Membranal Cholesterol Is Not Required for L-Selectin Adhesiveness in Primary Lymphocytes but Controls a Chemokine-Induced Destabilization of L-Selectin Rolling Adhesions

Dwir, Oren; Grabovsky, Valentin; Pasvolsky, Ronit; Manevich, Eugenia; Shamri, Revital; Gutwein, Paul; Feigelson, Sara W.; Altevogt, Peter; Alon, Ronen

doi:10.4049/jimmunol.179.2.1030

Cited by 6 publications

(6 citation statements)

References 61 publications

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“…It is also consistent with prior observation that faster rolling attenuates activation of integrin-mediated arrest (41), and that cholesterol extraction disrupts integrin-mediated adhesive process (16). …”

Section: Discussionsupporting

confidence: 92%

“…The prominent effect of cholesterol on both tether mechanics and cellular rigidity suggests a link between cholesterol and the actin cytoskeleton (8, 9). Since cholesterol removal by MβCD has been shown to inhibit polymerization (13) and disruption of actin cytoskeleton has been shown to interfere with intrinsic selectin adhesiveness (16), it is also possible that cholesterol effects on the actin network alter the mobility of adhesion molecules in the membrane which affects their binding ability. Also, disruption of the interaction between PSGL-1 and the actin cytoskeleton has been previously shown to reduce adhesion and rolling on P-selectin (39).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, cholesterol enrichment increases membrane stiffness in pure lipid (14). While Abbal et al found that cholesterol extraction reduced T-cell rolling (15), others did not detect changes in rolling behavior from cholesterol extraction (16). …”

Section: Introductionmentioning

confidence: 99%

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Membrane Cholesterol Is a Biomechanical Regulator of Neutrophil Adhesion

Mohler

Tian

et al. 2009

ATVB

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Objective-To evaluate the role of membrane cholesterol on human neutrophil and HL-60 biomechanics, capture, rolling, and arrest to P-selectin or IL-1-activated endothelium.Methods and Results-Methyl-β-cyclodextrin (MβCD) removed up to 73% and 45% of membrane cholesterol from HL-60 cells and neutrophils, while MβCD/cholesterol complexes resulted in maximum enrichment of 65% and 40%, respectively, above control levels. Cells were perfused at a venous wall shear rate of 100 s −1 over adherent P-selectin-coated 1-µm diameter beads, uncoated 10-µm diameter beads, P-selectin-coated surfaces, or activated endothelium. Elevated cholesterol enhanced capture efficiency to 1-µm beads and increased membrane tether growth rate by 1.5-to 2-fold, whereas cholesterol depletion greatly reduced tether formation. Elevated cholesterol levels increased tether lifetime by 17% in neutrophils and adhesion lifetime by 63% in HL-60 cells. Deformation of cholesterol-enriched neutrophils increased the contact time with 10-µm beads by 32% and the contact area by 7-fold. On both P-selectin surfaces and endothelial-cell monolayers, cholesterol-enriched neutrophils rolled more slowly, more stably, and were more likely to firmly arrest. Cholesterol depletion resulted in opposite effects.Conclusions-Increasing membrane cholesterol enhanced membrane tether formation and whole cell deformability, contributing to slower, more stable rolling on P-selectin and increased firm arrest on activated endothelium.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

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Membrane Cholesterol Is a Biomechanical Regulator of Neutrophil Adhesion

Mohler

Tian

et al. 2009

ATVB

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“…Collectively, these findings support the concept of lipid raft integrity as a precondition for microvillus receptor presentation. However, other studies seem to contradict: cell body CD44 in murine fibroblasts (38) but not microvillus L-selectin in human primary cells (39) was shown to be enriched in rafts using the detergent Triton X-100. This incongruence could be due to cell typespecific discrepancies, the existence of multiple mechanisms for protein sorting, or multiple types of rafts populating different surface subdomains.…”

Section: Discussionmentioning

confidence: 98%

The Transmembrane Domains of L-selectin and CD44 Regulate Receptor Cell Surface Positioning and Leukocyte Adhesion under Flow

Buscher

Riese

Shakibaei

et al. 2010

Journal of Biological Chemistry

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During inflammation and immune surveillance, initial contacts (tethering) between free-flowing leukocytes and the endothelium are vitally dependent on the presentation of the adhesion receptor L-selectin on leukocyte microvilli. Determinants that regulate receptor targeting to microvilli are, however, largely elusive. Therefore, we systematically swapped the extracellular (EC), transmembrane (TM), and intracellular (IC) domains of L-selectin and CD44, a hyaluronan receptor expressed on the cell body and excluded from microvilli. Electron microscopy of transfected human myeloid K562 cells showed that the highly conserved TM domains are responsible for surface positioning. The TM segment of L-selectin forced chimeric molecules to microvilli, and the CD44 TM domain evoked expression on the cell body, whereas the IC and EC domains hardly influenced surface localization. Transfectants with microvillus-based chimeras showed a significantly higher adhesion rate under flow but not under static conditions compared with cells with cell body-expressed receptors. Substitution of the IC domain of L-selectin caused diminished tethering but no change in surface distribution, indicating that both microvillus positioning and cytoskeletal anchoring contribute to leukocyte tethering. These findings demonstrate that TM domains of L-selectin and CD44 play a crucial role in cell adhesion under flow by targeting receptors to microvilli or the cell body, respectively.Neutrophil invasion at sites of inflammation as well as lymphocyte recirculation from blood into secondary lymphoid tissue require that free-flowing leukocytes become activated and subsequently exit the bloodstream. At the molecular level, the cells undergo a closely orchestrated cascade of interactions with the endothelium, each step consisting of a characteristic pattern of receptors, their ligands, cytokines, and chemoattractants (1, 2).Distinct cell surface receptor segregation is a widely observed phenomenon with effects on cell proliferation, migration, and tumorigenesis (3). Leukocyte membrane protrusions known as microvilli serve as active grouping sites for a variety of surface receptors including L-selectin (4), ␣4-integrins (5), P-selectin glycoprotein ligand 1 (PSGL-1) 3 (6, 7), CD4, and the chemokine receptors CCR5 and CXCR4 (8). In contrast, the hyaluronan receptor CD44 (4) and the integrins ␣M␤2 (Mac-1) and ␣L␤2 (LFA-1) (9, 10) show expression on the planar cell body excluding microvilli.Both L-selectin and CD44 are type I integral membrane glycoproteins expressed on most circulating leukocytes. L-selectin knock-out mice show a profound defect of lymphocyte accumulation in secondary lymphatic tissue and of neutrophils at sites of inflammation (11). CD44 is involved in many physiological processes, including lymphocyte activation, adhesion, and proliferation (12). There is also increasing evidence that CD44 and L-selectin contribute to tumor metastasis (13,14).Under many physiologic conditions, L-selectin initiates the first step of the adhesion cascade (15,1...

