Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis.

Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death.     

The central nervous system inflammatory response to neurotropic virus infection is peroxynitrite dependent.

We have recently demonstrated that increased blood-CNS barrier permeability and CNS inflammation in a conventional mouse model of experimental allergic encephalomyelitis are dependent upon the production of peroxynitrite (ONOO(-)), a product of the free radicals NO* and superoxide (O2*(-)). To determine whether this is a reflection of the physiological contribution of ONOO(-) to an immune response against a neurotropic pathogen, we have assessed the effects on adult rats acutely infected with Borna disease virus (BDV) of administration of uric acid (UA), an inhibitor of select chemical reactions associated with ONOO(-). The pathogenesis of acute Borna disease in immunocompetent adult rats results from the immune response to the neurotropic BDV, rather than the direct effects of BDV infection of neurons. An important stage in the BDV-specific neuroimmune response is the invasion of inflammatory cells into the CNS. UA treatment inhibited the onset of clinical disease, and prevented the elevated blood-brain barrier permeability as well as CNS inflammation seen in control-treated BDV-infected rats. The replication and spread of BDV in the CNS were unchanged by the administration of UA, and only minimal effects on the immune response to BDV Ags were observed. These results indicate that the CNS inflammatory response to neurotropic virus infection is likely to be dependent upon the activity of ONOO(-) or its products on the blood-brain barrier.     

IFN-gamma determines distinct clinical outcomes in autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS initiated by autoreactive CD4(+) T cells. EAE classically presents with a progressive ascending paralysis and is a model of multiple sclerosis that recapitulates some aspects of the disease. In this report we describe a mouse strain that spontaneously develops a severe, nonclassical form of EAE with 100% incidence. The distinct clinical phenotype is marked initially by a slight head tilt, progressing to a severe head tilt, spinning, or a rotatory motion. Classical EAE spontaneously occurs in myelin basic protein (MBP)-specific TCR transgenic RAG-1(-/-) mice (referred to as T/R(-)), whereas nonclassical EAE spontaneously occurs in T/R(-) IFN-gamma(-/-) mice (T/R(-)gamma(-)). Thus, the TCR recognizes the same Ag (MBP) and uses identical TCR in both cases. The cellular infiltrate in nonclassical EAE is predominantly found in the brainstem and cerebellum, with very little inflammation in the spinal cord, which is primarily affected in classical disease. Importantly, depending on the genetic makeup and priming conditions of the MBP-specific T cells, nonclassical disease can occur in the presence of an inflammatory infiltrate with eosinophilic, neutrophilic, or monocytic characteristics. Finally, we believe that nonclassical spontaneous EAE could be a useful model for the study of some characteristics of multiple sclerosis not observed in classical EAE, such as the inflammatory responses in the brainstem and cerebellum that can cause vertigo.     

Redirecting therapeutic T cells against myelin-specific T lymphocytes using a humanized myelin basic protein-HLA-DR2-zeta chimeric receptor

Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes implicated in MS. We generated two heterodimeric receptors that genetically link the human MBP84-102 epitope to HLA-DR2 and either incorporate or lack a TCRzeta signaling domain. The Ag-MHC domain serves as a bait, binding the TCR of MBP-specific target cells. The zeta signaling region stimulates the therapeutic cell after cognate T cell engagement. Both receptors were well expressed on primary T cells or T hybridomas using a tricistronic (alpha, beta, green fluorescent protein) retroviral expression system. MBP-DR2-zeta-, but not MBP-DR2, modified CTL were specifically stimulated by cognate MBP-specific T cells, proliferating, producing cytokine, and killing the MBP-specific target cells. The receptor-modified therapeutic cells were active in vivo as well, eliminating Ag-specific T cells in a humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP84-102/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to counteract pathologic, self-specific T lymphocytes, and specifically validate humanized MBP-DR2-zeta chimeric receptors as a potential therapeutic in MS.     

