APPLICATION OF IMMUNOGOLD LABELING FOR LIGHT AND ELECTRON-MICROSCOPICLOCALIZATION OF ENDOTHELIAL-LEUKOCYTE ADHESION MOLECULE-1 (ELAM-1) ONCULTURED HUMAN ENDOTHELIAL-CELLS

This study describes the expression characteristics of E-selectin mole cules using immunogold histochemical techniques on cultured human umbi lical vein endothelial cells (HUVEC). The expression of E-selectin was induced by tumour necrosis factor-alpha (TNF-alpha, 300 U/ml), phorbo l ester (PMA, 10 ng/ml) and bacterial lipopolysaccharide (LPS, 4 mug/m l). No expression was demonstrated on control cells. Using the silver- enhanced colloidal gold-labelling technique, at the light microscopica l level, HUVEC could be distinctively subdivided into three staining t ypes. The cell labelling index, expressed as the number of 'positively ' stained cells as a proportion of all viewed cells was the highest in the LPS group. For transmission electron microscopy (TEM) the preembe dding immunocytochemical staining method and embedding in epoxy resin (Agar 100) according to standard procedures was used. In TEM gold part icles were localized in close association with the apical plasma membr ane, as well as on the surface of microvillus-like projections (the la tter by TNF-alpha group). For high resolution scanning electron micros copy (HR-SEM) the secondary (SEI) and the backscattered electron imagi ng (BEI) modes were used. Gold particles were randomly distributed ove r the whole cell surface, although they appeared to be denser in the p erinuclear zone. The quantitative evaluation on SE and BE viewing (the number of gold particles per cell area in mum2) demonstrated the high est density of labelling in the LPS-treated group, but there was only a significant difference between LPS and TNF-alpha groups (P<0.01, t-t est). Furthermore, the ultrastructural studies indicated that treatmen t with substances which up-regulate E-selectin expression was not rela ted to toxic cell damage or significant alterations of cellular ultras tructure.