Chemokines and Chemokine Receptors as Novel Therapeutic Targets in Rheumatoid Arthritis （RA）： Inhibitory Effects of Traditional Chinese Medicinal Components

Chemokines belong to a large family of inflammatory cytokines responsible for migration and accumulation ofleukocytes at inflammatory sites.Over the past decade,accumulating evidence indicated a crucial role forchemokines and chemokine receptors in the pathophysiology of rheumatoid arthritis(RA).RA is a chronicautoimmune disease in which the synovial tissue is heavily infiltrated by leukocytes.Chemokines play animportant role in the infiltration,localization,retention of infiltrating leukocytes and generation of ectopicgerminal centers in the inflamed synovium.Recent evidence also suggests that identification of inhibitorsdirectly targeting chemokines or their receptors may provide a novel therapeutic strategy in RA.TraditionalChinese medicines(TCMs) have a long history in the treatment of inflammatory joint disease.The basis for theclinical benefits of TCM remains largely unclear.Our studies have led to the identification of numerous novelchemokine/chemokine receptor inhibitors present in anti-inflammatory TCMs.All of these inhibitors werepreviously reported by other researchers to have anti-arthritic effect,which may be attributable,at least inpart,to their inhibitory effect on chemokine and/or chemokine receptor.Therefore,identification of agentscapable of targeting chemokine/chemokine receptor interactions has suggested a mechanism of action forseveral TCM components and provided a means of identifying additional anti-RA TCM.Thus,this approachmay lead to the discovery of new inhibitors of chemokines or chemokine receptors that can be used to treatdiseases associated with inappropriately overactive chemokine mediated inflammatory reactions.Cellular &Molecular Immunology.2004;1(5):336-342.     

Role of chemokines in inflammation and immunoregulation

Chemokines are first noted for their ability to attract and activate leukocytes, as well as their potential role as mediators of inflammation. However, emerging data have shown that various chemokines may exert, other biologic effects both inside and outside the immune system. Inducible chemokines participate primarily in inflammatory responses and comprise the bulk of the chemokine family. Constitutive chemokines are expressed primarily in secondary lymphoid organs and some nonlymphoid organs, where they play amajor role in lymphocyte homing. Studies expanding to areas beyond inflammatory leukocyte recruitment, will likely give us a more complete picture of chemokine function, its regulation in lymphoid and nonlymphoid tissues and ways of utilizing endogenous chemokines to intervene with immune and inflammatory reactions.     

Knock out models to dissect chemokine receptor function in vivo

Chemokines are a family of small proteins involved in numerous biological processes ranging from hematopoiesis, angiogenesis and lymphocyte trafficking to the extravasation and tissue infiltration of leukocytes in response to inflammatory agents, tissue damage and bacterial or viral infection. Chemokines exert their effects by binding to specific G-protein-coupled seven-transmembrane receptors. In vitro studies suggest that the chemokine system is highly redundant in that most chemokines bind to more than one receptor and most receptors bind multiple chemokines. Therefore, targeted deletion of chemokine receptors has proved to be a useful tool for determining the distinct biological role of these molecules in vivo.     

Overview of the mechanisms regulating chemokine activity and availability

Physiological leukocyte homing and extravasation of leukocytes during inflammatory processes is directed by a number of proteins including adhesion molecules, proteases, cytokines and chemokines. Tight regulation of leukocyte migration is essential to ensure appropriate migration. A number of mechanisms exist that regulate leukocyte migration including up- or down-regulation of chemokine or chemokine receptor gene expression. However, chemokine availability in vivo also depends on the interaction of chemokines with specific glycosaminoglycans such as heparan sulfate on the surface of endothelial layers. Modification of the interaction of chemokines with these glycosaminoglycans alters the presentation of chemokines to chemokine receptors on circulating leukocytes. On top, binding of chemokines to atypical chemokine receptors that do not signal through G proteins affects chemokine availability on the endothelial layers. In addition to mechanisms that modulate chemokine availability, this review summarizes mechanisms that fine-tune chemokine function. These include synergy or antagonism between chemokines and alternative splicing of chemokine genes. Moreover, chemokines may be posttranslationally modified leading to molecules with enhanced or reduced potency to bind to G protein-coupled receptors or GAGs or generating chemokines with altered receptor specificity. Cross-talk between these different mechanisms generates a complex regulatory network that allows the organism to modulate leukocyte migration in a highly specific manner.     

