| Objective:To investigate the ocular experimental autoimmune myasthenia gravis (oEAMG) induced by E. coli plasmid expressing recombinant human acetycholine receptor (H-AChR) γ subunit immunization in HLA-DQ8transgenic mice, and to determine the role and characteristic of acetylcholine receptor antibody (AChR-Ab) in the pathogenesis of oEAMG.Methods:Twenty-seven HLA-DQ8transgenic mice were divided equally into H-AChR γ subunit-immunized, crude E. coli extract-immunized and only CFA-groups randomly. Mice were immunized with AChR y subunit in CFA emulsion,20μg of crude E. coli extract in CFA or CFA only on day0,30and60. After the2nd immunization, the gMG and oMG scores and were evaluated weekly, generalized muscle weakness was also measured by grip strength machine weekly. The study of vestibulo-ocular reflexes was performed before termination, serum samples from individual mice were collected on day15,45after the2nd immunization, and the serum anti-AChR antibody levels were tested by RIA and serum anti-AChR Ig (Ig G,IgG1,IgG2b,IgG2c,IgM) isotype levels were tested by ELISA.Results:As opposed to the crude E. coli extract-and CFA-immunized mice with no ocular or generalized symptoms,8of9γ subunit-immunized mice (89%) had ocular symptoms,6of9mice with H-AChR γ subunit-immunization had complete ptosis in one or both eyes at termination, Mild generalized muscle weakness (grade1) was observed in6of9H-AChR γ subunit-immunized mice at termination. The scores of ocular and generalized symptoms of the γ subunit-immunized group were significantly higher than those in other two groups (all P<0.001). The difference among the three groups in grip strengths attained statistical significance starting on day35after the2nd H-AChR γ subunit immunization (all p<0.05) and reached the lowest level at termination. The horizontal optokinetic response gains were significantly lower in H-AChR γ subunit immunization group mice as compared to other two group mice(all P<0.05). On day15and45after the2nd immunization, the levels of anti-AChR Abs and anti-AChR Ig (Ig G,IgG1,IgG2b, IgG2c,IgM) isotype levels were significantly higher in the sera of H-AChR γ subunit-immunized mice as compared to other two groups (all P<0.001)Conclusions:oEAMG can be generated in HLA-DQ8transgenic mice by H-AChR γ subunit immunization, and AChR-Abs played a key role in the pathogenesis of oEAMG. Objective:To explore the immunologic pathogenesis of oEAMG induced by recombinant H-AChR γ subunit immunization.Methods:DQ8mice were immunized as described above with20μg of AChR γ subunit,20μg of crude E. coli extract or CFA only. Seven days following y subunit immunization, mice were euthanized and draining lymph node cells (LNC) and spleen lymphocytes were cultured in vitro. The lymphocyte proliferation was estimated by the3H-TdR incorporation, and the supernatant were collected to observe the IL-2, IFN-y, IL-6, IL-10production by ELISA. The day30after the3rd immunization, The percentages of CD4+CD25+and CD19+cell in LNC and spleen lymphocytes of the mice from H-AChR γ subunit-immunized, crude E. coli extract-immunized and CFA-groups were detected by flow cytometry. IgG, C3and membrane attack complex (MAC) deposits at the NMJ from LM and EOM samples of the mice were detected by immunofluorescence assay. The levels of AChR α, γ and s subunit mRNA expression of LM and EOM samples of the mice were evaluated by RT-PCR.Results:LNCs and spleen lymphocytes of H-AChR γ subunit-immunized mice showed significantly increased proliferative responses as compared to E. coli extract-immunized and CFA-immunized mice (all P<0.05). LNCs and spleen lymphocytes of H-AChR γ subunit-immunized mice exhibited significantly enhanced IFN-γ and IL-2as compared to those of the other two groups (all P<0.05). There were no significant differences in IL-6, IL-10levels among three groups (all P>0.05). The percentages of CD4+CD25+and CD19+cell in LNC and spleen lymphocytes of the mice from H-AChR γ subunit-immunized were significantly higher than those in other two groups (all P<0.05). H-AChR γ subunit-immunized mice had abundant amounts of C3, IgG and MAC deposits in extraocular and limb muscle NMJs, whereas these deposits could hardly be detected on the NMJs of crude E. coli extract and CFA immunized mice (all P<0.001). The EOM and limb muscle samples from H-AChR γ subunit-immunized mice demonstrated significantly increased mRNA levels for AChR α, γ and ε subunit(all P<0.05)Conclusions:oEAMG and gEAMG could be triggered by autoimmunity to the recombinant H-AChR γ subunit. Multiple mechanisms including cellular Immunity, humoral immunity and complement are involved in the pathogenesis of oEAMG. Objective:To study the role of H-AChR ε immunity in the pathogenesis of oEAMG, and to further explore the relationship between the autoimmune response induced by AChR subunit and oEAMG.Methods:Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the levels of AChR α,γ andesubunit genes mRNA expression in the extra-ocular and limb muscles of HLA-DQ8transgenic mice of Ow,1w,2w,4w of their development. HLA-DQ8transgenic mice were immunized with AChR ε subunit in CFA emulsion,20μg of crude E. coli extract in CFA or CFA only on day0,30and60. Mice After the2nd immunization, the gMG and oMG scores and were evaluated weekly, and grip strength machine was also used to measure the generalized muscle weakness weekly, serum samples from individual mice were collected on day45after the2nd immunization, and the serum anti-AChR antibody levels were tested by RIA and serum anti-AChR Ig (Ig G,IgG1,IgG2b,IgG2c,IgM) isotype levels were tested by ELISA. IgG, C3and membrane attack complex (MAC) deposits at the NMJ from LM and EOM samples of the mice were detected by immunofluorescence assay before termination.Results:There were no significant differences in AChR α,γ and ε subunit expression of EOM samples from HLA-DQ8mice at different stages of ontogeny (P>0.05). At Ow after birth, the AChR Y subunit was detected whereas AChR ε subunit was not detected in the LM samples. The AChR ε subunit mRNA was initially detected at1w, the beginning of AChR ε subunit expression was coincident with the beginning of disappearance in AChR Y subunit expression. There were no significant differences in AChR a subunit expression of LM samples at different stages (P>0.05). As opposed to the crude E. coli extract-and CFA-immunized mice with no ocular or generalized symptoms,3of9ε subunit-immunized mice (33%) had ocular symptoms, Mild generalized muscle weakness (grade1) was observed in2of9H-AChR ε subunit-immunized mice at termination (P<0.05). The scores of ocular and generalized symptoms of the s subunit-immunized group were significantly higher than those in other two groups (all P<0.05). There were no significant differences in grip strengths among three groups (all P>0.05). On day45after the2nd immunization, the levels of anti-AChR Abs and anti-AChR Ig (Ig G,IgG,IgG2b,IgG2c,IgM) isotype levels were significantly higher in the sera of H-AChR ε subunit-immunized mice as compared to other two groups (all P<0.01). H-AChR ε subunit-immunized mice with prominent ocular symptoms had abundant amounts of C3, IgG and MAC deposits in extraocular and limb muscle NMJs, whereas these deposits could hardly be detected on the NMJs of crude E. coli extract and CFA immunized mice (all P <0.01).Conclusions:HLA-DQ8transgenic mice showed lower susceptibility to H-AChR ε subunit immunization, which can be explained by the weaker autoimmune response induced by the specific interaction between AChR ε subunit and HLA-DQ8molecular.
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