ELSEVIER Journal of Neuroimmunology 59 (1995) 35-40

Journal of Neuroimmunology

Increased levels of soluble vascular cell adhesion molecule-l (VCAM-1) in the cerebrospinal fluid and sera of patients with multiple sclerosis and

human T lymphotropic virus type-l-associated myelopathy

Masayuki Matsuda a, * , Naoyuki Tsukada b, Koichi Miyagi a, Nobuo Yanagisawa a

’ Department of Medicine (Neurology), Shinshu Vnitiersity School of Medicine, 3-l-l Asahi, Matsumoto 390, Japan b Health Medical Center, Shinshu University School of Medicine, 3-l-l Asahi, Matsumoto 390, Japan

Received 17 November 1994; revised 19 January 1995; accepted 19 January 1995

Abstract

We evaluated the relationship between the soluble form of vascular cell adhesion molecule-l (sVCAM-1) and disease activity in patients with multiple sclerosis (MS) or with human T lymphotropic virus type l-associated myelopathy (HAM), and measured levels of sVCAM-1 in their cerebrospinal fluid (CSF) and sera. Serum and CSF levels of sVCAM-1 were significantly increased in patients with acute relapsing MS during an exacerbation (P < 0.01 and P < O.OOl>, as well as in chronic progressive MS (P < 0.05 and P < O.OOl), compared with healthy individuals and patients with other neurological diseases, respectively. Patients with acute relapsing MS during an exacerbation also exhibited significantly higher serum and CSF levels of sVCAM-1 vs. patients with acute relapsing MS in remission (P < 0.001). Significantly higher serum levels of sVCAM-1 were observed in patients with HAM vs. either healthy individuals (P < 0.01) or non-HAM carriers (P < 0.01). These results suggest that the determination of sVCAM-1 in the sera and CSF may be useful in monitoring the activity of MS and HAM.

Keywords: Soluble vascular cell adhesion molecule-l; Multiple sclerosis; Human T lymphotropic virus type-l-associated myelopathy; Endothelial cell

1. Introduction

The central nervous system (CNS) is immunologi- cally unique in that it lacks areas of lymphatic drainage and is separated from the blood compartment by the blood-brain barrier (BBB). The latter is composed of CNS endothelial cells that line the brain microvessels.

A variety of CNS disorders are characterized by the destruction of the BBB with the infiltration of immune cells. Damage to the BBB in patients with multiple

sclerosis (MS) is probably due to an immune mecha- nism (Broman, 1964; Brown, 1978). It has been sug-

gested that the adhesion of lymphocytes to the brain endothelial cells is important in the early stage of the immune response (Male et al., 1990). Although the

* Corresponding author. Present address: Abteilung Neuroim-

munologie, Max-Planck-Institut fi.ir Psychiatric, Am Klopferspitz

18A, 82152 Planegg-Martinsried, Germany, Phone +49 (89) 8578

3551; Fax +49 (89) 8578 3790

0165-5728/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDI 0165.5728(95)00023-2

mechanisms by which the lymphocytes or leukocytes migrate across the BBB into the CNS are not well understood, an interaction between adhesion-related molecules on the endothelial cells and lymphocytes may facilitate such migration.

The neurological disease caused by human T- lymphotropic virus type 1 (HTLV-1) and that mainly involves the spinal cord is designated as HTLV-l-asso- ciated myelopathy (HAM)/tropical spastic paraparesis

(TSP) (Gassain et al., 1985; Osame et al., 1986). The pathological findings suggest that the BBB is also in- volved in HAM. A direct causal relationship between a viral infection and damage to the BBB has not been demonstrated.

