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Increase in Peripheral CD4 Bright+ CD8 Dull+ T Cells in Parkinson Disease
Kinya Hisanaga, MD;
Misa Asagi, MA;
Yasuto Itoyama, MD;
Yuzo Iwasaki, MD
Arch Neurol. 2001;58:1580-1583.
ABSTRACT
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Background Immune abnormalities are known to be involved in the pathogenesis of
sporadic Parkinson disease.
Objective To examine whether abnormalities in peripheral lymphocytes exist in
Parkinson disease.
Methods Immune mediators, including CD1a, CD3, CD4, CD8, CD45RO, and Fas (CD95),
were examined in peripheral lymphocytes of patients by 3-color flow cytometry.
Results Patients with Parkinson disease displayed a significantly greater population
of circulating CD3+ CD4 bright+ CD8 dull+
lymphocytes than age-matched control subjects (P
= .005) and patients with cerebrovascular disease (P
= .002). The increase in these cells appeared to continue for at least 17
months. These T cells also expressed CD45RO and Fas, markers for activated
T cells, while CD1a, a marker for thymic T cells, was negative, suggesting
that these cells are mature T cells with immune activities.
Conclusions As CD4+ CD8+ T cells are known to increase after
some specific viral infections, the continuous increase in CD4 bright+ CD8 dull+ T cells shown here may indicate postinfectious
immune abnormalities that are possibly associated with the pathogenesis of
this slowly progressive, multifactorial neurodegenerative disease.
INTRODUCTION
PARKINSON DISEASE (PD), characterized by various neurologic symptoms
including resting tremor, bradykinesia, rigidity, and pulsion, has been extensively
studied, but the cause is still unclear. Many factors, such as neurotoxins,
excitatory amino acids, oxidative stress, mitochondrial dysfunction, and genetic
abnormalities, appear to be involved.1
Immune abnormalities have also been observed in PD, such as the occurrence
of antineuronal antibodies,2, 3, 4, 5, 6
increases in HLA-DR+–activated microglia in the substantia
nigra,7 increases in HLA-DR expression on cerebrospinal
fluid monocytes,8 decreases in CD4+CD45RA+ (naive) T cells and increases in CD4+CD45RO+
(memory) T cells and TCR  + cells,8
and CD38+ cells (activated T) and interleukin 2 receptor (CD25)+ cells (activated T, B, and macrophage)9
in peripheral blood.
In the present study, we found that patients with PD displayed a significantly
high population of circulating CD4 bright+ CD8 dull+
lymphocytes, which are usually seen in the thymus, as compared with control
subjects. These cells appeared to be mature T cells with immune activities.
SUBJECTS AND METHODS
We studied 40 patients with idiopathic PD (20 men and 20 women) without
immune disorders and/or other neurologic disorders. For each patient, the
clinical diagnosis had been established on the basis of the medical history
and physical examinations. The age of patients was 68.9 ± 6.6 years
(mean ± SD). The patients were treated with levodopa, dopamine receptor
agonists (bromocriptine mesylate or pergolide mesylate), an anticholinergic
agent (trihexyphenidyl hydrochloride), amantadine hydrochloride, and/or droxidopa.
The clinical stage of each patient during his or her "on" period was estimated
as I to V according to the criteria described by Hoehn and Yahr.10
The other groups studied were 22 control subjects without any neurologic
disorders (8 men and 14 women, 60.4 ± 6.5 years old) and 33 patients
with mild cerebrovascular disease (CVD; 19 men and 14 women, 67.7 ±
8.4 years old).
