Adhesion Cascade Research Articles

Glycomechanics of the Metastatic Cascade: Tumor Cell–Endothelial Cell Interactions in the Circulation

Hydrodynamic shear force plays an important role in the leukocyte adhesion cascade that involves the tethering and rolling of cells along the endothelial layer, their firm adhesion or arrest, and their extravasation or escape from the circulatory system by inducing passive deformation, or cell flattening, and microvilli stretching, as well as regulating the expression, distribution, and conformation of adhesion molecules on leukocytes and the endothelial layer. Similarly, the dissemination of circulating tumor cells (CTCs) from the primary tumor sites is believed to involve tethering, rolling, and firm adhesion steps before their eventual extravasation which leads to secondary tumor sites (metastasis). Of particular importance to both the leukocyte adhesion cascade and the extravasation of CTCs, glycoproteins are involved in all three steps (capture, rolling, and firm adhesion) and consist of a variety of important selectin ligands. This review article provides an overview of glycoprotein glycosylation associated with the abnormal glycan expression on cancer cell surfaces, where well-established and novel selectin ligands that are cancer related are discussed. An overview of computational approaches on the effects of fluid mechanical force on glycoprotein mediated cancer cell rolling and adhesion is presented with a highlight of recent flow-based and selectin-mediated cell capturing/enriching devices. Finally, as an important branch of the glycoprotein family, mucins, specifically MUC1, are discussed in the context of their aberrant expression on cancer cells and their role as cancer cell adhesion molecules. Since metastasis relies heavily on glycoprotein interactions in the bloodstream where the fluid shear stress highly regulates cell adhesion forces, it is important to study and understand the glycomechanics of all relevant glycoproteins (well-established and novel) as they relate to the metastatic cascade.

Local Adrenergic Nerves Regulate Diurnal Leukocyte Adhesion: Impact In Sickle Cell Disease

