Regulation of CXC chemokine receptor 4-mediated migration by the Tec family tyrosine kinase ITK

Chemokines are critical in controlling lymphocyte traffic and migration. The CXC chemokine CXCL12/SDF-1alpha interacts with its receptor CXCR4 to induce the migration of a number of different cell types. Although an understanding of the physiological functions of this chemokine is emerging, the mechanism by which it regulates T cell migration is still unclear. We show here that the Tec family kinase ITK is activated rapidly following CXCL12/SDF-1alpha stimulation, and this requires Src and phosphatidylinositol 3-kinase activities. ITK regulates the ability of CXCL12/SDF-1alpha to induce T cell migration as overexpression of wild-type ITK-enhanced migration, and T cells lacking ITK exhibit reduced migration as well as adhesion in response to CXCL12/SDF-1alpha. Further analysis suggests that ITK may regulate CXCR4-mediated migration and adhesion by altering the actin cytoskeleton, as ITK null T cells were significantly defective in CXCL12/SDF-1a-mediated actin polymerization. Our data suggest that ITK may regulate the ability of CXCR4 to induce T cell migration.     

Rap1 translates chemokine signals to integrin activation,cell polarization,and motility across vascular endothelium under flow

Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.     

CXCL12 induces lung cancer cell migration by polarized mtDNA redistribution

Instability of mitochondrial DNA (mtDNA) has been associated with the initiation and development of cancer, but the specific role of mtDNA in the invasiveness and migration of cancer cells remains unclear. In this study, we investigated whether the chemokine CXCL12 causes intact mitochondria to redistribute in cancer cells and, in this way, to increase cell invasiveness and migration. A549 lung cancer cells with intact mtDNA (mtDNA+) and ρ⁰A549 cells depleted of mtDNA (mtDNA?) by long-term ethidium bromide incubation were examined for their responses to CXCL12 in a transwell migration assay and for mitochondrial distribution by fluorescence microscopy. Intact A549 cells showed significantly increased migration and increased polar distribution of mitochondria (asymmetry) in response to CXCL12. However, ρ⁰A549 cells showed no changes in mitochondrial distribution in response to CXCL12, and only a few ρ⁰A549 cells migrated across the transwell membrane after CXCL12 treatment. These results demonstrate that, in A549 lung cancer cells, intact mitochondrial DNA is necessary for mitochondrial redistribution and a chemotactic response to CXCL12.     

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.     

Vav1 and Rac control chemokine-promoted T lymphocyte adhesion mediated by the integrin alpha4beta1

The chemokine CXCL12 promotes T lymphocyte adhesion mediated by the integrin alpha4beta1. CXCL12 activates the GTPase Rac, as well as Vav1, a guanine-nucleotide exchange factor for Rac, concomitant with up-regulation of alpha4beta1-dependent adhesion. Inhibition of CXCL12-promoted Rac and Vav1 activation by transfection of dominant negative Rac or Vav1 forms, or by transfection of their siRNA, remarkably impaired the increase in T lymphocyte attachment to alpha4beta1 ligands in response to this chemokine. Importantly, inhibition of Vav1 expression by RNA interference resulted in a blockade of Rac activation in response to CXCL12. Adhesions in flow chambers and soluble binding assays using these transfectants indicated that initial ligand binding and adhesion strengthening mediated by alpha4beta1 were dependent on Vav1 and Rac activation by CXCL12. Finally, CXCL12-promoted T-cell transendothelial migration involving alpha4beta1-mediated adhesion was notably inhibited by expression of dominant negative Vav1 and Rac. These results indicate that activation of Vav1-Rac signaling pathway by CXCL12 represents an important inside-out event controlling efficient up-regulation of alpha4beta1-dependent T lymphocyte adhesion.     

Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells

Vascular adhesion protein-1 (VAP-1) is an amine oxidase and adhesion receptor that is expressed by endothelium in the human liver. The hepatic sinusoids are perfused by blood at low flow rates, and sinusoidal endothelium lacks selectin expression and has low levels of CD31, suggesting that VAP-1 may play a specific role in lymphocyte recruitment to the liver. In support of this we now report the constitutive expression of VAP-1 on human hepatic sinusoidal endothelial cells (HSEC) in vitro and demonstrate that VAP-1 supports adhesion and transmigration of lymphocytes across these cells under physiological shear stress. These are the first studies to report the function of VAP-1 on primary human endothelial cells. Under static conditions lymphocyte adhesion to unstimulated HSEC was dependent on VAP-1 and ICAM-2, whereas adhesion to TNF-alpha-stimulated HSEC was dependent on ICAM-1, VCAM-1, and VAP-1. Under conditions of flow, blocking VAP-1 reduced lymphocyte adhesion to TNF-alpha-treated HSEC by 50% and significantly reduced the proportion of adherent lymphocytes that transmigrated across cytokine or LPS-activated endothelium. In addition, inhibition of the amine oxidase activity of VAP-1 reduced both adhesion and transmigration of lymphocytes to a level similar to that seen with VAP-1 Ab. Thus, VAP-1 can support transendothelial migration as well as adhesion, and both functions are dependent on its enzymatic activity. In the absence of selectins and CD31, VAP-1 may play a specific role in lymphocyte recruitment via hepatic sinusoidal endothelium. Moreover, since VAP-1 is induced on nonhepatic endothelium in response to inflammation, its ability to support lymphocyte transendothelial migration may be an important systemic function of VAP-1.     

The Two Poles of the Lymphocyte: Specialized Cell Compartments for Migration and Recruitment

Chemotaxis, the directed migration of leukocytes towards a chemoattractant gradient, is a key phenomenon in the immune response. During lymphocyte-endothelial and – extracellular matrix interactions, chemokines induce the polarization of T lymphocytes. with generation of specialized cell compartments. The chemokine receptors involved in detection of the chemoattractant gradients concentrate at the leading edge (advancing front or anterior pole) of the cell. The adhesion molecules ICAM- 1, -3, CD44 and CD43 redistribute to the uropod, an appendage at the posterior pole of migrating T lymphocyte that protrudes from the contact area with endothelial or extracellular matrix substrates. Whereas chemokine receptors sense the direction of migration, the uropod is involved in the recruitment of bystander leukocytes through LFA-1/ICAM-dependent cell cell interactions. While β-actin concentrates preferentially at the cell's leading edge, the motor protein myosin II and a microtubule organizing center (MTOC) are packed in the uropod. The actin-binding protein moesin, which belongs to the ERM family of ezrin, radixin and moesin, redistributes to the distal portion of uropods and physically interacts with ICAM-3, CD44 and CD43, thus acting as a physical link between the membrane molecules and the actin cytoskeleton. Moreover, the moesin-ICAM-3 association correlates with the degree of cell polarity. The redistribution of the chemokine receptors and adhesion molecules to opposite poles of the cell in response to a chemoattractant gradient may guide cell migration and cell-cell interactions during lymphoid cell trafficking in immune and inflammatory responses.     

CXCR4 chemokine receptor engagement modifies integrin dependent adhesion of renal carcinoma cells

The mechanisms leading to renal cell carcinoma (RCC) metastasis are incompletely understood. Although evidence shows that the chemokine receptor CXCR4 and its ligand CXCL12 may regulate tumor dissemination, their role in RCC is not clearly defined. We examined CXCR4 expression and functionality on RCC cell lines, and explored CXCL12-triggered tumor adhesion to human endothelium (HUVEC) or extracellular matrix proteins. Functional CXCR4 was expressed on A498 tumor cells, enabling them to migrate towards a CXCL12 gradient. CXCR4 engagement by CXCL12 induced elevated cell adhesion to HUVEC, to immobilized fibronectin, laminin or collagen. Anti-CXCR4 antibodies or CXCR4 knock down by siRNA applied prior to CXCL12 stimulation impaired CXCL12-triggered tumor adhesion. However, blocking CXCR4 subsequent to CXCL12 stimulation did not. This pointed to an indirect control of tumor cell adhesion by CXCR4. In fact, CXCR4 engagement by CXCL12 also induced alterations of receptors of the integrin family, notably alpha3, alpha5, beta1 and beta3 subunits, and blocking beta1 integrins with a function-blocking antibody prevented CXCL12-induced A498 adhesion. Focal adhesion kinase (total and activated) and integrin-linked kinase significantly increased in CXCL12-treated A498 cells, accompanied by a distinct up-regulation of ERK1/2, JNK and p38 phosphorylation. Therefore, CXCR4 may be crucial in controlling adhesion of A498 cells via cross talking with integrin receptors. These data show that CXCR4 receptors contribute to RCC dissemination and may provide a novel link between CXCR4 chemokine receptor expression and integrin triggered RCC adhesion to the vascular wall and subendothelial matrix components.     

The VLA-4/VCAM-1 pathway is involved in lymphocyte adhesion to endothelium in rheumatoid synovium.

