Search

Since chemerin is an adipocytokine whose concentration in blood increases in the subjects with various cardiac diseases, chemerin may be involved in pathogenesis of cardiac diseases. In the present study, we examined the effects of chemerin-9, an active fragment of chemerin, on functions of cardiac fibroblasts, which are involved in pathophysiology of cardiac diseases. Primary cardiac fibroblasts were enzymatically isolated from adult male Wistar rats. Migration of cardiac fibroblasts was measured by a Boyden chamber assay and a scratch assay. Phosphorylation of Akt and extracellular signal-regulated kinase (ERK) was measured by Western blotting. Reactive oxygen species (ROS) production was measured by 2',7'-dichlorodihydrofluoresein staining. Chemerin-9 significantly stimulated migration in cardiac fibroblasts. Chemerin-9 significantly stimulated phosphorylation of Akt and ERK as well as ROS production. An Akt pathway inhibitor, LY294002, an ERK pathway inhibitor, PD98059, an antagonist of chemokine-like receptor 1 (CMKLR1), 2-(α-Napththoyl) ethyltrimethylammonium iodide, or an antioxidant, N-acetyl-L-cysteine prevented the migration induced by chemerin-9. In summary, we for the first time revealed that chemerin-9 stimulates migration perhaps through the ROS-dependent activation of Akt and ERK via CMKLR1 in cardiac fibroblasts. It is proposed that chemerin plays a role in the pathogenesis of cardiac diseases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Lymphocytes are some of the most motile cells of vertebrates, constantly navigating through various organ systems. Their specific positioning in the body is delicately controlled by site-specific directional cues such as chemokines. While it has long been suspected that an intrinsic molecular pilot, akin to a ship's pilot, guides lymphocyte navigation, the nature of this pilot is unknown. Here we show that the TIPE (TNF-α-induced protein 8-like) family of proteins pilot lymphocytes by steering them toward chemokines. TIPE proteins are carriers of lipid second messengers. They mediate chemokine-induced local generation of phosphoinositide second messengers, but inhibit global activation of the small GTPase Rac. TIPE-deficient T lymphocytes are completely pilot-less: they are unable to migrate toward chemokines despite their normal ability to move randomly. As a consequence, TIPE-deficient mice have a marked defect in positioning their T lymphocytes to various tissues, both at the steady-state and during inflammation. Thus, TIPE proteins pilot lymphocytes during migration and may be targeted for the treatment of lymphocyte-related disorders.

Thymocyte and T cell trafficking relies on signals initiated by G-protein coupled receptors. To address the importance of the G-proteins Gα i2 and Gα i3 in thymocyte and T cell function, we developed several mouse models. Gα i2 deficiency in hematopoietic progenitors led to a small thymus, a double negative (DN)1/DN2 thymocyte transition block, and an accumulation of mature single positive (SP) thymocytes. Loss at the double positive (DP) stage of thymocyte development caused an increase in mature cells within the thymus. In both models an abnormal distribution of memory and naïve CD4 T cells occurred, and peripheral CD4 and CD8 T cells had reduced chemoattractant responses. The loss of Gα i3 had no discernable impact, however the lack of both G-proteins commencing at the DP stage caused a severe T cell phenotype. These mice lacked a thymic medullary region, exhibited thymocyte retention, had a peripheral T cell deficiency, and lacked T cell chemoattractant responses. Yet a noteworthy population of CD4 + PD-1 + CXCR5 +/- cells resided in the spleen of these mice likely due to a loss of regulatory T cell function. Our results delineate a role for Gα i2 in early thymocyte development and for Gα i2/3 in multiple aspects of T cell biology.

Background: Thickening of the lamina reticularis, a feature of remodeling in the asthmatic airways, is now known to be present in young children who wheeze. Human rhinovirus (HRV) infection is a common trigger for childhood wheezing, which is a risk factor for subsequent asthma development. We hypothesized that HRV-infected epithelial cells release chemoattractants to recruit fibroblasts that could potentially contribute to thickening of the lamina reticularis., Objective: We sought to investigate whether conditioned medium from HRV-infected epithelial cells can trigger directed migration of fibroblasts., Methods: Human bronchial epithelial cells were exposed to medium alone or infected with HRV-16. Conditioned medium from both conditions were tested as chemoattractants for human bronchial fibroblasts in the xCELLigence cell migration apparatus., Results: HRV-conditioned medium was chemotactic for fibroblasts. Treatment of fibroblasts with pertussis toxin, an inhibitor of Gαi-coupled receptors, prevented their migration. Production of epithelial chemoattractants required HRV replication. Multiplex analysis of epithelial supernatants identified CXCL10, CXCL8, and CCL5 as Gαi-coupled receptor agonists of potential interest. Subsequent analysis confirmed that fibroblasts express CXCR3 and CXCR1 receptors and that CXCL10 and, to a lesser extent, CXCL8, but not CCL5, are major contributors to fibroblast migration caused by HRV-conditioned medium., Conclusion: CXCL10 and CXCL8 produced from HRV-infected epithelial cells are chemotactic for fibroblasts. This raises the possibility that repeated HRV infections in childhood could contribute to the initiation and progression of airway remodeling in asthmatic patients by recruiting fibroblasts that produce matrix proteins and thicken the lamina reticularis., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Blood chemerin concentration shows positive correlation not only with body mass index and serum triglyceride level but also with systolic blood pressure. While it seems likely that chemerin influences vascular smooth muscle cell (SMC) proliferation and migration, which are crucial to the development of hypertension, this remains to be clarified. In the present study, we investigated whether chemerin controls SMC proliferation and migration in vitro and also affects blood pressure in vivo. In vitro, chemerin significantly stimulated rat mesenteric arterial SMC proliferation and migration, as determined by a cell counting assay and Boyden chamber assay, respectively. The migratory effect of chemerin was confirmed in human aortic SMCs. Chemerin significantly increased ROS production in SMCs and phosphorylation of Akt (Ser(473)) and ERK, as measured by fluorescent staining and Western blot analysis, respectively. Various inhibitors (ROS inhibitor: N-acetyl-l-cysteine, phosphatidylinositol 3-kinase inhibitor: LY-294002, MAPKK inhibitor: PD-98059, NADPH oxidase inhibitor: gp91 ds-tat, and xanthine oxidase inhibitor: allopurinol) as well as chemokine-like receptor 1 small interfering RNA significantly inhibited chemerin-induced SMC proliferation and migration. Furthermore, chemerin-neutralizing antibody prevented carotid neointimal hyperplasia in the mouse ligation model. In vivo, chronic chemerin treatment (6 μg/kg, 6 wk) increased systolic blood pressure as well as phosphorylation of Akt and ERK in the mouse isolated aorta. In summary, we, for the first time, demonstrate that chemerin/chemokine-like receptor 1 stimulates SMC proliferation and migration via ROS-dependent phosphorylation of Akt/ERK, which may lead to vascular structural remodeling and an increase in systolic blood pressure., (Copyright © 2015 the American Physiological Society.)

