IL-8-induced L-selectin shedding regulates its binding kinetics to PSGL-1

L-selectin plays a crucial role in inflammation cascade by initiating the tethering and rolling of leukocytes on endothelium wall. While many L-selectin molecules are rapidly shed from the cell surface upon activation, the remaining membrane-anchored L-selectin may still play an important role in regulating leukocyte rolling and adhesion with different binding kinetics. Here we developed an in vitro model to activate Jurkat cells via interlukin-8 (IL-8) and quantified the two-dimensional (2D) binding kinetics, using a micropipette aspiration assay, of membrane-anchored L-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) ligand coupled onto human red blood cells (RBCs). The data indicated that L-selectin shedding reduced the amount of membrane-anchored L-selectin and lowered both its reverse and forward rates. These results suggested that the rolling dynamics of activated leukocytes was determined by two opposite impacts: reducing the surface presentation would enhance the rolling but lowering the kinetic rates would decrease the rolling. This finding provides a new insight into understanding how L-selectin shedding regulates leukocyte rolling and adhesion.

IL-8刺激L-选择素水解断裂对细胞膜拓扑结构和刚度的影响

目的 研究IL-8刺激所致的L-选择素水解断裂是否会引起细胞膜结构和刚度的变化,进而影响L广选择素-配体的反应动力学.方法 分别用IL-8、TAPI-0+IL-8和TAPI-0处理本构表达L-选择素的Jurkat细胞,用以构建L-选择素水解断裂、阻断水解断裂和阻断剂对照的细胞-分子模型.采用扫描电镜观察其表面拓扑结构,采用微管吸吮方法测量其刚度.结果 与正常组相比,水解断裂组和阻断水解断裂组细胞微绒毛减少、表面趋于光滑,而阻断剂对照组则无明显变化;无论是水解断裂组,阻断水解断裂组还是阻断剂对照组,细胞膜刚度与正常组相比均无显著性差异.结论 IL-8刺激所致L-选择素水解断裂会引发细胞膜拓扑结构的变化,进而影响L一选择素一配体反应动力学的变化;而细胞膜刚度未见明显变化,表明载体刚度不是水解断裂引发L-选择素-配体反应动力学变化的因索.

Probabilistic Modeling of Rosette Formation

英文摘要: Rosetting, or forming a cell aggregate between a single target nucleated cell and a number of red blood cells (RBCs), is a simple assay for cell adhesion-mediated by specific receptor-ligand interaction. For example, rosette formation between sheep RBC and human lymphocytes has been used to differentiate T cells from B cells. Rosetting assay is commonly used to determine the interaction of Fc gamma-receptors (Fc gamma R) expressed on inflammatory cells and IgG-coated on RBCs. Despite its wide use in measuring cell adhesion, the biophysical parameters of rosette formation have not been well characterized. Here we developed a probabilistic model to describe the distribution of rosette sizes, which is Poissonian. The average rosette size is predicted to be proportional to the apparent two-dimensional binding affinity of the interacting receptor-ligand pair and their site densities. The model has been supported by experiments of rosettes mediated by four molecular interactions: Fc gamma RIII interacting with IgG, T cell receptor and coreceptor CD8 interacting with antigen peptide presented by major histocompatibility molecule, P-selectin interacting with P-selectin glycoprotein ligand 1 (PSGL-1), and L-selectin interacting with PSGL-1. The latter two are structurally similar and are different from the former two. Fitting the model to data enabled us to evaluate the apparent effective two-dimensional binding affinity of the interacting molecular pairs: 7.19x10(-5) mu m(4) for Fc gamma RIII-IgG interaction, 4.66x10(-3) mu m(4) for P-selectin-PSGL-1 interaction, and 0.94x10(-3) mu m(4) for L-selectin-PSGL-1 interaction. These results elucidate the biophysical mechanism of rosette formation and enable it to become a semiquantitative assay that relates the rosette size to the effective affinity for receptor-ligand binding.

