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.     

Up-regulation of major histocompatibility complex class II antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis

Major histocompatibility complex class II (MHC II) and canine distemper virus (CDV) antigen expression were compared by immunohistochemistry in the cerebellar white matter of ten dogs with naturally occurring canine distemper encephalitis. In addition, infiltrating mononuclear cells were characterized by employing poly- and monoclonal antibodies directed against human CD3, canine MHC II, CD5, B cell antigen and CDV-specific nucleoprotein. Positive antigen-antibody reaction was visualized by the avidin-biotin-peroxidase complex method on frozen sections. Histologically, neuropathological changes were categorized into acute, subacute, and chronic. In control brains, MHC II expression was weak and predominantly detected on resident microglia of the white matter and on endothelial, perivascular and intravascular cells. In CDV antigen-positive brains, MHC II was mainly found on microglia and to a lesser extent on endothelial, meningeal, choroid plexus epithelial, ependymal and intravascular cells. In addition, virtually all of the perivascular cells expressed MHC II antigen. CDV antigen was demonstrated most frequently in astrocytes. Of the perivascular lymphocytes, the majority were CD3-positive cells, followed by B cells. Only a small proportion of perivascular cells expressed the CD5 antigen. In addition, B cells and CD3 and CD5 antigen-positive cells were found occasionally in subacute and frequently in chronic demyelinating plaques. In acute encephalitis, CDV antigen exhibited a multifocal or diffuse distribution, and MHC II was moderately up-regulated throughout the white matter and accentuated in CDV antigen-positive plaques. In subacute encephalitis, moderate multifocal CDV antigen and moderate to strong diffuse MHC II-specific staining, especially prominent in CDV antigen-positive lesions, were observed. In chronic encephalitis, CDV antigen expression was restricted to single astrocytes at the edge of the lesions or was absent, while MHC II expression, especially prominent on microglia, was strongly up-regulated throughout the white matter, most pronounced in demyelinated plaques. In summary, in acute and subacute lesions without perivascular cuffs, MHC II expression correlated with the presence of CDV antigen. In contrast, in chronic lesions, MHC II expression on microglial cells was the most prominent despite a few CDV antigen-positive astrocytes, indicating that nonviral antigens may play an important role as triggering molecules for the process of demyelination. Received: 13 September 1995 / Revised: 26 February 1996 / Accepted: 1 April 1996     

Ia-expressing microglial cells in experimental allergic encephalomyelitis in rats

Summary Monoclonal antibodies (MRC OX-6 and OX-17) recognized three types of cells expressing Ia antigen during the course of acute experimental allergic encephalomyelitis (EAE) in rats. In earlier stages of the disease, in animals with or without paralysis, Ia antigens were mostly localized to subarachnoidal and perivascular lymphocytic and histiocytic cell infiltrates, possibly serving as antigen-presenting cells. On the other hand, in convalescent rats, Ia antigens were expressed in a large number of cells with dendritic processes heavily populating the spinal gray matter. The appearance of these Ia-expressing cells in the convalescent stage coincided with the development of degenerating axon terminals in the spinal gray matter. These Ia-expressing cells possessed morphological features characteristic of microglia and were positive for ML-1 lectin but did not express glial fibrillary acidic protein. Immune electron microscopy disclosed the presence of Ia reaction products in the Golgi apparatus, endoplasmic reticulum and plasma membrane of these cells with dendritic processes, indicating active synthesis of Ia molecules in microglia. In addition, Ia antigens were localized to the cells with ultrastructural features of macrophages. Thus, Ia-expressing cells in EAE seems to play dual roles: the induction of immunological reactions during earlier stages and the participation in reparative processes during convalescence.Supported by Grants-in-aid from the Ministry of Health and Welfare for Intractable Neuroimmunological Diseases and from the Ministry of Education, Science and Culture (Project 61570380 to HK)     

Microglial involvement in experimental autoimmune inflammation of the central and peripheral nervous system