The ability of follicular T cells to migrate into B-cell follicles is central for them to participate in germinal center responses. The chemokine receptor CXCR5 is expressed by both Tfh and Tfr cells and is the defining marker for follicular T cells. In addition, Tfh and Tfr cells express additional chemokine receptors to enable them to interact with B cells and other cell types. CXCR5(+) Tfh and Tfr cells are divided into CCR7(+) perifollicular cells and CCR7(-) follicular cells. Most of the CXCR5(+) CCR7(-) Tfh cells reside in germinal centers and are called GC T cells. The methods to identify human Tfh and Tfr cell subsets based on chemokine receptors and other antigens and assess their migration potential are provided in this article.

Natural killer (NK) cells are innate immune cells able to rapidly kill virus-infected and tumor cells. Two NK cell populations are found in the blood; the majority (90%) expresses the CD16 receptor and also express the CD56 protein in intermediate levels (CD56(Dim) CD16(Pos)) while the remaining 10% are CD16 negative and express CD56 in high levels (CD56(Bright) CD16(Neg)). NK cells also reside in some tissues and traffic to various infected organs through the usage of different chemokines and chemokine receptors. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human virus that has developed numerous sophisticated and versatile strategies to escape the attack of immune cells such as NK cells. Here, we investigate whether the KSHV derived cytokine (vIL-6) and chemokines (vMIP-I, vMIP-II, vMIP-III) affect NK cell activity. Using transwell migration assays, KSHV infected cells, as well as fusion and recombinant proteins, we show that out of the four cytokine/chemokines encoded by KSHV, vMIP-II is the only one that binds to the majority of NK cells, affecting their migration. We demonstrate that vMIP-II binds to two different receptors, CX3CR1 and CCR5, expressed by naïve CD56(Dim) CD16(Pos) NK cells and activated NK cells, respectively. Furthermore, we show that the binding of vMIP-II to CX3CR1 and CCR5 blocks the binding of the natural ligands of these receptors, Fractalkine (Fck) and RANTES, respectively. Finally, we show that vMIP-II inhibits the migration of naïve and activated NK cells towards Fck and RANTES. Thus, we present here a novel mechanism in which KSHV uses a unique protein that antagonizes the activity of two distinct chemokine receptors to inhibit the migration of naïve and activated NK cells.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

Activated chemokine receptor initiates inside-out signaling to transiently trigger activation of integrins, a process involving multiple components that have not been fully characterized. Here we report that GM-CSF/IL-3/IL-5 receptor common beta-chain-associated protein (CBAP) is required to optimize this inside-out signaling and activation of integrins. First, knockdown of CBAP expression in human Jurkat T cells caused attenuated CXC chemokine ligand-12 (CXCL12)-induced cell migration and integrin α4β1- and αLβ2-mediated cell adhesion in vitro, which could be rescued sufficiently upon expression of murine CBAP proteins. Freshly isolated CBAP-deficient primary T cells also exhibited diminution of chemotaxis toward CC chemokine ligand-21 (CCL21) and CXCL12, and these chemokines-induced T-cell adhesions in vitro. Adoptive transfer of isolated naive T cells demonstrated that CBAP deficiency significantly reduced lymph node homing ability in vivo. Finally, migration of T cell-receptor-activated T cells induced by inflammatory chemokines was also attenuated in CBAP-deficient cells. Further analyses revealed that CBAP constitutively associated with both integrin β1 and ZAP70 and that CBAP is required for chemokine-induced initial binding of the talin-Vav1 complex to integrin β1 and to facilitate subsequent ZAP70-mediated dissociation of the talin-Vav1 complex and Vav1 phosphorylation. Within such an integrin signaling complex, CBAP likely functions as an adaptor and ultimately leads to activation of both integrin α4β1 and Rac1. Taken together, our data suggest that CBAP indeed can function as a novel signaling component within the ZAP70/Vav1/talin complex and plays an important role in regulating chemokine-promoted T-cell trafficking.

Forkhead box O (FOXO) transcription factors favor both T cell quiescence and trafficking through their control of the expression of genes involved in cell cycle progression, adhesion, and homing. In this article, we report that the product of the fam65b gene is a new transcriptional target of FOXO1 that regulates RhoA activity. We show that family with sequence similarity 65 member b (Fam65b) binds the small GTPase RhoA via a noncanonical domain and represses its activity by decreasing its GTP loading. As a consequence, Fam65b negatively regulates chemokine-induced responses, such as adhesion, morphological polarization, and migration. These results show the existence of a new functional link between FOXO1 and RhoA pathways, through which the FOXO1 target Fam65b tonically dampens chemokine-induced migration by repressing RhoA activity.

Objective: Vascular smooth muscle cell (VSMC) migration and proliferation are key components of vascular inflammation that may lead to atherosclerosis and restenosis, in which cytokines are considered as pivotal factors regarding recruitment of VSMC. A member of recently described family of chemokines, chemokine-like factor 1 (CKLF1), displays a wide spectrum of chemotaxis. This study investigated the role of CKLF1 in VSMC migration and proliferation during the process of vascular inflammation., Methods and Results: : The effects of CKLF1 on migration, proliferation and neointimal formation were investigated in cultured VSMCs, rat balloon injured arteries and human atherosclerotic plaques. CKLF1 overexpression greatly enhanced, whereas shRNA knockdown markedly retarded, VSMC migration and proliferation in vitro. In addition, CKLF1 protein accumulated preferentially in neointima of the injured rat arteries in vivo. CKLF1 overexpression resulted in a 2.5-fold increase in intimal thickness. In contrast, shRNA-mediated CKLF1 knockdown significantly suppressed neointima formation by 70% compared that in control group. Intriguingly, besides animal model, higher level of CKLF1 was observed in human atherosclerotic plaques than that in normal arteries., Conclusion: CKLF1 plays an essential role in migration and proliferation of VSMCs, which in turn facilitated neointimal hyperplasia and atherosclerosis. Inhibition of CKLF1 activity provides a potential target for the prevention of atherosclerosis and restenosis., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The gene encoding myopodin, an actin binding protein, is commonly deleted in invasive, but not in indolent, prostate cancers. There are conflicting reports on the effects of myopodin expression on prostate cancer cell migration and invasion. The recent recognition that myopodin is expressed as four different isoforms further complicates our understanding of how this potentially important invasive prostate cancer biomarker affects tumor cell migration and invasion. We now show that myopodin affects the chemokinetic, rather than the chemotactic, properties of PC3 prostate cancer cells. Furthermore, all myopodin isoforms can either increase or decrease PC3 cell migration in response to different chemokinetic stimuli. These migration properties were reflected by differences in cell morphology and the relative dependence on Rho-ROCK signaling pathways induced by the environmental stimuli. Truncation analysis determined that a unique 9-residue C-terminal sequence in the shortest isoform and the conserved, PDZ domain-containing N-terminal region of the long isoforms both contribute to the ability of myopodin to alter the response of PC3 cells to chemokinetic stimuli. Matrigel invasion assays also indicated that myopodin primarily affects the migration, rather than the invasion, properties of PC3 cells. The correlation between loss of myopodin expression and invasive prostate cancer therefore reflects complex myopodin interactions with pathways that regulate the cellular migration response to diverse signals that may be present in a tumor microenvironment.

