The purpose of this study was to identify the endothelial cell antigens that react with circulating antiendothelial antibody (AECA) in mixed connective tissue disease (MCTD). We screened serum AECA reactivity in 23 patients with MCTD using a human glomerular endothelial cell (HGEC) cellular ELISA. Proteomics, two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry were used to identify the endothelial cell antigens of HGECs that reacted with serum antibodies from MCTD patients. Sera from 12 patients (52.0%) were positive for anti-HGEC antibody based on cellular ELISA. MALDI-TOF mass spectrometry used in combination with immunoblotting using serum antibody revealed one protein spot that represented a 36-kDa cell component of HGECs, with an isoelectric point (IP) of about 9, which had a high homology with the voltage-dependent anion-selective channel 1 (VDAC-1). This protein spot was confirmed to react with the antibody specific to VDAC-1. This is the first report of the presence of antibody to VDAC-1 from HGECs in the sera from MCTD patients. Although future studies will be needed to clarify the disease specificity of the a-VDAC-1 antibody in MCTD, the results show that modern proteomics technology is useful for identifying antigens that react with AECA in autoimmune diseases such as MCTD.
Background: Acute post-streptococcal glomerulonephritis (APSGN) is induced by glomerular deposition of nephritogenic streptococcal antigen-antibody complexes. Recently, a streptococcal antigen, nephritis-associated plasminogen receptor (NAPlr) was purified from ruptured streptococcal cell supernatants (RCS). However, the cellular and molecular mechanisms of NAPlr action on the glomerular vas culature are still unknown. Methods: Expression of cell adhesion molecules were measured by cellular ELISA (enzyme-linked immunosorbent assay), immunofluorescence microscopy and Western blot analysis. Results: RCS and NAPlr significantly decreased the PECAM-1 expression in human glomerular endothelial cells (HGECs) as compared to that in the control cells. Plasminogen treatment reversed the RCS or NAPlr-induced decrease of PECAM-1 expression and increase of MCP-1 expression. Immunofluorescent microscopy and Western blot analysis also showed that PECAM-1 expression in HGECs was downregulated upon treatment with RCS or NAPlr and this effect was reversed by plasminogen treatment. Furthermore, we found that tumor necrosis factor-α production in culture medium of HGECs was increased at the lower level when the culture system was treated with RCS. Conclusion: RCS and NAPlr modulated PECAM-1 expression and MCP-1 production in HGECs, indicating the involvement of NAPlr in inflammatory cell accumulation in glomerular tufts and functional abnormality of glomerular microvasculature such as hyperpermeability.
The purpose of this study was to identify the endothelial cell antigens that react with circulating antiendothelial antibody (AECA) in mixed connective tissue disease (MCTD). We screened serum AECA reactivity in 23 patients with MCTD using a human glomerular endothelial cell (HGEC) cellular ELISA. Proteomics, two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry were used to identify the endothelial cell antigens of HGECs that reacted with serum antibodies from MCTD patients. Sera from 12 patients (52.0%) were positive for anti-HGEC antibody based on cellular ELISA. MALDI-TOF mass spectrometry used in combination with immunoblotting using serum antibody revealed one protein spot that represented a 36-kDa cell component of HGECs, with an isoelectric point (IP) of about 9, which had a high homology with the voltage-dependent anion-selective channel 1 (VDAC-1). This protein spot was confirmed to react with the antibody specific to VDAC-1. This is the first report of the presence of antibody to VDAC-1 from HGECs in the sera from MCTD patients. Although future studies will be needed to clarify the disease specificity of the a-VDAC-1 antibody in MCTD, the results show that modern proteomics technology is useful for identifying antigens that react with AECA in autoimmune diseases such as MCTD.
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