Experimental autoimmune myasthenia gravis in the mouse.

Myasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or the electric ray. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography. In addition, EAMG is frequently characterized by the presence of serum antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody-mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content. Another hallmark of the disease is complement and IgG deposits located at the neuromuscular junction, which can be visualized by immunofluorescence techniques.

Rad18 is a transcriptional target of E2F3.

The E2F family of transcription factors responds to a variety of intracellular and extracellular signals and, as such, are key regulators of cell growth, differentiation and cell death. The cellular response to DNA damage is a multistep process generally involving the initial detection of DNA damage, propagation of signals via posttranslational modifications (e.g., phosphorylation and ubiquitination) and, finally, the implementation of a response. We have previously reported that E2F3 can be induced by DNA damage, and that it plays an important role in DNA damage-induced apoptosis. Here, we demonstrate that E2F3 knockdown compromises two canonical DNA damage modification events, the ubiquitination of H2AX and PCNA. We find that the defect in these posttranscriptional modifications after E2F3 knockdown is due to reduced expression of important DNA damage responsive ubiquitin ligases. We characterized the regulation of one of these ligases, Rad18, and we demonstrated that E2F3 associates with the Rad18 promoter and directly controls its activity. Furthermore, we find that ectopic expression of Rad18 is sufficient to rescue the PCNA ubiquitination defect resulting from E2F3 knockdown. Our study reveals a novel facet of E2F3's control of the DNA damage response.

Experimental autoimmune myasthenia gravis in the mouse.

Myasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or the electric ray. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography. In addition, EAMG is frequently characterized by the presence of serum antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody-mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content. Another hallmark of the disease is complement and IgG deposits located at the neuromuscular junction, which can be visualized by immunofluorescence techniques.

E2F3 is a mediator of DNA damage-induced apoptosis.

The E2F transcription factors have emerged as critical apoptotic effectors. Herein we report that the E2F family member E2F3a can be induced by DNA damage through transcriptional and posttranslational mechanisms. We demonstrate that the posttranslational induction of human E2F3a is dependent on the checkpoint kinases. Moreover, we show that human E2F3a is a substrate for the checkpoint kinases (chk kinases) and that mutation of the chk phosphorylation site eliminates the DNA damage inducibility of the protein. Furthermore, we demonstrate that E2F1 and E2F2 are transcriptionally induced by DNA damage in an E2f3-dependent manner. Finally, using both in vitro and in vivo approaches, we establish that E2f3 is required for DNA damage-induced apoptosis. Thus, our data reveal the novel ability of E2f3 to function as a master regulator of the DNA damage response.

Cathepsin S is required for murine autoimmune myasthenia gravis pathogenesis.

Because presentation of acetylcholine receptor (AChR) peptides to T cells is critical to the development of myasthenia gravis, we examined the role of cathepsin S (Cat S) in experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. Compared with wild type, Cat S null mice were markedly resistant to the development of EAMG, and showed reduced T and B cell responses to AChR. Cat S null mice immunized with immunodominant AChR peptides showed weak responses, indicating failed peptide presentation accounted for autoimmune resistance. A Cat S inhibitor suppressed in vitro IFN-gamma production by lymph node cells from AChR-immunized, DR3-bearing transgenic mice. Because Cat S null mice are not severely immunocompromised, Cat S inhibitors could be tested for their therapeutic potential in EAMG.

Vaccination with purified Dr Fimbriae reduces mortality associated with chronic urinary tract infection due to Escherichia coli bearing Dr adhesin.

The vaccination of C3H/HeJ mice with Escherichia coli Dr fimbrial antigen reduced mortality associated with an experimental urinary tract infection due to a homologous strain bearing Dr adhesin. Immune sera with high titers of anti-Dr antibody inhibited bacterial binding to bladders and kidneys but did not affect the rate of renal colonization.

Circulating immune complexes augment severity of antibody-mediated myasthenia gravis in hypogammaglobulinemic RIIIS/J mice.

