Profiling heparin-chemokine interactions using synthetic tools.

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity.     

Chemokines and chemokine receptors in renal transplantation--from bench to bedside

Attraction of mononuclear cells to sites of inflammation requires a close interplay of the inflammatory signal presented via chemokines and specific receptors on effector cells. First studies on acute renal transplant rejection demonstrated the involvement of CC-chemokines, such as RANTES, MIP-1alpha, MIP-1beta and MCP-1, as well as CXC-chemokines such as IL-8 and IP-10, correlating with expression of the corresponding chemokine receptors, CCR1, CCR5 and CCR2 as well as CXCR3. Since then, the pathophysiologic relevance has been extended to chronic allograft nephropathy and transplant glomerulopathy. Chemokine expression can be triggered by different stimuli, e.g. brain death, ischemia, HLA-mismatch and infection. Furthermore, anti-inflammatory chemokines have been identified. Chemokine receptor 7, e.g. enhances homing of lymphocytes to lymphatic tissues and the Duffy antigen receptor, DARC, a non-specific receptor that binds and inactivates different chemokines. While measurement of chemokine expression in clinical transplantation may facilitate the differential diagnosis of allograft dysfunction, knowledge of the chemokine network has also widened the understanding of transplant rejection and opened novel therapeutic approaches. Observations from humans with mutations of the chemokine network as well as transplantation of animals with targeted deletions in this system suggest that manipulations of chemokine signalling may improve the success rates of transplantation. Blocking chemokines unselectively with Met-RANTES or specifically with small molecule inhibitors of various chemokine receptors has lead to improved outcome in animal models. Currently, first human trials are under way to investigate drugs that stimulate lymphocyte homing. Inhibitors of CCR1 and CCR5 are being tested for other human diseases and may eventually be available in transplantation. Nonetheless, chemokine blockade my rather serve as an adjunct in the management of transplant recipients than a new "magic bullet".     

Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood-brain barrier

Leukocyte transmigration across the blood-brain barrier (BBB) is a multistep process that can be mediated by chemokines. These low-molecular-weight chemoattractant proteins are secreted by cells within the central nervous system (CNS) in response to injury or on activation. Leukocytes transmigrate toward this chemokine gradient, crossing the BBB and gaining access to the CNS parenchyma. Depending on the chemokine, the nature of the insult, and the type of cell that transmigrates, the BBB integrity may be disrupted, leading to its increased permeability. Both the inflammation resulting from leukocyte transmigration and BBB perturbations contribute to CNS pathology. The mechanisms that mediate leukocyte transmigration and BBB disruption, as well as tissue culture models that are used to study leukocyte trafficking, are the focus of this review.     

Evasion and exploitation of chemokines by viruses

Chemokines and chemokine receptors play a critical role in the host defense against viruses by mobilizing leukocytes to sites of infection, injury and inflammation. In order to replicate successfully within their host organisms, viruses have devised novel strategies for exploiting or subverting chemokine networks. This review summarizes various mechanisms that are currently known to be used by viruses for modulating chemokine activities including viral homologs of chemokines and chemokine receptors and soluble viral chemokine binding proteins. Insight into these strategies is providing a wealth of information on viral-host interactions, the function of chemokines in host defense and may help to generate novel anti-chemokine agents for treating against viral diseases or inflammatory disorders.     

Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood–brain barrier

Leukocyte transmigration across the blood–brain barrier (BBB) is a multistep process that can be mediated by chemokines. These low-molecular-weight chemoattractant proteins are secreted by cells within the central nervous system (CNS) in response to injury or on activation. Leukocytes transmigrate toward this chemokine gradient, crossing the BBB and gaining access to the CNS parenchyma. Depending on the chemokine, the nature of the insult, and the type of cell that transmigrates, the BBB integrity may be disrupted, leading to its increased permeability. Both the inflammation resulting from leukocyte transmigration and BBB perturbations contribute to CNS pathology. The mechanisms that mediate leukocyte transmigration and BBB disruption, as well as tissue culture models that are used to study leukocyte trafficking, are the focus of this review.     

