Expression of Cell Adhesion Molecules and Cytokines in Murine Antigen-Induced Arthritis

Adhesion molecules and cytokines are important in chronic inflammatory conditions such as rheumatoid arthritis (RA) by virtue of their role in cell activation and emigration. Using immunohistochemical techniques we studied the expression of adhesion molecules and cytokines in cryopreserved sections of murine knee joint in the course of antigen-induced arthritis, an animal model of human RA. Various adhesion molecules and cytokines are expressed in the arthritic joint tissue. LFA-I, Mac-1, CD44, ICAM-I and P-selectin were strongly expressed in the acute phase and to a lesser degree in the chronic phase of arthritis. VLA-4 and VCAM-I appeared to be moderately expressed on day 1, L-selectin between days 1 and 3. LFA-I, Mac-I, CD44, a4-integrin, ICAM-I and the selectins were found expressed on cells of the synovial infiltrate, LFA-1, Mac-1 and ICAM-I on the synovial lining layer, and VCAM-I and P-selectin on endothelial cells. Expression of E-selectin could be demonstrated throughout the experiment at a low level in cells of the acute cell infiltrate. Cytokines, especially IL-2, IL-4, IL-6, TNF, and IFN-7, were heavily expressed during the acute phase of arthritis in cellular infiltrate. Taken together these data demonstrate that cytokines and their activation of adhesion molecules contribute to cell infiltration and activation during the initial phase of arthritis and to the induction and progression of tissue destruction in arthritic joints. These molecules might be potential targets for novel therapeutic strategies in inflammatory and arthritic disorders.     

IFN-β differentially regulates the function of T cell subsets in MS and EAE

Multiple sclerosis (MS) is considered as a T cell mediated autoimmune disease of the CNS, although a pathogenic role has also been attributed to other immune cell types as well as to environmental and genetic factors. Considering that T cells are interesting from an immunopathogenic point of view and consequently from a therapeutic perspective, various T cell targeted therapies have been approved for MS. Interferon beta (IFN-β) is widely used as first-line intervention for modulating T cell responses, although its pleiotropic and multifaceted activities influence its effectiveness on the disease development, with mechanisms that are not yet fully understood. Since different T cell populations, including pro-inflammatory and regulatory T cells, might affect the course of MS, the effects of IFN-β become even more complex.This review will summarize recent findings regarding the T cell targeted effect of IFN-β in MS and its animal model EAE, with emphasis on the direct actions of endogenous and exogenous IFN-β on each T cell subpopulation involved in CNS autoimmunity.Delineating how IFN-β exerts its action on different T cell types may eventually contribute to the designing of therapeutic strategies aiming to improve the effectiveness of this drug for MS treatment.     

Correlation of blood T cell and antibody reactivity to myelin proteins with HLA type and lesion localization in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS. The numbers of autoimmune T cells and Abs specific for proteins of CNS myelin are increased in the blood in some patients with MS. The aim of this study was to investigate whether there are correlations between the specificity of the autoimmune responses in the blood, the HLA molecules carried by the patient, and the clinical features of MS, because studies on experimental autoimmune encephalomyelitis, an animal model of MS, indicate that autoimmune responses targeting particular myelin proteins and the genetic background of the animal play a role in determining the pattern of lesion distribution. We tested blood T cell immunoreactivity to myelin proteins in 100 MS patients, 70 healthy controls, and 48 patients with other neurological disorders. Forty MS patients had strongly increased T cell reactivity to one or more myelin Ags. In these 40 patients, the most robust correlation was between CD4(+) T cell reactivity to myelin proteolipid protein residues 184-209 (PLP(184-209)) and development of lesions in the brainstem and cerebellum. Furthermore, carriage of HLA-DR4, -DR7, or -DR13 molecules by MS patients correlated with increased blood T cell immunoreactivity to PLP(184-209), as well as the development of lesions in the brainstem and cerebellum. Levels of PLP(190-209)-specific Abs in the blood also correlated with the presence of cerebellar lesions. These findings show that circulating T cells and Abs reactive against specific myelin Ags can correlate with lesion distribution in MS and suggest that they are of pathogenic relevance.     

