Chemokines and diseases

ARTICLE

Auteur(s) : Victor M. DONG¹, David H. McDERMOTT², Reza ABDI³

¹ Clinical Operations and Medical Affairs, Solvay Pharmaceuticals, Inc., 901 Sawyer Road, Marietta, GA 30062, USA.
² Molecular Signaling Section, Laboratory of Host Defenses of the National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA. 
³ Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Article accepted on 26/2/03

Chemokines (chemotactic cytokines) are the largest family of cytokines within the human genome and act through G protein-coupled receptors. They are basic proteins that have 20 to 70 percent homology in amino acid sequence but share a common structure. They avidly bind to cell surface glycoproteins on the endothelium via one binding site while a second site interacts with chemokine receptors expressed on the surface of the leukocyte (see Fig. 1). Given the abundance of heparin sulfate proteoglycans on the surface of endothelial cells, this interaction may serve to establish a local concentration gradient from the point source of chemokine secretion. The fact that chemokines are produced by essentially all somatic cells illustrates the vast scope of their function in a variety of immune responses. For example, chemokines synthesized by the endothelial cell may be released to the luminal surface or blood side following activation, or chemokines produced by the tissue may be transported across the endothelium to signal circulating leukocytes.

Chemokines play a vital role in leukocyte recruitment from the bloodstream into sites of inflammation and are also important regulators in the development, differentiation, and anatomic distribution of inflammatory cells [1-3]. Leukocyte extravasation (see Fig. 1) starts with the selectin-dependent process of leukocyte rolling. Conversion of rolling to firm adherence is mediated by the activation of leukocyte integrins, which may be triggered by chemokine receptor signaling or may also be directly mediated by the engagement of the adhesive chemokine receptor CX3CR1 by its ligand CX3CL1 (fractalkine) [3-5]. A newly recognized role of chemokines is the coordinated movement of dendritic cells and lymphocytes that are necessary for long-term antigen specific immunity [6, 7]. The secondary lymphoid tissue chemokine (SLC) that is produced by lymphoid organ endothelium is a ligand for the receptor CCR7 expressed by naïve T cells. Once the T cells have migrated across the endothelium, the same receptor responds to signals from ELC, a chemokine produced by dendritic cells, which is thought to promote interaction with these cells in the T cell areas of the lymph nodes. B cells express CXCR5, which allows them to respond to BCA-1, a chemokine produced by cells within the lymphoid follicles. Consequently, B cells are directed to the B cell areas of the tissue. It is known that mice lacking CXCR5 do not develop normal lymphoid follicles. Thus, chemokines are crucial to B cell homing and the lymphoid architecture required for adaptive immunity.

Almost 50 different chemokines and 15 chemokine receptors have been identified and characterized to date. Depending on the number of amino acids separating the first two of four conserved cysteine residues in the amino acid sequence, chemokines are classified into four subfamilies: CC, CXC CX3C, and XC families (where none, one, or three residues separate the cysteines and finally the C chemokines which lack both the first and third cysteines and where X is used to distinguish from complement receptor 1, CR1). Each chemokine receptor has a distinct chemokine and leukocyte specificity, but the specificities have considerable overlap since some chemokines may bind multiple receptors, and some receptors may bind multiple chemokines (see Table I). There are also differences in cell type expression. For example, CXCR1 is predominantly expressed on neutrophils, whereas CCR2 is more widely expressed on monocytes, T cells, natural killer cells, dendritic cells, and basophils [8]. A systematic classification of both chemokines and their receptors has been recently suggested and the reader is referred for further details [9-12].

Abbreviations – CTX-chemotaxis, PMN-neutrophil, NK-natural killer cell, CNS-central nervous system

Given the ubiquity of chemokines within inflammatory tissue destruction, it is not surprising that the involved human diseases therefore span numerous medical and surgical specialties. These include HIV infection, cancer, atherosclerosis, autoimmune / inflammatory diseases, transplant rejection and dermatological diseases (see Table II). We will briefly review these six major areas of interest and identify future potential targets of therapy.