Severe disease, unaltered leukocyte migration, and reduced IFN-gamma production in CXCR3-/- mice with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) Th1 T cell-mediated disease of the CNS, used to study certain aspects of multiple sclerosis. CXCR3, the receptor for CXCL10, CXCL9, and CXCL11, is preferentially expressed on activated Th1 T cells and has been proposed to govern the migration of lymphocytes into the inflamed CNS during multiple sclerosis and EAE. Unexpectedly, CXCL10-deficient mice were susceptible to EAE, leaving uncertain what the role of CXCR3 and its ligands might play in this disease model. In this study, we report that CXCR3(-/-) mice exhibit exaggerated severity of EAE compared with wild-type (CXCR3(+/+)) littermate mice. Surprisingly, there were neither quantitative nor qualitative differences in CNS-infiltrating leukocytes between CXCR3(+/+) and CXCR3(-/-) mice with EAE. Despite these equivalent inflammatory infiltrates, CNS tissues from CXCR3(-/-) mice with EAE showed worsened blood-brain barrier disruption and more von Willebrand factor-immunoreactive vessels within inflamed spinal cords, as compared with CXCR3(+/+) mice. Spinal cords of CXCR3(-/-) mice with EAE demonstrated decreased levels of IFN-gamma, associated with reduced inducible NO synthase immunoreactivity, and lymph node T cells from CXCR3(-/-) mice primed with MOG(35-55) secreted less IFN-gamma in Ag-driven recall responses than cells from CXCR3(+/+) animals. CXCR3(-/-) lymph node T cells also showed enhanced Ag-driven proliferation, which was reduced by addition of IFN-gamma. Taken with prior findings, our data show that CXCL10 is the most relevant ligand for CXCR3 in EAE. CXCR3 does not govern leukocyte trafficking in EAE but modulates T cell IFN-gamma production and downstream events that affect disease severity.     

Delta-like ligand 4 regulates central nervous system T cell accumulation during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for multiple sclerosis. Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the current study, we hypothesized that Notch ligand-receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid dendritic cells, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) after EAE induction. Treatment of mice with a DLL4-specific blocking Ab significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by Ag-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of Ag-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished Ag-specific CD4(+) T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.     

LF 15-0195 treatment protects against central nervous system autoimmunity by favoring the development of Foxp3-expressing regulatory CD4 T cells

Experimental autoimmune encephalomyelitis (EAE) is an instructive model for the human demyelinating disease multiple sclerosis. Lewis (LEW) rats immunized with myelin-basic protein (MBP) develop EAE characterized by a single episode of paralysis, from which they recover spontaneously and become refractory to a second induction of disease. LF 15-0195 is a novel molecule that has potent immunosuppressive effects in several immune-mediated pathological manifestations, including EAE. In the present study, we show that a 30-day course of LF 15-0195 treatment not only prevents MBP-immunized LEW rats from developing EAE but also preserves their refractory phase to reinduction of disease. This effect is Ag driven since it requires priming by the autoantigen during the drug administration. In contrast to other immunosuppressive drugs, short-term treatment with this drug induces a persistent tolerance with no rebound of EAE up to 4 mo after treatment withdrawal. This beneficial effect of LF 15-0195 on EAE does not result from the deletion of MBP-specific Vbeta8.2 encephalitogenic T cells. In contrast, this drug favors the differentiation of MBP-specific CD4 T cells into Foxp3-expressing regulatory T cells that, upon adoptive transfer in syngeneic recipients, prevent the development of actively induced EAE. Finally, we demonstrate that the tolerance induced by LF 15-0195 treatment is not dependent on the presence of TGF-beta. Together, these data demonstrate that short-term treatment with LF 15-0195 prevents MBP-immunized LEW rats from EAE by favoring the development of Foxp-3-expressing regulatory CD4 T cells.     

CD28 costimulation is crucial for the development of spontaneous autoimmune encephalomyelitis

Multiple sclerosis (MS) is a severe central nervous system disease. Experimental autoimmune encephalomyelitis (EAE) mimics MS in mice. We report that spontaneous development of EAE in RAG-1-deficient mice transgenic for a myelin basic protein (MBP)-specific TCR (TgMBP+/RAG-1-/-) requires expression of the T cell costimulatory molecule CD28. Surprisingly, T cells from CD28-/-TgMBP+/RAG-1-/- mice proliferate and produce IL-2 in response to MBP1-17 peptide in vitro, excluding clonal anergy as the mechanism of CD28-regulated pathogenesis. Proliferation of autoaggressive T cells was dependent on the concentration of the MBP peptide, as was the development of MBP-induced EAE in CD28-deficient PL/J mice. These results provide the first genetic evidence that CD28 costimulation is crucial for MBP-specific T cell activation in vivo and the initiation of spontaneous EAE.     