Middle-age male mice have increased severity of experimental autoimmune encephalomyelitis and are unresponsive to testosterone therapy

Treatment with sex hormones is known to protect against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, little is known about how age affects the course of EAE or response to hormone treatment. This study demonstrates striking differences between middle-age vs young C57BL/6 male mice in the clinical course of EAE and response to both testosterone (T4) and estrogen (E2) hormone therapy. Unlike young males that developed an acute phase of EAE followed by a partial remission, middle-age males suffered severe chronic and unremitting EAE that was likely influenced by alterations in the distribution and function of splenic immunocytes and a significant reduction in suppressive activity of CD4+CD25+ regulatory T cells in the spleen and spinal cord. Middle-age males had reduced numbers of splenic CD4+ T cells that were generally hypoproliferative, but enhanced numbers of splenic macrophages and MHC class II-expressing cells, and increased secretion of the proinflammatory factors IFN-gamma and MCP-1. Surprisingly, middle-age males were unresponsive to the EAE-protective effects of T4 and had only a transient benefit from E2 treatment; young males were almost completely protected by both hormone treatments. T4 treatment of young males inhibited proliferation of myelin oligodendrocyte glycoprotein 35-55-specific T cells and secretion of TNF-alpha and IFN-gamma. The effects of T4 in vivo and in vitro were reversed by the androgen receptor antagonist, flutamide, indicating that the regulatory effects of T4 were mediated through the androgen receptor. These data are the first to define age-dependent differences in EAE expression and response to hormone therapy.     

Essential role of CD8+CD122+ regulatory T cells in the recovery from experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is one of the best-documented animal models of autoimmune disease. We examined the role of CD8+CD122+ regulatory T cells, which we previously identified as naturally occurring regulatory T cells that effectively regulate CD8+ T cells, in EAE. Depletion of CD8+CD122+ regulatory T cells by in vivo administration of anti-CD122 mAb resulted in persistent EAE symptoms. Transfer of CD8+CD122+ regulatory T cells into EAE mice at the peak EAE score clearly improved symptoms, indicating an important role of CD8+CD122+ regulatory T cells in the recovery phase of EAE. This was further confirmed by an increase and a decrease in the number of infiltrating T cells in the CNS and T cell cytokine production in mice that were depleted of or complemented with CD8+CD122+ cells. Furthermore, transfer of preactivated CD8+CD122+ regulatory T cells resulted in diminished EAE symptoms, especially in the recovery phase of EAE. These results elucidate the essential role of CD8+CD122+ regulatory T cells in the recovery phase of EAE and suggest the preventive effect of preactivated CD8+CD122+ regulatory T cells for EAE.     

Complementarity-determining region 3 spectratyping analysis of the TCR repertoire in multiple sclerosis

Multiple sclerosis (MS) is considered to be an autoimmune disease mediated by T cells reactive with Ags in the CNS. Therefore, it has been postulated that neuroantigen-reactive T cells bearing particular types of TCRs are expanded clonally during the course of the disease. However, there is a controversy with regard to the TCR usage by T cells associated with the development of MS. By the use of complementarity-determining region 3 spectratyping analysis that is shown to be a useful tool for identification of pathogenic TCR in autoimmune disease models, we tried to demonstrate that spectratype was T cells bearing particular types of TCR are activated in MS patients. Consequently, it was found that Vbeta5.2 were often oligoclonally expanded in peripheral blood of MS patients, but not of healthy subjects. Sequence analysis of the complementarity-determining region 3 region of spectratype-derived TCR clones revealed that the predominant TCR clone was different from patient to patient, but that similar results were obtained in a patient examined at different time points. More importantly, examination of cerebrospinal fluid T cells and longitudinal studies of PBLs from selected patients revealed that Vbeta5.2 expansion was detectable in the majority of patients examined. These findings suggest that Vbeta5.2 spectratype expansion is associated with the development of MS and that TCR-based immunotherapy can be applicable to MS patients if the TCR activation pattern of each patient is determined at different stages of the disease.     

Degenerate self-reactive human T-cell receptor causes spontaneous autoimmune disease in mice

Thyroid autoimmune disorders comprise more than 30% of all organ-specific autoimmune diseases and are characterized by autoantibodies and infiltrating T cells. The pathologic role of infiltrating T cells is not well defined. To address this issue, we generated transgenic mice expressing a human T-cell receptor derived from the thyroid-infiltrating T cell of a patient with thyroiditis and specific for a cryptic thyroid-peroxidase epitope. Here we show that mouse major histocompatibility complex molecules sustain selection and activation of the transgenic T cells, as coexpression of histocompatibility leukocyte antigen molecules was not needed. Furthermore, the transgenic T cells had an activated phenotype in vivo, and mice spontaneously developed destructive thyroiditis with histological, clinical and hormonal signs comparable with human autoimmune hypothyroidism. These results highlight the pathogenic role of human T cells specific for cryptic self epitopes. This new 'humanized' model will provide a unique tool to investigate how human pathogenic self-reactive T cells initiate autoimmune diseases and to determine how autoimmunity can be modulated in vivo.     

