HISTOLOGY AND HISTOPATHOLOGY

From Cell Biology to Tissue Engineering

 

Ethanol enhances thymocyte apoptosis and autophagy in macrophages of rat thymi

Tsubasa Betsuyaku1, Nabil Eid1, Yuko Ito1, Yoshihisa Tanaka1, Yoshinori Otsuki2 and Yoichi Kondo1

1Department of Anatomy and Cell Biology, Division of Life Sciences, Osaka Medical College, Osaka, Japan and 2Osaka Medical College, Osaka, Japan

Offprint requests to: Dr. Nabil Eid, Division of Life Sciences, Department of Anatomy and Cell Biology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan. e-mail: nabil@osaka-med.ac.jp or nabileidm@yahoo.com


Summary. Tingible body macrophages (TBMs) play essential roles in the phagocytosis of apoptotic lymphocytes, specifically under exposure to various stressors. Although excessive ethanol consumption may enhance thymocyte apoptosis, reports investigating the autophagic response of the thymus to ethanol toxicity are still lacking. We investigated apoptosis and autophagy in thymi of an animal model of binge ethanol exposure. Adult male Wistar rats were injected intraperitoneally either with 5 g/kg ethanol or phosphate buffer saline (for the control group) and sacrificed 0, 3, 6 and 24 hours after injection. Light and transmission electron microscopy (TEM) studies revealed enhanced formation of TBMs phagocytosing many apoptotic thymocytes in the thymic cortex of the ethanol-treated rats (ETRs), and this formation was particularly marked at 24 h. The macrophages showed signs of activation under TEM and immunofluorescence double labeling with RM4 (a macrophage marker) and iNOS. Additionally, in comparison to the control group, autophagy was enhanced in ETR thymic TBMs as evidenced ultrastructurally by accumulation of autophagic vacuoles, immunohistochemical increases in LC3 puncta, Western blot analysis of the latter protein, and colocalization of LC3 and RM4 in immunofluorescence double labeling. Immunoelectron microscopy also revealed LC3-labeled autophagic vacuoles and apoptotic cell phagosomes in ETR TBMs, suggesting the possibility of LC3-related phagocytosis. This was confirmed by enhanced colocalization of LC3 with lysosomal cathepsins in double labeling. These results indicate that enhanced autophagy in ETR thymic TBMs is not only a cytoprotective mechanism but could also be involved in the clearance of apoptotic thymocytes, thus preventing autoimmune reactions and suppressing inflammatory response. Histol Histopathol 32, 963-975 (2017)

Key words: Autophagy, LAP, Apoptosis, Ethanol, Thymus, TBMs

DOI: 10.14670/HH-11-861