Microglial cells form a network of potential antigen presenting cells throughout the nervous system. Much progress has recently been made towards a better understanding of their immunological properties. This study examines their activation in 2 models of T cell-mediated autoimmune inflammation of the nervous system, experimental autoimmune encephalomyelitis (EAE) and its peripheral counterpart, experimental autoimmune neuritis (EAN), induced by the transfer of antigen-specific T cell lines. In both models microglial activation occurs at early stages of the disease. Activated microglial cells show an increased expression of MHC class I and II antigens. In EAE ultrastructural analysis revealed that MHC antigen expression is pronounced on perivascular microglial cells, suggesting this cell population may be important for antigen presentation at a site close to the blood-brain barrier. In contrast to EAE, the microglial reaction in EAN occurs at sites remote from the inflammatory response in the peripheral nerve, not only in the spinal cord but also in the terminal projection fields of primary sensory neurons in the lower brainstem. This early microglial activation in EAN suggests that a rapid and remote signaling mechanism can operate following peripheral inflammation. Immuno-electron microscopy revealed that activated microglial cells are also involved in the synaptic deafferentation of spinal cord motoneurons during autoimmune reactions. The rapid involvement of microglial cells in experimental autoimmune inflammation of the nervous system further points to their role as the main intrinsic immuneffector cell population of the central nervous system.     

Granulocyte-macrophage colony stimulating factor inhibits class II major histocompatibility complex expression and antigen presentation by microglia

Granulocyte-macrophage colony stimulating factor (GM-CSF) modulates various functions of monocytes/ macrophages including antigen-presenting capacity. Recently it was found that astrocytes produce GM-CSF in the central nervous system (CNS) and that GM-CSF can induce proliferation and morphological changes of microglia. Here we show that GM-CSF can down regulate the interferon-γ-mediated induction of major histocompatibility complex (MHC) class II antigens in microglia, but not in astrocytes. GM-CSF pretreatment completely prevents myelin basic protein-specific T cell proliferation induced by microglia but not astrocytes. GM-CSF did not affect the cell surface expression on microglia of either MHC class I or cell adhesion molecules. The inhibition of microglial MHC class II expression and antigen-presenting function is specific for GM-CSF, as treatment with a different CSF (interleukin-3) did not modulate microglial phenotype or functional capacity. These data suggest that GM-CSF might be involved in the regulation of immune responses within the central nervous system.     

An immunoelectron microscopical study of the expression of class II major histocompatibility complex during chronic relapsing experimental allergic encephalomyelitis in Biozzi AB/H mice

Immunoelectron microscopical techniques have been used to study class II major histocompatibility complex (MHC) expression by cells in the spinal cords of Biozzi AB/H mice with chronic relapsing experimental allergic encephalomyelitis. Throughout the course of disease both astrocytes and endothelia failed to express significant levels of class II MHC antigens. The major central nervous system resident cell types found to express class II MHC antigens were the perivascular microglia, with infiltrating macrophages and some lymphocytes being strongly positive.     

实验性变态反应性脑脊髓炎大鼠星形胶质细胞的变化

目的观察实验性变态反应性脑脊髓炎(EAE)大鼠脊髓中星形胶质细胞的变化,探讨EAE大鼠的发病相关生物学机制。方法采用免疫组化法,对豚鼠全脊髓匀浆诱导的Wistar大鼠EAE的过程中,脊髓内星形胶质细胞变化情况进行研究。结果EAE大鼠症状高峰期时星形胶质细胞开始激活,恢复期时激活达到高峰,而且活化的星形胶质细胞未见表达主要组织相容性抗原(MHC)。结论活化的星形胶质细胞可能与EAE大鼠的恢复有关。     

Restraint stress-induced suppression of major histocompatibility complex class II expression by murine peritoneal macrophages

The macrophage plays a central role in the development of immune responses. Macrophages take up and process antigen which is presented to antigen-responsive T lymphocytes in association with major histocompatibility complex class II (Ia) glycoproteins. We have investigated the effect of restraint stress on Ia expression by murine peritoneal macrophages. Stress resulted in a suppression of Ia expression which coincided with an increase in plasma corticosterone levels. In vitro experiments indicate that suppression of Ia expression can occur within 2 h after exposure to corticosterone. The suppression of this important aspect of macrophage function by stressors has important implications regarding the possible immunosuppressive effects of stress on the response of lymphocytes to antigens that depend on intact macrophage function.     