Mechanisms of leukocyte trafficking in allergic diseases: insights into new therapies targeting chemokines and chemokine receptors

Marked inflammatory infiltration by activated leukocytes is a characteristic feature of allergic diseases. Elucidation of the mechanisms of leukocyte trafficking in allergic diseases would identify targets to establish novel anti-inflammatory strategies for treatment of these diseases. Leukocyte trafficking is controlled by tissue-specific expression of chemokines and chemokine receptor expression on the leukocyte surface. Here, we review the role of chemokines and their receptors in leukocyte trafficking to inflammatory sites in allergic diseases and discuss therapeutic strategies targeting chemokine networks for treatment of these diseases.     

Borrelia burgdorferi induces chemokines in human monocytes.

Lyme disease is clinically and histologically characterized by strong inflammatory reactions that contrast the paucity of spirochetes at lesional sites, indicating that borreliae induce mechanisms that amplify the inflammatory response. To reveal the underlying mechanisms of chemoattraction and activation of responding leukocytes, we investigated the induction of chemokines in human monocytes exposed to Borrelia burgdorferi by a dose-response and kinetic analysis. Lipopolysaccharide (LPS) derived from Escherichia coli was used as a positive control stimulus. The release of the CXC chemokines interleukin-8 (IL-8) and GRO-alpha and the CC chemokines MIP-1alpha, MCP-1, and RANTES was determined by specific enzyme-linked immunosorbent assays, and the corresponding gene expression patterns were determined by Northern blot analysis. The results showed a rapid and strong borrelia-inducible gene expression which was followed by the release of chemokines with peak levels after 12 to 16 h. Spirochetes and LPS were comparably effective in stimulating IL-8, GRO-alpha, MCP-1, and RANTES expression, whereas MIP-1alpha production preceded and exceeded chemokine levels induced by LPS. Unlike other bacteria, the spirochetes themselves did not bear or release factors with intrinsic chemotactic activity for monocytes or neutrophils. Thus, B. burgdorferi appears to be a strong inducer of chemokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation and tissue damage observed in Lyme disease.     

Possible mechanisms involved in chemokine synergy fine tuning the inflammatory response

The arrest and directed migration of leukocytes during homeostasis, tumour development and inflammation is orchestrated by a multitude of chemokines, which govern leukocyte migratory activities. Immune cells are particularly adept at adjusting rapidly to changes within the environment by migration in response to chemokines. The confrontation of leukocytes with different combination of chemokines that are concomitantly produced under physiological or pathological conditions in vivo is complex. There are different ways to enhance or reduce leukocyte migration mediated by chemokines such as posttranslational modifications. Here, we described a positive regulatory mechanism in leukocyte trafficking, by the synergism between chemokines to rapidly augment the local leukocyte influx, thereby enhancing the outcome of an inflammatory response in vivo. The cellular mechanisms involved in chemokine synergy are still debated, but probably include chemokine and/or receptor heterodimerization and subsequent cooperation in signal transduction.     

Targeting the chemokine network in renal inflammation

Chemokines and their receptors are involved in the pathogenesis of renal diseases. They mediate leukocyte recruitment and activation during initiation as well as progression of renal inflammation. Infiltrating leukocyte subpopulations contribute to renal damage by releasing inflammatory and profibrotic cytokines. All intrinsic renal cells are capable of chemokine secretion on stimulation in vitro. Expression of inflammatory chemokines correlates with renal damage and local accumulation of chemokine receptor-bearing leukocytes in a variety of animal models of renal diseases as well as in human biopsy studies. Chemokines and their respective receptors could represent new targets for therapeutic intervention in renal inflammatory disease states that often tend to progress to end-stage renal disease. This article summarizes the present data on the role of chemokines and their receptors in renal inflammation with special emphasis on our efforts to identify the chemokine receptors CCR1 and CCR2 as promising targets for therapeutic intervention.     

