The response of intestinal stem cells and epithelium after alemtuzumab administration

Abstract

Intestinal stem cells may have important roles in the maintenance of epithelial integrity during tissue repair. Alemtuzumab is a humanized anti-CD52 lymphocytic antibody that is increasingly being used to induce immunosuppression; intestinal barrier function is impaired during treatment with alemtuzumab. We investigated the response of intestinal stem cells to epithelial damage resulting from alemtuzumab treatment. Intestinal epithelial cell loss and abnormal Paneth cell morphology were found following a single dose of alemtuzumab. The animals receiving alemtuzumab exhibited increased apoptosis in the villi 3 days after alemtuzumab treatment and in the crypt on day 9, but apoptosis was scarce on day 35. We assessed expression of Musashi-1- and Lgr5-positive stem cells following alemtuzumab treatment. Increased numbers of cells staining positive for both Musashi-1 and Lgr5 were found in the stem cell zone after alemtuzumab treatment for 3 and 9 days. These data indicated that the epithelial cells were injured following alemtuzumab treatment, with the associated expansion of intestinal stem cells. After alemtuzumab treatment for 35 days, the numbers of intestinal epithelial cells and intestinal stem cells returned to normal. This study suggests that alemtuzumab treatment induced the increase in stem cells, resulting in the availability of more enterocytes for repair.

Figure 1

Alemtuzumab affected intestinal homeostasis. The shortened villi in the intestines of alemtuzumab-treated animals (a). The asterisks indicate the Paneth cells (scale bar=100 µm). (b) Quantification of intestinal epithelial cells.

Figure 2

Alemtuzumab affected renewal of the epithelium. (a) The nuclear DNA was stained with DAPI and examined by immunofluorescence. Note the loss of nuclear localization of the DAPI signal in the boxed area of alemtuzumab-treated animals (scale bar=20 µm). (b) Quantification of the nuclei. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

Depletion of secretory granules from Paneth cells after alemtuzumab treatment. Secretory granules were abundant in normal Paneth cells. After alemtuzumab treatment, the secretory granules decreased in number (scale bar=25 µm). Arrows indicate Paneth cells; dashed arrows indicate intestinal stem cells.

Figure 4

Ultrastructural alteration in the crypts induced by alemtuzumab. Electron micrographs of normal and alemtuzumab-treated crypts. Note the reduced number of secretory granules in the Paneth cells after alemtuzumab treatment. Scale bars=2 µm. CBC, crypt base columnar cells; P, Paneth cells.

Figure 5

Alemtuzumab induced apoptosis in the villi and crypts of the intestine. (a) Apoptotic cells within the intestinal villi and crypts were determined by TUNEL staining using the In Situ Apoptosis Detection Kit. The apoptotic cells were detected by localized green fluorescence, and the nuclei were visualized by DAPI stain. A magnified view (lower panel) of the designated area (white lozenges in the upper panel) is also shown (scale bar=20 µm). (b) Quantification of apoptotic cells. Data are presented as mean±s.e.m. Asterisks indicate significant differences compared with control. *P<0.05; **P<0.01. DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 6

Localization of Msi-1 in the intestine of normal and alemtuzumab-treated animals. (a) The animals were treated with alemtuzumab and immunofluorescence was performed to investigate the distribution of Msi-1. Green fluorescent signal indicated the cells positive for Msi-1 at the base of the crypts. Nuclei were stained with DAPI. Crypt borders were depicted with white dashed lines (scale bar=20 µm). (b) The number of Msi-1 positive cells in the crypts was counted. Asterisks indicate significant differences. *P<0.05; **P<0.01. DAPI, 4′,6-diamidino-2-phenylindole; Msi-1, Musashi-1.

Figure 7

The expression pattern of Lgr5 in the crypts. (a) Intestinal sections stained for Lgr5 (green). The section was counterstained with DAPI. The crypt unit was outlined with white dashed lines (scale bar=20 µm). (b) Quantification of the Lgr5-positive cells in the crypts. Data are shown as mean±s.e.m., and asterisks indicate significant differences. *P<0.05; **P<0.01. DAPI, 4′,6-diamidino-2-phenylindole.