Abstract 1099Leukocyte recruitment is a crucial component of immune homeostasis. The sequential multistep process leading to leukocyte migration is thought to be regulated on demand at the inflammatory site. Here, we show that broad systemic programs involving long-range signals from the sympathetic nervous system (SNS) delivered by adrenergic nerves regulate rhythmic recruitment of leukocytes in tissues. While oscillations in the numbers of circulating leukocytes ranged from 4.1 to 7.8 × 106 cells/ml blood (P<0.001) with a peak ∼5h after the onset of light (zeitgeber time, ZT5), numbers of extravascular leukocytes in skeletal muscle exhibited circadian fluctuations whose pattern ran in antiphase with that of blood, peaking at ZT13 (night) and reaching a trough at ZT5 (ZT5 / ZT13: 5.3 ± 0.3 / 7.3 ± 0.3 cells per 100 × 50 μm2 extravascular vessel segment, p<0.001). Investigations of leukocyte-endothelial cell interactions in real-time by multi-channel fluorescence intravital microscopy (MFIM) of the cremasteric muscle microvasculature revealed increased myeloid cell recruitment at night (1.7-fold enhanced adhesion, p<0.05). Migratory oscillations were implemented by local SNS fibers since unilateral neurectomy of the genitofemoral nerve (GFNx), the nerve that innervates this muscle, completely (100%) ablated the night increase in leukocyte recruitment, suggesting an essential role for local innervation of the tissue in this process. Bone marrow chimeras revealed that signals acting on β2 and β3 adrenoreceptors expressed by non-hematopoietic cells were responsible for circadian recruitment leading to fluctuations of intercellular cell adhesion molecule-1 (ICAM-1) expression on endothelial cells (1.7- and 2-fold increase at ZT13 in ICAM-1 mRNA and protein levels, p<0.001 and p<0.05 respectively). ICAM-1 plays a critical role in this activity since Icam1-deficient animals did not exhibit an apparent rhythm in leukocyte infiltration. GFNx also completely abolished the night increase of ICAM-1 expression providing a direct link between SNS input and leukocyte infiltration. Since circadian rhythms are synchronized by light we tested whether alterations in photic input by induction of experimental jetlag would be sufficient to modify rhythms in leukocyte recruitment to the cremaster muscle. Indeed, circadian rhythms in extravascular leukocyte numbers in the tissue and fluctuations of ICAM-1 expression were completely abolished after a 12h jetlag. Oscillations were mediated by the molecular clock since rhythms of leukocyte numbers in both blood and tissue ceased to exist in Bmal1−/− animals kept in constant darkness for three weeks, in contrast to normal rhythms observed in heterozygous and wild-type littermates. Remarkably, circadian rhythms in leukocyte recruitment persisted during inflammation. After administration of TNF-α, circadian rhythms in ICAM-1 expression (1.4-fold increase, p<0.05) and leukocyte recruitment remained apparent, with ZT13 showing significantly higher leukocyte infiltration than ZT5. In contrast, TNF-α-induced leukocyte infiltration (82%) and adhesion molecule expression were strongly dampened when mice were subjected to a jetlag. To test the relevance in a disease model, we evaluated mice with sickle cell disease (SCD), a genetic disorder in which leukocyte adhesion and activation play a critical role in acute vaso-occlusion. Intravital microscopy revealed an increase in the numbers of adherent and extravasated leukocytes when the experiment was performed at night. In addition, we observed a 2-fold increase in the interactions between circulating RBCs and adherent leukocytes mediated by enhanced β2 integrin (Mac-1) activity (1.9-fold, p<0.05) on leukocytes and resulting in a 31% reduction of the mean venular blood flow rates. Strikingly, the overall survival of SCD mice was significantly reduced in mice challenged at night compared to the daytime (mean survival night: 305 min, day: 489 min, p<0.05, Log rank test). In conclusion, these data suggest that circadian rhythms in leukocyte recruitment can influence disease outcome and provide a novel paradigm in the leukocyte adhesion cascade with the potential for time-based therapeutics in inflammatory diseases. Disclosures:No relevant conflicts of interest to declare.

Talin-1 and Kindlin-3 Regulate α4β1 Integrin-Mediated Adhesion Stabilization, but Not G Protein-Coupled Receptor-Induced Affinity Upregulation

Chemokine/chemoattractant G protein-coupled receptors trigger an inside-out signaling network that rapidly activates integrins, a key step in inflammatory leukocyte recruitment. Integrins mediate leukocyte arrest and adhesion to endothelium through multivalent binding, and they transmit outside-in signals to stabilize adhesion and coordinate cell spreading and migration. In the present study, we used RNA interference in the U937 monocytic cell line to investigate the role of talin-1, kindlin-3, and α-actinin-1 in the fMLF- and SDF-1α-induced upregulation of α(4)β(1) integrin affinity and consequent adhesive events. Affinity upregulation of α(4)β(1) integrin was not impaired by small interfering RNA knockdown of talin-1, kindlin-3, or α-actinin-1. Only kindlin-3 knockdown increased flow-induced detachment from VCAM-1-coated surfaces in response to fluid flow, whereas knockdown of either talin-1 or kindlin-3 increased detachment from ICAM-1-coated surfaces. Biochemical analyses revealed that α(4)β(1) expression was highly enriched in U937 cell microridges and murine lymphocyte microvilli. Kindlin-3 was present throughout the cell, whereas talin-1 was largely excluded from microridges/microvilli. The subcellular colocalization of α(4)β(1) and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α(4)β(1) after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α(4)β(1)-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement.