Venous blood samples of patients were collected with informed consent
between 6 AM and 10 AM to minimize the influence of diurnal fluctuations of
lymphocyte subsets, and mixed with citrate. Mononuclear cells were isolated
from the blood by density gradient centrifugation on Ficoll–sodium diatrizoate
solution (Ficoll-Paque Plus; Amersham Pharmacia Biotech, Uppsala, Sweden)
at 300g for 30 minutes at room temperature, and diluted
with phosphate-buffered saline (pH 7.4) containing 2% calf serum. The cells
were stained with fluorescein isothiocyanate (FITC)–, phycoerythrin
(PE)-, or phycoerythrin-cyanin 5 (PC5)–conjugated antibodies to CD1a
(PE), CD3 (PC5), CD4 (FITC), CD8 (PE or PC5), CD25 (PE), CD28 (PE), CD38 (PE),
CD45RO (PE), CD54 (PE), CD95 (PE), and CD126 (PE) (Beckman-Coulter, Fullerton,
Calif). The CD3, CD4, and CD8 were studied in all the patients and control
subjects as described above, and the other markers were used for the 3-color
analysis of CD4 bright+ CD8 dull+ lymphocytes. Control
staining was performed with FITC-IgG1, PE-IgG1, PE-IgG2a, or PC5-IgG1. All
monoclonal antibodies were applied at saturating concentrations. The population
of stained cells was calculated by flow cytometric analysis with the use of
a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson, San Jose,
Calif). A sample gate that contained lymphocytes but excluded granulocytes
and monocytes was used to acquire data. At least 10 000 mononuclear cells
were analyzed. The populations of stained cells in patients with PD were compared
with those in control subjects and patients with CVD by statistical analysis
using the Mann-Whitney test.
RESULTS
Table 1 shows proportions
of peripheral lymphocyte subsets in PD and the other groups. Peripheral lymphocytes
were slightly lower in patients with PD than in control subjects. The percentages
of CD4-CD8-, CD4+CD8-, and CD4-CD8+ lymphocytes in peripheral
blood remained unchanged in patients with PD. On the other hand, we found
significantly higher proportions of the CD4+CD8+ lymphocytes
in patients with PD compared with control subjects or patients with CVD. The
CD4+CD8+ lymphocytes could be clearly separated into
CD4 bright+ CD8 dull+ lymphocytes and CD4 dull+ CD8 bright+ lymphocytes according to the density of CD4
and CD8 immunoreactivity11 (Figure 1). The expression of CD4 in CD4 bright+ CD8 dull+cells was identical to that of CD4+ cells in the same sample.
The expression of CD8 in these cells was lower than that of CD8+
cells. The situation in CD4 dull+ CD8 bright+ cells
was the opposite. In the present study, the most remarkable differences were
demonstrated in CD4 bright+ CD8 dull+ lymphocytes, that
is, the proportions in patients with PD were significantly higher than those
in control subjects and patients with CVD. There was no sex predominance in
the above results (Figure 2). There
was a trend for patients with PD in the early stages to demonstrate higher
proportions of CD4 bright+ CD8 dull+ lymphocytes, which,
however, failed to achieve significance (Table 2). The results were not significantly correlated with any
medications (not shown). No significant differences were observed in CD4 dull+ CD8 bright+ lymphocytes (Table 1).
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Table 1. Lymphocytic Subpopulations in Peripheral Blood of Control
Subjects and Patients With Cerebrovascular Disease (CVD) or Parkinson Disease
(PD)*
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Figure 1. Flow cytometry of lymphocytes
(A, control subject; B, patient with Parkinson disease). Cells in the square
indicated by an arrow were counted as CD4 bright+ CD8 dull+ lymphocytes. Cells in the square above were counted as CD4 dull+ CD8 bright+ lymphocytes. PE indicates phycoerythrin; FITC,
fluorescein isothiocyanate.
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Figure 2. Percentages of CD4 bright+ CD8 dull+ lymphocytes in control subjects, patients with
cerebrovascular disease (CVD), and patients with Parkinson disease (PD). Note
that the cell population in patients with PD is significantly greater than
that in control subjects and patients with CVD.
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Table 2. Effects of Severity of Parkinson Disease on Size of CD4 Bright+ CD8 Dull+ T-Cell Population*
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Three-color flow cytometry showed that these CD4 bright+
CD8 dull+ lymphocytes were also CD3+, indicating that
these cells were T cells (not shown). These T cells demonstrated CD54 (intercellular
adhesion molecule 1)+ (not shown), CD45RO dull+, and
CD95 (Fas) dull+ (Figure 3).
On the other hand, CD1a, CD25 (interleukin 2 receptor  ), CD28, and
CD126 (interleukin 6 receptor ) were negative (not shown).
