Analysis of the Roles of ICAM-1 in Neutrophil Transmigration Using a Reconstituted Mammalian Cell Expression Model: Implication of ICAM-1 Cytoplasmic Domain and Rho-Dependent Signaling Pathway

Abstract

Interaction between ICAM-1 (CD54) and fibrinogen (fg) has been shown to enhance leukocyte adhesion, but its specific role in the process of migration across endothelial cell junctions remains unclear. To overcome the problem of multiple adhesion receptors found on endothelial cells, we have engineered stable Chinese hamster ovary cell lines expressing ICAM-1 (Chinese hamster ovary ICAM-1). The transfection of ICAM-1 alone in these cells is sufficient to recapitulate the entire process of neutrophil adhesion and transmigration. This phenomenon was mediated by fg-ICAM-1 interactions, as depletion of fg, as well as the use of an Ab that specifically inhibits ICAM-1-fg interaction (2D5), completely abolished the effect of ICAM-1 expression on PMN transmigration. In addition, this ICAM-1-mediated transmigration is clearly dependent on the occurrence of fg-ICAM-1 interactions on the monolayer, and not on neutrophils, as the preincubation of the PMN with the mAb was ineffective. Furthermore, PMN transmigration, but not adhesion, is totally abolished when the ICAM-1 cytoplasmic domain is deleted, indicating that signaling inside the cell is required to mediate the fg-ICAM-1 effect on transmigration. Using a specific inhibitor of the small GTP-binding protein Rho, we have obtained evidence that this signaling cascade is involved. Thus, our results clearly show that ICAM-1 plays a key role in the migration of leukocytes across cell junctions, and indicate that this phenomenon is not a direct consequence of the enhanced adhesion mediated by the expression of ICAM-1.

RKIKK Motif in the Intracellular Domain Is Critical for Spatial and Dynamic Organization of ICAM-1: Functional Implication for the Leukocyte Adhesion and Transmigration

“…We also generated COS-7 cells that express the ICAM-1 lacking intracellular domain (C-terminal 28 amino acids; IC1⌬CTD_GFP) in order to investigate potential function of intracellular domain on the distribution and localization of ICAM-1. Although earlier studies have utilized the CHO cell system to overcome the problem of multiple adhesion receptors found on endothelial cells (Sans et al, 2001;Carman et al, 2003), others have found that Chinese hamster ovary (CHO) cells express endogenous cell surface molecules that mediate T-cell costimulation (Gaglia et al, 2001). We also noticed that this uncharacterized molecule could mediate T-cell adhesion potentially through its interaction with LFA-1 (data not shown).…”

“…To this end, peptides comprising the 14 C-terminal amino acids or RKIKK residues were used to antagonize the binding potential of ICAM-1-binding/signal partners. Such ICAM-1 peptides were fused to the penetratin sequence to facilitate the uptake of peptides into cells (Hall et al, 1996;Sans et al, 2001). We assessed the effects of penetratin-ICAM-1 peptides (P-CTD, RQRKIKKYRLQQAQ; P-RKIKK, and RKIKK) for the leukocyte adhesion to and transmigration through HUVECs under the static flow condition.…”

“…These proteins all have the potential to perform signaling functions. In addition, several reports have described that the deletion of intracellular domain of ICAM-1 abolishes T-lymphocyte migration (Greenwood et al, 2003) as well as PMN migration (Sans et al, 2001).…”

“…In addition to the important role of the extracellular domain of ICAM-1, the intracellular domain also plays a pivotal role in leukocyte transendothelial migration (TEM), but has no catalytic activity (Sans et al, 2001;Greenwood et al, 2003). After ICAM-1 cross-linking or coculture with leukocytes, endothelial ICAM-1 initiates intracellular calcium flux and cytoskeletal signal transduction events, including activation of Rho GTPase and phosphorylation of cortactin, paxillin, p130 cas , and focal-adhesion kinase (DurieuTrautmann et al, 1994;Etienne et al, 1998;Adamson et al, 1999;Thompson et al, 2002).…”

Reactive carbonyl compounds (RCCs) cause aggregation and dysfunction of fibrinogen

“…al., 2001;Sans et. al., 2001), phagocytosis (Rubel et al, 2001(Rubel et al, , 2002, cytokine and chemokine expression (Fan and Edgington, 1993;RL Perez 1995;Smiley et.…”