Interleukin 6 production in the central nervous system during experimental autoimmune encephalomyelitis

Interleukin 6 (IL6) is one of the major inflammation-associated cytokines. Elevated serum or tissue levels of IL6 have been reported to occur in several human diseases, including infections of the central nervous system (CNS), but not in non-infectious CNS inflammation, e.g. multiple sclerosis. While studying experimental autoimmune encephalomyelitis (EAE) as an animal model for autoimmune inflammation of the CNS, we found increased IL6 levels in the CNS of mice suffering from a lethal form of the disease. IL6 levels in the spleens and sera were not significantly increased. These findings are indicative of local production of IL6 in the CNS during EAE, and represent the first demonstration of IL6 production in non-infectious CNS inflammatory disease.     

IVIG enters the central nervous system during treatment of experimental autoimmune encephalomyelitis and is localised to inflammatory lesions

Intravenous immunoglobulin (IVIG) treatment reduces the relapse rate in relapsing–remitting multiple sclerosis (MS) and may interfere with MS pathology through its various anti-inflammatory and immunomodulatory properties. It is presently unknown whether IVIG enters the central nervous system (CNS) in sufficient amounts to influence the local immune response within the brain and spinal cord, or if the treatment effects are entirely due to peripheral actions of IVIG. The purpose of the present study was to evaluate if IVIG radiolabeled with ^99mTc enters the CNS during treatment of experimental autoimmune encephalomyelitis (EAE) in the susceptible rat strain Dark Agouti. After in vivo administration of ^99mTc-IVIG we observed significantly increased accumulation in the brain and spinal cord from rats with EAE. Accumulation of ^99mTc-IVIG was not detectable in CNS tissue from control animals. In peripheral tissue samples minor increases in ^99mTc-IVIG organ binding were observed in the liver and kidney during EAE. Localisation of ^99mTc-IVIG in the brain tissue was visualised by autoradiography and revealed significant accumulation of IVIG only in areas also affected by perivascular inflammation and leakage of serum proteins. In conclusion, the results indicate that significant extravasation of IVIG to the CNS only occurs when blood–brain barrier function is compromised during EAE.     

The critical role of antigen-presentation-induced cytokine crosstalk in the central nervous system in multiple sclerosis and experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a debilitating disease of the central nervous system (CNS) that has been extensively studied using the animal model experimental autoimmune encephalomyelitis (EAE). It is believed that CD4(+) T lymphocytes play an important role in the pathogenesis of this disease by mediating the demyelination of neuronal axons via secretion of proinflammatory cytokines resulting in the clinical manifestations. Although a great deal of information has been gained in the last several decades about the cells involved in the inflammatory and disease mediating process, important questions have remained unanswered. It has long been held that initial neuroantigen presentation and T cell activation events occur in the immune periphery and then translocate to the CNS. However, an increasing body of evidence suggests that antigen (Ag) presentation might initiate within the CNS itself. Importantly, it has remained unresolved which antigen presenting cells (APCs) in the CNS are the first to acquire and present neuroantigens during EAE/MS to T cells, and what the conditions are under which this takes place, ie, whether this occurs in the healthy CNS or only during inflammatory conditions and what the related cytokine microenvironment is comprised of. In particular, the central role of interferon-γ as a primary mediator of CNS pathology during EAE has been challenged by the emergence of Th17 cells producing interleukin-17. This review describes our current understanding of potential APCs in the CNS and the contribution of these and other CNS-resident cells to disease pathology. Additionally, we discuss the question of where Ag presentation is initiated and under what conditions neuroantigens are made available to APCs with special emphasis on which cytokines may be important in this process.     

Dynamics and fate of USPIO in the central nervous system in experimental autoimmune encephalomyelitis

Signal loss observed in the brain by MRI following the administration of ultrasmall superparamagnetic particles of iron oxide (USPIO) has been correlated with immune cell activity in inflammatory areas during multiple sclerosis. Uptake of USPIO by circulating monocytes and their migration towards inflammatory areas have been considered as the most important mechanism for USPIO uptake by the brain parenchyma. However, the involvement of a damaged blood–brain barrier is also debated as a possible mechanism for cerebral USPIO uptake. Compared with these uptake‐associated issues, little is known about the clearance of USPIO from the brain. The acute uptake and chronic clearance of USPIO in the brain were therefore studied with MRI in an animal model of multiple sclerosis. Lewis Hannover rats with acute experimental autoimmune encephalomyelitis received a single intravenous injection of USPIO (300 µmol Fe/kg), and repetitive MRI of the brain and cervical lymph nodes, a possible drainage pathway, was performed. USPIO were detected in the brain within 1 h after injection independent of the severity of experimental autoimmune encephalomyelitis, and histological analysis revealed extracellular iron clusters colocalising with a leaky blood–brain barrier. Loss of signal was not present 72 h after USPIO injection, irrespective of the disease state. MR images of cervical lymph nodes showed USPIO accumulation at 24 h after administration, which stabilised at 72 h. Histological analyses revealed that USPIO accumulated in infiltrated macrophages in the medulla and subcapsular sinus. The current study demonstrates that USPIO enter the central nervous system directly after administration, pointing to the involvement of a damaged blood–brain barrier in the appearance of USPIO‐associated MR abnormalities. Furthermore, a possible role of the cervical lymph nodes as a drainage pathway of USPIO is suggested. These data shed new light on the use of USPIO in neuroinflammatory diseases, identifying USPIO as a marker for both cellular infiltration and blood–brain barrier damage. Copyright © 2010 John Wiley & Sons, Ltd.     

Transgenic expression of CCL2 in the central nervous system prevents experimental autoimmune encephalomyelitis

CC chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, a member of the CC chemokine family, is a chemoattractant for monocytes and T cells through interaction with its receptor CCR2. In the present study, we examined a T helper cell type 1 (Th1)-dependent disease, proteolipid protein-induced experimental autoimmune encephalomyelitis, in a transgenic mouse line that constitutively expressed low levels of CCL2 in the central nervous system (CNS) under control of the astrocyte-specific glial fibrillary acidic protein promoter. CCL2 transgenic mice developed significantly milder clinical disease than littermate controls. As determined by flow cytometry, mononuclear cell infiltrates in the CNS tissues of CCL2 transgenic and littermate-control mice contained equal numbers of CD4+ and CD8+ T cells, and the CCL2 transgenic mice showed an enhanced number of CNS-infiltrating monocytes. CNS antigen-specific T cells from CCL2 transgenic mice produced markedly less interferon-gamma. Overexpression of CCL2 in the CNS resulted in decreased interleukin-12 receptor expression by antigen-specific T cells. Collectively, these results indicate that sustained, tissue-specific expression of CCL2 in vivo down-regulates the Th1 autoimmune response, culminating in milder clinical disease.     

Adhesion-related molecules in the central nervous system. Upregulation correlates with inflammatory cell influx during relapsing experimental autoimmune encephalomyelitis.

The expression of a battery of adhesion-related molecules and cytokines was investigated by immunocytochemistry in the central nervous system (CNS) of SJL/J mice sensitized for experimental autoimmune encephalomyelitis (EAE). These molecules consisted of the ligands MECA-325, intercellular adhesion molecule-1, and major histocompatibility complex molecules I and II, plus the receptors lymphocyte function-associated antigen-1, CD8, and CD4. The cytokines comprised interferon-gamma and tumor necrosis factor-alpha. EAE was induced by the adoptive transfer of myelin basic protein-sensitized lymphocytes. MECA-325, a marker for murine high endothelial venules in lymph node tissue, was absent from normal CNS tissue, was expressed at low levels on venules 24 to 48 hours before the onset of clinical signs, rose to maximal levels during acute disease, decreased to preclinical levels during remissions, and rose again during relapses. Intercellular adhesion molecule-1, major histocompatibility antigen-I, and major histocompatibility antigen-II showed similar fluctuations around CNS vessels. The receptors lymphocyte function-associated antigen-1 and CD4 fluctuated in parallel with the above molecules, whereas CD8 remained at a similar low level. Interferon-gamma was present during the acute, remitting, and relapsing phases and was localized to inflammatory cells, whereas tumor necrosis factor occurred at low levels only. Thus, several molecules associated with lymphocyte traffic in lymphoid tissue are selectively expressed in a stage-specific manner within the target organ, the CNS, during EAE. This suggests that the CNS may act as an ancillary organ of the immune system, and that cellular traffic into the CNS during EAE is related to the fluctuating expression of several distinct adhesion-related molecules, frequently co-expressed on the same vessel. The findings may have relevance to the sequence of events in the developing CNS lesion of multiple sclerosis.     