Specific assays for measuring the levels of secretory proteins produced by the various inflammatory cells in the blood and other body fluids now facilitate our ability to assess the activity of inflammatory disorders. The endothelial cells upregulate various adhesion molecules after being activated by various cytokines (Albelda and Buck, 1990). Such molecules, including

36 M. Matsuda et al. /Journal of Neuroimmunologv 59 (1995) 35-40

the intercellular adhesion molecule-l (ICAM-11, en- dothelial leukocyte adhesion molecule-l (ELAM-11, and the vascular cell adhesion molecule-l (VCAM-11 are shed from the membrane and may be measured in body fluids by sensitive immunoassays. Patients with acute relapsing MS demonstrate higher levels of solu- ble ICAM- (slCAM-1) in their sera or cerebrospinal fluid (CSF) as compared with control subjects (Hartung et al., 1993; Sharief et al., 1993; Tsukada et al., 1993a, Tsukada et al., 1993b). The serum level of sICAM-1 is also significantly increased in HAM as compared with controls (Tsukada et al., 1993a). These findings suggest that the level of sICAM-1 in the sera or CSF may be a marker for inflammatory disease of the CNS, and that ICAM- is involved in the evolution and worsening of MS or HAM. There are at least four molecular path- ways for lymphocyte adhesion to endothelial cells (Shimizu et al., 19911, so that the clinical importance of those adhesion molecules is still unclear.

including the presence of demyelinating lesions in the CNS as confirmed by serial studies of magnetic reso- nance imaging (MRI) and by oligoclonal bands (Tour- tellotte and Ma, 1978) and increased levels of IgG in the CSF. Remission was defined as a definite improve- ment of signs, symptoms or both, that was present for at least 24 h (Poser et al., 1983). Analysis of antibodies showed no anti-HTLV-1 in the sera of the MS patients. All patients had been withdrawn from the administra- tion of corticosteroids or other immunosuppressive agents for at least 6 months at the time of sampling. We measured serum and CSF concentrations of sV- CAM-l in five patients with acute relapsing MS before and after the relapse.

2.2. HAM

We measured the serum and CSF levels of soluble VCAM-1 (sVCAM-1) in patients with MS and HAM, and evaluated the relationship between this mediator and disease activity.

2. Materials and methods

Serum samples were obtained from ten patients with HAM, six men and four women, age range 26-67 years (mean 48.7 years). Diagnosis of HAM was based on the criteria of Osame et al. (1986). Their sera showed high antibody titers to HTLV-1 as determined by the indirect immunofluorescence (IF) method. Those anti-HTLV-1 antibody titers in the sera in HAM pa- tients exceeded 80 IF. All serum samples were ob- tained before the administration of corticosteroids.

2.1. MS 2.3. Non-HAM carriers

Paired samples of CSF and sera were collected from 32 Japanese patients with MS, including 14 men and 18 women whose ages ranged from 28 to 72 years (mean 44.8 years). They were diagnosed as laboratory-sup- ported definite MS by the clinical criteria of Poser et al. (1983). Patients were classified as having the chronic progressive (mean duration of disease, 6.5 years) or the relapsing type of disease (mean number of exacerba- tions, 2.6 per year) according to Schumacher et al. (1965). Patients with acute relapsing MS were divided into two groups: sera and CSF were sampled either within 3 days after onset of a relapse (active phase) or during a remission (days 86-189, inactive phase). We analyzed patients with acute relapsing MS in the active phase (clinical exacerbation) (sera: )z = 17, mean dura- tion of disease 3.7 years; CSF: n = 16, mean duration of disease 4.1 years) and in the inactive phase (clinical remission) (sera: n = 12, mean duration of remission 11.3 months; CSF: II = 13, mean duration of remission 10.9 months), and those with primary (n = 7) or sec- ondary (n = 4) chronic progressive MS (sera: n = 10, CSF: II = 11). Patients with concomitant infections were excluded from study. Exacerbation was defined as the occurrence the clinical signs or symptoms of neurologi- cal dysfunction lasting more than 24 h (Poser et al., 1983). The diagnosis of an exacerbation of acute re- lapsing MS was supported by the objective findings

For comparison, we analyzed sera collected from 10 sex- and age-matched non-HAM carriers who were seropositive for HTLV-1. These were five men and five women, age range 32-62 years (mean 47.3 years).

2.4. Other neurological diseases

Paired samples of CSF and serum were also ob- tained from 11 patients with other neurological dis- eases (OND), seven men and four women, age range 33-66 years (mean 44.1 years). Diagnoses included spinocerebellar degeneration (three patients), amy- atrophic lateral sclerosis (three patients) and Parkin- son’s disease (five patients). Patients with OND served as controls for the detection of CSF levels of sVCAM-1.

