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Detection of tumor necrosis factor-α-positive cells in cerebrospinal fluid of patients with HTLV-I-associated myelopathy
Journal of Neuroimmunology, 42 (1993) 127-130
127
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-5728/93/$06.00 JNI 02275
Detection of tumor necrosis factor-a-positive cells in cerebrospinal fluid of patients with HTLV-I-associated myelopathy S h o z o N a k a m u r a , Isao N a g a n o , M a s a r u Yoshioka, S h i g e r u Shimazaki, Junichi O n o d e r a and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku Unicersity School of Medicine, Sendai, Japan
(Received 21 April 1992) (Revision received8 July 1992) (Accepted 8 July 1992) Key words': Tumor necrosis factor-a-positivecell; Cerebrospinal fluid; HTLV-l-associatedmyelopathy;Immunocytochemistry
Summary Tumor necrosis factor (TNF)-a-posltive cells constituted 1.6-18% and 8.2-23.5% of the total number of cerebrospinal fluid cells from six of 12 patients with HTLV-I-associated myelopathy and in all samples obtained from inflammatory cases, respectively. However, in non-inflammatory cases no TNF-a-positive cells were detected. These results suggest that some of the infiltrating CSF cells produce TNF-a, which plays a role in host immune defenses against causative agents including HTLV-I and in lesion formation within the central nervous system in inflammatory diseases.
Introduction Immunological studies of the cerebrospinal fluid (CSF) of HTLV-I-associated myelopathy (HAM) patients have shown an increased number of activated T lymphocytes with H L A - D R (Ijichi et al., 1989), an elevated intra-blood-brain IgG synthesis rate with an elevated IgG index in the majority of patients (Gessain et al., 1988), and increased interleukin (IL)-6 levels in the CSF (Nishimoto et al., 1990; Ohbo et al., 1991). These results indicate that an inflammatory reaction may take place in the central nervous system including the cerebrospinal fluid (CSF) and may be associated with the pathogenesis of HAM. Recent studies have revealed that TNF-a has a wide variety of biological activities such as direct tumoricidal activity (Mathews et al., 1983; Helson et al., 1986), antiviral effects (Mestan et al., 1986; Wong et al., 1986) and immunomodulation (Talmage et al., 1988). TNF-a concentration was clinically measured in the serum and CSF of patients with various infectious diseases of the central nervous system (CNS), including acquired Correspondence to: S. Nakamura, Department of Neurology,Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi,Aoba-ku, Sendai 980, Japan.
immunodeficiency syndrome (AIDS) (Lahdevirta et al., 1988), cerebral malaria (Gran et al., 1989) and bacterial meningitis (Waage et al., 1989). The elevated TNF-a concentration in CSF shown in these studies may play an important role in immunological responses to pathogenic organisms and the process of inflammation, and may be due to local production of TNF-a within the CNS (Waage et al., 1989). In order to detect cells which are immunopositive for TNF-a, we studied CSF cell smears obtained from patients with HAM and inflammatory diseases using immunocytochemistry. In this report, we present data that show the presence of immuno-reactivity for TNF-a in CSF cells from patients with HAM in spite of normal CSF cell counts or mild pleocytosis.
Subjects and Methods Subjects
The subjects were 12 patients with HAM clinically diagnosed by Osame's criteria (Osame et al., 1986). Five of the 12 were female. The age range was 27-69 years and the mean duration of illness was 10 years. Control cases included nine patients with Parkinson's disease (PD), eight patients with subacute cerebellar degeneration (SCD), and ten patients with motor neu-
128
ron disease (MND). In addition, three patients with herpes simplex encephalitis (HSE), one patient with EB virus polyradiculoneuritis (EBV) and three patients with viral meningitis (VM) were also examined. Ten CSF samples were obtained from the 12 H A M patients before steroid treatment. Twelve samples were also obtained from the 12 patients with H A M during administration of prednisolone. Nine samples were obtained from patients with PD, eight from patients with SCD, ten from patients with MND, three from patients with HSE, two from patients with EBV, and three from patients with VM.
Preparation of CSF cell smears Immediately after lumbar puncture, the number of CSF cells was counted by Fuchs-Rosenthal's chamber, and then CSF cell smears were obtained from 2 - 3 ml of CSF after routine examination by centrifugation at 800 rpm for 5 min at 4°C using a cytocentrifuge apparatus (Model SC2, Tomy Seiko Corp., Tokyo). Immediately after drying, the slides were fixed in cold acetone for 10 min, and then stored at -7(I°C until immunostaining.
mersion in l
Enumeration of TNF-a-positice cells The number of cells immunostained with TNF-a, approximately 100 cells per slide, were counted and classified to be lymphocytes or macrophages under a microscope.