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Figure 3. Three-color flow cytometry for
CD4, CD8, and CD45RO (A-C) or CD95 (Fas) (D-F). Left, center, and right graphs
show CD8 bright+, CD8 dull+, and CD8-,
respectively. Arrows indicate CD4 bright+ CD8 dull+
lymphocytes. Note that these cells are CD45RO+ dull and CD95 (Fas)
dull+. PE indicates phycoerythrin; FITC, fluorescein isothiocyanate.
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Seven patients with PD who demonstrated greater populations of peripheral
CD4 bright+ CD8 dull+ T cells were reexamined 17 to
25 months later. All of these patients showed similar percentages of this
cell subpopulation (not shown).
COMMENT
In this study, we found further evidence of the presence of abnormalities
of immune function in PD; that is, patients with PD demonstrated increased
CD4 bright+ CD8 dull+ T cells in peripheral blood as
compared with control subjects and patients with CVD.
T cells differentiate from CD4-CD8-cells
to CD4+CD8+ cells during maturation in the thymus. After
selection, the surviving T cells lose the expression of 1 of these 2 molecules
and become either CD4+ or CD8+ T cells, and then normally
enter the circulation. Therefore, the concomitant expression of CD4 and CD8
on the cell surface of the T lymphocytes has been regarded as representing
immaturity.11 However, the increased peripheral
CD4 bright+ CD8 dull+ T cells in the present results
were not immature thymocytes, since they lacked CD1a expression, a characteristic
of later developmental stages of thymocytes.11
CD4+ CD45RO+ T cells and CD8+ CD45RO+ T cells are known to be memory T cells and cytotoxic T cells, respectively.
CD95 (Fas) has been shown to be increased in activated T cells. Therefore,
these CD4 bright+ CD8 dull+ lymphocytes may be mature
T cells with immune activities.
CD4 dull+ CD8 bright+ T cells were found for less
than a month in association with Epstein-Barr virus.12
Cytomegalovirus, human herpesvirus 6, human T-cell leukemia virus type 1,
and human immunodeficiency virus are also known to induce this phenotype.13 On the other hand, CD4 bright+ CD8 dull+ T cells were observed in patients with neoplasms as well as in small
populations of healthy adults and were continuously present in similar percentages
for a long period (about 3 years).12 An increase
in CD4+CD8+ lymphocytes in peripheral blood also has
been demonstrated during the rejection of renal transplants and in patients
with multiple sclerosis or myasthenia gravis. However, the origin and functional
characteristics of this immunophenotype are unknown.14, 15
A previous report showed that interleukin 4 can induce the expression of CD8
on CD4+ lymphocytes, resulting in the induction of the cytotoxic
activity to cells in culture.16
Why CD4 bright+ CD8 dull+ T cells increase in
PD and whether these circulating lymphocytes contribute to the pathogenesis
of neuronal death in PD remain to be investigated. Wekerle et al17
demonstrated that the nervous system is constantly patrolled by small numbers
of T lymphocytes, which penetrate the blood-brain barrier nonspecifically
and play a major role in the initiation and subsequent regulation of the intracerebral
immune response. Therefore, CD4 bright+ CD8 dull+ T
cells may contact central nervous system cells. As CD4+ CD8+ T cells are known to increase after some specific viral infections
as described above, the continuous increase of CD4 bright+ CD8
dull+ T cells shown herein may indicate postinfectious immune abnormalities
that are possibly associated with the pathogenesis of PD, which is a slowly
progressive, multifactorial neurodegenerative disease.
AUTHOR INFORMATION
Accepted for publication June 12, 2001.
This study was supported in part by a grant from the Research Committee
on Neuroimmunological Diseases, Ministry of Health and Welfare, Tokyo, Japan.
We thank Brent Bell, MA, for reading the manuscript.
From the Departments of Neurology and Clinical Research, Miyagi National
Hospital, Miyagi, Japan (Drs Hisanaga and Iwasaki and Mr Asagi), and Department
of Neurology, Tohoku University School of Medicine, Tohoku, Japan (Dr Itoyama).
Corresponding author and reprints: Kinya Hisanaga, MD, Department
of Neurology, Miyagi National Hospital, 100 Kassenhara, Takase, Yamamoto,
Watari, Miyagi 989-2202, Japan.
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