CCL25 and CCL28 promote alpha4 beta7-integrin-dependent adhesion of lymphocytes to MAdCAM-1 under shear flow

Inflammatory bowel disease is characterized by the recruitment of lymphocytes to the gut via mucosal vessels. Chemokines are believed to trigger alpha(4)beta(1)- and alpha(4)beta(7)-integrin-mediated adhesion to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on mucosal vessels, although the contribution of each pathway and the chemokines involved are not well characterized. These interactions occur under conditions of hemodynamic shear, which is critical in determining how lymphocytes integrate chemokine signals to promote transmigration. To define the role of specific chemokines in mediating lymphocyte adhesion to VCAM-1 and MAdCAM-1, we studied the ability of immobilized chemokines to activate adhesion of human lymphocytes in a flow-based adhesion assay. Adhesion to immobilized MAdCAM-1 was alpha(4)beta(7) dependent, with no contribution from alpha(4)beta(1), whereas alpha(4)beta(1) mediated rolling and static adhesion on VCAM-1. Immobilized CC-chemokine ligand (CCL) 25 and CCL28 were both able to trigger alpha(4)beta(7)-dependent lymphocyte arrest on MAdCAM-1 under shear, highlighting a potential role for these chemokines in the arrest of lymphocytes on postcapillary venules in the gut. Neither had any effect on adhesion to VCAM-1, suggesting that they selectively trigger alpha(4)beta(7)-mediated adhesion. Immobilized CCL21, CCL25, CCL28, and CXC-chemokine ligand (CXCL) 12 all converted rolling adhesion to static arrest on MAdCAM-1 by activating lymphocyte integrins, but only CCL21 and CXCL12 also triggered a motile phenotype characterized by lamelipodia and uropod formation. Thus alpha(4)beta(1)/VCAM-1 and alpha(4)beta(7)/MAdCAM-1 operate independently to support lymphocyte adhesion from flow, and chemokines may act in concert with one chemokine triggering integrin-mediated arrest and a second chemokine promoting motility and transendothelial migration.     

Modeling the reversible kinetics of neutrophil aggregation under hydrodynamic shear.

Neutrophil emigration into inflamed tissue is mediated by beta 2-integrin and L-selectin adhesion receptors. Homotypic neutrophil aggregation is also dependent on these molecules, and it provides a model system in which to study adhesion dynamics. In the current study we formulated a mathematical model for cellular aggregation in a linear shear field based on Smoluchowski's two-body collision theory. Neutrophil suspensions activated with chemotactic stimulus and sheared in a cone-plate viscometer rapidly aggregate. Over a range of shear rates (400-800 s-1), approximately 90% of the single cells were recruited into aggregates ranging from doublets to groupings larger than sextuplets. The adhesion efficiency fit to these kinetics reached maximum levels of > 70%. Formed aggregates remained intact and resistant to shear up to 120 s, at which time they spontaneously dissociated back to singlets. The rate of cell disaggregation was linearly proportional to the applied shear rate, and it was approximately 60% lower for doublets as compared to larger aggregates. By accounting for the time-dependent changes in adhesion efficiency, disaggregation rate, and the effects of aggregate geometry, we succeeded in predicting the reversible kinetics of aggregation over a wide range of shear rates and cell concentrations. The combination of viscometry with flow cytometry and mathematical analysis as presented here represents a novel approach to differentiating between the effects of hydrodynamics and the intrinsic biological processes that control cell adhesion.     

Carboxypeptidase cathepsin X mediates beta2-integrin-dependent adhesion of differentiated U-937 cells

Cathepsin X is a lysosomal carboxypeptidase with a potential role in processes of inflammation and immune response. The integrin-binding motifs RGD and ECD, present in the pro- and in mature forms of cathepsin X, respectively, suggest that this enzyme might have a function in cell signaling and adhesion. In this study, we report that cysteine protease inhibitors E-64 and CA-074 and 2F12 monoclonal antibody, all of which inhibit cathepsin X activity, significantly reduced adhesion of differentiated U-937 cells to polystyrene- and fibrinogen-coated surfaces via Mac-1 integrin receptor, whereas their binding to vitronectin, fibronectin or Matrigel was not affected. On the other hand, cathepsin X, added to differentiating U-937 cells, stimulated their adhesion. Using confocal microscopy, we demonstrated that the pro-form of cathepsin X was co-localized with beta(2) and beta(3) integrin subunits and its mature form solely with the beta(2) integrin subunit with the most intense signal in cell-cell junctions in differentiated U-937 cells and in co-cultures with endothelial cells. Our results indicate that active cathepsin X mediates the function of beta(2) integrin receptors during cell adhesion and that it could also be involved in other processes associated with beta(2) integrin receptors such as phagocytosis and T cell activation.     