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

Bacteroides fragilis Enterotoxin Upregulates Intercellular Adhesion Molecule-1 in Endothelial Cells via an Aldose Reductase-, MAPK-, and NF-κB–Dependent Pathway, Leading to Monocyte Adhesion to Endothelial Cells

Enterotoxigenic Bacteroides fragilis (ETBF) produces a ∼ 20-kDa heat-labile enterotoxin (BFT) that plays an essential role in mucosal inflammation. Although a variety of inflammatory cells is found at ETBF-infected sites, little is known about leukocyte adhesion in response to BFT stimulation. We investigated whether BFT affected the expression of ICAM-1 and monocytic adhesion to endothelial cells (ECs). Stimulation of HUVECs and rat aortic ECs with BFT resulted in the induction of ICAM-1 expression. Upregulation of ICAM-1 was dependent on the activation of IκB kinase (IKK) and NF-κB signaling. In contrast, suppression of AP-1 did not affect ICAM-1 expression in BFT-stimulated cells. Suppression of NF-κB activity in HUVECs significantly reduced monocytic adhesion, indicating that ICAM-1 expression is indispensable for BFT-induced adhesion of monocytes to the endothelium. Inhibition of JNK resulted in a significant attenuation of BFT-induced ICAM-1 expression in ECs. Moreover, inhibition of aldose reductase significantly reduced JNK-dependent IKK/NF-κB activation, ICAM-1 expression, and adhesion of monocytes to HUVECs. These results suggest that a signaling pathway involving aldose reductase, JNK, IKK, and NF-κB is required for ICAM-1 induction in ECs exposed to BFT, and may be involved in the leukocyte-adhesion cascade following infection with ETBF.

L-Selectin – A dynamic regulator of leukocyte migration

The leukocytic cell adhesion receptor l-selectin mediates the initial step of the adhesion cascade, the capture and rolling of leukocytes on endothelial cells. This event enables leukocytes to migrate out of the vasculature into surrounding tissues during inflammation and immune surveillance. Distinct domains of l-selectin contribute to proper leukocyte migration. In this review, we discuss the contributions of these domains with respect to l-selectin function: the regulation by serine phosphorylation of the cytoplasmic tail, the role of the transmembrane domain in receptor positioning on the cell surface as well as the N-glycosylation of the extracellular part and the identification of novel binding partners.

Hepatic consequences of vascular adhesion protein-1 expression

The liver is constantly exposed to antigens present in the blood and to particulate antigens delivered from the gut. To maintain effective levels of immune surveillance and yet tolerate food antigens, the hepatic environment has become highly specialised. A low flow environment exists within the hepatic sinusoids that not only facilitates the exchange of toxins and nutrients within the liver parenchyma, but also provides an ideal niche for the recruitment of leukocytes. One such adhesion molecule involved in this process, the vascular adhesion protein-1 (VAP-1), is unusual in the context of the leukocyte adhesion cascade in that it is both an adhesion molecule and a primary amine oxidase. In this review, we examine the biological functions of VAP-1 and examine what role this molecule might play in the establishment and progression of chronic liver disease.

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressions) displayed on endothelial cells (1 2). Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment. Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Mimicking the inflammatory cell adhesion cascade by nucleic acid aptamer programmed cell‐cell interactions

Nature has evolved effective cell adhesion mechanisms to deliver inflammatory cells to inflamed tissue; however, many culture-expanded therapeutic cells are incapable of targeting diseased tissues following systemic infusion, which represents a great challenge in cell therapy. Our aim was to develop simple approaches to program cell-cell interactions that would otherwise not exist toward cell targeting and understanding the complex biology of cell-cell interactions. We employed a chemistry approach to engineer P- or L-selectin binding nucleic acid aptamers onto mesenchymal stem cells (MSCs) to enable them to engage inflamed endothelial cells and leukocytes, respectively. We show for the first time that engineered cells with a single artificial adhesion ligand can recapitulate 3 critical cell interactions in the inflammatory cell adhesion cascade under dynamic flow conditions. Aptamer-engineered MSCs adhered on respective selectin surfaces under static conditions >10 times more efficiently than controls including scrambled-DNA modified MSCs. Significantly, engineered MSCs can be directly captured from the flow stream by selectin surfaces or selectin-expressing cells under flow conditions (≤2dyn/cm²). The simple chemistry approach and the versatility of aptamers permit the concept of engineered cell-cell interactions to be generically applicable for targeting cells to diseased tissues and elucidating the biology of cell-cell interactions.