Immunoo, tochemistry
Results
Detection of T N F - a by polyclonal anti-human T N F - a rabbit serum was performed using the avidinbiotin-peroxidase complex (ABC) method. The acetone-fixed CSF cell smears were washed three times in PBS, and intrinsic peroxidase was inactivated by ira-
The TNF-a-positive cells immunostained with specific antibody showed brown-colored deposits in the cytoplasm, whereas no deposits were seen in the nucleus. Morphologically, these TNF-a-positive cells
TABLE 1 Percent and phenotypes of TNF-a-positive cells in the CSF and anti-HTLV-I titers in HAM patients Patient
Age/sex
Anti-HTLV-I triers in
CSF findings
5
10
15
i
J
i
i
1
59/M
NO
56/F 43/M 53/F 69/M 58/M 43/M
4 I 4 I 3 I NO
63/F
21
9 10 tI 12
55/F 27/F 49/M 43/M
16 21 13
NO, not obtained. a Values are means +_SD. i ,
macrophages
[~,
lymphocytes.
74
10
c,: and phenotypes of TNF-a '+ cells
0
2 3 4 5 6 7 8
CSF
Serum
5.4 + 2.1 1) 7.0 + 2.5 0 1.6 _+ 0.2
32 16 8 8 16
11.0 + 3.4
8
0 IS.0 _+ 5.0
2 64 16 8 4 64
640 1280 321) 1280 1280 160 32{) 2561) 1280 1280 321) 1280
After steroid treatment
Before treatment Cell count ( n / 3 / m m 3)
(O) "
20 i
0
5
10
15
2O
i
i
i
i
i
61 22
51 2t I-I 12.4 8.0 14.5 0 6.0 5.0 0
_+ 2.8 _+ 2.3 -t- 3.8
36
81 12
6i _+ 2.0 + 1.7
% and phenotypes of TNF-a t cells
Cell count ( n / 3 / m m 3)
51 14 I t0
0
0 0 6.7 + 2.6
129
the range of CSF cell counts was 5 4 - 1 2 4 0 / 3 / m m 3 in the inflammatory diseases including HSE, VM and EB virus polyradiculoneuritis. In all three samples from HSE, two samples from EBV and three samples from VM, TNF-a-positive cells were found to constitute 8.2-23.5% of the total cells. In contrast, no TNF-apositive cells were detected in the non-inflammatory cases of PD, SCD and MND (Table 2). Moreover, there was no correlation between the CSF cell counts and percent of TNF-a-positive cells in 21 samples from patients with HAM.
a
8
Fig. 1. Immunostaining of CSF cell smears with anti-human TNF-a rabbit serum. Two immunopositive cells for TNF-a, morphologically identified as an activated lymphocyte and a macrophage can be seen. The deposit of immunoreactivity is located only within cytoplasm.
were identified to be macrophages or lymphocytes (Fig. 1). In contrast, there was no detection of positive cells immunostained with normal rabbit serum as a primary antibody. The range of CSF cell counts was 3 7 4 / 3 / m m 3 in both untreated and treated patients with HAM in the present study. In five of ten samples from untreated patients, TNF-a-positive cells were found to constitute 5.0-14.5% of total CSF cells. Similarly, in six of 12 CSF cell smears obtained from HAM patients with prednisolone treatment, TNF-a-positive cells constituted 1.6-18% of the cells. Based on morphological analysis, the macrophage : lymphocyte ratios in TNF-apositive cells are summarized in Table 1. Furthermore, TABLE 2 Percent of TNF-a-positive cells in the CSF in the inflammatory and non-inflammatory patients Patient
DAO
Cell count ( n / 3 / m m 3)
TNF-a (%) mean + SD
Herpes simplex encephalitis
13 14 15
22 9 21
135 392 54
8.2+2.1 10.8+3.4 12.0+_2.8
Viral meningitis
16 17 18
14 10 7
214 1240 182
13.5+4.6 9.4 + 3.9 11.5+5.1
EB virus polyradiculoneuritis
19 19
180 190
86 54
23.5 + 6.8 18.6 _+5.8
Parkinson's disease
n= 9
ND
0-5
0
Spinocerebellar degeneration
n= 8
ND
3-10
0
Motor neuron disease
n = 10
ND
0-8
0
DAO, days after onset; ND, not determined.