The chemokine receptor CCR7 is essential for lymphocyte and dendritic cell homing to secondary lymphoid organs. Owing to the ability to induce directional migration, CCR7 and its ligands CCL19 and CCL21 are pivotal for the regulation of the immune system. Here, we identify a novel function for receptor ubiquitylation in the regulation of the trafficking process of this G-protein-coupled seven transmembrane receptor. We discovered that CCR7 is ubiquitylated in a constitutive, ligand-independent manner and that receptor ubiquitylation regulates the basal trafficking of CCR7 in the absence of chemokine. Upon CCL19 binding, we show that internalized CCR7 recycles back to the plasma membrane via the trans-Golgi network. An ubiquitylation-deficient CCR7 mutant internalized normally after ligand binding, but inefficiently recycled in immune cells and was transiently retarded in the trans-Golgi network compartment of HEK293 transfectants. Finally, we demonstrate that the lack of CCR7 ubiquitylation profoundly impairs immune cell migration. Our results provide evidence for a novel function of receptor ubiquitylation in the regulation of CCR7 recycling and immune cell migration.

Chemokines regulate T cell trafficking into secondary lymphoid organs and migration across endothelial cells in response to inflammatory signals. The small guanosine triphosphatase Rap1 is a critical regulator of chemokine signaling in T cells, but how chemokines activate Rap1 has been unclear. A study showed that Abl family tyrosine kinases were essential for chemokine-induced Rap1 activation, T cell polarization, and migration. Abl family kinases promoted Rap1 activation by phosphorylating the adaptor protein human enhancer of filamentation 1 (HEF1), thus establishing a critical Abl-HEF1-Rap1 signaling axis for chemokine-induced T cell migration.

We have previously demonstrated a role for the chemokines MCP-1, MIP-1α and GRO-α in directing subventricular zone (SVZ)-derived neural precursor cell migration towards the site of cell death in the adult rodent brain. However the influence of chemokines such as MCP-1, MIP-1α and GRO-α on the differentiation of adult neural precursor cells has not previously been investigated. Further, as the majority of neurological disorders and injuries occur during ageing, it is important to investigate the effect of chemokines on adult neural precursor cell cultures obtained from the ageing brain. This study therefore examined the effect of MCP-1, MIP-1α and GRO-α on SVZ-derived neural precursor cell differentiation in vitro, and assessed whether precursor cells from the middle-aged rat brain (13 months old) follow the same migratory and differential profile as neural precursor cells obtained from the young adult rat brain (2 months old). We observed that each of the chemokines examined generated differing effects in regards to neuronal or glial differentiation. Further, both MIP-1α and GRO-α increased total cell number, suggesting an effect on precursor cell proliferation and/or survival. In agreement with cultures obtained from young adult brains, SVZ-derived neural precursor cells cultured from the middle-aged brain exhibited chemotactic migration in response to a concentration gradient. These results indicate that the chemokines MCP-1, MIP-1α and GRO-α can influence both the migration and fate choice of SVZ-derived neural precursor cells, as well as promoting cell viability. While a response to each of these chemokines is maintained in the middle-aged brain, a distinct age-related alteration in differential fate can be identified., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rac1 and Rac2 GTPases transduce signals from multiple receptors leading to cell migration, adhesion, proliferation, and survival. In the absence of Rac1 and Rac2, B cell development is arrested at an IgD- transitional B cell stage that we term transitional type 0 (T0). We show that T0 cells cannot enter the white pulp of the spleen until they mature into the T1 and T2 stages, and that this entry into the white pulp requires integrin and chemokine receptor signaling and is required for cell survival. In the absence of Rac1 and Rac2, transitional B cells are unable to migrate in response to chemokines and cannot enter the splenic white pulp. We propose that loss of Rac1 and Rac2 causes arrest at the T0 stage at least in part because transitional B cells need to migrate into the white pulp to receive survival signals. Finally, we show that in the absence of Syk, a kinase that transduces B cell antigen receptor signals required for positive selection, development is arrested at the same T0 stage, with transitional B cells excluded from the white pulp. Thus, these studies identify a novel developmental checkpoint that coincides with B cell positive selection.

In this issue of Immunity, Shulman et al. (2009) describe shear-facilitated chemokine-induced adhesive filopodia on crawling T lymphocytes that scan the endothelial surface for potential sites of transendothelial migration. These filopodia facilitate a novel millipede-like mode of lymphocyte locomotion over endothelial cells prior to extravasation.

Background Aims: The transmigratory capacity of bone marrow (BM) mesenchymal stromal cells (MSC) through the endothelial cell barrier into various tissues and their differentiation potential makes them ideal candidates for cell therapy. Nevertheless, the mechanisms and agents promoting their migration are not fully understood. We evaluated the effects of several inflammatory cytokines on the migration of BM MSC and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) production., Methods: The migratory potential of BM MSC was evaluated using a Boyden chamber coated with Matrigel in the presence and absence of stromal cell-derived (SDF)-1alpha, platelet-derived growth factor (PDGF)bb, insulin-like growth factor (IGF)-I and interleukin (IL)-6. The ability of inflammatory cytokines to induce MSC migration was tested in presence of their respective Ab or blocking peptide. We used immunofluorescence to check the expression of cytokine receptors, and MMP/TIMP production was analyzed at the protein (human cytokine array, enzyme-linked immunosorbent assay (ELISA), gelatine zymography and Western blot) and mRNA quantitative real-time polymerase chain reaction (qRT-PCR) levels., Results: We have demonstrated that inflammatory cytokines promote the migratory capacity of BM MSC according to the expression of their respective receptors. Higher migration through Matrigel was observed in response to IL-6 and PDGFbb. qRT-PCR and cytokine array revealed that migration was the result of the variable level of MMP/TIMP in response to inflammatory stimuli., Conclusions: Our observations suggest that chemokines and cytokines involved in the regulation of the immunity or inflammatory process promote the migration of MSC into BM or damaged tissues. One of the mechanisms used by MSC to promote their migration though the extracellular matrix is modulation of the production of MMP-1, MMP-2, MMP-13, TIMP-1 and TIMP-2.

