Control of glial immune function by neurons

The immune status of the central nervous system (CNS) is strictly regulated. In the healthy brain, immune responses are kept to a minimum. In contrast, in a variety of inflammatory and neurodegenerative diseases, including multiple sclerosis, infections, trauma, stroke, neoplasia, and Alzheimer's disease, glial cells such as microglia gain antigen-presenting capacity through the expression of major histocompatibility complex (MHC) molecules. Further, proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF), interleukin-1beta (IL-1beta), and interferon-gamma (IFN-gamma), as well as chemokines, are synthesized by resident brain cells and T lymphocytes invade the affected brain tissue. The proinflammatory cytokines stimulate microglial MHC expression in the lesioned CNS areas only. However, the induction of brain immunity is strongly counterregulated in intact CNS areas. For instance, recent work demonstrated that microglia are kept in a quiescent state in the intact CNS by local interactions between the microglia receptor CD200 and its ligand, which is expressed on neurons. Work done in our laboratory showed that neurons suppressed MHC expression in surrounding glial cells, in particular microglia and astrocytes. This control of MHC expression by neurons was dependent on their electrical activity. In brain tissue with intact neurons, the MHC class II inducibility of microglia and astrocytes by the proinflammatory cytokine IFN-gamma was reduced. Paralysis of neuronal electric activity by neurotoxins restored the induction of MHC molecules on microglia and astrocytes. Loss of neurons or their physiological activity would render the impaired CNS areas recognizable by invading T lymphocytes. Thus, immunity in the CNS is inhibited by the local microenvironment, in particular by physiologically active neurons, to prevent unwanted immune mediated damage of neurons.     

Functional expression of CCL6 by rat microglia: a possible role of CCL6 in cell-cell communication

There is growing evidence that chemokines play important roles in the immune surveillance of central nervous system (CNS). In the CNS, microglia are primary immune effector cells and secrete various chemokines in response to their microenvironment. Using the RT-PCR procedure and indirect immunofluorescence analysis, we found that CCL6 (known as C10/MRP-1 in mouse) was expressed in rat primary microglia without any stimulation, but not in primary astrocytes, although both cell types expressed CCR1 mRNA, which is a receptor for CCL6. Furthermore, immunohistochemical analysis demonstrated that microglia produced CCL6 protein in a normal brain, suggesting that microglia may be the primary source of CCL6 in a normal brain. Recombinant rat CCL6 mediated the migration of microglia and astrocytes in vitro. The CCL6-mediated cell migration was blocked by treating the cells with LY294002, a PI3-kinase inhibitor and Western blot analysis showed that the phosphorylation of Akt could be induced by treating microglia with a recombinant CCL6, suggesting that CCL6 functions by activating the PI3-kinase/Akt pathway. A proinflammatory cytokine, interferon-gamma enhanced the expression of both CCL6 mRNA and protein in microglia, while other proinflammatory cytokines, interleukin-6 and tumor necrosis factor-alpha and an anti-inflammatory cytokine, transforming growth factor-beta exerted no effect on CCL6 expression in microglia. These findings suggest that CCL6 may be a mediator released by microglia for cell-cell communication under physiological as well as pathological conditions of CNS.     

Th1 polarization of CD4+ T cells by Toll-like receptor 3-activated human microglia

ABSTRACT: Toll-like receptors (TLRs) are expressed by human microglia and translate environmental cues into distinct activation programs. We addressed the impact of TLR ligation on the capacity of human microglia to activate and polarize CD4 T cell responses. As microglia exist under distinct states of activation, we examined both ramified and ameboid microglia isolated from adult and fetal CNS, respectively. In vitro, ligation of TLR3 significantly increased major histocompatibility complex and costimulatory molecule expression on adult microglia and induced high levels of interferon-alpha, interleukin-12p40, and interleukin-23. TLR4 and, in particular, TLR2 had a more limited capacity to induce such responses. Coculturing allogeneic CD4 T cells with microglia preactivated with TLR3 did not increase T cell proliferation above basal levels but consistently led to elevated levels of interferon-gamma secretion and Th1 polarization. Fetal microglial TLR3 responses were comparable; in contrast, TLR2 and TLR4 decreased major histocompatibility complex class II expression on fetal cells and reduced CD4 T cell proliferation to levels below those found in untreated cocultures. All 3 TLRs induced comparable interleukin-6 secretion by microglia. Our findings illustrate how activation of human microglia via TLRs, particularly TLR3, can change the profile of local CNS immune responses by translating Th1 polarizing signals to CD4 T cells.     

