Dooyoung Lee

Dendritic Cells Distinguish Individual Chemokine Signals through CCR7 and CXCR4

Dendritic cells (DCs) respond to chemotactic signals to migrate from sites of infection to secondary lymphoid organs where they initiate the adaptive immune response. The key chemokines directing their migration are CCL19, CCL21, and CXCL12, but how signals from these chemokines are integrated by migrating cells is poorly understood. Using a microfluidic device, we presented single and competing chemokine gradients to murine bone-marrow derived DCs in a controlled, time-invariant microenvironment. Experiments performed with counter-gradients revealed that CCL19 is 10–100-fold more potent than CCL21 or CXCL12. Interestingly, when the chemoattractive potencies of opposing gradients are matched, cells home to a central region in which the signals from multiple chemokines are balanced; in this region, cells are motile but display no net displacement. Actin and myosin inhibitors affected the speed of crawling but not directed motion, whereas pertussis toxin inhibited directed motion but not speed. These results provide fundamental insight into the processes that DCs use to migrate toward and position themselves within secondary lymphoid organs.

The Adapter Protein SLP-76 Mediates “Outside-In” Integrin Signaling and Function in T Cells

ABSTRACT The adapter protein SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an essential mediator of signaling from the T-cell antigen receptor (TCR). We report here that SLP-76 also mediates signaling downstream of integrins in T cells and that SLP-76-deficient T cells fail to support adhesion to integrin ligands. In response to both TCR and integrin stimulation, SLP-76 relocalizes to surface microclusters that colocalize with phosphorylated signaling proteins. Disruption of SLP-76 recruitment to the protein named LAT (linker for activation of T cells) inhibits SLP-76 clustering downstream of the TCR but not downstream of integrins. Conversely, an SLP-76 mutant unable to bind ADAP (adhesion and degranulation-promoting adapter protein) forms clusters following TCR but not integrin engagement and fails to support T-cell adhesion to integrin ligands. These findings demonstrate that SLP-76 relocalizes to integrin-initiated signaling complexes by a mechanism different from that employed during TCR signaling and that SLP-76 relocalization corresponds to SLP-76-dependent integrin function in T cells.

Endothelial cell specific adhesion molecule (ESAM) localizes to platelet–platelet contacts and regulates thrombus formation in vivo

Summary.  Background: In resting platelets, endothelial cell specific adhesion molecule (ESAM) is located in alpha granules, increasing its cell surface expression following platelet activation. However, the function of ESAM on platelets is unknown. Objective: To determine whether ESAM has a role in thrombus formation. Methods and results: We found that following platelet activation ESAM localizes to the junctions between adjacent platelets, suggesting a role for this protein in contact‐dependent events that regulate thrombus formation. To test this hypothesis we examined the effect of ESAM deletion on platelet function. In vivo, ESAM−/− mice achieved more stable hemostasis than wild‐type mice following tail transection, and developed larger thrombi following laser injury of cremaster muscle arterioles. In vitro, ESAM−/− platelets aggregated at lower concentrations of G protein‐dependent agonists than wild‐type platelets, and were more resistant to disaggregation. In contrast, agonist‐induced calcium mobilization, αIIbβ3 activation, alpha‐granule secretion and platelet spreading, were normal in ESAM‐deficient platelets. To understand the molecular mechanism by which ESAM regulates platelet activity, we utilized a PDZ domain array to identify the scaffold protein NHERF‐1 as an ESAM binding protein, and further demonstrated that it associates with ESAM in both resting and activated platelets. Conclusions: These findings support a model in which ESAM localizes to platelet contacts following platelet activation in order to limit thrombus growth and stability so that the optimal hemostatic response occurs following vascular injury.

