engleski

Immunohistological and flow cytometry analysis of glycoconjugate expression in mouse lymphoid organs

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in GM1b-biosynthetic pathway were assigned to T lymphocytes, whereas B cell gangliosides and GSLs have been poorly characterized by former publications. We used specific polyclonal antibodies in immunohistology and flow cytometry to analyze the distribution of globotriaosylceramide (Gb3Cer), globoside (Gb4Cer), gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb4Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg4Cer, and Gb4Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blot analysis only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometry analysis of spleen and lymph node cells revealed that B cells carried epitopes recognized by all antibodies, whereas T cell GSL repertoire was mostly oriented to ganglio-series neutral GSLs and GM1b-type gangliosides. The results of immunohistology and flow cytometry were not always identical, possibly because of cross reactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment.

flow cytometry; gangliosides; glycosphingolipids; immunohistology; in vivo; lymph node; mouse; spleen; thymus

nije evidentirano