ROS production [41].
Fig. 4. Levels of iGSH in leukocyte subsets. Patients with RA (n44) have
significantly lower mean levels of iGSH, as measured by AlexaFluor647-
8.1GSH (anti-GS-NEM) intracellular staining, after fixation and NEM treat-
ment, than do the normal, healthy controls (n17). Levels are expressed as the
MFI of the AlexaFluor-647. In terms of relative quantity of iGSH, the pattern
seen is monocytes CD8 CD4 CD19; this pattern differs from that seen
for surface-thiol levels. The data are mean SD; *, significant difference
(P0.05) from the appropriate control subset.
Fig. 5. Patients with RA show a trend to-
ward decreasing iGSH levels with increas-
ing age, although the trend reached signif-
icance only for monocytes and B cells. This
trend was not seen in a healthy, aging pop-
ulation; there was no significant positive or
negative correlation of iGSH and age in the
control population (data not shown).
938 Journal of Leukocyte Biology Volume 81, April 2007 http://www.jleukbio.org
The available amount of iGSH depends on the equilibrium
between processes, during which GSH is expended and regen-
erated, and the process of its biosynthesis. These processes are
associated closely with Meister’s -glutamyl cycle, in which
the membrane enzyme GGT plays a pivotal role in the salvage
pathway of extracellular GSH. GGT initiates the breakdown of
GSH into its amino acid components with cysteine transported
into the cell for GSH biosynthesis [13, 45]. Studies have shown
that elevated GGT activity is involved in the prevention of
NO-induced apoptosis in human Th2 cells [46] and the U937
cell line [47], regardless of the iGSH levels. In RA, the
mechanisms that elicit and/or propagate chronic inflammation
remain unclear, but accumulating evidence indicates that in-
sufficient apoptosis represents at least one underlying process
[48, 49]. Although not designed specifically to promote apo-
ptosis, several medications currently used in the treatment of
RA may function in part through their induction of apoptosis
[50]. As it was beyond the scope of this study to quantify GGT
activity directly, as a result of time restrictions in specimen
arrivals, we chose to examine CD53 levels. GGT is physically
associated with the TM4 protein CD53, as indicated by the
coimmunoprecipitation of GGT activity with CD53, although
little is known about this association [15]. In addition, a CD53
gene has been identified among the set of those genes that
regulates apoptosis [18]. Ligation of CD53, through interac-
tions with the extracellular environment, triggers a survival
response and reduces the number of cells that enter apoptosis,
most likely, as a result of the transient activation of the c-Jun
N-terminal kinase [19]. Increasing evidence implicates c-Jun
in the protection of cells against stress-induced apoptosis [51].
A previous study implicated CD53 in adhesion by showing that
stimulation of rat B cells with antibodies to CD53 triggered a
homotypic adhesion reaction related to cellular migration [52].
In the present study, we have demonstrated that RA patients
have significantly higher levels of CD53 on the majority of PBL
subpopulations than do control subjects. In light of what is
currently known about the function of CD53, it is plausible that
this protein plays important roles, not only in the migration of
PBL into synovia of RA joints, but also in establishing the
apoptosis-resistant nature of these cells.
Cumulative oxidative effects play a significant role in the
aging process [33]. A host of diseases, including cardiovascular
and neurodegenerative disorders, as well as RA, certainly
increase in frequency exponentially with age; however, the
basis for the steep rise in disease incidence with age is unex-
plained. An underlying factor common to aging and disease
states is inflammation [53], which is most likely related to the
increase of ROS and a decrease in efficiency of the redox
balance mechanisms. The results of the work presented here
show that a decline in surface-thiols and iGSH levels with
increasing age is restricted to the RA patients. If we take thiol
levels as an indicator of redox balance, we can posit that the
accelerated decrease in thiol levels, as a result of chronic
inflammation in RA patients, is compounded by normal age-
related decreases.
Fig. 6. CD53 expression by leukocyte subsets. Patients with RA (n26) had
significantly higher mean levels of CD53 on the cell surface of most leukocyte
populations than did the healthy controls (n24). The CD53 levels are
expressed as MFI. It is interesting that the relative expression of CD53 follows
the same pattern as for surface-thiols (monocytesCD19
CD8
CD4
).
