Basic FGF and FGF receptor 1 are expressed in microglia during experimental autoimmune encephalomyelitis: Temporally distinct expression of midkine and pleiotrophen

Heparin-binding growth factors have been implicated in central nervous system development, regeneration and pathology. To assess the expression pattern and possible function in multiple sclerosis, the heparin-binding growth factors pleiotrophin (PTN), midkine (MK), basic fibroblast growth factor (FGF-2) and one of its receptors (FGFR1/ flg) mRNA and protein levels were examined in an experimental autoimmune encephalomyelitis (EAE) model in the Lewis rat. We assessed the time course of expression of PTN, MK and FGF-2 during EAE and determined the cellular origin of FGF-2 and FGFR1 in normal spinal cord and during inflammatory demyelination. Basal expression of PTN and MK mRNAs in normal spinal cords was significantly upregulated after induction of EAE. MK expression was upregulated two to threefold correlating with disease progression, whereas PTN expression reached peak levels threefold above basal levels during the clinical recovery period. FGF-2 mRNA expression was low in normal spinal cord and dramatically increased in correlation with progressive demyelination. FGF-2 was confined to neurons in normal tissue and shifted dramatically to microglia, paralleling their activation during EAE. Double immunohistochemistry revealed co-localization of FGF-2 to activated microglia/ macrophages with strongest expression in the macrophage-rich perivascular core area and microglial expression at the edges of white and gray matter perivascular regions. FGFR1, like its ligand, was induced in activated macrophages/ microglia. Growth factor expression in demyelinating diseases could serve several functions, e.g., to modulate the activity of microglia/ macrophage in an autocrine fashion, to induce the expression of other factors like insulin-like growth factor 1 or plasminogen activator, which can effect regeneration or degeneration, respectively, and finally to stimulate directly localized proliferation and/ or regeneration of oligodendrocytes within the lesion area. GLIA 24:390–397, 1998. © 1998 Wiley-Liss, Inc.     

Involvement of neuropsin in the pathogenesis of experimental autoimmune encephalomyelitis

Inflammation, demyelination, and axonal damage of the central nervous system (CNS) are major pathological features of multiple sclerosis (MS). Proteolytic digestion of the blood-brain barrier and myelin protein by serine proteases is known to contribute to the development and progression of MS. Neuropsin, a serine protease, has a role in neuronal plasticity, and its expression has been shown to be upregulated in response to injury to the CNS. To determine the possible involvement of neuropsin in demyelinating diseases of the CNS, we examined its expression in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a recognized animal model for MS. Neuropsin mRNA expression was induced in the spinal cord white matter of mice with EAE. Combined in situ hybridization and immunohistochemistry demonstrated that most of the cells expressing neuropsin mRNA showed immunoreactivity for CNPase, a cell-specific marker for oligodendrocytes. Mice lacking neuropsin (neuropsin-/-) exhibited an altered EAE progression characterized by delayed onset and progression of clinical symptoms as compared to wild-type mice. Neuropsin-/- mice also showed attenuated demyelination and delayed oligodendroglial death early during the course of EAE. These observations suggest that neuropsin is involved in the pathogenesis of EAE mediated by demyelination and oligodendroglial death.     

Serum pro-inflammatory and anti-inflammatory cytokines and the pathogenesis of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) as an experimental model of multiple sclerosis (MS) is characterized by demyelination, infiltration of inflammatory cells into the nervous system and dysregulation of serum inflammatory cytokines. We investigated the correlation of serum cytokines and other inflammatory markers with the EAE pathogenesis. After EAE induction, the levels of different serum cytokine/inflammatory mediators were measured. Furthermore, motor functions, myelination, and lymphocyte infiltration in EAE mice were also assessed. Our results revealed that the serum concentrations of T-helper 1 (Th1) and Th17 cytokines, interleukin (IL)-6, IL-1β, IL-1α and prostaglandin E2 in EAE mice were significantly higher than controls. The ratios of pro- to anti-inflammatory cytokines were different between the EAE and the control group. A statistically significant positive correlation was found between the IL-6/IL-10 ratio and the EAE severity, demyelination rate, and lymphocyte infiltration in EAE mice. Results indicate that the profiles of serum pro- and anti-inflammatory cytokines might be useful as biomarkers for monitoring the pathological manifestation of EAE. Furthermore, evaluating the dynamic interplay of serum cytokine levels and the correlation with pathogenic mechanisms of EAE may provide diagnostic and therapeutic insights for MS and some other inflammatory disorders.     