Resident and infiltrating central nervous system APCs regulate the emergence and resolution of experimental autoimmune encephalomyelitis

During experimental autoimmune encephalomyelitis (EAE), autoreactive Th1 T cells invade the CNS. Before performing their effector functions in the target organ, T cells must recognize Ag presented by CNS APCs. Here, we investigate the nature and activity of the cells that present Ag within the CNS during myelin oligodendrocyte glycoprotein-induced EAE, with the goal of understanding their role in regulating inflammation. Both infiltrating macrophages (Mac-1(+)CD45(high)) and resident microglia (Mac-1(+)CD45(int)) expressed MHC-II, B7-1, and B7-2. Macrophages and microglia presented exogenous and endogenous CNS Ags to T cell lines and CNS T cells, resulting in IFN-gamma production. In contrast, Mac-1(-) cells were inefficient APCs during EAE. Late in disease, after mice had partially recovered from clinical signs of disease, there was a reduction in Ag-presenting capability that correlated with decreased MHC-II and B7-1 expression. Interestingly, although CNS APCs induced T cell cytokine production, they did not induce proliferation of either T cell lines or CNS T cells. This was attributable to production by CNS cells (mainly by macrophages) of NO. T cell proliferation was restored with an NO inhibitor, or if the APCs were obtained from inducible NO synthase-deficient mice. Thus, CNS APCs, though essential for the initiation of disease, also play a down-regulatory role. The mechanisms by which CNS APCs limit the expansion of autoreactive T cells in the target organ include their production of NO, which inhibits T cell proliferation, and their decline in Ag presentation late in disease.     

Naringenin attenuates experimental autoimmune encephalomyelitis by protecting the intact of blood-brain barrier and controlling inflammatory cell migration

Targeting pathogenic immune cell trafficking poses an attractive opportunity to attenuate autoimmune disorders such as multiple sclerosis (MS). MS and its animal model, experimental autoimmune encephalomyelitis (EAE), are characterized by the immune cells-mediated demyelination and neurodegeneration of the central nervous system (CNS). Our previous study has proven that dietary naringenin ameliorates EAE clinical symptoms via reducing the CNS cell infiltration. The present study examined the beneficial effects of naringenin on maintaining the blood-brain barrier in EAE mice via dietary naringenin intervention. The results showed that naringenin-treated EAE mice had an intact blood-CNS barrier by increasing tight junction-associated factors and decreasing Evans Blue dye in the CNS. Naringenin decreased the accumulation and maturation of conventional dendritic cells (cDCs), CCL19, and CCR7 in the CNS. Also, naringenin blocked the chemotaxis and antigen-presenting function of cDCs that resulted in reducing T-cell secreting cytokines (IFN-γ, IL-17, and IL-6) in the spleen. Importantly, naringenin blocked pathogenic T cells infiltrated into the CNS and attenuates passive EAE. Therefore, by blocking chemokine-mediated migration of DCs and pathogenic T cells into the CNS, naringenin attenuates EAE pathogenesis and might be a potential candidate for the treatment of autoimmune diseases, such as MS and other chronic T-cell mediated autoimmune diseases.     

In vivo blockade of macrophage migration inhibitory factor ameliorates acute experimental autoimmune encephalomyelitis by impairing the homing of encephalitogenic T cells to the central nervous system

Macrophage migration inhibitory factor (MIF) is a cytokine that plays a critical role in the regulation of macrophage effector functions and T cell activation. However, its role in the pathogenesis of T cell-mediated autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), has remained unresolved. In this study, we report that anti-MIF Ab treatment of SJL mice with acute EAE improved the disease severity and accelerated the recovery. Furthermore, the anti-MIF treatment impaired the homing of neuroantigen-reactive pathogenic T cells to the CNS in a VCAM-1-dependent fashion. Interestingly, MIF blockade also decreased the clonal size of the neuroantigen-specific Th1 cells and increased their activation threshold. Taken together, the results demonstrate an important role for MIF in the pathogenesis of EAE/multiple sclerosis and suggest that MIF blockade may be a promising new strategy for the treatment of multiple sclerosis.     