The data are mean
SD; *, significant difference (P0.05) from the appro-
priate control subset.
Fig. 7. Teleomere length analyses. PBMC from RA patients have significantly
shorter telomeres than do PBMC from the non-RA controls (A). Two cell lines
(Jurkat and CEM-CCRF) were used as internal controls for short and long
telomere controls. The telomere fluorescence signal is defined as the mean
fluorescence signal in G
0
/G
1
cells after subtraction of the background fluores
-
cence signal (FISH procedure without probe); results are expressed in molec-
ular equivalents of soluble fluorochrome units. Telomere length was calculated
[29] as described in Materials and Methods. The data are mean
SD;*,
significant difference from the controls (P0.003). When adjusted for age, the
mean length of telomeres of PBMC is shorter in RA patients than in the
non-RA controls, and the difference is more pronounced after the second
decade (B).
Pedersen-Lane et al. Cellular thiol changes associated with arthritis 939
With the ever-increasing interest in the aging process, re-
search has turned its attention toward telomeres as biomarkers
for aging and age-related diseases such as RA [21, 22]. Telo-
meres in most human cells shorten with each round of DNA
replication, as a result of decreased telomerase activity; how-
ever, telomerase activity is not the only determinant of rate of
telomeric DNA loss. Damage to DNA as a result of oxidative
stress is repaired less well in the telomeric region than else-
where on the chromosome, and the result is that telomere loss
is accelerated, and cellular, replicative senescence is triggered
[54, 55]. Given our hypothesis that cellular thiol levels are
indicative of cellular oxidative stress, we looked at telomere
length in PBMC of RA patients and control subjects. Although
the correlation between thiol levels and reduced telomere
length was not significant (0.05), our results were consistent
with findings of other studies that indicated a premature ero-
sion of telomere length in RA [56].
In conclusion, surface-thiols and iGSH play important roles
in redox buffering of cells, providing protection from oxidative
stress and the resultant cellular damage. However, chronic
stress, such as the inflammation associated with RA, leads to a
decline in the levels of this protection. We hypothesize that the
loss of cellular thiols of lymphocytes from the RA patients
causes increased expression of GGT with a concomitant in-
crease of CD53. Paradoxically, CD53 has been suggested to
interfere with apoptosis [18, 19], which should be increased as
a result of a cell’s loss of reducing equivalents [5]. Apoptotic
resistance has been suggested to be related to the pathogenesis
of RA [20]. Currently, our aim is to examine adjunct therapies
with antioxidants, which may aid in maintaining or increasing
iGSH and decreasing CD53 expression, thus promoting the
potential for lessening the functional changes that occur as the
result of the severe stress of inflammation and aging.
ACKNOWLEDGMENTS
Studies were, in part, supported by National Institutes of
Health grant RO1-AT00309. The authors thank the staff of the
Immunology Core of the Wadsworth Center for their assistance
with the flow cytometry.
REFERENCES
1. Firestein, G. S. (2003) Evolving concepts of rheumatoid arthritis. Nature
423, 356 –361.
2. Goronzy, J. J., Weyand, C. M. (2005) Rheumatoid arthritis. Immunol. Rev.
204, 55–73.
3. Darlington, L. G., Stone, T. W. (2001) Antioxidants and fatty acids in the
amelioration of rheumatoid arthritis and related disorders. Br. J. Nutr. 85,
251–269.
4. Winyard, P. G., Moody, C. J., Jacob, C. (2005) Oxidative activation of
antioxidant defense. Trends Biochem. Sci. 30, 453– 461.
5. Dro¨ge, W. (2002) Free radicals in the physiological control of cell func-
tion. Physiol. Rev. 82, 47–95.
6. Dickinson, D. A., Forman, H. J. (2002) Cellular glutathione and thiols
metabolism. Biochem. Pharmacol. 64, 1019 –1026.
7. Cuozzo, J. W., Kaiser, C. A. (1999) Competition between glutathione and
protein thiols for disulfide-bond formation. Nat. Cell Biol. 1, 130 –135.