Two-Dimensional Kinetics Of Beta(2)-Integrin And Icam-1 Bindings Between Neutrophils And Melanoma Cells In A Shear Flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta(2)-integrin ( lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta(2)-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta(2)-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Kinetics and Force Dependence of P-selectin/PSGL-1 Interactions Measured by AFM

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory and thrombotic processes. The rolling under hydrodynamic shear forces is a first step in directing leukocytes out of the blood stream into sites of inflammation and is mediated by the selectins, a family of extended, modular, and calcium-dependent lectin receptors. The interactions between P-, E-or L-selectins and their count.

Impact of carrier stiffness and microtopology on two-dimensional kinetics of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interactions

Mechanics and surface microtopology of the molecular carrier influence cell adhesion, but the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the carrier stiffness and microtopology affected two-dimensional kinetics of interacting adhesion molecules on two apposing surfaces. Interactions of P-selectin with P-selectin glycoprotein ligand-1 (PSGL-1) were used to demonstrate such effects by presenting the molecules on three carrier systems: human red blood cells (RBCs), human promyelocytic leukemia HL-60 cells, and polystyrene beads. Stiffening the carrier alone or in cooperation with roughing the surface lowered the two-dimensional affinity of interacting molecules by reducing the forward rate but not the reverse rate, whereas softening the carrier and roughing the surface had opposing effects in affecting two-dimensional kinetics. In contrast, the soluble antibody bound with similar three-dimensional affinity to surface-anchored P-selectin or PSGL-1 constructs regardless of carrier stiffness and microtopology. These results demonstrate that the carrier stiffness and microtopology of a receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding. This provides new insights into understanding the rolling and tethering mechanism of leukocytes onto endothelium in both physiological and pathological processes.

Mechano-Chemical Coupling at Molecular Level: Forced Dissociation of Selectin/Ligand Binding

Mechano-chemical coupling is a common phenomenon that exists in various biological processes at different physiological levels. Bone tissue remodeling strongly depends on the local mechanical load. Leukocytes are sheared to form the transient aggregates with platelets or other leukocytes in the circulation. Flow pattern affects the signal transduction pathways in endothelial cells. Receptor/ligand interactions are important to cell adhesion since they supply the physical linkages...

应用光阱技术测量P-selectin/PSGL-1键解离的断裂力

选择素(selectin)与配体相互作用在诸如炎症反应、肿瘤转移等生物学过程中具有重要作用;作用力影响受体-配体键解离.本文发展了基于光阱技术的新实验方法,用于考察P-选择素(P-selectin)与P-选择素糖蛋白配体-1(P-selectin Glycoprotein Ligand 1, PSGL-1)相互作用的解离过程.采用黏滞力法对光阱刚度系数进行标定,并通过分子在玻璃小球表面的功能化表征,研究力作用下P-selectin/PSGL-1键的解离,得到了在较低加载率(<25 pN/s)下键解离的断裂力分布,发现键的最可几断裂力随加载率而增加.实验结果在较低加载率下补充和验证了已有的结论.

Adlayer Structures of DL-Homocysteine and L-Homocysteine Thiolactone on Au(111) Surface: an in situ STM Study

The adsorption Of DL-homocysteine (Hcy) and L-homocysteine thiolactone (HTL) on Au(1 1 1) electrode was investigated in 0.1 M HClO4 by cyclic voltammetry and in situ scanning tunneling microscopy (STM). Hcy and HTL molecules formed highly ordered adlayers on Au(1 1 1) surface. High-resolution STM images revealed the orientation and packing arrangement in the ordered adlayers. Hcy molecules formed (2root3 x 3root3)R30degrees adlayer structure and H-bonds between carboxyl groups were assumed to be responsible for the origin of tail-to-tail or head-to-head molecular arrangement, while HTL molecules formed (4 x 6) adlayer structure, and two different orientations and appearances in the ordered adlayer were found. Structural models were proposed for the two adlayers.

Quantifying the Effects of Contact Duration, Loading Rate, and Approach Velocity on P-Selectin-PSGL-1 Interactions Using AFM

Kinetics and its regulation by extrinsic physical factors govern selectin-ligand interactions that mediate tethering and rolling of circulating cells on the vessel wall under hemodynamic forces. While the force regulation of off-rate for dissociation of selectin-ligand bonds has been extensively studied, much less is known about how transport impacts the on-rate for association of these bonds and their stability. We used atomic force microscopy (AFM) to quantify how the contact duration, loading rate, and approach velocity affected kinetic rates and strength of bonds of P-selectin interacting with P-selectin glycoprotein ligand I (PSGL-1). We found a saturable relationship between the contact time and the rupture force, a biphasic relationship between the adhesion probability and the retraction velocity, a piece-wise linear relationship between the rupture force and the logarithm of the loading rate, and a threshold relationship between the approach velocity and the rupture force. These results provide new insights into how physical factors regulate receptor-ligand interactions.