2.5. Normal controls

We also evaluated serum samples from 12 healthy individuals, seven men and five women, age range 27-65 years (mean 42.3 years).

Blood samples were allowed to clot at room temper- ature for 60 min followed by separation by centrifuga- tion. To prevent the degradation of protein, 1000 kallikrein inhibitory units of protease inhibitor (aprotinin, Sigma, St. Louis, MO) was added to each ml of CSF and sera at the time of collection. Samples

M. Matsuda et al. /Journal of Neuroimmunology 59 (1995) 35-40 37

were subsequently frozen and stored at -70°C until assayed for sVCAM-1. Informed consent was obtained from each subject before the assay. Assays were per- formed on coded sterile samples in a blinded fashion.

2.6. sVCAh4-1 assay

The concentration of sVCAM-1 in each sample was determined using a commercially available enzyme-lin- ked immunosorbent assay (ELISA) kit (British Bio- technology Products Ltd., Abingdon, UK). We mea- sured sVCAM-1 according to the procedure supplied with the ELISA kit. Serum samples diluted 1:50 with buffered protein matrix or non-diluted CSF samples were added to polystyrene microwell strips coated with a murine monoclonal antibody to human sVCAM-1. After being washed twice with a buffer, horseradish peroxidase (HRP)-conjugated murine monoclonal anti- body with neutralizing properties against sVCAM-1 was added and bound to the sVCAM-1 captured by the first antibody. Unbound enzyme-conjugated anti- sVCAM-1 was removed by washing, and a substrate solution reactive to HRP was added to the wells. A coloured product was formed in proportion to the amount of circulating sVCAM-1 present in the sample. The reaction was terminated by the addition of 4 N sulfuric acid, and absorbance was measured at 450 nm with a Beckman (Fullerton, CA) LS-5800 spectro- photometer. A standard curve was prepared from seven sVCAM-1 standard dilutions in duplicate. Sample val- ues were determined from the standard curve and the measurement of sVCAM-1 was performed in dupli- cate. The limit of detection for sVCAM-1 was 2.8 ng/ml in CSF and 140 ng/ml in sera.

2.7. Statistical evaluation

Data are reported as mean & standard deviation. Statistical analysis utilized Student’s t-test and a paired t-test, with P < 0.05 accepted as statistically significant.

3. Results

Serum levels of sVCAM-1 were significantly in- creased in patients with acute relapsing MS during an exacerbation compared with healthy individuals (691.8 f 288.6 ng/ml vs. 402.5 + 161.5 ng/ml, P < 0.01) (Fig. 1). There was also a significant difference between patients with acute relapsing MS during an exacerba- tion vs. a remission (387.6 &- 118.6 ng/ml, P < 0.001). Serum levels of sVCAM-1 were measured in both the active phase and the inactive phase in five patients with acute relapsing MS. Those patients showed high serum levels of sVCAM-1 in the active phase, and exhibited a decrease during a remission. Patients with

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active inactive chronic HAM non-HAM OIND normal phr ph,ase progressive

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Fig. 1. Serum levels of sVCAM-1. Levels were significantly increased

in patients with acute relapsing MS during an exacerbation or

chronic progressive MS as compared with controls (P < 0.01 and

P < 0.05, respectively). Five patients with acute relapsing MS showed

high serum levels of sVCAM-1 in the active phase, with a decrease

seen during a remission (dashed lines). Patients with HAM also

exhibited significantly higher serum levels of sVCAM-1 than the

non-HAM carriers or controls (P < 0.01).

chronic progressive MS (1254.0 _t 885.3 ng/ml) had higher levels of sVCAM-1 than healthy individuals (P < 0.05). Although the mean level of sVCAM-1 in patients with chronic progressive MS was markedly elevated, the standard deviation was large. Patients with HAM (1556.5 + 953.1 ng/ml> also exhibited a markedly high mean level of sVCAM-1, and signifi- cantly higher levels than healthy individuals (P < 0.01). There was a significant difference between patients with HAM and the non-HAM carriers (512.0 * 143.5 ng/ml, P < 0.01). In patients with OND (343.6 f 102.6 ng/ml), levels of sVCAM-1 did not differ significantly from those of healthy individuals. Serum levels of sV- CAM-1 were also significantly increased in patients with acute relapsing MS during an exacerbation (P < O.Ol), those with chronic progressive MS (P < 0.05) and those with HAM (P < 0.01) as compared with those with OND. Although three patients in the chronic progressive MS group and two patients in the HAM group showed markedly high serum levels of sVCAM-1, we could not find any distinguishing clinical features about these individuals.