The efficient migration of mesenchymal stem cells (MSCs) to diseased tissues is required for the fulfillment of their regenerative potential. Recruitment of circulating cells into the damaged tissues is regulated by a complex network, which includes the non-neural cholinergic system. We found that human MSCs (hMSCs) express nicotinic acetylcholine receptor subunits alpha 7, beta 2 and beta 4. The receptor agonist nicotine caused calcium (Ca(++)) influx into hMSCs suggesting that the calcium ion channel alpha 7 homopolymer mediated this response. While high concentrations of nicotine (10(5)M) induced hMSC apoptosis, physiological concentrations (10(7)M) did not interfere with cell survival. At non-toxic concentrations, nicotine increased spontaneous migration of hMSCs, whereas chemotaxis of hMSCs toward C3a and bFGF in vitro and migration of intravenously infusion hMSCs into bone marrow and spleen in vivo were inhibited. The antagonist for the alpha 7 homopolymer, bungarotoxin, blocked the inhibitory effect of nicotine on chemotactic factor-induced migration of hMSCs. These findings reveal an involvement of the non-neural cholinergic system in regulation of hMSC migration.

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.

Trafficking of immune cells is controlled by directed migration of relevant cells toward chemotactic signals. Actin cytoskeleton undergoes continuous remodeling and serves as machinery for cell migration. The mDia family of formins and the Wiskott-Aldrich syndrome protein (WASP)-Arp2/3 system are two major actin nucleating-polymerizing systems in mammalian cells, with the former producing long straight actin filaments and the latter producing branched actin meshwork. Although much is known about the latter, the physiological functions of mDia proteins are unclear. We generated mice deficient in one mDia isoform, mDia1. Although mDia1(-/-) mice were born and developed without apparent abnormality, mDia1(-/-) T lymphocytes exhibited impaired trafficking to secondary lymphoid organs in vivo and showed reduced chemotaxis, little actin filament formation, and impaired polarity in response to chemotactic stimuli in vitro. Similarly, mDia1(-/-) thymocytes showed reduced chemotaxis and impaired egression from the thymus. These results suggest that mDia1 plays a distinct role in chemotaxis in T lymphocyte trafficking.

Resolvin E1 (RvE1) is a potent anti-inflammatory and proresolving mediator derived from omega-3 eicosapentaenoic acid generated during the resolution phase of inflammation. RvE1 possesses a unique structure and counterregulatory actions that stop human polymorphonuclear leukocyte (PMN) transendothelial migration and PMN infiltration in several murine inflammatory models. To examine the mechanism(s) underlying anti-inflammatory actions on PMNs, we prepared [(3)H]RvE1 and characterized its interactions with human PMN. Results with membrane fractions of human PMN demonstrated specific binding with a K(d) of 48.3 nM. [(3)H]RvE1 specific binding to human PMN was displaced by leukotriene B(4) (LTB(4)) and LTB(4) receptor 1 (BLT1) antagonist U-75302, but not by chemerin peptide, a ligand specific for another RvE1 receptor ChemR23. Recombinant human BLT1 gave specific binding with [(3)H]RvE1 with a K(d) of 45 nM. RvE1 selectively inhibited adenylate cyclase with BLT1, but not with BLT2. In human PBMC, RvE1 partially induced calcium mobilization, and blocked subsequent stimulation by LTB(4). RvE1 also attenuated LTB(4)-induced NF-kappaB activation in BLT1-transfected cells. In vivo anti-inflammatory actions of RvE1 were sharply reduced in BLT1 knockout mice when given at low doses (100 ng i.v.) in peritonitis. In contrast, RvE1 at higher doses (1.0 mug i.v.) significantly reduced PMN infiltration in a BLT1-independent manner. These results indicate that RvE1 binds to BLT1 as a partial agonist, potentially serving as a local damper of BLT1 signals on leukocytes along with other receptors (e.g., ChemR23-mediated counterregulatory actions) to mediate the resolution of inflammation.

Objective: Although several methods for the generation of dendritic cells (DCs) exist, little is known about the transmigration capacities of the cells developed. Their ability to migrate to the adjacent lymphatic system is relevant since their efficacy does also rely on their potential to interact with lymphocytes., Methods: We studied the transmigration of DCs derived from hematopoietic progenitor cells (HPC), from peripheral blood monocytes, and from leukemic cells. DCs from monocytes and leukemic cells could be generated within 1 week, whereas DCs from HPC needed 2 weeks for maturation., Results: While DCs from all sources showed similar morphologic features and allostimulatory capacities, their transmigration capacities varied: HPC-derived DCs showed the highest migratory response to macrophage inflammatory protein (MIP)-3alpha and beta. Monocyte-derived DCs were equally attracted to MIP-3beta and stroma-derived factor (SDF)-1alpha. Only few leukemic DCs migrated in response to SDF-1. Other chemoattractants tested included MIP-1alpha and RANTES. Replacement of fetal bovine by human serum did not change the DC's overall migratory capacities. It did, however, influence the responsiveness to certain chemokines., Conclusion: Although DCs from all three sources are immunocompetent antigen-presenting cells, our findings suggest that HPC and monocyte-derived DCs can be administered subcutaneously and intravenously, but that leukemic DCs should be injected into the lymph node.

We have earlier shown that the rat neural progenitor cell line HiB5 is capable of suppressing intracranial growth of glioma cells in Fisher rats. Unlike some neural progenitor cells, HiB5 cells have not shown homing capacity towards glioma cells growing intracranially. In this study, we have genetically modified HiB5 progenitor cells to over-express the chemokine receptor CXCR3. We show that the introduced receptor is functionally responding to ligand stimulation with increased phosphorylation levels of ERK and SAPK/JNK and a transcriptional response of an AP-1 reporter system introduced into HIB5 cells. These transfected progenitor cells migrate in vitro in response to IP-10 and I-TAC. Further, we show an enhanced in vivo migration of the CXCR3 transfected HiB5 cells over the corpus callosum towards an IP-10 and I-TAC expressing glioma, as compared to wild type HiB5 cells. Our data indicate that it is possible to take advantage of chemokines natural capacity to initiate migratory responses, and to use this ability to enhance tumor-inhibitory neural progenitor cells to target an intracranially growing glioma.

In humans, fetal cytotrophoblasts leave the placenta and enter the uterine wall, where they preferentially remodel arterioles. The fundamental mechanisms that govern these processes are largely unknown. Previously, we have shown that invasive cytotrophoblasts express several chemokines, as well as the receptors with which they interact. Here, we report that these ligand-receptor interactions stimulate cytotrophoblast migration to approximately the same level as a growth factor cocktail that includes serum. Additionally, cytotrophoblast commitment to uterine invasion was accompanied by rapid downregulation of EPHB4, a transmembrane receptor associated with venous identity, and upregulation of ephrin B1. Within the uterine wall, the cells also upregulated expression of ephrin B2, an EPH transmembrane ligand that is associated with arterial identity. In vitro cytotrophoblasts avoided EPHB4-coated substrates; upon co-culture with 3T3 cells expressing this molecule, their migration was significantly inhibited. As to the mechanisms involved, cytotrophoblast interactions with EPHB4 downregulated chemokine-induced but not growth factor-stimulated migration. We propose that EPHB4/ephrin B1 interactions generate repulsive signals that direct cytotrophoblast invasion toward the uterus, where chemokines stimulate cytotrophoblast migration through the decidua. When cytotrophoblasts encounter EPHB4 expressed by venous endothelium, ephrin B-generated repulsive signals and a reduction in chemokine-mediated responses limit their interaction with veins. When they encounter ephrin B2 ligands expressed in uterine arterioles, migration is permitted. The net effect is preferential cytotrophoblast remodeling of arterioles, a hallmark of human placentation.

