Zahra Mamdouh

CD99 plays a major role in the migration of monocytes through endothelial junctions

CD99 is a heavily O-glycosylated 32-kD type I transmembrane protein that is expressed on most hematopoietic cells. We show here that CD99 is expressed on endothelial cells and is concentrated at the borders between confluent cells. We found that a monoclonal antibody to CD99, hec2, selectively inhibited diapedesis of monocytes across endothelial cells by >90%. Diapedesis involved the homophilic interaction of CD99 on monocytes with CD99 on endothelial junctions. CD99 functioned distally to the point at which platelet-endothelial cell adhesion molecule 1 (PECAM-1, also known as CD31), another adhesion molecule involved in transmigration, played its critical role. Confocal microscopy showed that anti–PECAM-1 arrested leukocytes on the apical surface of endothelium, whereas blocking CD99 arrested monocytes at a point where they were partially through the junction. Therefore, diapedesis, the forward migration of leukocytes through endothelial junctions, is regulated sequentially by two distinct molecules, PECAM-1 and CD99.

Locomotion of monocytes on endothelium is a critical step during extravasation

Monocytes, like all leukocytes, undergo a series of sequential steps during extravasation from blood into tissues: tethering, rolling, adhesion and diapedesis. We have discovered an essential step, which we call locomotion, in which the monocyte moves from a site of firm adhesion to the nearest junction to begin diapedesis. Blocking CD11a-CD18 and CD11b-CD18 on human monocytes or adhesion molecules ICAM-1 and ICAM-2 on endothelial cells prevented the monocytes from reaching junctions. The blocked monocytes spun in circles as if they were unable to direct their movement despite being able to adhere and polarize normally. This step fills a gap in the paradigm of extravasation as a multistep process.

Targeted recycling of PECAM from endothelial surface-connected compartments during diapedesis

Leukocytes enter sites of inflammation by squeezing through the borders between endothelial cells that line postcapillary venules at that site. This rapid process, called transendothelial migration (TEM) or diapedesis, is completed within 90 s after a leukocyte arrests on the endothelial surface. In this time, the leukocyte moves in ameboid fashion across the endothelial borders, which remain tightly apposed to it during transit. It is not known how the endothelial cell changes its borders rapidly and reversibly to accommodate the migrating leukocyte. Here we show that there is a membrane network just below the plasmalemma at the cell borders that is connected at intervals to the junctional surface. PECAM-1, an integral membrane protein with an essential role in TEM, is found in this compartment and constitutively recycles evenly along endothelial cell borders. During TEM, however, recycling PECAM is targeted to segments of the junction across which monocytes are in the act of migration. In addition, blockade of TEM with antibodies against PECAM specifically blocks the recruitment of this membrane to the zones of leukocyte migration, without affecting the constitutive membrane trafficking.

Leukocyte transmigration requires kinesin-mediated microtubule-dependent membrane trafficking from the lateral border recycling compartment

Mamdouh et al. 2008. J. Exp. Med. doi:10.1084/jem.20072328[OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft.jtitle%253DJ.%2BExp.%2BMed.%26rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20072328%26rft_id%253Dinfo%253Apmid%252F18378793%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%

Unique cellular events occurring during the initial interaction of macrophages with matrix-retained or methylated aggregated low density lipoprotein (LDL). Prolonged cell-surface contact during which ldl-cholesteryl ester hydrolysis exceeds ldl protein degradation.

A critical event in atherogenesis is the interaction of arterial wall macrophages with subendothelial lipoproteins. Although most studies have investigated this interaction by incubating cultured macrophages with monomeric lipoproteins dissolved in media, arterial wall macrophages encounter lipoproteins that are mostly bound to subendothelial extracellular matrix, and these lipoproteins are often aggregated or fused. Herein, we utilize a specialized cell-culture system to study the initial interaction of macrophages with aggregated low density lipoprotein (LDL) bound to extracellular matrix. The aggregated LDL remains extracellular for a relatively prolonged period of time and becomes lodged in invaginations in the surface of the macrophages. As expected, the degradation of the protein moiety of the LDL was very slow. Remarkably, however, hydrolysis of the cholesteryl ester (CE) moiety of the LDL was 3–7-fold higher than that of the protein moiety, in stark contrast to the situation with receptor-mediated endocytosis of acetyl-LDL. Similar results were obtained using another experimental system in which the degradation of aggregated LDL protein was delayed by LDL methylation rather than by retention on matrix. Additional experiments indicated the following properties of this interaction: (a) LDL-CE hydrolysis is catalyzed by lysosomal acid lipase; (b) neither scavenger receptors nor the LDL receptor appear necessary for the excess LDL-CE hydrolysis; and (c) LDL-CE hydrolysis in this system is resistant to cellular potassium depletion, which further distinguishes this process from receptor-mediated endocytosis. In summary, experimental systems specifically designed to mimic thein vivo interaction of arterial wall macrophages with subendothelial lipoproteins have demonstrated an initial period of prolonged cell-surface contact in which CE hydrolysis exceeds protein degradation.

A Novel and Critical Role for Tyrosine 663 in Platelet Endothelial Cell Adhesion Molecule-1 Trafficking and Transendothelial Migration

PECAM-1/CD31 is required for leukocyte transendothelial migration (TEM) under most inflammatory conditions. A critical pool of PECAM-1 resides in the lateral border recycling compartment (LBRC). During TEM, membrane from the LBRC is redirected to surround the leukocyte, and this targeted recycling per se is required for TEM. The cytoplasmic domain of PECAM-1 contains two tyrosine residues that have been implicated in PECAM-1 signaling in other cells but never examined in the context of TEM. We found that expression of PECAM-1 imparts on cells the ability to support TEM and that tyrosine 663 (but not tyrosine 686) is required. Furthermore, tyrosine 663 is required for PECAM-1 to efficiently enter and exit the LBRC. Most important, mutation of tyrosine 663 abolishes the ability of the endothelial cells to support targeted recycling of the LBRC. These data define a novel role for tyrosine 663 and suggest that it is part of a recognition motif for trafficking to and/or from the LBRC.