Publications
2009
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(2009) Small. 5, 15, p. 1723-1726 Abstract
A biochip model of lymphocyte locomotion on confined chemokine tracks is described. Micropatterned chemokine biochips were constructed with the help microscope glass coverslips coated with daisy molecular films. Chemically reactive primary amines were fully de-protected at the solution termini of daisy by shining UV light. Biochips were assembled on slides mounted on an automated stage of an inverted microscope. The slides were assembled within a hermetically sealed chamber apparatus 0.3 mm in height to avoid fluid convection. Human T lymphocytes freshly isolated from blood were perfused through the chamber. Upon entry to the chamber flow was stopped, allowing cells to randomly settle on the CXCL12-presenting biochip. Movies were recorded over 30-min periods in multiple fields of view using Softmorx 3.5 at four frames per minute using a 20x/0.95 NA differential interference contrast objective.
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(2009) Journal of Neuroimmunology. 209, 1-2, p. 87-95 Abstract
We have previously demonstrated that the chemokine IFN-γ inducible protein 10 (IP-10) and its receptor CXCR3, are overexpressed in myasthenia gravis (MG) and its animal model experimental autoimmune MG (EAMG). We now studied the potential of modulating rat EAMG by interference in CXCR3/IP-10 signaling. Two different approaches were used: 1) blocking IP-10 by IP-10-specific antibodies and 2) inhibiting the CXCR3 chemokine receptor by a CXCR3 antagonist. Treatment by either of these reagents led to suppression of EAMG suggesting that inhibition of CXCR3/IP-10 signaling can be considered as a potential treatment modality for MG.
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(2009) Cancer Research. 69, 7, p. 3121-3130 Abstract
Homing to secondary lymphoid organs and bone marrow (BM) is a central aspect of leukemic pathophysiology. We LFA-I and VLA-4 on B-cell chronic lymphocytic leukemia (CLL) cells in these processes. We found that the majority of CLL cells expressed significantly reduced LFA-I due to low ß2 integrin transcripts. VLA-4 expression was heterogenous but underwent rapid activation by the BM chemokine CXCL,2. CLL cells failed to transmigrate across VCAM-1-expressing, ICAM-I-expressing, and CXCL,2-expressing endothelium, whereas when LFA-I expression was regained in subsets of CLL cells, these lymphocytes rapidly transmigrated the endothelium. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LN) in a LFA-1-dependent manner, whereas CLL cells did not. Nevertheless, only residual CLL subsets could reenter BM, whereas both normal and CLL cells homed to the mice spleen in an LFA-1-independent and VLA-4-independent manner. Our results suggest that CLL cells have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than spleen. Integrin blocking could thus be an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM but not in spleen.
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(2009) Immunity. 30, 3, p. 384-396 Abstract
Endothelial chemokines are instrumental for integrin-mediated lymphocyte adhesion and transendothelial migration (TEM). By dissecting how chemokines trigger lymphocyte integrins to support shear-resistant motility on and across cytokine-stimulated endothelial barriers, we found a critical role for high-affinity (HA) LFA-1 integrin in lymphocyte crawling on activated endothelium. Endothelial-presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath crawling lymphocytes. Shear forces applied to endothelial-bound lymphocytes dramatically enhanced filopodia density underneath crawling lymphocytes. A fraction of the adhesive filopodia invaded the endothelial cells prior to and during TEM and extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. Memory T cells generated more frequent invasive filopodia and transmigrated more rapidly than their naive counterparts. We propose that shear forces exerted on HA-LFA-1 trigger adhesive and invasive filopodia at apical endothelial surfaces and thereby promote lymphocyte crawling and probing for TEM sites.
