CD63 is an essential cofactor to leukocyte recruitment by endothelial P-selectin

Abstract

The activation of endothelial cells is critical to initiating an inflammatory response. Activation induces the fusion of WeibelPalade Bodies (WPB) with the plasma membrane, thus transferring P-selectin and VWF to the cell surface, where they act in the recruitment of leukocytes and platelets, respectively. CD63 has long been an established component of WPB, but the functional significance of its presence within an organelle that acts in inflammation and hemostasis was unknown. We find that ablating CD63 expression leads to a loss of P-selectindependent function: CD63-deficient HUVECs fail to recruit leukocytes, CD63-deficient mice exhibit a significant reduction in both leukocyte rolling and recruitment and we show a failure of leukocyte extravasation in a peritonitis model. Loss of CD63 has a similar phenotype to loss of P-selectin itself, thus CD63 is an essential cofactor to P-selectin. (Blood. 2011; 118(15):4265-4273) IntroductionEndothelial cells contain specialized regulated secretory organelles, Weibel-Palade bodies (WPB), 1 which play an important role in inflammation and hemostasis. The best-known components of WPB are the major cargo protein von Willebrand Factor (VWF), 2 P-selectin, 3 and CD63. 4 On WPB fusion the VWF tubules unfurl into long platelet-catching strings at a site of injury, 5 playing a major role in hemostasis. VWF also has an important function in inflammation, because activated platelets make a major contribution to the inflammatory response. 6 Further, VWF binds the leukocyte receptor PSGL-1 and integrin ␣ M ␤ 2 7 implicating a more direct role in leukocyte recruitment to the endothelium.The integral membrane protein leukocyte receptor P-selectin is stored within the membrane of WPB of endothelial cells 3,8 from where it is delivered to the cell surface within minutes after secretagogue-triggered exocytosis. 9 P-selectin plays a key early role in the inflammatory trafficking of leukocytes, being the first receptor involved in recruiting leukocytes from flowing plasma to the endothelial surface. 10,11 CD63 12 is the third long-established component of WPB, 4 but this protein has no identified endothelial-specific function in either hemostasis or inflammation. This universally expressed membrane protein of the tetraspanin family is best known as a marker of the intralumenal vesicles within multivesicular endosomes. The phenotype of CD63 knockout mice suggests a redundant role for CD63 in development and distribution of immune system cells, very mild effects on platelet adhesion, and a role in kidney physiology. 13 It has been reported that CD63 might modulate the trafficking of other membrane proteins, including altering internalization from the plasma membrane, and most recently, in targeting synaptotagmin VII to the lysosome, thus facilitating lysosome-plasma membrane fusion. 12,14 Whether CD63 is involved in the hemostatic or inflammatory roles of WPB is unknown, but if so, it might operate by modulating trafficking events that underpin WPB formation and function. CD63 is found only...

Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

“…Contrary to the increased presence of P-selectin in WPB, we found strongly decreased amounts of CD63 in WPB of anxA8-depleted HUVEC. These findings point to a regulatory function of anxA8 in delivering CD63 to WPB, which is in line with the suggestion that WPB-derived CD63 stabilizes cell surface levels of P-selectin 6 . The fact that we did not detect any effects of either increased or reduced levels of anxA8 on the formation, overall morphology or regulated exocytosis of WPB (defined by the histamine-evoked release of VWF) argues against a contribution of direct VWF-PMN interactions 28 to the impaired leukocyte recruitment seen in anxA8-ablated HUVEC.…”

“…Recently, the secretagogue-induced cell surface presentation of P-selectin was reported to critically depend on the tetraspanin CD63 (ref. 6). Thus, the lower P-selectin cell surface presentation observed upon anxA8 depletion might be caused by reduced CD63 cell surface levels.…”

“…Recent evidence indicates that CD63, another WPB component, acts as a P-selectin retention factor at the plasma membrane. Ablation of CD63 was reported to cause decreased P-selectin levels at the cell surface, resulting in impaired leukocyte recruitment 6 . Indeed, we found that levels of CD63 were significantly reduced on the plasma membrane of anxA8-silenced cells when the cells had been stimulated with histamine.…”

“…Considering the role of WPB-associated CD63 in P-selectinmediated leukocyte adhesion, this transport process is, however, of prime importance, as it delivers CD63 to WPB and thus guarantees the simultaneous cell surface presentation of CD63 and P-selectin and their function in leukocyte recruitment 6 . Therefore, we set out to identify cellular factors required for the delivery of CD63 to WPB, focussing on endosome-associated components.…”

“…Recently, the tetraspanin CD63 (also known as lysosome-associated membrane protein 3 (LAMP3)) was shown to function as an essential cofactor for P-selectinmediated leukocyte recruitment, presumably through stabilization of larger P-selectin clusters that are only formed in the presence of CD63 (ref. 6).…”