8. Deneke, S. M. (2000) Thiol-based antioxidants. Curr. Top. Cell. Regul.
36, 151–180.
9. Remans, P. H. J., van Oosterhout, M., Smeets, T. J. M., Sanders, M.,
Frederiks, W. M., Reesquist, K. A., Tak, P. P., Breedveld, F. C., van Laar,
J. M. (2005) Intracellular free radical production in synovial T lympho-
cytes from patients with rheumatoid arthritis. Arthritis Rheum. 52, 2003–
2009.
10. Brown-Galatola, C. H., Hall, N. D. (1992) Impaired suppressor cell
activity to surface sulfydryl oxidation in rheumatoid arthritis. Br. J.
Rheumatol. 31, 599 603.
11. Lawrence, D. A., Song, R., Weber, P. (1996) Surface thiols in human
lymphocytes and their changes after in vitro and in vivo activation.
J. Leukoc. Biol. 60, 611– 618.
12. Kanner, S. B., Kvanagh, T. J., Grossmann, A., Hu, S-L., Bolen, J. B.,
Rabinovitch, P. S., Ledbetter, J. (1992) Sulfhydryl oxidation down-regu-
lates T-cell signaling and inhibits tyrosine phosphorylation of phospho-
lipase C1. Proc. Natl. Acad. Sci. USA 89, 300 –304.
13. Hanigan, M. H., Ricketts, W. A. (1993) Extracellular glutathione is a
source of cysteine for cells that express -glutamyl transpeptidase. Bio-
chemistry 32, 6302– 6306.
14. Carlisle, M. L., King, M. R., Karp, D. R. (2003) -Glutamyl transpeptidase
activity alters the T cell response to oxidative stress and FAS-induced
apoptosis. Int. Immunol. 15, 17–27.
15. Nichols, T. C., Guthridge, J. M., Karp, D. R., Molina, H., Fletcher, D. R.,
Holers, V. M. (1998) -Glutamyl transpeptidase, an ecto-enzyme regulator
of intracellular redox potential, is a component of TM4 signal transduction
complexes. Eur. J. Immunol. 28, 4123– 4129.
16. Maecker, H. T., Todd, S. C., Levy, S. (1997) The tetraspanin superfamily:
molecular facilitators. FASEB J. 11, 428 442.
17. Kim, T-R., Yoon, J-H., Kim, Y-C., Yook, Y-H., Kim, I-G., Kim, Y-S., Lee,
H., Paik, S-G. (2004) LPS-induced CD53 expression: a protection mech-
anism against oxidative and radiation stress. Mol. Cells 17, 125–131.
18. Voehringer, D. W., Hircschberg, D. L., Xiao, J., Roederer, M., Lock, C. B.,
Herzenberg, L. A., Steinman, L., Herzenberg, L. A. (2000) Gene microar-
ray identification of redox and mitochondrial elements that control resis-
tance or sensitivity to apoptosis. Proc. Natl. Acad. Sci. USA 97, 2680
2685.
19. Yunta, M., Lazo, P. A. (2003) Apoptosis protection and survival signal by
the CD53 tetraspanin antigen. Oncogene 22, 1219 –1224.
20. Liu, H., Pope, R. M. (2003) The role of apoptosis in rheumatoid arthritis.
Curr. Opin. Pharmacol. 3, 317–322.
21. Epel, E. S., Balckburn, E. H., Lin, J., Dhabhar, F. S., Adler, N. E.,
Morrow, J. D., Cawthon, R. M. (2004) Accelerated telomere shortening in
response to life stress. Proc. Natl. Acad. Sci. USA 101, 17312–17315.
22. Goronzy, J. J., Fujii, H., Weyand, C. M. (2006) Telomeres, immune aging
and autoimmunity. Exp. Gerontol. 41, 246 –251.
23. Kurz, D. J., Decary, S., Hong, Y., Trivier, E., Akhmedov, A., Eruslimsky,
J. D. (2004) Chronic oxidative stress compromises telomere integrity and
accelerates the onset of senescence in human endothelial cells. J. Cell Sci.
