The role of macrophages in demyelination

Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS). In particular in the CsA-induced chronic relapsing form (CREAE), pronounced demyelination occurs, in temporal association with relapses. It is still a matter of discussion which cell type ultimately is responsible for the actual process of demyelination. Macrophages, cytotoxic T lymphocytes and also astrocytes are possible candidates. In this short overview, the role of macrophages in the pathogenesis of EAE is discussed. It is shown that in particular, newly recruited macrophages play a crucial role in the generation of clinical signs. Possible mechanisms by which macrophages are involved in the pathogenesis of demyelinating diseases are presented.     

Morphology of demyelination in the human central nervous system

The principles of the neuropathological classification of disorders of central nervous system myelin are recalled. They are illustrated by a few selected examples. Dysmyelination is characterized by the production of an abnormal and unstable myelin sheath; it is often associated with hypomyelination (paucity of myelin formation) and is due to metabolic disorders. It is the main process in leukodystrophies. Storage of different lipids (e.g. sulfatides, long-chain fatty acids) or associated pathology of various cell types (in Alexander's disease, for example) are used for classifying these disorders. Biochemical and genetic characterizations are presently ongoing. Demyelination is the destruction of apparently normal myelin. It is often followed by remyelination. Our present knowledge on the neuropathology of multiple sclerosis, the most common demyelinating disease, is summarized. Cell-mediated demyelination affects the myelin sheaths for an obscure reason. The causes of the multifocal and sharply demarcated plaques, and of the fading of the remyelination process at the edge of some plaques, are not clear. A few examples of demyelinating diseases of known etiology and of various mechanisms are given. The similarities between acute disseminated leukoencephalitis and experimental autoimmune encephalitis are stressed. In progressive multifocal leukoencephalopathy, chronic infection of oligodendrocytes by JC virus induces poorly defined areas of demyelination. In AIDS, the pathogenesis of the myelin change is unclear. Macrophages may be responsible. Toxic and vascular disorders provide also good models for the understanding of mechanisms of demyelination.     

Preliminary analysis of cell and serum-induced demyelination in vitro using a syngeneic system

Previous studies on immune-mediated demyelination in vitro have usually tested sera and lymphoid cells in heterologous systems. The present study involved the examination of CNS cultures of SJL/J mouse spinal cord exposed to sera and lymphoid cells isolated from animals of the same strain previously injected with syngeneic spinal cord homogenate (SSCH) to induce acute experimental autoimmune encephalomyelitis (EAE). Examination of treated versus control cultures by light and electron microscopy at varying time points after exposure showed that spleen cells from animals with EAE produced significant demyelination and oligodendroglial cell destruction. Lymph node cells and sera from the same animals showed the same type of demyelination without marked oligodendroglial cell damage. The degree of myelin damage induced by spleen cells and sera did not correlate with the clinical status of the animal but a slight positive correlation was noted with lymph node cells. Cells and sera from control animals did not induce significant demyelination. These results suggest that in this syngeneic mouse system, there was a differential effect among cells from spleens and lymph nodes, and serum. This syngeneic system might allow for more meaningful pathologic and genetic analyses of immune-mediated demyelination.     

Evidence for the production of peroxynitrite in inflammatory CNS demyelination

Peroxynitrite, which is generated by the reaction of nitric oxide (NO) with superoxide, is a strong oxidant that can damage subcellular organelles, membranes and enzymes through its actions on proteins, lipids, and DNA, including the nitration of tyrosine residues of proteins. Detection of nitrotyrosine (NT) serves as a biochemical marker of peroxynitrite-induced damage. In the present studies, NT was detected by immunohistochemistry in CNS tissues from mice with acute experimental autoimmune encephalomyelitis (EAE). NT immunoreactivity was displayed by many mononuclear inflammatory cells, including CD4+ cells. It was also observed in astrocytes near EAE lesions. Immunostaining for the inducible isoform of NO synthase (iNOS) was also observed, particularly during acute EAE. These data strongly suggest that peroxynitrite formation is a major consequence of NO produced via iNOS, and implicate this powerful oxidant in the pathogenesis of EAE.     

