Pathogenesis Of Experimental Allergic Encephalomyelitis Research Articles

The role of costimulation in autoimmune demyelination

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated, autoimmune disorder characterized by central nervous system (CNS) inflammation and demyelination, features reminiscent of the human disease, multiple sclerosis (MS). In addition to the signal the encephalitogenic T cell receives through the T cell receptor (TCR), a second signal, termed costimulation, is required for complete T cell activation. The B7 family of cell surface molecules expressed on antigen presenting cells (APC) is capable of providing this second signal to T cells via two receptors, CD28 and CTLA-4. Our studies have shown that costimulation provided by B7 molecules to its ligand CD28 is important in the initiation of the autoimmune response in EAE. Further, it appears the costimulation provided by B7-1 is important in disease development, while B7-2 may play an important regulatory role. We and others later showed that B7/CTLA-4 interaction plays a critical role in down-regulating the immune response. Previous work has shown that activated T cells and T cells of a memory phenotype are less dependent on costimulation than naive T cells. T cells reactive with myelin components that are involved in the pathogenesis of EAE and possibly MS would be expected to have been activated as part of the disease process. Building upon our prior work in the EAE model, we have tested the hypothesis that myelin-reactive T cells, which are relevant to the pathogenesis of CNS inflammatory demyelination, can be distinguished from naive myelin-reactive T cells by a lack of dependence upon costimulation for activation and that the costimulatory requirements of these myelin-reactive T cells change during the course of disease. Our studies in the EAE model have also addressed the mechanisms of extrathymic (peripheral) T cell tolerance following intravenous (i.v.) administration of high dose antigen. It is believed that TCR signaling in the absence of costimulation is a vital component of peripheral tolerance mechanisms. However, recent evidence suggests that peripheral tolerance of antigen-specific T cells induced in vivo may require CTLA-4 engagement of the tolerized T cells. We have begun to examine the molecular mechanisms of tolerance induction following intravenous and intraperitoneal administration of myelin antigens in the EAE model and test the hypothesis that tolerance induction is dependent on the B7:CD28/CTLA-4 pathway. The results from our studies will enhance our understanding of the role that myelin-reactive T cells may play in the pathogenesis of MS. We have determined that MBP-reactive T cells in MS patients are less dependent upon CD28 costimulation than in normal controls, suggesting that these T cells were previously primed in vivo. Characterization of these CD28-independent myelin-specific T cells will have broad implications for a variety of immunologically based therapies in diseases such as MS.

Activation of nuclear factor-kB in the spinal cord of experimental allergic encephalomyelitis

The DNA-binding activity of nuclear factor (NF-kB) was found to be induced in the spinal cord of rats with experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), from the onset of the disease. This activation of NF-kB persisted throughout the disease period and decreased thereafter in the recovery phase. Supershift analysis of NF-kB DNA-binding activity in nuclear extracts of spinal cords showed that RelA/p65 and p50 subunits but not c-Rel/p75, RelB/p68 and p52 subunits were involved in DNA binding. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kB activation, markedly inhibited the in vivo activation of NF-kB in the spinal cord of EAE rats and attenuated the clinical symptoms of EAE. These studies suggest that activation of NF-kB plays an important role in the pathogenesis of EAE and inhibitors of NF-kB activation may have therapeutic importance in MS.

Chemokines and chemokine receptors in the pathogenesis of multiple sclerosis.

In recent years we have seen growing evidence for the role of chemokines in the pathogenesis of several infectious and non-infectious inflammatory CNS disease states, including Multiple Sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE). An increase in proinflammatory chemokines has been associated with demyelinating lesions and clinical neurological dysfunction in patients with MS; these chemokines could be potential targets for MS therapy. Besides a clearly defined role in mediating leukocyte migration, these and other chemokines may act as immunoregulatory molecules in the driving to Th1/Th2 responses, switch of cytokine profiles, and the induction of tolerance. Since chemokine receptors have now been identified on macrophages, microglia, astrocytes, and endothelial cells as well as neurons in the CNS, chemokine/receptor interactions may mediate functional responses in a variety of CNS cell types during the course of inflammatory disease states. Therefore, clarification of the roles of chemokines and their receptors in the pathogenesis of EAE and MS will be useful in establishing immunotherapeutic strategies for these neurological autoimmune disorders.

B cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide.

While the pathology of multiple sclerosis implicates a role for B cells and antibodies in the disease process, results from animal models have yielded conflicting results. To further characterize the role of B cells in experimental allergic encephalomyelitis (EAE), wild-type and B cell-deficient C57BL/6 mice were immunized with either a recombinant form of myelin oligodendrocyte glycoprotein (MOG) or with the encephalitogenic MOG(35-55) peptide. B cell-deficient mice did not develop EAE when immunized with MOG, although they were susceptible to MOG(35-55)-induced disease. In contrast, wild-type mice were fully susceptible to both MOG and MOG(35-55)-induced EAE. B cell-deficient mice immunized with MOG were primed to the encephalitogenic MOG(35- 55) epitope, as their spleen cells responded with Th1 cytokine production in a fashion similar to WT cells when challenged in vitro with MOG protein or MOG(35-55) peptide. These results demonstrate that the form of inducing antigen (protein vs. peptide) plays a role in the pathogenesis of EAE and may be relevant when applying results from the EAE model to multiple sclerosis.

Amelioration of experimental allergic encephalomyelitis in Lewis rats by lovastatin

Proinflammatory cytokines and inducible nitric oxide synthase (iNOS) are involved in the pathogenesis of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We have previously reported that lovastatin (Pahan, K., Sheikh., F.G., Namboodiri, A. and Singh, I., Lovastatin and Phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia and macrophages. J. Clin. Invest., 100 (1997) 2671–2679.), an inhibitor of the mevalonate pathway, inhibits the expression of iNOS and proinflammatory cytokines in rat primary glial cells (astroglia and microglia) and macrophages. The present study underlines the therapeutic importance of lovastatin in ameliorating the neuroinflammatory disease process in the central nervous system of EAE rats. Immunohistochemical results show a higher degree of expression of iNOS, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ ) in brains of rats with acute monophasic EAE relative to the control animals. Administration of lovastatin inhibited the expression of iNOS, TNF-α and IFN-γ in the CNS of EAE rats and improved the clinical signs of EAE suggesting that this compound may have therapeutic potential in the treatment of neuroinflammatory diseases like MS.

Dual Role for Fas Ligand in the Initiation of and Recovery from Experimental Allergic Encephalomyelitis

We have previously demonstrated a role for Fas and Fas ligand (FasL) in the pathogenesis of experimental allergic encephalomyelitis (EAE). However, using an active induction paradigm we could not distinguish between FasL expressed on activated CD4(+) T cells from that expressed on other inflammatory or resident central nervous system (CNS) cells. To address this issue, we have conducted reciprocal adoptive transfer experiments of nontransgenic or myelin basic protein-specific T cell receptor transgenic wild-type, lpr, or gld lymphocytes into congenic wild-type, lpr, and gld hosts. We found that FasL expressed on donor cells is important for the development of EAE, as FasL-deficient lymphocytes transfer attenuated disease. Furthermore, Fas expressed in the recipient animals is important for the progression of EAE, as clinical signs of disease in lpr recipients were dramatically attenuated after transfer of either wild-type or lpr T cells. Surprisingly, these experiments also identified CNS cells as a source of functional FasL. Host-derived FasL appears to be especially important in the recovery from EAE, as many gld recipients of wild-type lymphocytes develop prolonged clinical signs of disease. Thus it appears that FasL plays distinct roles in EAE during the initiation of and recovery from disease.

