Myasthenia gravis is a T cell-dependent, antibody-mediated autoimmune disease. A dual altered peptide ligand (APL) that is composed of the tandemly arranged two single amino acid analogs of two myasthenogenic peptides, p195–212 and p259–271, was demonstrated to down-regulate in vitro and in vivo myasthenia gravis-associated autoreactive responses. The aims of this study were to demonstrate the suppressive properties and to elucidate the mechanism of action of the dual APL on a T cell line specific to the myasthenogenic peptide p195–212. We demonstrate here that incubation of cells of the line with the dual APL resulted in the inhibition of proliferation and secretion of IL-2 and IFN-γ triggered by p195–212. In contrast, secretion of TGF-β and IL-10 was upregulated. The dual APL induced the generation of CD4+CD25+ cells that were characterized by the expression of CD45Rblow, cytotoxic T lymphocyte-associated antigen-4, TGF-β, CD62L, Foxp3, and neuropilin. In addition, the dual APL-treated cells were capable of inhibiting the proliferation response of the line when the two sets of cells were cocultured. The role of CD4+CD25+ cells was further confirmed by demonstrating that the suppression was abrogated by blocking/neutralization of CD25. Thus, the dual APL acts by inducing the formation of CD4+CD25+ regulatory cells. By using a T cell line, we could show that the immunosuppressive CD4+CD25+ cells were indeed induced by the dual APL and are not part of the naturally occurring regulatory cells
SummaryMyasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent, antibody-mediated autoimmune diseases. A dual altered peptide ligand (APL) that is composed of the tandemly arranged two single amino acid analogues of two myasthenogenic peptides, p195-212 and p259-271, was demonstrated to down-regulate in vitro and in vivo MG-associated autoreactive responses. The aims of this study were to investigate the possible role of Fas-FasL-mediated apoptosis in the downregulatory mechanism of the dual APL. We demonstrate here the effect of the dual APL on expression of key molecules involved in the Fas-FasL pathway, in a p195-212-specific T cell line, in mice immunized with Torpedo acetylcholine receptor and in mice afflicted with EAMG (induced with the latter). In vitro and in vivo results show that the dual APL up-regulated expression of Fas and FasL on the CD4 cells. Expression of the pro-apoptotic molecules, caspase 8 and caspase 3, was significantly up-regulated, while anti-apoptotic cFLIP and Bcl-2 were down-regulated upon treatment with the dual APL. The dual APL also increased phosphorylation of the mitogen-activated protein kinases, c-Jun-NH2-terminal kinase and p-38, known to play a role in the regulation of FasL expression. Further, in the T cell line incubated with the dual APL as well as in mice of the SJL inbred strain immunized with the myasthenogenic peptide and treated concomitantly with the dual APL, the percentage of apoptotic cells increased. Results strongly indicate that up-regulation of apoptosis via the Fas-FasL pathway is one of the mechanisms by which the dual APL reverses EAMG manifestations in C57BL/6 mice.
Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are T cell-dependent antibody-mediated autoimmune diseases. A dual altered peptide ligand (APL) that is composed of the tandemly arranged two single amino acid analogues of two myasthenogenic peptides, p195-212 and p259 -271, down-regulated in vitro and in vivo MG-associated autoreactive responses. The dual APL was shown to exert its beneficial effects by up-regulating ERK1,2 in CD4 ؉ CD25 ؉ regulatory cells. In this study, we investigated a novel 50-kDa ERK-like protein (ERK-50) that is up-regulated significantly in addition to ERK1,2 after treatment with the dual APL. We report here that ERK-50 was upregulated in LN cells and in LN-derived T cells of mice that were immunized with the myasthenogenic peptides and treated with the dual APL. Moreover, ERK-50 was up-regulated in dual-APLtreated mice that were immunized with the Torpedo acetylcholine receptor. ERK-50 was demonstrated to be recognized by antibodies directed against the C and N termini of ERK1, against the C terminus of ERK2, and against general ERK. The 50-kDa ERK was shown to be stimulated by Con A, and inhibition of MEK1 down-regulated the 50-kDa ERK as was shown for ERK1,2. However, 4-phorbol 12-myristate 13-acetate (TPA) did not stimulate ERK-50. Finally, the activated ERK-50 was up-regulated in the dual-APL-induced CD4 ؉ CD25 ؉ regulatory cells. Thus, ERK-50 is suggested to be a novel ERK isoform, being up-regulated in response to treatment with the dual APL.autoimmunity ͉ immunomodulation ͉ kinase
Myasthenia gravis (MG) and its animal model experimental autoimmune MG (EAMG), are T‐cell dependent, antibody‐mediated autoimmune disorders. A dual altered peptide ligand (APL) composed of the tandemly arranged two single amino acids analogs of two myasthenogenic peptides, p195–212 and p259–271, was demonstrated to downregulate, in vitro and in vivo, MG‐associated autoimmune responses. Upregulation of regulatory CD4+CD25+ cells plays a key role in the mechanism of action of the dual APL. The objectives of the present study were to address the involvement of extracellular‐regulated kinase (ERK)1,2 in the mechanisms by which the dual APL‐induced CD4+CD25+ cells suppress MG‐associated autoimmune responses. We demonstrate here that administration of the dual APL increased activated ERK1,2 in the CD4+CD25+‐enriched population. Further, inhibition of ERK1,2 by its inhibitor, U0126, in dual APL‐induced CD4+CD25+ cells, abrogated their ability to suppress interferon (IFN)‐γ secretion by lymph node (LN) cells of mice that were immunized with the myasthenogenic peptide. Moreover, inhibition of ERK1,2 in the dual APL‐induced regulatory CD4+CD25+ cells, resulted in downregulation of the forkhead box p3 (Foxp3) gene and protein expression levels, as well as in the downregulation of CD4+CD25+ development, suggesting that the active suppression exerted by the dual APL via CD4+CD25+ cells depends on ERK1,2 activity.
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