Protein expression of sialolith with antrolith and tonsillolith examined by immunohistochemistry and...

Ultrastructural and proteomic analyses have been performed to elucidate the growth mechanism of sialoliths. However, the role of salivary/blood proteins, inflammatory signaling proteins, inflammatory cytokines, and antimicrobial proteins has not been elucidated. This study examined eight sialoliths, one antrolith, and one tonsillolith which underwent radiological, histological, immunohistochemical (IHC), and immunoprecipitation-based high-performance liquid chromatography (IP-HPLC) analyses. The IHC showed that BMP2/4 were strongly positive for the exosome-like vesicles in the central nidus zone and diffusely positive in the intermediate compact calcification zone. The TGFβ1 was strongly positive in the proximal intermediate compact zone and dispersed in the distal direction. Finally, the KL1 was occasionally positive in the peripheral multilayer zone with a linear or spotty appearance. The stones were divided into three groups based on protein expression determined through IP-HPLC. Group A showed dominant expression of saliva/blood and cross-linking/calcification proteins, while Group B showed increased expression of inflammation signaling proteins, NFkB and TNFα, inflammatory cytokines, and antimicrobial proteins in the absence of bacterial infection as determined by LPS expression. Group C exhibited a severe bacterial infection, along with the protein expression observed in Groups A and B. One case of antrolith and one case of tonsillolith belonged to Group A. The data indicated that TGFβ1 and TNFα cooperated in the expression of cross-linking/calcifying proteins admixed with exfoliated epithelial cells; also, bacterial infection might not only aggravate salivary inflammation, but also facilitate the additional formation of sialoliths with thick biofilm and results in lamellar calcification reinforced with organic materials, becoming partially resistant to lithotripsy. Therefore, it has been suggested that sialoliths form through the expression of saliva and blood proteins, as well as cross-linking and calcification proteins, via TGFβ1/BMP signaling. In addition, inflammation and bacterial infection can paradoxically accelerate lithogenesis through TGFβ1/NFkB signaling.