CC类趋化因子亚家族与心肌梗死的研究进展

CC类趋化因子亚家族是趋化因子家族中成员最多、研究最广泛的一大类细胞因子,其主要功能参与炎症细胞激活、迁移、粘附等病理生理过程。大量研究表明,CC类趋化因子亚家族成员参与了心肌梗死后病理过程的各个阶段。其中研究最为深入的为单核细胞趋化蛋白-1（monocyte chemoattractant protein-1,MCP-1）及其受体CC趋化因子受体2（CC chemokine receptor 2,CCR2）,在心肌梗死后炎症期、增殖期及疤痕愈合期都发挥了重要作用从而影响梗死后心室重构。近年来,CC类趋化因子亚家族其他成员亦被逐渐揭示参与了心肌梗死的发展。本文结合以往大量文献将对CC类趋化因子亚家族在心肌梗死各个阶段中尤其是梗死后各期对于心室重构的影响进行综述,以期为今后的实验研究提供方向及疾病的预防和治疗提供药物靶点。     

The chemokine system and its role in obesity

The chemokine system is a complex arrangement of molecules that attract leukocytes to the site of injury or inflammation. This chemotactic behavior gives the system the name “Chemokine.” The intricate and redundant nature of the chemokine system has made it a subject of ongoing scientific investigation. Obesity is characterized as low-grade systemic or chronic inflammation that is responsible for the release of cytokines, adipokines, and chemokines. Excessive tissue fat expansion triggers the release of chemokines, which in turn attract various leukocytes and activate the resident immune surveillance system, eventually leading to worsening of obesity and other related comorbidities. To date, 50 chemokines and 20 chemokine receptors that belong to the G-protein-coupled receptor family have been discovered, and over the past two decades, the physiological and pathological roles of many of these chemokines and their receptors have been elucidated. The objective of this review is to present an update on the link between chemokines and obesity under the light of recent knowledge.     

趋化因子对肿瘤生长的影响

虽然趋化因子早先被认为主要影响炎症和造血过程时白细胞的迁移,但现在有大量的事实显示它们也影响许多肿瘤过程,像白细胞浸润、血管生成、肿瘤细胞生长、存活、浸润和转移。控制肿瘤细胞内趋化因子网将为肿瘤治疗提供一种选择。     

Expression of chemokine receptors by lung T cells from normal and asthmatic subjects

The lung is an important tertiary lymphoid organ with constant trafficking of T cells through the lung in both health and disease. T cell migration is controlled by a combination of adhesion receptors and chemokines expressed on vascular endothelium and in the tissue, often in an organ-specific manner. This leads to selective accumulation of different T cell subsets, a process called lymphocyte homing. There is evidence for a distinct lung-homing pathway, but no specific lung-homing receptors have been described. We analyzed the chemokine receptor profile of lung T cells to determine the extent to which lung T cells shared homing pathways with other organs such as the gut and skin. In addition, we compared expression of receptors in normal and asthmatic individuals to determine whether different pathways were used in health and disease. We observed that lung T cells expressed a profile of chemokine and adhesion receptors distinct from that of gut- and skin-homing T cells although no chemokine receptor specific for the lung was found. In particular, lung T cells expressed CCR5 and CXCR3, but not CCR9 or cutaneous lymphocyte Ag, and only low levels of CCR4 and alpha(4)beta(7). No differences were observed between lung T cells from normal vs asthmatic subjects. This study provides added support for the concept of a lung-homing pathway separate from other mucosal organs such as the gut and suggests that the chemokine pathways that control T cell migration in normal homeostasis and Th2-type inflammatory responses are similar.     