Adoptive transfer of experimental allergic encephalomyelitis from immature guinea pig donors

In immature Strain 13 guinea pigs sensitized to syngeneic spinal cord, a chronic allergic encephalomyelitis is elicited reminiscent of demyelinating diseases of man and which features relapses or progressive downhill course and extensive areas of demyelination in the central nervous system. However, juvenile recipients of syngeneic lymphocytes from similarly sensitized juveniles show only the acute form of experimental allergic encephalomyelitis. Neuropathologically, the CNS of affected animals displayed mild changes only and minimal demyelination. These observations indicate that the age-dependent differences seen between the acute disease of adults and the chronic disease of juveniles may be due to differences in availability of modulating or reparatory factors, rather than differences in the central nervous system organ or in the immune response itself.     

Chronic experimental allergic encephalomyelitis in strain 2 guinea pigs : Absence of resistance to nervous system changes and sex dependence of clinical disease

Experimental autoimmune (allergic) encephalomyelitis (EAE) was induced in Strain 2 guinea pigs, a strain usually regarded as resistant to EAE. The development of disease in groups of juvenile male or female Strain 2/N guinea pigs was irregular with clinical EAE manifesting only in some groups of females. All animals examined morphologically (12 females and 9 males), between 3 and 18 months postinoculation, showed extensive changes in the central nervous system, although 12 of these had displayed no neurologic signs. This silent central nervous system disease, reminiscent of similar phenomena in man, indicates that Strain 2/N is fully competent immunologically at the level of the target organ.     

About demyelinating properties of humoral antibodies in experimental allergic encephalomyelitis

Summary In order to elucidate the role of humoral antibodies in the pathogenesis of myelin lesions in experimental allergic encephalomyelitis (EAE) a combined in vivo and in vitro study was done using rabbits immunized with the purified A₁ basic protein. Rabbits injected with whole white matter were used for comparison. Demyelinating activity appeared in the rabbit sera 5 days after injection, as tested in myelinated organotypic tissue cultures. In spite of this no lesions of the myelin preceded the appearance of inflammatory cells in the living animals. In the spinal cord changes in vascular permeability, as revealed by leakage of Evans blue-albumin complex, appeared at the same times as the cells. In contrast to in vitro, the mere presence of circulating antibodies in vivo does not appear to be enough to cause structural changes of the myelin. Possible reasons for this discrepancy are discussed; it is emphasized that the inflammatory changes develope first in areas where the so-called blood-brain barrier to diffusion of proteins is lacking.     

Immunohistological analysis of macrophages in the central nervous system of Lewis rats with acute experimental allergic encephalomyelitis

Inflammatory cells were characterized by immunohistochemistry, utilizing monoclonal antibodies specific for T cell subsets, B cells, Ia-positive cells and cells of the mononuclear phagocyte system, in cryostat sections of central nervous system of Lewis rats, sacrificed during the course of actively induced experimental allergic encephalomyelitis. The present study provides interesting information about the presence and distribution of cells of the monocyte-macrophage lineage in this immunologically mediated disease. Using these monoclonal antibodies different subpopulations of macrophages having varying distribution patterns in the central nervous system can be recognized.     

Complement depletion affects demyelination and inflammation in experimental allergic neuritis

The effect of systemic complement depletion by cobra venom factor (CVF) on experimental allergic neuritis (BAN) was studied in rats immunized with variable amounts of bovine peripheral nerve myelin. Low-dose myelin EAN rats treated with CVF i.p. (n = 10) had lower clinical scores (0.3 ± 0.7 vs. 1.1 ± 1.1), less demyelination (0.4 ± 0.8 vs. 1.9 ± 1.1) and inflammation (0.6 ± 1.2 vs. 2 ± 1) than EAN animals treated with i.p. saline (n = 10). Endoneurial infiltrates had fewer EDI-positive (phagocytic) macrophages (if0.4 ± 0.5 vs.> 1.6 ± 1.1) and CD11bc-positive (expressing iC3b receptor or CR3) cells (1 ± 0.8 vs. 2.5 ± 0.8) (mean ± S.D.) detected by immunocytochemistry. This effect was partially abrogated by immunizing animals with a higher dose of myelin. Our studies suggest that complement may play a role in the recruitment of macrophages into the endoneurium and in opsonizing myelin for phagocytosis.     