Mast cells are the major effector cells for immediate hypersensitivity and chronic allergic reactions. These cells accumulate in mucosal tissues of allergic reactions, where immunoglobulin E (IgE) is produced locally. Here we provide evidence that, in addition to antigen that can attract IgE-bound mast cells, the type of IgE molecules that efficiently activate mast cells can promote the migration of mast cells in the absence of antigen. IgE- and IgE+Ag-mediated migration involves an autocrine/paracrine secretion of soluble factors including adenosine, leukotriene B4, and several chemokines. Their secretion depends on 2 tyrosine kinases, Lyn and Syk, and they are agonists of G-protein-coupled receptors and signal through phosphatidylinositol 3-kinase gamma, leading to mast cell migration. In mouse experiments, naive mast cells are attracted to IgE, and IgE-sensitized mast cells are attracted to antigen. Therefore, IgE and antigen are implicated in mast cell accumulation at allergic tissue sites with local high IgE levels.

Mobilization of endogenous endothelial progenitor cells (EPCs) from the bone marrow may be an alternative way to increase neovascularization and may be used as therapeutic option for the treatment of ischemic cardiovascular diseases. In this review, we discuss the EPC mobilizing effects of pro-inflammatory cytokines such as granolocyte monocyte colony-stimulating factor and granulocyte colony-stimulating factor, growth factors such as vascular endothelial growth factor, placental growth factor, erythropoietin, and angiopoietin-1, chemokines such as stromal cell-derived factor-1, hormones such as estrogens and lipid-lowering and anti-diabetic drugs, as well as physical activity.

The migration of tumor cells is a prerequisite for tumor cell invasion and metastasis development, which accounts for over 90% of cancer mortality. Therefore a major focus of current tumor biological research is the study of those factors that regulate tumor cell migration. Those chemokines and neurotransmitters that bind to G-protein coupled receptors (also known as serpentine receptors) are the most prominent of these factors. Neurotransmitters have been identified that have not only a stimulatory (e.g. norepinephrine) effect, but an inhibitory effect (e.g. GABA) as well. This is an especially fortuitous development, because many known agonists and antagonists of neurotransmitter receptors are currently being successfully used in the treatment of other pathological conditions (e.g. beta-blockers in the treatment of cardiovascular diseases). Likewise, chemokine receptor antagonists, which are under development for the treatment of HIV or rheumatoid arthritis, may be effective tools for the inhibition of chemokine-driven tumor cell migration as well. A further approach to inhibit tumor cell migration arises from the investigation of the relevant signal transduction pathways. The PKC alpha, for example, is a key enzyme in the regulation of tumor cell migration, but not of leukocyte migration. It thus offers a selective target opportunity for specific pharmacological agents to interfere with tumor cell migration. In this review we therefore summarize the current findings on those serpentine receptors involved in the neurotransmitter- and chemokine-regulated tumor cell migration, on the underlying signal transduction pathways, and on the opportunities to inhibit tumor cell migration and ultimately metastasis development with pharmaceutical agents.

Plasmacytoid dendritic cells (pDCs) recognize microbes, viruses in particular, and provide unique means of innate defense against them. The mechanism of pDC tissue recruitment remained enigmatic because the ligands of CXCR3, the cardinal chemokine receptor on pDCs, have failed to induce in vitro chemotaxis of pDCs in the absence of additional chemokines. In this study, we demonstrate that CXCR3 is sufficient to induce pDC migration, however, by a migratory mechanism that amalgamates the features of haptotaxis and chemorepulsion. To mediate "haptorepulsion" of pDCs, CXCR3 requires the encounter of its cognate ligands immobilized, optimally by heparan sulfate, in a form of a negative gradient. This is the first report of the absolute requirement of chemokine immobilization and presentation for its in vitro promigratory activity. The paradigmatic example of pDC haptorepulsion described here may represent a new pathophysiologically relevant migratory mechanism potentially used by other cells in response to other chemokines.

Cancer starts as a localised disease, which, if detected early, can often be treated successfully by removal of the primary tumour. A pernicious progression is the invasion of tumour cells into surrounding tissues, resulting in development of distant metastases. Because active migration of tumour cells is a prerequisite for tumour-cell invasion and metastasis, a pressing goal in tumour biology has been the elucidation of factors regulating the migratory activity of these cells. The most prominent regulatory factors are ligands to serpentine receptors-eg, chemokines and neurotransmitters. Many types of neurotransmitter receptors are expressed on tumour cells, supporting the theory that psychosocial factors are involved in the progression of cancer. Understanding how such receptors regulate migration and the availability of specific receptor antagonists could open up new avenues for chemoprevention of tumour-cell migration and metastatic development.

Development of allergic contact dermatitis to haptens depends upon a balance between CD8(+) T lymphocytes with pathogenic activity and CD4(+) T cells, which comprise both effector and regulatory cells. Thus, differential recruitment of CD8(+) and CD4(+) lymphocytes to sites of hapten challenge may have considerable impact on disease expression. Here the migration of cutaneous lymphocyte-associated antigen+, nickel-specific CD8(+) and CD4(+) T cell lines were compared with a panel of chemokines produced in the skin during allergic contact dermatitis. CCL17/TARC and CCL22/MDC induced a 3-fold higher migration of CD4(+) compared with CD8(+) lymphocytes. In contrast, CXCL10/IP-10 was 2-fold more potent in attracting CD8(+) cells. These findings were consistent with the higher expression of CCR4 and CXCR3 on CD4(+) and CD8(+) T cell lines, respectively. Moreover, CCR4 expression was high on nickel-specific T helper 2, intermediate on T helper 1 and T cytotoxic 2, and almost undetectable on T cytotoxic 1 clones. On the contrary, CXCR3 was expressed by T cytotoxic 1 and 2 and T helper 1, but not T helper 2 clones. Reverse transcription-polymerase chain reaction analysis of the skin before and after hapten challenge revealed the constitutive presence of TARC, and the early appearance of CCL2/MCP-1, followed by IP-10, CCL4/MIP-1beta, and MDC mRNA. Supernatants from activated keratinocytes induced a strong migration of CD8(+) lymphocytes, which was blocked by neutralization of IP-10. Conversely, supernatants from immature and mature dendritic cells attracted mostly CD4(+) lymphocytes in a TARC- and MDC-dependent manner. Our data indicate that distinct chemokines and cell types control the accumulation of CD8(+) and CD4(+) T cells within inflamed skin.