IFN-gamma signaling in the central nervous system controls the course of experimental autoimmune encephalomyelitis independently of the localization and composition of inflammatory foci

Background  

Murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, presents typically as ascending paralysis. However, in mice in which interferon-gamma (IFNγ) signaling is disrupted by genetic deletion, limb paralysis is accompanied by atypical deficits, including head tilt, postural imbalance, and circling, consistent with cerebellar/vestibular dysfunction. This was previously attributed to intense cerebellar and brainstem infiltration by peripheral immune cells and formation of neutrophil-rich foci within the CNS. However, the exact mechanism by which IFNγ signaling prohibits the development of vestibular deficits, and whether the distribution and composition of inflammatory foci within the CNS affects the course of atypical EAE remains elusive.     

Pre‐existing central nervous system lesions negate cytokine requirements for regional experimental autoimmune encephalomyelitis development

In region‐specific forms of experimental autoimmune encephalomyelitis (EAE), lesion initiation is regulated by T‐cell‐produced interferon‐γ (IFN‐γ) resulting in spinal cord disease in the presence of IFN‐γ and cerebellar disease in the absence of IFN‐γ. Although this role for IFN‐γ in regional disease initiation is well defined, little is known about the consequences of previous tissue inflammation on subsequent regional disease, information vital to the development of therapeutics in established disease states. This study addressed the hypothesis that previous establishment of regional EAE would determine subsequent tissue localization of new T‐cell invasion and associated symptoms regardless of the presence or absence of IFN‐γ production. Serial transfer of optimal or suboptimal doses of encephalitogenic IFN‐γ‐sufficient or ‐deficient T‐cell lines was used to examine the development of new clinical responses associated with the spinal cord and cerebellum at various times after EAE initiation. Previous inflammation within either cerebellum or spinal cord allowed subsequent T‐cell driven inflammation within that tissue regardless of IFN‐γ presence. Further, T‐cell IFN‐γ production after initial lesion formation exacerbated disease within the cerebellum, suggesting that IFN‐γ plays different roles at different stages of cerebellar disease. For the spinal cord, IFN‐γ‐deficient cells (that are ordinarily cerebellum disease initiators) were capable of driving new spinal‐cord‐associated clinical symptoms more than 60 days after the initial acute EAE resolution. These data suggest that previous inflammation modulates the molecular requirements for new neuroinflammation development.     

Anti-TWEAK monoclonal antibodies reduce immune cell infiltration in the central nervous system and severity of experimental autoimmune encephalomyelitis

TWEAK is a member of the TNF family, constitutively expressed in the central nervous system (CNS), with pro-inflammatory, proliferative or apoptotic effects depending upon cell types. Its receptor, Fn14, is expressed in CNS by endothelial cells, reactive astrocytes and neurons. We showed that TWEAK and Fn14 mRNA expression increased in spinal cord during experimental autoimmune encephalomyelitis (EAE). We investigated the role of TWEAK during EAE using neutralizing anti-TWEAK antibody in myelin oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice. We observed a reduction of disease severity and leukocyte infiltration when mice were treated after the priming phase.     

Functional interleukin-17 receptor A is expressed in central nervous system glia and upregulated in experimental autoimmune encephalomyelitis

Background  

Interleukin-17A (IL-17A) is the founding member of a novel family of inflammatory cytokines that plays a critical role in the pathogenesis of many autoimmune diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). IL-17A signals through its receptor, IL-17RA, which is expressed in many peripheral tissues; however, expression of IL-17RA in the central nervous system (CNS) and its role in CNS inflammation are not well understood.     

gammadelta T cells express activation markers in the central nervous system of mice with chronic-relapsing experimental autoimmune encephalomyelitis.

In this study, we assessed the expression of activation markers on gammadelta T cells in central nervous system (CNS) lesions of SJL mice adoptively sensitized to develop experimental autoimmune encephalomyelitis (EAE) using myelin basic protein-reactive T cells. Although disease expression is known to be dependent upon T cells that express the alphabeta T cell receptor (TCR), a role for gammadelta T cells has been implicated in some studies but not in others. Using three-color flow cytometric analysis of both total and gammadelta T cells in spleen and CNS, the data showed that expression of CD69 (early activation marker), CD62L (lymphocyte homing receptor), CD25 (IL-2Ralpha), CD122 (IL-2Rbeta) and CD95/CD95L (Fas/FasL), fluctuated on gammadelta T cells in EAE lesions in a disease-related fashion. Furthermore, the pattern of expression for these markers on gammadelta T cells was distinct from that found on the total lymphocyte population. Cytokine analysis of gammadelta T cells in the CNS demonstrated a bias towards a Th1-like cytokine profile. From these data, we conclude that gammadelta T cells in EAE lesions display an activated phenotype and form a dynamic component of the total lymphocyte population in the CNS, supporting a contributory role for these cells.     