Lungkine, a novel CXC chemokine, specifically expressed by lung bronchoepithelial cells

We describe a novel mouse CXC chemokine that is selectively expressed in lung epithelial cells and up-regulated in various lung inflammation models. Although this chemokine clusters with other ELR-CXC chemokines, none of them can confidently be assigned to be its human homologue based on sequence identity. In addition, the highly restricted mRNA tissue distribution of this chemokine differentiates it from all previously described chemokines: Lungkine could not be detected in any of the 70 cDNA libraries analyzed corresponding to specific murine cell populations and tissues. High levels of Lungkine mRNA were specifically detected in the lung and at lower levels in fetal lung tissue by Northern blot and in situ hybridization, suggesting a potential role for this chemokine during lung development. Moreover, Lungkine protein is secreted into the airway spaces and induces the in vitro and in vivo migration of neutrophils, suggesting that it is involved in lung-specific neutrophil trafficking. Using fluorescent in situ hybridization, we show that Lungkine maps to mouse chromosome 5.     

Plasma-cell homing

Recent studies indicate that chemoattractant cytokines (chemokines), together with tissue-specific adhesion molecules, coordinate the migration of antibody-secreting cells (ASCs) from their sites of antigen-driven differentiation in lymphoid tissues to target effector tissues. Developing ASCs downregulate the expression of receptors for lymphoid tissue chemokines and selectively upregulate the expression of chemokine receptors that might target the migration of IgA ASCs to mucosal surfaces, IgG ASCs to sites of tissue inflammation and both types of ASC to the bone marrow - an important site for serum antibody production. By directing plasma-cell homing, chemokines might help to determine the character and efficiency of mucosal, inflammatory and systemic antibody responses.     

Cutting edge: selective usage of chemokine receptors by plasmacytoid dendritic cells

The existence of dendritic cell (DC) subsets is firmly established, but their trafficking properties are virtually unknown. In this study, we show that myeloid (M-DCs) and plasmacytoid (P-DCs) DCs isolated from human blood differ widely in the capacity to migrate to chemotactic stimuli. The pattern of chemokine receptors expressed by blood M-DCs and P-DCs, with the exception of CCR7, is similar. However, most chemokine receptors of P-DCs, in particular those specific for inflammatory chemokines and classical chemotactic agonists, are not functional in circulating cells. Following maturation induced by CD40 ligation, the receptors for inflammatory chemokines are down-regulated, and CCR7 on P-DCs becomes coupled to migration. The drastically impaired capacity of blood P-DCs to migrate in response to inflammatory chemotactic signals contrasts with the response to lymph node-homing chemokines, indicating a propensity to migrate to secondary lymphoid organs rather than to sites of inflammation.     

The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory protein-3 beta are potent chemoattractants for in vitro- and in vivo-derived dendritic cells

Dendritic cell migration to secondary lymphoid tissues is critical for Ag presentation to T cells necessary to elicit an immune response. Despite the importance of dendritic cell trafficking in immunity, at present little is understood about the mechanisms that underlie this phenomenon. Using a novel transwell chemotaxis assay system, we demonstrate that the CC chemokine receptor-7 (CCR7) ligands 6Ckine and macrophage inflammatory protein (MIP)-3 beta are selective chemoattractants for MHC class IIhigh B7-2high bone marrow-derived dendritic cells at a potency 1000-fold higher than their known activity on naive T cells. Furthermore, these chemokines stimulate the chemotaxis of freshly isolated lymph node dendritic cells, as well as the egress of skin dendritic cells ex vivo. Because these chemokines are expressed in lymphoid organs and 6Ckine has been localized to high endothelial venules and lymphatic endothelium, we propose that they may play an important role in the homing of dendritic cells to lymphoid tissues.     

