Identification of HnRNP-A2/B1 as a Target Antigen of Anti-Endothelial Cell IgA Antibody in Behçet's Disease  Sung Bin Cho, Keun Jae Ahn, Do Hee Kim, Zhenlong Zheng, Suhyun Cho, Shin-Wook Kang, Ju Hee Lee, Yong-Beom Park, Kwang Hoon Lee, Dongsik Bang  Journal of Investigative Dermatology  Volume 132, Issue 3, Pages 601-608 (March 2012) DOI: 10.1038/jid.2011.397 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 ELISA, western blotting, and immunoprecipitation using human dermal microvascular endothelial cell (HDMEC) antigen. (a) Serum samples from 10 Behçet's disease (BD) patients, presenting relatively high optical density values on ELISA of extracts of HDMECs with serum samples from 30 BD patients, were selected (above the bar). (b) Western blotting using extracts of HDMECs with selected serum samples of 10 BD patients presented a band of HDMEC protein between 36 and 40kD in all 10 patients but not in normal controls or patients with other rheumatic diseases. (c) Immunoprecipitation was performed to identify the anti-endothelial cell IgA antibody–binding antigen and revealed a 36–40kD protein band of HDMEC antigen reacting with IgA antibodies of BD patients. Journal of Investigative Dermatology 2012 132, 601-608DOI: (10.1038/jid.2011.397) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Western blotting using recombinant human heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1. Western blotting using recombinant human hnRNP-A2/B1 was performed with sera from patients with Behçet's disease (BD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Takayasu's arteritis (TA), IgA nephropathy (IgAN), and from healthy controls (HC) as a primary antibody and goat anti-human IgA antibody as a secondary antibody. Reactivity of serum IgA against human recombinant hnRNP-A2/B1 was detected in (a, b) 83.3% of BD patients, (b) 13.3% of SLE patients, (c) 26.7% of RA patients, (c) 30% of TA patients, and (a) 20% of healthy controls on western blots, whereas (c) none of the 30 IgAN patients presented reactivity against recombinant human hnRNP-A2/B1. Journal of Investigative Dermatology 2012 132, 601-608DOI: (10.1038/jid.2011.397) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Western blotting using recombinant human α-Synuclein as recombinant protein control. Western blotting using recombinant human α-Synuclein was performed with sera from patients with Behçet's disease (BD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Takayasu's arteritis (TA), IgA nephropathy (IgAN), and from healthy controls (HC) as a primary antibody and goat anti-human IgA antibody as a secondary antibody. Reactivity of serum IgA against human recombinant α-Synuclein was detected in (a, b) 16.7% of BD patients, (b) 30% of SLE patients, (c) 20% of RA patients, (c) 10% of TA patients, (c) 3.3% of IgAN patients, and (a) 13.3% of the healthy controls on western blots. Journal of Investigative Dermatology 2012 132, 601-608DOI: (10.1038/jid.2011.397) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Correlation of ELISA using recombinant proteins. Optical densities (ODs) obtained from ELISAs against recombinant human heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 were significantly correlated with those against recombinant streptococcal streptococcal 65-kD heat shock protein (hsp-65) in (a) healthy controls (HC; r=0.738, P<0.0001), (b) Behçet's disease (BD; r=0.549, P=0.002), and (c) systemic lupus erythematosus (SLE; r=0.742, P<0.0001), but not in (d) rheumatoid arthritis (RA), (e) Takayasu's arteritis (TA), or (f) IgA nephropathy (IgAN) patients. (b)Ten BD sera samples with relatively high OD values on ELISA of extracts of HDMECs are presented as triangle. Trend lines were analyzed by linear regression with 95% confidence intervals (dotted lines) using the GraphPad Prism software. Journal of Investigative Dermatology 2012 132, 601-608DOI: (10.1038/jid.2011.397) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Cross-immunoabsorption against recombinant human heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and recombinant streptococcal 65-kD heat shock protein (hsp-65). (a) Western blotting using recombinant human hnRNP-A2/B1 was performed using Behçet's disease (BD) patients’ sera preincubated with 0.5 and 2μg of recombinant streptococcal hsp-65, as well as 0.5 and 2μg of recombinant human α-Synuclein. (b) Serum IgA reactivity against recombinant human hnRNP-A2/B1 was notably reduced in BD patients’ sera preincubated with 0.5 and 2μg of recombinant streptococcal hsp-65 in a dose-dependent manner. (c) Significant cross-immunoabsorption was not observed in BD patients’ sera preincubated with 0.5μg (P>0.05) and 2μg (P>0.05) of recombinant human α-Synuclein. *P=0.046; **P<0.0001. Journal of Investigative Dermatology 2012 132, 601-608DOI: (10.1038/jid.2011.397) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions