TSP)

Journal of Neuroimmunology, 25 (1989) 251-254

251

Elsevier JNI 00865

Activated T lymphocytes in cerebrospinal fluid of patients with HTLV-I-associated myelopathy (HAM/TSP) Shinji Ijichi 1 Nobutaka Eiraku 1, Mitsuhiro Osame 1, Shuji Izumo 1, Ryuji Kubota 1, Ikuro Maruyama a, Makoto Matsumoto 2, Tatsuru Niimura 3 and Shunro Sonoda 4 I Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima, Japan, 2 Institute of Cancer Research, Faculty of Medicine, Kagoshima University, Kagoshima, Japan, 3 Kagoshima Red Cross Hospital, Kagoshima, Japan, and 4 Department of Virology, Kagoshima University, Kagoshima, Japan

(Received21 December1988) (Revised, received30 May 1989) (Accepted 23 June 1989)

Key words: Human T lymphotropicvirus type I (HTLV-I); Myelopathy;T lymphocyte,activated; Cerebrospinal fluid; HTLV-I-as-

sociated myelopathy/tropical spastic paraparesis (HAM/TSP) Summary In order to detect activated T lymphocytes in the cerebrospinal fluid (CSF) of patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis ( H A M / T S P ) , we studied CSF lymphocytes in untreated patients with H A M / T S P and other neurological diseases (OND). Dual-immunofluorescence staining technique was performed using fluorescence microscopy. No significant difference in the C D 4 - / C D 8 + ratio of CSF lymphocytes was observed between H A M / T S P patients and patients with OND. However, both CD4 + and CD8 ÷ CSF lymphocytes of H A M / T S P patients contained higher percentages of HLA-DR-positive cells than those of patients with O N D ( P < 0.05), suggesting that the activated CSF T lymphocytes were composed of both CD4 ÷ and CD8 ÷ subsets in patients with H A M / T S P .

Introduction

Human T lymphotropic virus type I (HTLV-I), which was best known for its relation to adult T cell leukemia/lymphoma, has been implicated in a chronic progressive myelopathy: HTLV-I-associated myelopathy (HAM) (Osame et al., 1986, 1987)

Address for correspondence:Mitsuhiro Osame, MD, Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, 1208-1 Usuki-Cho, Kagoshima 890, Japan.

and HTLV-I-associated tropical spastic paraparesis (TSP) (Gessain et al., 1985; Rodgers-Johnson et al., 1985). Furthermore, the identity of these two entities ( H A M / T S P ) has recently been reported (Roman and Osame, 1988). Despite a clear association of HTLV-I infection with this chronic neurologic disease, the underlying pathogenetic mechanisms remain to be elucidated. T cell activation in peripheral blood (Itoyama et al., 1988; Jacobson et al., 1988; Kitajima et al., 1988) and cerebrospinal fluid (CSF) (Nonoyama et al., 1989) of patients with H A M / T S P has been

0165-5728/89/$03.50 © 1989 Elsevier SciencePublishers B.V. (Biomedical Division)

252 observed. I n addition, Mori a n d colleagues (1988) reported u n u s u a l l y high p r o p o r t i o n s of C D 4 + H L A - D R + cells a n d C D 8 + H L A - D R + cells in peripheral blood of H A M / T S P patients, by a d o u b l e - s t a i n i n g method. Here we report that H L A - D R b e a r i n g C D 4 + a n d C D 8 + lymphocytes in C S F increase in H A M / T S P patients.

o b t a i n e d were then resuspended in 1% fetal call s e r u m / 0 . 1 % N a N 3 / R P M I 1640, a n d washed twice. Cells were divided in five aliquots, each with a volume of a b o u t 50 /zl, a n d were mixed with an o p t i m a l c o n c e n t r a t i o n of the m o n o c l o n a l a n t i b o d ies, which had been tagged with either fluorescein i s o t h i o c y a n a t e ( F I T C ) or p h y c o e r y t h r i n (PE). For d u a l - i m m u n o f l u o r e s c e n c e staining, the PE-conj u g a t e d a n t i b o d y a n d the F I T C - c o n j u g a t e d one were paired to the following: C D 2 / C D 4 , C D 2 / CD8, H L A - D R / C D 4 , H L A - D R / C D 8 (all obtained from Coulter I m m u n o l o g y , Hialeah. FL, U.S.A.). After i n c u b a t i o n at 4 ° C for 30 min, cells were washed 3 times a n d suspended in 5 #1 glycerine buffer, a n d prepared. Usually more than 100 lymphocytes were c o u n t e d u n d e r fluorescence microscopy. C S F lymphocytes were detected morphologically, a n d then each fluorescein on them was evaluated. S t u d e n t ' s t-test was used to c o m p a r e differences b e t w e e n groups. P values less than 0.05 were considered significant.

