Yoh Matsumoto

Microglia: intrinsic immuneffector cell of the brain.

Microglia form a regularly spaced network of resident glial cells throughout the central nervous system (CNS). They are morphologically, immunophenotypically and functionally related to cells of the monocyte/macrophage lineage. In the ultimate vicinity of the blood-brain barrier two specialized subsets of macrophages/microglia can be distinguished: firstly, perivascular cells which are enclosed within the basal lamina and secondly juxtavascular microglia which make direct contact with the parenchymal side of the CNS vascular basal lamina but represent true intraparenchymal resident microglia. Bone marrow chimera experiments indicates that a high percentage of the perivascular cells undergoes replacement with bone marrow-derived cells. In contrast, juxtavascular microglia like other resident microglia form a highly stable pool of CNS cells with extremely little turnover with the bone marrow compartment. Both the perivascular cells and the juxtavascular microglia play an important role in initiating and maintaining CNS autoimmune injury due to their strategic localization at a site close to the blood-brain barrier, their rapid inducibility for MHC class II antigens and their potential scavenger role as phagocytic cells. The constantly replaced pool of perivascular cells probably represents an entry route by which HIV gets access to the brain. Microglia are the first cell type to respond to several types of CNS injury. Microglial activation involves a stereotypic pattern of cellular responses, such as proliferation, increased or de-novo expression of immunomolecules, recruitment to the site of injury and functional changes, e.g., the release of cytotoxic and/or inflammatory mediators. In addition, microglia have a strong antigen presenting function and a pronounced cytotoxic function. Microglial activation is a graded response, i.e., microglia only transform into intrinsic brain phagocytes under conditions of neuronal and or synaptic/terminal degeneration. In T-cell-mediated autoimmune injury of the nervous system, microglial activation follows these lines and occurs at an early stage of disease development. In experimental autoimmune encephalomyelitis (EAE), microglia proliferate vigorously, show a strong expression of MHC class I and II antigens, cell adhesion molecules, release of reactive oxygen intermediates and inflammatory cytokines and transform into phagocytic cells. Due to their pronounced antigen presenting function in vitro, activated microglia rather than astrocytes or endothelial cells are the candidates as intrinsic antigen presenting cel of the brain. In contrast to microglia, astrocytes react with a delay, appear to encase morphologically the inflammatory lesion and may be instrumental in downregulating the T-cell-mediated immune injury by inducing T-cell apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel experimental model of giant cell myocarditis induced in rats by immunization with cardiac myosin fraction

It is suspected that autoimmune disease processes are involved in the pathogenesis of a part of giant cell myocarditis. However, evidence for autoimmunity has rarely been demonstrated in clinical investigations. In this study, we have demonstrated a new animal model of autoimmune myocarditis characterized by the appearance of multinucleated giant cells. Lewis rats were immunized twice with human cardiac myosin fraction in complete Freunds adjuvant. Cardiac myosin fraction was prepared from the ventricular muscle of human hearts. Three weeks after the first immunization, acute and severe myocarditis was elicited in all rats. This myocarditis was characterized by massive pericardial effusion, enlargement of the heart, and gray discoloration of the cardiac muscle. Microscopically, there was marked cellular infiltration consisting of mononuclear cells, neutrophils, fibroblasts, and a considerable number of multinucleated giant cells. Extensive myocardial necrosis was also present. The heart weights increased from the third week to the fourth week and then gradually decreased. The titer of anti-myosin antibodies began to elevate from the second week and remained high until the sixth week. In the sixth week, inflammation became smoldering and the multinucleated giant cells disappeared. These findings indicate that the cardiac myosin fraction contains myocarditogenic antigen and that giant cell myocarditis can be induced by autoimmune involvement. To our knowledge, this is the first report of experimental giant cell myocarditis, which is closely similar to human giant cell myocarditis in its histology and clinical course.

