Yoshihiro Ishihara

Competitive PCR quantification of pro- and anti-inflammatory cytokine mRNA in the central nervous system during autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system that can be induced by immunization with myelin basic protein (MBP)/complete Freunds adjuvant and serves as a model for multiple sclerosis. Recent studies have suggested that cytokines play a crucial role in the clinical course of EAE. To clarify the roles of cytokines in EAE, we examined levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) mRNA in isolates from infiltrating inflammatory cells in EAE lesions induced in Lewis rats. The non-radioactive and sensitive competitive PCR method was employed to quantify the relative amounts of cytokine mRNA. Levels of both IFN-gamma and TNF-alpha mRNA were increased at the early stage of EAE and rapidly decreased at the peak stage. On the other hand, TGF-beta1 mRNA was demonstrated throughout the course of EAE as well as under normal conditions and its amount paralleled the severity of EAE. IL-10 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) under normal conditions, but was below the level of detection of competitive PCR. IL-10 mRNA expression peaked at the early stage of EAE and declined gradually thereafter. Taken together, these results suggest that IFN-gamma and TNF-alpha might play a crucial role in the development of EAE. Furthermore, it appears that the peak expression of IL-10 mRNA at the early stage and the following marked TGF-beta1 expression at the peak stage might represent an important endogenous mechanism to limit the extent of inflammation and to prevent relapse in the course of acute monophasic EAE.

THE SUBARACHNOID SPACE AS A SITE FOR PRECURSOR T CELL PROLIFERATION AND EFFECTOR T CELL SELECTION IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

To characterize the phenotype of inflammatory cells in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), Lewis rats were immunized with guinea pig myelin basic protein and frozen sections of the spinal cord with EAE were examined immunohistochemically using a panel of monoclonal antibodies against T cells and adhesion molecules. In addition, double immunostaining was performed with glial and T cells markers to examine the interaction between infiltrating T cells and reactive brain cells during the course of EAE. In the early stage of EAE, inflammatory cells first appeared in the subarachnoid space (SAS) and infiltrated the subpial region. The majority of inflammatory cells in SAS expressed TCR alpha beta and either CD4 or CD8 molecules. However, only CD4+ T cells infiltrated the parenchyma while the majority of CD8+ cells remained in SAS. A similar differential localization of T cells was observed with regard to CD45RC molecules. Inflammatory cells in SAS consisted of both CD45RC+ and CD45RC- population, while those in the parenchyma were largely CD45RC-. With regard to adhesion molecules, the leptomeninges constitutively expressed fibronectin (FN) and intercellular adhesion molecule 1 (ICAM-1). Most SAS inflammatory cells expressed very late activation antigen 4 (VLA-4) and, to lesser extent, lymphocyte function-associated antigen 1 (LFA-1) in the early stage of EAE. On the other hand, parenchymal infiltrating cells expressed LFA-1 more strongly in the peak stage. Double staining for V beta 8.2 TCR and microglia demonstrated an increase in the number of microglia together with morphological changes into rod-shape cells in the vicinity of infiltrating T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrin deposition in the central nervous system correlates with the degree of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

We examined the role of coagulation-fibrinolysis system in Theilers murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). The degree of fibrin deposition around the vessels in the spinal cord was significantly higher in susceptible SJL/J mice on 30 days post intracerebral injection (i.c.) than resistant C57BL/6 mice on 30 days post i.c. or mock infected SJL/J mice. Treatment with batroxobin (30 BU/kg/day), which is a thrombin-like defibrinogenating enzyme, causing a profound degree of afibrinogenemia, suppressed clinical signs of TMEV-IDD. Plasma fibrinogen concentration was significantly decreased in batroxobin-treated mice. Histologically, though the degree of perivascular mononuclear cell infiltration in the spinal cord was not suppressed in batroxobin-treated mice compared to saline-treated control mice, fibrin deposition was markedly suppressed in batroxobin-treated mice. These findings suggest that batroxobin suppresses TMEV-IDD through its defibrination effect, and provide evidence that CNS-associated deposition of fibrin and ensuing fibrinolysis, together with increased permeability of the blood-brain barrier (BBB), are prerequisite events for clinical manifestations of TMEV-IDD.

SUPPRESSION OF THEILER'S MURINE ENCEPHALOMYELITIS VIRUS INDUCED DEMYELINATING DISEASE BY ADMINISTRATION OF GANGLIOSIDES

Intracerebral (i.c.) inoculation of susceptible strains of mice with Theilers murine encephalomyelitis virus (TMEV) results in immune-mediated demyelinating disease. Gangliosides are membrane components of essentially all eukaryotic cells and are abundant in plasma membranes. Endogenous gangliosides have been implicated in cell recognition, cell adhesion, cell differentiation and neurite outgrowth. We studied the effect of gangliosides on TMEV-induced demyelinating disease (TMEV- IDD). We injected TMEV intracerebrally into susceptible SJL/J mice and induced TMEV-IDD. Gangliosides were injected subcutaneously and examined for various immunological indicators. The results show that when gangliosides were administered in the effector phase, TMEV-IDD was suppressed both clinically and histologically. Cellular immunity such as delayed-type hypersensitivity, and the proliferative response of T cells against TMEV and mitogens were decreased, and only in this group anti-TMEV IgG2a antibody was not detected. Taken together, these data suggest that administration of gangliosides suppressed the function of pathogenic Th1 cells and suppressed TMEV-IDD. Additionally, this study proposes the possibility of a new therapy in multiple sclerosis.

PHOSPHATIDYLSERINE SUPPRESSES THEILER'S MURINE ENCEPHALOMYELITIS VIRUS-INDUCED DEMYELINATING DISEASE

Intracerebral inoculation of susceptible strains of mice with Theilers murine encephalomyelitis virus (TMEV) results in immune-mediated demyelinating disease. Various cytokines may play an important role in the pathogenesis of this disease. Tumor necrosis factor (TNF)-alpha contributes to the further development of perivascular cellular infiltration and demyelination in the central nervous system (CNS). Phosphatidylserine (PS), a major anionic phospholipid of mammalian cells, has been proposed to function as a regulator of immune and inflammatory responses, especially reducing TNF-alpha production and release in mice. We studied the effect of PS on TMEV-induced demyelinating disease (TMEV-IDD). We injected TMEV intracerebrally into susceptible SJL/J mice and induced TMEV-IDD. PS were injected intraperitonealy, and clinical course and various immunological indicators were closely studied. The results show that when PS were administered in the effector phase. TMEV-IDD was significantly (P < 0.01) suppressed both clinically and histologically. In an ELISPOT assay, the number of TNF-alpha producing spleen cells was low in PS treated mice compared with saline treated control mice. mRNA of TNF-alpha was not detected in spleen cells of mice PS treated in the effector phase. These data suggest that administration of PS suppresses TMEV-IDD by suppressing TNF-alpha production in the effector phase.