The role of CD4+CD25+ immunoregulatory T cells in the induction of autoimmune gastritis

A number of experimental models of organ-specific autoimmunity involve a period of peripheral lymphopenia prior to disease onset. There is now considerable evidence that the development of autoimmune disease in these models is due to the absence of CD4+CD25+ regulatory T cells. However, the role of CD4+CD25+ regulatory T cells in the prevention of autoimmune disease in normal individuals has not been defined. Here we have assessed the affect of depletion of CD4+CD25+ regulatory T cells in BALB/c mice on the induction of autoimmune gastritis. The CD4+CD25+ T cell population was reduced to 95% of the original population in adult thymectomized mice by treatment with anti-CD25 mAb. By 48 days after the anti-CD25 treatment, the CD4+CD25+ regulatory T cell population had returned to a normal level. Treatment of thymectomized adult mice for up to 4 weeks with anti-CD25 mAb did not result in the development of autoimmune gastritis. Furthermore, we have demonstrated that depletion of CD4+CD25+ regulatory T cells, together with transient CD4+ T lymphopenia, also did not result in the development of autoimmune gastritis, indicating that peripheral expansion of the CD4+ T cell population, per se, does not result in autoimmunity in adult mice. On the other hand, depletion of CD4+CD25+ T cells in 10-day-old euthymic mice resulted in a 30% incidence of autoimmune gastritis. These data suggest that CD4+CD25+ regulatory T cells may be important in protection against autoimmunity while the immune system is being established in young animals, but subsequently other factors are required to initiate autoimmunity.     

Galectin-1 suppresses autoimmune retinal disease by promoting concomitant Th2- and T regulatory-mediated anti-inflammatory responses

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4(+) regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology.     

Down-regulation of cell adhesion molecules LFA-1 and ICAM-1 after in vitro treatment with the anti-TNF-alpha agent thalidomide.

The tumor necrosis factor-alpha (TNF-alpha) inhibitor thalidomide is known to be a potent modulator of host immunity, a potential treatment for autoimmune disorders such as rheumatoid arthritis (RA) and a treatment for complications of HIV-1 infection. RA is an autoimmune disease of the joints that has been associated with hyperactivity of lymphocytes and other leukocytes, over-expression of pro-inflammatory cytokines (TNF-alpha and IL-1) and chronic debilitating inflammation. Thalidomide may play a role in RA treatment by altering leukocyte function through down-modulation of cell adhesion molecules necessary for leukocyte migration to inflammatory sites. The present study investigates down-regulation of cell adhesion molecules (ICAM-1 and LFA-1) and decreases in cell-cell contacts between human T leukemic (CEM) cells and human umbilical vein endothelial cells (HUVEC) after thalidomide exposure. CEM cells were cultured in RPMI 1640 medium with 0, 10 or 50 microg/ml thalidomide, stained with fluorescent monoclonal antibodies specific to ICAM-1 and LFA-1 and expression was measured with flow cytometry. For cell-cell adhesion measurements, monolayers of HUVEC cultured in Kaign's F-12 medium were incubated with thalidomide treated CEM cells stained with calcein AM. Specific cell adhesion between the two cell types was visualized with fluorescence microscopy. Thalidomide treatment significantly reduced cell adhesion molecule expression in a dose-dependent fashion and inhibited HUVEC/CEM cell adhesion. These data support the hypothesis that thalidomide has modulatory actions on leukocyte functions through expression of cell adhesion molecules.     

Refractory juvenile idiopathic arthritis: using autologous stem cell transplantation as a treatment strategy

Cellular immune therapy for severe autoimmune diseases can now be considered when such patients are refractory to conventional treatment. The use of autologous stem cell transplantation (ASCT) to treat human autoimmune diseases has been initiated following promising results in a variety of animal models. Anecdotal observations have been made of autoimmune disease remission in patients who have undergone allogeneic bone marrow transplantation as a result of coincidental haematological malignancies. The possibility of inducing immunological self-tolerance by ASCT is particularly attractive as a means for treating juvenile idiopathic arthritis (JIA). In this disease, ASCT restores self-tolerance both through a cell-intrinsic mechanism, involving the reprogramming of autoreactive T cells, and through a cell-extrinsic mechanism, involving a renewal of the immune balance between CD4+CD25+ regulatory T cells and other T cells. This review describes the clinical results of ASCT performed for this disease and the possible underlying immunological mechanisms.     

