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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.

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.

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.

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.

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.

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.

Table 1 
Lymphocytic Subpopulations in Peripheral Blood of Control Subjects and Patients With Cerebrovascular Disease (CVD) or Parkinson Disease (PD)*
Lymphocytic Subpopulations in Peripheral Blood of Control Subjects and Patients With Cerebrovascular Disease (CVD) or Parkinson Disease (PD)*
Table 2 
Effects of Severity of Parkinson Disease on Size of CD4 Bright+ CD8 Dull+ T-Cell Population*
Effects of Severity of Parkinson Disease on Size of CD4 Bright+ CD8 Dull+ T-Cell Population*
1.
Jenner  PSchapira  AHMarsden  CD New insights into the cause of Parkinson's disease. Neurology.1992;42:2241-2250.
2.
Pouplard  AEmile  JPouplard  FHurez  D Parkinsonism and autoimmunity: antibody against human sympathetic ganglion cells in Parkinson's disease. Adv Neurol.1979;24:321-326.
3.
McRae-Degueurce  MGottfries  CGKarlsson  ISvennerholm  LDahlstrom  A Antibodies in the CSF of a Parkinson patient recognize neurons in rat mesencephalic regions. Acta Physiol Scand.1986;126:313-315.
4.
McRae-Degueurce  ARosengren  LHaglid  K  et al Immunocytochemical investigations on the presence of neuron-specific antibodies in the CSF of Parkinson's disease cases. Neurochem Res.1988;13:679-684.
5.
Barker  RACahn  AP Parkinson's disease: an autoimmune process. Int J Neurosci.1988;43:1-7.
6.
Chen  SLe  WDXie  WJ  et al Experimental destruction of substantia nigra initiated by Parkinson disease immunoglobulins. Arch Neurol.1998;55:1075-1080.
7.
McGeer  PLItagaki  SBoyes  BEMcGeer  EG Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology.1988;38:1285-1291.
8.
Fiszer  UMix  EFredrikson  SKostulas  VLink  H Parkinson's disease and immunological abnormalities: increase of HLA-DR expression on monocytes in cerebrospinal fluid and of CD45RO+ T cells in peripheral blood. Acta Neurol Scand.1994;90:160-166.
9.
Chiba  SMatsumoto  HSaitoh  MKasahara  MMatsuya  MKashiwagi  M A correlation study between serum adenosine deaminase activities and peripheral lymphocyte subsets in Parkinson's disease. J Neurol Sci.1995;132:170-173.
10.
Hoehn  MMYahr  MD Parkinsonism: onset, progression and mortality. Neurology.1967;17:427-442.
11.
Lanier  LLAllison  JPPhillips  JH Correlation of cell surface antigen expression on human thymocytes by multi-color flow cytometric analysis: implications for differentiation. J Immunol.1986;137:2501-2507.
12.
Ortolani  CForti  ERadin  ECibin  RCossarizza  A Cytofluorimetric identification of two populations of double positive (CD4+, CD8+) T lymphocytes in human peripheral blood. Biochem Biophys Res Commun.1993;191:601-609.
13.
Rentenaar  RJWever  PCvan Diepen  FNJSchellekens  PTAWertheim  PMEten Berge  IJM CD4dullCD8bright double-positive T-lymphocytes have a phenotype of granzyme Bpos CD8pos memory T-lymphocytes. Nephrol Dial Transplant.1999;14:1430-1434.
14.
Munschauer  FEStewart  CJacobs  L  et al Circulating CD3+ CD4+ CD8+ T lymphocytes in multiple sclerosis. J Clin Immunol.1993;13:113-118.
15.
Berrih  SGaud  CBach  M-ABrigand  HLBinet  JPBach  JF Evaluation of T cell subsets in myasthenia gravis using anti–T cell monoclonal antibodies. Clin Exp Immunol.1981;45:1-8.
16.
Paliard  XMalefijt  RWde Vries  JESpits  H Interleukin-4 mediates CD8 induction on human CD4+ T-cell clones. Nature.1988;335:642-644.
17.
Wekerle  HLinington  CLassmann  HMeyermann  R Cellular immune reactivity within the CNS. Trends Neurosci.1986;9:271-277.
Original Contribution
October 2001

Increase in Peripheral CD4 Bright+ CD8 Dull+ T Cells in Parkinson Disease

Author Affiliations

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).

