Activation of fibroblasts by plasma cells via PDGF/PDGFR signaling in IgG4-related sialadenitis

Abstract

IgG4-related sialadenitis (IgG4-SA) is one of the IgG4-related disease. The histological features of IgG4-SA include dense lymphoplasmacytic infiltrates and fibrosis. This study aimed to reveal the involvement of plasma cells in the development of fibrosis and the mechanism underlying fibrosis in IgG4-SA. Hematoxylin-eosin staining, Azan staining, silver staining, and immunohistochemistry (IHC) were performed on IgG4-SA and chronic sialadenitis specimens, and theses samples were analyzed by image analysis software. Histological spatial analysis was used to analyze the localization of IHC-positive cells and the distances between these cells. In the IgG4-SA group, many secondary lymphoid follicles with germinal centers were found, and many collagen fibers developed around these germinal centers. Collagen fibers composed mainly of type I collagen was abundant at sites away from secondary lymphoid follicles, and reticular fibers composed of type III collagen was abundant near secondary lymphoid follicles. Many FAP⁺ fibroblasts and MUM1⁺ plasma cells were localized near secondary lymphoid follicles. Histological spatial analysis demonstrated that 90.4% of MUM1⁺ plasma cells accumulated within 20 µm of FAP⁺ fibroblasts. Multiple immunofluorescence assays revealed that MUM1⁺ plasma cells expressed platelet-derived growth factor (PDGF) β, and FAP⁺ fibroblasts expressed PDGF receptor (PDGFR) β and pSTAT3 in IgG4-SA. We have shown that fibrosis is localized around secondary lymphoid follicles and that fibroblasts are activated by plasma cells via PDGF/PDGFR signaling in IgG4-SA.

Keywords: IgG4-related sialadenitis; PDGF-PDGFR signaling; fibroblast; histological spatial analysis; plasma cell.

Fig. 1

Fibrosis was localized around the secondary lymphoid follicles in IgG4-related sialadenitis a: Low-power field image of the IgG4-related sialadenitis (IgG4-SA) sample. b: High-power field image of the IgG4-SA sample. c: Collagen fibers developed around the secondary lymphoid follicles. d: Low-power field image of the chronic sialadenitis (chronic-SA) sample. e: High-power field image of the acinar area in the chronic-SA sample. f: A few collagen fibers were observed around the secondary lymphoid follicles. g: The area of collagen fibers around secondary lymphoid follicles in the IgG4-SA group was significantly larger than that in the chronic-SA group. The area closed by the dashed line showed lymphoid follicles (b, c, e, and f). Scale bars, 500 μm (a and d) and 250 μm (b, c, e, and f). ***, P < 0.001.

Fig. 2

Localization of collagen fibers, reticular fibers, and fibroblasts a: Localization of fibrosis around secondary lymph follicle. The green rectangle was set near the secondary lymphoid follicle, and the black rectangle was set away from the secondary lymphoid follicle. b: Collagen fibers were sparse near the secondary lymph follicles. c: Collagen fibers were dense away from the secondary lymphoid follicles. d: Reticular fibers developed near the secondary lymphoid follicles. e: Collagen fibers in red were predominant away from the secondary lymphoid follicles. f: Many FAP⁺ fibroblasts were observed near the secondary lymphoid follicles. g: Few FAP⁺ fibroblasts were observed away from the secondary lymphoid follicles. h: The number of FAP⁺ fibroblasts around the secondary lymphoid follicles in IgG4-related sialadenitis (IgG4-SA) group was significantly higher than in chronic sialadenitis (chronic-SA) group. a, b, and c were Azan staining; d and e were silver staining; g and h were immunohistochemistry of FAP. Scale bars, 500 µm. ***, P < 0.001.

Fig. 3

Inflammatory cells in fibrotic regions of IgG4-related sialadenitis and chronic sialadenitis a: Many MUM1⁺ cells were observed around the secondary lymphoid follicles in IgG4-related sialadenitis (IgG4-SA). b: Only a few MUM1⁺ cells were observed around the dilated ducts in chronic sialadenitis (chronic-SA). c, d, and e: The number of plasma cells (c), macrophages (d), and Tregs (e) per a fibroblast was compared around the secondary lymphoid follicles in IgG4-SA and dilated ducts in chronic-SA. Scale bar, 100 µm. ***, P < 0.001.

Fig. 4

Histological spatial analysis of fibroblasts and plasma cells in IgG4-related sialadenitis FAP⁺ fibroblasts in the fibrotic region around secondary lymphoid follicles in IgG4-related sialadenitis (IgG4-SA) were recognized by image analysis software (a) and allocated yellow triangles (b). Yellow triangles were plotted on the grid chart (c). MUM1⁺ plasma cells at the same location as the FAP-immunohistochemistry specimen were recognized by image analysis software (d) and allocated green circles (e). Green circles were plotted on the grid chart (f). Yellow triangles (c) and green circles (f) were synchronized and overlaid into one grid chart (g). The schematic diagram (h) shows the distribution of MUM1⁺ plasma cells around a fibroblast. The histogram (i) shows the density of MUM1⁺ cells at each distance from the fibroblasts. Approximately 90% of the plasma cells were present within 20 µm of the fibroblasts. Scale bars, 50 µm.

Fig. 5

Identification of platelet-derived growth factor β expression in plasma cells and platelet-derived growth factor β receptor expression in fibroblasts by multiple immunofluorescence assay a: Multiplex immunofluorescence (IF) staining of MUM1. MUM1⁺ cells were stained with TRITC (red). b: Multiplex IF staining of platelet-derived growth factor (PDGF)β. PDGFβ⁺ cells were stained with FITC (green). c: DAPI staining. d: Merged image of a, b, and c. e: Multiplex IF staining of FAP. FAP⁺ cells were stained with FITC (green). f: Multiplex IF staining of PDGF receptor (PDGFR)β. PDGFRβ⁺ cells were stained with TRITC (red). g: DAPI staining. h: Merged image of e, f, and g. i: Multiplex IF staining of FAP. FAP⁺ cells were stained with FITC (green). j: Multiplex IF staining of phosphorylated signal transducer and activator of transcription-3 (pSTAT3). pSTAT3⁺ cells were stained with TRITC (red). k: DAPI staining. l: Merged image of i, j, and k.