Microglia are more susceptible than macrophages to apoptosis in the central nervous system in experimental autoimmune encephalomyelitis through a mechanism not involving Fas (CD95)

Morphological studies have shown that macrophages and microglia undergo apoptosis in the central nervous system (CNS) in acute experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. To assess the relative levels of macrophage and microglial apoptosis, and the molecular mechanisms involved in this process, we used three-colour flow cytometry to identify CD45lowCD11b/c+ microglial cells and CD45highCD11b/c+ macrophages in the inflammatory cells isolated from the spinal cords of Lewis rats 13 days after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Simultaneously, we analyzed the DNA content of these cell populations to assess the proportions of cells undergoing apoptosis and in different stages of the cell cycle or examined their expression of three apoptosis- regulating proteins, i.e. Fas (CD95), Fas ligand (FasL) and Bcl-2. Microglia were highly vulnerable to apoptosis and were over-represented in the apoptotic population. Macrophages were less susceptible to apoptosis than microglia and underwent mitosis more frequently than microglia. The different susceptibilities of microglia and macrophages to apoptosis did not appear to be due to variations in Fas, FasL or Bcl- 2 expression, as the proportions of microglia and macrophages expressing these proteins were similar, and were relatively high. Furthermore, in contrast to T cell apoptosis, apoptosis of microglia/macrophages did not occur more frequently in cells expressing Fas or FasL, or less frequently in cells expressing Bcl-2. These results indicate that the apoptosis of microglia and CNS macrophages in EAE is not mediated through the Fas pathway, and that Bcl-2 expression does not protect them from apoptosis. Expression of FasL by macrophages and microglia may contribute to the pathogenesis and immunoregulation of EAE through interactions with Fas+ oligodendrocytes and Fas+ T cells. The high level of microglial apoptosis in EAE indicates that microglial apoptosis may be an important homeostatic mechanism for controlling the number of microglia in the CNS following microglial activation and proliferation.      

FTY720 rescue therapy in the dark agouti rat model of experimental autoimmune encephalomyelitis: expression of central nervous system genes and reversal of blood-brain-barrier damage

FTY720 (fingolimod) is an oral sphingosine-1 phosphate (S1P) receptor modulator in phase III development for the treatment of multiple sclerosis. To further investigate its mode of action, we analyzed gene expression in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE). FTY720 downregulated inflammatory genes in addition to vascular adhesion molecules. It decreased the matrix metalloproteinase gene MMP-9 and increased its counterregulator—tissue inhibitor of metalloproteinase, TIMP-1—resulting in a proteolytic balance that favors preservation of blood-brain-barrier (BBB) integrity. Furthermore, FTY720 reduced S1P lyase that increases the S1P concentration in the brain, in line with a marked reversal of neurological deficits and raising the possibility for enhanced triggering of S1P receptors on resident brain cells. This is accompanied by an increase in S1P₁ and S1P₅ in contrast with the attenuation of S1P₃ and S1P₄. Late-stage rescue therapy with FTY720, even up to 1 month after EAE onset, reversed BBB leakiness and reduced demyelination, along with normalization of neurologic function. Our results indicate rapid blockade of ongoing disease processes by FTY720, and structural restoration of the CNS parenchyma, which is likely caused by the inhibition of autoimmune T cell infiltration and direct modulation of microvascular and/or glial cells.     