The chemokine receptor CXCR2 is differentially regulated on glial cells in vivo but is not required for successful remyelination after cuprizone-induced demyelination

Unravelling the factors that can positively influence remyelination is one of the major challenges in multiple sclerosis research. Expression of the chemokine receptor CXCR2 on oligodendrocytes both in vitro and in MS lesions has suggested a possible role for CXCR2 in the recruitment of oligodendrocyte precursor cells (OPC). To investigate the function of CXCR2 during remyelination in vivo, we studied this receptor in cuprizone-induced demyelination and subsequent remyelination. We found that CXCR2 is constitutively expressed on OPC, whereas on macrophages/microglia CXCR2 is upregulated upon activation during demyelination. Hence, the expression of CXCR2 is differentially regulated in oligodendrocytes and macrophages/microglia. Furthermore, we subjected CXCR2-/- mice to the cuprizone model demonstrating that remyelination was not altered in comparison to wildtype controls. In addition, the number of OPC and the amount of microglial accumulation were similar in both CXCR2-/- and wildtype animals during the whole demyelination and remyelination process. These results suggest that despite expression on OPC and microglia CXCR2 plays only a minor role during remyelination.     

Gelatinase B is present in the cerebrospinal fluid during experimental autoimmune encephalomyelitis and cleaves myelin basic protein

Gelatinases in inflammatory demyelinating diseases of the central nervous system (CNS) were studied using actively induced experimental autoimmune encephalomyelitis (EAE) in mice as a model system. Clinical disease scores correlated in time and in intensity with pathology parameters such as cytosis in the cerebrospinal fluid (CSF), inflammatory infiltrates, and demyelination in the CNS. Zymographic analysis was employed to measure gelatinases A and B in the CSF from individual animals. According to their apparent molecular weight (MW), gelatinases A and B appeared with a MW of 65 and 95 kDa, respectively. The 65 kDa form was present in all samples, even in those derived from non-induced animals, whereas the 95 kDa form was present only in samples from animals developing EAE. The levels of 95 and 65 kDa gelatinase correlated with the CSF cytosis. In vitro digestion of myelin basic protein (MBP) with gelatinase B and analysis of the cleavage products by protein sequence analysis pinpointed two cleavage sites in conserved regions of MBP. Gelatinase production within the CNS may constitute an important pathogenic mechanism for both the disruption of the blood-brain barrier and the destruction of myelin, as observed in several neuroinflammatory disorders. © 1993 Wiley-Liss, Inc.     

Induction of glial L-CCR mRNA expression in spinal cord and brain in experimental autoimmune encephalomyelitis

Chemokines and chemokine receptors are important regulators of leukocyte trafficking and immune response. It is well established that chemokines and their receptors are also expressed in the central nervous system (CNS), where their expression has been associated with various neuroinflammatory diseases, such as multiple sclerosis (MS). One of the most important chemokines involved in MS pathology is CCL2 (previously known as MCP-1). CCL2, released by glial cells, activates the chemokine receptor CCR2, causing the infiltration of blood monocytes in tissues affected by MS. There is evidence, however, that CCL2 also has local effects on CNS cells, including induction or modulation of cytokine release and synthesis of matrix metalloproteinases, that might contribute to CNS pathology. These effects are most likely independent of CCR2, since CCR2 expression in glial cells is rarely observed. We have recently provided evidence for the presence of an alternative CCL2 receptor in glial cells called L-CCR and have investigated the expression of L-CCR mRNA in a murine EAE model. It is shown that L-CCR mRNA is expressed in infiltrating macrophages during EAE, but not in infiltrating T cells. Prominent expression of L-CCR mRNA was detected in astrocytes and microglia already at early time points throughout the brain and spinal cord supporting the hypothesis that L-CCR expression in glial cells is related to CNS inflammation.     

