LFA-1 signals to promote actin polymerization and upstream migration in T cells.

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the phosphoinositide 3-kinase (PI3K) and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, whereas VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl (also known as CBL), failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior.This article has an associated First Person interview with the first author of the paper.

Migration against the direction of flow is LFA-1-dependent in human hematopoietic stem and progenitor cells.

The recruitment of immune cells during inflammation is regulated by a multi-step cascade of cell rolling, activation, adhesion and transmigration through the endothelial barrier. Similarly, hematopoietic stem and progenitor cells (HSPCs) use this pathway to migrate and home to the bone marrow. After selectin-mediated braking, HSPCs migrate on adhesion ligands presented by the vascular endothelium including ICAM-1, VCAM-1 or MAdCAM-1. Here, we report that both the KG1a stem cell line and primary bone marrow CD34+ HSPCs can migrate against the direction of fluid flow on surfaces coated with cell adhesion molecules (CAMs), a behavior thus far only reported in T lymphocytes. We demonstrate that KG1a cells and primary HSPCs migrate upstream on surfaces presenting ICAM-1, downstream on surfaces presenting VCAM-1, and both upstream and downstream on surfaces presenting MAdCAM-1. In addition, we demonstrate that KG1a cells and HSPCs display upstream migration both on surfaces with multiple CAMs, as well as on human umbilical vein endothelial cell (HUVEC) monolayers. By blocking with monoclonal antibodies, we show that lymphocyte function-associated antigen-1 (LFA-1) is the key receptor responsible for upstream migration on the endothelium during the trafficking of HSPCs to the bone marrow.This article has an associated First Person interview with the first author of the paper.

Human Neutrophils Will Crawl Upstream on ICAM-1 If Mac-1 Is Blocked.

The recruitment of neutrophils to sites of inflammatory insult is a hallmark of the innate immune response. Neutrophil recruitment is regulated by a multistep process that includes cell rolling, activation, adhesion, and transmigration through the endothelium commonly referred to as the leukocyte adhesion cascade. After selectin-mediated braking, neutrophils migrate along the activated vascular endothelium on which ligands, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are expressed. Previous studies have shown that two cells that commonly home from blood vessel to tissue-T cells and hematopoietic stem and progenitor cells-use the integrin lymphocyte functional antigen-1 (LFA-1) to migrate against the direction of shear flow once adherent on ICAM-1 surfaces. Like T cells and hematopoietic stem and progenitor cells, neutrophils express LFA-1, but they also express macrophage-1 antigen (Mac-1), which binds to ICAM-1. Previous reports have shown that neutrophils will not migrate against the direction of flow on ICAM-1, but we hypothesized this was due to the influence of Mac-1. Here, we report that both the HL-60 neutrophil-like cell line and primary human neutrophils can migrate against the direction of fluid flow on ICAM-1 surfaces via LFA-1 if Mac-1 is blocked; otherwise, they migrate downstream. We demonstrate this both on ICAM-1 surfaces and on activated endothelium. In sum, both LFA-1 and Mac-1 binding ICAM-1 play a critical role in determining the direction of neutrophil migration along the endothelium, and their interaction may play an important role in controlling neutrophil trafficking during inflammation.

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes.

The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 α(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galß1,4GlcNAc) to create sialyl Lewis-X (sLe(X)) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe(X) epitope on both leukocyte N- and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34(+) hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function.

Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin-Dependent Rolling in the Multistep Leukocyte Adhesion Cascade.

OBJECTIVE: Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids expressed on human myeloid cells in regulating E-selectin-mediated cell adhesion. APPROACH AND RESULTS: A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase using either lentivirus-delivered shRNA or CRISPR-Cas9-based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UDP-Glucose Ceramide Glucosyltransferase disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics-based flow chamber studies demonstrated that both the UDP-Glucose Ceramide Glucosyltransferase knockouts and knockdowns display ≈60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells, without altering cell adhesion to P-selectin. Consistent with the concept that the glycosphingolipids support slow rolling and the transition to firm arrest, inhibiting UDP-Glucose Ceramide Glucosyltransferase activity resulted in frequent leukocyte detachment events, skipping motion, and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished. CONCLUSIONS: Glycosphingolipids likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.

Silencing α1,3-fucosyltransferases in human leukocytes reveals a role for FUT9 enzyme during E-selectin-mediated cell adhesion.