Stem cells within the bone marrow (BM) exist in a quiescent state or are instructed to differentiate and mobilize to circulation following specific signals. Matrix metalloproteinase-9 (MMP-9), induced in BM cells, releases soluble Kit-ligand (sKitL), permitting the transfer of endothelial and hematopoietic stem cells (HSCs) from the quiescent to proliferative niche. BM ablation induces SDF-1, which upregulates MMP-9 expression, and causes shedding of sKitL and recruitment of c-Kit+ stem/progenitors. In MMP-9-/- mice, release of sKitL and HSC motility are impaired, resulting in failure of hematopoietic recovery and increased mortality, while exogenous sKitL restores hematopoiesis and survival after BM ablation. Release of sKitL by MMP-9 enables BM repopulating cells to translocate to a permissive vascular niche favoring differentiation and reconstitution of the stem/progenitor cell pool.

At the time of organ transplantation, a variety of non-parenchymal cells are transplanted simultaneously with the allograft. Recognition of the importance of these cells as potential immunostimulatory cells lead to the concept of 'passenger leukocytes' as the principal instigators of rejection. Passenger leukocytes include interstitial dendritic cells (DCs) and blood-derived monocytes/macrophages. As investigators have discovered the significance of DCs in influencing graft outcome, so have they begun to determine the best ways to influence DCs themselves. This review discusses the role of DCs in transplantation and then focuses on three different approaches for manipulating DCs to improve allograft survival: (1) targeting of chemokines involved in DC migration, (2) pharmacological arrest of DC maturation, and (3) genetic engineering of DCs.

Niuhuang is a commonly used Chinese traditional medicine with immunoregulatory and anti-inflammatory properties. Deoxycholic acid (DCA) is a major active constituent of Niuhuang. The reaction of human leukocytes to chemoattractants is an important part of the host immune response and also plays a crucial role in the development of inflammation. We, therefore, investigated the in vitro effects of DCA on human monocyte and neutrophil responses to classic chemoattractants [fMet-Leu-Phe (fMLP), complement fraction 5a (C5a)], CC chemokine [monocyte chemoattractant protein-1 (MCP-1/CCL2)], and/or CXC chemokines [stromal cell-derived factor-1 (SDF-1alpha/CXCL12), interleukin-8 (IL-8/CXCL8)]. The results showed that DCA significantly inhibited fMLP-induced monocyte and neutrophil chemotaxis and calcium mobilization, and also blocked the binding of [3H]fMLP and anti-formyl peptide receptor (FPR) monoclonal antibodies (mAb) to the cells. The inhibitory effects of DCA on calcium mobilization and anti-FPR-mAb binding to the receptor could be abrogated by washing DCA out of the cell suspension, suggesting that DCA blocked fMLP receptors via a steric hindrance mechanism, not via receptor internalization. DCA had no significant inhibitory effects on MCP-1-, SDF-1alpha-, or C5a-induced monocyte function, or C5a- or IL-8-induced neutrophil function. Taken together, our experimental results suggest that blockade of fMLP receptors may contribute to the anti-inflammatory effects of traditional medicine containing DCA.

The hematopoietic stem cell (HSC) is an attractive target for gene therapy of genetic diseases of the immune and hematopoietic system, and for drug-resistance strategies in which genes conferring resistance to a variety of chemotherapeutic agents can be transduced. Stem cells are relatively easy to obtain; e.g., by marrow aspiration or G-CSF mobilization into the peripheral blood, and can be enriched e.g., by the use of anti-CD34 + monoclonal antibody. For conventional retroviral transduction, normally quiescent HSC must be activated into the cell cycle by priming with appropriate cytokines, and it has been critical to identify cytokine combinations that preserve the self-renewal capacity of long-term repopulating HSC. It has become apparent that strategies designed to optimize HSC cycling and proviral integration can compromise the capacity of transduced HSC to compete in vivo against endogenous HSC or HSC that have not been activated into cell cycle. Lentiviral vectors can integrate genes into non-cycling cells but there is an increased efficiency of transduction if Go HSC are activated into G1-phase of the cell cycle. This reduced efficiency of long-term engraftment of ex vivo cultured HSC may be due to impaired self-renewal capacity or reduced marrow homing efficiency. The latter may be attributed to down modulation of chemokine receptors necessary for chemotactic homing to the marrow. Alternatively, or in addition, there may be down modulation of (1) HSC adhesion molecules necessary for endothelial adhesion and egress from the circulation: (2) metalloproteinases secreted by HSC that facilitate their migration through extracellular matrix and promote release of critical soluble regulatory factors in the marrow microenvironment. A more controversial view is that cell death pathways, for example those involving FasR (CD95) may be activated in cycling HSC, resulting in their selective destruction upon transplantation and localization to sites rich in Fas ligand such as the liver.

Endothelial cells of the blood-brain barrier (BBB) have the ability to regulate and restrict the passage of cells and molecules from the periphery to the CNS. We have used an in vitro assay of lymphocyte migration across monolayers of human adult brain endothelial cells (HBEC) as a model of lymphocyte migration across the BBB. We found that human allogeneic or MBP-reactive Th2-polarized lymphocytes migrate more avidly than Th1-polarized lymphocytes. Migration of Th2 but not Th1 cells across brain endothelium was inhibited by antibodies directed at MCP-1, a chemokine produced by HBECs. We could detect CCR2, a chemokine receptor that recognizes MCP-1 on Th2 but not Th1 lymphocytes. ICAM-1 and VCAM-1 molecules were expressed on the surface of HBECs under basal conditions and were upregulated by Th1 but not Th2 cell-derived supernatants. Migration of both lymphocyte subsets was dependent on LFA-1/ICAM-1 interactions. Blocking VLA-4/VCAM-1 binding did not influence actual trans-endothelial migration. These results suggest that HBECs composing the BBB favor the migration of Th2 cells. We postulate that this selectivity may help prevent activated Th1 lymphocytes, the putative CNS autoimmune disease initiating cells, from reaching the CNS parenchyma and favor entry of Th2 cells, a putative means to induce bystander suppression in the CNS.

