Koichi Miyagi

Tumor necrosis factor and interleukin-1 in the CSF and sera of patients with multiple sclerosis

Serum and cerebrospinal fluid (CSF) from 31 patients with multiple sclerosis (MS) were examined to determine the levels of tumor necrosis factor (TNF) and interleukin (IL)-1 alpha (or IL-1 beta) by an enzyme-linked immunosorbent assay. TNF was detected in 29 (93.5%) of CSF from 31 cases of MS. TNF was also detectable in 100% of CSF from patients with acute relapsing MS in exacerbation. Patients with acute relapsing MS in exacerbation showed significantly higher CSF levels of TNF as compared with either those in remission or the controls (P less than 0.001 and P less than 0.0001, respectively). Increased levels of TNF were also detected in 35.5% of the MS sera, and especially in those with acute relapsing MS in exacerbation. Increased TNF levels were also frequent in the CSF and sera of patients with Guillain-Barré syndrome (GBS), which is also a demyelinating disease. No IL-1 alpha (or IL-1 beta) was detected in either CSF or sera of 31 MS patients. It is considered likely that TNF CSF levels may reflect disease activity in MS.

Increased levels of soluble vascular cell adhesion molecule-1 ( VCAM-1) in the cerebrospinal fluid and sera of patients with multiple sclerosis and human T lymphotropic virus type-1-associated myelopathy

We evaluated the relationship between the soluble form of vascular cell adhesion molecule-1 (sVCAM-1) and disease activity in patients with multiple sclerosis (MS) or with human T lymphotropic virus type 1-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 < 0.001), as well as in chronic progressive MS (P < 0.05 and P < 0.001), 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.

Cytotoxicity of T cells for cerebral endothelium in multiple sclerosis

We investigated the cytotoxic effect of peripheral blood T cells on cerebral endothelium in patients with MS. We examined in vitro the damage to 51Cr-labelled dissociated human brain endothelial cells produced by mitogen-stimulated T cell lines from patients with MS and controls. Endothelial targets were lysed by T-lymphocytes from patients with acute relapsing MS during an exacerbation at every target-effector cell ratio tested compared with controls (P < 0.001). The percentage of endothelial targets lysed was not significantly increased by incubation with T cells from patients with acute relapsing MS in remission and chronic progressive MS, compared with that of normal subjects. Relapsing MS patients during an exacerbation had significantly higher interleukin-1 (IL-1)-alpha concentrations in cultures of targets with effector cells than normal subjects (P < 0.02). Experiments of major histocompatibility complex (MHC)-restricted cytotoxicity in MS demonstrated incomplete blocking of specific lysis by either anti-MHC class I or class II monoclonal antibody (mAb). These results indicate that cytotoxicity of T cells for cerebral endothelial cells may play a role in the initiation of immune response in acute relapsing MS during an exacerbation which appears to cause an increase in blood-brain barrier (BBB) permeability.

Increased levels of soluble tumor necrosis factor receptor in patients with multiple sclerosis and HTLV-1-associated myelopathy

Tumor necrosis factor-alpha (TNF-alpha) is a potent mediator produced by activated T lymphocytes and macrophages, which may play a role in the pathogenesis and development of multiple sclerosis (MS) and HTLV-1-associated myelopathy (HAM). The first step in the induction of many biological effects elicited by TNF-alpha is its binding to specific cell surface receptors. A soluble form of TNF receptor (sTNF-R) can be detected in the body fluid. We measured sTNF-R levels in the sera and cerebrospinal fluid (CSF) of patients with either MS or HAM, and evaluated the correlation between this mediator and disease activity. The levels of sTNF-R in the sera and CSF of patients with MS were significantly increased compared with controls, particularly patients with acute relapsing MS during an exacerbation (P < 0.001). CSF levels of sTNF-R showed a strong correlation with those of TNF (r = 0.716, P < 0.001). Higher levels of sTNF-R in the sera of HAM patients were detected as compared with those of either controls (P < 0.001) or non-HAM carriers (P < 0.001). Patients with HAM exhibited significantly higher CSF levels of sTNF-R than those with other neurological diseases (P < 0.0001). These results suggest that the detection of sTNF-R in the sera and CSF may predict disease progression. Availability of such a marker would be useful in monitoring disease activity.