实验性自身免疫性脑脊髓炎大鼠听神经损害研究

目的建立实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)大鼠模型,研究其脑干听觉诱发电位(brainstem auditory evoked potential,BAEP)Ⅰ波和听神经组织学改变。方法40只Wistar大鼠分为实验组和对照组,实验组(EAE组)用含卡介苗的豚鼠全脊髓匀浆为抗原免疫Wistar大鼠建立EAE动物模型,对照组用生理盐水混合完全福氏佐剂注射,采用诱发电位仪检测EAE组急性发病的大鼠和对照组大鼠的BAEP,通过透射电镜观察发病大鼠听神经外周段组织学改变。结果急性发病实验组大鼠BAEP的Ⅰ波潜伏期较对照组显著延长(P<0.01)。透射电镜下观察听神经外周段髓鞘松散,板层分离,局部变薄,呈不完全性脱髓鞘改变,轴突完整。对照组大鼠未见组织学改变。结论急性发病EAE大鼠听神经外周段脱髓鞘可能引起脑干听觉诱发电位Ⅰ波的改变。     

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。     

Dendritic cells in experimental allergic encephalomyelitis and multiple sclerosis

The mechanisms by which autoimmune diseases are triggered and by which the activation of autoreactive T cells is initiated and maintained are not yet fully understood. As the most potent antigen presenting cells (APC), and also being responsible for antigen transport as well as primary sensitisation of T cells, dendritic cells (DC) are capable of breaking the state of self-ignorance and inducing aggressive autoreactive T cells. In the development of autoimmune diseases, different types of DC exhibit distinct properties for inducing Th1/Th2 cell responses. Appropriate cytokines can convert immunogenic DC to tolerogenic DC. Utilizing the possibility to promote the tolerogenic effects of DC, a new therapeutic tool might soon become available to treat multiple sclerosis and other autoimmune diseases.     

Immunohistochemical localisation of terminal complement component C9 in experimental allergic encephalomyelitis

Summary The deposition of terminal complement component C9 within the central nervous system (CNS) has been studied immunohistochemically in three models of experimental allergic encephalomyelitis (EAE) in the rat; inflammatory EAE induced by the passive transfer of myelin basic protein-specific T cells (tEAE), antibody-mediated, demyelinating tEAE and a subacute/chronic model induced by active immunisation with guinea pig spinal cord tissue in adjuvant. Two distinct patterns of C9 reactivity were observed, a diffuse staining of the tissue adjacent to inflammatory lesions, similar to that seen for other extra-vasculated serum proteins, and also granular, sometimes fibrillar C9 deposits around some inflammed vessels and in areas of active demyelination. The latter staining pattern was most pronounced in animals with acute antibody-mediated demyelinating tEAE, in which extensive, but transient, subpial and perivascular granular deposits of C9 were associated with regions of acute demyelination. A similar pattern of granular C9 reactivity was also associated with demyelinating lesions in animals with actively induced chronic progressive EAE. However, these C9 deposits were not observed in rats with purely inflammatory, clinically mild tEAE, although C9 deposition was occasionally observed around a small number of inflammed vessels in animals with hyperacute, lethal tEAE. These observations demonstrate that deposition of C9, the major component of the cytolytic membrane attack complex, in EAE is related to myelin injury rather than CNS inflammation.Supported by the Science Research Fund (Austria), Project P6438M and the Multiple Sclerosis Society of Great Britain and Northern Ireland     

Electrophysiological follow-up of acute and chronic experimental allergic encephalomyelitis in the Lewis rat

Summary Cortical somatosensory evoked potentials (c-SEP) and flash visual evoked potentials (f-VEP) were serially recorded in acute monophasic and chronic relapsing experimental allergic encephalomyelitis (EAE) in the Lewis rat. In acute EAE, a significantly delayed latency and broadened peak of the c-SEP were observed corresponding to the clinical onset, and then returned to normal with the disappearance of clinical signs. In chronic EAE, the c-SEP showed the same changes as in acute EAE, also reflecting the first attack, remission and relapsing phase. However, chronic EAE, when paralysis had recovered in the relapsing phase, showed c-SEP abnormalities suggestive of subclinical active lesions. In contrast, the f-VEP showed no obvious abnormalities in acute or chronic EAE. These findings suggest that the c-SEP is an objective and sensitive index for detecting clinical and pathological changes in acute and chronic EAE in the Lewis rat.     