Discussion We detected TNF-a-positive cells in six of 12 patients, and such cells were found to constitute 1.618% of the total CSF cells in both untreated and treated patients with H A M in spite of normal CSF cell counts or mild pleocytosis, whereas in all samples obtained at the early stages of inflammatory cases, TNF-a-positive cells were found to constitute 8.223.5% of the total number of CSF cells. In contrast, no TNF-a-positive cells were detected in patients with non-inflammatory diseases. The results obtained here indicate that not only lymphocytes but also macrophages in the infiltrating CSF cells are activated in inflammatory diseases including HAM. With regard to cytokines in CSF of HAM patients, Nishimoto et al. (1990) and Ohbo et al. (1991) reported significant titers of interleukin-6 (IL-6) in CSF from patients with HAM when compared to controls. On the contrary, the levels of other cytokines such as IL-1, TNF-a (Ohbo et al., 1991), IL-2 and lymphotoxin (Iwasaki et al., 1989) were not detectable in CSF of HAM patients. However, Iwasaki et al (1989) reported the presence of myelinotoxicity in CSF of HAM patients using murine CNS explant tissue culture. In addition, it has been reported that not only m o n o c y t e / m a c r o p h a g e lineage and T and B cells, but also glial cells such as astrocytes (Lieberman et al., 1989; Selmaj et al., 1991) and microglia (Hofman et al., 1990) also produce TNF-a under these pathological conditions. Although the primary role of TNF-a is tumoricidal in murine or human tumors (Mathews et al., 1983; Helson et al., 1986), there is accumulating evidence of other TNF-a activities including inhibition of virus replication (Mestan et al., 1986), synergizing anti-viral activity with interferon-y (IFN-T) (Wong et al., 1986) and direct effects on mitogen-stimulated B cell differentiation and proliferation (Jelinek et al., 1987; Kehrl et al., 1987). Additionally, T N F - a has been shown to be toxic to oligodendroglia in vitro (Selmaj and Raine, 1988) and to have multiple effects on endothelia, in-
13u
ducing vasodilatation, thrombosis and leukocyte adhesion (Mantovani and Dejana, 1989). Therefore, it is strongly suggested that TNF-o~ secreted by activated macrophages and lymphocytes detected in CSF may participate in the above-mentioned pleiotropic activities within the CSF compartment in inflammatory diseases and HAM. In addition to the present findings, the presence of perivascular cuffing with lymphocytes, abundant foamy macrophages and astrocytosis observed in pathological studies of HAM (Akizuki et al., 1989) may support the possibility of an immune reaction involving CSF cells and the endogenous ceils against HTLV-I within the CNS. Further studies for detection of cytokines including IL-1, IL-6, IFN-7 and lymphotoxin positive cells in the CSF and the spinal cord lesions of autopsied HAM patients are required to clarify the pathogenic mechanism of HAM.
Acknowledgements This work was supported by a grant from the lmmunoneurological Diseases Research Committee, Ministry of Health and Welfare of Japan.
