Alteration of Cell-Cell Junctions in Cultured Human Lymphatic Endothelial Cells with Inflammatory Cytokine Stimulation

Background: To maintain normal function, the lymphatic endothelium is regulated by cell-cell junctions. There have been few studies of lymphatic endothelial cell junctions using standard cell biological methods. This study had two purposes: to characterize cell junctions in cultured lymphatic endothelial cells and to investigate the effects of the inflammatory cytokine TNF-alpha on altered cell-cell junctions. Methods and Results: Cultured human dermal lymphatic endothelial cells (HDLEC) were immunostained with the tight junction marker, ZO-1, and adherens junction markers, VE-cadherin and PECAM-1. In TNF-alpha-treated HDLEC, we evaluated changes in endothelial cell junctions by immunostaining and through the use of transendothelial electrical resistance (TER). Immunofluorescence staining of HDLEC revealed heterogeneity among the endothelial cell junctions, which could be classified into continuous and discontinuous junctions. In these cell junctions, ZO-1 and VE-cadherin were co-localized. Double immunofluorescence staining revealed the broad distribution of VE-cadherin at the cell periphery, where VE-cadherin and PECAM-1 were co-localized. TNF-alpha treatment decreased TER, caused a predominance in the appearance of discontinuous junctions with a reduction in the broad distribution of VE-cadherin at the cell periphery in HDLEC. Conclusions: The results indicate a heterogeneous distribution of cell junctions in HDLEC involving continuous and discontinuous junctions. Our data also suggest that TNF-alpha alters the normal distribution of cell junctions and affects the endothelial barrier of cultured lymphatic endothelial cells. The broad distribution of VE-cadherin at the cell periphery may reflect the lymphatic permeability.