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Objective: Multiple sclerosis (MS) has affected over 2 million people worldwide and it is thought to be initiated by the activated central nervous system (CNS). Reactive CD4+ T cells (TH1, TH17, and Treg phenotypes) are crucial to MS. The TH1 phenotype can promote major histocompatibility complex-II expression and TH17 can induce inflammatory gene expression. Curcumin, a yellow pigment, is found in turmeric rhizomes and has been reported to have various activities, such as anti-proliferative and anti-inflammatory activity. Curcumin has great potential in MS treatment. Little is known about the effect of curcumin on MS. Therefore, we investigated the effect of curcumin on MS, especially on CD4+ T cells., Materials and Methods: CD4+ T cells (TH1, TH17, and Treg cells) were cultured in Iscove's Modified Dulbecco's Medium (IMDM) medium. Cell proliferation was evaluated by MTT assay. The ability of individual CD4+ T cells to aggregate into viable colony clusters was assessed by clonogenic survival assay. Apoptosis of CD4+ T cells was determined by flow cytometry. The expression of Bcl-2, Bax, and active caspase-3 was detected by Western blotting. The effect of curcumin on the activation molecule was also evaluated by flow cytometry., Results: MTT assay showed that curcumin significantly inhibited CD4+ T cell viability. Furthermore, TH1, TH17, and Treg all showed a dose-dependent but not time-dependent. The results of clonogenic survival assay revealed that curcumin markedly decreased the colony formation ability of CD4+ T cells. Flow cytometry results indicated that curcumin-induced remarkable apoptosis in TH1, TH17, and Treg cells. After treatment with curcumin, the expression of Bcl-2 was decreased and that of Bax and active caspase-3 was increased. Western blotting results also showed that curcumin-induced apoptosis in CD4+ T cells. Hence, our results demonstrated that curcumin inhibited CD4+ T cell proliferation via inducing apoptosis in CD4+ T cells. Meanwhile, flow cytometry results also showed that curcumin directly inhibited CD4+ T cell activation., Conclusions: Curcumin could inhibit CD4+ T cell proliferation and effector cell activation.

CD62L (l-selectin, mel 14) regulates naïve T cell homing into lymph nodes and the migration of leucocytes to sites of inflammation. The requirement of CD62L in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has been demonstrated previously. However, it remains controversial as to whether CD62L is required for the induction or the effector phase of EAE. It is also unclear whether other non-T effector cells need CD62L to enter the central nervous system (CNS) parenchyma and exert their damaging effects on myelin. We report that mice with a targeted mutation of CD62L are resistant to Myelin oligodendrocyte glycoprotein peptide-induced EAE. CD62L-deficient mice had no peptide-specific T cell responses in the draining lymph nodes and had lower levels of peptide-specific T cell responses in spleens at a later time point. Adoptive transfer studies showed that CD62L-deficient mice were fully susceptible to adoptive transfer EAE induced by either wildtype or CD62L-deficient T cells. Moreover, CD62L-deficient, F4/80(+) macrophages can be efficiently recruited into the CNS parenchyma. These data suggest that CD62L is required for the induction of encephalitogenic T cells during EAE development, but is not required by T and non-T effector cells to attack the CNS parenchyma.

Induction of myelin-specific CD4 T cells is a pivotal event in the development of experimental autoimmune encephalomyelitis (EAE). Other checkpoints in EAE pathogenesis have not been clearly defined, although multiple genetic loci are known to influence EAE development. We report here that targeted mutation of the heat-stable antigen (HSA) abrogates development of EAE despite a complete lack of effect on induction of autoimmune T cells. To test whether T-cell expression of HSA is sufficient, we created transgenic mice in which HSA is expressed exclusively in the T-cell lineage. We found that these mice remain resistant to EAE induction. Adoptive transfer studies demonstrate that both T cells and non-T cells must express HSA in order for the pathogenic T cells to execute their effector function. Moreover, HSAIg, a fusion protein consisting of the extracellular domain of the HSA and the Fc portion of immunoglobulin, drastically ameliorates the clinical sign of EAE even when administrated after self-reactive T cells had been expanded. Thus, identification of HSA as a novel checkpoint, even after activation and expansion of self-reactive T cells, provides a novel approach for immunotherapy of autoimmune neurologic diseases, such as multiple sclerosis.

