In this study we demonstrated by RNA in situ hybridization (RNAscope) that IL-6 mRNA-expressing cells were mainly present in the stroma both in MCD and in IgG4-LD, and that IL-6 expression tended to be higher in MCD compared with IgG4-LD, which may help distinguish MCD from IgG4-LD.
Because IL-6 expression is an important factor in MCD and is related to symptoms , discoveries of IL-6 expression in tissues may be useful for the accurate diagnosis of MCD. Although IL-6 expression was also observed in IgG4-LD, there was a relatively different IL-6 H-score between MCD and IgG4-LD; therefore, expression analysis by RNAscope may be useful for differentiating between MCD and IgG4-LD, especially when serum IL-6 data are not available.
There have been several reports of increased IgG4-positive plasma cells by the histopathological analysis of clinicopathologigally diagnosed with MCD [6, 11]. In these reports, serum IgG4 levels were also elevated. Furthermore, high serum IgG4 levels and numbers of IgG4-positive plasma cells were found in tissues of clinicopathologigally diagnosed with MCD in the lung [12, 13]. However, there is no clear explanation of the mechanism involved in the increase in IgG4-positive plasma cell numbers and serum IgG4 levels in MCD. However, it might be related to activation of the Th2 cascade, which induces the secretion of IgG4. Indeed, Th2 lymphocytes are induced in MCD [14, 15]. Analysis of the relationship between Th2 lymphocytes and IgG4 in MCD is warranted.
Clinicopathologically, increased IL-6 levels were reported in IgG4-RD  and IL-6 was elevated in IgG4-related dacryoadenitis and sialadenitis . The reason for high IL-6 levels in IgG4-RD is not well understood, but IL-6 is thought to directly promote the development of fibrosis in damaged tissues [18, 19]. Zongfei et al. reported elevated levels of IL-6 and IL-6R in the serum and tissues of IgG4-RD patients and that serum IL-6 was positively correlated with ESR, CRP, and IgG4-RD responder index, but not with serum IgG4 . In addition, Tsukuda et al. reported that in IgG4-RD, the high IL-6 group was older, with lower albumin levels, and higher CRP and AST levels . Liver swelling and splenomegaly were also significantly more common. Serum IL-6 levels in IgG4-RD may be significantly correlated with clinical inflammatory parameters. In addition, Tsukuda et al. concluded that serum IL-6 levels may be associated with the spread of disease to the bile ducts, liver, and spleen . Previous studies reported cases that met the diagnostic criteria for IgG4-RD in the lung but with high IL-6 levels . Therefore, IL-6 may only be increased for a specific period during IgG4-RD or in an organ-specific manner.
The concept of an “MCD-like” subtype of IgG4-RD, which has the characteristics of MCD and IgG4-LD, has been proposed . Further studies on the clinicopathological features of the mixed type are required. Some of these cases did not respond to steroid administration suggesting they might represent a heterogeneous disease group.
Several studies have reported IL-6 expression in MCD and IgG4-RD by immunostaining and RNA in situ [7, 21]. Although the detection sensitivity of proteins such as cytokines by IHC may be insufficient, the detection of mRNA may be an effective alternative because it is localized in cells . Otani et al. reported MCD cases that were IL-6 negative by the RNA in situ method . In detail, only 1 out of 8 cases of MCD showed focal IL-6 positivity. Another study reported that IL-6 was positive in situ . However, H-scores were not measured in either study and the RNA in situ method used was different from ours. The difference in stainability due to IL-6 in situ depends on the conditions at the time of staining, including the number of storage years and storage conditions.
This study had some limitations. These diseases are rare and the number of MCD and IgG4-LD samples was low in this study; therefore, further case accumulation is desired. IL-6 is secreted by various immunocompetent cells including T/B lymphocytes, monocytes, fibroblasts, and endothelial cells; however, its role and secreting cell type in MCD are unknown . Therefore, it will be necessary to clarify which cells secrete IL-6 in MCD. In addition, it is unclear which factors induce the elevation of IL-6 in MCD and IgG4-LD. Confirmation using RNA Next-Generation Sequencing will be necessary.