Endogenous myelin basic protein is presented in the periphery by both dendritic cells and resting B cells with different functional consequences

Multiple sclerosis is an inflammatory disease believed to be triggered by erroneous activation of self-reactive T cells specific for myelin proteins such as myelin basic protein (MBP). Inflammation is limited to the CNS, suggesting that the myelin-specific T cells encounter their Ags only after they cross the blood-brain barrier. However, our previous studies in mice showed that MBP epitopes are constitutively presented in lymphoid tissues. Here we identified which APCs in lymph nodes present endogenous MBP epitopes and determined the functional consequences of this presentation for both naive and activated MBP-specific T cells. Both CD8alpha+ and CD8alpha- dendritic cells were potent stimulators of proliferation for both naive and previously activated/memory MBP-specific T cells. Surprisingly, resting B cells also presented endogenous MBP that was acquired using a BCR-independent mechanism. Interaction with resting B cells triggered proliferation of both naive and activated MBP-specific T cells. Activated/memory MBP-specific T cells proliferating in response to resting B cells presenting endogenous MBP did not produce cytokines and became more refractory to subsequent stimulation. Interestingly, cytokine production by activated/memory T cells was triggered by resting B cells if the number of MBP epitopes presented was increased by adding exogenous MBP peptide. These results suggest that activated MBP-specific T cells may become less pathogenic in vivo following encounter with resting B cells presenting steady-state levels of endogenous MBP but can expand and remain pathogenic if the amount of MBP presented by B cells is increased, which could occur during chronic demyelinating disease.     

LF 15-0195 inhibits the development of rat central nervous system autoimmunity by inducing long-lasting tolerance in autoreactive CD4 T cells

Experimental autoimmune encephalomyelitis (EAE) is a T cell-dependent autoimmune disease induced in susceptible animals by a single immunization with myelin basic protein (MBP). LF 15-0195 is a novel immunosuppressor that has been shown to have a potent immunosuppressive effect in several pathological manifestations. The purpose of this study was to investigate the effect of this drug on the induction and progression of established rat EAE and to dissect the mechanisms involved. We show that LF 15-0195 administration at the time of MBP immunization reduces the incidence and severity of EAE in Lewis rats. This drug also inhibits ongoing and passively induced EAE, indicating that LF 15-0195 affects already differentiated pathogenic lymphocytes. Compared with lymph node cells from untreated rats, lymphocytes from MBP-immunized rats treated with LF 15-0195 proliferated equally well in response to MBP in vitro, while their ability to produce effector cytokines and to transfer EAE into syngeneic recipients was significantly reduced. This phenomenon is stable and long-lasting. Indeed, neither IL-12 nor repeated stimulation with naive APC and MBP in vitro rendered MBP-specific CD4 T cells from protected rats encephalitogenic. In conclusion, LF 15-0195 treatment suppresses EAE by interfering with both the differentiation and effector functions of autoantigen-specific CD4 T cells.     

Inhibition of calpain attenuates encephalitogenicity of MBP-specific T cells

Multiple sclerosis (MS) is a T-cell mediated autoimmune disease of the CNS, possessing both immune and neurodegenerative events that lead to disability. Adoptive transfer (AT) of myelin basic protein (MBP)-specific T cells into naïve female SJL/J mice results in a relapsing–remitting (RR) form of experimental autoimmune encephalomyelitis (EAE). Blocking the mechanisms by which MBP-specific T cells are activated before AT may help characterize the immune arm of MS and offer novel targets for therapy. One such target is calpain, which is involved in activation of T cells, migration of immune cells into the CNS, degradation of axonal and myelin proteins, and neuronal apoptosis. Thus, the hypothesis that inhibiting calpain in MBP-specific T cells would diminish their encephalitogenicity in RR-EAE mice was tested. Incubating MBP-specific T cells with the calpain inhibitor SJA6017 before AT markedly suppressed the ability of these T cells to induce clinical symptoms of RR-EAE. These reductions correlated with decreases in demyelination, inflammation, axonal damage, and loss of oligodendrocytes and neurons. Also, calpain : calpastatin ratio, production of truncated Bid, and Bax : Bcl-2 ratio, and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated. Thus, these data suggest calpain as a promising target for treating EAE and MS.     