Major histocompatibility antigens and lymphocyte subsets during experimental allergic neuritis in the Lewis rat

Summary Major histocompatibility antigens were identified in frozen sections of normal Lewis rat peripheral nerve tissue with monoclonal antibodies and an avidin-biotin-peroxidase complex system. Class I antigen is normally required for cytotoxic/suppressor T lymphocyte function and class II antigen for activation of helper T lymphocytes. In the sciatic nerves class I antigen was expressed diffusely by most endoneurial and perineurial cells but class II antigen only by a minority. In the cauda equina class I antigen was expressed by all arachnoid and some endoneurial cells, while class II antigen was expressed by a smaller proportion of arachnoid cells in the endoneurium of spinal roots and interstitial cells surrounding dorsal root ganglion neurons. The endothelium of endoneurial, perineurial and meningeal vessels uniformly expressed class I but not class II antigen. Experimental allergic neuritis was induced in Lewis rats by immunisation with bovine intradural root myelin. Early lesions consisted of multifocal infiltration of the nerve roots by cells expressing leucocyte common antigen. Surrounding endoneurial cells showed markedly increased expression of major histocompatibility antigens. In inflammatory lesions about 10% of the cells were stained with pan T cell antibodies. T lymphocyte subsets were identified with antibody W3/25 for helper cells and MRC OX-8 for cytotoxic/suppressor cells. The W3/25 positive cells were usually slightly in excess of OX-8 positive cells and their relative proportions did not alter during the disease. The presence of class I antigen on normal endothelium and its increased expression on endoneurial cells in the early phase of inflammation suggest an important role for class I restricted lymphocytes in the pathogenesis of the early stages of experimental allergic neuritis.     

Microglial and astroglial reactions to inflammatory lesions of experimental autoimmune encephalomyelitis in the rat central nervous system

Gliosis is a repair process of lesions appearing in the central nervous system (CNS). Although gliosis by astrocytes (astrocytic gliosis) has been well documented, that by microglia (microglial gliosis) remains poorly understood. In the present study we induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats and examined microglial and astroglial reactions to EAE lesions at various stages of the disease by immunohistochemistry. For the demonstration of microglia and astrocytes, antibodies against complement receptor type 3 (OX42) and glial fibrillary acidic protein (GFAP) were used, respectively. It was revealed that the whole course of microglial and astroglial reactions to EAE lesions is divisible into three stages, i.e., initial, peak and recovery stages. Microglial and astroglial reactions to EAE lesions at each stage correspond well with the clinical and histological stages of EAE. At the initial stage, rats showed mild clinical signs and a few inflammatory foci were found in the CNS. Microglia were increased in number in close association with inflammatory cell aggregates, whereas astrocytes showed no significant reaction in spite of the presence of inflammatory cells. At the peak stage, rats showed full-blown EAE and the number of inflammatory cells reached maximum. The most characteristic finding at this stage was 'encasement' of inflammatory lesions by astrocytic fibers. Microglia were increased in number, but association of microglia with lesions was prevented by astrocytes. Interestingly, however, such characteristic distribution of microglia and astrocytes was not observed at the recovery stage. Residual inflammatory cell aggregates were intermingled with dense microglial and astrocytic gliosis, forming 'micro-astroglial scars'. Double immunofluorescence staining with anti-GFAP and anti-bromodeoxyuridine (BrdU), or with OX42 and anti-BrdU revealed that BrdU-incorporated microglia, but not astrocytes, were present mainly at the initial and peak stages, suggesting that microglia would proliferate by cell division to create gliosis, whereas astrocytic gliosis would be a result of migration of astrocytes and/or up-regulation of expression of GFAP molecule. Taken together with previous in vitro findings that microglia, but not astrocytes, stimulate encephalitogenic T cell proliferation, these in vivo findings suggest that microglia augment, whereas astrocytes suppress, inflammatory processes in the CNS.     

