Naoyuki Tanuma

Role of natural killer cells and TCRγ δ T cells in acute autoimmune encephalomyelitis

To elucidate the role of NK cells and TCRγ δ + T cells in acute experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats, the distribution, number and function of these cells were studied using several methods. Immunohistochemical and flow cytometric analysis revealed that a certain number of NK cells (17 % of the total inflammatory cells) infiltrated the central nervous system (CNS) at the peak stage of EAE and were mainly located in the perivascular region. On the other hand, virtually no TCRγ δ + T cells were found in the CNS. NK‐T (NKR‐P1+ TCRα β + ) cells were few and did not increase in number in the CNS and lymphoid organs. In the cytotoxic assay using YAC‐1 cells, effector cells isolated from the spleen of rats at the peak of EAE showed essentially the same cytotoxicity as those isolated from normal controls although the total number of NK cells decreased to one fifth of that of normal rats. Furthermore, in vivo administration of anti‐NK cell (3.2.3 and anti‐asialo GM1), but not of anti‐TCRγ δ (V65), antibodies exacerbated the clinical features of EAE and induced fatal EAE in some rats. These findings suggest that NK cells play a suppressive role in acute EAE whereas TCRγ δ + T cells are not involved in the development of or recovery from the disease.

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)

Upregulation of monocyte chemotactic protein-1 and CC chemokine receptor 2 in the central nervous system is closely associated with relapse of autoimmune encephalomyelitis in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is a disease model of multiple sclerosis (MS) that is characterized by remittance and relapse of the disease and autoimmune and demyelinating lesions in the central nervous system (CNS). To better understand the mechanism of disease relapse, we induced acute and chronic relapsing (CR)-EAE in Lewis rats and examined the differences between the two groups. An immunohistochemical study revealed that significantly higher numbers of macrophages infiltrated the spinal cord during the first and second attacks of CR-EAE than at the peak of acute EAE, whereas the number of infiltrating T cells was essentially the same in acute and CR-EAE. In accordance with this finding, monocyte chemoattractant protein-1 (MCP-1) mRNA, but not MIP-1alpha and RANTES mRNA, increased significantly in CR-EAE lesions rather than in acute EAE lesions. More importantly, the level of MCP-1 during the remission of CR-EAE was significantly higher than during the recovery phase of acute EAE, suggesting that this high level of MCP-1 in CR-EAE is associated with relapse of the disease. CC chemokine receptor 2 (CCR2), the main receptor for MCP-1, was expressed on astrocytes, macrophages and T cells and the number of positive cells was higher in CR-EAE than in acute EAE. Collectively, these findings suggest that high expression of MCP-1 and its receptor, CCR2, in the CNS play important roles in relapse of EAE.

Expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

The expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was analyzed. Western blot analysis showed that three isotypes of caveolins including caveolin-1, -2 and -3 increased significantly in the spinal cords of rats during the early stage of EAE, as compared with the levels in control animals (p<0.05); the elevated level of each caveolin persisted during the peak and recovery stage of EAE. Immunohistochemistry demonstrated that caveolin-1 and -2 were expressed constitutively in the vascular endothelial cells and ependymal cells of the normal rat spinal cord, whereas caveolin-3 was almost exclusively localized in astrocytes. In EAE lesions, the immunoreactivity of caveolin-1 was increased in the ependymal cells, some astrocytes, and some inflammatory cells of the spinal cord, while that of caveolin-2 showed an intense immunoreactivity. Caveolin-3 was expressed constitutively in some astrocytes, but not in endothelial cells; its immunoreactivity was increased in reactive astrocytes in EAE lesions. The results of the Western blot analysis largely confirmed the observations obtained with immunohistochemistry. Taking all the findings into consideration, we postulate that the expression levels of each caveolin begin to increase when EAE is initiated, possibly contributing to the modulation of signal transduction pathways in the affected cells.

Analysis of neurotrophic effects of hepatocyte growth factor in the adult hypoglossal nerve axotomy model.

Recent studies have shown that hepatocyte growth factor (HGF) promotes the survival of embryonic motor neurons. However, it remains unclear whether HGF has trophic effects on mature motor neurons. In the present study, we examined the effects of HGF on adult motoneurons using the hypoglossal nerve transection model. In adult rats, neurons in the hypoglossal nucleus show a dramatic loss of choline acetyltransferase (ChAT) protein and mRNA after the axotomy. This reduction of ChAT was markedly prevented when HGF was administered continuously at the cut end of the nerve using an osmotic pump. The HGF receptor, c‐met, protein and mRNA, which were faintly expressed in hypoglossal neurons under normal conditions, gradually increased and reached maximal levels 2 weeks after the axotomy. Administration of HGF reduced this c‐met upregulation almost to normal levels. We also quantified HGF mRNA in the tongue and hypoglossal nucleus. The tongue contained abundant HGF mRNA, whereas the nucleus contained only low levels. Interestingly, the HGF mRNA level in the nucleus did not increase after the axotomy. These findings suggest that HGF is principally produced in the tongue and contributes to maintain ChAT expression in the nucleus. HGF produced in the hypoglossal nucleus alone after disconnection from the tongue may not be sufficient for the maintenance of the motor neuron function. Thus, exogenously applied HGF was effective to prevent the downregulation of ChAT activities. These findings provide a strong rationale for the potential clinical use of HGF for the treatment of motor neuron degenerative disease.