117, 2417–2426.
24. Townsend, D. M., Tew, K. D., Tapiero, H. (2003) The importance of
glutathione in human disease. Biomed. Pharmacother. 57, 145–155.
25. Franceschi, C., Bonafe`, M. (2003) Centenarians as a model for healthy
aging. Biochem. Soc. Trans. 31, 457– 461.
26. Hochberg, M. C., Chang, R. W., Dwosh, I., Lindsey, S., Pincus, T., Wolfe,
F. (1992) The American College of Rheumatology 1991 revised criteria for
the classification of global functional status in rheumatoid arthritis. Ar-
thritis Rheum. 35, 498 –502.
27. Messina, J. P., Mazurkiewicz, J., Lawrence, D. A. (1987) Production and
characterization of monoclonal antibodies to thiol-modified glutathione. In
Anticarcinogenesis and Radiation Protection (P. A. Cerutti, M. G. Nygaard,
M. G. Simic, eds.), New York, NY, USA, Plenum, 407– 412.
28. Ault, J. G., Lawrence, D. A. (2003) Glutathione distribution in normal and
oxidatively stressed cells. Exp. Cell Res. 285, 9 –14.
29. Kapoor, V., Telford, W. G. (2004) Telomere length measurement by
fluorescence in situ hybridization and flow cytometry. In Methods in
Molecular Biology: Flow Cytometry Protocols, 2nd ed. (T. S. Hawley, R. G.
Hawley, eds.), Totowa, NJ, USA, Humana. pp. 385–398.
30. Donoghue, N., Yam, P. T. W., Jiang, X-M., Hogg, P. (2000) Presence of
closely spaced protein thiols on the surface of mammalian cells. Protein
Sci. 9, 2436 –2445.
31. Duncan, D. D., Lawrence, D. A. (1991) Residual activation events func-
tional after irradiation of mouse splenic lymphocytes. Radiat. Res. 125,
6 –13.
32. Duncan, D. D., Lawrence, D. A. (1988) Four sulfhydryl-modifying com-
pounds cause different structural damage but similar functional damage in
murine lymphocytes. Chem. Biol. Interact. 68, 137–152.
33. Finkel, T., Holbrook, N. J. (2000) Oxidants, oxidative stress and the
biology of aging. Nature 408, 239 –247.
940 Journal of Leukocyte Biology Volume 81, April 2007 http://www.jleukbio.org
34. Ha¨rle, P., Straub, R. H. (2005) Neuroendocrine-immune aspects of accel-
erated aging in rheumatoid arthritis. Curr. Rheumatol. Rep. 7, 389 –394.
35. Dro¨ge, W., Breitkreutz, R. (2000) Glutathione and immune function. Proc.
Nutr. Soc. 59, 595– 600.
36. Roederer, M., Staal, F. J. T., Osada, H., Herzenberg, L. A., Herzenberg,
L. A. (1991) CD4 and CD8 cells with high intracellular glutathione levels
are selectively lost as the HIV infection progresses. Int. Immunol. 3,
933–937.
37. Tohami, T., Drucker, L., Radnay, J., Shapira, H., Lishner, M. (2004)
Expression of tetraspanins in peripheral blood leukocytes: a comparison
between normal and infectious conditions. Tissue Antigens 64, 235–242.
38. Koetz, K., Bryl, E., Spickschen, K., O’Fallon, W. M., Goronzy, J. J.,
Weyand, C. M. (2000) T cell homeostasis in patients with rheumatoid
arthritis. Proc. Natl. Acad. Sci. USA 97, 9203–9208.
39. Castro, L., Freeman, B. A. (2001) Reactive oxygen species in human
health and disease. Nutrition 17, 161–165.
40. Lunec, J., Halloran, S. P., White, A. G., Dormandy, T. L. (1981) Free-
radical oxidation (peroxidation) products in serum and synovial fluid in
rheumatoid arthritis. J. Rheumatol. 8, 233–245.
41. Gambhir, J. K., Lali, P., Jain, A. L. (1997) Correlation between blood
antioxidant levels and lipid peroxidation in rheumatoid arthritis. Clin.