Phagocytosis of apoptotic inflammatory cells by microglia and modulation by different cytokines: mechanism for removal of apoptotic cells in the inflamed nervous system

Apoptosis of autoaggressive T cells in the central nervous system (CNS) is an effective, nonphlogistic mechanism for the termination of autoimmune inflammation in experimental autoimmune encephalomyelitis (EAE). The clearance of apoptotic leukocytes by tissue-specific phagocytes is a critical event in the resolution of the inflammatory attack. To investigate the role of microglia in the removal of apoptotic cells and potential regulatory mechanisms of microglial phagocytosis, an in vitro phagocytosis assay was established, using Lewis rat microglia. Microglia exhibited a high capacity for the uptake of apoptotic autologous thymocytes, as well as apoptotic encephalitogenic myelin basic protein (MBP)-specific T cells, in contrast to nonapoptotic target cells. Pretreatment of microglia with interferon-gamma (IFN-gamma) raised the proportion of microglia capable of phagocytosing apoptotic cells to 75% above the untreated controls. The increased phagocytic activity was selective for apoptotic target cells and was not dependent on phosphatidylserine-mediated recognition mechanisms. In contrast, preincubation of microglia with interleukin-4 (IL-4) inhibited the uptake of apoptotic cells, whereas tumor-necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) did not alter phagocytosis. Phagocytic clearance of apoptotic inflammatory cells by microglia may be an important mechanism for the termination of autoimmune inflammation in the CNS. Augmentation of microglial phagocytosis by the Th-1-type cytokine IFN-gamma suggests a feedback mechanism for the accelerated clearance of the inflammatory infiltrate in the CNS.     

Impact of IVIg on the interaction between activated T cells and microglia

When human microglia are co-cultured with activated human T lymphocytes, several cytokines become up-regulated in significant quantities. This condition can also occur at sites of inflammation in autoimmune inflammatory diseases of the central nervous system (CNS), including multiple sclerosis (MS), where T cells infiltrate the brain tissue and come in proximity to microglia. Therefore, T cell-microglia interaction is a potential avenue of drug therapy to decrease neuroinflammation. An immunomodulator used in autoimmune disorders is intravenous immunoglobulins (IVIg). The mechanisms of IVIg activity in diseases such as MS remain unclear. Here, we report that the application of IVIg to activated T cells leads to their decreased ability to engage microglia. As a result of IVIg treatment of T cells, there were reduced levels of tumor necrosis factor-alpha a and interleukin-10 in T cell-microglia co-culture. Our results add to the understanding of how IVIg may affect inflammation of the CNS.     

Effects of methylmercury on the secretion of pro-inflammatory cytokines from primary microglial cells and astrocytes

Glial cells, including oligodendrocytes, astrocytes and microglia are important to proper central nervous system (CNS) function. Deregulation or changes to CNS populations of astrocytes and microglia in particular are expected to play a role in many neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). Previous studies have reported methylmercury (MeHg) induced changes in glial cell function; however, the effects of MeHg on these cells remains poorly understood. This study aims to examine the effect of MeHg on the secretion of pro-inflammatory cytokines from microglia and astrocytes. The impact of the microglia/astrocyte ratio on cytokine secretion was also examined. Microglia and astrocytes were cultured from the brains of neo-natal BALB/C mice and dosed with MeHg (0-1 μM) and stimulated with PAM(3)CSK(4) (PAM(3)), a toll-like receptor (TLR) ligand. After this, the secretion of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) was measured by ELISA. MeHg reduced the secretion of IL-6 in a dose dependant manner but did not effect the secretion of TNF-α. No change in IL-1β was observed in any treatments, indicating that PAM(3) cannot induce the secretion of this cytokine from glial cells. Additionally, the ratio of microglia/astrocyte had an effect on the secretion of IL-6 but not TNF-α. These results indicate that MeHg can modify the response of glial cells and the interactions with astrocytes can affect the response of the microglia cells in culture. These results are significant in understanding the potential relationship with MeHg and neurodegenerative diseases and for the interpretation of results of future in vitro studies using monoculture.     