CSF levels of sVCAM-1 were significantly increased in patients with acute relapsing MS during an exacer- bation (14.3 f 4.5 ng/ml) as compared with patients with OND patients (4.8 k 1.0 ng/ml, P < 0.001) (Fig. 2). There was a significant difference in CSF levels in patients with acute relapsing MS during an exacerba-

38 M. Matsuda et al. /Journal of Neuroimmunology 59 (1995) 35-40

p<o.o01

( P<O.OOl , , p<o.o01 1

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3 30

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.

.

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L active InactIve chronic OND

progressive

phase MS acute relapsing MS

Fig. 2. CSF levels of sVCAM-1. Levels were significantly increased in patients with acute relapsing MS during an exacerbation or chronic progressive MS as compared with patients with OND (P < 0.001). Five patients with acute relapsing MS showed high CSF levels of sVCAM-1 in the active phase, with a decrease observed during a remission (dashed lines).

tion vs. a remission (5.4 + 2.3 ng/ml, P < 0.001). CSF levels of sVCAM-1 were measured in both the active phase and the inactive phase in five patients with acute relapsing MS. Those patients showed high serum levels of sVCAM-1 in the active phase, and showed a de- crease during a remission. Patients with chronic pro- gressive MS (20.9 + 9.3 ng/ml) also exhibited higher CSF levels of sVCAM-1 than did the patients with OND (P < 0.001).

4. Discussion

The endothelial cells are believed to be fundamen- tally altered by trauma, acute and chronic inflamma- tion, and vasculitis (Wilder et al., 1991). One of the earliest changes may be a microvascular activation, including an enhanced surface expression of the mark- ers for endothelial cell activation such as major histo- compatibility complex and adhesion molecules (Kaha- leh, 1990; Wilder et al., 1991). Several adhesion molecules are implicated in the migration of lympho- cytes (Duijvestijn and Hamann, 1989; Mackay, 1991; Shimizu et al., 1992) and in the activation of T cells (Dustin and Springer, 1989; Matsuyama et al., 1989; Nojima et al., 1990; Shimizu et al., 1990). Interaction between the adhesion molecules on endothelial cells and their counterreceptors on the leukocytes is be- lieved to be a primary event in initiation of the inflam- matory response (Shimizu et al., 1992).

In MS, an inflammatory demyelinating disease of the CNS, a breakdown of the BBB is thought to be an

early event in the immune response (Adams et al., 1989; James, 1989; Kermode et al., 1990). Although the interaction between circulating lymphocytes and acti- vated endothelial cells may be involved in the damage to the BBB, the role of the activated endothelium or of the expression of endothelial cell adhesion molecules has not been thoroughly evaluated.

Some of these endothelial adhesion molecules are reportedly shed from the membrane and can be mea- sured in body fluids by sensitive immunoassays (Rothlein et al., 1991; Gearing et al., 1992). VCAM-1, a member of the immunoglobulin superfamily, is such a membrane surface antigen, which is expressed on a number of cells including endothelium, macrophages, kidney epithelia, dendritic cells, myoblasts, and bone marrow fibroblasts. VCAM-1 is expressed on the vas- cular endothelial cells in response to cytokines (Osborn et al., 1989) and plays a major role in the adhesion of leukocytes to the endothelium by interaction with its ligand very late activation antigen-4 (VLA-4), a mem- ber of the /31 integrin family. This agent is expressed on the lymphocytes, monocytes, thymocytes, and my- oblasts. VCAM-1 may exist in a soluble form suggested by the observation that it has been found in the super- natants of cytokine-activated endothelial cells in cul- ture, but not in the supernatants of control, non- activated cells (Pigott et al., 1992). The use of ELISA techniques has demonstrated the presence of sVCAM-1 in the sera of normal individuals (Gearing et al., 1992). Research models suggest that concentrations of sV- CAM-l in the sera may be elevated above the normal level (Gearing et al., 1992; Mason et al., 1993; Welli- come et al., 1993).