The Role of the Antigen-Presenting Cell in Fas-Mediated Direct and Bystander Killing: Potential In Vivo Function of Fas in Experimental Allergic Encephalomyelitis

Costimulatory molecules are critical in mediating Fas-dependent direct and bystander lysis. In direct lysis, the APC is the Fas-positive target. It presents Ag to the T cell, thereby activating the T cell. The activated T cell then up-regulates FasL, allowing it to kill the APC. In bystander lysis, the APC again induces FasL expression on the T cell, but the target is a third Fas-positive cell that may lack the appropriate MHC-restricting element to activate the T cell. This study shows that ICAM-1 and B7-1 can serve as important adhesion molecules in direct killing using CD4+ T cell effectors. In bystander killing, B7-1 appears to act as a signaling molecule as well. It has been demonstrated that lpr and gld mice are less susceptible to experimental allergic encephalomyelitis than their wild-type counterparts. In this study, we show that although microglia are poor targets of direct killing, they are capable of stimulating myelin basic protein-specific T cells to kill innocent Fas-positive targets. This presents a possible mechanism for the pathogenesis of experimental allergic encephalomyelitis.

Cytokine/chemokine networks regulate the pathogenesis of experimental allergic encephalomyelitis (EAE)

Cytokine/chemokine networks regulate the pathogenesis of experimental allergic encephalomyelitis (EAE)

Fas and Fas ligand enhance the pathogenesis of experimental allergic encephalomyelitis, but are not essential for immune privilege in the central nervous system.

Mutations of CD95 and CD95L, lpr and gld, respectively, are associated with spontaneous autoimmune disease and alteration of immune privilege. In lpr or gld animals these processes would be expected to exacerbate experimental allergic encephalomyelitis (EAE), an animal model of the autoimmune demyelinating disease multiple sclerosis. However, here we show that the lpr and gld mutations did not overcome the MHC-defined limits of disease and, surprisingly, did not exacerbate the pathology of EAE on a sensitive haplotype. In fact, the mutations dramatically ameliorated clinical signs of EAE without affecting the development of a Th1 response or inflammatory cell infiltration into the central nervous system. Fewer apoptotic cells were detected in inflammatory lesions of lpr mice than in wild-type lesions of similar severity. Our results indicate that CD95L is not an instrumental component of immune privilege in the central nervous system, and that functional CD95 and CD95L are important for the progression of clinical disease.

The generation of encephalitogenic T cell lines from experimental allergic encephalomyelitis-resistant strains of mice.

While only a few strains of mice are susceptible to the primary induction or passive transfer of experimental allergic encephalomyelitis (EAE), the basis of EAE resistance remains unclear. In the present studies, we have defined two approaches that allow for the generation of encephalitogenic, myelin basic protein-reactive, T cell lines from EAE-resistant strains of mice. The first approach, based on the putative relevance of apoptosis to autoimmune disease, involves repeat antigenic stimulation of recently initiated T cell lines. The second approach involves the initiation of lymph node cultures in the absence of exogenous splenocytes as antigenic-presenting cells and the use of a higher antigen concentration. Both approaches lead to the generation of encephalitogenic T cell lines from EAE-resistant mouse strains and will be useful for identifying factors relevant to the pathogenesis of EAE.

Treatment of experimental allergic encephalomyelitis by selective removal of CD4+ T cells.

Experimental allergic encephalomyelitis (EAE) is a major animal model of human multiple sclerosis (MS). The pathogenesis of both MS and EAE directly involves CD4+ helper T cells. To remove CD4+ T cells selectively from the circulation, we designed a new column in which anti-CD4 monoclonal antibody was immobilized on the activated substance. Most of the CD4+ T cells, including pathogenic T cells, were selectively adsorbed from whole blood with a direct perfusion through the column in vitro, resulting in depletion of the antigen-specific T cell responses. A preliminary trial of ex vivo treatment with the column in EAE rats lowered the percentages of CD4+ populations but failed to alter the course of the disease, suggesting repeated treatment would be necessary to suppress the development of the disease. We conclude that this new column is potentially useful, and repeated treatment would be beneficial in MS and other CD4+ T cell dependent autoimmune diseases.