α4β1 integrin-dependent cell sorting dictates T-cell recruitment in oral submucous fibrosis

Aim:The biological mechanism(s) that guide the immunological effectors of lymphocytes to sites of inflammatory response, a feature consistently seen in oral submucous fibrosis (OSF) was evaluated. It is envisaged that endothelial/lymphocyte adhesion cascades involving VCAM-1/α4β1 integrins control the migration of lymphocytes across the vascular endothelium resulting in their homing in these locales.Materials and Methods:The study group comprised 28 OSF cases (M:F = 12:16, age range 18-65 years; mean 55.4 ± 8.5 SD) divided into early (n=17) and advanced (n=11) disease groups. Biopsy specimens of normal buccal mucosa (site compatible) were obtained from 10 healthy volunteers (age and sex matched) who served as control. All the samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Immunolocalization of β1 subunit associated with α4 integrin was performed by a mouse heterodimer (clone 4B7R, Ig G, R & D Systems Inc., dilution 1:100) using a peroxidase labeled streptavidin–biotin technique. The immunocompetent cell density was expressed as the number of positive cells per mm2. The Mann–Whitney U-test and Fischer exact test were used to evaluate differences. P<0.05 was considered to be significant.Results:The median percentage of “T” lymphocytes with positive integrin α4β1 expression was 77.7 (an interquartile range of 73.3–83.4) for the test cases and for the controls, it was 28.2 (IQR 24.0–38.3). This difference was significant at 0.001 level. For the endothelial cells the positive expression was 82.8 (IQR 77–90.6) and 22.3 (IQR 18.3–29.2) respectively (P<0.001). When the intensity of integrin expression was considered 26/28 cases (96%) and 2/10 (20%) of controls showed intense expression of integrins α4β1 on T lymphocytes (P<0.001). Similarly, 27/28 cases (92.9%) and 2/10 (20%) of controls showed intense expression on endothelial cells (P<0.001). T lymphocyte–endothelial cell interactions were assessed by evaluating the overexpression of integrins on both the endothelial cells and lymphocytes together. The interaction was positive in 15/17 and 11/11 early and advanced OSF cases respectively (P=0.51).Conclusion:Following leukocyte activation, the interaction between leukocyte integrin heterodimers and endothelial superfamily adhesion ligands results in a firm adherence of leukocytes to endothelium, leading to leukocyte migration and homing to sites of mucosal inflammation consistently seen in OSF.

N-Glycan Analysis of Recombinant L-Selectin Reveals Sulfated GalNAc and GalNAc−GalNAc Motifs

The leukocytic adhesion receptor L-selectin plays a crucial role in the first step of the adhesion cascade, enabling leukocytes to migrate into surrounding tissues during inflammation and immune surveillance. We analyzed the site-specific N-glycosylation of the lectin and EGF-like domain of L-selectin using recombinant variants ("LEHis"). The three glycosylation sites of LEHis were mutated to obtain singly glycosylated variants that were expressed in HEK293F cells. alpha1-Acid glycoprotein (AGP), expressed in the same system, was used to distinguish between cell type- and protein-specific glycosylation. Using mass spectrometry and exoglycosidase digestions, we established that LEHis was mostly bearing multifucosylated diantennary N-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could also show that parts of the GalNAc residues were sulfated. Furthermore, we identified novel diantennary glycan structures terminating with the motif GalNAc-GalNAc or SO(4)-GalNAc-GalNAc, which have not been described for N-glycans yet. Interestingly, none of these specific features were found in the N-glycan profile of AGP. This indicates that protein intrinsic information of L-selectin leads to decoration with specific N-glycans, which in turn may be related to L-selectin function.