Chemokine Ligand and Receptor Expression in the Pregnant Uterus : Reciprocal Patterns in Complementary Cell Subsets Suggest Functional Roles

During human pregnancy, the uterus is infiltrated by a population of maternal leukocytes that co-exist with fetal cytotrophoblasts occupying the decidua and uterine blood vessels. These immune cells, termed "decidual granulated leukocytes," are composed predominantly (70%) of the CD56(bright) subset of natural killer cells, accompanied by T cells (15%) and macrophages (15%). The mechanisms underlying the recruitment of these cells are unknown, but by analogy to other systems, chemokines are likely to be involved. We examined the expression patterns of 14 chemokines in the decidualized uterine wall by in situ hybridization, and the expression of chemokine receptors on decidual leukocytes by RNase protection. The striking concordance between the expression of chemokines in the uterus and their receptors on decidual leukocytes allowed us to identify numerous receptor-ligand pairs that may recruit the latter cells to the uterus during pregnancy. Additionally, chemokine expression patterns suggested other, nonimmune functions for these molecules, including a role in cytotrophoblast differentiation. Together, our results imply that chemokine networks serve important functions at the maternal-fetal interface.     

Chemokine receptor trafficking and viral replication

Summary: Chemokines and chemokine receptors have emerged as crucial factors controlling the development and function of leukocytes. Recent studies have indicated that, in addition to these essential roles, both chemokines and chemokine receptors play critical roles in viral infection and replication. Not only are chemokine receptors key components of the receptor/fusion complexes of primate immunodeficiency viruses, hut chemokines can also influence virus entry and infection. Many viruses, in particular herpesviruses, encode chemokines and chemokine receptors that influence the replication of both the parent virus and other unrelated viruses. The cell surface expression of the chemokine receptors is regulated through their interaction with membrane trafficking pathways, ligands induce receptor internalization and downmodulation through endocytosis, and recycling is regulated within endosomes. Pan of the mechanism through which chemokines protect cells from HIV infection is through ligand-induced internalization of the specific chemokine receptor co-receptors. In addition, mechanisms may exist to regulate the trafficking of newly synthesized receptors to the cell surface. Here we discuss aspects of the mechanisms through which chemokine receptors interact with membrane-trafficking pathways and the influence of these interactions on viral replication.     

New Drug Targets in Rheumatoid Arthritis

Rheumatoid arthritis is a chronic inflammatory disease where the synovial tissue is characterized by heavy infiltration of leukocytes. Chemokines and chemokine receptors play an important role in cell migration and positioning of leukocytes within the inflamed rheumatoid synovium. There is now much focus on the specific contribution and role of each chemokine and chemokine receptor in the chronic inflammatory process in the synovial tissue. Recent evidence indicates that interference with the chemokines released from the inflamed synovial cells or the chemokine receptors expressed on the cells infiltrating the synovial tissue may lead to discovery of new therapeutics for this disease.     

C and CX3C chemokines: cell sources and physiopathological implications

Within the fascinating world of chemokines, C and CX3C chemokines have long been regarded as two minor components, even though they present unique features and show less redundancy than the other chemokine families. Nevertheless, the body of data on their expression and role in various inflammatory disorders has grown in the past few years. The C chemokine family is represented by two chemokines, XCL1/lymphotactin-alpha and XCL2/lymphotactin-beta, whereas the CX3C chemokine family contains only one member, called CX3CL1/ fractalkine. In this review, we present an overview on the structure, expression and signaling properties of these chemokines and their respective receptors and examine how they contribute to inflammation and the regulation of leukocyte trafficking, as well as their potential role in the pathophysiology of human inflammatory diseases. Taken together, these data expand the biological importance of C and CX3C chemokines from that of simple immune modulators to a much broader biological role, even though their precise commitment within the framework of immune responses has still to be determined.     

Chemokine scavenging by D6: a movable feast?