Biochem. 30, 351–355.
42. Giustarini, D., Lorenzini, S., Rossi, R., Chindamo, D., DiSimplicio, P.,
Marcolongo, R. (2005) Altered thiol patterns in plasma of subjects affected
by rheumatoid arthritis. Clin. Exp. Rheumatol. 23, 205–212.
43. Halliwell, B., Gutteridge, J. M. (1990) The antioxidants of human extra-
cellular fluids. Arch. Biochem. Biophys. 280, 1– 8.
44. Sahaf, B., Heydrai, K., Herzenberg, L. A., Herzenberg, L. A. (2005) The
extracellular microenvironment plays a key role in regulating the redox
status of cell surface proteins in HIV-infected subjects. Arch. Biochem.
Biophys. 434, 26 –32.
45. Meister, A. (1988) Glutathione metabolism and its selective modification.
J. Biol. Chem. 263, 17205–17208.
46. Roozendaal, R., Vellenga, E., de Jong, M. A., Traanberg, K. F., Postma,
D. S., de Monchy, J. G. R., Kauffman, H. F. (2001) Resistance of activated
human Th2 cells to NO-induced apoptosis is mediated by -glutamyl
transpeptidase. Int. Immunol. 13, 519 –528.
47. Del Bello, B., Paolicchi, A., Comporti, M., Pompella, A., Maellaro, E.
(1999) Hydrogen peroxide produced during -glutamyl transpeptidase
activity is involved in prevention of apoptosis and maintenance of prolif-
eration in U937 cells. FASEB J. 13, 69 –79.
48. Pope, R. M. (2002) Apoptosis as a therapeutic tool in rheumatoid arthritis.
Nat. Rev. Immunol. 2, 527–535.
49. Peng, S. L. (2006) FAS (CD95)-related apoptosis and rheumatoid arthritis.
Rheumatology (Oxford) 45, 26 –30.
50. Liu, H., Pope, R. (2004) Apoptosis in rheumatoid arthritis: friend or foe.
Rheum. Dis. Clin. North Am. 30, 603– 625.
51. Leppa, S., Bohmann, D. (1999) Diverse functions of JNK signaling and
c-Jun in stress response and apoptosis. Oncogene 18, 6158 6162.
52. Lazo, P. A., Cuevas, L., Gutierrez del Arroyo, A., Orue, E. (1997) Ligation
of CD53/OX44, a tetraspan antigen, induces homotypic adhesion medi-
ated by specific cell-cell interactions. Cell. Immunol. 178, 132–140.
53. Sarkar, D., Fisher, P. B. (2006) Molecular mechanisms of aging-associated
inflammation. Cancer Lett. 236, 13–23.
54. Von Zglinicki, T., Martin-Ruiz, C. M. (2005) Telomeres as biomarkers for
ageing and age-related diseases. Curr. Mol. Med. 5, 197–203.
55. Saretzki, G., von Zglinicki, T. (2002) Replicative aging, telomeres, and
oxidative stress. Ann. N. Y. Acad. Sci. 959, 24 –29.
56. Scho¨nland, S. O., Lopez, C., Wildman, T., Zimmer, J., Bryl, E., Goronsky,
J. J., Weyand, C. M. (2003) Premature telomeric loss in rheumatoid
arthritis is genetically determined and involves both myeloid and lym-
phoid cell lineages. Proc. Natl. Acad. Sci. USA 100, 13471–13476.
Pedersen-Lane et al. Cellular thiol changes associated with arthritis 941
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    • "Increased surface levels of CD53 in response to IL-2 were detected on both NK cells and T cells. Interestingly, up-regulation of CD53 under inflammatory conditions is also observed on macrophages in response to lipopolysaccharide [35], as well as on leukocytes obtained from rheumatoid arthritis patients [36], patients with atopic eczema [37], or in lesions after spinal cord injury [38]. The up-regulation of CD53 under inflammatory conditions is suggested to protect leukocytes from apoptosis duringFigure 4. Increased proliferative activity of NK cells in response to CD53 ligation. "
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