Suppression of experimental autoimmune encephalomyelitis by selective blockade of encephalitogenic T-cell infiltration of the central nervous system

Multiple sclerosis (MS) is a devastating neuroinflammatory disorder of the central nervous system (CNS) in which T cells that are reactive with major components of myelin sheaths have a central role. The receptor for advanced glycation end products (RAGE) is present on T cells, mononuclear phagocytes and endothelium. Its pro-inflammatory ligands, S100-calgranulins, are upregulated in MS and in the related rodent model, experimental autoimmune encephalomyelitis (EAE). Blockade of RAGE suppressed EAE when disease was induced by myelin basic protein (MBP) peptide or encephalitogenic T cells, or when EAE occurred spontaneously in T-cell receptor (TCR)-transgenic mice devoid of endogenous TCR-alpha and TCR-beta chains. Inhibition of RAGE markedly decreased infiltration of the CNS by immune and inflammatory cells. Transgenic mice with targeted overexpression of dominant-negative RAGE in CD4+ T cells were resistant to MBP-induced EAE. These data reinforce the importance of RAGE-ligand interactions in modulating properties of CD4+ T cells that infiltrate the CNS.     

Antigen processing of myelin basic protein is required prior to recognition by T cells inducing EAE.

The processing and presentation of whole myelin basic protein (MBP) and a 12 amino acid encephalitogenic peptide were investigated using MBP-immune and peptide-immune murine T cell lines. Myelin basic protein is the major component of central nervous system (CNS) white matter capable of inciting an autoimmune response which leads to the disease, experimental allergic encephalomyelitis (EAE), in a number of animal species. MBP-immune T cell lines caused a form of adoptively transferred EAE when injected into naive, syngeneic recipients. It has been found that both whole MBP and peptide required processing in order to induce proliferation of the T cell lines. The proliferative response was greatest when MBP was processed under conditions in which proteolysis was prevented. The demonstration that activation of encephalitogenic MBP immune T cells requires a processed form of MBP may have relevance to the human inflammatory CNS demyelinating condition, multiple sclerosis, for which EAE is the EAE is the prime animal model.     

Competition between foreign and self proteins in antigen presentation. Ovalbumin can inhibit activation of myelin basic protein-specific T cells.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory neurological disease initiated by activated T cells specific for the autoantigen, myelin basic protein (MBP). The ability of Lewis rat splenic T cells to transfer EAE after in vitro incubation with MBP-pulsed dendritic cells (DC) was used as an index of MBP-specific T cell activation. OVA, previously processed by macrophages, was incubated with MBP and DC at the pulsing stage to determine whether it could inhibit presentation of the autoantigen. At molar equivalents of 2.5:1 and 20:1 relative to MBP, processed OVA increasingly inhibited the ability of DC to activate MBP-specific T cells for EAE transfer. Unprocessed OVA, which cannot be presented immunogenically by Lewis rat DC, was much less effective. However, processed OVA added to DC after they had been pulsed with MBP could not compete. OVA also blocked appearance of EAE when mixed with MBP/CFA in the inoculum used for active induction of the disease. Splenic T cells from MBP + OVA/CFA-immunized rats transferred EAE with a substantially delayed onset, suggesting that a reduced number of MBP-specific T cells was generated by immunizing with the OVA + MBP mixture compared with MBP alone. Overall, the data indicate that fragments of a foreign protein, OVA, which can be bound by APC, can also inhibit presentation of encephalitogenic determinants of MBP to T cells.     