CD1 expression is differentially regulated by microglia, macrophages and T cells in the central nervous system upon inflammation and demyelination

Expression of CD1 by microglia, macrophages and T cells was investigated ex vivo. In the healthy central nervous system (CNS), resident microglia, macrophages and T cells express levels of CD1 significantly lower than that expressed by splenic macrophages and T cells. During experimental autoimmune encephalomyelitis (EAE), CD1 expression by microglia and the number of CD1+ microglia increase. Macrophages and T cells strongly upregulate CD1 expression in the CNS, but not in the spleen. Whereas the function of CD1 expressed by T cells remains unclear, the expression by microglia and macrophages provides the CNS with a (glyco)lipidic-presenting molecule in an inflammatory and demyelinating environment.     

Recent advances in the immunopathology of experimental autoimmune encephalomyelitis: With special reference to cytokine production in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease that can be induced by immunization with encephalitogenic antigens such as myelin basic protein. Recent in vitro studies have demonstrated that cytokines play an important role in immune reactions in the central nervous system (CNS), suggesting that cytokines released by infiltrating cells and glial cells may contribute to the pathogenesis of EAE. In this review, we focus on the interactions between infiltrating cells and brain cells during the inflammatory process in EAE and discuss the roles of cytokines in the CNS. After immunization with proper myelin antigens, encephalitogenic T cells increase in number and infiltrate the CNS parenchyma via the subarachnoid space or the blood vessels. Once inflammatory cells infiltrate the CNS, microglia and astrocytes are activated, and some of these cells proliferate in response to cytokines released by infiltrating cells. Following this, activated microglia present antigens to induce T cell proliferation and cytokine production. In contrast, astrocytes induce T cell unresponsiveness, probably due to a lack of costimulatory signals. Furthermore, infiltrating T cells are the main producers of Th1 cytokines and are involved in T cell-brain cell interactions. This cascade of events indicates that immune reactions take place in the CNS, although the CNS has previously been considered to be an immunologically privileged site. Based on these findings, we also discuss the feasibility of using various cytokines to stimulate the immunomodulation of brain inflammation as a treatment for autoimmune demyelinating diseases.     

Interferon beta-1b increases interleukin-10 in a model of T cell-microglia interaction: relevance to MS

BACKGROUND: The modes of action of interferon beta (IFN-beta) in MS remain unclear, but enhanced levels of the anti-inflammatory cytokine interleukin-10 (IL-10) in the CSF of patients with MS may be a marker of its prognostic efficacy. OBJECTIVE: To examine potential mechanisms by which IL-10 may be increased by IFN-ss in the milieu of the CNS. METHODS: A model of T cell interaction with microglia in vitro was used. Production of cytokines was monitored by measuring the levels of various cytokine proteins, using ELISA. RESULTS: Pretreatment of T cells with IFN-beta potentiates the production of IL-10 when they interact with adult human microglia, human fetal microglia, or U937 cells treated with phorbol-12-myristate-13-acetate (PMA) and IFN-gamma. The enhancing effect of IFN-beta on IL-10 requires cell-cell contact, but does not seem to depend on pathways implicated in microglia-T cell interactions, involving CD40, CD23, and B7. In contrast to IL-10, IFN-beta inhibits the production of other cytokines, including tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-4, IL-12, and IL-13. CONCLUSIONS: The increase of IL-10 in microglia-T cell interaction by IFN-beta together with a decrease of other cytokines may lead to a noninflammatory milieu in the CNS. This mechanism could contribute to the efficacy of IFN-beta in MS.     

C1q, the recognition subcomponent of the classical pathway of complement, drives microglial activation

Microglia, central nervous system (CNS) resident phagocytic cells, persistently police the integrity of CNS tissue and respond to any kind of damage or pathophysiological changes. These cells sense and rapidly respond to danger and inflammatory signals by changing their cell morphology; by release of cytokines, chemokines, or nitric oxide; and by changing their MHC expression profile. We have shown previously that microglial biosynthesis of the complement subcomponent C1q may serve as a reliable marker of microglial activation ranging from undetectable levels of C1q biosynthesis in resting microglia to abundant C1q expression in activated, nonramified microglia. In this study, we demonstrate that cultured microglial cells respond to extrinsic C1q with a marked intracellular Ca(2+) increase. A shift toward proinflammatory microglial activation is indicated by the release of interleukin-6, tumor necrosis factor-alpha, and nitric oxide and the oxidative burst in rat primary microglial cells, an activation and differentiation process similar to the proinflammatory response of microglia to exposure to lipopolysaccharide. Our findings indicate 1) that extrinsic plasma C1q is involved in the initiation of microglial activation in the course of CNS diseases with blood-brain barrier impairment and 2) that C1q synthesized and released by activated microglia is likely to contribute in an autocrine/paracrine way to maintain and balance microglial activation in the diseased CNS tissue.     