Effect of thalidomide and pentoxifylline on experimental autoimmune encephalomyelitis (EAE)

Background

Autoimmune encephalomyelitis (EAE) in Lewis rats is a classical experimental model of demyelinating inflammatory disease of the central nervous system. EAE is widely accepted for study of immune-inflammatory mechanisms in the CNS related to multiple sclerosis (MS) due to similar clinical evolution.

Objectives

In the present study we investigated the effects of Thalidomide and pentoxifylline during EAE development in Lewis rats.

Methods

EAE was induced in Lewis rats and treatment with Thalidomide or pentoxifylline was performed. Clinical evaluation was carried out daily. Histopathological analysis of the brain tissue and spinal cord was performed. Griess method was used for determination of NO serum levels. TNF-alpha and IFN-gamma serum levels were investigated using ELISA method.

Results

Thalidomide and pentoxifylline treatment is associated with significant reduction of neuroinflammation in CNS. Serum levels of NO, IFN-gamma and TNF-alpha showed a marked reduction. Such findings were correlated with improvement of clinical symptoms, particularly in thalidomide treated rats.

Conclusions

Taken together the data suggested that thalidomide and pentoxifylline may be therapeutic options for the treatment of MS, however further experiments must be performed to investigate this hypothesis.     

Differential expression of fibroblast growth factor-2 and fibroblast growth factor receptor 1 in a scarring and nonscarring model of CNS injury in the rat

Injury to the adult brain results in abortive axon regeneration and the deposition of a dense fibrous glial scar. Therapeutic strategies to promote postinjury axon regeneration are likely to require antiscarring strategies. In neonatal brain wounds, scar material is not laid down and axons grow across the lesion site, either by de novo growth or regeneration. To achieve the therapeutic goal of recapitulating the nonscarring neonatal response in the injured adult, an understanding of how ontogenic differences in scarring reflect developmental diversities in the trophic response to injury is required. Fibrobast growth factor-2 (FGF-2) expression is developmentally regulated and has been implicated as a regulator of the wounding response of the adult rat central nervous system. We have investigated the expression of FGF-2 and fibroblast growth factor receptor 1 (FGFR1) after penetrating lesions to the cerebral cortex of 5 days post partum (dpp) (nonscarring) and 16 dpp and adult (scarring) rats. In situ hybridization, immunohistochemistry and Western blotting showed robust and sustained increases in FGF-2 and FGFR1 mRNA and protein in reactive astrocytes around the lesion in scarring rats, a response that was attenuated substantially in the nonscarring neonate. These results demonstrate that changes in astrocyte FGF-2 and FGFR1 expression are coincident with the establishment of a mature pattern of glial scarring after injury in the maturing central nervous system, but it is premature to infer a causal relationship without further experiments.     

LPS-induced expression of a novel chemokine receptor (L-CCR) in mouse glial cells in vitro and in vivo

There is increasing evidence that chemokines, specialized regulators of the peripheral immune system, are also involved in the physiology and pathology of the CNS. It is known that glial cells (astrocytes and microglia) express various chemokine receptors like CCR1, -3, -5, and CXCR4. We have investigated the possible expression of the known CC chemokine receptors (CCR1-8 and D6) in murine glial cells. In addition, we examined possible glial expression of the orphan CC chemokine receptor L-CCR that has been identified previously in murine macrophages. We report here expression of L-CCR mRNA in murine astrocytes and microglia. Furthermore, L-CCR mRNA expression was strongly induced after application of bacterial lipopolysaccharide (LPS), both in vitro and in vivo. Functional studies and binding experiments using biotinylated monocyte chemoattractant protein (MCP)-1 (CCL2) indicate that CCL2 could be a candidate chemokine ligand for glial L-CCR. Based on the data presented, it is suggested that L-CCR is a functional glial chemokine receptor that is important in neuroimmunology.     