Experimental autoimmune myasthenia gravis (EAMG) is severe in RIIIS/J mice, despite a significant B cell immunodeficiency and a massive TCR V beta gene deletion. Severity of EAMG in RIIIS/J mice is greater than MHC-identical (H-2(r)) B10.RIII mice, suggesting the influence of non-MHC genes as an EAMG-potentiating factor in this strain. To delineate the role of deleted TCR V beta genes in RIIIS/J mice, we obtained (RIIIS/J x B10.RIII)F(1) (V beta(b/c)) x RIIIS/J (V beta(c)) backcross mice using Mendelian genetic methods and immunized them with acetylcholine receptor. EAMG susceptibility was not elevated in mice with V beta(c) genotype having 70% V beta gene deletion. Next, we performed microarray analysis on 12,488 spleen cDNAs obtained from spleens of naive RIIIS/J and B10.RIII mice. In RIIIS/J mice, 263 cDNAs were overexpressed and 303 cDNAs were underexpressed greater than 2-fold, compared with B10.RIII mice. TCR gene expression was augmented, whereas NK receptor, C1q, and C3 gene expressions were diminished in RIIIS/J mice. RIIIS/J mice also had increased lymph node T cell counts, elevated serum anti-AChR Ab levels, and serum C3 and C1q-conjugated circulating immune complex levels. A direct correlation between increased serum C1q-conjugated circulating immune complex levels and disease severity was observed in RIIIS/J mice.

Induction of myasthenia gravis in HLA transgenic mice by immunization with human acetylcholine receptors.

We utilized HLA transgenic mice to identify the dominant epitopes on the human (H)-AChR alpha subunit. The cytoplasmic H-AChR peptide alpha320-337 was the dominant T cell epitope for DQ8, DR3, and DQ8xDQ6 F1 mice. The H-AChR-immunized HLA-DQ8, DR3, DQ8xDR3 F1 and DQ8xDQ6 F1 mice developed clinical EAMG, whereas HLA-DQ6 mice were less susceptible.

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis.

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.

B7-1 costimulatory molecule is critical for the development of experimental autoimmune myasthenia gravis.

Following immunization with acetylcholine receptor (AChR), MHC class II-restricted, AChR-specific CD4 cell activation is critical for the development of experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 mice. To study the contributions of B7-1 and B7-2 costimulatory molecules in EAMG, B7-1, B7-2, and B7-1/B7-2 gene knockout (KO) mice were immunized with Torpedo AChR in CFA. Compared with wild-type C57BL6 mice, B7-1 and B7-1/2 KO mice were resistant to EAMG development. B7-1 KO mice had reduced anti-AChR Ab compared with C57BL/6 mice. However, neither B7-1 nor B7-2 gene disruption impaired AChR-induced or dominant alpha(146-162) peptide-induced in vitro lymphoproliferative responses. Blocking of the B7-1 or B7-2 molecule by specific mAbs in vivo led to a reduction in the AChR-specific lymphocyte response, and the reduction was more pronounced in mice treated with anti-B7-2 Ab. The findings implicate B7-1 molecules as having a critical role in the induction of EAMG, and the resistance of B7-1 KO mice is associated with suppressed humoral, rather than suppressed AChR-specific, T cell responses. The data also point to B7-2 molecules as being the dominant costimulatory molecules required for AChR-induced lymphocyte proliferation.

Suppressed clinical experimental autoimmune myasthenia gravis in bm12 mice is linked to reduced intracellular calcium mobilization and IL-10 and IFN-gamma release by acetylcholine receptor-specific T cells.

Class II MHC mutant bm12 mice have an increased resistance to experimental autoimmune myasthenia gravis (EAMG) compared to C57BL/6 mice. In vitro, this relative resistance was mainly associated with a reduced cytokine response to acetylcholine receptor (AChR) and its dominant pathogenic peptide alpha 146-162, whereas the response to the epitope alpha 111-126 remained intact. Calcium mobilization after stimulation of AChR-immune T cells with AChR or alpha 146-162 peptide, but not alpha 111-126 peptide, was decreased in bm12 compared to C57BL/6. Thus, the reduced incidence of clinical EAMG in bm12 is linked to lower IFN-gamma and IL-10 release, and intracellular calcium mobilization by alpha 146-162-specific T cells.

Resistance to experimental autoimmune myasthenia gravis in IL-6-deficient mice is associated with reduced germinal center formation and C3 production.

To provide direct genetic evidence for a role of IL-6 in experimental autoimmune myasthenia gravis (EAMG), IL-6 gene KO (IL-6(-/-)) mice in the C57BL/6 background were immunized with Torpedo californica acetylcholine receptor (AChR) and evaluated for EAMG. Only 25% of AChR-immunized IL-6(-/-) mice developed clinical EAMG compared to 83% of C57BL/6 (wild-type) mice. A significant reduction in the secondary anti-AChR Ab of IgG, IgG(2b), and IgG(2c), but not the primary or secondary IgM response was observed in AChR-immunized IL-6(-/-) mice, suggesting a possible defect in T cell help and class switching to anti-AChR IgG(2) isotype. The AChR-specific lymphocyte proliferative response, IFN-gamma, and IL-10 production were suppressed in AChR-immunized IL-6(-/-) mice. EAMG resistance in IL-6(-/-) mice was associated with a significant reduction in germinal center formation and decreased serum complement C3 levels. The data provide the first direct genetic evidence for a key role of IL-6 in the autoimmune response to AChR and in EAMG pathogenesis.