Changes in Blood B Cell-Activating Factor (BAFF) Levels in Multiple Sclerosis: A Sign of Treatment Outcome

Multiple sclerosis (MS) is mediated primarily by autoreactive T cells. However, evidence suggesting the involvement of humoral immunity in brain diseases has increased interest in the role of B cells and their products during MS pathogenesis. The major survival factor for B cells, BAFF has been shown to play a role in several autoimmune conditions. Elevated BAFF levels have been reported in MS animal model and during MS relapse in patients. Moreover, disease-modifying treatments (DMT) reportedly influence blood BAFF levels in MS patients, but the significance of these changes remains unclear. The present study addresses how blood BAFF levels are associated with the clinical course of relapsing-remitting MS and the effectiveness of DMT and short-term steroid treatment. During a prospective longitudinal follow-up of 2.3 years, BAFF was measured in the blood of 170 MS patients in the stable phase and within 186 relapses. BAFF levels were significantly higher in MS patients compared to healthy controls. However, stable MS patients without relapses exhibited significantly higher BAFF levels than relapsing patients. Treatment with interferon-β and immunosuppressants raised BAFF blood levels. Interestingly, a similar effect was not seen in patients treated with glatiramer acetate. Short-term treatment with high doses of intravenous methylprednisolone did not significantly alter plasma BAFF levels in 65% of relapsing-remitting MS patients. BAFF were correlated weakly but significantly with monocyte and basophil counts, but not with other blood cell types (neutrophils, lymphocytes, or eosinophils) or inflammatory biomarkers. To our knowledge, this is the first report demonstrating that higher blood BAFF levels may reflect a more stable and effective MS treatment outcome. These results challenge hypotheses suggesting that elevated blood BAFF levels are associated with more severe disease presentation and could explain the recent failure of pharmaceutical trials targeting BAFF with soluble receptor for MS treatment.     

G protein-coupled receptor kinase 2 in multiple sclerosis and experimental autoimmune encephalomyelitis

Many modulators of inflammation, including chemokines, neuropeptides, and neurotransmitters signal via G protein-coupled receptors (GPCR). GPCR kinases (GRK) can phosphorylate agonist-activated GPCR thereby promoting receptor desensitization. Here we describe that in leukocytes from patients with active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS, GRK2 levels are significantly reduced. Unexpectedly, cells from patients during remission express even lower levels of GRK2. The level of GRK2 in leukocytes of patients after stroke, a neurological disorder with paralysis but without an autoimmune component, was similar to GRK2 levels in cells from healthy individuals. In addition, we demonstrate that the course of recombinant myelin oligodendrocyte glycoprotein (1-125)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is markedly different in GRK2(+/-) mice that express 50% of the GRK2 protein in comparison with wild-type mice. Onset of EAE was significantly advanced by 5 days in GRK2(+/-) mice. The earlier onset of EAE was associated with increased early infiltration of the CNS by T cells and macrophages. Although disease scores in the first phase of EAE were similar in both groups, GRK2(+/-) animals did not develop relapses, whereas wild-type animals did. The absence of relapses in GRK2(+/-) mice was associated with a marked reduction in inflammatory infiltrates in the CNS. Recombinant myelin oligodendrocyte glycoprotein-induced T cell proliferation and cytokine production were normal in GRK2(+/-) animals. We conclude that down-regulation of GRK2 expression may have important consequences for the onset and progression of MS.     

Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis

Autoreactive CD4(+) T cells exist in normal individuals and retain the capacity to initiate autoimmune disease. The current study investigates the role of CD4(+)CD25(+) T-regulatory (T(R)) cells during autoimmune disease using the CD4(+) T cell-dependent myelin oligodendrocyte glycoprotein (MOG)-specific experimental autoimmune encephalomyelitis model of multiple sclerosis. In vitro, T(R) cells effectively inhibited both the proliferation of and cytokine production by MOG(35-55)-specific Th1 cells. In vivo, adoptive transfer of T(R) cells conferred significant protection from clinical experimental autoimmune encephalomyelitis which was associated with normal activation of autoreactive Th1 cells, but an increased frequency of MOG(35-55)-specific Th2 cells and decreased CNS infiltration. Lastly, transferred T(R) cells displayed an enhanced ability to traffic to the peripheral lymph nodes and expressed increased levels of the adhesion molecules ICAM-1 and P-selectin that may promote functional interactions with target T cells. Collectively, these findings suggest that T(R) cells contribute notably to the endogenous mechanisms that regulate actively induced autoimmune disease.     