Kinetics of beta2-integrin and L-selectin bonding during neutrophil aggregation in shear flow.

Activated neutrophils aggregate in a shear field via bonding of L-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) followed by a more stable bonding of LFA-1 (CD11a/CD18) to intercellular adhesion molecule 3 (ICAM-3) and Mac-1 (CD11b/CD18) to an unknown counter receptor. Assuming that the Mac-1 counter receptor is ICAM-3-like in strength and number, rate processes were deconvoluted from neutrophil homoaggregation data for shear rates (G) of 100-3000 s-1 with a two-body hydrodynamic collision model (. Biophys. J. 73:2819-2835). For integrin-mediated aggregation (characteristic bond strength of 5 microdynes) in the absence of L-selectin contributions, an average forward rate of kf = 1.57 x 10(-12) cm2/s predicted the measured efficiencies for G = 100-800 s-1. For a selectin bond formation rate constant equal to the integrin bond formation rate constant, the colloidal stability of unactivated neutrophils was satisfied for a reverse rate of the L-selectin-PGSL bond corresponding to an average bond half-life of 10 ms at a characteristic bond strength of 1 microdyne. Colliding neutrophils initially bridged by at least one L-selectin-PSGL-1 bond were calculated to rotate from 8 to 50 times at G = 400 to 3000 s-1, respectively, before obtaining mechanical stability in sheared fluid of either 0.75 or 1.75 cP viscosity. Thus for G > 400 s-1, the interaction time needed for the rotating aggregates to become stable was relatively constant at 52.5 +/- 8.5 ms, largely independent of shear rate or shear stress. Aggregation data and the colloidal stability criterion can provide a consistent set of forward and reverse rate constants and characteristic bond strengths for a known time-dependent stoichiometry of receptors on cells interacting in a shear flow field.     

S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.     

Characteristics of new Staphylococcus aureus-RBC adhesion mechanism independent of fibrinogen and IgG under hydrodynamic shear conditions

Staphylococcus aureus infection begins when bacterial cells circulating in blood adhere to components of the extracellular matrix or endothelial cells of the host and initiate colonization. S. aureus is known to exhibit extensive interactions with platelets. S. aureus is also known to bind to red blood cells (RBCs) in the presence of plasma proteins, such as fibrinogen and IgG. Herein we report a new binding mechanism of S. aureus to RBC independent of those plasma proteins. To characterize the new adhesion mechanism, we experimentally examine the binding kinetics and molecular constituents mediating the new adhesive interactions between S. aureus and RBCs under defined shear conditions. The results demonstrate that the receptors for fibrinogen (clumping factor A) and IgG (protein A) of S. aureus are not involved in the adhesion. S. aureus binds to RBCs with maximal adhesion at the shear rate 100 s^–1 and decreasing adhesion with increasing shear. The heteroaggregates formed after shear are stable when subjected to the shear rate 2,000 s^–1, indicating that intercellular contact time rather than shear forces controls the adhesion at high shear. S. aureus binding to RBC requires plasma, and 10% plasma is sufficient for maximal adhesion. Plasma proteins involved in the cell-cell adhesion, such as fibrinogen, fibronectin, von Willebrand factor, IgG, thrombospondin, laminin, and vitronectin are not involved in the observed adhesion. The extent of heteroaggregation is dramatically reduced on RBC treatment with trypsin, chymotrypsin, or neuraminidase, suggesting that the receptor(s) mediating the heteroaggregation process is a sialylated glycoprotein on RBC surface. Adhesion is divalent cation dependent and also blocked by heparin. This work demonstrates a new mechanism of S. aureus-RBC binding under hydrodynamic shear conditions via unknown RBC sialoglycoprotein(s). The binding requires plasma protein(s) other than fibrinogen or IgG and does not involve the S. aureus adhesins clumping factor A or protein A. adhesion; red blood cell     

DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.     

Simulation of cell rolling and adhesion on surfaces in shear flow: general results and analysis of selectin-mediated neutrophil adhesion.