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“…In contrast, L-selectin was mostly in the nonraft fractions, confirming previous observations. 42 Both WT and ⌬CD PSGL-1 were located primarily in the buoyant fractions ( Figure 2B). Treatment of extracts with methyl-␤-cyclodextrin, a cholesterol chelator that disrupts raft structures, 7 moved both WT and ⌬CD PSGL-1 to the less buoyant fractions.…”

Section: ⌬Cd Psgl-1 Is Concentrated Normally On Leukocyte Microvillimentioning

confidence: 97%

Separable requirements for cytoplasmic domain of PSGL-1 in leukocyte rolling and signaling under flow

Miner

Xia

Yago

et al. 2008

Blood

142

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In inflamed venules, leukocytes use Pselectin glycoprotein ligand-1 (PSGL-1) to roll on P-selectin and E-selectin and to activate integrin ␣L␤2 (lymphocyte function-associated antigen-1, LFA-1) to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Studies in cell lines have suggested that PSGL-1 requires its cytoplasmic domain to localize in membrane domains, to support rolling on Pselectin, and to signal through spleen tyrosine kinase (Syk). We generated "⌬CD" mice that express PSGL-1 without the cytoplasmic domain. Unexpectedly, neutrophils from these mice localized PSGL-1 normally in microvilli, uropods, and lipid rafts. ⌬CD neutrophils expressed less PSGL-1 on their surfaces because of inefficient export from the endoplasmic reticulum. Limited digestion of wild-type neutrophils with O-sialoglycoprotein endopeptidase was used to reduce the PSGL-1 density to that on ⌬CD neutrophils. At matched PSGL-1 densities, both ⌬CD and wild-type neutrophils rolled similarly on P-selectin. However, ⌬CD neutrophils rolling on P-selectin did not trigger Sykdependent activation of LFA-1 to slow rolling on ICAM-1. These data demonstrate that the PSGL-1 cytoplasmic domain is dispensable for leukocyte rolling on P-selectin but is essential to activate ␤2 integrins to slow rolling on ICAM-1. IntroductionDuring inflammation, leukocytes tether to and roll on the vessel wall. They then roll more slowly until they arrest. Finally, they crawl through or between endothelial cells into the underlying tissues. Interactions of selectins with glycosylated ligands mediate tethering and rolling. Interactions of ␤2 integrins with ligands, such as intercellular adhesion molecule-1 (ICAM-1), mediate slow rolling and arrest. 1,2 These interactions occur in blood flow, which exerts force on adhesive bonds that affects bond lifetimes. 3,4 Furthermore, engagement of adhesion receptors transmits signals that intersect with chemokine receptor signals to influence the adhesion cascade. 1 Binding of integrin cytoplasmic domains to signaling and cytoskeletal proteins is critical for integrin function. 1,5 Interactions of selectin cytoplasmic domains with cytosolic proteins also contribute to their adhesive properties. E-selectin and P-selectin are expressed on activated endothelial cells and/or platelets, whereas L-selectin is expressed on the microvilli of leukocytes. 2 The cytoplasmic domains of E-selectin and P-selectin interact with clathrin-coated pits. These interactions cluster E-selectin and P-selectin on the endothelial cell surface, enhancing leukocyte rolling under flow. 6,7 The cytoplasmic domain anchors L-selectin to the cytoskeleton by binding to ␣-actinin 8 and to the ezrin/radixin/ moesin (ERM) family. 9 Mutation of the ERM-binding site in the cytoplasmic domain shifts L-selectin out of microvilli onto the cell body of transfected cells and impairs tethering to L-selectin ligands under flow. 10 Removal of the ␣-actinin-binding site markedly impairs rolling of transfected cells on L-selectin ligands, and deletion of the cytoplas...

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Membranal Cholesterol Is Not Required for L-Selectin Adhesiveness in Primary Lymphocytes but Controls a Chemokine-Induced Destabilization of L-Selectin Rolling Adhesions

Cited by 6 publications

References 61 publications

Membrane Cholesterol Is a Biomechanical Regulator of Neutrophil Adhesion

Membrane Cholesterol Is a Biomechanical Regulator of Neutrophil Adhesion

The Transmembrane Domains of L-selectin and CD44 Regulate Receptor Cell Surface Positioning and Leukocyte Adhesion under Flow

Separable requirements for cytoplasmic domain of PSGL-1 in leukocyte rolling and signaling under flow

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