High immunogenicity of intracellular myelin oligodendrocyte glycoprotein epitopes

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS with associated axonal loss. There is strong evidence for an autoimmune pathogenesis driven by myelin-specific T cells. Myelin oligodendrocyte glycoprotein (MOG) induces a type of experimental autoimmune encephalomyelitis in animals which is very MS-like since there are demyelinating CNS lesions and axonal loss. This underscores the potential role of MOG in MS pathogenesis. We performed a T cell reactivity pattern analysis of MS patients at the onset of relapse or progression of neurological deficits and controls that were stratified for the genetic risk factor HLA-DRB1*1501. For the first time, we show that there is an HLA-DR-restricted promiscuous dominant epitope for CD4(+) T cells within the transmembrane/intracellular part of MOG comprising aa 146-154 (FLCLQYRLR). Surprisingly, controls had broader T cell reactivity patterns toward MOG peptides compared with MS patients, and the transmembrane and intracellular parts of MOG were much more immunogenic compared with the extracellular part. Measurements of in vitro binding affinities revealed that HLA-DRB1*1501 molecules bound MOG 146-154 with intermediate and HLA-DRB1*0401 molecules with weak affinities. The binding of MOG 146-154 was comparable or better than myelin basic protein 85-99, which is the dominant myelin basic protein epitope in context with HLA-DRB1*1501 molecules in MS patients. This is the first study in which the data underscore the need to investigate the pathogenic or regulatory role of the transmembrane and intracellular part of MOG for MS in more detail.     

Glutamate excitotoxicity in a model of multiple sclerosis

Glutamate excitotoxicity mediated by the AMPA/kainate type of glutamate receptors damages not only neurons but also the myelin-producing cell of the central nervous system, the oligodendrocyte. In multiple sclerosis, myelin, oligodendrocytes and some axons are lost as a result of an inflammatory attack on the central nervous system. Because glutamate is released in large quantities by activated immune cells, we expected that during inflammation in MS, glutamate excitotoxicity might contribute to the lesion. We addressed this by using the AMPA/kainate antagonist NBQX to treat mice sensitized for experimental autoimmune encephalomyelitis, a demyelinating model that mimics many of the clinical and pathologic features of multiple sclerosis. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced dephosphorylation of neurofilament H, an indicator of axonal damage. Despite the clinical differences, treatment with NBQX had no effect on lesion size and did not reduce the degree of central nervous system inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect on the immune system. Thus, glutamate excitotoxicity seems to be an important mechanism in autoimmune demyelination, and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for multiple sclerosis.     

Suppression of Th1-Mediated Autoimmunity by Embryonic Stem Cell-Derived Dendritic Cells

We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs) using two models of autoimmune disease, namely non-obese diabetic (NOD) mice and experimental autoimmune encephalomyelitis (EAE). Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases.     

The antiepileptic drug valproic Acid restores T cell homeostasis and ameliorates pathogenesis of experimental autoimmune encephalomyelitis

Maintaining a constant number and ratio of immune cells is one critical aspect of the tight regulation of immune homeostasis. Breakdown of this balance will lead to autoimmune diseases such as multiple sclerosis (MS). The antiepileptic drug valproic acid (VPA) was reported to regulate the growth, survival, and differentiation of many cells. However, its function in T cell homeostasis and MS treatment remains unknown. In this study, VPA was found to reduce spinal cord inflammation, demyelination, and disease scores in experimental autoimmune encephalomyelitis, a mouse model of MS. Further study indicated that VPA induces apoptosis in activated T cells and maintains the immune homeostasis. This effect was found to be mainly mediated by the caspase-8/caspase-3 pathway. Interestingly, this phenomenon was also confirmed in T cells from normal human subjects and MS patients. Considering the long history of clinical use and our new findings, we believe VPA might be a safe and effective therapy for autoimmune diseases, such as multiple sclerosis.