We demonstrated that sVCAM-1 levels in the CSF and sera were significantly elevated in patients with acute relapsing MS during an exacerbation vs. a remis- sion, or in the control subjects. Five patients with acute relapsing MS showed a high serum and CSF levels of sVCAM-1 during an exacerbation, and a decrease of those levels during a remission. Those results suggest that CSF and serum levels of sVCAM-1 may reflect the activity of acute relapsing MS.

We also demonstrated that patients with chronic progressive MS or HAM exhibited significantly higher serum or CSF levels of sVCAM-1 than controls, and that the mean levels of that substance were markedly elevated in those patients as compared with acute relapsing MS. Although these results may be due, in part, to variations in sampling time of acute relapsing MS group, a differing pattern of expression of adhe- sion molecules may be responsible. Washington et al. (1994) demonstrated that the majority of microvessels from the CNS of MS patients expressed high levels of VCAM-1 and ICAM-1. ELAM-1 was sometimes ex- pressed transiently on endothelial cells on exposure to inflammatory cytokines. While ELAM-1 is involved in

M. Matsuda et al. /Journal of Neuroimmunology 59 (1995) 35-40 39

early inflammation, VCAM-1 may be important in both acute and chronic inflammation.

Endothelial adhesion molecules such as ICAM- and VCAM-1 are upregulated by inflammatory cy- tokines and thereby enable the lymphocytes to be recruited to sites of inflammation (Wawryk et al., 1989; Carlos et al., 1990). Evidence suggests that ICAM- may be important in the pathogenesis of MS or other demyelinating diseases. For instance, immunohisto- chemical studies demonstrate that the expression of ICAM- is increased in the CNS of patients with MS (Sobel et al., 1990) and in experimental allergic en- cephalomyelitis (EAE) (Cannella et al., 1990). Also, blocking of the ICAM-l/lymphocyte function-associ- ated antigen-l (LFA-1) interaction inhibits the signs of EAE in the rat (Archelos et al., 1993). Finally, serum levels of soluble ICAM- (sICAM-1) are increased in the sera and CSF of patients with acute relapsing MS during an exacerbation, and levels of sICAM-1 in the serum and the CSF were associated with a clinical exacerbation of MS (Hartung et al., 1993; Sharief et al., 1993; Tsukada et al., 1993a, Tsukada et al., 1993b).

Recent reports demonstrate that the expression of VCAM-1 is increased in the CNS of patients with MS or of animals with EAE (Steffen et al., 1994; Washing- ton et al., 19941, and that the blocking of the VCAM- l/VLA-4 interaction inhibits both the adhesion of lymphocyte to the cerebral endothelium and the devel- opment of signs of EAE in the rat (Yednock et al., 1992). Our data showed that sVCAM-1 was increased in the sera and the CSF of patients with MS and HAM. These findings support the idea that VCAM-1 may be involved in directing the T cells to the sites of inflammation in the CNS.

The biological function of sVCAM-1 is not known. Its increased levels may result from its release from cells during inflammation, or it may provide a means of controlling adhesive interactions between the endothe- lial cells and the immunocompetent cells. The mea- surement of sVCAM-1 in sera and CSF may be useful in monitoring the activity and course of such inflamma- tory diseases as MS and HAM.

Acknowledgements

We thank Professor M. Osame, Department of Neu- rology, Kagoshima University, and Professor Y. Itoyama, Department of Neurology, Tohoku Univer- sity, for providing clinical specimens from patients with HAM and from the non-HAM carrier patients. We are also indebted to Dr. N. Hanyu (Nagano Red Cross Hospital), Dr. S. Nakagawa and Dr. K. Tabata (Saku General Hospital) for collecting samples from patients with MS. This study was supported by grants from the Intractable Disease Division (Neuroimmunology) of the

Ministry of Public Health and Welfare of Japan and from the Shinshu Research Foundation of Critical Ill- ness and Incurable Diseases.

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