Leucocyte/endothelium interactions and microvessel permeability: coupled or uncoupled?

In response to infections or tissue injury, circulating leucocytes adhere to and migrate from the vessel lumen to interstitial inflammatory sites to combat invading pathogens. However, these defensive actions may also cause host tissue injury and microvascular dysfunction through oxidative bursts or enzyme release. For decades, the interaction between leucocytes and microvessel walls has been considered as a critical event leading to organ dysfunction. Extensive investigations have therefore focused on blocking specific adhesive ligands to prevent tissue injury. However, anti-adhesion therapies have shown limited success in preventing vascular dysfunction in clinical trials. Numerous studies have demonstrated temporal and spatial dissociations of leucocyte adhesion and/or emigration from permeability increases. The mechanisms that initiate the adhesion cascade have been found to be distinct from those that trigger the leucocyte oxidative burst responsible for increasing microvessel permeability. Recent studies demonstrated that endothelial activation by inflammatory mediators is critical for initiating platelet adhesion and platelet-dependent leucocyte recruitment resulting in augmented increases in microvessel permeability. These new developments suggest that targeting endothelial activation via directly enhancing endothelial barrier function might be a more efficient strategy than focusing on anti-adhesion or platelet/leucocyte depletion to prevent vascular damage during inflammation. Owing to space limitations and the wide range of studies in the field, this article will not serve as a comprehensive review. Instead, it will highlight the emerging evidence of adhesion-uncoupled permeability changes and establish a basis for re-evaluating the coupled relationship between leucocyte/platelet activation and microvessel permeability to achieve a better understanding of permeability regulation during inflammation.

Immobilized Chemokine Fields and Soluble Chemokine Gradients Cooperatively Shape Migration Patterns of Dendritic Cells

Chemokines orchestrate immune cell trafficking by eliciting either directed or random migration and by activating integrins in order to induce cell adhesion. Analyzing dendritic cell (DC) migration, we showed that these distinct cellular responses depended on the mode of chemokine presentation within tissues. The surface-immobilized form of the chemokine CCL21, the heparan sulfate-anchoring ligand of the CC-chemokine receptor 7 (CCR7), caused random movement of DCs that was confined to the chemokine-presenting surface because it triggered integrin-mediated adhesion. Upon direct contact with CCL21, DCs truncated the anchoring residues of CCL21, thereby releasing it from the solid phase. Soluble CCL21 functionally resembles the second CCR7 ligand, CCL19, which lacks anchoring residues and forms soluble gradients. Both soluble CCR7 ligands triggered chemotactic movement, but not surface adhesion. Adhesive random migration and directional steering cooperate to produce dynamic but spatially restricted locomotion patterns closely resembling the cellular dynamics observed in secondary lymphoid organs.

Abstract 5174: Characterization of glycolipid ligands for E-selectin

Abstract The prevention, treatment, and curing of cancer are three of the most researched and highly anticipated areas of modern medicine. One of the greatest barriers to making advances within these fields is the lack of understanding of the molecular mechanisms responsible for cancer metastasis. Head and neck cancer (HNC) is the fifth most diagnosed cancer in the United States, according to the American Cancer Society, and claims the lives of many of its victims because it is often not diagnosed until metastasis has already occurred. After the onset of metastasis, the five-year survival rate drops to below thirty-five percent. Therefore, the study of how metastasis occurs is essential to finding a treatment for HNC. It is believed that metastasis occurs when cancer cells break free from the primary tumor and travel through the blood stream, attaching themselves onto endothelium of blood vessel walls and in turn, forming secondary tumors in new organs. The cell adhesion mediated by glycolipids on cancer cells to E-selectin on vascular endothlelium may be a significant contributor to cancer metastasis. E-selectin plays a significant role in the first two steps of the adhesion cascade, tethering and rolling, which initiate the recruitment process. In our preliminary studies, it has been shown that HNC cells express glycolipids that can bind to E-selectin. Flow cytometry shows that HNC cells express sialofucosylated molecules that could act as E-selectin ligands, and flow chamber adhesion assays confirm that HNC cells bind to cytokine-stimulated endothelial cells in a wholly E-selectin-dependent manner. Enzyme treatment further reveals that sialofucosylated glycolipids are major adhesive ligand(s) for E-selectin. Through the use of specialized flow chambers mimicking physiological conditions, optical tweezers, gas chromatography-mass spectrometry, the mediators of these adhesive interactions can be studied in depth, leading to the discovery of characteristics of cancer glycolipids that promote adhesion. The purpose of this investigation is to gain further insight as to which specific glycolipids located on HNCcells are potent ligands for E-selectin binding. With increased understanding of selectin-ligand adhesion interactions, the identity and properties of molecules responsible for metastasis can be used to determine possible therapeutic methods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5174.

