The recruitment of specific leukocyte subtypes to the site LX 1606 Hippurate of tissue injury is the cornerstone of inflammation LX 1606 Hippurate and disease progression. between the endothelial cells and leukocytes. Several of the endothelial cell proteins that function in TEM are localized to the Lateral Border Recycling Compartment (LBRC) an interconnected reticulum of membrane that recycles selectively to the endothelial borders. The recruitment of the LBRC to surround the migrating leukocyte is required for efficient TEM. This review will focus on the proteins and mechanisms that mediate TEM and specifically how the LBRC functions in the context of these molecular interactions and membrane movements. demonstrated Cnp a role for the molecules PVR and DNAM-1 in TEM showing that monoclonal antibodies against either molecule could block the transmigration of isolated monocytes across a monolayer of cultured HUVEC [97]. More recently Manes and Pober used antibodies against PVR and DNAM-1 to block TEM of effector memory T cells [96]. However characterization of the mechanism of action and the relationship of these molecules to other components of the TEM machinery was only recently uncovered (discussed below) [95]. LX 1606 Hippurate CD99 and CD99L2 CD99 is a small 32 kDa Type 1 membrane protein that is highly O-glycosylated [105]. It does not belong to a characterized superfamily and only exhibits sequence similarity to one other protein CD99 antigen-like protein 2 (CD99L2). CD99L2 is usually a 45 kDa Type 1 membrane protein that has 32% amino acid similarity to CD99 and is similarly highly O-glycosylated [106 107 Both have short (<40 amino acids) but divergent cytoplasmic domains. Like LX 1606 Hippurate PECAM CD99 and CD99L2 are expressed at endothelial cell junctions and diffusely on leukocytes. CD99 facilitates TEM through homophilic interactions between the two cell types [108]. Similarly CD99L2 has been reported to function through homophilic interactions but it may have another unidentified ligand [109-111]. Disruption of CD99 and CD99L2 interactions using function blocking antibodies or genetic knock out (in the case of CD99L2) impairs leukocyte extravasation in vitro and in vivo. Interestingly unlike PECAM blockade which arrests leukocytes on the apical surface blocking CD99 function in vitro traps the migrating leukocytes midway through the junctions [108 112 While the requirement for CD99 and CD99L2 in TEM is established the mechanism by which they control TEM is unknown. In mice blocking either CD99 or CD99L2 by polyclonal antibody arrested leukocytes in vivo a similar step in extravasation suggesting that the two proteins functional to facilitate the same step [113]. Although there are several regions that are highly conserved between the two molecules and across species there are no known relevant interactions with other proteins that have been reported. Interestingly though in a study using the mouse homologues of CD99 and CD99L2 Nam and coworkers showed that the two molecules interact with LX 1606 Hippurate each other heterophilically through their cytoplasmic tails [111]. Through this interaction CD99 appears to facilitate the trafficking of CD99L2 to the plasma membrane. Key Events in TEM Sequential functions of molecules in TEM One interesting finding that is beginning to be appreciated in the field is that several of these molecules have been observed to function in a sequential manner during TEM [18 114 115 Current understanding of the process is that ICAM-1 and VCAM-1 function upstream of PECAM and CD99. This finding is well supported in the literature both in vivo and in vitro [55]. It also fits with the subcellular localization of these proteins with ICAM-1 and VCAM-1 both localized to the apical surface of endothelial cells where they can function in the activation and adherence of captured leukocytes whereas PECAM and CD99 are localized to the cell border where they facilitate the subsequent migration though the junction. Furthermore antibody blockade studies have highlighted an additional level of sequential function for PECAM PVR and CD99 operating in that order [95 108 These assays took advantage of the observation that the antibody blockade of one molecule can be relieved by extensive washing of unbound antibody and allowing additional time for TEM to recover. For example incubating transmigrating leukocytes with anti-PECAM antibodies halts TEM at the PECAM-requiring step. After washing out the antibody the.