Occlusive thrombi arise in mammals but not birds in response to arterial injury: evolutionary insight into human cardiovascular disease

Mammalian platelets are small, anuclear circulating cells that form tightly adherent, shear-resistant thrombi to prevent blood loss after vessel injury. Platelet thrombi that form in coronary and carotid arteries also underlie common vascular diseases such as myocardial infarction and stroke and are the target of drugs used to treat these diseases. Birds have high-pressure cardiovascular systems like mammals but generate nucleated thrombocytes rather than platelets. Here, we show that avian thrombocytes respond to many of the same activating stimuli as mammalian platelets but are unable to form shear-resistant aggregates ex vivo. Avian thrombocytes are larger than mammalian platelets, spread less efficiently on collagen, and express much lower levels of the α(₂b)β₃ integrin required for aggregate formation, features predicted to make thrombocyte aggregates less resistant than platelets are to the high fluid shear forces of the arterial vasculature. In vivo carotid vessel injury stimulates the formation of occlusive platelet thrombi in mice but not in the size- and flow-matched carotid artery of the Australian budgerigar. These studies indicate that unique physical and molecular features of mammalian platelets enable them to form shear-resistant arterial thrombi, an essential element in the pathogenesis of human cardiovascular diseases.

Diminished contact-dependent reinforcement of Syk activation underlies impaired thrombus growth in mice lacking Semaphorin 4D.

We recently reported that Semaphorin 4D (Sema4D) and its receptors are expressed on the platelet surface and showed that Sema4D((-/-)) mice have a selective defect in collagen-induced platelet aggregation and an impaired vascular injury response. Here we investigated the mechanisms involved, tested the role of platelet-platelet contacts in Sema4D-mediated events, and examined the relationship between Sema4D-dependent signaling and integrin α(IIb)β(3) outside-in signaling. The results show that spleen tyrosine kinase (Syk) activation, an early step in collagen signaling via the glycoprotein VI (GPVI)/FcRγ complex, is greatly reduced in Sema4D((-/-)) platelets and can be restored by adding soluble Sema4D. Earlier events, including FcRγ phosphorylation, occur normally; later events are impaired. In contrast, when engagement of α(IIb)β(3) was blocked, Sema4D((-/-)) and control platelets were indistinguishable in assays of Syk activation, adhesion, spreading on collagen, and activation of α(IIb)β(3). Finally, we found that, unlike the Sema4D knockout, α(IIb)β(3) blockade inhibited FcRγ phosphorylation and that stimulating aggregation with Mn(2+) failed to normalize Syk activation in the absence of Sema4D. Collectively, these results show that α(IIb)β(3) and Sema4D jointly promote collagen responses by amplifying Syk activation, partly by forming integrin-mediated contacts that enable the binding of Sema4D to its receptors and partly through integrin outside-in signaling. These 2 processes are interdependent, but distinguishable.

Effects of membrane rheology on leuko-polymersome adhesion to inflammatory ligands

A strategy for treating inflammatory disease is to create micro-particles with the adhesive properties of leukocytes. The underlying rheology of deformable adhesive microspheres would be an important factor in the adhesive performance of such particles. In this work the effect of particle deformability on the selectin-mediated rolling of polymer vesicles (polymersomes) is evaluated. The rheology of the polymersome membrane was modulated by cross-linking unsaturated side-chains within the hydrophobic core of the membrane. Increased membrane rigidity resulted in decreased rates of particle recruitment rather than decreased average rolling velocities. Reflective interference contrast microscopy of rolling vesicles confirmed that neither flaccid nor rigid vesicles sustained close contacts with the substrate during rolling adhesion. A variable-shear rate parallel-plate flow chamber was employed to evaluate individual vesicles rolling on substrates under different flow conditions. Analysis of the trajectories of single flaccid vesicles revealed several distinct populations of rolling vesicles; however, some of these populations disappear when the vesicle membranes are made rigid. This work shows that membrane mechanics affects the capture, but not the rolling dynamics, of adherent leuko-polymersomes.

Microcontact printing of P‐selectin increases the rate of neutrophil recruitment under shear flow