IL-5-deficient mice are susceptible to experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model commonly used to investigate mechanisms involved in the activation of self-reactive T cells. Whereas auto-reactive T(h)1 cells are believed to be involved in the generation of EAE, T(h)2 cells can induce EAE in immunocompromised hosts. Since the T(h)2 cytokine IL-5 can influence the nature and severity of disease, we investigated the role of IL-5 in the EAE model. Wild-type C57BL/6J and IL-5(-/-) mice were immunized with myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide and the development of EAE observed. Our results show that IL-5(-/-) mice developed EAE with a similar day of onset and comparable severity to wild-type mice. Primed T cells isolated from IL-5(-/-) mice proliferated equally to wild-type cells in response to antigen challenge with MOG(35-55). Antigen-specific T cells from IL-5(-/-) mice produced IFN-gamma and tumor necrosis factor-alpha, but no IL-4 or IL-10, indicating that a predominant T(h)1 environment was induced following immunization. No differences in the types of cells infiltrating into the central nervous system were observed between IL-5(-/-) and wild-type mice. Our results suggest that IL-5 is not directly involved in the initiation or effector phase of MOG(35-55)-induced EAE in immunocompetent C57BL/6J mice.     

Tumor necrosis factor blockade in actively induced experimental autoimmune encephalomyelitis prevents clinical disease despite activated T cell infiltration to the central nervous system

Recently, we demonstrated that experimental autoimmune encephalomyelitis (EAE) in the rat, passively transferred using myelin basic protein (MBP)-reactive encephalitogenic CD4⁺ T cells, was preventable by administration of a p55-tumor necrosis factor-IgG fusion protein (TNFR-IgG). This was despite quantitatively and qualitatively normal movement of these MBP-specific T cells to the central nervous system (CNS). To extend these findings, the effect of TNFR-IgG on EAE actively induced by injection of MBP in complete Freund's adjuvant was examined. This form of EAE in the rat typically involves an acute, self-limiting neurological deficit, substantial CNS inflammation, but minimal demyelination. Here we show that administration of TNFR-IgG prior to onset of disease signs completely prevented the neurological deficit or markedly reduced its severity. This blockade of clinical disease was dissociated from weight loss which occurred at the same tempo and magnitude as in control rats exhibiting neurological signs of disease such as paralysis. The timing of TNF blockade was critical as established clinical disease was relatively refractory to TNFR-IgG treatment. Activated CD4⁺ T cells expressing normal or elevated levels of VLA4, major histocompatibility complex class II, MRC OX40 and CD25 were isolated from or immunohistochemically localized in the CNS of clinically healthy rats treated before disease onset. There was a reduction of the amount of other inflammatory leukocytes in the CNS of these treated animals but, more importantly, the activation state of inflammatory leukocytes, as well as that of microglia isolated from treated animals, was reduced. Thus, TNFR-IgG, when administered before disease onset, appears to act by inhibiting an effector function of activated T cells and possibly other inflammatory leukocytes necessary to bring about the neurological deficit. However, while TNF is a critically important cytokine for the early events leading to initiation of EAE, it is not a necessary factor in the acute neurological deficit characteristic of this form of EAE, once disease onset has occurred.     

Differential expression of neurotrophic factors and inflammatory cytokines by myelin basic protein-specific and other recruited T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis

Recent evidence suggests that autoimmune reactions in the central nervous system (CNS) not only have detrimental consequences but can also be neuroprotective, and that this effect is mediated by the expression of neuronal growth factors by infiltrating leucocytes. Here we dissect these two phenomena in guinea pig myelin basic protein peptide (gpMBP 63-88)-induced experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. Real-time TaqMan polymerase chain reaction (PCR) was used to measure mRNA for the nerve growth factors, brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3. As reference, the well-known proinflammatory mediator molecules interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha were quantified. In whole lumbar cord tissue, both the nerve growth factors and the proinflammatory cytokines, IFN-gamma and TNF-alpha, displayed similar expression patterns, peaking at the height of the disease. Among the infiltrating inflammatory cells isolated and sorted from the CNS, alphabeta+/T-cell receptor (TCR)BV8S2+, but not alphabeta+/TCRBV8S2-, recognized the encephalitogenic MBP peptide. Interestingly, these two populations displayed contrasting expression patterns of nerve growth factors and proinflammatory cytokines with higher inflammatory cytokine mRNA levels in alphabeta+/TCRBV8S2+ cells at all time intervals, whereas the levels of BDNF and NT3 were higher in alphabeta+/TCRBV8S2- cells. We conclude that a potentially important neuroprotective facet of CNS inflammation dominantly prevails within other non-MBP peptide-specific lymphoid cells and that there are independent regulatory mechanisms for neurotrophin and inflammatory cytokine expression during EAE.