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(2009) Blood. 113, 23, p. 5801-5810 Abstract
Adhesion and motility of mammalian leukocytes are essential requirements for innate and adaptive immune defense mechanisms. We show here that the guanine nucleotide exchange factor cytohesin-1, which had previously been demonstrated to be an important component of beta-2 integrin activation in lymphocytes, regulates the activation of the small GTPase RhoA in primary dendritic cells (DCs). Cytohesin-1 and RhoA are both required for the induction of chemokine-dependent conformational changes of the integrin beta-2 subunit of DCs during adhesion under physiological flow conditions. Furthermore, use of RNAi in murine bone marrow DCs (BM-DCs) revealed that interference with cytohesin-1 signaling impairs migration of wild-type dendritic cells in complex 3D environments and in vivo. This phenotype was not observed in the complete absence of integrins. We thus demonstrate an essential role of cytohesin-1/RhoA during ameboid migration in the presence of integrins and further suggest that DCs without integrins switch to a different migration mode.
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(2009) Blood. 114, 11, p. 2344-2353 Abstract
Leukocyte adhesion deficiency (LAD)-III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor-stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3-null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3-null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative β1 Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4-adhesive functions in human lymphocytes.
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(2009) Chemokines, Part B. Hamel D. & Handel T.(eds.). B ed. Vol. 461. p. 311-332 Abstract
The mechanisms underlying leukocyte migration across endothelial barriers are still largely elusive. Integrin activation by chemokine signals is a key checkpoint in this process. Most of the current knowledge on transendothelial migration (TEM) of leukocytes has been derived from in vitro modified Boyden-chamber transfilter migration assays. In these assays, leukocyte migration toward chemokine gradients established across an endothelial barrier is measured under shear-free conditions. Consequently, these assays do not address the critical contribution of shear forces to dynamic integrin activation and redistribution at focal lymphocyte-endothelial contacts. Endothelial chemokines are displayed at high levels on blood vessel walls in vivo and play critical roles in both integrin activation and polarization of leukocytes on blood vessels, yet transwell assays do not assess the role of these chemokines in leukocyte TEM. To overcome these two drawbacks, several laboratories, including our group, developed assays based on in vitro live imaging microscopy to follow leukocyte migration across endothelial barriers that display defined compositions of integrin-stimulatory chemokines. These assays not only successfully simulate physiologic TEM processes but also enable the tracking and dissection of leukocyte adhesion, motility, and crossing of endothelial barriers in real time and under physiologic flow conditions. In addition, fluorescent tagging of membranes, adhesion molecules, and cytoskeletal regulatory elements on the endothelial barrier or the leukocyte can provide key spatial and temporal information on the mode of activity of these elements during distinct stages of leukocyte TEM. After fixation, subcellular changes in the redistribution of these key molecules can be further dissected by immunofluorescence tools and by ultrastructural analysis based on scanning and transmission electron microscopy.
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(2009) Microcirculation. 16, 1, p. 3-16 Abstract
The arrest of lymphocytes at target vascular sites depends on the rapid activation of their integrins by specialized endothelial chemokines. For over a decade, the mechanisms by which these chemokines trigger initial integrin-mediated adhesiveness and subsequent adhesion strengthening and crawling over endothelial surfaces have been dissected in vitro using flow chamber setups. These studies revealed that lymph node chemokines and subsets of inflammatory chemokines, collectively termed "arrest chemokines," can trigger the fastest measurable inside-out integrin activation events. Recent studies indicate that shear forces applied on lymphocytes are instrumental in these rapid activation processes. Different GTPases have been implicated in these activation processes. As these enzymes contribute to successive integrin activation and redistribution processes in both early and prolonged contacts there is a growing need to dissect in vitro and validate in vivo specific signaling routes involved in early and late integrin activation events controlling lymphocyte arrest and their subsequent crawling to sites of diapedesis. In this article, we present an overview of both early and recent shear-flow studies of integrin activation in lymphocytes and discuss future perspectives of integrin activation research in vitro and in vivo.