Activation of microglia cells is dispensable for the induction of rat retroviral spongiform encephalopathy

In the course of retroviral CNS infections, microglia activation has been observed frequently, and it has been hypothesized that activated microglia produce and secrete neurotoxic products like proinflammatory cytokines, by this promoting brain damage. We challenged this hypothesis in a rat model for neurodegeneration. In a kinetic study, we found that microglia cells of rats neonatally inoculated with neurovirulent murine leukemia virus (MuLV) NT40 became infected in vivo to maximal levels within 9-13 days postinoculation (d.p.i.). Beginning from 13 d.p.i., degenerative alterations, i.e., vacuolization of neurons and neuropil were found in cerebellar and other brain-stem nuclei. Elevated numbers of activated microglia cells--as revealed by immunohistochemical staining with monoclonal antibody ED1--were first detected at 19 d.p.i. and were always locally associated with degenerated areas but not with nonaltered, yet infected, brain regions. Both neuropathological changes and activated microglia cells increased in intensity and numbers, respectively, with ongoing infection but did not spread to other than initially affected brain regions. By ribonuclease protection assays, we were unable to detect differences in the expression levels of tumor-necrosis-factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) in microglia cells nor in total brains from infected versus uninfected rats. Our results suggest that the activation of microglia in the course of MuLV neurodegeneration is rather a reaction to, and not the cause of, neuronal damage. Furthermore, overt expression of the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 within the CNS is not required for the induction of retroviral associated neurodegeneration in rats.     

Production of interleukin-12 and expression of its receptors by murine microglia

Production of interleukin-12 (IL-12) by cultured murine microglia and astrocytes was examined, by means of ELISA to detect heterodimeric p70 and RT-PCR to analyze the expression of mRNA encoding p35 and p40. Microglia, but not astrocytes, produced IL-12 p70 in response to lipopolysaccharide and interferon-γ. The microglial cell line, Ra2, produced only p40, but not p35, upon above stimulation. Thus, it is possible that some population of microglia induce helper 1 type T cell response via producing IL-12 in the CNS. Microglia were induced to express mRNA encoding IL-12 receptors which were exclusively expressed in activated T and NK cells.     

Cytokine production in T lymphocyte-microglia interaction is attenuated by glatiramer acetate: a mechanism for therapeutic efficacy in multiple sclerosis

The efficacy of glatiramer acetate in multiple sclerosis (MS) is thought to involve the production of Th2 regulatory lymphocytes that secrete anti-inflammatory cytokines; however, other mechanisms cannot be excluded Given that activated T lymphocytes infiltrate into the CNS and become in dose proximity to microglia, we evaluated whether glatiramer acetate affects the potential interaction between T cells and microglia. We report that the co-culture of activated T lymphocytes with microglia led to the induction of several cytokines, and that these were reduced by glatiramer acetate treatment Morphological transformation of bipolar/ramified microglia into an activated ameboid form was attenuated by glatiramer acetate. These results reveal a novel mechanism for glatiramer acetate: the impairment of activated T cells to effectively interact with microglia to produce cytokines. The net result of a non-inflammatory milieu within the CNS, in spite of T cell infiltration, may help account for the amelioration of disease activity in MS patients on glatiramer acetate therapy.     

Glia-T cell dialogue

Interactions of CD4(+) T helper (Th) cells with microglia and astrocytes are likely to play an important role in regulating immune responses as well as tissue damage and repair during infectious and autoimmune central nervous system (CNS) diseases. T cells secreting Th1-type cytokines provide inducing signals for microglia to mature into functional antigen presenting cells (APC). The ability of microglia to act as efficient APC for the restimulation of Th1 cells suggests a role for these cells in the local amplification of pro-inflammatory immune responses. Conversely, the Th2-inducing capacity of microglia and astrocytes together with their ability to produce anti-inflammatory mediators could play a role in providing counter-regulatory signals limiting CNS inflammation. In this article, we review recent studies addressing the functional significance of T cell-CNS glia interactions and present new data on the expression of cyclooxygenase-2, the inducible enzyme involved in prostanoid biosynthesis, in microglia and astrocytes during the course of experimental allergic encephalomyelitis.     