Role of IL-5 during primary and secondary immune response to acetylcholine receptor.

To analyze the role of the Th2 cytokine interleukin-5 (IL-5) in experimental autoimmune myasthenia gravis (EAMG) pathogenesis, we induced clinical EAMG in C57BL/6 and IL-5 gene-knockout (KO) mice in the C57BL/6 background. IL-5 KO mice had a significantly reduced incidence and severity of EAMG. Despite their increased resistance to EAMG, IL-5 KO mice displayed intact secondary antibody and lymphoproliferative responses to acetylcholine receptor (AChR) after immunization with this molecule. However, the relative resistance of IL-5 KO mice was associated with a reduced primary lymphocyte response to AChR, and reduced C3 levels in muscle extracts compared to those in C57BL/6 mice.

Mapping myasthenia gravis-associated T cell epitopes on human acetylcholine receptors in HLA transgenic mice.

Susceptibility to myasthenia gravis (MG) is positively linked to expression of HLA-DQ8 and DR3 molecules and negatively linked to expression of the DQ6 molecule. To elucidate the molecular basis of this association, we have induced experimental autoimmune MG (EAMG) in mice transgenic for HLA-DQ8, DQ6, and DR3, and in DQ8xDQ6 and DQ8xDR3 F(1) transgenic mice, by immunization with human acetylcholine receptor (H-AChR) in CFA. Mice expressing transgenes for one or both of the HLA class II molecules positively associated with MG (DQ8 and DR3) developed EAMG. T cells from DQ8 transgenic mice responded well to three cytoplasmic peptide sequences of H-AChR (alpha320-337, alpha304-322, and alpha419-437), of which the response to alpha320-337 was the most intense. DR3 transgenic mice also responded to this sequence very strongly. H-AChR- and alpha320-337 peptide-specific lymphocyte responses were restricted by HLA class II molecules. Disease resistance in DQ6 transgenic mice was associated with reduced synthesis of anti-AChR IgG, IgG(2b), and IgG(2c) Ab's and reduced IL-2 and IFN-gamma secretion by H-AChR- and peptide alpha320-337-specific lymphocytes. Finally, we show that DQ8 imparts susceptibility to EAMG and responsiveness to an epitope within the sequence alpha320-337 as a dominant trait.

Tumor necrosis factor receptor p55 and p75 deficiency protects mice from developing experimental autoimmune myasthenia gravis.

The precise pathogenic role of proinflammatory cytokines belonging to the tumor necrosis factor (TNF) family has not been investigated yet in antibody-mediated myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG). In this study we report that TNF receptor p55(-/-) p75(-/-) mice were resistant to the development of clinical EAMG induced by acetylcholine receptor (AChR) immunizations. The resistance was associated with reduced serum levels of IgG, IgG(1), IgG(2a), and IgG(2b) anti-AChR antibody isotypes. However, IgM anti-AChR antibodies were not reduced, suggesting defective anti-AChR IgG class switching in TNF receptor p55(-/-) p75(-/-) mice. We thus demonstrate the genetic evidence for the role of TNF receptor p55 and p75 in EAMG pathogenesis, and their requirement for the generation of anti-AChR IgG antibodies.

Treatment of experimental autoimmune myasthenia gravis with recombinant human tumor necrosis factor receptor Fc protein.

Lymphotoxin-alpha (TNF-beta) and TNF receptor p55 gene knockout mice are resistant to the development of antibody and complement mediated experimental autoimmune myasthenia gravis (EAMG), suggesting a possible role of TNF in mediating EAMG. Therefore, we tested the hypothesis that blocking the functional interaction of TNF with their receptors by soluble recombinant human TNFR:Fc would suppress the ongoing clinical EAMG. Recombinant human TNFR:Fc administered daily for 2 weeks to C57BL6 mice with ongoing clinical EAMG significantly improved clinical EAMG when compared with placebo-treated mice. A clinical trial of selected myasthenia gravis patients with recombinant human TNFR:Fc could be attempted.