Relative susceptibility of SJL/J and B10.S mice to experimental allergic encephalomyelitis (EAE) is determined by the ability of prethymic cells in bone marrow to develop into EAE effector T cells

SJL/J mice are highly susceptible to actively induced experimental allergic encephalomyelitis (EAE), whereas B10.S mice are resistant. However, both strains share the H-2^s haplotype. We have previously shown that the relative susceptibility of SJL/J and B10.S mice to acute EAE correlates, respectively, with high and low responsiveness to myelin basic protein (MBP), as determined by cloning and limiting dilution analysis of in vitro T cell proliferation. Here, we have investigated the ability of SJL/J and B10.S mice to generate EAE-effector T cells in vivo. We have developed a new mouse strain, B10.S Thy 1.1, that differs at the Thy 1 locus from SJL/J and B10.S mice (both Thy 1.2) but has the same MHC and resistance pattern to EAE as do B10.S mice. Using radiation bone marrow chimeras formed between SJL/J and B10.S Thy 1.1 mice, we have shown that a population of radiosensitive prethymic cells in SJL/J bone marrow has an intrinsic potential to generate EAE-effector T cells, whereas that in B10.S Thy 1.1 bone marrow does not. This lack of detectable EAE effector cells in B10.S Thy 1.1 mice does not appear to be due to the generation of suppressor T cells or to a detect in antigen-presenting cells. Moreover, the potential of SJL/J bone marrow to generate EAE-effector T cells is not inhibited by the concomitant presence of B10.S Thy 1.1 bone marrow cells, thymocytes or dendritic cells in mixed chimeras. Hence, the relative susceptibility of SJL/J and B10.S mice to EAE appears to be directly related to the respective responder status of their T cells to MBP, as evidenced by their ability (or inability) to generate EAE-effector T cells. This high and low responder status appears in turn to be linked to non-MHC background genes, although this has not been established formally.     

Immunohistochemical study of microglia in the Creutzfeldt-Jakob diseased brain

Immunohistochemical techniques have been used to investigate microglial reaction in Creutzfeldt-Jakob diseased (CJD) brains. Autopsy cases of six patients with CJD and age-matched controls were studied. Formalin-fixed, paraffin-embedded brain tissue samples were stained with antibodies against major histocompatibility complex (MHC) class II antigen (Ag), leukocyte common antigen (LCA), CDw75, CD68 and glial fibrillary acidic protein. Of the patients with CJD, two with a subacute spongiform encephalopathic type and short-survival periods after onset of the disease showed an increased number of reactive microglia labeled with anti-MHC class II Ag or LCA in the affected cerebral cortex. In advanced cases of the panencephalopathic type of CJD, in which both cerebral atrophy and astrocytosis were marked, the increase of reactive microglia was small. Some vacuoles developing in the neuropil of the CJD patients were surrounded by MHC class II Ag- or LCA-immunoreactive microglial cells. The number of ramified microglia in the affected lesions was decreased, although their number in the hippocampus was not affected. These results indicate that microglia can frequently be involved in the process of CJD and may be activated at the early stage of the disease.     

Distribution of major histocompatibility complex class II-positive microglia and cytokine profile of Parkinson's disease brains

There are numerous observations confirming that microglia expressing major histocompatibility complex (MHC) class II molecules are associated with the central nervous system (CNS) in aging and pathological conditions. In this study, we investigated the distribution of MHC class II-positive microglia in Parkinson's disease (PD) brains. The number of MHC class II-positive microglia in the substantia nigra (SN) and putamen increased as the neuronal degeneration of the SN proceeded. These cells were also ICAM-1 (CD54) and LFA-1 (CD11a) positive. The number of activated microglia not only in the SN and putamen but also in the hippocampus, transentorhinal cortex, cingulate cortex and temporal cortex in PD was significantly higher than that in the normal control. Most activated microglia persisted regardless of the presence or absence of Lewy bodies. They were frequently associated not only with -synuclein-positive Lewy neurites, but also with TH-16-positive dopaminergic and WH-3-positive serotonergic neurites, as well as MAP-2- and SMI-32-positive neurites. These activated microglia were also positive for TNF- and interleukin-6, which are known to have a neuroprotective function. We conclude that MHC class II-positive microglia are a sensitive index of neuropathological change and are actively associated with damaged neurons and neurites.     