Aggravation of murine experimental allergic encephalomyelitis by administration of T-cell receptor γδ-specific antibody

Experimental allergic encephalomyelitis (EAE) is thought to be dominantly mediated by Ag-specific CD4 + MHC class II-restricted T-cells. Recent reports demonstrated accumulation of γ δ T-cells in active multiple sclerosis (MS) plaque and infiltration into brains with EAE. However, the role of γ δ T-cells in pathogenesis of EAE remains unknown. In the present study we have examined EAE mice administered T-cell receptor (TCR) γ δ-specific mAb (UC7-13D5) to elucidate the potential role of γ δ T-cells in the pathogenesis of EAE. MAb treatment led to transient depleting γ δ T-cells in vivo. MAb-treated EAE mice showed aggravation and disease recurrence and also increased Ag-specific proliferative responses. Semiquantitative PCR analysis demonstrated an increased level of IFN- γ mRNA expression in splenocytes from mAb-treated EAE mice during the induction and pre-relapse phase, however, aggravation and disease recurrence have not been suggested to be directly mediated by IFN- γ in the present study. Our results imply that γ δ T-cells play a preventing role in the recurrence of EAE.

A CD4-CDR3 peptide analog inhibits both primary and secondary autoreactive CD4+ T cell responses in experimental allergic encephalomyelitis.

A structure-based design approach was used to develop a cyclized peptide analog of the murine CD4-CDR3-like region as a potential inhibitor of autoimmune CD4+ T cells responsible for the pathogenesis of experimental allergic encephalomyelitis (EAE). Our results indicate that this peptide, referred to as rD-mPGPtide, is able to significantly inhibit the clinical and pathologic symptoms of EAE in the SJL mouse model when administered on day 12 of induction. The optimum effective dosage range for the peptide, injected i.v., was between 0.125 and 0.5 mg and dosages of as high as 5 mg had no observable toxic effects. Treated mice had normal levels of lymphocytes less than 2 wk later and exhibited normal in vitro primary responses to alloantigen and secondary responses to keyhole limpet hemocyanin Ag. The specificity of the rD-mPGPtide treatment for autoreactive T cells was demonstrated by inhibiting proteolipid protein (p139-151)-induced EAE and finding that the lymph node T cells from these mice had suppressed responses to this Ag, but normal responses to alloantigen or other nominal Ag. Importantly, rD-mPGPtide was found to be effective on secondary T cell responses in an EAE rechallenge situation and was able to establish conditions for long-term resistance to further Ag exposure. Analysis of the cytokine profile of responding T cells during late effector stages of disease revealed that the levels of IFN-gamma and IL-4 are significantly reduced in rD-mPGPtide-treated mice. These results strongly suggest that the administration of a CD4-CDR3 peptide analog is an effective therapeutic approach for the inhibition of the CD4+ T cell-mediated autoimmune response in EAE.

Preferential association of Vλx light chains with γ2a heavy chains in naturally occurring human myelin basic protein reactive antibodies

Active immunization with myelin basic protein (MBP) induces experimental allergic encephalomyelitis (EAE) in a variety of animal species, including rats and mice. We have previously described the ability of the newly described mouse lambda (λ) variable (V) region, Vλx, to confer MBP reactivity to an Ab. In this report, we have evaluated the heavy (H) chain isotype distribution of Vλx-bearing Abs in normal mouse serum. We demonstrate a biased H chain isotype association with Vλx light (L) chains with a skewing towards γ2a and 2b isotypes. The IgG2a restriction in normal mouse Igs is even more evident in Vλx-containing Abs that bind MBP. This was confirmed by the ability of purified polyclonal IgG2a Abs to bind MBP and the finding that most or all of the IgG2a Abs that bind MBP seem to harbor a Vλx L chain. The specificity of naturally-occurring Vλx-bearing Abs with MBP can be localized to a particular epitope encompassing residues 25–34 of the MBP molecule. Furthermore, virtually all of the reactivity of Vλx-containing Abs with MBP peptide 25–34 is associated with the γ2a isotype. Collectively, these results suggest that the interaction of Vλx with MBP seems to be facilitated by an association with γ2a which may reflect preferred V H usage by this isotype. Such unique pairing of particular H chains with Vλx L chains in Abs that bind MBP may be indicative of a new B-cell component involved in the pathogenesis of EAE.