High level expression, activation, and antagonism of CC chemokine receptors CCR2 and CCR3 in Chinese hamster ovary cells

The specificity of leukocyte trafficking in inflammation is controlled by the interactions of chemokines with chemokine receptors. Reliable structure-function studies of chemokine-receptor interactions would benefit from cell lines that express consistent high levels of chemokine receptors. We describe herein two new Chinese hamster ovary (CHO) cell lines in which the genes for chemokine receptors CCR2 and CCR3 have been incorporated into identical positions in the host genome. CCR2 is the primary receptor for the chemokine monocyte chemoattractant protein-1 (MCP-1) whereas CCR3 is the primary receptor for the chemokines eotaxin-1, eotaxin-2 and eotaxin-3. Both receptors are expressed at >5,000,000 copies per cell, substantially higher levels than in previous cell lines, and both are competent for binding and activation by the cognate chemokines for these receptors. Using these cell lines we confirm that eotaxin-1 and eotaxin-3 can act as an agonist and an antagonist, respectively, of CCR2. In addition, we show that eotaxin-2 is an antagonist of CCR2 and MCP-1 is an agonist of CCR3. Comparison of the chemokine sequences reveals several positions that are identical in MCP-1 and eotaxin-1 but different in eotaxin-2 and eotaxin-3, suggesting that these amino acids play a role in CCR2 activation.     

Maltose binding protein facilitates high-level expression and functional purification of the chemokines RANTES and SDF-1alpha from Escherichia coli

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1α (stromal cell-derived factor-1α) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1α fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP–RANTES and MBP–SDF-1α was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1α.     

Abundant expression of chemokines in malignant and infective human lymphadenopathies.

Lymph nodes can be the primary target of infection or malignant transformation and may exhibit characteristic patterns of leukocyte infiltration analogous to those seen in inflammation of other tissues. Leukocyte migration to lymph nodes in vivo is a highly regulated, multi-step process that depends upon adhesion molecules and as yet, uncharacterized chemotactic signals. Chemokines are a key part of the orchestrated code of signals that directs leukocyte subsets to sites of inflammation or immune response. The potential role of these chemoattractants in selective trafficking of leukocyte subsets into lymph nodes was assessed by determining the expression of chemokines on a range of pathological and normal human lymph nodes and by evaluating the cellular composition of each lymph node. In situ hybridization using chemokine riboprobes and immunohistochemistry using specific antibodies were performed in order to correlate the mRNA and protein expression of the chemokines. The cellular source(s) of each chemokine was assessed by immunohistochemical staining of adjacent sections using antibodies directed against distinctive cellular markers. Substantial, but varied, expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, macrophage chemotactic protein (MCP)-1, eotaxin, and interleukin 8 (IL-8) were detected in the pathological lymph nodes by diverse cell types. Control lymph nodes showed expression only of RANTES, mainly by high endothelial venules. In all lymph nodes, except the nodes infiltrated with breast cancer, chemokine mRNA expression was highly concordant with the corresponding protein. In contrast with in vitro studies that have suggested discrete target cell specificity of chemokines, this study showed that with the possible exception of the neutrophil chemoattractant, IL-8, no chemokine appeared to be uniquely associated with the accumulation of a specific leukocyte subset. These data implicate chemokines in the recruitment of leukocytes to lymph nodes affected by diverse disease states.     

Chemokines at mucosal barriers and their impact on HIV infection

Aside from representing a physical barrier and providing an unfavorable chemical milieu to viral and bacterial infections, mucosae of gut and female genital tract also contain organized lymphoid structures that support the initiation of anti-microbial immune responses, and more diffuse lymphoid tissues that represent immune effector mucosal sites. Local expression of specific chemokines orchestrates lymphoid cell trafficking and positioning in the mucosa-associated lymphoid tissues, leading to their efficient priming during antigenic stimulations as well as their specific homing back where they were primed. This review examines productions and roles of mucosae-specific chemokines in healthy and pathological conditions, as well as their possible positive and deleterious effects during mucosal HIV infection.     

