Experimental Encephalomyelitis Research Articles

Histamine H3 Receptor-Mediated Signaling Protects Mice from Cerebral Malaria

BackgroundHistamine is a biogenic amine that has been shown to contribute to several pathological conditions, such as allergic conditions, experimental encephalomyelitis, and malaria. In humans, as well as in murine models of malaria, increased plasma levels of histamine are associated with severity of infection. We reported recently that histamine plays a critical role in the pathogenesis of experimental cerebral malaria (CM) in mice infected with Plasmodium berghei ANKA. Histamine exerts its biological effects through four different receptors designated H1R, H2R, H3R, and H4R.Principal FindingsIn the present work, we explored the role of histamine signaling via the histamine H3 receptor (H3R) in the pathogenesis of murine CM. We observed that the lack of H3R expression (H3R−/− mice) accelerates the onset of CM and this was correlated with enhanced brain pathology and earlier and more pronounced loss of blood brain barrier integrity than in wild type mice. Additionally tele-methylhistamine, the major histamine metabolite in the brain, that was initially present at a higher level in the brain of H3R−/− mice was depleted more quickly post-infection in H3R−/− mice as compared to wild-type counterparts.ConclusionsOur data suggest that histamine regulation through the H3R in the brain suppresses the development of CM. Thus modulating histamine signaling in the central nervous system, in combination with standard therapies, may represent a novel strategy to reduce the risk of progression to cerebral malaria.

Kinetics of myelin antigen presentation in the CNS during EAE (137.8)

Abstract Despite the plethora of information on immune mechanisms derived from studies of autoimmune experimental encephalomyelitis (EAE), the animal model of multiple sclerosis, little information is available in terms of the kinetics of presentation of myelin antigens by APCs in the CNS. The purpose of this study was to show at which time points and by which APCs myelin antigen (MBP, MOG, PLP) was acquired and presented after induction of EAE. To address this issue, mice were immunized with MOG 35-55 peptide and injected with pertussis toxin on day 0 and 2 and followed for disease. Representative mice were sacrificed every day over forty days and brain tissue was recovered for immunofluorescence analysis. Immunofluorescence and confocal microscopy was used to determine expression of APC cell surface markers and myelin antigens. The results show that T cells or dendritic cells (DC) were rare (<1 cell per 500 fields) in non-immunized mice. In contrast, numbers of CD4+ T cells in immunized mice began to increase at day 3, and numbers of DCs showed a notable increase around day 9. Myelin laden DCs appeared between day 11 and 13. Inflammatory infiltrates peaked between days 15 to day 20, coinciding with peak EAE scores. In conclusion, our data suggest that infiltration of CD4+ T cells precedes substantial uptake and presentation of myelin antigens by CNS APCs, which could have important treatment implications.

Advanced Intercross Line Mapping Suggests That Ncf1 (Ean6) Regulates Severity in an Animal Model of Guillain-Barré Syndrome

We here present the first genetic fine mapping of experimental autoimmune neuritis (EAN), the animal model of Guillain-Barré syndrome, in a rat advanced intercross line. We identified and refined a total of five quantitative trait loci on rat chromosomes 4, 10, and 12 (RNO4, RNO10, RNO12), showing linkage to splenic IFN-gamma secretion and disease severity. All quantitative trait loci were shared with other models of complex inflammatory diseases. The quantitative trait locus showing strongest linkage to clinical disease was Ean6 and spans 4.3 Mb on RNO12, harboring the neutrophil cytosolic factor 1 (Ncf1) among other genes. Polymorphisms in Ncf1, a member of the NADPH oxidase complex, have been associated with disease regulation in experimental arthritis and encephalomyelitis. We therefore tested the Ncf1 pathway by treating rats with a NADPH oxidase complex activator and ameliorated EAN compared the oil-treated control group. By proving the therapeutic effect of stimulating the NADPH oxidase complex, our data strongly suggest the first identification of a gene regulating peripheral nervous system inflammation. Taken together with previous reports, our findings suggest a general role of Ncf1 and oxidative burst in pathogenesis of experimental autoimmune animal models.