Leukocyte adhesion during inflammation is initiated by the binding of sialofucosylated carbohydrates expressed on leukocytes to endothelial E/P-selectin. Although the glycosyltransferases (glycoTs) constructing selectin-ligands have largely been identified using knock-out mice, important differences may exist between humans and mice. To address this, we developed a systematic lentivirus-based shRNA delivery workflow to create human leukocytic HL-60 cell lines that lack up to three glycoTs. Using this, the contributions of all three myeloid α1,3-fucosyltransferases (FUT4, FUT7, and FUT9) to selectin-ligand biosynthesis were evaluated. The cell adhesion properties of these modified cells to L-, E-, and P-selectin under hydrodynamic shear were compared with bone marrow-derived neutrophils from Fut4(-/-)Fut7(-/-) dual knock-out mice. Results demonstrate that predominantly FUT7, and to a lesser extent FUT4, forms the selectin-ligand at the N terminus of leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) in humans and mice. Here, 85% reduction in leukocyte interaction was observed in human FUT4(-)7(-) dual knockdowns on P/L-selectin substrates. Unlike Fut4(-/-)Fut7(-/-) mouse neutrophils, however, human knockdowns lacking FUT4 and FUT7 only exhibited partial reduction in rolling interaction on E-selectin. In this case, the third α1,3-fucosyltransferase FUT9 played an important role because leukocyte adhesion was reduced by 50-60% in FUT9-HL-60, 70-80% in dual knockdown FUT7(-)9(-) cells, and ∼85% in FUT4(-)7(-)9(-) triple knockdowns. Gene silencing results are in agreement with gain-of-function experiments where all three fucosyltransferases conferred E-selectin-mediated rolling in HEK293T cells. This study advances new tools to study human glycoT function. It suggests a species-specific role for FUT9 during the biosynthesis of human E-selectin ligands.

Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and "roll" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).

Not all (cells) who wander are lost: Upstream migration as a pervasive mode of amoeboid cell motility.

Leukocytes possess the ability to migrate upstream-against the direction of flow-on surfaces of specific chemistry. Upstream migration was first characterized in vitro for T-cells on surfaces comprised of intracellular adhesion molecule-1 (ICAM-1). Upstream migration occurs when the integrin receptor αLß2 (also known as lymphocyte function-associated antigen-1, or LFA-1) binds to ICAM-1. LFA-1/ICAM-1 interactions are ubiquitous and are widely found in leukocyte trafficking. Upstream migration would be employed after cells come to arrest on the apical surface of the endothelium and might confer an advantage for both trans-endothelial migration and tissue surveillance. It has now been shown that several other motile amoeboid cells which have the responsibility of trafficking from blood vessels into tissues, such as Marginal zone B cells, hematopoietic stem cells, and neutrophils (when macrophage-1 antigen, Mac-1, is blocked), can also migrate upstream on ICAM-1 surfaces. This review will summarize what is known about the basic mechanisms of upstream migration, which cells have displayed this phenomenon, and the possible role of upstream migration in physiology and tissue homeostasis.

Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins.

Novel strategies to control the binding of adhesion molecules belonging to the selectin family are required for the treatment of inflammatory diseases. We tested the possibility that synthetic monosaccharide analogs can compete with naturally occurring sugars to alter the O-glycan content on human leukocyte cell surface selectin-ligand, P-selectin glycoprotein ligand-1 (PSGL-1). Resulting reduction in the sialyl Lewis-X-bearing epitopes on this ligand may reduce cell adhesion. Consistent with this hypothesis, 50muM per-acetylated 4F-GalNAc added to the growth media of promyelocytic HL-60 cells reduced the expression of the cutaneous lymphocyte associated-antigen (HECA-452 epitope) by 82% within 2 cell doubling cycles. Cell binding to all 3 selectins (L-, E-, and P-selectin) was reduced in vitro. 4F-GalNAc was metabolically incorporated into PSGL-1, and this was accompanied by an approximately 20% reduction in PSGL-1 glycan content. A 70% to 85% reduction in HECA-452 binding epitope and N-acetyl lactosamine content in PSGL-1 was also noted on 4F-GalNAc addition. Intravenous 4F-GalNAc infusion reduced leukocyte migration to the peritoneum in a murine model of thioglycolate-induced peritonitis. Thus, the compound has pharmacologic activity. Overall, the data suggest that 4F-GalNAc may be applied as a metabolic inhibitor to reduce O-linked glycosylation, sialyl Lewis-X formation, and leukocyte adhesion via the selectins.