References Akizuki, S., Nakazato, O., Higuchi, Y., Tanabe, K., Setoguchi, M., Yoshida, S., Miyazaki, Y., Yamamoto, S. and Sudou, S. (1987) Necropsy findings in HTLV-I associated myelopathy. Lancet i, 156-157. Gessain, A., Caudie, C., Gout, O., Vernant, J., Maurs, S., Kodano, C., Malone, G., Tournier-Lasserve, E. and de-The G. (1988) Intrathecal synthesis of antibodies to human T lymphotrophic virus type 1 and the presence of IgG oligoclonal bands in the cerebrospinal fluid of patients with endemic tropical spastic paraparesis. J. Infect. Dis. 157, 1226-1234. Gran, G.E., Pignet, P.F., Vassalli, P. and Lambert, P.H. (1989) Tumor necrosis factor and other eytokines in cerebral malaria: experimental and clinical data. Immunol. Rev. 112, 49-70. Helson, L., Helson, C. and Green, S. (1986) Effects of murine tumor necrosis factor on heterotransplanted human tumors. Exp. Cell. Biol. 47, 53-60. Hofmann, F.M., Hinton, D.R, Johnson, K. and Merrill. J.E. (1989) Tumor necrosis factor identified in multiple sclerosis. J. Exp. Med. 170, 607-612. Ijichi, S., Eiraku, N., Osame, M., Izumo, K., Kubota, R., Maruyama, I., Matsumoto, M., Niimura, T. and Sonoda, S. (1989) Activated
T I,vmphocytes in ccrebrospinal fluid of patient~, with HTLV-I-associated myelopathy (HAM/TSPt ,1. Neuroinmmnol. 25. 251 254. Iwasaki, Y., Tsukamoto, T.. Terunuma, H. and Osame. M. 119891 Mechanisms of neural tissue damage in human retrovirus infections: cytopathic activit:~ in CSF of patients with HTLV-l-associated myelopathy. In: G.C. Roman, J.C. Vernant and M. Osame (Eds.), HTLV-1 and nervous ~ystem. Alan R. Liss., Ne~ Y¢~rk. NY. pp. 325-332. Jelinek, D.F. and Lips,', D.E. (1987) Enhancement o1 human B cell proliferation and differentiation by tumor necrosis factor-or, and interleukin-1. J. Immuno[. 139, 2970-2976. Kehrl. J.H., Miller, A. and Fauchi. A.S. (1987) Effect of tumor necrosis factor-a on mitogen-aetivated human B cells. J. Exp. Med. 166, 786 791. Lahdevirta, J., Maul', C.P., Teppo. A. and Repo, H. (1988l Elevated levels of circulating cacheetin/tumor necrosis factor in patients with acquired immunodeficienc~ syndrome. Am. J. Med. 85, 289-291. Lieberman, A.P., Pitha, P.M.. Shin. H.S. and Shin, M.L. ~1989l Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neutrophic virus. Proc. Natl. Acad. Sci. USA 86, 6348-0352. Mantoviani, A. and Dejana, E. (1989) Cytokines as communication signals between leukocytes and endothelial cell. Immunol. Today 10, 370 375. Mathews, N. (1083)Anti-tumor cytotoxin produced by human monocytes. Studies on its mode of action. Br. J. Cancer. 48, 405-410. Mestan, J., Digel, W., Mittnacht. S., Hillen. H., Blohm. D., Moiler, A., Jacobson. H. and Kirchner, H. (1986) Antiviral effects of recombinant tumor necrosis factor in vitro. Nature 323, 816-819. Nishimoto, N., Yoshizaki, K., Eiraku, N., Machigashira, K., Tagoh, H., Ogata. A., Kuritani, T., Osame, M. and Kishimoto, T. (1990) Elevated levels of interleukin 6 in serum and cerebrospinal fluid of ttTLV-I-associated myelopathy/tropical spastic paraparesis. J. Neurol. Sci. 97, 183-193. Ohbo, K.. Sugamura, K., Sekizawa, T. and Kogure, K. (1991) Interleukin-6 in cerebrospinal fluid of HTLV-I-associated myelopathy. Neurology 41,594-595. Osame, M., Usuku. K.. lzumo, S., Ichiji, N.. ,~nitani, H., lgata, A.. Matsumoto, M. and Tara. M. (1986) HTLV-I-associated myelopathy. A new entity. Lancet ii, 1031-1032. Selmaj. K.W. and Raine, C.S. (1988) Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann. Neurol. 23, 339-346. Selmaj, K.W., Raine, C.S., Cannella, B, and Brosnan, C.F. (1991) Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions. J. Clin. Invest. 87, 949-954. Talmage, J.E., Phillips, H.. Schneider, M., Auhmann, A.J.. Figari, T.S.. Ranger. G.E. and Palladion, M.A. Jr. (1988) Immunomodulatory properties of recombinant mouse and human tumor necrosis factor. Cancer Res. 48, 544-550. Waage, A., Halstensen, A.. Shalaby, R., Brandtaeg, P. and Espevik, T. (1989) Local production of tumor necrosis factor a, interleukin 1, and interleukin-6 in meningococcal meningitis. Relation to the inflammatory, response. J. Exp. Med. 170, 1859-1867. Wong, G.H.W. and Goeddel, D.V. (1988.1 Tumor necrosis factor a and /3 inhibit virus replication and synergize with interferons. Nature 323, 819-822.