Treatment of rat hemiparkinson model with xenogeneic neural transplantation: Tolerance induction by anti-T-cell antibodies

To obtain basic knowledge for the application of xenogeneic neural transplantation to patients with Parkinsons disease, the rejection process of xenogeneic neural grafts in rats was examined and a therapy to control it was developed. Tissues including the ventral mesencephalon were taken from mouse embryos and transplanted into the right lateral ventricle of mature male rats. Transplanted xenografts were usually rejected by day 15. To prevent the graft rejection, host rats were treated with anti‐T‐cell receptor αβ (anti‐TCRαβ) or anti‐CD2 monoclonal antibody (mAb) or by a combination of the two. Anti‐TCRαβ (1 mg/kg) and anti‐CD2 (7 mg/kg) mAb were administered for 3 consecutive days (day ‐2, ‐1, and 0 of transplantation). Although the administration of mAb against either CD2 or TCRαβ did not induce tolerance, the combination therapy with anti‐CD2 and anti‐TCR αβ mAb produced graft survival for more than 100 days. The tolerance induced by this combined antibody therapy is antigen specific because rats with long‐term surviving neural xenograft accepted a second neural graft from the same donor strain C3H/He mouse, but not from a third‐party strain BALB/c mouse, without additional treatment. In addition, T cells isolated from these rats did not respond to cultured C3H/He brain cells, but did respond vigorously to BALB/c brain cells in mixed lymphocyte reaction. More importantly, the finding that xenograft transplantation with the proper treatment reduced the rotation rate of 6‐OHDA‐lesioned rats confirmed that surviving grafts functioned properly. The results of the present study suggest that xenogeneic neural transplantation in combination with T‐cell‐targeted immunotherapy is an effective approach for treatment of Parkinsons disease. J. Neurosci. Res. 48:385–396, 1997.

Interaction between apoptotic cells and reactive brain cells in the central nervous system of rats with autoimmune encephalomyelitis.

To elucidate the role of brain cells in the immune regulation in the central nervous system (CNS), acute and chronic relapsing experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats and the location of apoptotic inflammatory cells and their interaction with astrocytes and microglia was investigated at various stages of the disease. Apoptotic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) were few in number at day 10-12 post-immunization (PI), increased and peaked at day 13 PI. Then, these cells decreased gradually by day 21 PI. The most characteristic finding was that apoptotic cells were mainly distributed in the CNS parenchyma with only a few cells present in perivascular cuffs. Double staining by the TUNEL method and immunocytochemistry for astrocytes and microglia revealed that astrocytes were more closely associated with apoptotic cells than microglia. Apoptotic cell death may be one mechanism by which T cells are eliminated from the CNS. Furthermore, the present study suggests that astrocytes, rather than microglia, induce programmed cell death of infiltrating inflammatory cells.

STAT expression and localization in the central nervous system during autoimmune encephalomyelitis in Lewis rats

Autoimmune inflammation in the central nervous system (CNS) is maintained by secretion of a large number of cytokines. To elucidate its molecular mechanisms, we examined the expression and localization of STAT1, STAT3, STAT4 and STAT6 molecules, which are the downstream molecules of the cytokine signal transduction pathway, in the CNS during acute experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats. Western blot analysis demonstrated that STAT1 protein increased gradually till the recovery stage, whereas STAT4 protein showed abrupt increase at the early stage followed by gradual decrease. STAT3 and STAT6 showed stable expression throughout the course of the disease. The kinetics of the phosphorylated form of STAT1 and STAT4 roughly paralleled that of the total protein although the peak of STAT3 phosphorylation was recognized at the preclinical stage. Immunohistochemical examinations revealed that STAT3 and STAT4, but not STAT1 and STAT6, immunoreactivities were mainly expressed in astrocytes and microglia, respectively, and were closely associated with inflammatory lesions. Taken together, these findings suggest that STAT3 and STAT4 play an important role in the formation of, and recovery from, autoimmune inflammation in the CNS.

Increased expression of p53 and Bax in the spinal cords of rats with experimental autoimmune encephalomyelitis.

The expression of pro-apoptotic molecules p53 and Bax in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) was examined. Apoptosis was confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. TUNEL (+) apoptotic cells were mainly either ED1 (+) macrophages or T-cells in the parenchyma of EAE. Western blot analysis showed that both p53 and Bax expression significantly (P<0. 01) increased in the spinal cords of EAE rats at the peak stage, and thereafter declined. An immunohistochemical study showed that inflammatory cells (notably T cells) in the parenchyma express p53 and Bax, while brain cells, including neurons and glia, were devoid of nuclear staining for these molecules. The nuclear expression of p53 largely matches apoptotic cells in the parenchyma of EAE. These findings suggest that the pro-apoptotic molecules p53 and Bax may play an important role in eliminating T cells in the parenchyma in EAE.