Lymphocyte migration to inflammatory sites is an essential factor in the pathogenesis of chronic inflammation. An ensemble of adhesion receptors mediating lymphocyte-endothelial cell recognition and binding are thought to play a crucial role in this process. In the present study, we have explored the molecular basis of lymphocyte adhesion to endothelium in the synovial membrane of patients with rheumatoid arthritis. We established that the very late antigen-4 [VLA-4 (CD49d)] and the vascular cell adhesion molecule-1 (VCAM-1) are important mediators of binding to synovial endothelium of resting and, to a greater extent, of activated T lymphocytes, whereas the leukocyte-function associated antigen-1 [LFA-1 (CD11a/18)]/intercellular adhesion molecule-1 [ICAM-1 (CD54)] pathway is less important in this interaction. In contrast to its prominent role in lymphocyte interaction with endothelium in rheumatoid synovium, the VLA-4/VCAM-1 pathway does not significantly contribute to lymphocyte adhesion to peripheral lymph node high endothelial venule. Thus, the VLA-4/VCAM-1 pathway may be of primary importance in mediating lymphocyte adhesion to inflamed endothelium and in lymphocyte homing to rheumatoid synovium.     

Annexin 1 modulates monocyte-endothelial cell interaction in vitro and cell migration in vivo in the human SCID mouse transplantation model

The effect of the glucocorticoid inducible protein annexin 1 (ANXA1) on the process of monocytic cell migration was studied using transfected U937 cells expressing variable protein levels. An antisense (AS) (36.4AS; approximately 50% less ANXA1) and a sense (S) clone (15S; overexpressing the bioactive 24-kDa fragment) together with the empty plasmid CMV clone were obtained and compared with wild-type U937 cells in various models of cell migration in vitro and in vivo. 15S-transfected U937 cells displayed a reduced (50%) degree of trans-endothelial migration in response to stromal cell-derived factor-1alpha (CXC chemokine ligand 12 (CXCL12)). In addition, the inhibitory role of endogenous ANXA1 on U937 cell migration in vitro was confirmed by the potentiating effect of a neutralizing anti-ANXA1 serum. Importantly, overexpression of ANXA1 in clone 15S inhibited the extent of cell migration into rheumatoid synovial grafts transplanted into SCID mice. ANXA1 inhibitory effects were not due to modifications in adhesion molecule or CXCL12 receptor (CXCR4) expression as shown by the similar amounts of surface molecules found in transfected and wild-type U937 cells. Likewise, an equal chemotactic response to CXCL12 in vitro excluded an intrinsic defect in cell motility in clones 15S and 36.4AS. These data strongly support the notion that ANXA1 critically interferes with a leukocyte endothelial step essential for U937 cell, and possibly monocyte, transmigration both in vitro and in vivo.     

Platelet-derived chemokines CXC chemokine ligand (CXCL)7, connective tissue-activating peptide III,and CXCL4 differentially affect and cross-regulate neutrophil adhesion and transendothelial migration

In this study, we have examined the major platelet-derived CXC chemokines connective tissue-activating peptide III (CTAP-III), its truncation product neutrophil-activating peptide 2 (CXC chemokine ligand 7 (CXCL7)), as well as the structurally related platelet factor 4 (CXCL4) for their impact on neutrophil adhesion to and transmigration through unstimulated vascular endothelium. Using monolayers of cultured HUVEC, we found all three chemokines to promote neutrophil adhesion, while only CXCL7 induced transmigration. Induction of cell adhesion following exposure to CTAP-III, a molecule to date described to lack neutrophil-stimulating capacity, depended on proteolytical conversion of the inactive chemokine into CXCL7 by neutrophils. This was evident from experiments in which inhibition of the CTAP-III-processing protease and simultaneous blockade of the CXCL7 high affinity receptor CXCR-2 led to complete abrogation of CTAP-III-mediated neutrophil adhesion. CXCL4 at substimulatory dosages modulated CTAP-III- as well as CXCL7-induced adhesion. Although cell adhesion following exposure to CTAP-III was drastically reduced, CXCL7-mediated adhesion underwent significant enhancement. Transendothelial migration of neutrophils in response to CXCL7 or IL-8 (CXCL8) was subject to modulation by CTAP-III, but not CXCL4, as seen by drastic desensitization of the migratory response of neutrophils pre-exposed to CTAP-III, which was paralleled by selective down-modulation of CXCR-2. Altogether our results demonstrate that there exist multiple interactions between platelet-derived chemokines in the regulation of neutrophil adhesion and transendothelial migration.     

