Table 1 summarises the results of the immunohistochemical analysis.
Table 1Immunohistochemical analysis of the study participants
All synovial biopsy specimens from patients with rheumatoid arthritis showed signs of inflammation, with a median histological score of 2 for synovial lining thickness (range 1–4, 4 missing data due to absence of the lining layer in the biopsy specimen), 2 for vascularity (range 1–4) and 2.5 for inflammatory cell infiltrate (range 1–4). Citrullinated proteins were present in all investigated synovial biopsy specimens (100%) from patients with rheumatoid arthritis (fig 1A), with large variations between different patients resulting in a median value of 1 (range 0.5–3). Citrullination was present both in the lining and in the sublining layer, intracellularly as well as extracellularly (fig 2). Endothelial cells, fibroblasts and mononuclear cells were positive for citrullinated proteins, whereas extracellular citrullination was mainly located in the amorphous fibrin areas. 3 of 10 (30%) healthy synovia contained low amounts of citrullinated proteins (fig 1B), resulting in a median value of 0, with a range of 0–1 (p<0.05 compared with specimens from patients with rheumatoid arthritis). Citrullinated proteins were present in isolated mononuclear cells in the lining and sublining, few endothelial cells and sublining fibroblasts.
Figure 1Citrullinated proteins are preferentially expressed in inflammatory tissues. Images represent immunohistochemistry pictures of citrulline proteins in (A) rheumatoid arthritis synovium, (B) healthy synovium, (C) polymyositis, (D) normal (more ...)
Figure 2Citrullinated proteins are detected both extracellularly and intracellularly in the lining (A) and in the sublining (B) layers in rheumatoid arthritis synovium.
All samples from patients with polymyositis (100%) were positive (fig 1C), with citrullinated proteins present both in endothelial and inflammatory cells, whereas muscle fibres were negative. By contrast, muscle biopsy specimens from healthy controls (fig 1D) were completely negative (p<0.05 compared with samples from patients with polymyositis).
Colonic biopsy specimens from patients with IBD showed various amounts of citrullinated proteins with no significant differences between macroscopically affected (fig 1E) and non‐affected (fig 1F) areas when assessed semiquantitatively. However, in patients with IBD, 70% of the macroscopically affected samples showed the presence of citrullinated proteins compared with only 40% of the macroscopically non‐affected samples (p>0.05). Citrullination was present mainly in the lamina propria cells and also in some epithelial cells and infiltrating mononuclear cells of the Lieberkuhn's crypts, with the same pattern distribution as PAD‐2 (data not shown). In agreement with these data, histological examination showed that all macroscopically non‐affected biopsy specimens showed minimal to moderate signs of inflammation. Even though less inflammation was seen in the macroscopically non‐affected biopsy specimens, no major differences were observed between the two groups.
All four chronically inflamed tonsil samples were positive (data not shown), with citrullinated proteins and PAD‐2 expression in macrophage‐like cells, both in the T cell and B cell areas.