Selective CC chemokine receptor expression by central nervous system-infiltrating encephalitogenic T cells during experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell disease of the central nervous system (CNS) characterized by mononuclear cell infiltration, demyelination, and paralysis. Recent studies describing the relationship of chemokine expression with development of clinical disease have led to the hypothesis that distinct chemokine receptors corresponding to specific ligands are expressed by CNS-infiltrating antigen-specific encephalitogenic T cells as well as host-derived bystander T cells and monocytes. In an effort to study encephalitogenic T cell chemokine receptor expression, we examined CC chemokine receptor expression from resting, activated, and CNS-isolated CD4(+) T cells. CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, and CCR8 mRNA is expressed by normal CD4(+) T cells. In vitro activated T cells expressed CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, and CCR8 mRNA as well as CCR4. After EAE induction, CCR1 mRNA was expressed by donor-derived encephalitogenic and host-derived CD4(+) T cells isolated only from CNS and not from spleen. In vivo neutralization of the CCR1 ligand, macrophage inflammatory protein-1alpha (CCL3), resulted in less encephalitogenic CD4(+) T cell CNS infiltration. These results demonstrate the importance of CC chemokine receptor expression by CD4(+) encephalitogenic T cells for CNS infiltration and subsequent disease development.     

Mice overexpressing Bcl-2 in their neurons are resistant to myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by destruction of myelin. Recent studies have indicated that axonal damage is involved in the pathogenesis of the progressive disability of this disease. To study the role of axonal damage in the pathogenesis of MS-like disease induced by myelin oligodendrocyte glycoprotein (MOG), we compared experimental autoimmune encephalomyelitis (EAE) in wild-type (WT) and transgenic mice expressing the human bcl-2 gene exclusively in neurons under the control of the neuron-specific enolase (NSE) promoter. Our study shows that, following EAE induction with pMOG 35-55, the WT mice developed significant clinical manifestations with complete hind-limb paralysis. In contrast, most of the NSE-bcl-2 mice (16/27) were completely resistant, whereas the others showed only mild clinical signs. Histological examination of CNS tissue sections showed multifocal areas of perivascular lymphohistiocytic inflammation with loss of myelin and axons in the WT mice, whereas only focal inflammation and minimal axonal damage were demonstrated in NSE-bcl-2 mice. No difference could be detected in the immune potency as indicated by delayed-type hypersensitivity (DTH) and T-cell proliferative responses to MOG. We also demonstrated that purified synaptosomes from the NSE-bcl-2 mice produce significantly lower level of reactive oxygen species (ROS) following exposure to H₂0₂ and nitric oxide (NO) than WT mice. In conclusion, we demonstrated that the expression of the antiapoptotic gene, bcl-2, reduces axonal damage and attenuates the severity of MOG-induced EAE. Our results emphasize the importance of developing neuroprotective therapies, in addition to immune-specific approaches, for treatment of MS. D.O. and J.F.K. contributed equally.     

Suppression of experimental autoimmune encephalomyelitis by sulfasalazine

It has recently been suggested that the sulfidopeptide leukotriene C4 (LTC4), a 5-lipoxygenase product of the arachidonic acid metabolism and one of the most potent mediators of vascular permeability, might be involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). Subsequently, 20 guinea pigs with EAE were treated with sulfasalazine, a substance with a proved leukotriene inhibiting effect, which has previously been described as exerting beneficial effects in patients with inflammatory bowel disease and rheumatoid arthritis. The sulfasalazine-treated guinea pigs showed a significantly better clinical outcome, as well as a significantly lower histological inflammation score compared with 19 controls.     

Expression of citrullinated proteins in murine experimental autoimmune encephalomyelitis

In this study, we demonstrate for the first time the immunohistochemical expression of citrullinated proteins in the central nervous system (CNS) of mice with myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). By using an established monoclonal antibody (F95) against natural and synthetic citrullinated proteins (Nicholas and Whitaker [2002] Glia 37:328-336), numerous, small, previously unrecognized "patches" of citrullinated proteins were discovered throughout EAE brains, whereas EAE spinal cords showed similar but much larger lesions. On dual color immunofluorescence, these lesions were found to contain citrullinated myelin basic protein (MBP) and were surrounded by astrocytes immunoreactive for both glial fibrillary acidic protein (GFAP) and F95. These lesions became evident about the time when EAE mice became symptomatic and increased in size and number with increasing disease severity. In some sections of spinal cord but not brains of severely debilitated EAE mice, a widespread gliotic response was seen, with astrocytes containing citrullinated GFAP spread throughout the gray and white matter. Western blot analysis of acidic proteins from the brains and spinal cords of EAE mice had higher levels of multiple citrullinated GFAP isoforms compared with controls, with more F95-positive bands in the EAE brains vs. spinal cords. These results raise the possibility that citrullination of both GFAP and MBP may contribute to the pathophysiology of EAE and that the brains of EAE mice may contain more pathology than previously realized.     

Distribution in ocular structures and optic pathways of immunocompetent and glial cells in an experimental allergic encephalomyelitis (EAE) relapsing model

Relapsing experimental allergic encephalomyelitis (EAE) was induced in DA rats and the ocular pathologic events were examined at the various phases of the illness. About 80% of EAE rats presented anterior uveitis (AU), even after complete EAE recovery. We studied the phenotype and localization of immunocompetent cells, the major histocompatibility complex (MHC) class I and II antigen expression, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1) appearance. In control animals, there were many glial fibrillary acidic protein (GFAP)(+) cells and OX42(+) cells in the ciliary body, retina, optic nerve and chiasma. Except in retina, we observed constitutive MHC class I and II expression. During the EAE acute phase, there was up-regulation of MHC class II and GFAP antigens in iris, ciliary body, limbus, and optic pathways. MHC class I and ED2 antigens were expressed in meninges and in the prechiasmatic cisterna, by cells which could have a role in immune surveillance. MCP-1 mRNA was highly expressed in optic pathways during the acute phase and the protein was expressed by astrocytes, macrophages, and lymphocytes. During the relapsing phase, MCP-1 was weakly expressed to disappear almost completely during the final recovery phase. The expression of MHC class II on astrocytes was increased during the relapsing and final recovery phase in which the inflammatory lesions persisted. These findings suggest that ocular areas and optic pathways, mainly optic chiasma, are important targets in the relapsing EAE.     

法舒地尔通过诱导EAE小鼠Occludin表达抑制其炎性细胞浸润

目的观察法舒地尔(Fasudil)对实验性自身免疫性脑脊髓炎(EAE)小鼠模型的治疗效果并探讨其作用机制。方法采用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)免疫建立国际标准的小鼠EAE模型。将免疫后的42只雌性C57BL/6小鼠随机分为EAE组、Fasudil早期治疗组和Fasudil晚期治疗组。Fasudil早期治疗组即免疫后第3天按体质量40mg/(kg.d)腹腔注射Fasudil,1次/d,至免疫后30d;Fasudil晚期治疗组即免疫后首只小鼠出现症状即开始腹腔注射Fasudil,注射方法和剂量同Fasudil早期治疗组;与晚期治疗组给药同一时间,给予EAE组等量生理盐水作为对照。免疫后隔天观察各组小鼠临床评分和体质量变化。于免疫30d后处死动物,分离小鼠脊髓腰膨大和脑冠状位中部1/3处做冷冻切片,行HE染色和免疫荧光染色。同时提取小鼠脊髓蛋白检测Occludin蛋白的表达。结果 Fasudil可改善EAE的临床症状,抑制脑血管内皮细胞Rho激酶(ROCK)活性。与EAE组相比,Fasudil早期治疗组和晚期治疗组CD31阳性内皮细胞上p-MYPT1表达均减弱(P<0.01,P<0.05),起病时间均延长(P<0.01,P<0.05),脊髓Occludin蛋白表达均增加(P<0.05)。结论 Fasudil对EAE具有治疗的潜能,其机制可能为通过抑制血管内皮细胞ROCK活性,参与诱导内皮细胞紧密连接蛋白Occludin的表达,从而抑制炎性细胞的中枢浸润,最终阻止疾病的发生和发展。     