Don't sugarcoat it: How glycocalyx composition influences cancer progression.

Mechanical interactions between tumors and the extracellular matrix (ECM) of the surrounding tissues have profound effects on a wide variety of cellular functions. An underappreciated mediator of tumor-ECM interactions is the glycocalyx, the sugar-decorated proteins and lipids that act as a buffer between the tumor and the ECM, which in turn mediates all cell-tissue mechanics. Importantly, tumors have an increase in the density of the glycocalyx, which in turn increases the tension of the cell membrane, alters tissue mechanics, and drives a more cancerous phenotype. In this review, we describe the basic components of the glycocalyx and the glycan moieties implicated in cancer. Next, we examine the important role the glycocalyx plays in driving tension-mediated cancer cell signaling through a self-enforcing feedback loop that expands the glycocalyx and furthers cancer progression. Finally, we discuss current tools used to edit the composition of the glycocalyx and the future challenges in leveraging these tools into a novel tractable approach to treat cancer.

Proteoglycans as Mediators of Cancer Tissue Mechanics.

Proteoglycans are a diverse group of molecules which are characterized by a central protein backbone that is decorated with a variety of linear sulfated glycosaminoglycan side chains. Proteoglycans contribute significantly to the biochemical and mechanical properties of the interstitial extracellular matrix where they modulate cellular behavior by engaging transmembrane receptors. Proteoglycans also comprise a major component of the cellular glycocalyx to influence transmembrane receptor structure/function and mechanosignaling. Through their ability to initiate biochemical and mechanosignaling in cells, proteoglycans elicit profound effects on proliferation, adhesion and migration. Pathologies including cancer and cardiovascular disease are characterized by perturbed expression of proteoglycans where they compromise cell and tissue behavior by stiffening the extracellular matrix and increasing the bulkiness of the glycocalyx. Increasing evidence indicates that a bulky glycocalyx and proteoglycan-enriched extracellular matrix promote malignant transformation, increase cancer aggression and alter anti-tumor therapy response. In this review, we focus on the contribution of proteoglycans to mechanobiology in the context of normal and transformed tissues. We discuss the significance of proteoglycans for therapy response, and the current experimental strategies that target proteoglycans to sensitize cancer cells to treatment.

T lymphocytes migrate upstream after completing the leukocyte adhesion cascade.

The leukocyte adhesion cascade is of critical importance for both the maintenance of immune homeostasis and the ability of immune cells to perform effector functions. Here, we present data showing CD4+ T cells migrate upstream (against the direction of flow) after completing the leukocyte adhesion cascade on surfaces displaying either ICAM-1 or ICAM-1 and VCAM-1, but migrate downstream on surfaces displaying only VCAM-1. Cells completing the cascade on HUVECs initially migrate upstream before reverting to more random migration, partly caused by transmigration of cells migrating against the flow. Furthermore, cells migrating upstream transmigrate faster than cells migrating downstream. On HUVECs, blocking interactions between LFA-1 and ICAM-1 resulted in downstream migration and slower transmigration. These results further suggest a possible physiological role for upstream migration in vivo.

The blood-borne sialyltransferase ST6Gal-1 is a negative systemic regulator of granulopoiesis.

Responding to systemic demands in producing and replenishing end-effector blood cells is predicated on the appropriate delivery and interpretation of extrinsic signals to the HSPCs. The data presented herein implicate the systemic, extracellular form of the glycosyltransferase ST6Gal-1 in the regulation of late-stage neutrophil development. ST6Gal-1 is typically a membrane-bound enzyme sequestered within the intracellular secretory apparatus, but an extracellular form is released into the blood from the liver. Both human and murine HSPCs, upon exposure to extracellular ST6Gal-1 ex vivo, exhibited decreased proliferation, diminished expression of the neutrophilic primary granule protein MPO, and decreased appearance of CD11b+ cells. HSPC suppression was preceded by decreased STAT-3 phosphorylation and diminished C/EBPα expression, without increased apoptosis, indicating attenuated G-CSF receptor signaling. A murine model to raise systemic ST6Gal-1 level was developed to examine the role of the circulatory enzyme in vivo. Our results show that systemic ST6Gal-1 modified the cell surface of the GMP subset of HSPCs and decreased marrow neutrophil reserves. Acute airway neutrophilic inflammation by LPS challenge was used to drive demand for new neutrophil production. Reduced neutrophil infiltration into the airway was observed in mice with elevated circulatory ST6Gal-1 levels. The blunted transition of GMPs into GPs in vitro is consistent with ST6Gal-1-attenuated granulopoiesis. The data confirm that circulatory ST6Gal-1 is a negative systemic regulator of granulopoiesis and moreover suggest a clinical potential to limit the number of inflammatory cells by manipulating blood ST6Gal-1 levels.

