Table 1.
Characteristics of patients.
Fig 1.
Pepsin detected in the tonsillar hypertrophy tissues.
A) Pepsin was expressed in the tonsil tissue. Stomach tissues were used as positive control for pepsin detection. Other tissues were used as negative control. B) Pepsin staining levels were different from each patient. β-actin was used for housekeeping control. LN, lymph node. Thy, thyroid. Parotid g, parotid gland. SG, salivary gland. C) Pepsin-positive cells localized below the surface epithelium, mainly in the crypt regions (a and b), also surrounding the more negative germinal centers (c and d), and surrounding the lymphoid follicle with excessive developing fibrosis (e and f). D) Stomach sections showed a typical pattern of pepsin staining (a), but not in the lymph node (b) and thyroid (c).
Fig 2.
The injured tonsil site and pepsin-positive cells in the tonsil sections.
No positive signals were detected in the non-injured tonsillar squamous epithelium (left insert in A and B). Pepsin-positive cells were strongly found in the damaged tonsillar squamous epithelium (right insert in A and C, D, E, and F). Dashed lines show damaged clefts and broken squamous epithelium. Inserts in A and D were magnified as “B and C” and “E and F”, respectively. Scale bar, 50 μm (A) and 100 μm (D).
Fig 3.
Localization of TGF- β1 and iNOS-positive cells in the tonsil sections.
TGF- β1 and iNOS-positive cells were found in the crypt epithelium (A and B), surrounding germinal centers (C and D), and surrounding the lymphoid follicle with excessive developing fibrosis (E and F). Inserts in each image was magnified (A’-F’). Scale bar, 50 μm.
Fig 4.
Colocalization of TGF-β1, pepsin and macrophage and lymphocyte-positive cells in the tonsil sections and mRNA expression for inflammatory mediators in tonsil tissues with tonsillar hypertrophy.
A) Dual pepsin and CD68-positive cells and dual TGF-β1and CD68-positive cells were detected in the crypt. B) Dual pepsin and CD68-positive cells and dual TGF- β1and CD68-positive cells were also detected in the surrounding germinal centers. C) Few dual pepsin and CD20 or CD45-positive cells were detected. Scale bar, 50 μm. D) The mRNAs of IL-1, IL-6, and TNF- β in the tonsil tissues were determined by RT-PCR. GAPDH housekeeping gene was used as a control.
Fig 5.
Flow cytometric analysis of monocyte population and monocyte differentiation from PBMNCs from tonsillar hypertrophy.
A) Lymphocytes and monocytes were identified with side and forward scatter profiles. Lymphocytes and monocytes were also confirmed by staining with CD4 and CD8 and CD14 antibodies. B) PBMNCs were cultivated in macrophage-specific culture conditions with or without activated pepsin for 15 days. Monocyte population was identified with side and forward scatter profile on flow cytometry for each condition. Each level was based on the value from day 8 (pepsin-untreated, control cells) and given a value of “1” as baseline. C) Monocyte to macrophage differentiation was examined by staining with CD11c and CD163 antibodies.
Fig 6.
Direct effects of pepsin on macrophage viability and migration using the RAW264.7 cells.
A) RAW264.7 cells were treated with various doses of pepsin for 24 hours. Viability was measured by WST-1 assy. The fold changes are calculated as the ratio of the final value in the each presence of pepsin to the value in absence of pepsin (set as “1”). Values are represented as the mean ± SEM. *P < 0.05 vs. the corresponding absence of pepsin (0 μg/ml). B) Migration of RAW264.7 cells was increased with 0.1 μg/ml of activated pepsin by scratch wound migration assay. C) Migration of RAW264.7 cells was also induced with 0.1 μg/ml of activated pepsin by transwell migration system.
Fig 7.
Two hypotheses for pepsin-mediated tonsillar hypertrophy.