It is still not for the old iron: adjuvant effects of carbonyl iron in experimental autoimmune encephalomyelitis induction

Experimental autoimmune encephalomyelitis (EAE) is a model of multiple sclerosis. Dark Agouti rats immunized with spinal cord homogenate (SCH) and carbonyl iron (CI), as an adjuvant, develop severe hyperacute form of EAE. They succumb to EAE earlier and have higher clinical scores and lethality rate in comparison to counterparts immunized with SCH + complete Freund's adjuvant. There is no difference in the number of cells or in histological presentation of the CNS infiltrates of rats immunized with the two adjuvants. However, there are more granulocytes, NK and NKT cells, and less CD4(+) T cells in the spinal cord infiltrates of SCH + CI-immunized animals. Nitric oxide (NO)-generating enzyme inducible NO synthase have higher expression in spinal cord of SCH + CI-immunized rats, and this corresponds to more intensive nitrotyrosine formation in the CNS tissue of these rats. Abundant infiltration of granulocytes and NK cells into the CNS and excessive generation of peroxynitrite within the CNS of SCH + CI-immunized rats might account for the severe neurological deficits induced by immunization with CI. These factors should be closely examined in the fulminant forms of multiple sclerosis and acute disseminated encephalomyelitis, as they could represent a promising targets for therapy.     

Does the frequency and avidity spectrum of the neuroantigen-specific T cells in the blood mirror the autoimmune process in the central nervous system of mice undergoing experimental allergic encephalomyelitis?

In humans, studies of autoreactive T cells that mediate multiple sclerosis have been largely confined to testing peripheral blood lymphocytes. Little is known how such measurements reflect the disease-mediating autoreactive T cells in the CNS. This information is also not available for murine experimental allergic encephalomyelitis (EAE); the low number of T cells that can be obtained from the blood or the brain of mice prevented such comparisons. We used single-cell resolution IFN-gamma ELISPOT assays to measure the frequencies and functional avidities of myelin basic protein (MBP:87-99)-specific CD4 cells in SJL mice immunized with this peptide. Functional MBP:87-99-specific IFN-gamma-producing cells were present in the CNS during clinical signs of EAE, but not during phases of recovery. In contrast, MBP:87-99-specific T cells persisted in the blood during all stages of the disease, and were also present in mice that did not develop EAE. Therefore, the increased frequency of MBP:87-99-reactive T cells in the blood reliably reflected the primed state, but not the inflammatory activity of these cells in the brain. The functional avidity of the MBP:87-99-reactive T cells was identical in the brain and blood and did not change over 2 mo as the mice progressed from acute to chronic EAE. Therefore, high-affinity T cells did not become selectively enriched in the target organ, and avidity maturation of the MBP:87-99-specific T cell repertoire did not occur in the observation period. The data may help the interpretation of measurements made with peripheral blood lymphocytes of multiple sclerosis patients.     

Oral administration of myelin basic protein is superior to myelin in suppressing established relapsing experimental autoimmune encephalomyelitis

Oral administration of a myelin component, myelin basic protein (MBP), induces immunological unresponsiveness to CNS Ags and ameliorates murine relapsing experimental autoimmune encephalomyelitis (REAE). However, a recent clinical trial in which multiple sclerosis patients were treated with repeated doses of oral myelin was unsuccessful in reducing disease exacerbations. Therefore, we directly compared the tolerizing capacity of myelin vs MBP during REAE in B10.PL mice. Oral administration of high doses of myelin, either before disease induction or during REAE, did not provide protection from disease or decrease in vitro T cell responses. In contrast, repeated oral administration of high doses of MBP suppressed established disease and MBP-specific T cell proliferation and cytokine responses. The frequency of IL-2-, IFN-gamma-, and IL-5-secreting MBP-specific T cells declined with MBP feeding, implicating anergy and/or deletion as the mechanism(s) of oral tolerance after high Ag doses. We have previously shown that the dosage and timing of Ag administration are critical parameters in oral tolerance induction. Studies presented here demonstrate that Ag homogeneity is also important, i.e., homogeneous Ag (MBP) is more effective at inducing oral tolerance than heterogeneous Ag (myelin).