The receptor-mediated adhesion of cells to ligand-coated surfaces in viscous shear flow is an important step in many physiological processes, such as the neutrophil-mediated inflammatory response, lymphocyte homing, and tumor cell metastasis. This paper describes a calculational method which simulates the interaction of a single cell with a ligand-coated surface under flow. The cell is idealized as a microvilli-coated hard sphere covered with adhesive springs. The distribution of microvilli on the cell surface, the distribution of receptors on microvilli tips, and the forward and reverse reaction between receptor and ligand are all simulated using random number sampling of appropriate probability functions. The velocity of the cell at each time step in the simulation results from a balance of hydrodynamic, colloidal and bonding forces; the bonding force is derived by summing the individual contributions of each receptor-ligand tether. The model can simulate the effect of many parameters on adhesion, such as the number of receptors on microvilli tips, the density of ligand, the rates of reaction between receptor and ligand, the stiffness of the resulting receptor-ligand springs, the response of springs to strain, and the magnitude of the bulk hydrodynamic stresses. The model can successfully recreate the entire range of expected and observed adhesive phenomena, from completely unencumbered motion, to rolling, to transient attachment, to firm adhesion. Also, the method can generate meaningful statistical measures of adhesion, including the mean and variance in velocity, rate constants for cell attachment and detachment, and the frequency of adhesion. We find a critical modulating parameter of adhesion is the fractional spring slippage, which relates the strain of a bond to its rate of breakage; the higher the slippage, the faster the breakage for the same strain. Our analysis of neutrophil adhesive behavior on selectin-coated (CD62-coated) surfaces in viscous shear flow reported by Lawrence and Springer (Lawrence, M.B., and T.A. Springer 1991. Cell. 65:859-874) shows the fractional spring slippage of the CD62-LECAM-1 bond is likely below 0.01. We conclude the unique ability of this selectin bond to cause neutrophil rolling under flow is a result of its unique response to strain. Furthermore, our model can successfully recreate data on neutrophil rolling as function of CD62 surface density.     

Promotion of neutrophil chemotaxis through differential regulation of beta 1 and beta 2 integrins.

Migration of neutrophils requires sequential adhesive and deadhesive interactions between beta 1 and beta 2 integrins and components of the extracellular matrix. Prompted by reports that describe interaction of soluble beta-glucan with the beta 2 integrin Mac-1, a role for beta-glucan in regulation of integrin-mediated migration was investigated. Neutrophil migration in response to fMLP was assessed using an agarose overlay method with slides precoated with fibronectin (Fn) +/- beta-glucan. On Fn, random migration in excess of directed migration was observed. In contrast, migration on Fn + beta-glucan was directional, with marked diminution of random migration. This conversion of random to directed migration was seen neither when Fn was supplemented with alternative polysaccharides nor when beta-glucan was applied to other components of the extracellular matrix. This effect of beta-glucan was shown to be cation dependent and to be effected by Arg-Gly-Asp-containing peptides consistent with an integrin-mediated event. mAb inhibition studies demonstrate that beta-glucan effects this shift toward directed migration through suppression of migration mediated by Mac-1 and very late Ag 5 and enhancement of very late Ag 3-mediated migration. Adhesion assays suggest that the prochemotactic influence of beta-glucan is due, in part but not entirely, to modulation of PMN adhesion to Fn. In summary, these data support a novel role for beta-glucan in regulation of beta 1- and beta 2-mediated neutrophil migration on Fn.     

Molecular dynamics simulations from putative transition states of alpha-spectrin SH3 domain

A series of molecular dynamics simulations in explicit solvent were started from nine structural models of the transition state of the SH3 domain of alpha-spectrin, which were generated by Lindorff-Larsen et al. (Nat Struct Mol Biol 2004;11:443-449) using molecular dynamics simulations in which experimental Phi - values were incorporated as restraints. Two of the nine models were simulated 10 times for 200 ns and the remaining models simulated two times for 200 ns. Complete folding was observed in one case, while in the other simulations partial folding or unfolding events were observed, which were characterized by a regularization of elements of secondary structure. These results are consistent with recent experimental evidence that the folding of SH3 domains involves low populated intermediate states.     

Chemoattractant signals and beta 2 integrin occupancy at apical endothelial contacts combine with shear stress signals to promote transendothelial neutrophil migration

Lymphocyte transendothelial migration (TEM) is promoted by fluid shear signals and apical endothelial chemokines. Studying the role of these signals in neutrophil migration across differently activated HUVEC in a flow chamber apparatus, we gained new insights into how neutrophils integrate multiple endothelial signals to promote TEM. Neutrophils crossed highly activated HUVEC in a beta(2) integrin-dependent manner but independently of shear. In contrast, neutrophil migration across resting or moderately activated endothelium with low-level beta(2) integrin ligand activity was dramatically augmented by endothelial-presented chemoattractants, conditional to application of physiological shear stresses and intact beta(2) integrins. Shear stress signals were found to stimulate extensive neutrophil invaginations into the apical endothelial interface both before and during TEM. A subset of invaginating neutrophils completed transcellular diapedesis through individual endothelial cells within <1 min. Our results suggest that low-level occupancy of beta(2) integrins by adherent neutrophils can mediate TEM only if properly coupled to stimulatory shear stress and chemoattractant signals transduced at the apical neutrophil-endothelial interface.     