127
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-5728/93/$06.00 JNI 02275
Detection of tumor necrosis factor-a-positive cells in cerebrospinal fluid of patients with HTLV-I-associated myelopathy S h o z o N a k a m u r a , Isao N a g a n o , M a s a r u Yoshioka, S h i g e r u Shimazaki, Junichi O n o d e r a and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku Unicersity School of Medicine, Sendai, Japan
(Received 21 April 1992) (Revision received8 July 1992) (Accepted 8 July 1992) Key words': Tumor necrosis factor-a-positivecell; Cerebrospinal fluid; HTLV-l-associatedmyelopathy;Immunocytochemistry
Summary Tumor necrosis factor (TNF)-a-posltive cells constituted 1.6-18% and 8.2-23.5% of the total number of cerebrospinal fluid cells from six of 12 patients with HTLV-I-associated myelopathy and in all samples obtained from inflammatory cases, respectively. However, in non-inflammatory cases no TNF-a-positive cells were detected. These results suggest that some of the infiltrating CSF cells produce TNF-a, which plays a role in host immune defenses against causative agents including HTLV-I and in lesion formation within the central nervous system in inflammatory diseases.
Introduction Immunological studies of the cerebrospinal fluid (CSF) of HTLV-I-associated myelopathy (HAM) patients have shown an increased number of activated T lymphocytes with H L A - D R (Ijichi et al., 1989), an elevated intra-blood-brain IgG synthesis rate with an elevated IgG index in the majority of patients (Gessain et al., 1988), and increased interleukin (IL)-6 levels in the CSF (Nishimoto et al., 1990; Ohbo et al., 1991). These results indicate that an inflammatory reaction may take place in the central nervous system including the cerebrospinal fluid (CSF) and may be associated with the pathogenesis of HAM. Recent studies have revealed that TNF-a has a wide variety of biological activities such as direct tumoricidal activity (Mathews et al., 1983; Helson et al., 1986), antiviral effects (Mestan et al., 1986; Wong et al., 1986) and immunomodulation (Talmage et al., 1988). TNF-a concentration was clinically measured in the serum and CSF of patients with various infectious diseases of the central nervous system (CNS), including acquired Correspondence to: S. Nakamura, Department of Neurology,Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi,Aoba-ku, Sendai 980, Japan.
immunodeficiency syndrome (AIDS) (Lahdevirta et al., 1988), cerebral malaria (Gran et al., 1989) and bacterial meningitis (Waage et al., 1989). The elevated TNF-a concentration in CSF shown in these studies may play an important role in immunological responses to pathogenic organisms and the process of inflammation, and may be due to local production of TNF-a within the CNS (Waage et al., 1989). In order to detect cells which are immunopositive for TNF-a, we studied CSF cell smears obtained from patients with HAM and inflammatory diseases using immunocytochemistry. In this report, we present data that show the presence of immuno-reactivity for TNF-a in CSF cells from patients with HAM in spite of normal CSF cell counts or mild pleocytosis.
Subjects and Methods Subjects
The subjects were 12 patients with HAM clinically diagnosed by Osame's criteria (Osame et al., 1986). Five of the 12 were female. The age range was 27-69 years and the mean duration of illness was 10 years. Control cases included nine patients with Parkinson's disease (PD), eight patients with subacute cerebellar degeneration (SCD), and ten patients with motor neu-
128
ron disease (MND). In addition, three patients with herpes simplex encephalitis (HSE), one patient with EB virus polyradiculoneuritis (EBV) and three patients with viral meningitis (VM) were also examined. Ten CSF samples were obtained from the 12 H A M patients before steroid treatment. Twelve samples were also obtained from the 12 patients with H A M during administration of prednisolone. Nine samples were obtained from patients with PD, eight from patients with SCD, ten from patients with MND, three from patients with HSE, two from patients with EBV, and three from patients with VM.
Preparation of CSF cell smears Immediately after lumbar puncture, the number of CSF cells was counted by Fuchs-Rosenthal's chamber, and then CSF cell smears were obtained from 2 - 3 ml of CSF after routine examination by centrifugation at 800 rpm for 5 min at 4°C using a cytocentrifuge apparatus (Model SC2, Tomy Seiko Corp., Tokyo). Immediately after drying, the slides were fixed in cold acetone for 10 min, and then stored at -7(I°C until immunostaining.
mersion in l
Enumeration of TNF-a-positice cells The number of cells immunostained with TNF-a, approximately 100 cells per slide, were counted and classified to be lymphocytes or macrophages under a microscope.