Lysophospholipids control integrin-dependent adhesion in splenic B cells through G(i) and G(12)/G(13) family G-proteins but not through G(q)/G(11)

Integrin-mediated adhesion is a crucial step in lymphocyte extravasation and homing. We show here that not only the chemokines CXCL12 and CXCL13 but also the lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) enhance adhesion of murine follicular and marginal zone B cells to ICAM-1 in vitro. This process involves clustering of integrin LFA-1 and is blocked by pertussis toxin, suggesting that G(i) family G-proteins are involved. In addition, lysophospholipid-induced adhesion on ICAM-1 depends on Rho and Rhokinase, indicative of an involvement of G(12)/G(13), possibly also G(q)/G(11) family G-proteins. We used G(12)/G(13)- or G(q)/G(11)-deficient B cells to study the role of these G-protein families in lysophospholipid-induced adhesion and found that the pro-adhesive effects of LPA and S1P are completely abrogated in G(12)/G(13)-deficient marginal zone B cells, reduced in G(12)/G(13)-deficient follicular B cells, and normal in G(q)/G(11)-deficient B cells. We also show that loss of lysophospholipid-induced adhesion results in disinhibition of migration in response to the follicular chemokine CXCL13, which might contribute to the abnormal localization of splenic B cell populations observed in B cell-specific G(12)/G(13)-deficient mice in vivo. Taken together, this study shows that lysophospholipids regulate integrin-mediated adhesion of splenic B cells to ICAM-1 through G(i) and G(12)/G(13) family G-proteins but not through G(q)/G(11).     

G12/G13 family G proteins regulate marginal zone B cell maturation, migration, and polarization

G protein-coupled receptors play an important role in the regulation of lymphocyte functions such as migration, adhesion, proliferation, and differentiation. Although the role of G(i) family G proteins has been intensively studied, no in vivo data exist with respect to G12/G13 family G proteins. We show in this study that mice that lack the G protein alpha-subunits G alpha12 and G alpha13 selectively in B cells show significantly reduced numbers of splenic marginal zone B (MZB) cells, resulting in a delay of Ab production in response to thymus-independent Ags. Basal and chemokine-induced adhesion to ICAM-1 and VCAM-1, two adhesion molecules critically involved in MZB localization, is normal in mutant B cells, and the same is true for chemokine-induced migration. However, migration in response to serum and sphingosine 1-phosphate is strongly increased in mutant MZB cells, but not in mutant follicular B cells. Live-cell imaging studies revealed that G alpha12/G alpha13-deficient MZB cells assumed more frequently an ameboid form than wild-type cells, and pseudopod formation was enhanced. In addition to their regulatory role in serum- and sphingosine 1-phosphate-induced migration, G12/G13 family G proteins seem to be involved in peripheral MZB cell maturation, because also splenic MZB cell precursors are reduced in mutant mice, although less prominently than mature MZB cells. These data suggest that G12/G13 family G proteins contribute to the formation of the mature MZB cell compartment both by controlling MZB cell migration and by regulating MZB cell precursor maturation.     

The role of the gap junction protein connexin43 in B lymphocyte motility and migration

The gap junction family of proteins is widely expressed in mammalian cells and form intercellular channels between adjacent cells, as well as hemichannels, for transport of molecules between the cell and the surrounding environment. In addition, gap junction proteins have recently been implicated as important for the regulation of cell adhesion and migration in a variety of cell types. The gap junction protein connexin43 (Cx43) regulates B lymphocyte adhesion, BCR- and LFA-1-mediated activation of the GTPase Rap1, and cytoskeletal rearrangements resulting in changes to cell shape and membrane spreading. We demonstrate here that the actin cytoskeleton is important for the distribution of Cx43 in the B cell plasma membrane and for other cell processes involving the cytoskeleton. Using shRNA knockdown of Cx43 in B lymphoma cells we show that Cx43 is also necessary for chemokine-mediated Rap 1 activation, motility, CXCL12-directed migration, and movement across an endothelial cell monolayer. These results demonstrate that in addition to its role in B cell spreading, Cx43 is an important regulator of B-cell motility and migration, processes essential for normal B-cell development and immune responses.     