The atypical chemokine receptor, D6, is efficient at sequestering and scavenging inflammatory CC chemokines. The absence of D6 blocks the successful resolution of immune responses in models of inflammation, suggesting that CC-chemokine scavenging by D6 is an important component of the resolution phase of in vivo inflammatory responses. Most studies have suggested that lymphatic endothelial cells are the main vehicles for D6 function in vivo. Here, we propose that leukocytes, which also express D6, could be more-effective vehicles for D6 scavenging function. Thus, leukocytes might be the primary cell type that removes inflammatory chemokines from inflamed tissues. We also propose that lymphatic endothelial cell-expressed D6 might have a distinct but complementary role in restricting inflammatory leukocyte access to the lymphatic vasculature.     

Synchronous synthesis of alpha- and beta-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis.

Chemokines are secreted peptides that exhibit selective chemoattractant properties for target leukocytes. Two subfamilies, alpha- and beta-chemokines, have been described, based on structural, genetic, and functional considerations. In acute experimental autoimmune encephalomyelitis (EAE), chemokines are up-regulated systemically and in central nervous system (CNS) tissues at disease onset. Functional significance of this expression was supported by other studies; intervention with an antichemokine antibody abrogated passive transfer of EAE, and chemokines expressed in brains of transgenic mice recruited appropriate leukocyte populations into the CNS compartment. Chemokine expression in the more relevant circumstance of chronic EAE has not been addressed. We monitored the time course and cellular sources of chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha, interferon-gamma-inducible protein of 10 kd, KC, and regulated on activation, normal T-cell expressed and secreted cytokine) in CNS and peripheral tissues during spontaneous relapses of chronic EAE. We found coordinate chemokine up-regulation in brain and spinal cord during clinical relapse, with expression confined to CNS tissues. Monocyte chemoattractant protein-1, interferon-gamma-inducible protein of 10 kd, and KC were synthesized by astrocytic cells, whereas macrophage inflammatory protein-1 alpha and regulated on activation, normal T-cell expressed and secreted cytokine were elaborated by infiltrating leukocytes. The results demonstrate stringent regulation of multiple chemokines in vivo during a complex organ-specific autoimmune disease. We propose that chemokine expression links T-cell antigen recognition and activation to subsequent CNS inflammatory pathology in chronic relapsing EAE.     

The roles of cytokine receptors in diseases

Cytokines are produced by various types of cells and have profound effects on the regulation of immune reactions, hematopoiesis, and inflammation. Herein, we will discuss the pathophysiological relevance of cytokine receptor expression, particularly focusing on chemokine receptor expression. Chemokines are cytokines with 4 cysteines at the well-conserved positions and exhibit potent chemotactic activities for various types of leukocytes. To date, accumulating evidence has indicated the potential involvement of these chemokines in inflammatory reactions through regulating inflammatory cell infiltration. Moreover, several lines of evidence demonstrate that different sets of chemokine receptors are expressed by T helper type 1 (Th1) and Th2 cells and that Th1 and Th2 cells respond to distinct sets of chemokines. These observations establish the essential roles of chemokines in helper T lymphocyte migration in vivo. Furthermore, several chemokine receptors are utilized as co-factors for human immunodeficiency virus entry and mutation in one chemokine receptor confers marked resistance to HIV infection. Therefore, the determination of chemokine receptors may provide invaluable information on the immune status and susceptibility to HIV infection.     

Chemokine receptors and molecular mimicry

Chemokines are small pro-inflammatory peptides that are best known for their leukocyte-chemoattractant activity. The cloned leukocyte chemokine receptors, interleukin 8 receptor (IL-8R) types A and B and the macrophage inflammatory protein 1α (MIP-1α/RANTES receptor, are related by sequence and chemokine binding to two herpesvirus products, and to the Duffy antigen that mediates erythrocyte invasion by the malaria-causingg parasite H smodium vivax. Here, Sunil Abuja, Ji-Liang Gao and Philip Murphy suggest that in addition to the activation of leukocytes, chemokines may be important in the function of erythrocytes and, through molecular mimicry, in microbial pathogenesis.     

