Lipid Raft Is Required for PSGL-1 Ligation Induced HL-60 Cell Adhesion on ICAM-1

Tingshuang Xu,Jixian Luo,Yong Jiang,Xianlu Zeng,Xiaoguang Wang,Chunfeng Li,Wenai Liu,Xueqing Ba,Khamal Kwesi Ampah,Robert Dettman

doi:10.1371/journal.pone.0081807

Abstract

P-selectin glycoprotein ligand-1 (PSGL-1) and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD), we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation) to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These ﬁndings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

Highlights

IntroductionThe recruitment of neutrophils to a site of infection or tissue injury entails a cascade of cellular adhesive events, including tethering, rolling, adhesion, diapedesis, transmigration and chemotaxis [1,2,3]
Neutrophils are essential component of the innate immune system
We quantitatively investigated the effect of P-selectin glycoprotein ligand 1 (PSGL-1) ligation on HL-60 cell adhesion and morphological changes using time-lapsed videomicroscopy to record HL-60 adhesion in ICAM-1 coated plate for 10 min (Figure 1C)

Summary

Introduction

The recruitment of neutrophils to a site of infection or tissue injury entails a cascade of cellular adhesive events, including tethering, rolling, adhesion, diapedesis, transmigration and chemotaxis [1,2,3]. PSGL-1 binds to P- or E-selectin expressed on activated endothelium, and mediates leukocyte rolling on endothelium. Integrins are activated and mediate the cell arrest and stable adhesion to endothelium under shear flow conditions [2,7]. PSGL-1/selectin binding appears to be involved in the activation of integrin in a gradual manner [8,9]. Investigating the molecular mechanism underlying the PSGL-1-induced signals and integrin activation is of great importance for understanding the relationship between the initial rolling and subsequent adhesion

Methods

Results

Conclusion