Pathologic Findings and Immunohistochemical Distribution of Viral Antigen in Experimental Avian Encephalomyelitis of the Chick and Quail

Summary In this study, the differences and diagnostic importances of the pathological and immunohistochemical findings in avian encephalomyelitis were investigated comparatively in chicks and quails by inoculating van Roekel strain (reference strain) via intracerebral and intramuscular routes. Histopathologically, nonpurulent encephalomyelitis characterized with infiltration of perivascular mononuclear cells and gliosis was described in both species. Neuronal lesion in the chicks consisted of central chromatolysis and neuronal shrinkage (neuronal pyknosis), whereas in quails it was composed of only neuronal shrinkage. Lymphoid aggregates and/or infiltration of mononuclear cells were observed in the proventriculus, gizzard, intestines, liver, pancreas, kidneys, myocardium, lungs, spleen and eyes. However, these lesions in quails were restricted only in the proventriculus, intestines, myocardium and eyes. In the immunofluorescein examination, viral antigen was found especially in neurons of the midbrain, cerebral hemispheres, cerebellum, and occasionally in medulla oblongata and medulla spinalis in both species. Viral antigen in the brain and spinal cord samples of the chicks decreased gradually by increasing inflammatory reaction. Viral antigen in both species was continously detected in the proventriculus, gizzard, intestines, myocardium, pancreas, spleen, liver, kidneys, lungs and eyes..

DP IV/CD26, APN/CD13 and related enzymes as regulators of T cell immunity: implications for experimental encephalomyelitis and multiple sclerosis

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system. Peptidases like dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) play a regulatory role in T cell activation and represent potential targets for the treatment of inflammatory disorders. Synthetic inhibitors of DP IV and/or APN enzymatic activity induce production of the immunosuppressive cytokine TGF-beta1 and subsequently suppress DNA synthesis and Th1 cytokine production of activated human T cells. Compelling evidence has demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to the pathogenesis of autoimmune inflammation. Here, we report that inhibitors of DP IV-like activity as well as of APN activity inhibit IL-17 production in activated human and mouse T cells. Combining inhibitors of DP IV and APN increases the suppressive effect on T cell specific IL-17 production in vitro compared to a single peptidase inhibitor. In the following, we summarize the evidence for the role of both ectoenzymes in T cell activation in vitro and in vivo and provide a rationale for the use of combined or dual ectopeptidase inhibitors to treat autoimmune diseases like MS.

Monomeric DR2/MOG-35–55 recombinant TCR ligand treats relapses of experimental encephalomyelitis in DR2 transgenic mice

Treatment of human autoimmune diseases such as multiple sclerosis (MS) will likely require agents that can prevent or reverse the inflammatory process that results in clinical relapses and disease progression. We evaluated the ability of a newly designed monomeric recombinant TCR ligand (RTL342M) containing HLA-DR2 peptide-binding domains covalently linked to MOG-35–55 peptide to prevent and treat both the initial episode and subsequent relapses of experimental autoimmune encephalomyelitis (EAE) in HLA-DR2 transgenic mice. Single doses of RTL342M given either i.v. or s.c. to HLA-DR2 mice produced a rapid (within 24 h) and dose-dependent reversal of clinical signs of paralytic EAE, and even a single dose ≤ 2 μg could produce a significant treatment effect. Multiple daily doses were even more effective than the same total amount of RTL given as a single dose. By establishing the minimal effective dose, we determined that RTLs may be 50 times more potent than molar equivalent doses of myelin peptide alone. RTL342M given prior to induction of EAE prevented disease in most mice, and the remainder could be successfully retreated with RTL. Most important for clinical application, RTL342M was highly effective for treating EAE relapses when given periodically prior to the relapse or even after relapses had occurred. These data demonstrate the rapid and potent clinical effects of RTL342M at disease onset and during relapses in EAE and establish important principles governing the application of this novel approach as a possible therapy for patients with MS.