CC chemokine receptor 7 and 9 double-deficient hematopoietic progenitors are severely impaired in seeding the adult thymus

AbstractT-cell development depends on recruitment of bone marrow–derived precursor cells to the thymus via a multistep adhesion cascade involving the chemokine receptor CCR9. However, CCR9 deficiency does not result in complete abrogation of progenitor entry into the adult thymus. Therefore, we tested the hypothesis that additional chemokine/chemokine receptor systems might play a role in this process. To this end, we generated mice deficient in both CCR9 and CCR7. Deficiency in both chemokine receptors resulted in severely reduced numbers of early T-cell progenitors and in near-complete abrogation of thymus reconstitution. Progenitors in bone marrow and peripheral blood remained largely unaffected in CCR7−/−CCR9−/− mice, and direct intrathymic transfer of precursors from CCR7−/−CCR9−/− mice as well as single-mutant mice showed that intrathymic differentiation of these precursors remained functional. Thus, our data reveal a previously unrecognized role of CCR7 in progenitor seeding of the adult thymus, which is largely masked by compensatory effects of CCR9 signals. In turn, CCR7 signals can partially compensate for CCR9 signals, thus explaining the rather mild phenotype of CCR9−/− mice with respect to progenitor seeding.

Distinct roles for LFA-1 affinity regulation during T-cell adhesion, diapedesis, and interstitial migration in lymph nodes

AbstractDuring the course of homing to lymph nodes (LNs), T cells undergo a multistep adhesion cascade that culminates in a lymphocyte function-associated antigen 1 (LFA-1)–dependent firm adhesion to the luminal surface of high endothelial venules (HEVs). The importance of LFA-1 affinity regulation in supporting T-cell arrest on HEVs has been well established, however, its importance in the postadhesion phase, which involves intraluminal crawling and diapedesis to the extravascular space, remains elusive. Here we have shown that LFA-1 affinity needs to be appropriately regulated to support these essential steps in the homing cascade. Genetically engineered T cells that were unable to properly down-regulate LFA-1 affinity underwent enhanced, chemokine-independent arrest in HEVsbut showed perturbed intravascular crawling to transmigration sites and compromised diapedesis across HEVs. By contrast, the extravascular migration of T cells was insensitive to the affinity-enhancing LFA-1 mutation. These results highlight the requirement for balanced LFA-1 affinity regulation in intravascular and transvascular, but not extravascular, T-cell migration in LNs.

Selectin-blocking semisynthetic sulfated polysaccharides as promising anti-inflammatory agents.