Immunoo, tochemistry
Results
Detection of T N F - a by polyclonal anti-human T N F - a rabbit serum was performed using the avidinbiotin-peroxidase complex (ABC) method. The acetone-fixed CSF cell smears were washed three times in PBS, and intrinsic peroxidase was inactivated by ira-
The TNF-a-positive cells immunostained with specific antibody showed brown-colored deposits in the cytoplasm, whereas no deposits were seen in the nucleus. Morphologically, these TNF-a-positive cells
TABLE 1 Percent and phenotypes of TNF-a-positive cells in the CSF and anti-HTLV-I titers in HAM patients Patient
Age/sex
Anti-HTLV-I triers in
CSF findings
5
10
15
i
J
i
i
1
59/M
NO
56/F 43/M 53/F 69/M 58/M 43/M
4 I 4 I 3 I NO
63/F
21
9 10 tI 12
55/F 27/F 49/M 43/M
16 21 13
NO, not obtained. a Values are means +_SD. i ,
macrophages
[~,
lymphocytes.
74
10
c,: and phenotypes of TNF-a '+ cells
0
2 3 4 5 6 7 8
CSF
Serum
5.4 + 2.1 1) 7.0 + 2.5 0 1.6 _+ 0.2
32 16 8 8 16
11.0 + 3.4
8
0 IS.0 _+ 5.0
2 64 16 8 4 64
640 1280 321) 1280 1280 160 32{) 2561) 1280 1280 321) 1280
After steroid treatment
Before treatment Cell count ( n / 3 / m m 3)
(O) "
20 i
0
5
10
15
2O
i
i
i
i
i
61 22
51 2t I-I 12.4 8.0 14.5 0 6.0 5.0 0
_+ 2.8 _+ 2.3 -t- 3.8
36
81 12
6i _+ 2.0 + 1.7
% and phenotypes of TNF-a t cells
Cell count ( n / 3 / m m 3)
51 14 I t0
0
0 0 6.7 + 2.6
129
the range of CSF cell counts was 5 4 - 1 2 4 0 / 3 / m m 3 in the inflammatory diseases including HSE, VM and EB virus polyradiculoneuritis. In all three samples from HSE, two samples from EBV and three samples from VM, TNF-a-positive cells were found to constitute 8.2-23.5% of the total cells. In contrast, no TNF-apositive cells were detected in the non-inflammatory cases of PD, SCD and MND (Table 2). Moreover, there was no correlation between the CSF cell counts and percent of TNF-a-positive cells in 21 samples from patients with HAM.
a
8
Fig. 1. Immunostaining of CSF cell smears with anti-human TNF-a rabbit serum. Two immunopositive cells for TNF-a, morphologically identified as an activated lymphocyte and a macrophage can be seen. The deposit of immunoreactivity is located only within cytoplasm.
were identified to be macrophages or lymphocytes (Fig. 1). In contrast, there was no detection of positive cells immunostained with normal rabbit serum as a primary antibody. The range of CSF cell counts was 3 7 4 / 3 / m m 3 in both untreated and treated patients with HAM in the present study. In five of ten samples from untreated patients, TNF-a-positive cells were found to constitute 5.0-14.5% of total CSF cells. Similarly, in six of 12 CSF cell smears obtained from HAM patients with prednisolone treatment, TNF-a-positive cells constituted 1.6-18% of the cells. Based on morphological analysis, the macrophage : lymphocyte ratios in TNF-apositive cells are summarized in Table 1. Furthermore, TABLE 2 Percent of TNF-a-positive cells in the CSF in the inflammatory and non-inflammatory patients Patient
DAO
Cell count ( n / 3 / m m 3)
TNF-a (%) mean + SD
Herpes simplex encephalitis
13 14 15
22 9 21
135 392 54
8.2+2.1 10.8+3.4 12.0+_2.8
Viral meningitis
16 17 18
14 10 7
214 1240 182
13.5+4.6 9.4 + 3.9 11.5+5.1
EB virus polyradiculoneuritis
19 19
180 190
86 54
23.5 + 6.8 18.6 _+5.8
Parkinson's disease
n= 9
ND
0-5
0
Spinocerebellar degeneration
n= 8
ND
3-10
0
Motor neuron disease
n = 10
ND
0-8
0
DAO, days after onset; ND, not determined.