Molecular dynamics simulations of the unfolding of beta(2)-microglobulin and its variants

In this study, we examined the unfolding processes of native beta(2)-microglobulin and two related variants, one with an N-terminal hexapeptide deletion DeltaN6 and another with Lys57-Asp58 cleavage, by high-temperature molecular dynamics simulations. Three simulation models were used, molecular dynamics (MD) simulations with explicit water solvation, MD simulations with the CHARMM EEF1 force field and Langevin dynamics with the CHARMM EEF1 force field. Our simulations reproduce many of the experimentally observed structural changes. The most striking agreement is in the beta-strands to alpha-helix transition. In our simulations, strands beta(3), beta(4) and beta(5) consistently change to alpha-helix, whereas beta(8) changes to an alpha-helix only briefly. Through comparisons of the conformational behavior of the native, the DeltaN6 and the Lys-cut beta(2)-m, using the three simulation methods, we identified the consensus conformational changes that differentiate between the native beta(2)-m and its two variants. We found that the main effect of the removal of the N-terminal hexapeptide is to increase the separation between strands beta(2) and beta(6) and to facilitate the beta to alpha transition. On the other hand, the lysine cleavage only increases the flexibility of strand beta(5) and does not affect the interactions between strands beta(2) and beta(6). These conformational changes may relate to polymerization tendencies of these variants.     

Characterization of the enhanced adhesion of neutrophil leukocytes to thrombin-stimulated endothelial cells.

We have characterized the mechanisms by which thrombin enhances neutrophil leukocyte (PMN) adhesion to human endothelial cells in vitro. Thrombin rapidly and transiently increased PMN adhesion by an action on the endothelial cells. The transience of the response was due to at least two factors: desensitization of the endothelial cell responsiveness to thrombin in the continued presence of the agonist; and the lability (t1/2 less than 15 min) of the effector molecules expressed by the endothelium. Experiments with exogenous platelet-activating factor (PAF) and with PAF antagonists demonstrated that PAF production, although it may facilitate the enhanced PMN adhesion seen in response to thrombin, is not sufficient to explain the reaction. By using a variety of antibodies directed against cell surface ligands, and comparing adhesion of PMN to endothelium and to protein-coated surfaces, we deduce that several endothelial ligands not previously reported as playing a role in PMN adhesion are involved in these interactions. Of particular interest was the finding that antibodies recognizing two thrombin-regulated endothelial cell surface ligands, GMP-140 and the CD63-related Ag, both inhibited adhesion of PMN to thrombin- or LPS-pretreated endothelium. We conclude that thrombin acts to enhance PMN adhesion to endothelium at least in part by transiently altering the conformation or level of expression of these ligands.     

Cell adhesion and migration properties of beta 2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation

Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.     

Cell adhesion molecules and the transition from short- to long-term memory

Training chicks on a one-trial passive avoidance task results in a cascade of molecular and cellular processes in two forebrain regions, culminating within 60–90 min in post-translational glycosylation of synaptic membrane proteins and expression of immediate early genes c-fos and c-jun. We have now found a second window of vulnerability of memory to the protein synthesis inhibitor anisomycin, 4 h downstream of training. By 5.5 h post-training this window closes, to be replaced by a window of sensitivity to blockade of glycoprotein synthesis, presumably representing post-translational modification of the newly synthesised proteins. Amongst the pre- and post-synaptic membrane glycoproteins involved at both first and second time windows are the cell adhesion molecules, L1 (at both times) and NCAM (at the later). Molecular dissection of the external membrane domains of L1 distinguishes between a requirement for the IgG domain at the early time, the fibronectin-like domain at the later. The second time window only occurs if the animal is trained on a stimulus strong enough to be remembered for a long period. Weak memories do not persist beyond 6–8 h and the second wave of glycoprotein synthesis does not occur. Thus the second wave may represent the molecular processes required for the alterations in synaptic configuration, by way of the adhesion molecules amongst others, required for the morphological changes in neuronal connectivity hypothesised to encode memory.     

Dynamic study of the transition from hyaluronan- to integrin-mediated adhesion in chondrocytes

Membrane-bound hyaluronan mediates the initial adhesive interactions between many cell types and external surfaces. In RCJ-P chondrocytes, such early contacts are mediated through a thick hyaluronidase-sensitive coat. The early adhesion is followed by integrin-mediated interactions and the formation of stable focal adhesions. During this process, the distance between the cell membrane and the surface is reduced from micrometers to few tens of nanometers. The transition from hyaluronan- to integrin-mediated adhesion was studied on glass surfaces by total internal reflection fluorescence microscopy. Hyaluronan-mediated adhesion precedes focal adhesions formation by 2-10 min. After these initial interactions, the pericellular hyaluronan remains sequestered into discrete pockets between the cell and the surface, which are a few hundreds nanometers thick and a few micrometers wide, and are flanked by focal adhesions. The hyaluronan coat facilitates the nucleation of small paxillin-rich contacts, which later mature into focal adhesions. These dynamic studies demonstrate that pericellular hyaluronan mediates initial cell-surface adhesion, and regulates the formation of focal adhesions.