Adhesion of Cerebral Endothelial Cells to Lymphocytes from Patients with Multiple Sclerosis

To investigate the factors regulating the entry of lymphocytes into the brain, we assessed the adhesion in vitro of 51Cr labelled lymphocytes from peripheral blood of patients with multiple sclerosis (MS) to human cerebral endothelial cells, and evaluated the effect on the adhesion of endothelium activated by interferon-gamma (IFN-gamma), lipopolysaccharides (LPS), interleukin-1 (IL-1) and tumor necrosis factor (TNF). Patients with acute relapsing MS during an exacerbation showed significant increase in MNC adherence to cerebral endothelial cells as compared with controls (p < 0.001). MNC adherence to cerebral endothelial cells activated by IFN-gamma or LPS, was significantly increased as compared to the controls (p < 0.01). MNC adherence to endothelial cells was not blocked by antibodies against the intercellular adhesion molecule-1 (ICAM-1), but was blocked by lymphocyte function-associated antigen-1 (LFA-1). The increased adherence observed in patients with acute relapsing MS during an exacerbation would modulate the migration of lymphocytes across the blood-brain barrier (BBB).

Increased interleukin-1 production by peripheral blood mononuclear cells in patients with multiple sclerosis

The production of interleukin-1 (IL-1) by peripheral blood mononuclear cells (MNC) was assessed in patients with relapsing multiple sclerosis (MS) in both the active and inactive phase, in chronic progressive MS patients, in other neurological diseases, and in healthy subjects. Production was determined by measuring the IL-1 concentration in cultures with MNC supernatants using enzyme-linked immunosorbent assay (ELISA). IL-1 in sera of MS patients and healthy subjects also was investigated. MNC IL-1 alpha production was significantly higher in MS patients (180.2 +/- 177.5 pg/ml) than in healthy subjects (66.2 +/- 66.0 pg/ml) (P less than 0.05). Relapsing MS patients in the active phase had significantly higher MNC IL-1 alpha concentrations (360.1 +/- 130.0 pg/ml) than normal subjects (P less than 0.001), but MNC IL-1 alpha production in patients with relapsing MS in the inactive phase (65.3 +/- 52.8 pg/ml) or chronic progressive MS (80.9 +/- 71.9 pg/ml) was not increased significantly. MNC IL-1 beta production in MS patients was not elevated significantly. IL-1 alpha and -1 beta were not detected in sera of MS patients. The correlation between increased IL-1 alpha production and the clinical course of MS suggests that activated MNC may play a role in the pathogenesis of MS.

Adhesion of Lymphocytes to Endothelial Cells in Experimental Allergic Encephalomyelitis before and after Treatment with Endotoxin Lipopolysaccharide

We investigated the in vitro adhesion of 51Cr-labeled lymphocytes to cultured brain endothelial cells and the in vivo expression of intercellular adhesion molecule-1 (ICAM-1) on cerebral endothelial cells in a rat model of experimental allergic encephalomyelitis (EAE) before and after treatment with lipopolysaccharide (LPS). Adhesion of lymphocytes to cerebral endothelial cells was significantly increased in EAE compared with controls (p < 0.01), and was significantly correlated with the percentage of major histocompatibility complex class II antigen-positive cells in lymph node cells (p < 0.001). LPS enhanced ICAM-1 expression on endothelial cells and lymphocyte adhesion to those cells, and caused a significant increase in the in vivo expression of ICAM-1 compared with controls (p < 0.001). Lymphocyte adhesion to endothelial cells was significantly blocked by monoclonal antibodies against ICAM-1, lymphocyte function-associated antigen-1, or very late activation antigen-4. Our findings suggest that lymphocyte adhesion to brain endothelial cells may contribute to lymphocyte migration across the blood-brain barrier in EAE and that LPS may cause progression of EAE lesions.

Expression of FcϵR2/CD23 and p55 IL-2R/CD25 on peripheral blood macrophages/monocytes in multiple sclerosis

Macrophages have been found histologically to be activated in multiple sclerosis. We analyzed the expression of CD23 and CD25 on monocytes/macrophages in peripheral blood obtained from patients with multiple sclerosis (MS) to investigate their role in the demyelinating process. Peripheral blood mononuclear cells were obtained from 30 patients with MS including for Balos diseases (24 with acute relapsing type disease, six with chronic progressive type disease) and 12 healthy controls. The percentage of CD14+ CD23+ monocytes/macrophages and CD14+ CD25+ monocytes/macrophages were determined by two-color flow cytometry. The percentage of CD14+ CD23+ monocytes was significantly higher in patients with MS in the active phase as compared with controls (P < 0.01). Six patients with acute relapsing MS, who had received no therapy, had higher CD14+ CD23+ cells than did controls (P < 0.0001). CD14+ CD25+ monocytes/macrophages were not detected in peripheral blood monocytes/macrophages of patients with MS except Balos concentric sclerosis. The four patients with Balos concentric sclerosis had markedly elevated levels of CD14+ CD25+ monocytes/macrophages. Our findings suggest that monocytes/macrophages are activated during an exacerbatiion of MS. and that they may play an important role in the process of demyelination.