Experimental allergic encephalomyelitis: the balance between encephalitogenic T lymphocytes and demyelinating antibodies determines size and structure of demyelinated lesions

Summary The effect of a circulating monoclonal antibody recognizing an antigen located on the surface of myelin sheaths (myelin/oligodendroglia glycoprotein, MOG) on clinical and histopathological expression of experimental allergic encephalomyelitis (EAE) was tested in a model of EAE passively transferred by monospecific T lymphocytes. Intravenous injection of anti-MOG antibody at the onset of the disease massively augmented clinical impairment as well as primary demyelination. The structure of the CNS lesions depended on the balance between encephalitogenic T cells and anti-MOG antibody: when EAE was induced with high numbers of T cells, circulating anti-MOG antibody resulted in ubiquitous perivenous demyelination in the spinal cord and medulla oblongata. On the contrary, focal confluent demyelinated lesions were observed in animals injected with low numbers of T cells (even as few as 10⁴) and anti-MOG antibody. Our studies, thus, indicate that the formation of inflammatory demyelinating lesions may be due to a synergistic action of cellular and humoral immune mechanisms.Supported by the Science Research Fund (Austria), Project P5354 and P6438M     

Experimental Usutu virus infection of suckling mice causes neuronal and glial cell apoptosis and demyelination

Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus group has been responsible for avian mortality in Austria since 2001. In the present study, the neuropathogenicity and neuroinvasiveness of USUV for 1-week-old suckling mice was investigated. After intraperitoneal inoculation, clinical signs like depression, disorientation, paraplegia, paralysis and coma were observed between 6 and 11 days post infection. Histologically, there was widespread neuronal apoptosis especially in the brain stem. Inflammatory infiltrates were scarce. Apoptosis was also present in white matter of cerebellum, medulla and spinal cord, and was frequently accompanied by primary demyelination. While apoptosis of neurons was clearly associated with presence of viral signals, the cause of apoptosis of white matter cells was more ambiguous. However, focal immunostaining was found in the white matter, especially in the spinal cord. As with all flaviviruses, USUV proved to be neuropathogenic for mice. In contrast to other flaviviruses, neuroinvasion occurred only in animals that were not older than 1 week at the time of inoculation. While neuronal apoptosis is a general aspect of flavivirus pathogenicity, demyelination seems to be a unique feature of USUV infection.     

Pathogenic MOG-reactive CD8+ T cells require MOG-reactive CD4+ T cells for sustained CNS inflammation during chronic EAE

XIncreasing evidence supports a role for CD8+ T cells in multiple sclerosis. In an attempt to isolate the contribution of CD8+ T cells in a murine model of MS, we immunized mice with a dominant CD8 epitope MOG37–46, a truncated version of MOG35–55. The data presented here show mild disease induced with MOG37–46, characterized by lower clinical scores, a decrease in CNS infiltration and a decrease in microglial activation. CD8+ T cells reactive to MOG37–46 are pro-inflammatory and traffic to the CNS; however, the presence of CD4+ T cells elicits more severe disease and sustained inflammation of the CNS.     

Astrocytes support mast cell viability in vitro

Mast cells are normally found adjacent to blood vessels in the nervous system, and have been implicated in the development of inflammatory central nervous system (CNS) diseases such as experimental allergic encephalomyelitis. To further study mast cell-CNS interactions, we have developed a model in which viable rat peritoneal mast cells can be maintained in culture for up to 30 days on a monolayer of rat astrocytes. In this microenvironment, mast cells maintain their phenotype, morphology, and ability to degranulate in response to appropriate stimuli.