Induction of mucosal tolerance by inhalation of soluble peptides with defined T cell epitopes is receiving much attention as a means of specifically down-regulating pathogenic T cell reactivities in autoimmune and allergic disorders. Experimental autoimmune encephalomyelitis (EAE) induced in the Lewis rat by immunization with myelin basic protein (MBP) and Freund's adjuvant (CFA) is mediated by CD4+ T cells specific for the MBP amino acid sequences 68-86 and 87-99. To further define the principles of nasal tolerance induction, we generated three different MBP peptides (MBP 68-86, 87-99 and the non-encephalitogenic peptide 110-128), and evaluated whether their nasal administration on day -11, -10, -9, -8 and -7 prior to immunization with guinea pig MBP (gp-MBP) + CFA confers protection to Lewis rat EAE. Protection was achieved with the encephalitogenic peptides MBP 68-86 and 87-99, MBP 68-86 being more potent, but not with MBP 110-128. Neither MBP 68-86 nor 87-99 at doses used conferred complete protection to gp-MBP-induced EAE. In contrast, nasal administration of a mixture of MBP 68-86 and 87-99 completely blocked gp-MBP-induced EAE even at lower dosage compared to that being used for individual peptides. Rats tolerized with MBP 68-86 + 87-99 nasally showed decreased T cell responses to MBP reflected by lymphocyte proliferation and IFN-gamma ELISPOT assays. Rats tolerized with MBP 68-86 + 87-99 also had abrogated MBP-reactive IFN-gamma and tumor necrosis factor-alpha mRNA expression in lymph node cells compared to rats receiving MBP 110-128 nasally, while similar low levels of MBP-reactive transforming growth factor-beta and IL-4 mRNA expressing cells were observed in the two groups. Nasal administration of MBP 68-86 + 87-99 only slightly inhibited guinea pig spinal cord homogenate-induced EAE, and passive transfer of spleen mononuclear cells from MBP 68-86 + 87-99-tolerized rats did not protect naïve rats from EAE. Finally, we show that nasal administration of MBP 68-86 + 87-99 can reverse ongoing EAE induced with gp-MBP, although higher doses are required compared to the dosage needed for prevention. In conclusion, nasal administration of encephalitogenic MBP peptides can induce antigen-specific T cell tolerance and confer incomplete protection to gp-MBP-induced EAE, and MBP 68-86 and 87-99 have synergistic effects. Non-regulatory mechanisms are proposed to be responsible for tolerance development after nasal peptide administration.

Nasal administration of soluble antigens is an exciting means of specifically down-regulating pathogenic T-cell reactivities in autoimmune diseases. The mechanisms by which nasal administration of soluble antigens suppresses autoimmunity are poorly understood. To define further the principles of nasal tolerance induction, we studied the effects of nasal administration of myelin basic protein (MBP) on experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. EAE is a CD4+ T-cell-mediated animal model for human multiple sclerosis. Nasal administration of guinea-pig (gp)-MBP at a dose as low as 30 micrograms/rat can completely prevent gp-MBP-induced EAE, whereas nasal administration of bovine (b)-MBP is not effective even at a much higher dosage. Cellular immune responses, as reflected by T-cell proliferation and interferon-gamma (IFN-gamma)-ELISPOT, were suppressed in rats receiving the two different doses (30 and 600 micrograms/rat) of gp-MBP, but not after administration of b-MBP. Rats tolerized with both doses of gp-MBP had also abrogated MBP-induced IFN-gamma mRNA expression in popliteal and inguinal lymph node mononuclear cells compared with rats receiving phosphate-buffered saline nasally. However, adoptive transfer revealed that only spleen mononuclear cells from rats pretreated with a low dose, but not from those pretreated with a high dose, of gp-MBP transferred protection to actively induced EAE. Low-dose (30 micrograms/rat) gp-MBP-tolerized rats also had high numbers of interleukin-4 (IL-4) mRNA-expressing lymph node cells, while high-dose (600 micrograms/rat) gp-MBP-tolerized rats had low numbers of IL-4 mRNA-expressing lymph node cells. Our data suggest an exquisite specificity of nasal tolerance. Dose-dependent mechanisms also relate to nasal tolerance induction and protection against EAE in the Lewis rat.

EAE is an autoimmune disease of the central nervous system (CNS) that serves as an experimental model for the human inflammatory demyelinating disease multiple sclerosis (MS). Antigen-based immunotherapy including soluble antigen administration via feeding has been shown to be successful in treating EAE in rodents. In the present study, we explore nasal administration of small amounts of myelin basic protein (MBP) as a potential means of treatment of protracted, relapsing EAE (PR-EAE) in a novel DA rat system. We found that nasal administration of MBP prevented EAE induced with whole spinal cord homogenate + Freund's incomplete adjuvant (FIA), and strongly down-regulated levels of MBP-reactive interferon-gamma (IFN-gamma)-secreting Th1-like cells. However, in rats with ongoing PR-EAE receiving the same regimen of MBP, a trend of aggravated disease was recorded, in conjunction with augmented levels of MBP-reactive IFN-gamma-secreting Th1-like splenocytes during the acute phase of EAE. These data have implications for the clinical application of nasal tolerance to autoimmune diseases.