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Abstract
Antibody conjugates directed against intercellular adhesion molecule (ICAM-1) or platelet-endothelial
cell adhesion molecule (PECAM-1) have formed the basis for drug delivery vehicles
that are specifically recognized and internalized by endothelial cells. There is increasing
evidence that ICAM-1 and PECAM-1 may also play a role in cell scavenger functions
and pathogen entry. To define the mechanisms that regulate ICAM-1 and PECAM-1 internalization,
we examined the uptake of anti-PECAM-1 and anti-ICAM-1 conjugates by endothelial cells.
We found that the conjugates must be multimeric, because monomeric anti-ICAM-1 and
anti-PECAM-1 are not internalized. Newly internalized anti-ICAM-1 and anti-PECAM-1
conjugates did not colocalize with either clathrin or caveolin, and immunoconjugate
internalization was not reduced by inhibitors of clathrin-mediated or caveolar endocytosis,
suggesting that this is a novel endocytic pathway. Amiloride and protein kinase C
(PKC) inhibitors, agents known to inhibit macropinocytosis, reduced the internalization
of clustered ICAM-1 and PECAM-1. However, expression of dominant-negative dynamin-2
constructs inhibited uptake of clustered ICAM-1. Binding of anti-ICAM-1 conjugates
stimulated the formation of actin stress fibers by human umbilical vein endothelial
cells (HUVEC). Latrunculin, radicicol and Y27632 also inhibited internalization of
clustered ICAM-1, suggesting that actin rearrangements requiring Src kinase and Rho
kinase (ROCK) were required for internalization. Interestingly, these kinases are
part of the signal transduction pathways that are activated when circulating leukocytes
engage endothelial cell adhesion molecules, suggesting the possibility that CAM-mediated
endocytosis is regulated using comparable signaling pathways.