Brain microglia and blood-derived macrophages: molecular profiles and functional roles in multiple sclerosis and animal models of autoimmune demyelinating disease

Microglia and macrophages, one a brain-resident, the other a mostly hematogenous cell type, represent two related cell types involved in the brain pathology in multiple sclerosis and its autoimmune animal model, the experimental allergic encephalomyelitis. Together, they perform a variety of different functions: they are the primary sensors of brain pathology, they are rapidly recruited to sites of infection, trauma or autoimmune inflammation in experimental allergic encephalomyelitis and multiple sclerosis and they are competent presenters of antigen and interact with T cells recruited to the inflamed CNS. They also synthesise a variety of molecules, such as cytokines (TNF, interleukins), chemokines, accessory molecules (B7, CD40), complement, cell adhesion glycoproteins (integrins, selectins), reactive oxygen radicals and neurotrophins, that could exert a damaging or a protective effect on adjacent axons, myelin and oligodendrocytes.The current review will give a detailed summary on their cellular response, describe the different classes of molecules expressed and their attribution to the blood derived or brain-resident macrophages and then discuss how these molecules contribute to the neuropathology. Recent advances using chimaeric and genetically modified mice have been particularly telling about the specific, overlapping and nonoverlapping roles of macrophages and microglia in the demyelinating disease. Interestingly, they point to a crucial role of hematogenous macrophages in initiating inflammation and myelin removal, and that of microglia in checking excessive response and in the induction and maintenance of remission.     

Determination of immune cells and expression of major histocompatibility complex class II antigen in encephalitic lesions of experimental Borna disease

Summary After intracerebral infection with Borna disease virus adult Lewis rats develop a virus-induced immunopathological reaction resulting in severe neurological symptoms and a non-purulent meningoencephalitis. The composition of inflammatory cells and major histocompatibility complex (MHC) class II antigen expression during the course of the infection was investigated using immunocytochemistry with a panel of monoclonal antibodies (mAb). Macrophages and lymphocytes of the T helper phenotype (CD4⁺) were dominant at all stages of infection, whereas T suppressor/cytotoxic lymphocytes (CD8⁺) were less frequent. B lymphocytes and plasma cells occurred mainly during later stages of the disease and marked parenchymal deposition of immunoglobulin developed. Beginning 10 days after infection massive expression of MHC class II antigen was noted up to the termination of experiments 70 days after infection. Besides lymphatic cells and macrophages, cells morphologically resembling microglia expressed this antigen. Furthermore, ependymal cells were found positive for MHC class II expression during infection whereas astrocytes remained negative. These findings are consistent with previous results which provide evidence for a delayed-type hypersensitivity reaction being operative in the pathogenesis of Borna disease.Supported by Deutsche Forschungsgemeinschaft     

Constitutive and visna virus induced expression of class I and II major histocompatibility complex antigens in the central nervous system of sheep and their role in the pathogenesis of visna lesions