Kinetic measurements of cell surface E-selectin/carbohydrate ligand interactions

Selectin/ligand interactions initiate the multistep adhesion and signaling cascades in the recruitment of leukocytes from circulation to inflamed tissues and may also play a role in tumor metastasis. Kinetic properties of these interactions are essential determinants governing blood-borne cells' tethering to and rolling on the vessel wall. Extending our recently developed micropipette method, we have measured the kinetic rates of E-selectin/ligand interactions. Red cells coated with an E-selectin construct were allowed to bind HL-60 or Colo-205 cells bearing carbohydrate ligands. Specific adhesions were observed to occur at isolated points, the frequency of which followed a Poisson distribution. These point attachments were formed at the same rate with both the HL-60 and Colo-205 cells (0.14 +/- 0.04 and 0.13 +/- 0.03 mum(2) s(-1) per unit density of E-selectin, respectively) but dissociated from the former at a rate twice as fast as did from the latter (0.92 +/- 0.23 and 0.44 +/- 0.10 s(-1), respectively). The reverse rates agree well with those measured by the flow chamber. The forward rates are orders of magnitude higher than those of Fc gamma receptors interacting with IgG measured under similar conditions, consistent with the rapid kinetics requirement for the function of E-selectin/ligand binding, which is to capture leukocytes on endothelial surfaces from flow.

Effects of Surface Microtopology and Membrane Stiffness on Kinetics of Selectin/Ligand interactions by a Modified BFP

More and more evidences come out to support that the functionality of adhesion molecules are influenced by the surface microtopology of cell carrier or substrate. Adhesive molecules usually express on the microvilli of a cell, providing a well-defined spatial configuration to mediate the adhesions to the counterpart molecules on the apposed surface.

Forced Dissociation of Selectin-ligand Complexes Using Steered Molecular Dynamics Simulation

Selectin-ligand interactions are crucial to such biological processes as inflammatory cascade or tumor metastasis. How transient formation and dissociation of selectin-ligand bonds in blood flow are coupled to molecular conformation at atomic level, however, has not been well understood. In this study, steered molecular dynamics (SMD) simulations were used to elucidate the intramolecular and intermolecular conformational evolutions involved in forced dissociation of three selectin-ligand systems: the construct consisting of P-selectin lectin (Lec) and epidermal growth factor (EGF)-like domains (P-LE) interacting with synthesized sulfoglycopeptide or SGP-3, P-LE with sialyl Lewis X (sLeX), and E-LE with sLeX. SMD simulations were based on newly built-up force field parameters including carbohydrate units and sulfated tyrosine(s) using an analogy approach. The simulations demonstrated that the complex dissociation was coupled to the molecular extension. While the intramolecular unraveling in P-LESGP-3 system mainly resulted from the destroy of the two anti-parallel sheets of EGF domain and the breakage of hydrogen-bond cluster at the Lec-EGF interface, the intermolecular dissociation was mainly determined by separation of fucose (FUC) from Ca2+ ion in all three systems. Conformational changes during forced dissociations depended on pulling velocities and forces, as well as on how the force was applied. This work provides an insight into better understanding of conformational changes and adhesive functionality of selectin-ligand interactions under external forces.

Effects of Molecular Length, Ortientation and Amino Acid Mutation on Kinetics of Selectin/Ligand interactions

Receptor/ligand interactions are basic issues to cell adhesion, which are important to many physiological and pathological processes such as lymphocyte-mediated cytotoxicity, tumor metastasis and inflammatory reactionl. Selectin/carbohydrate ligand bindings have been found to mediate the fast rolling of leukocytes on activated endothelial monolayer. Kinetic rate and binding affinity constants are essential determinants of cell adhesion...