Corticotropin-releasing hormone and the blood-brain-barrier

Increased blood-brain-barrier (BBB) permeability precedes any clinical or pathologic signs and is critical in the pathogenesis of multiple sclerosis (MS) and brain metastases. CD4+ TH1 cells mediate demyelination in MS, but how they get sensitized and enter the brain to induce brain inflammation remains obscure. TH2 cytokines associated with allergic disorders have recently been implicated in MS, while genes upregulated in MS plaques include the mast cell-specific tryptase, the IgE receptor (Fc-epsilon-RI) and the histamine-1 receptor. Mast cell specific tryptase is elevated in the CSF of MS patients, induces microvascular leakage and stimulates protease-activated receptors (PAR), leading to widespread inflammation. BBB permeability, MS and brain metastases appear to worsen in response to acute stress that leads to the local release of corticotropin-releasing hormone (CRH), which activates brain mast cells to selectively release IL-6, IL-8 and vascular endothelial growth factor (VEGF). Acute stress increases BBB permeability that is dependent on CRH and mast cells. Acute stress shortens the time of onset of experimental alleric encephalomyelitis (EAE) that does not develop in W/W mast cell deficient or CRH -/- mice. Brain mast cell inhibition and CRHR antagonists offer novel therapeutic possibilities.

Lactoferrin ameliorates symptoms of experimental encephalomyelitis in Lewis rats

Lactoferrin (LF) is a multifunctional protein present in secretory fluids of mammals and circulating neutrophils. Beside anti-inflammatory properties, LF was found to inhibit some autoimmune disorders. In this investigation we studied effects of oral administration of LF on experimental autoimmune encephalomyelitis (EAE) in Lewis rats. LF was given in drinking water as 0.25% solution beginning the day of elicitation of EAE or with a seven-day delay. The effects of LF were evaluated by the following criteria: clinical score, lymph node cell number, serum cytokine levels and histopathological changes. We found that LF treatment led to a significant acceleration of the recovery process, particularly on days 16–18 following elicitation of EAE. The delayed administration of LF was less effective in reducing the score of EAE. In addition, cell number of the inguinal lymph nodes of untreated EAE rats, almost 3 times higher as compared with control, naïve rats, was normalized by LF treatment. Furthermore, LF decreased elevated serum concentrations of tumor necrosis factor alpha and transforming growth factor beta. The histological analysis of the spinal cord revealed reduction in the number and size of inflammatory foci in LF-treated rats. In summary, treatment of EAE Lewis rats with LF reduced the clinical symptoms and accelerated the recovery of animals.

Experimental Encephalomyelitis Induces Changes in DJ-1: Implications for Oxidative Stress in Multiple Sclerosis

DJ-1 plays an important role in oxidative stress, and is involved in various neurodegenerative diseases. Accumulating evidence suggests a central role for oxidative stress in multiple sclerosis (MS). The aim of this study was to examine whether changes occur in DJ-1 expression in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We found upregulation of DJ-1 mRNA and protein expression levels in EAE and a correlation between disease severity and increased DJ-1 levels. Although DJ-1 isoforms were more alkaline in controls, in EAE, a shift was noted toward acidic isoforms. ROS induced by SIN-I exposure led to an increase in DJ-1 mRNA and protein levels in human glioma U-87 cells. Immunocytochemical staining demonstrated that DJ-1 is present both in the cytoplasm and the nuclei of these cells. This is the first report of modulation of DJ-1 expression in EAE. Upregulation of DJ-1 was noted in EAE, and similar results were observed in glioma cells exposed to ROS. In view of the accumulating evidence on the central role of oxidative stress in MS, and the importance of DJ-1 in oxidative stress management by the CNS, we believe that DJ-1 will be found to have a central role in MS.