CXCR4–SDF-1 Signalling, Locomotion, Chemotaxis and Adhesion

Chemokines, small pro-inflammatory chemoattractant cytokines, that bind to specific G-protein-coupled seven-span transmembrane receptors present on plasma membranes of target cells are the major regulators of cell trafficking. In addition some chemokines have been reported to modulate cell survival and growth. Moreover, compelling evidence is accumulating that cancer cells may employ several mechanisms involving chemokine-chemokine receptor axes during their metastasis that also regulate the trafficking of normal cells. Of all the chemokines, stromal-derived factor-1 (SDF-1), an alpha-chemokine that binds to G-protein-coupled CXCR4, plays an important and unique role in the regulation of stem/progenitor cell trafficking. First, SDF-1 regulates the trafficking of CXCR4+ haemato/lymphopoietic cells, their homing/retention in major haemato/lymphopoietic organs and accumulation of CXCR4+ immune cells in tissues affected by inflammation. Second, CXCR4 plays an essential role in the trafficking of other tissue/organ specific stem/progenitor cells expressing CXCR4 on their surface, e.g., during embryo/organogenesis and tissue/organ regeneration. Third, since CXCR4 is expressed on several tumour cells, these CXCR4 positive tumour cells may metastasize to the organs that secrete/express SDF-1 (e.g., bones, lymph nodes, lung and liver). SDF-1 exerts pleiotropic effects regulating processes essential to tumour metastasis such as locomotion of malignant cells, their chemoattraction and adhesion, as well as plays an important role in tumour vascularization. This implies that new therapeutic strategies aimed at blocking the SDF-1-CXCR4 axis could have important applications in the clinic by modulating the trafficking of haemato/lymphopoietic cells and inhibiting the metastatic behaviour of tumour cells as well. In this review, we focus on a role of the SDF-1-CXCR4 axis in regulating the metastatic behaviour of tumour cells and discuss the molecular mechanisms that are essential to this process.     

Bovine CCL28 Mediates Chemotaxis via CCR10 and Demonstrates Direct Antimicrobial Activity against Mastitis Causing Bacteria

In addition to the well characterized function of chemokines in mediating the homing and accumulation of leukocytes to tissues, some chemokines also exhibit potent antimicrobial activity. Little is known of the potential role of chemokines in bovine mammary gland health and disease. The chemokine CCL28 has previously been shown to play a key role in the homing and accumulation of IgA antibody secreting cells to the lactating murine mammary gland. CCL28 has also been shown to act as an antimicrobial peptide with activity demonstrated against a wide range of pathogens including bacteria, fungi and protozoans. Here we describe the cloning and function of bovine CCL28 and document the concentration of this chemokine in bovine milk. Bovine CCL28 was shown to mediate cellular chemotaxis via the CCR10 chemokine receptor and exhibited antimicrobial activity against a variety of bovine mastitis causing organisms. The concentration of bovine CCL28 in milk was found to be highly correlated with the lactation cycle. Highest concentrations of CCL28 were observed soon after parturition, with levels decreasing over time. These results suggest a potential role for CCL28 in the prevention/resolution of bovine mastitis.     

Human pregnancy: the role of chemokine networks at the fetal-maternal interface

Chemokines are multifunctional molecules initially described as having a role in leukocyte trafficking and later found to participate in developmental processes such as differentiation and directed migration. Similar events occur in pregnancy during development of the fetal-maternal interface, where there is extensive leukocyte trafficking and tissue morphogenesis, and this is accompanied by abundant chemokine expression. The relationship between chemokines, leukocytes and placental development is beginning to be delineated. During pregnancy a specialised population of maternal leukocytes infiltrates the implantation site. These leukocytes are thought to sustain the delicate balance between protecting the developing embryo/fetus and tolerating its hemiallogeneic tissues. A network of chemokine expression by both fetal and maternal components in the pregnant uterus functions in establishing this leukocyte population. Intriguingly, experiments investigating immune cell recruitment revealed the additional possibility that chemokines influence aspects of placental development. Specifically, cytotrophoblasts, the effector cells of the placenta, express chemokine receptors that can bind ligands found at key locations, implicating chemokines as regulators of cytotrophoblast differentiation and migration. Thus, as in other systems, at the fetal-maternal interface chemokines might regulate multiple functions.