Recent reports revealed that myogenic progenitors, derived from either bone marrow or muscle can migrate into muscle tissue and participate in myofiber regeneration, when injected in the peripheral circulation. This observation might open a new strategy for the treatment of muscular dystrophies. The signals involved in myoblast recruitment from circulation are at present poorly understood. To investigate myoblast migration we used a transwell assay in which murine myoblasts and myogenic cell lines were seeded on microporous membrane covered by an endothelial monolayer and chemotactic factors were added in the lower chamber. We demonstrated that myoblasts are able to cross the endothelium and that this process can be modulated. In particular among tested factors, we observed a gradient of chemotactic activity as follows: HGF >> RANTES > PDGF-A > PDGF-B > FGF >> TNF-alpha > IFN-gamma > EGF. Endothelial and myoblast expression of Pax3 (a transcription factor expressed by embryonic migrating myogenic cells) and cytokine transcripts (TNF-alpha, IFN-gamma) was also monitored either at the basal level and after transmigration. We observed increased Pax3 expression after interaction of C2C12 myoblasts with endothelial cells. We consider that any new report elucidating the molecular signals involved in myoblast migration may be useful toward the development of systemic cellular-mediated gene therapy of muscle diseases., (Copyright 2001 Academic Press.)

Eosinophils play a central role in the pathophysiology of allergic disease. The mechanisms that regulate eosinophil migration are complex; however, chemokines and cytokines produced in both the early and late phases of the asthmatic response appear to cooperate in eosinophil recruitment. In particular, there exists a unique synergy between eotaxin and IL-5. The role of chemokine/cytokine cooperativity has been investigated in the extracellular matrix, adhesion molecule/integrin interactions, receptor polarization and aggregation and the convergence and divergence of intracellular signalling pathways. Understanding the mechanisms whereby eosinophils migrate will allow the development of specific therapeutic strategies aimed at attenuating specific components of the allergic response.

Opioid users having acquired human immunodeficiency syndrome (AIDS) are at a greater risk than non-users of contracting opportunistic infections. Opioid-administered and simian immunodeficiency virus (SIV)-infected rhesus monkeys have been an excellent model for studying AIDS and drug abuse in humans. In this study, chemotaxis of monkey leukocytes was evaluated using the chemokines interleukin-8 (IL-8) and regulated upon activation, normal T cell expressed (RANTES) as the chemoattractants, and the effects of various opioid agonists and antagonists on the efficiency of chemotaxis were examined. Opioids were either incubated with monkey leukocytes or added directly to chemokines, and the number of cells migrating toward IL-8 (for neutrophils) or RANTES (for monocytes) was scored. Inhibition of chemotaxis was seen with both assay conditions, and the inhibition was mediated by opioids binding to mu or kappa receptors. Binding to delta opiod receptors was rarely, if ever, observed. Although opioids themselves may act as weak chemoattractants for monkey leukocytes, addition of opioid agonists to chemokines would reduce the chemoattractant ability of the chemokines. Opioids did not cause the same inhibitory effect on the chemotactic migration of neutrophils when the complement component C5a or the chemotactic peptide N-formyl-MET-LEU-PHE (fMLP) was used as chemoattractant. These studies suggest that the presence of opioids during SIV infection immediately alters chemokine-mediated immune functions.

The blood - brain barrier (BBB) plays a crucial role in central nervous system (CNS) homeostasis. Serving as the brain's protective shield it regulates soluble factor and cellular exchanges from blood to brain. Critical to its function, the BBB is composed of brain microvascular endothelial cells (BMVEC), a collagen matrix, and astrocytes. Astrocytic endfeet surround the BMVEC abluminal surface and influence the 'tightness' and trafficking role of the barrier. In neurodegenerative disorders (for example stroke, multiple sclerosis and HIV encephalitis) the BBB becomes compromised. This is, in part, immune mediated. An accumulating body of evidence demonstrates that the cellular components of the BBB are themselves immunocompetent. Perivascular cells (astrocytes, macrophages and microglial cells) and BMVEC produce inflammatory factors that affect BBB permeability and expression of adhesion molecules. These affect cell trafficking into the CNS. Leukocyte BBB migration can be influenced by cytokines and chemokines produced by glia. Astrocytes and macrophages secrete a multitude of factors that affect brain immune responses. Interactions between BMVEC, leukocytes and/or glia, immunological activation and noxious (infectious, toxic and immune-mediated) brain insults all appear to play important roles in this BBB cell trafficking. New information gained into the mechanisms of leukocyte-brain penetration may provide novel insights in the pathogenesis and treatment strategies of neurodegenerative disorders.

Microglia, the resident macrophages of the central nervous system, are the primary cells to respond to injury in the brain, both in inflammation, e.g., in multiple sclerosis, and trauma. Chemokines are potential mediators of microglial cell recruitment to sites of injury; thus, the ability of microglia to migrate in response to a number of chemokines was assessed. The chemokines monocyte chemoattractant protein 1, macrophage inflammatory protein 1alpha, macrophage inflammatory protein 1beta, RANTES (regulated upon activation normal T cell expressed and secreted), interleukin 8, and IP-10 (interferon gamma inducible protein-10), induce migration and changes in the distribution of f-actin in adult rat microglia and a human microglial cell line, CHME3, in vitro. Both cell types show a significant migration response, above control levels, to all the chemokines tested in a typical dose-dependent manner. These chemokines also induced a reorganization of the actin cytoskeleton of the cells. This study indicates that chemokines play an important role in the recruitment of microglia to areas of central nervous system inflammation.

DCs (dendritic cells) function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. They then leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. This suggestive link between DC traffic pattern and functions led us to investigate the chemokine responsiveness of DCs during their development and maturation. DCs were differentiated either from CD34(+) hematopoietic progenitor cells (HPCs) cultured with granulocyte/macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-alpha or from monocytes cultured with GM-CSF plus interleukin 4. Immature DCs derived from CD34(+) HPCs migrate most vigorously in response to macrophage inflammatory protein (MIP)-3alpha, but also to MIP-1alpha and RANTES (regulated on activation, normal T cell expressed and secreted). Upon maturation, induced by either TNF-alpha, lipopolysaccharide, or CD40L, DCs lose their response to these three chemokines when they acquire a sustained responsiveness to a single other chemokine, MIP-3beta. CC chemokine receptor (CCR)6 and CCR7 are the only known receptors for MIP-3alpha and MIP-3beta, respectively. The observation that CCR6 mRNA expression decreases progressively as DCs mature, whereas CCR7 mRNA expression is sharply upregulated, provides a likely explanation for the changes in chemokine responsiveness. Similarly, MIP-3beta responsiveness and CCR7 expression are induced upon maturation of monocyte- derived DCs. Furthermore, the chemotactic response to MIP-3beta is also acquired by CD11c+ DCs isolated from blood after spontaneous maturation. Finally, detection by in situ hybridization of MIP-3alpha mRNA only within inflamed epithelial crypts of tonsils, and of MIP-3beta mRNA specifically in T cell-rich areas, suggests a role for MIP-3alpha/CCR6 in recruitment of immature DCs at site of injury and for MIP-3beta/CCR7 in accumulation of antigen-loaded mature DCs in T cell-rich areas.