The local variation of P‐selectin expression on inflamed endothelial layers affects leukocyte recruitment in vivo. As an initial study of the spatially heterogeneous presentation of P‐selectin in vitro, the influence of microcontact printing (μCP) of P‐selectin on a planar surface in neutrophil recruitment was investigated using a parallel‐plate flow chamber. Microline patterned and nonpatterned P‐selectin were prepared using PDMS, Poly(dimethylsiloxane) (PMDS) stamps and isolated neutrophils perfused over the surface to quantify the level of neutrophil recruitment. We first found a significant increase in cell rolling flux and a decrease in cell rolling velocity on the microcontact printed P‐selectin‐surfaces compared with a randomly adsorbed P‐selectin‐surface. However, the increase in rolling adhesion under shear on the surfaces prepared by μCP was not proportional to the number of functional sites of P‐selectin transferred using immunofluorescent labeling. Interestingly, the relative immunofluorescent intensities of the nonfunctional regions of microcontact printed P‐selectin‐surfaces were substantially lower than that that of randomly adsorbed P‐selectin. Taken together, these data indicate that the μCP of selectin increases the transfer rate of the adhesion molecule on a surface in the functionally correct orientation and, consequently, improves the recruitment of leukocytes to the selectin surface under flow. It is concluded that μCP may be a more general technique to control protein orientation on a substrate.

An Integrated Stochastic Model of “Inside-Out” Integrin Activation and Selective T-Lymphocyte Recruitment

The pattern of T-lymphocyte homing is hypothesized to be controlled by combinations of chemokine receptors and complementary chemokines. Here, we use numerical simulation to explore the relationship among chemokine potency and concentration, signal transduction, and adhesion. We have developed a form of adhesive dynamics-a mechanically accurate stochastic simulation of adhesion-that incorporates stochastic signal transduction using the next subvolume method. We show that using measurable parameter estimates derived from a variety of sources, including signaling measurements that allow us to test parameter values, we can readily simulate approximate time scales for T-lymphocyte arrest. We find that adhesion correlates with total chemokine receptor occupancy, not the frequency of occupation, when multiple chemokine receptors feed through a single G-protein. A general strategy for selective T-lymphocyte recruitment appears to require low affinity chemokine receptors. For a single chemokine receptor, increases in multiple cross-reactive chemokines can lead to an overwhelming increase in adhesion. Overall, the methods presented here provide a predictive framework for understanding chemokine control of T-lymphocyte recruitment.

SLP-76 is required for optimal CXCR4-stimulated T lymphocyte firm arrest to ICAM-1 under shear flow

Rapid arrest of T cells at target sites upon engagement of chemokine receptors is crucial to the proper functioning of the immune system. Although T‐cell arrest always occurs under hydrodynamic forces in vivo, most studies investigating the molecular mechanisms of arrest have been performed under static conditions. While the requirement of the adapter protein SLP‐76 (Src homology 2‐domain containing leukocyte‐specific phosphoprotein of 76 kDa) in TCR‐induced integrin activation has been demonstrated, its role in chemokine‐triggered T‐cell adhesion is unknown. Using a flow chamber system, we show that SLP‐76 plays an important role in regulating the transition from tethering and rolling to firm adhesion of T cells under physiological shear flow in response to CXCL12α (stromal cell‐derived factor‐1α); SLP‐76‐deficient primary T cells exhibited defective adhesion with a significant decrease in the number of firmly arrested cells. We further demonstrate the N‐terminal phosphotyrosines of SLP‐76 play a critical role in T‐cell adhesion under flow. These findings reveal a novel role for SLP‐76 in CXCR4‐mediated T lymphocyte trafficking.

Adhesive dynamics simulations of the mechanical shedding of L-selectin from the neutrophil surface.

Here we accurately recreate the mechanical shedding of L-selectin and its effect on the rolling behavior of neutrophils in vitro using the adhesive dynamics simulation by incorporating the shear-dependent shedding of L-selectin. We have previously shown that constitutively expressed L-selectin is cleaved from the neutrophil surface during rolling on a sialyl Lewis x-coated planar surface at physiological shear rates without the addition of exogenous stimuli. Utilizing a Bell-like model to describe a shedding rate which presumably increases exponentially with force, we were able to reconstruct the characteristics of L-selectin-mediated neutrophil rolling observed in the experiments. First, the rolling velocity was found to increase during rolling due to the mechanical shedding of L-selectin. When most of the L-selectin concentrated on the tips of deformable microvilli was cleaved by force exerted on the L-selectin bonds, the cell detached from the reactive plane to join the free stream as observed in the experiments. In summary, we show through detailed computational modeling that the force-dependent shedding of L-selectin can explain the rolling behavior of neutrophils mediated by L-selectin in vitro.