Participation of the Fas and Fas ligand systems in apoptosis during atrophy of the rat submandibular glands

Abstract

Most acinar cells and some duct cells undergo apoptosis during atrophy of the submandibular gland. The present study was designed to elucidate whether Fas and its receptor ligand (FasL) are involved during apoptotic atrophy of the gland. The excretory duct of the right submandibular gland of rats was doubly ligated with metal clips from 1 to 14 days for induction of gland atrophy. Control rats were untreated. Fas and FasL expression in the atrophied submandibular gland was detected using immunohistochemistry (IHC) and Western immunoblot. Expression of activated caspase 8 and activated caspase 3 was also detected with IHC. Fas-positive acinar and duct cells and FasL-positive duct cells increased in the atrophic glands at 3 and 5 days after duct ligation when apoptotic cells were commonly observed. Thereafter, Fas- and FasL-positive cells declined in number. Patterns of expression of Fas and FasL using Western immunoblots concurred with the IHC results. Activated caspase 8-positive cells were present at every time interval but peaked at 3 and 5 days following duct ligation. The cells showing immunoreaction for activated caspase 3 first appeared on day 3, with the peak in apoptosis, after which they decreased. The results indicate that the Fas/FasL systems likely play an important role in apoptotic pathways during atrophy of the submandibular gland.

Figure 1

Histology (HE). (a) Control. Normal submandibular gland. (b) Day 1. Ducts dilate. (c) Day 3. Many apoptosis in acini (arrowheads). (d) Day 14. Most acini disappear, leaving many ducts. Bars = 50 μm.

Figure 2

Fas immunohistochemistry. (a) Control. Several intercalated duct cells are positive (arrowheads). (b) Day 5. Many acinar cells show positive reaction. (c) Day 14. The positivity for Fas (arrowheads) decreases in the atrophic submandibular gland. Bars = 50 μm.

Figure 3

FasL immunohistochemistry. (a) Control. The positivity for FasL is identified in a large interlobular duct (arrowheads). (b) Day 5. FasL-positive cells are observed in small ducts. (c) Day 14. A few FasL-positive reactions are observed in small ducts (arrowheads). Bars = 50 μm.

Figure 4

Activated caspase 8 immunohistochemistry. (a) Control. The normal submandibular gland shows no reaction. (b) Day 3. Activated caspase 8-positive acinar cells are commonly identified (arrowheads). (c) Day 14. The positive reaction (arrowhead) decreases in number. Bars = 25 μm.

Figure 5

Activated caspase 3 immunohistochemistry. (a) Control. No immunoreaction is identified in the normal gland. (b) Day 3. There are many positive reactions against activated caspase 3. (c) Day 14. The number of FasL-positive cells (arrowhead) is small. Bar = 25 μm.

Figure 6

Western blot analysis of proteins from different intervals of submandibular glands for Fas. P, positive control (mouse thymus extract); C, control (normal submandibular gland); 1d, 1 day after duct ligation; 5d, day 5; 14d, day 14. The positive reactions are identified in positive control and day 5 at 45 kDa.

Figure 7

Western blot analysis for FasL. P, positive control (whole cell lysate of human chronic myelogenous leukaemia); C, control (normal submandibular gland); 1d, 1 day after duct ligation; 5d, day 5; 14d, day 14. The immunoreactions are identified at 40 kDa in positive control and day 5.