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(2009) Leukocyte Adhesion. Ley K.(eds.). p. 157-193 (trueCurrent Topics in Membranes). Abstract
The arrest of rolling leukocytes at target vascular sites depends on rapid activation of their vascular integrins at endothelial contacts enriched with specific Ig superfamily (IgSF) ligands. Accumulating data suggest that these integrins acquire high affinity to these endothelial ligands in a process tightly regulated by a variety of cytoskeletal conformational changes in their α- and β-subunits. These alterations are controlled by inside-out signals that are induced primarily by chemokines displayed on the endothelium and transduced by G protein-coupled receptors (GPCRs) on the responding leukocytes. The GPCR signals can propagate within milliseconds at submicron ranges. Upon binding their ligands, GPCR-stimulated integrins also undergo outside-in conformational activation, which is accelerated by applied forces. The integrin activator, talina universal cytoskeletal adaptor in integrinmediated focal adhesionsplays an instrumental role in this bidirectional activation process, which is critical for leukocyte arrest on endothelial IgSF ligands. Multiple effectors in the immediate vicinity to integrintalin complexes can determine the extent to which talin may translate a given chemokine signal into a fully productive integrin activation. Substantial heterogeneity exists in the pathways used by different cell types to translate a given combination of chemokines and integrin ligands into leukocyte arrest and subsequent adhesion strengthening on various target endothelia. This chapter highlights the main molecular players in the earliest events of chemokine signaling to lymphocyte integrins and proposes modalities used by rapid chemokine signals to trigger cytoskeletal rearrangements in integrin tails, leading to rapid integrin activation and leukocyte arrest on vascular endothelium.
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(2009) Soft Matter. 5, 21, p. 4141-4151 Abstract
Cell adhesion is a complex event dictated by the properties of individual adhesion molecules. It is desirable to link their individual properties to the adhesive behavior of a whole cell. Here, we examine with atomic force microscopic (AFM) and flow chamber experiments how the exogenous activation of the major T cell integrin VLA-4 affects the adhesiveness of T-lymphocytes to the endothelial VLA-4 ligand, VCAM-1. The atomic force measurements are performed on substrates with different VCAM-1 densities to compare the properties of single adhesive bonds on low-density ligands with the effect of multiple bonds on higher ligand densities. We determine the basal off-rate and the potential width of the single adhesive VLA-4-VCAM-1 bond by Monte-Carlo simulations. We show that activating VLA-4 with magnesium increases the potential width of the bond from 0.18 nm to 0.44 nm, but hardly affects the basal off-rate of single VLA-4-VCAM-1 bonds measured at low VCAM-1 density, lowering it only from 1.2 s-1 to 0.9 s-1. At higher VCAM-1 densities, the apparent off-rate of high avidity VLA-4-VCAM-1 interactions is significantly reduced by magnesium to
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(2009) Blood. 114, 5, p. 1073-1082 Abstract
Leukocyte extravasation involves interdependent signaling pathways underlying the complex dynamics of firm adhesion, crawling, and diapedesis. While signal transduction by agonist-bound chemokine receptors plays a central role in the above responses, it is unclear how it contributes to the sustained and concurrent nature of such responses, given the rapid kinetics of chemokine-induced trimeric G protein coupling and homologous desensitization. Our findings unveil a novel role of β-arrestins in regulating the activation of signaling pathways underlying discrete integrin-mediated steps in CXCR2-driven leukocyte extravasation. By combining in vivo approaches in β-arrestin knockout mice with in vitro studies in engineered cellular models, we show that membrane-recruited β-arrestin 2 is required for the onset and maintenance of shear stress-resistant leukocyte adhesion mediated by both β1 and β2 integrins. While both β-arrestin isoforms are required for rapid keratinocyte-derived chemokine (KC)-induced arrest onto limiting amounts of vascular cell adhesion molecule-1 (VCAM-1), adhesion strengthening under shear is selectively dependent on β-arrestin 2. The latter synergizes with phospholipase C in promoting activation of Rap1A and B, both of which cooperatively control subsecond adhesion as well as postarrest adhesion stabilization. Thus, receptor-induced Gαi and β-arrestins act sequentially and in spatially distinct compartments to promote optimal KC-induced integrin-dependent adhesion during leukocyte extravasation.