Leukocyte-borne α(1,3)-fucose is a negative regulator of ß2-integrin-dependent recruitment in lung inflammation.

Leukocyte recruitment in inflammation is a multistep, sequential cascade where the initial step is the selectin-dependent tethering, followed by the formation of firmer integrin-mediated adhesive forces leading to extravasation. The α(1,3)-fucose-containing sialyl-Lewis X (sLeX) is the archetypical ligand on leukocyte surfaces mediating selectin interactions. Canonically, disruption of α(1,3)-fucose formation ablates selectin-mediated adhesion, dramatically reducing trafficking. We report a paradoxical response to α(1,3)-fucose deficiency in which the loss exacerbated rather than attenuated leukocyte recruitment in a murine model of acute airway inflammation. The architecture of the capillary-dominated vasculature in the lung minimized the importance of the selectin dependent step, and we observed that α(1,3)-fucose deficiency augmented CXCR2-mediated Rap1-GTP signaling to enhance the ß2-integrin-ICAM-1-binding axis. The data disclose a previously unknown function for α(1,3)-fucose, in which this structure negatively regulates the integrin activation step in leukocyte recruitment.

Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion.

There is often interest in dissecting the relative contributions of the N-glycans, O-glycans and glycosphingolipids (GSLs) in regulating complex biological traits like cell signaling, adhesion, development and metastasis. To address this, we developed a CRISPR-Cas9 toolkit to selectively truncate each of these commonly expressed glycan-types. Here, O-glycan biosynthesis was truncated by knocking-out Core 1 ß3Gal-T Specific Molecular Chaperone (COSMC), N-glycans by targeting the ß1,2 GlcNAc-transferase (MGAT1) and GSLs by deleting UDP-glucose ceramide glucosyltransferase (UGCG). These reagents were applied to reveal the glycoconjugates regulating human myeloid cell adhesion to selectins under physiological shear-flow observed during inflammation. These functional studies show that leukocyte rolling on P- and L-selectin is ablated in cells lacking O-glycans, with N-glycan truncation also increasing cell rolling velocity on L-selectin. All three glycan families contributed to E-selectin dependent cell adhesion with N-glycans contributing to all aspects of the leukocyte adhesion cascade, O-glycans only being important during initial recruitment, and GSLs stabilizing slow cell rolling and the transition to firm arrest. Overall, the genome editing tools developed here may be broadly applied in studies of cellular glycosylation.

Distinct glycosyltransferases synthesize E-selectin ligands in human vs. mouse leukocytes.

The binding of selectins to carbohydrate epitopes expressed on leukocytes is the first step in a multi-step cell adhesion cascade that controls the rate of leukocyte recruitment at sites of inflammation. The glycans that function as selectin-ligands are post-translationally synthesized by the serial action of Golgi resident enzymes called glycosyltransferases (glycoTs). Whereas much of our current knowledge regarding the role of glycoTs in constructing selectin-ligands comes from reconstituted biochemical investigations or murine models, tools to assess the impact of these enzymes on the human ligands are relatively underdeveloped. This is significant since the selectin-ligands, particularly those that bind E-selectin, vary between different leukocyte cell populations and they are also different in humans compared with mice. To address this shortcoming, a recent study by Buffone et al. (2013) outlines a systematic strategy to knockdown upto three glycoTs simultaneously in human leukocytes. The results suggest that the fucosyltransferases (FUTs) regulating selectin-ligand synthesis may be species-specific. In particular, they demonstrate that FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Overall, this article discusses the relative roles of the FUTs during human L-, E-, and P-selectin-ligand biosynthesis, and the potential that the knockdown strategy outlined here may assess the role of other glycoTs in human leukocytes also.