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The means by which the lectin soybean agglutinin (SBA) binds to the corneal endothelium cell surface following explantation into organ culture was investigated using Sprague-Dawley rats. SBA binding does not occur in freshly isolated and fixed rat corneal endothelium. However, after 48 h in organ culture, SBA binding occurs in a punctate pattern that clearly outlines all endothelial cells of the tissue monolayer. To determine what cell surface component was responsible for this binding, a series of experiments were employed that focused on the possibility that SBA bound to a nectin molecule(s). To this extent we performed a series of immunocytochemical localizations using antibodies against either nectin-2, nectin-3 or nectin-4. Of these, only nectin-3 bound to the endothelium in a manner that mimicked SBA binding. To further verify that nectin-3 bound SBA, displacement experiments employing non-labeled SBA were undertaken. Following a 48 h organ culture, tissues were fixed and incubated with SBA followed by exposure to nectin-3 antibody. No subsequent immunofluorescence could be detected, indicating that anti-nectin-3 binding was prevented. Likewise, when organ-cultured tissues were fixed and incubated in anti-nectin-3 antibody, followed by SBA exposure, no SBA binding could be detected. These results suggest that stresses accompanying explantation of the tissue into organ culture promote the appearance of nectin-3 around the cell periphery. The emergence of nectin-3 along the peripheral endothelial cell membrane in organ culture may imply a necessary role for this molecule in maintaining monolayer integrity and barrier function during either a pathologic condition, wound repair, or in organ storage. |
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