Chemokine stimulation promotes enterocyte migration through laminin-specific integrins

Intestinal homeostasis is regulated in part by the single cell layer of the mucosal epithelium. This physical barrier is a prominent part of the innate immune system and possesses an intrinsic ability to heal damage and limit infection. The restitutive epithelial migration phase of healing requires dynamic integrin adhesion to the extracellular matrix. Previously, we have shown that the homeostatic chemokine CXCL12 utilizes intracellular calcium to increase enterocyte migration on laminin. The aim of these studies was to investigate integrin specificity and, in turn, functional responses elicited by CXCL12 stimulation. Analysis of cellular adhesion and spreading revealed CXCL12 preferentially activated laminin-specific integrins compared with collagen IV-binding integrins. Laminin-specific cell adhesion and spreading elicited by CXCL12 was dependent on intracellular calcium. CXCL12 increased activated β1-integrins on the surface of epithelial cells compared with untreated cells. RT-PCR confirmed expression of the laminin-binding integrins-α3β1, -α6β1, and -α6β4. Interestingly, shRNA-mediated depletion of laminin-specific α3- or α6-integrin subunits revealed differential functions. α3-Integrin knockdown reduced basal as well as inducible restitution. Depletion of α6-integrin specifically abolished CXCL12-stimulated, but not TGF-β1 or basal, migration. Depletion with either shα3-integrin or shα6-integrin prevented CXCL12-evoked cell spreading. Our data indicate that CXCL12 stimulates the inside-out activation of laminin-specific integrins to promote cell migratory functions. Together, our findings support the notion that extracellular mediators within the gastrointestinal mucosa coordinate cell-matrix interactions during epithelial restitution.     

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.     

The atypical Rho GTPases Miro-1 and Miro-2 have essential roles in mitochondrial trafficking

We recently described the atypical Rho GTPases Miro-1 and Miro-2. These proteins have tandem GTP-binding domains separated by a linker region with putative calcium-binding motives. In addition, the Miro GTPases have a C-terminal transmembrane domain, which confers targeting to the mitochondria. It was reported previously that a constitutively active mutant of Miro-1 induced a clustering of the mitochondria. This response can be separated into two distinct phenotypes: a formation of aggregated mitochondria and the appearance of thread-like mitochondria probably caused by defects in mitochondrial trafficking. The first GTPase domain is required for the clustering of the mitochondria, but the effect is not dependent on the EF-hands. Miro-2 only induces aggregation and not the formation of thread-like mitochondria. Moreover, we show that Miro interacts with the Kinesin-binding proteins, GRIF-1 and OIP106, suggesting that the Miro GTPases form a link between the mitochondria and the trafficking apparatus of the microtubules.     

The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis

The human CXC-chemokine CXCL4 is a potent inhibitor of tumor-induced angiogenesis. Considering that CXCL4 is sequestered in platelet alpha-granules and released following platelet activation in the vicinity of vessel wall injury, we tested the hypothesis that CXCL4 might function as a ligand for integrins. Integrins are a family of adhesion receptors that play a crucial role in angiogenesis by regulating early angiogenic processes, such as endothelial cell adhesion and migration. Here, we show that CXCL4 interacts with alphavbeta3 on the surface of alphavbeta3-CHO. More importantly, human umbilical vein endothelial cells adhere to immobilized CXCL4 through alphavbeta3 integrin, and also through other integrins, such as alphavbeta5 and alpha5beta1. We further demonstrate that CXCL4-integrin interaction is of functional significance in vitro, since immobilized CXCL4 supported endothelial cell spreading and migration in an integrin-dependent manner. Soluble CXCL4, in turn, inhibits integrin-dependent endothelial cell adhesion and migration. As a whole, our study identifies integrins as novel receptors for CXCL4 that may contribute to its antiangiogenic effect.     

CXCL12-stimulated lymphocytes produce secondary stimulants that affect the surrounding cell chemotaxis

Chemotactic factors locally secreted from tissues regulate leukocyte migration via cell membrane receptors that induce intracellular signals. It has been suggested that neutrophils stimulated by bacterial peptides secrete a secondary stimulant that enhances the chemotactic cell migration of the surrounding cells. This paracrine mechanism contributes to chemokine-dependent neutrophil migration, however, it has not yet been extensively studied in lymphocytes. In this study, we provide evidence that lymphocytes stimulated by the chemokine, CXCL12, affect the CXCR4-independent chemotactic response of the surrounding cells. We found that CXCR4-expressing lymphocytes or the conditioned medium from CXCL12-stimulated cells promoted CXCR4-deficient cell chemotaxis. In contrast, the conditioned medium from CXCL12-stimulated cells suppressed CCR7 ligand-dependent directionality and the cell migration speed of CXCR4-deficient cells. These results suggest that paracrine factors from CXCL12-stimulated cells navigate surrounding cells to CXCL12 by controlling the responsiveness to CCR7 ligand chemokines and CXCL12.