A pharmacological study on the role of nitric oxide in the pathogenesis of experimental allergic encephalomyelitis.

The study examines the effects of nitric oxide synthase (NOS) inhibitors on the development of neurological EAE and the levels of nitrite in the central nervous system (CNS) during established disease. EAE was induced in male Lewis rats (200-250 g). Rats received NG-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg body weight) day 7 to 12 post-inoculation (P.I.), 7-nitroindazole (10 mg/kg) day 7 to 11 P.I. or aminoguanidine (200 or 400 mg/kg) day 1 to 12 P.I. Neurological symptoms were assessed and CNS cytosol nitrite and protein levels measured. Results were analysed using the Mann Whitney U-test and the Fischer exact probability test. Symptoms of EAE were associated with a significant elevation in CNS nitrite (p < 0.001). Treatment with NOS inhibitors caused a marked reduction in nitrite levels (p < 0.01). However, in some experiments, vehicle administration also reduced CNS nitrite content (p < 0.05). Although neurological disease was suppressed in EAE-sensitised rats receiving L-NAME (2 +/- 0.3 vs 3 +/- 0.3 mean peak severity +/- SEM) and 7-nitroindazole (1 +/- 0.3 vs 3 +/- 0.3, p < 0.01) comparable inhibition was achieved by respective vehicle treatment (p < 0.01). In contrast, neither aminoguanidine nor corresponding vehicle altered disease development. Drug-induced reductions in CNS nitrite levels during EAE are not always associated with a suppression of neurological symptoms, which suggests NO is not of primary importance in disease pathogenesis. In addition, the study emphasises the strict requirement for appropriate controls when assessing the efficacy of drugs on the course of EAE.

A pathogenic role for gamma delta T cells in relapsing-remitting experimental allergic encephalomyelitis in the SJL mouse.

Previous studies have detected gamma delta T cells in multiple sclerosis and experimental allergic encephalomyelitis (EAE) lesions but their role remains obscure. In the present study, we assessed gamma delta T cell dynamics and distribution in spleen and central nervous system (CNS) from mice with relapsing-remitting EAE, and studied the effect of depleting these cells on clinical and pathologic expression of disease using the mAb GL3. By immunohistochemistry and FACS analysis, striking disease-related changes were observed in the gamma delta T cell population in the CNS. FACS analysis showed that while gamma delta T cells remained low in the spleen (approximately 2% total CD3+ T cells) at all stages, in the CNS they increased to approximately 12% at the height of the acute attack, fell to approximately 5% during the recovery phase, but rose again to approximately 12% during the chronic phase. In animals in which gamma delta T cells were depleted immediately before the onset of acute disease, or during the chronic stage, a striking and significant reduction in the severity of the clinical signs was observed that was associated with a decrease in the percentage of CD3+/gamma delta T cells in the CNS. In depleted animals a statistically significant reduction in inflammation and demyelination was noted during the acute stage, but only marginal effects on these disease parameters were found in the chronic phase. Taken together, the data support the conclusion that gamma delta T cells play an important role in the pathogenesis of EAE in mice during both acute and chronic/progressive phases of the disease process.