Animal models of multiple sclerosis: Focus on experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic, progressive disorder of the central nervous system (CNS) that affects more than two million people worldwide. Several animal models resemble MS pathology; the most employed are experimental autoimmune encephalomyelitis (EAE) and toxin‐ and/or virus‐induced demyelination. In this review we will summarize our knowledge on the utility of different animal models in MS research. Although animal models cannot replicate the complexity and heterogeneity of the MS pathology, they have proved to be useful for the development of several drugs approved for treatment of MS patients. This review focuses on EAE because it represents both clinical and pathological features of MS. During the past decades, EAE has been effective in illuminating various pathological processes that occur during MS, including inflammation, CNS penetration, demyelination, axonopathy, and neuron loss mediated by immune cells.     

Treatment of relapsing experimental autoimmune encephalomyelitis with T cell receptor peptides

Restricted T cell receptor (TCR) VB gene usage by T cells for recognition of antigens involved in the production of experimental autoimmune encephalomyelitis (EAE) offers the possibility of selective immunotherapy. We determined the preferential VB gene usage of lymph node-derived clones from SJL/J mice to recognize the encephalitogenic epitope PLP 139-151 and from PL/J mice to recognize the newly described encephalitogenic epitope PLP 43-64. In addition, the VB gene usage for recognition of PLP 139-151 by T cell lines derived from SJL/J spinal cords was analyzed. Lymph node-derived SJL/J lines and clones specific for PLP 139-151 expressed VB2, VB4, and VB17a preferentially, and PL/J lines and clones specific for PLP 43-64 expressed VB2 and VB8.2 preferentially. A VB4+ SJL/J clone and a VB8.2+ PL/J clone were encephalitogenic. Encephalitogenic SJL/J lines derived from spinal cord expressed VB2, VB10, VB16, and VB17a preferentially, with a predominance of VB2. Candidate TCR peptides were synthesized and tested from the VB gene families VB4, VB8.2, and VB17a, based on our data and previous data on BP-induced EAE in mice. Treatment of relapsing EAE (R-EAE) in SJL/J mice with VB4 and VB17a peptides reduced clinical and histological disease severity, and treatment of R-EAE in (PLxSJL)F1 mice with VB4 and VB8.2 peptides also reduced clinical and histological disease. The use of TCR peptide therapy may have applications for the treatment of human autoimmune diseases such as multiple sclerosis. © 1993 Wiley-Liss, Inc.     

Expression of the HGF receptor c‐met by macrophages in experimental autoimmune encephalomyelitis

Hepatocyte growth factor (HGF) is a pleiotropic cytokine able to evoke a wide array of cellular responses including proliferation, migration, and survival through activation of its receptor c‐met. Various types of leukocytes have been described to express c‐met suggesting that HGF/c‐met signaling may directly influence leukocyte responses in inflammation. We have investigated the HGF/c‐met pathway in experimental autoimmune encephalomyelitis (EAE), a common mouse model of multiple sclerosis (MS), in which macrophages play a dual role, contributing directly to CNS damage at disease onset but promoting recovery during remission by removing myelin debris. Here we show that during EAE both HGF and c‐met are expressed in the CNS and that c‐met is activated. We subsequently demonstrate that c‐met is primarily expressed in inflammatory lesions by macrophages and a small number of dendritic cells (DCs) and oligodendrocyte progenitor cells (OPCs) but not by microglia or T cells. Complementary in vitro experiments show that only LPS and TNFα, but not IL‐6, IL‐10, or IL‐13, are able to induce c‐met expression in macrophages. In addition, using TNF signaling deficient macrophages we demonstrate that LPS and TNFα induce c‐met through distinct pathways. Furthermore, TNFα‐ and LPS‐induced c‐met is functional because treatment of macrophages with recombinant HGF results in rapid phosphorylation of c‐met. Interestingly, HGF/c‐met signaling does not modulate cytokine expression, phagocytosis, or antigen presentation but promotes proliferation of activated macrophages. Taken together, our data indicate a pro‐inflammatory role for the HGF/c‐met pathway in EAE rather than a role in the initiation of repair mechanisms. © 2009 Wiley‐Liss, Inc.