PMC6038191_01
An inflammatory cyst within the submandibular gland. Coronal (a) or axial (b) T2-weighted MRI images of the head and neck demonstrating the existence of a cyst within the right submandibular gland (yellow arrows). Ultrasonic imaging of the inflamed submandibular gland (c) showed the inflammatory cyst as a black hole with sharp borders, measured 39.8 mm x 19.7 mm in diameter
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Fig1
Ultrasonography and MRI imaging of the head and neck revealed a well-defined cystic lesion with sharp regular borders measuring 39.8 mm x 19.7 mm within the right submandibular gland (Fig. 1). RNAs were collected from formalin-fixed paraffin-embedded (FFPE) samples (NucleoSpin total RNA FFPE XS, Macherey-Nagel, Catalog No: 740969.10) and then reverse-transcribed into cDNA (ReverTraAce qPCR RT Master Mix, Toyobo, Catalog No: F0937K). RT-PCR analysis on these samples showed elevated levels of IL-34 mRNA in addition to several inflammatory cytokines such as interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor alpha (TNFalpha) in the inflammatory cyst of the submandibular gland compared to normal salivary gland tissues (Additional file 1: Figure S1).
Enhanced IL-34 expression in an inflammatory cyst of the submandibular gland compared to normal salivary gland tissues. Representative data of immunohistochemistry staining of IL-34 in normal salivary gland tissues (a) or inflamed submandibular gland (b). IL-34 staining was performed on sections obtained from five different regions of the surgically resected inflammatory cyst (from S1 to S5) in addition to one sample from the adjacent swollen lymph nodes (S6). H & E staining of sections is shown in the left column, followed by two representative data of IL-34 staining in tissues surrounding the inflammatory cyst. Squares within H & E photography indicate the correspondent positions of IHC data. Scale bars, 20 muM. The specific staining of IL-34 in keratinocytes of normal skin is shown in c at different magnifications as a positive control to confirm the specificity of the anti-IL-34 antibody
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Fig2
To examine the possible involvement of IL-34 in the inflammatory response observed in this case, 5-mum thickness sections were prepared from FFBE tissue samples obtained from five different regions of the surgically resected inflammatory cyst in addition to one sample from the adjacent swollen lymph nodes (Fig. 2). By microscopic observation, normal salivary gland tissues showed typical histology, composing of serous acini mixed with adipocytes and ducts lined by cuboidal epithelial cells (Fig. 2a). IL-34 expression was undetectable in serous acini or endothelium, but can be observed at very weak levels in the epithelium of the ductal system (Fig. 2a). IL-34 showed enhanced expression levels in tissues of the submandibular gland surrounding the inflammatory cyst (Fig. 2b). In particular, IL-34 showed remarkable enhancement in the cuboidal epithelial cells of the ductal system and interestingly in endothelial cells lining blood vessels within theaffected gland (Fig. 2b). IL-34 showed very weak staining in some areas within the adjacent swollen lymph nodes (Fig. 2b). Catalog No: T2234218, Lot. C105169) (Fig. 2c).
High expression of IL-34 in ductal epithelial cells and endothelial cells of blood vessels correlates with high infiltration of immune cells. Representative data of IL-34 staining by IHC showing the localized expression of IL-34 in ductal epithelial cells and endothelial cells of blood vessels. Hematoxylin staining demonstrates lymphocyte accumulation around IL-34-expressing epithelial (red arrows) or endothelial (green arrows) cells. Scale bars, 20 muM
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Fig3
Consistent with this inflammatory condition, high expression of IL-34 in the affected gland showed a tendency to be accompanied with high infiltration of lymphocytes, as confirmed in both cases of the epithelium of the ductal system as well as endothelium of blood vessels (Fig. 3).