Chemokines play a central role in immune and inflammatory responses. It has been observed recently that certain viruses have evolved molecular piracy and mimicry mechanisms by encoding and synthesizing proteins that interfere with the normal host defense response. One such viral protein, vMIP-II, encoded by human herpesvirus 8, has been identified with in vitro antagonistic activities against CC and CXC chemokine receptors. We report here that vMIP-II has additional antagonistic activity against CX3CR1, the receptor for fractalkine. To investigate the potential therapeutic effect of this broad-spectrum chemokine antagonist, we studied the antiinflammatory activity of vMIP-II in a rat model of experimental glomerulonephritis induced by an antiglomerular basement membrane antibody. vMIP-II potently inhibited monocyte chemoattractant protein 1-, macrophage inflammatory protein 1beta-, RANTES (regulated on activation, normal T cell expressed and secreted)-, and fractalkine-induced chemotaxis of activated leukocytes isolated from nephritic glomeruli, significantly reduced leukocyte infiltration to the glomeruli, and markedly attenuated proteinuria. These results suggest that molecules encoded by some viruses may serve as useful templates for the development of antiinflammatory compounds.

Chemokines have been shown to chemoattract and activate different leukocyte populations. Here we report the in vitro effect of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation, normal T-cells, expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), interferon inducible protein-10 (IP-10), neutrophil-activating peptide-2 (NAP-2), growth-related protein (GRO)-alpha and GRO-gamma, on the migration of 3 human breast carcinoma cell lines, MCF-7, T47D and ZR-75-1, using a microchemotaxis chamber to assess migration across fibronectin-coated polycarbonate membranes. MCF-7 cells responded chemotactically to all chemokines tested in a pattern which was dose and time dependent, although responses to the different chemokines were variable. ZR-75-1 responded to MIP-1beta and GRO-alpha, giving maximum migration indices of 3.7 and 5.3, respectively, and exhibited a migratory response to MIP-1alpha, IL-8 and MCP-1 although to a lower degree. T47D was unresponsive to the chemokines tested, but both MCF-7 and T47D cells bound radiolabelled ligands with binding constants (Kd) ranging from 0.6 to 2.2 nM and 0.6 to 2.1 nM, respectively. The specificity of the chemotactic response of MCF-7 to MIP-1alpha and MIP-1beta was confirmed using chemokine-specific neutralising antibodies and heat denaturation, and was demonstrated to involve G protein and cyclic AMP signalling pathways. MIP-1beta and MIP-1alpha were shown to induce changes in the organisation of the actin cytoskeleton and the level of F-actin in MCF-7 cells, as determined using flow cytometric analysis and confocal microscopy. Our results show that breast carcinoma cells can respond to chemokines, and suggests a potential role for these molecules in the process of tumour cell migration, invasion and metastasis.

Chemokines are a family of chemotactic cytokines which attract different types of leukocytes. This property, combined with some additional inflammatory and growth-regulatory activities, demonstrate their crucial role in the immune system. Chemokines are low molecular weight proteins and possess a typical positioning of four conserved cysteines. This family is further subdivided in two subfamilies depending on whether the first two cysteines are adjacent or not (CC and CXC chemokines, respectively). The CXC chemokines (including interleukin-8) predominantly attract neutrophils, whereas CC chemokines induce migration of monocytes, as well as other leukocyte cell types. In this article, the general characteristics of chemokines are reviewed. Furthermore, the murine CC chemokines, JE/MCP-1, MCP-3/MARC, MIP-1 alpha, MIP-1 beta, RANTES, TCA3, C10/MRP-1, MRP-2, and eotaxin, are discussed more in detail.

Previous studies from this laboratory have demonstrated that the chemokines RANTES (recombinant human regulated upon activation, normally T cell expressed and presumably secreted), macrophage chemotactic peptide-1, recombinant human macrophage inflammatory protein-1 alpha (rhMIP-1 alpha) IL-8, and IP-10 are capable of inducing human T cell infiltration into the injection site of severe combined immunodeficiency (SCID) mice reconstituted with human PBL. However, the ability of these chemokines to facilitate T cell homing into various lymphoid tissues has not been examined. Initial studies focused on the ability of rhMIP-1 beta to induce human T cell infiltration into injection sites in human PBL-SCID mice. SCID mice received s.c. injections of rhMIP-1 beta or PBS (1 microgram/injection) in the hindflank for 4 h or sequential injections for 3 days. Biopsies of the MIP-1 beta injection site revealed the presence of significant mononuclear cell accumulation 72 h after injection. Immunohistologic evaluation determined that significant numbers of human CD3+ T cells were recruited in response to MIP-1 beta injections, and this infiltration could be specifically blocked by co-administration of anti-MIP-1 beta antiserum. We subsequently examined these chemokine-injected mice for the effect of trafficking of human T cells to peripheral lymphoid organs. Flow cytometric analysis of the thymus in human PBL-SCID mice revealed that treatment with rhMIP-1 beta or rhRANTES, but not platelet factor-4, resulted in improved thymic homing of the human T cells after 72 h. This trafficking effect was shown to be direct, as pretreatment of the human T cells with the chemokines in vitro also improved peripheral lymphoid trafficking of the human cells. In addition, co-injection of rhMIP-1 beta with anti-1 beta antiserum abrogated the increase in T cell homing to the thymus. These data demonstrate that MIP-1 beta and RANTES directly augment human T cell trafficking to peripheral murine lymphoid tissues. Chemokines may, therefore, under either isogeneic or xenogeneic conditions, play a role in normal lymphocyte recirculation and homing, and may be of potential clinical use in promoting immune cell trafficking and function.

Chemokines have been shown to play an important role in both the adhesion and migration of numerous leukocytic cell types, including granulocytes, monocytes, mast cells, and T lymphocytes. However, the biologic effects of chemokines on NK cells remain to be defined. Chemotaxis studies using purified human NK cells and a panel of human recombinant chemokines revealed that macrophage inflammatory protein (MIP)-1 alpha and IFN-inducible protein-10 (IP-10) are potent NK cell chemoattractants in vitro. Modest but significant chemotactic (not chemokinetic) responses were also observed in response to RANTES, MCP-1, MCP-2, MCP-3, and MIP-1 beta. Chemokine receptor expression on human NK cells was determined through displacement and Scatchard analyses, using a panel of radiolabeled chemokines, and revealed the presence of both distinct and shared chemokine receptors with affinities similar to those previously described for other cell types. Functional studies have also revealed that the beta chemokines and IP-10 are capable of augmenting NK- but not LAK- or ADCC-specific cytolytic responses in both a dose- and donor-dependent fashion. Neutralization analysis using Abs specific for various adhesion molecules revealed that NK:tumor cell conjugate formation is required for chemokine-induced NK killing. In addition, NK cells incubated in the presence of beta chemokines and IP-10 for 4 h induced the release of granule-derived serine esterases, suggesting a possible mechanism for chemokine-mediated NK killing. These results suggest that chemokines not only play an important role in the recruitment of NK cells, but also may be important mediators of NK cell degranulation augmenting local tumor cell destruction.