Interferon beta-1b exacerbates multiple sclerosis with severe optic nerve and spinal cord demyelination

To evaluate the effect of interferon beta-1b (IFNB-1b) on multiple sclerosis (MS) with severe optic nerve and spinal cord demyelination, we examined the relationship between IFNB-1b treatment outcome and the clinical and genetic characteristics of three types of demyelinating diseases of the central nervous system, i.e., neuromyelitis optica (NMO), MS and MS with severe optic-spinal demyelination. Japanese MS frequently carried HLA DPB1*0501, which is associated with NMO. MS with DPB1*0501 showed severe optic-spinal demyelination represented by longitudinally extensive spinal cord lesion, blindness and CSF pleocytosis. IFNB-1b treatment did not succeed in these patients because of the increase of optic nerve and spinal cord relapse and other severe side effects. IFNB-1b should not be administered to demyelinating patients with genetic and clinical characteristics mimicking NMO such as HLA DPB1*0501 allele, longitudinally extensive spinal cord lesion, blindness and CSF pleocytosis even if they have symptomatic cerebral lesions as typically seen in MS. The present study strongly suggests that these patients should be diagnosed as having NMO.

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.

Chemokine expression by astrocytes plays a role in microglia/ macrophage activation and subsequent neurodegeneration in secondary progressive multiple sclerosis

The pathological hallmarks of secondary progressive (SP) multiple sclerosis (MS) include slowly expanding demyelination and axonal damage with less inflammation. To elucidate the pathomechanisms of secondary progressive (SP) multiple sclerosis (MS), we have investigated the expression of chemokines, chemokine receptors, matrix metalloproteinase-9 (MMP-9) and immunoglobulins in the demyelinating plaques. Immunohistochemical analysis revealed that numerous hypertrophic astrocytes were observed at the rim, but not in the center, of the chronic active lesions. Microglia/macrophages phagocytosing myelin debris were also found at the lesion border. In contrast, T cell infiltration was minimal in these plaques. Characteristically, at the rim of the lesions, there were abundant immunoreactivities for monocyte chemoattractant protein-1 (MCP-1)/CCL2 and interferon-γ inducible protein-10 (IP-10)/CXCL10 and their receptors, CCR2 and CXCR3, while these immunoreactivities were weak in the center, thus forming a chemokine gradient. Double immunofluorescense staining demonstrated that cellular sources of MCP-1/CCL2 and IP-10/CXCL10 were hypertrophic astrocytes and that both astrocytes and microglia/macrophages expressed CCR2 and CXCR3. MMP-9 was also present at the rim of the lesions. These results suggest that MCP-1/CCL2 and IP-10/CXCL10 produced by astrocytes may activate astrocytes in an autocrine or paracrine manner and direct reactive gliosis followed by migration and activation of microglia/macrophages as effector cells in demyelinating lesions. Targeting chemokines in SPMS may therefore be a powerful therapeutic approach to inhibit lesional expansion.

In vivo lymphocyte-mediated myocardial injuries demonstrated by adoptive transfer of experimental autoimmune myocarditis.

BackgroundTo elucidate the mechanisms of immune-related myocardial injuries, we examined whether autoimmune myocarditis was passively transferable by use of humoral or cellular factors. Methods and ResultsActive myocarditis was elicited in Lewis rats by immunization with human cardiac myosin fraction in complete Freunds adjuvant. This experimental myocarditis was characterized by macroscopic features such as pericardial effusion, enlargement of the heart, and gray discoloration of the cardiac surface. Histologically, extensive myocardial necrosis and numerous inflammatory cell infiltrations were observed. Interestingly, multinucleated giant cells were frequently observed in the lesions. Transfer of the disease by the humoral factor was examined by use of fresh sera and immunoglobulin fraction of pooled sera from rats with severe myocarditis, and transfer by the cellular factor was tested by use of spleen cells and lymph node cells from the diseased rats. When naive Lewis rats were given 15.75 mg of immunoglobulin fraction, no particular change was observed in the hearts. Fresh sera also could not elicit myocarditis in recipient rats. In contrast, intravenous injection of spleen cells or lymph node cells that were cultured for 3 days in the presence of 1 μg/ml of concanavalin A elicited severe myocarditis. The macroscopic and microscopic findings of passively transferred myocarditis are essentially the same as those found in actively induced myocarditis. Multinucleated giant cells were also observed in the lesions of transferred myocarditis. ConclusionsThis study demonstrates direct evidence for in vivo lymphocyte-mediated myocardial injuries.