Experimental allergic encephalomyelitis in the rat is inhibited by aminoguanidine, an inhibitor of nitric oxide synthase

This study assessed the role of de novo nitric oxide (NO) production in the pathogenesis of experimental allergic encephalomyelitis (EAE) by using aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS). which preferentially inhibits the cytokine- and endotoxin-inducible isoform of NOS versus the constitutive isoforms consisting of endothelial and neuronal NOS. The maximum clinical severity of EAE and the duration of illness were significantly reduced or totally inhibited by twice daily subcutaneous injection of 100 mg/kg body weight AG. Histochemical staining for NADPH diaphorase, which detects enzymatic activity of NOS, revealed positive reactivity in untreated EAE rats both in parenchymal blood vessel walls and in anterior horn cell neurons, while normal rats and rats with EAE treated with AG showed predominantly the neuronal positivity. Moreover, this NADPH staining pattern was further supported by the immunohistochemical findings that endothelial NOS (eNOS) expression was increased in blood vessels in the inflamed lesions of untreated EAE rats and that inducible NOS (iNOS) was detected in some infiltrating inflammatory cells, while treatment with AG could significantly reduce both iNOS and eNOS production. These results suggest that: (i) both iNOS and eNOS are upregulated in inflamed areas of the rat central nervous system in EAE; (ii) increased NO production plays a role in the development of clinical signs in EAE; and (iii) selective inhibitors of iNOS and/or eNOS may have therapeutic potential for the treatment of certain autoimmune diseases.

Murine V lambda x and V lambda x-containing antibodies bind human myelin basic protein.

Myelin basic protein (MBP) is highly immunogenic and a known autoantigen capable of inducing experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis. We have previously described a murine monoclonal antibody (mAb), F28C4, directed against the encephalitogenic MBP peptide acetyl (Ac) 1-9, which contains a V lambda x light chain. Considering the rarity of V lambda x usage, we determined whether other Abs having V lambda x light chains shared similar antigen (Ag) specificity. We screened a panel of V lambda x-containing monoclonal and polyclonal Abs, of unknown specificity for reactivity with MBP. All such Ab, but not heavy chain isotype matched controls, bound MBP but were not polyreactive with other potential self Ags. The binding of a recombinant form of V lambda x alone to MBP demonstrated the important contribution of the V lambda x light chain to the reaction. With the exception of mAb F28C4 which recognizes MBP Ac1-9, the epitope specificity of all other V lambda x-bearing Abs was localized to MBP residues 25-34. These results demonstrate a unique association between V lambda x expression and MBP reactivity. Given that V lambda x shares sequence homology with T cell receptors (TCR) from encephalitogenic T lymphocytes, these results imply a potential role for V lambda x in the pathogenesis of EAE.

Macrophages and microglial cells in the pathogenesis of experimental allergic encephalomyelitis

Macrophages and microglial cells in the pathogenesis of experimental allergic encephalomyelitis

Reactive oxygen species are involved in the pathogenesis of experimental allergic encephalomyelitis in Lewis rats

During experimental allergic encephalomyelitis (EAE), both blood-borne macrophages as well as activated, resident microglial cells are considered to be involved in inflammatory reactions in the central nervous system (CNS), resulting in the neurological deficits common to EAE. Both cell types can produce multiple mediators of tissue damage, among which are the reactive oxygen species (ROS). In this study we show that macrophages and microglial cells, isolated from the CNS of Lewis rats with clinical signs of EAE, exhibited significantly elevated spontaneous and phorbol myristate acetate (PMA)-inducible levels of ROS compired to similar cells isolated from healthy controls, sham (complete Freund's adjuvant, CFA)-immunized rats as well as rats sacrificed before the manifestation of clinical signs of EAE. However, during clinical EAE, peripheral blood mononuclear cells (PBMC) did not show increased spontaneous nor PMA-inducible ROS production compared to controls. In vivo treatment of EAE with catalase, which scavenges the ROS H 2O 2, markedly suppressed the severity of the disease as compared to sham (albumin)-treated controls. In contrast, Superoxide dismutase had no effect on clinical signs. Our studies point at a putative functional role for ROS, and in particular H 2O 2, in the pathogenesis of EAE.