Dynamic changes in cell-surface expression of mannose in the oral epithelium during the development of graft-versus-host disease of the oral mucosa in rats

Abstract

Background: The role of cell-surface glycoconjugates in oral mucosal graft-versus-host disease (GVHD) is still unclear, even though molecular changes in the oral epithelium are essential for the pathogenesis of these lesions. In this study, we investigated changes in the binding of mannose (Man)-specific Lens culinaris lectin (LCA) in the oral mucosa of rats with GVHD.

Methods: Lewis rat spleen cells were injected into (Lewis x Brown Norway) F1 rats to induce systemic GVHD, including oral mucosal lesions. Tongue and spleen samples were evaluated using lectin histochemistry, immunohistochemistry, Western blotting, transwell migration assays and Stamper-Woodruff binding assays.

Results: Binding of Man-specific LCA expanded to the epithelial layers of the tongue in GVHD-rats. An expansion of LCA binding was related to the increased expression of mannosyltransferase in the oral mucosa. CD8+ cells, effector cells of oral mucosal GVHD, expressed mannose-binding protein (MBP) and migrated to the medium containing Man in the transwell migration assay. Adherence of CD8+ cells to the oral epithelium could be inhibited by pretreating CD8+ cells with MBP antibody and/or by pretreating sections with Man-specific LCA.

Conclusions: Increased expression of Man on keratinocytes leads to the migration and/or adhesion of CD8+ cells in the surface epithelium, which is mediated in part by the MBP/Man-binding pathway during the development of oral mucosal GVHD.

Figure 1

Immunohistochemical expression of intercellular adhesion molecule-1 (ICAM-1) and CD8⁺ in graft-versus-host disease (GVHD)-related oral mucosa. a and b: No obvious changes are observed in hematoxylin and eosin (H&E)-stained control tongue sections (a). In tongue tissue sections from GVHD rats, epithelial destruction is observed (b). c and d: In tongue specimens from control rats, ICAM-1 is expressed only in the vascular slits (arrows) (c). Epithelial ICAM-1 expression is observed in the basal to spinous layers of the oral mucosa from GVHD rats (d). e and f: Only a few CD8⁺ cells are found in the control (e). In the GVHD rats, increased numbers of CD8⁺ cells are observed in both lamina propria and surface epithelium (f). The dotted line shows a junction between the surface epithelium and lamina propria of the tongue. Bar = 100 μm.

Figure 2

Lens culinaris lectin (LCA) binding in graft-versus-host disease (GVHD) oral mucosa. LCA binding occurs on the cell surface of basal to parabasal cells in control tongues (a). Staining extends to the spinous layers of the surface epithelium in GVHD rats (b). The white dotted line circumscribes the surface epithelial layer of the tongue. Bar = 100 μm.

Figure 3

Immunohistochemical detection of mannosyltransferase complex in graft-versus-host disease (GVHD)-affected oral mucosa. ALG11, antibody (Ab) of mannosyltransferase complex, is weakly and faintly reactive in basal and parabasal epithelial cells of control tongues (a). In contrast, staining with ALG11 extends to keratinocytes (KCs) in the spinous layers of tongues from GVHD rats (b). Bar = 100 μm.

Figure 4

Mannose-binding protein (MBP) expression in tongue and spleen specimens of graft-versus-host disease (GVHD) rats. Infiltrating cells are expressed immunohistochemically by anti-MBP antibody (Ab) (a). Western blot analysis of MBP levels of the tongue and spleen from control and GVHD rats (b). Both tongue and spleen from the GVHD-mediated rats show remarkable reaction with MBP. β-actin (ACTB) was similarly analyzed as a loading control. Bar = 100 μm.

Figure 5

Induction of mannose (Man) to CD8⁺ cell migration. CD8⁺ cell migration was examined by the transwell migration assay. Man, Man with Lens culinaris lectin (LCA), or galactose was placed in the lower chamber well. The wells of the upper chamber receive isolated CD8⁺ cells, pretreated or not with anti-mannose-binding protein (MBP) antibody (Ab). RPMI was used as a negative control. The figure represents results of three different experiments expressed as mean ± standard deviation. Same symbols show no statistically significant differences. Others, significantly different at P < 0.05.

Figure 6

Effect of lectins and anti-mannose-binding protein (MBP) antibody (Ab) on CD8⁺ cell adhesion to the oral epithelium in GVHD. Adhesion of CD8⁺ cells to the oral keratinocytes (KCs) were investigated by Stamper-Woodruff binding assay (SWBA). Binding of CD8⁺ cells was significantly decreased when the cells were pretreated with anti-MBP Ab, as well as when epithelia were pretreated with Lens culinaris lectin (LCA). The figure represents the results of three different experiments and expressed as mean ± standard deviation. Same symbols show no statistically significant differences. Others, significantly different at P < 0.05.