Selectin-induced leucocytes rolling along the endothelial surface of blood vessels initiate a complex adhesion cascade, which is an essential step in the cellular immune response. Consequently, blocking the binding between the selectins and their ligands represents a promising strategy for suppressing pathological inflammatory reactions. This study describes the effects of an unfractionated heparin and a low-molecular-weight heparin and a series of structurally well-defined semisynthetic glucan sulfates on selectin-mediated cell-rolling with respect to inhibition. To simulate the blood flow characteristics of postcapillary venules, the rolling experiments were performed in a dynamic-flow-chamber system with immobilized selectins and selectin ligand-carrying U937 cells. The influence of the test compounds on cell rolling was measured by the percentage of adherent cells after a certain flow time and the velocity of the rolling cells. Whereas the test compounds displayed no inhibitory effect on E-selectin-mediated cell rolling, they efficiently blocked the rolling induced by P-selectin. The glucan sulfates were much more active than either unfractionated heparin or low-molecular-weight heparin, or the standard inhibitor Sialyl Lewis(X). Their inhibitory potency turned out to be strongly dependent on various structural parameters, such as sulfation pattern and molecular weight. In conclusion, the semisysnthetic glucan sulfates represent promising candidates in the development of selectin blocking agents.

Rap1 controls lymphocyte adhesion cascade and interstitial migration within lymph nodes in RAPL-dependent and -independent manners

AbstractThe small GTPase Rap1 and its effector RAPL regulate lymphocyte adhesion and motility. However, their precise regulatory roles in the adhesion cascade preceding entry into lymph nodes and during interstitial migration are unclear. Here, we show that Rap1 is indispensably required for the chemokine-triggered initial arrest step of rolling lymphocytes through LFA-1, whereas RAPL is not involved in rapid arrest. RAPL and talin play a critical role in stabilizing lymphocyte arrest to the endothelium of blood vessels under flow or to the high endothelial venules of peripheral lymph nodes in vivo. Further, mutagenesis and peptide studies suggest that release of a trans-acting restraint from the β2 cytoplasmic region of LFA-1 is critical for Rap1-dependent initial arrest. Rap1 or RAPL deficiency severely impaired lymphocyte motility over lymph node stromal cells in vitro, and RAPL deficiency impaired high-velocity directional movement within lymph nodes. These findings reveal the several critical steps of Rap1, which are RAPL-dependent and -independent, in lymphocyte trafficking.

Inhibitors of Platelet Adhesion

Platelet aggregation, in fundamental terms, is considered a biological end point that contributes to the occurrence of clinical events among patients with advanced atherosclerotic coronary artery disease. Acute coronary syndrome, including non–ST elevation myocardial infarction (NSTEMI) and ST elevation myocardial infarction (STEMI), accounts for upward of 733 000 hospital admissions yearly in the United States.1 The primary pathophysiological mechanism responsible for the majority of acute coronary syndromes is endothelial plaque disruption with and subsequent platelet adhesion, activation, and thrombus formation.2 The end result is formation of thrombus within a coronary artery, leading to subtotal vessel occlusion with NSTEMI and complete occlusion of the artery with STEMI. In this review, we provide a contemporary view of platelet adhesion as a highly coordinated and teleologically conserved process achieved by surface receptors, protein ligands, and matrix proteins operating at the platelet–subendothelium interface. We also discuss drugs in development, including monoclonal antibodies, inhibitory peptides, and oligonucleotides; preclinical data; and, where available, clinical trial results, highlighting the potential translation of fundamental constructs in platelet biology to patient care. Platelets are derived from a hematopoietic bone marrow stem cell precursor.3 Through a highly conserved program of cellular differentiation, this stem cell precursor becomes a megakaryocyte at a rate of approximately 100 000 000 megakaryocytes produced per day.3 Subsequently, each individual megakaryocyte will give rise to approximately 500 platelets via additional developmental steps within the bone marrow that involve a proplatelet intermediate stage.3 Once released into circulation, a mature platelet has an expected life span of 7 to 10 days.3 Platelet maturation and development involve the expression of receptors on the platelet cell surface. These receptors facilitate platelet adhesion and activation, and they promote thrombus development through receptor–ligand interactions, with several ligands expressed on the surface of endothelial cells, within the …