Chemokines and Their Receptors in the Allergic Airway Inflammatory Process

The development of the allergic airway disease conveys several cell types, such as T-cells, eosinophils, mast cells, and dendritic cells, which act in a special and temporal synchronization. Cellular mobilization and its complex interactions are coordinated by a broad range of bioactive mediators known as chemokines. These molecules are an increasing family of small proteins with common structural motifs and play an important role in the recruitment and cell activation of both leukocytes and resident cells at the allergic inflammatory site via their receptors. Trafficking and recruitment of cell populations with specific chemokines receptors assure the presence of reactive allergen-specific T-cells in the lung, and therefore the establishment of an allergic inflammatory process. Different approaches directed against chemokines receptors have been developed during the last decades with promising therapeutic results in the treatment of asthma. In this review we explore the role of the chemokines and chemokine receptors in allergy and asthma and discuss their potential as targets for therapy.     

Chemokines

Motility is a hallmark of leukocytes, and breakdown in the control of migration contributes to many inflammatory diseases. Chemotactic migration of leukocytes largely depends on adhesive interaction with the substratum and recognition of a chemoattractant gradient. Chemokines are secreted proteins and have emerged as key controllers of integrin function and cell locomotion. Numerous distinct chemokines exist that target all types of leukocytes, including hematopoietic precursors, leukocytes of the innate immune system, as well as naive memory, and effector lymphocytes. The combinatorial diversity in responsiveness to chemokines ensures the proper tissue distribution of distinct leukocyte subsets under normal and pathological conditions. Inflammatory chemokines are readily detected in lesional tissue and local cellular infiltrates carry corresponding chemokine receptors. Blocking of inflammatory chemokines represents a promising strategy for the development of novel anti-inflammatory therapeutics.This review focuses on a separate class of chemokines, termed homeostatic chemokines, with steady-state production at diverse sites, including primary and secondary lymphoid tissues as well as peripheral (extralymphoid) tissues. More precisely, we discuss the chemokines involved in T-cell traffic during the initiation of adaptive immunity and compare the distinct migration properties of short-lived effector T cells and long-lived memory T cells. Memory T cells are currently classified according to the presence of the lymph node-homing receptor CCR7 into CCR7+ central memory T (T(CM)) cells and CCR7- effector memory T (T(EM)) cells. For better understanding memory T-cell function, we propose the distinction of a third category, termed peripheral immune surveillance T (T(PS)) cells, which typically reside in healthy peripheral tissues, such as skin, lung, and gastrointestinal tract.Localization and relocation of memory T cells is strictly related to their function in recall responses. Therefore, detailed knowledge of their generation and tissue distribution may help to design better vaccination strategies.     

Regulation of Atherogenesis by Chemokines and Chemokine Receptors

Atherosclerosis is a chronic inflammatory and metabolic disorder affecting large- and medium-sized arteries, and the leading cause of mortality worldwide. The pathogenesis of atherosclerosis involves accumulation of lipids and leukocytes in the intima of blood vessel walls creating plaque. How leukocytes accumulate in plaque remains poorly understood; however, chemokines acting at specific G protein-coupled receptors appear to be important. Studies using knockout mice suggest that chemokine receptor signaling may either promote or inhibit atherogenesis, depending on the receptor. These proof of concept studies have spurred efforts to develop drugs targeting the chemokine system in atherosclerosis, and several have shown beneficial effects in animal models. This study will review key discoveries in basic and translational research in this area.     

Chemokine and cytokine production during Orientia tsutsugamushi infection in mice

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus which is histopathologically characterized by inflammatory manifestations, indicating that rickettsiae induce mechanisms that amplify the inflammatory response. To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production after infection with O. tsutsugamushi in mice. The mRNAs that were upregulated included lymphotactin, RANTES (regulated upon activation, normal T-cell expressed and secreted), macrophage inflammatory proteins 1alpha/beta (MIP-1alpha/beta), MIP-2, monocyte chemoattractant protein 1, lymphotoxin beta, tumor necrosis factor alpha, interleukin-6, gamma-interferon, transforming growth factor beta1, and migration inhibition factor. Peak expression of these chemokines and cytokines was observed between 4 and 8 days after infection. Gene induction was followed by the secretion of chemokine and cytokine proteins. Chemokine profile in infected mice was well correlated with kinetics of inflammatory cell infiltration. Thus, O. tsutsugamushi appears to be a strong inducer of chemokines and cytokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation observed in scrub typhus.