Arch Neurol. 2001;58(10):1580-1583. doi:10.1001/archneur.58.10.1580
Abstract

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.

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,26 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 TCRγδ+ 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 CD4CD8, CD4+CD8, and CD4CD8+ 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).

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 α), CD28, and CD126 (interleukin 6 receptor α) were negative (not shown).

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 CD4CD8cells 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.

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Article 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.

Corresponding author and reprints: Kinya Hisanaga, MD, Department of Neurology, Miyagi National Hospital, 100 Kassenhara, Takase, Yamamoto, Watari, Miyagi 989-2202, Japan.

References
1.
Jenner  PSchapira  AHMarsden  CD New insights into the cause of Parkinson's disease. Neurology.1992;42:2241-2250.
2.
Pouplard  AEmile  JPouplard  FHurez  D Parkinsonism and autoimmunity: antibody against human sympathetic ganglion cells in Parkinson's disease. Adv Neurol.1979;24:321-326.
3.
McRae-Degueurce  MGottfries  CGKarlsson  ISvennerholm  LDahlstrom  A Antibodies in the CSF of a Parkinson patient recognize neurons in rat mesencephalic regions. Acta Physiol Scand.1986;126:313-315.
4.
McRae-Degueurce  ARosengren  LHaglid  K  et al Immunocytochemical investigations on the presence of neuron-specific antibodies in the CSF of Parkinson's disease cases. Neurochem Res.1988;13:679-684.
5.
Barker  RACahn  AP Parkinson's disease: an autoimmune process. Int J Neurosci.1988;43:1-7.
6.
Chen  SLe  WDXie  WJ  et al Experimental destruction of substantia nigra initiated by Parkinson disease immunoglobulins. Arch Neurol.1998;55:1075-1080.
7.
McGeer  PLItagaki  SBoyes  BEMcGeer  EG Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology.1988;38:1285-1291.
8.
Fiszer  UMix  EFredrikson  SKostulas  VLink  H Parkinson's disease and immunological abnormalities: increase of HLA-DR expression on monocytes in cerebrospinal fluid and of CD45RO+ T cells in peripheral blood. Acta Neurol Scand.1994;90:160-166.
9.
Chiba  SMatsumoto  HSaitoh  MKasahara  MMatsuya  MKashiwagi  M A correlation study between serum adenosine deaminase activities and peripheral lymphocyte subsets in Parkinson's disease. J Neurol Sci.1995;132:170-173.
10.
Hoehn  MMYahr  MD Parkinsonism: onset, progression and mortality. Neurology.1967;17:427-442.
11.
Lanier  LLAllison  JPPhillips  JH Correlation of cell surface antigen expression on human thymocytes by multi-color flow cytometric analysis: implications for differentiation. J Immunol.1986;137:2501-2507.
12.
Ortolani  CForti  ERadin  ECibin  RCossarizza  A Cytofluorimetric identification of two populations of double positive (CD4+, CD8+) T lymphocytes in human peripheral blood. Biochem Biophys Res Commun.1993;191:601-609.
13.
Rentenaar  RJWever  PCvan Diepen  FNJSchellekens  PTAWertheim  PMEten Berge  IJM CD4dullCD8bright double-positive T-lymphocytes have a phenotype of granzyme Bpos CD8pos memory T-lymphocytes. Nephrol Dial Transplant.1999;14:1430-1434.
14.
Munschauer  FEStewart  CJacobs  L  et al Circulating CD3+ CD4+ CD8+ T lymphocytes in multiple sclerosis. J Clin Immunol.1993;13:113-118.
15.
Berrih  SGaud  CBach  M-ABrigand  HLBinet  JPBach  JF Evaluation of T cell subsets in myasthenia gravis using anti–T cell monoclonal antibodies. Clin Exp Immunol.1981;45:1-8.
16.
Paliard  XMalefijt  RWde Vries  JESpits  H Interleukin-4 mediates CD8 induction on human CD4+ T-cell clones. Nature.1988;335:642-644.
17.
Wekerle  HLinington  CLassmann  HMeyermann  R Cellular immune reactivity within the CNS. Trends Neurosci.1986;9:271-277.
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