Enhanced tryptophan catabolism in the absence of the molecular adapter DAP12

DAP12 is an immunoreceptor tyrosine-based activation motif-bearing membrane adapter molecule expressed by different cell types. Although several receptors associate with DAP12 in murine dendritic cells (DC), the function of these receptors is as yet unknown. Here we report that splenic mature DC with DAP12 overexpression are characterized by an impaired tolerogenic potential. In contrast, inhibition of DAP12 function results in enhanced tolerogenesis and constitutive expression of immunosuppressive tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO). Increased resistance to experimental encephalomyelitis is observed in DAP12 knockin mice, which is dependent on IDO expression. Therefore, DAP12-related receptors act as negative regulators of IDO-mediated tolerance in vivo.

Profiling of Alopecia Areata Autoantigens Based on Protein Microarray Technology

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.

Immune Regulatory Mechanisms Influence Early Pathology in Spinal Cord Injury and in Spontaneous Autoimmune Encephalomyelitis

Injuries to the central nervous system (CNS) trigger an inflammatory reaction with potentially devastating consequences. In this report we compared the characteristics of the inflammatory response on spinal cord injury (SCI) caused by a stab wound between the T7 and T9 vertebrae and spontaneous experimental autoimmune encephalomyelitis (EAE). SCI and EAE were compared in two types of myelin basic protein Ac1-11-specific T-cell receptor transgenic mice: T/R+ mice harbor regulatory T cells, and T/R- mice lack regulatory T cells. Our results show that 8 days after SCI, T/R- mice developed a strong T-cell infiltrate in the spinal cord, with remarkable down-modulation of CD4 expression that was accompanied by a local increase in Mac-3+ and F4/80+ reactivity and diffuse local and distal astrogliosis. In contrast, T/R+ mice exhibited a modest increase in CD4+ cells localized to the site of injury, without CD4 down-modulation; focal astrogliosis was restricted to the site of the lesion, although Mac-3+ and F4/80+ cells were also present. Similarly to T/R- mice that underwent SCI, T cells displaying down-modulated CD4 expression were found in the CNS of older T/R- mice afflicted by spontaneous EAE. Overall, our results suggest that common mechanisms regulate T-cell accumulation in CNS lesions of different causes, such as mechanic lesion or autoimmune-mediated damage.

TCR Peptide Vaccination in Multiple Sclerosis: Boosting a Deficient Natural Regulatory Network that may Involve TCR-Specific CD4+CD25+ Treg Cells

Vaccination with self peptides contained within T cell receptor (TCR) chains, expressed by pathogenic Th1 cells can induce a second set of regulatory T cells that can reverse paralysis in rodents with experimental encephalomyelitis, and similarly, may have the potential to regulate myelin-reactive Th1 cells in patients with multiple sclerosis (MS). In this review, we discuss our recent discovery that TCR-reactive T cells generally possess classical inhibitory activity associated with Treg cells. CD4+CD25+ TCR-reactive T cells can inhibit CD4+CD25- indicator cells stimulated with anti-CD3/anti-CD28 antibody in a dose-dependent and cell-contact-dependent manner. Additionally, CD4+CD25+ T cells from blood of healthy control donors have significant responses to a pool of discriminatory TCR peptides, including BV10S1P, BV19S20, BV13S7, BV12S2A2T, BV11S1A1T, BV21S3A1T, AV15S1, and BV12S1A1N1. Patients with MS have varying degrees of deficient responses to TCR peptides, and by association, a defect in Treg cell function as well. TCR peptide vaccination using a new tripeptide mixture emulsified in IFA produced strong T cell responses in 100% of MS recipients, a dramatic improvement over previous vaccines given i.d. in saline that induced TCR-reactive T cell responses in about 50% of recipients. Responders to vaccination had a tendency towards reduced MRI lesions, and an early indication of enhanced Treg activity mediated by TCR-reactive T cells that could provide suppression of target as well as bystander T cells. These data provide a strong foundation for future TCR vaccination studies that will critically test the ability of the tripeptide mixture to induce significantly enhanced Treg activity and possible clinical and MRI benefits in vivo.

Inhibiting leukocyte recruitment to the brain by IVIg: is it relevant to the treatment of demyelinating CNS disorders?