β-Lapachone ameliorization of experimental autoimmune encephalomyelitis

β-Lapachone is a naturally occurring quinine, originally isolated from the bark of the lapacho tree (Tabebuia avellanedae) which is currently being evaluated in clinical trials for the treatment of cancer. In addition, recent investigations suggest its potential application for treatment of inflammatory diseases. Multiple sclerosis (MS) is an autoimmune disorder characterized by CNS inflammation and demyelination. Reactive T cells including IL-17 and IFN-γ-secreting T cells are believed to initiate MS and the associated animal model system experimental autoimmune encephalomyelitis (EAE). IL-12 family cytokines secreted by peripheral dendritic cells (DCs) and CNS microglia are capable of modulating T-cell phenotypes. The present studies demonstrated that β-lapachone selectively inhibited the expression of IL-12 family cytokines including IL-12 and IL-23 by DCs and microglia, and reduced IL-17 production by CD4⁺ T-cells indirectly through suppressing IL-23 expression by microglia. Importantly, our studies also demonstrated that β-lapachone ameliorated the development on EAE. β-Lapachone suppression of EAE was associated with decreased expression of mRNAs encoding IL-12 family cytokines, IL-23R and IL-17RA, and molecules important in Toll-like receptor signaling. Collectively, these studies suggest mechanisms by which β-lapachone suppresses EAE and suggest that β-lapachone may be effective in the treatment of inflammatory diseases such as MS.     

T cells contribute to lysophosphatidylcholine-induced macrophage activation and demyelination in the CNS

We have previously shown that intraspinal microinjection of lysophosphatidylcholine (LPC), a potent demyelinating agent, results in a rapid but brief influx of T cells (between 6 and 12 h). This is accompanied by a robust activation of macrophages/microglia that leads to demyelination by 48 h. In the present study, we examined whether this brief influx of T cells contributes to the activation of macrophages/microglia and demyelination by injecting LPC into the dorsal column white matter of athymic Nude mice that lack T cells. We show that there is a significant reduction in macrophage/microglial activation and myelin clearance after LPC injection in Nude mice as compared with wildtype controls. We also show that there is no difference in the recruitment of hematogenous macrophages into the spinal cord after LPC injection in the two mouse strains. Of the T cell cytokines assessed, there was a marked reduction in the mRNA expression of interleukin-2 (IL-2) in Nude mice compared with wildtype animals. Neutralizing IL-2 with function-blocking antibodies in wildtype animals resulted in a significant decrease in the number of phagocytic macrophages/microglia and a reduction in demyelination induced by LPC. While there may be other defects in Nude mice that might contribute to the effects shown here, these data suggest that the brief influx of T cells in this model of chemically-induced demyelination could play a role in macrophage/microglial activation and demyelination. These results may also have implications for remyelination in this and other types of CNS damage.     

Pathogenesis of chronic progressive myelopathy associated with human T-cell lymphotropic virus type I

Human T-cell lymphotropic virus type I (HTLV-I) induces a chronic demyelinating disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). While only 0.25% of HTLV-I-infected individuals develop HAM/TSP, the mechanisms responsible for the progression of an HTLV-I carrier state to clinical disease are not clear. In particular, no specific sequence differences have been found between HTLV-I recovered from HAM patients and HTLV-I-infected carriers. Since CD4 T cells are the major reservoir of the virus, at least three hypotheses implicating CD4 T cells directly or indirectly have been proposed: 1) The cytotoxic hypothesis predicts that activated and HTLV-I-infected CD4 T cells migrate to the CNS and infect resident cells. Cytotoxic CD8 T cells may then recognize viral antigens on HTLV-I-infected CNS cells causing a cellularly mediated cytotoxic demyelination. 2) The autoimmune hypothesis predicts that either (a) virally reactive T cells cross-react with a CNS antigen, or (b) random infection of CD4 T cells eventually results in the infection of CNS-autoreactive CD4 T cells that, by virtue of the productive HTLV-I infection, become activated, expand and migrate to the CNS, where they encounter their antigen. This results in a specific immune response and demyelination, as is known to occur in experimental autoimmune encephalomyelitis. 3) The bystander damage hypothesis does not implicate a specific response against CNS cells. Instead this hypothesis suggests that the presence of IFN-γ-secreting HTLV-I-infected CD4 T cells and their recognition by virally specific CD8 T cells in the CNS induce microglia to secrete cytokines, such as TNF-α, which may be toxic for the myelin.