Table II. Chemokines associated with human diseases

Chemokines and diseases

Human Immunodeficiency Virus (HIV) Disease

Chemokine research was the exclusive domain of immunologists and pathologists studying inflammation until 1995 [13]. However, since that time research in HIV pathophysiology has made significant contributions to the growing literature of chemokines. HIV enters its host cell through membrane fusion. The viral gp120/gp41 membrane glycoprotein complex binds to the cellular transmembrane protein CD4 on T helper cells and macrophages. This binding then induces a conformational change in gp120/gp41, and subsequent binding of another coreceptor that is typically a molecule of the chemokine receptor class. CXCR4 is the principal coreceptor for HIV-1 isolates that infect T cell lines (T-tropic strains) and CCR5 is a coreceptor for HIV-1 isolates that infect macrophages and activated T cells (M-tropic strains) [2]. The central role of chemokine receptors in the pathophysiology of HIV infection was emphasized by the discovery of a mutation of CCR5 (CCR5 Δ32) that allowed persons at high risk for HIV-1 infection to have substantially lower infection rates [14-18]. In CCR5 Δ32 homozygotes, a functional CCR5 protein cannot be made and such individuals rarely become HIV-1 positive. These homozygotes do not display any immune response impairment. The majority of exposed but uninfected individuals are deficient in cell surface CCR5 expression due to homozygous carriage of the Δ32 deletion. In persons who are heterozygous for the deletion, the rate of progression of infection is slower than in those without the mutation [14-16]. Interestingly, CCR5 Δ32 has also been associated in some studies with a reduced risk of asthma, less severe course of rheumatoid arthritis and later onset of multiple sclerosis [13].
Orally active small molecule CCR5 antagonists have been developed to block HIV entry. Schering Plough’s SCH-C is the first of these to reach clinical testing and has been demonstrated to have potent in vitro activity against primary HIV-1 isolates [19]. Unfortunately, the drug was found to prolong cardiac conduction (QT interval) and has been withdrawn from further testing. However, a new candidate that reportedly lacks this side effect and has similar or better potency (SCH-D) is now in testing [20]. AnorMED is currently developing an oral CXCR4 antagonist, AMD-3100, that also blocks HIV entry.

Cancer

Chemokines are important in both tumor growth and angiogenesis, which are considered the mainstays of tumor physiology. Human melanoma cell lines have been reported to constitutively express CXCL1 (growth-related oncogene-, GRO-α), CXCL8 (IL-8) and CCL5 (RANTES). This overexpression in melanoma tumor cells is associated with the promotion of tumor growth and metastatic potential. Targeting these ligands with antibodies retards the growth of melanoma tumors in mice [21].
Although the role of leukocyte infiltration in cancer remains unclear, chemokines may affect the tumor growth by recruiting leukocytes to the tumor. This in turn may stimulate tumor growth by supplying growth factors and promoting angiogenesis, or inhibit tumor growth by enhancing the host response to the tumor. CXCL8 (IL-8) was found to function as a direct autocrine growth factor in a variety of cancers such as malignant melanoma, liver and pancreatic tumors, and colon cancers [22]. The receptors for CXCL8, namely CXCR1 and 2, have been shown to act closely with the epidermal growth factor receptor (EGFR) [23], indicating the links between this growth-factor pathway with chemokines. The overexpression of CXCL2(GRO-β) and CXCL3 (GRO-γ) in melanoma cells has been found to promote their growth both in vitro and in vivo [24].
Inflammatory signaling pathways involving NF-kB may also be corrupted in tumors and impact the chemokine system. The expression of CCL5 (RANTES) is induced by NF-kB; therefore the down-modulation of CCL5 through the inhibition of NF-kB offers a novel therapeutic application [25]. The constitutive activation of NF-kB in melanoma cells helps the tumor cells to constitutively express both angiogenic and angiostatic chemokines, and cytokines such as VEGF, IL-1 and IL-6 which promote melanoma growth and resistance to apoptosis. The constitutive expression of CXCL1 (GRO-α) and CXCL8 (IL-8) leads to dysregulated NF-kB activity which in turn translates into accelerated melanoma tumor growth, and increased metastasis [26-28].
The role of chemokines in angiogenesis has been shown to be even more complex. Chemokines may play roles as both angiogenic and angiostatic factors. For instance, CXC chemokines containing the glutamate-leucine-arginine (ELR) motif are angiogenic in corneal micropocket assays, whereas CXC chemokines without this motif are angiostatic [29]. Platelet factor 4 and CXCL10 (interferon- (IFN-) inducible protein-10, IP-10) inhibit neovascularization, tumor growth and metastasis, whereas CXCL8 (IL-8) promotes angiogenesis and tumor metastasis [2, 30, 31]. Nevertheless, CXCL12 (stromal-cell-derived factor-1, SDF-1), an ELR^- CXC chemokine, was recently shown to be a direct chemoattractant for endothelial cells [32, 33]. CXCL12 has been shown to augment the expression of VEGF by endothelial cells and VEGF can in turn upregulate CXCR4 on endothelial cell surfaces.
Chemokines may also be associated with metastatic behavior [34]. CXCL8 can increase matrix metalloproteinase activity which then facilitates the transmigration of tumor cells through more permeable basement membranes leading to enhanced metastasis [35]. Specific expression of chemokine receptors on breast cancer cells is a critical event that leads to homing and metastasis of these cells in a chemokine ligand and receptor-dependent, organ-specific manner [36]. CXCR4 and CCR7 and their respective ligands CXCL12 (SDF 1-α) and CCL21 (6Ckine) showed peak levels of expression in organs representing the destinations of breast cancer metastasis. Neutralization of the CXCL12/CXCR4 interaction by blocking antibody significantly impaired metastasis of breast cancer cells to regional lymph nodes and lung.