Recruitment and migration of activated leukocytes into the brain is a fundamental process in the induction of various autoimmune CNS disorders, most notably multiple sclerosis. Although not all tissues use the same molecules for leukocyte recruitment (Alter et al ., 2003; Ransohoff et al ., 2003), the information derived from experimental allegic encephalomyelitis (EAE), a T-cell-mediated animal model that resembles multiple sclerosis, has provided useful data on the key molecules associated with T-cell transmigration into the brain. The initial contact between a leukocyte and an endothelial cell is referred to as ‘tethering’, while the immediate subsequent interaction is described as ‘rolling’ \[Fig. 1(1) and (2)\] (von Andrian and MacKay, 2000). These steps, which allow the leukocyte to slow down and roll along the vascular wall, are mediated predominantly by selectins and facilitated by two α-4 integrins, α4β1 [very late antigen-4 (VLA-4)] and αLβ2 [lymphocyte function-associated antigen-1 (LFA-1)]. The rolling leukocyte can stop only if it receives an activating signal, such as that provided by a chemokine on the endothelial surface. This signal switches the two α-4 integrins to a high affinity state [Fig. 1(3)]. Thereafter, the α-4 integrins bind to the counter-receptors vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) on the endothelial cells and adhere irreversibly [Fig. 1(4)]. Leukocytes can then transmigrate out of the microvasculature. Fig. 1 The CNS microvasculature constitutively expresses selectins, such as P selectin, low levels of adhesion molecules ICAM-1 and VCAM, and immobilized chemokines; the activated T cells that enter the CNS express selectin ligands, α-4 intergrins such as α4β1 (VLA-4) and αLβ2 (LFA-1) and various …

Immunological Unresponsiveness Characterized by Increased Expression of CD5 on Peripheral T Cells Induced by Dendritic Cells In Vivo

In the steady state, interaction between T cells and antigen-presenting dendritic cells (DCs) leads to T cell tolerance. To examine the role of DC regulated peripheral tolerance in a model autoimmune disease, we delivered an encephalitogenic oligodendrocyte glycoprotein (MOG) peptide to DCs in vivo. We found that targeting MOG peptide to DCs resulted in a novel form of peripheral T cell tolerance that was sufficiently profound to prevent autoimmune experimental acute encephalomyelitis (EAE). The tolerized T cells were severely impaired in specific secondary responses to antigen in vivo but they were not intrinsically anergic since they remained highly responsive to T cell receptor (TCR) stimulation in vitro. The mechanism that mediates this dynamic antigen-specific T cell unresponsiveness differs from previously described forms of tolerance in that it requires that DCs induce CD5 expression on activated T cells.

Effects of peripheral application of LPS on serum hormone levels and expression of interleukin 1 and its receptor antagonist in the brain of the common marmoset Callithrix jacchus

Callithrix jacchus (CJ) is a primate which is susceptible to a form of experimental encephalomyelitis that resembles multiple sclerosis (MS). Human studies seeking disease related genes for MS have identified candidates including interleukin 1ß (IL1ß) and its receptor antagonist IL1ra. We therefore attempted to measure expression of IL1ß and IL1ra in the brain of CJ and examined whether mRNA levels are affected by peripheral application of lipopolysaccharide (LPS). For cloning of partial sequences, RNA was extracted from frontal cortex. RT-PCR was performed with human primers, products were cloned for sequencing. CJ – specific primers/probes were developed for mRNA determinations by TaqMan PCR. Seven adult male CJ were subcutaneously injected with 40µg LPS, while 7 animals received NaCl injections. 3 hours later, animals were sacrificed by an overdose of ketamine. Brain sections were cut on a cryostat and specific areas (frontal Cortex, Hippocampus, Claustrum) were dissected using the micropunch technique. These areas were chosen because human studies have shown, that MS is associated with degenerative changes in these parts of the CNS. LPS reduced serum testosterone levels (1.3±0.4 vs. 0.5±0.09 ng/dl), while prolactin and IL1ß levels increased (9.2±1 vs. 20.4±4.9 ng/ml and 0.098±0.03 vs. 1.75±0.4 pg/ml, respectively, all results p<0.05). These endocrine changes were associated with significant increases of IL1ß- mRNA levels in the frontal cortex (15.3 fold), hippocampus (13.2 fold) and claustrum (12.8 fold). In contrast, mRNA levels of IL1ra remained unchanged in all three areas.

Optic neuritis, a new variant of experimental encephalomyelitis, a durable model for all seasons, now in its seventieth year.

One of the most enduring models of human disease now celebrates the seventieth anniversary of its publication in The Journal of Experimental Medicine . Thomas Rivers, working at the Hospital of the Rockefeller Institute for Medical Research, along with his colleagues D.H. Sprunt and G.P. Berry,

Treatment of relapsing paralysis in experimental encephalomyelitis by targeting Th1 cells through atorvastatin.

Statins, known as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, exhibit numerous functions related to inflammation, such as MHC class II down-regulation, interference with T cell adhesion, and induction of apoptosis. Here we demonstrate that both subcutaneous and oral administration of atorvastatin inhibit the development of actively induced chronic experimental autoimmune encephalomyelitis in SJL/J mice and significantly reduce the inflammatory infiltration into the central nervous system (CNS). When treatment was started after disease onset, atorvastatin reduced the incidence of relapses and protected from the development of further disability. Both the reduced autoreactive T cell response measured by proliferation toward the encephalitogenic peptide PLP139–151 and the cytokine profile indicate a potent blockade of T helper cell type 1 immune response. In in vitro assays atorvastatin not only inhibited antigen-specific responses, but also decreased T cell proliferation mediated by direct TCR engagement independently of MHC class II and LFA-1. Inhibition of proliferation was not due to apoptosis induction, but linked to a negative regulation on cell cycle progression. However, early T cell activation was unaffected, as reflected by unaltered calcium fluxes. Thus, our results provide evidence for a beneficial role of statins in the treatment of autoimmune attack on the CNS.

High-dose intravenous immunoglobulin treatment of multiple sclerosis.

A review of the pathological basis of multiple sclerosis is presented to see whether the many immunological effects if IVIg may exert some benefit and at what level. This is probably due to the wide spectrum of interactions between IVIg and the immune system, which are analyzed in this review. Macrophage Fc receptor saturation and block, anti-idiotypic effect, reduction of endothelial cell activation and superantigen-neutralizing antibodies are probably involved in the action of IVIg in dysimmune demyelinating diseases. Furthermore, IVIg promote remyelination in virus-induced experimental encephalomyelitis. Trials on IVIg in MS demonstrated a reduction of relapse rate (RR) and appearance of gadolinium-enhancing lesions on magnetic resonance imaging. Furthermore IvIg are regarded as a promising treatment to reduce RR in post-partum period. However, studies on secondary-progressive MS failed to demonstrate an IVIg effect on disability and IVIg failed to improve stabilized visual and motor deficits in two large trials.

Estrogen Inhibits Systemic T Cell Expression of TNF-α and Recruitment of TNF-α+ T Cells and Macrophages into the CNS of Mice Developing Experimental Encephalomyelitis

Estrogen treatment has been found to have suppressive activity in several models of autoimmunity. To investigate the mechanism of 17β-estradiol (E2) suppression of experimental autoimmune encephalomyelitis, we evaluated E2 effects on TNF-α expression in the central nervous system (CNS) and spleen of C57BL/6 mice immunized with MOG 35-55/CFA. Kinetic analysis demonstrated that E2 treatment drastically decreased the recruitment of total inflammatory cells as well as TNF-α+ macrophages and T cells into the CNS at disease onset. In contrast, E2 had only moderate effects on the relatively high constitutive TNF-α expression by resident CNS microglial cells. E2 treatment also had profound inhibitory effects on expression of TNF-α by splenic CD4+ T cells, including those responsive to MOG 35–55 peptide. We propose that the mechanism of E2 protection may involve both systemic inhibition of TNF-α expression and local (CNS) recruitment of inflammatory cells, with modest effects on TNF-α expression by resident CNS microglial cells.