Fig. 2
Identification of citrullinated inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) in sera and increase of ITIH4 in arthritic joints in peptide glucose-6-phosphate isomerase-induced arthritis (pGIA). a Serum samples from pGIA and control mice at day 14 separated by two-dimensional PAGE, then stained with Coomassie brilliant blue (left) or subjected to Western blot analysis using anti-modified citrulline (AMC) antibodies (right). Citrullinated proteins were detected at approximately 120 kDa and pH 5.3 in pGIA but not in controls. b The MS spectrum of ITIH4429–438 and modified peptides bearing the citrullinated arginine (R438) in pGIA. Citrullinated residues were identified by the unmodified b4 ion and the modified y6, y7, y8, y9, and y10 ion confirmed a mass increase of 1.0D. The experiments depicted in (a) and (b) were performed on at least three different samples with similar results. c Joint sections at day 0 and day 14 were immunohistochemically stained with anti-ITIH4 antibodies in pGIA or control mice. ITIH4 were detected at day 14 in pGIA (indicated by boxed area) but not in naïve or control mice at day 14. The image on the fourth column is a magnified view of the boxed area in the joint image at day 14 of pGIA. Original magnification × 100 and × 400 as marked. d Joint sections at day 14 were stained with anti-F4/80 antibodies for detection of macrophages (bottom panel) and isotype control antibodies (top panel) in pGIA. Macrophages colocalized with ITIH4 in some areas in inflamed synovium of pGIA. e Joint sections at day 14 were stained with anti-ITIH4 antibodies (left bottom panel), AMC antibodies (right bottom panel), and respective isotype control antibodies (top panels) in pGIA. ITIH4 colocalized with citrullinated proteins in inflamed synovium of pGIA in comparative serial sections. Original magnification × 400