Patients and methods Seven H A M / T S P patients a n d five seronegative patients with other neurological diseases ( O N D ) were studied. The patients with O N D i n c l u d e d two patients with spinocerebellar degeneration, a p a t i e n t with progressive s u p r a n u c l e a r palsy and two patients with cerebrovascular disease. The diagnosis of H A M / T S P was based on clinical a n d serological criteria as described (Osame et al., 1987). The clinical features of the patients are shown in T a b l e 1. The percentages of T cell subsets in the C S F l y m p h o c y t e p o p u l a t i o n s were d e t e r m i n e d according to the technique described by K u r o d a a n d Shibasaki (1985, 1987) with some modifications. I n brief, a b o u t 10 ml C S F was o b t a i n e d within half an hour of a t r a u m a t i c l u m b a r puncture. C S F was centrifuged at 300 × g for 10 min. Cells thus

Results T a b l e 2 shows the p r o p o r t i o n s of C S F l y m p h o c y t e s positive for each m o n o c l o n a l anti-

TABLE 1 CLINICAL FEATURES OF PATIENTS WITH HAM/TSP AND OTHER NEUROLOGICAL DISEASES Patient No./age/sex/ duration of symptoms (years)

Blood transfusion

CSF cells/mm3

HAM 1/25/F/0.7 HAM 2/33/F/3 HAM 3/46/F/13 HAM 4/50/F/5 HAM 5/52/F/3 HAM 6/53/M/13 HAM 7/64/F/12

-+

5/3 17/3 14/3 36/3 29/3 7/3 15,/3

-

5,/3

-

6/3 5/3 5/3

-

9,/3

SCD b 1/34/M/20 SCD 2/64/M/9 PSP ~ 1/76/M/3 CVD d 1/47/M/2 CVD 2/76/M/0.5

~l Ab(PA), antibody titer to HTLV-I (particle agglutination). h SCD, spinocerebellar degeneration. c PSP, progressive supranuclear palsy. d CVD, cerebrovascular disease.

HTLV-I Ab(PA) " Serum ( × ) 1 024 2 048 1 024 2 048 2 048 1024 1 024 < < < < <

16 16 16 16 16

CSF ( × ) 16 256 128 64 256 32 32 < 16 < 16 < 16 < 16 < 16

253 TABLE 2 PHENOTYPIC ANALYSIS OF CSF LYMPHOCYTES IN H A M / T S P PATIENTS A N D PATIENTS WITH OTHER N E U R O LOGICAL DISEASES (OND) Data are mean _+ SD.

H A M / T S P (n = 7) OND (n = 5)

CD4+/CD2 + (%)

CD8+/CD2 + (%)

CD4+/CD8 + (ratio)

CD4 + H L A - D R + / CD4 + (%)

CD8 + H L A - D R + / CD8 + (%)

58.1_+16.5 69.3 +_ 9.7

42.8___10.5 37.4 + 10.2

1.48_+0.64 2.05 _+ 0.89

11.8_+9.4 * 2.6 +_ 3.2

23.4_+18.7 * 1.3 _+ 1.8

• Differences were statistically significant between H A M / T S P and OND, P < 0.05.

body. No significant differences in the percentage of T cell subsets (CD4+/CD2 +, CD8+/CD2 ÷) and CD4÷/CD8 ÷ ratio of CSF lymphocytes were observed between HAM/TSP patients and patients with OND. However, the percentages of CD4 ÷ H L A - D R + / C D 4 ÷ and CD8 ÷ HLADR+/CD8 ÷ of CSF lymphocytes increased significantly more in HAM/TSP patients than in patients with OND (P < 0.05).

Discussion In previous reports about CSF findings in neurological diseases, the central nervous system (CNS) parenchyme and subarachnoid space are regarded as a compartment and it is suggested that abnormalities detected in CSF mirror events that occur in the CNS parenchyme (Noronha et al., 1980; Hommes and Brinkman, 1984; Hailer and Weiner, 1987). Nonoyama et al. (1989) reported that CD3 ÷ lymphocytes in CSF of HAM/TSP patients had activation markers including Tal and HLA-DR. The results of our study demonstrated that the phenotype of HLA-DR-positive T lymphocytes in CSF of HAM/TSP patients was not only CD4 ÷ but also CD8 +. These findings provide evidence of an ongoing T cell response in the CNS in HAM/TSP. However, it has remained unknown whether these changes play a primary role in the pathogenesis of HAM/TSP or represent only an epiphenomenon. Hirose et al. (1986) established a T cell line by co-culturing of cells in CSF of an HAM/TSP patient with cord blood cells, and demonstrated the presence of C-type particles in the cultured T

cells. Nishimura et al. (1988) recently reported the isolation of HTLV-I-infected T cell lines with CD4 phenotype from CSF of a patient with HAM/TSP. These findings indicate the presence of HTLV-I-infected CD4 subsets in the CSF of the patients with HAM/TSP. In addition, Usuku et al. (1988) demonstrated that peripheral lymphocytes of H A M / T S P patients had a genetically determined immunological potency that caused a high immune response against HTLV-I in vitro. We have proposed (Ijichi et al., 1989) a hypothetical pathogenesis that lymphocyte proliferations occur in the CNS following the supply of infected (CD4 ÷) T cells from peripheral blood to CNS, and that cytokines derived from activated lymphocytes (CD4 ÷ and CD8-) and macrophages produce the CNS lesions of HAM/TSP. The presence of activated CD4 ÷ and CD8 ÷ cells in CSF of patients with HAM/TSP is compatible with this hypothesis.

Acknowledgement We thank Mr. S. Yashiki for his helpful suggestions.

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