Expression of major histocompatibility complex (MHC) antigens was studied in the brains of 10 healthy sheep 2 months to 5 years old and 13 sheep infected with visna virus by intracerebral inoculation and killed one and 6 months post infection (p.i.). In healthy sheep there was prominent expression of class I, mainly on endothelial cells but also detected on ependyma, choroid plexus and in the leptomeninges. Class II expression was sparse. It was observed on perivascular cells, in choroid plexus, leptomeninges and on microglial cells in the white matter. No definite increase with age in the constitutive expression of class I and II was observed, confirming that we are dealing with a true constitutive expression. In visna-infected sheep a considerable induction of MHC antigens on microglia was observed, which correlated with severity of lesions and was mainly found in or adjacent to inflammatory infiltrates of the white matter. Increase in class II antigen expression was detected in all sheep but class I only in sheep with the most severe lesions 6 months p.i., an indication of a higher threshold for induction of class I than class II antigens on microglia. Few cells expressed viral antigens, indicating that direct immune-mediated destruction of infected cells plays a minor role in evolution of lesions. Since the preferential induction of MHC antigens on microglia in the white matter correlated with the lesion pattern, activated microglia may play a considerable role in the pathogenesis of lesions.     

Strain-specific expression of microglial keratan sulfate proteoglycans in the normal rat central nervous system: Inverse correlation with constitutive expression of major histocompatibility complex class II antigens

We have recently demonstrated that a novel type of keratan sulfate proteoglycan (KSPG) identified by the monoclonal antibody (mAb) 5D4 is expressed on ramified microglia but downregulated coincident with T-cell-mediated autoimmune inflammation of the spinal cord in Lewis (LEW) rats. In this study we show by immunocytochemistry and Western blot analysis that various inbred rat strains differ significantly in their constitutive expression of KSPG on ramified microglia in the normal CNS. Microglial KSPG was high in LEW and Fischer 344 rats but low in DA, Brown Norway (BN), and PVG rats. The KSPG low-expressing strains exhibited constitutive expression of major histocompatibility complex (MHC) class II antigens on ramified microglia that was not detectable in the KSPG high-expressing strains. Thus, an inverse correlation between constitutive KSPG and MHC class II expression was present. The KSPG-low-/MHC class II-positive phenotype is associated with resistance to experimental autoimmune encephalomyelitis in BN and PVG, but not DA rats. These findings suggest a significant impact of genetic factors on the molecular differentiation of resident macrophages in the CNS. © 1996 Wiley-Liss, Inc.     

The expression of major histocompatibility complex (MHC) class I antigens in the brain differs markedly in acute and persistent infections with lymphocytic choriomeningitis virus (LCMV)

Intracranial inoculation of immunocompetent mice with lymphocytic choriomeningitis virus (LCMV) induces a fatal neurologic illness. In this disease a marked increase in MHC class I expression was found, closely associated with viral antigens and inflammatory infiltrates, in meninges, choroid plexus and ventricular ependyma but not within the brain parenchyma. Immunosuppression prevented MHC induction. Mice inoculated at birth had persistent infections, with LCMV antigens found primarily in neurons, but no inflammatory cells or focal increase in MHC class I. Failure of infected neurons to express MHC class I allows them to escape destruction by cytotoxic T cells (CTL) but may increase their susceptibility to be persistently infected by non-lytic viruses.     

Interleukin-12 induces mild experimental allergic encephalomyelitis following local central nervous system injury in the Lewis rat

The major pathological feature in the central nervous system (CNS) following traumatic brain injury is activation of microglia both around and distant from the injury site. Intraperitoneal administration of interleukin-12 (IL-12) after brain injury resulted in a 7% weight loss, clinical signs of mild EAE and significant myelin basic protein (MBP)-specific splenic cell proliferation. The extent of pathology, in terms of the number of inflammatory perivascular cuffs and activation of microglia was greatest if IL-12 was administered immediately compared to a week following brain injury, whether at one or two sites. Specifically immunostaining for MHC class II and iNOS on macrophages and microglia, ICAM-1 on endothelial cells and macrophages was observed around the site of injury. A degree of myelin processing was apparent from immunostaining of MBP in inflammatory cells distant from the lesion. Inflammatory cuffs comprising macrophages, activated microglia, CD4⁺ T cells and iNOS⁺ cells were also detected distant to the injury site in the medulla and spinal cord of animals treated with IL-12. These results suggest that immune-mediated events in which IL-12 production is stimulated as for example viral infection, superimposed on a brain injury, could provide a trigger for a MS-like pathology.