Absence of donor-type major histocompatibility complex class I antigen-bearing microglia in the rat central nervous system of radiation bone marrow chimeras

Localization of bone marrow-originated cells in the central nervous system (CNS) of the rat was investigated by using bone marrow chimeras. In order to do this, Lewis rats which carry major histocompatibility complex (MHC) class I antigens haplotype 1 (RT1.Al) were reconstituted with (Lew X PVG)F1 (RT1.Al/c) bone marrow cells after lethal irradiation. Transferred bone marrow cells were detected by immunohistochemical staining using a monoclonal antibody, OX27, specific for haplotype c of rat MHC class I antigens (RT1.Ac). The spleen and thymus of chimeric rats were fully reconstituted with transferred F1 cells 4 weeks after bone marrow transplantation. At this stage, mononuclear cells in the subarachnoid space of the CNS expressed OX27 antigen indicating that they were of bone marrow origin. A few OX27-positive blood cells were scattered in the CNS parenchyma 4-12 weeks after reconstitution. Ramified microglia, however, remained OX27-negative. Bone marrow-derived microglia were not observed throughout the period of examination until 24 weeks. In addition, experimental allergic encephalomyelitis (EAE) was induced in chimeric rats in order to augment the expression of MHC class I antigens on microglia. Even under this condition, no OX27-positive microglia were observed. Taken together, ramified microglia might be of neuroectodermal origin and there is little possibility that the microglia are derived from the bone marrow. However, if the ramified microglia are derived from blood cells, the microglia may be expected to have characteristic cell kinetics from the following points: (1) the precursor cells of the microglia may enter the CNS only at the perinatal stage; and (2) even under the condition in which lymphocytes and macrophages enter the CNS as observed in EAE, the precursor cells of the microglia are not supplied from the blood.

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.

In situ detection of class I and II major histocompatibility complex antigens in the rat central nervous system during experimental allergic encephalomyelitis: an immunohistochemical study

To determine in situ localization of cells bearing major histocompatibility complex (MHC) class I or II antigens in the central nervous system (CNS), immunohistochemical examination was performed on CNS sections of Lewis rats sensitized for experimental allergic encephalomyelitis (EAE). Class I antigens identified by OX18 were detected on endothelial cells (EC) and cells with dendritic morphology (DC) of normal rats. OX18+ DC increased in number as the clinical signs of EAE became more severe, while the number of OX18+ EC in clinical EAE rats was not different from that of normal control rats. Infiltrating lymphocytes were always observed around OX18+ vessels. Double staining showed that OX18+ DC was negative for glial fibrillary acidic protein (GFAP). Cells with morphological features of oligodendroglia were not detected with OX18 in both normal control and EAE rats. MHC class II antigens (Ia antigens) were detected using three MAbs: OX3, OX6 and OX17. These three different MAbs essentially showed the same staining pattern. In normal controls, mononuclear cells in the subarachnoid space were stained positively, but no Ia+ parenchymal cells were detected. In EAE rats, Ia+ DC were first detectable in the white matter of the spinal cord at the preclinical stage, and increased in number as the disease progressed. On the other hand, double-staining with OX6 and anti-factor VIII-related antigen antiserum, or with OX3 and anti-vimentin antiserum demonstrated that endothelial cells even with lymphocyte cuffing were negative for Ia antigens. Based on the data obtained in the present study, the possible role of MHC class I and II antigens in the development of EAE is discussed.

Regulation of the severity of neuroinflammation and demyelination by TLR-ASK1-p38 pathway

Apoptosis signal‐regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen‐activated protein kinase (MAPK) kinase kinase which plays important roles in stress and immune responses. Here, we show that ASK1 deficiency attenuates neuroinflammation in experimental autoimmune encephalomyelitis (EAE), without affecting the proliferation capability of T cells. Moreover, we found that EAE upregulates expression of Toll‐like receptors (TLRs) in activated astrocytes and microglia, and that TLRs can synergize with ASK1‐p38 MAPK signalling in the release of key chemokines from astrocytes. Consequently, oral treatment with a specific small molecular weight inhibitor of ASK1 suppressed EAE‐induced autoimmune inflammation in both spinal cords and optic nerves. These results suggest that the TLR‐ASK1‐p38 pathway in glial cells may serve as a valid therapeutic target for autoimmune demyelinating disorders including multiple sclerosis.