Discussion We detected TNF-a-positive cells in six of 12 patients, and such cells were found to constitute 1.618% of the total CSF cells in both untreated and treated patients with H A M in spite of normal CSF cell counts or mild pleocytosis, whereas in all samples obtained at the early stages of inflammatory cases, TNF-a-positive cells were found to constitute 8.223.5% of the total number of CSF cells. In contrast, no TNF-a-positive cells were detected in patients with non-inflammatory diseases. The results obtained here indicate that not only lymphocytes but also macrophages in the infiltrating CSF cells are activated in inflammatory diseases including HAM. With regard to cytokines in CSF of HAM patients, Nishimoto et al. (1990) and Ohbo et al. (1991) reported significant titers of interleukin-6 (IL-6) in CSF from patients with HAM when compared to controls. On the contrary, the levels of other cytokines such as IL-1, TNF-a (Ohbo et al., 1991), IL-2 and lymphotoxin (Iwasaki et al., 1989) were not detectable in CSF of HAM patients. However, Iwasaki et al (1989) reported the presence of myelinotoxicity in CSF of HAM patients using murine CNS explant tissue culture. In addition, it has been reported that not only m o n o c y t e / m a c r o p h a g e lineage and T and B cells, but also glial cells such as astrocytes (Lieberman et al., 1989; Selmaj et al., 1991) and microglia (Hofman et al., 1990) also produce TNF-a under these pathological conditions. Although the primary role of TNF-a is tumoricidal in murine or human tumors (Mathews et al., 1983; Helson et al., 1986), there is accumulating evidence of other TNF-a activities including inhibition of virus replication (Mestan et al., 1986), synergizing anti-viral activity with interferon-y (IFN-T) (Wong et al., 1986) and direct effects on mitogen-stimulated B cell differentiation and proliferation (Jelinek et al., 1987; Kehrl et al., 1987). Additionally, T N F - a has been shown to be toxic to oligodendroglia in vitro (Selmaj and Raine, 1988) and to have multiple effects on endothelia, in-
13u
ducing vasodilatation, thrombosis and leukocyte adhesion (Mantovani and Dejana, 1989). Therefore, it is strongly suggested that TNF-o~ secreted by activated macrophages and lymphocytes detected in CSF may participate in the above-mentioned pleiotropic activities within the CSF compartment in inflammatory diseases and HAM. In addition to the present findings, the presence of perivascular cuffing with lymphocytes, abundant foamy macrophages and astrocytosis observed in pathological studies of HAM (Akizuki et al., 1989) may support the possibility of an immune reaction involving CSF cells and the endogenous ceils against HTLV-I within the CNS. Further studies for detection of cytokines including IL-1, IL-6, IFN-7 and lymphotoxin positive cells in the CSF and the spinal cord lesions of autopsied HAM patients are required to clarify the pathogenic mechanism of HAM.
Acknowledgements This work was supported by a grant from the lmmunoneurological Diseases Research Committee, Ministry of Health and Welfare of Japan.
References Akizuki, S., Nakazato, O., Higuchi, Y., Tanabe, K., Setoguchi, M., Yoshida, S., Miyazaki, Y., Yamamoto, S. and Sudou, S. (1987) Necropsy findings in HTLV-I associated myelopathy. Lancet i, 156-157. Gessain, A., Caudie, C., Gout, O., Vernant, J., Maurs, S., Kodano, C., Malone, G., Tournier-Lasserve, E. and de-The G. (1988) Intrathecal synthesis of antibodies to human T lymphotrophic virus type 1 and the presence of IgG oligoclonal bands in the cerebrospinal fluid of patients with endemic tropical spastic paraparesis. J. Infect. Dis. 157, 1226-1234. Gran, G.E., Pignet, P.F., Vassalli, P. and Lambert, P.H. (1989) Tumor necrosis factor and other eytokines in cerebral malaria: experimental and clinical data. Immunol. Rev. 112, 49-70. Helson, L., Helson, C. and Green, S. (1986) Effects of murine tumor necrosis factor on heterotransplanted human tumors. Exp. Cell. Biol. 47, 53-60. Hofmann, F.M., Hinton, D.R, Johnson, K. and Merrill. J.E. (1989) Tumor necrosis factor identified in multiple sclerosis. J. Exp. Med. 170, 607-612. Ijichi, S., Eiraku, N., Osame, M., Izumo, K., Kubota, R., Maruyama, I., Matsumoto, M., Niimura, T. and Sonoda, S. (1989) Activated
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