Atherosclerosis

Inflammation is being increasingly recognized as a key pathogenic mechanism in atherosclerosis, and macrophages and lymphocytes are the main inflammatory cells found in the diseased blood vessels. Furthermore, macrophages are the progenitors of lipid laden foam cells and a source of growth factors for intimal hyperplasia. Although many chemokines including CXCL8 (IL-8), CXCL12 (SDF-1), CXCL10 (IP-10), CCL1 (I-309) and CXCR2 have been found in lesions in animal models, the best evidence for a chemokine role in atherosclerosis has been the ligand receptor pair of CCL2 (MCP-1) and CCR2, since either MCP-1 or CCR2 knockout results in markedly smaller lesions with less lipid deposition than what develops in mice genetically susceptible to atherosclerosis [37-39]. CCL2 (MCP-1) has been found in diseased human carotid arteries but not in normal ones [40, 41]. Interestingly, members of the statin family of HMG CoA reductase inhibitors also inhibit expression of MCP-1 [42, 43]. There is also some evidence linking MCP-1 and infectious agents associated with restenosis and atherosclerosis. Cytomegalovirus (CMV) encodes a CCL2 (MCP-1), CCL5 (RANTES), and CX3CL1 (fractalkine) responsive chemokine receptor called US28 on infected smooth muscle cells, and Chlamydia pneumoniae activates CCL2 (MCP-1) expression after infection of endothelial cells [44-46].
Another chemokine receptor (CX3CR1) has been recently implicated in human atherosclerosis by two epidemiologic studies. CX3CL1 (fractalkine), a chemokine expressed by inflamed endothelium, induces leukocyte adhesion and migration through the receptor CX3CR1. In a study of patients undergoing cardiac catheterization, the prevalence and severity of CAD was less in the group that was heterozygous or homozygous for polymorphism of CX3CR1[47] and in another study CX3CR1 polymorphism was associated with a lower risk of heart attack/unstable angina [48]. Recently, Horvath and colleagues were successful in selectively targeting neutrophil and monocyte recruitment after angioplasty or stent implantation in an animal model by targeting the MCP-1 receptor CCR2 [49]. This study demonstrated the subtleties of vascular injury and feasibility of potential anti-inflammatory strategies in both atherosclerotic diseases and coronary interventions.

Autoimmune/Inflammatory Diseases

Chemokines have been implicated in a variety of autoimmune / inflammatory diseases such as rheumatoid arthritis [13, 50], asthma [51-55], and inflammatory bowel disease (ulcerative colitis and Crohn’s disease) [56, 57]. The CCR5 Δ32 allele has been found less frequently in patients with rheumatoid arthritis [58] and also associated with a milder course [59]. It is also associated with a reduced risk of developing asthma [60]. Two studies recently examined the role of the CCL5 (RANTES) polymorphism in the development of atopy and asthma [61, 62]. The 403 allele was associated with increased susceptibility and is one of several genes associated with asthma and atopy.
Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS). CNS lesions in EAE have increased expression of CCL5 (RANTES), CCL3 (MIP 1-α), CCL4 (MIP 1-β), CXCL10 (IP-10) and CCL2 (MCP-1) mRNA and proteins [13]. CCL2, 7 and 8 (MCP-1, 2 and 3) have also been found in active human MS lesions on autopsy [63]. CXCR3, the receptor for CXCL10 (IP-10), has been noted in virtually all perivascular T cells and astrocytes associated with active lesions [64]. Also, the inactive CCR5 Δ32 allele was associated with an approximate 3 year delay in the onset of MS [65].
A growing body of evidence also indicates that chemokines are implicated in the pathogenesis of insulitis and diabetes. Cameron et al have demonstrated that temporal expression of CCL3 (MIP 1-α) was associated with a more severe insulitis and higher likelihood of developing diabetes in NOD mice and that NOD CCL3 (MIP 1-α) knockout mice were less susceptible to disease [66, 67]. The serum concentration of CXCL10 (IP-10) has been reported to be elevated in the patients with early stage diabetes. Serum concentrations of CXCL10 (IP-10) are augmented in both newly diagnosed Type I diabetes mellitus patients and patients at risk [68].

Organ Transplant Rejection

The central importance of chemokines in transplant rejection is likely because of their roles in leukocyte recruitment, Th1 and Th2 cell differentiation, and dendritic cell movement and maturation. Chemokines are activated at the initial stage of transplantation when ischemia reperfusion injury occurs. Studies on human renal biopsies revealed that the expression of Th1 chemokine receptors (CCR5 and CXCR3) and their ligands (CXCL10 (IP 10), CXCL9 (Mig), CXCL11 (I-TAC) and CCL5 (RANTES)) are associated with acute rejection [69, 70]. In addition, the susceptibility of human renal allograft recipients to acute rejection episodes is associated with their CCR5 and CCR2 receptor genotypes [71]. Targeting CCR5 also results in significant prolongation of islet allograft survival [72]. In this study, a prominent Th2 response was observed in the CCR5 – / – model, which was regarded as a plausible protective mechanism. Targeting CXCR3 also results in significant prolongation of cardiac allograft survival [73].
CCR7 plays a major role in the migration of dendritic cells to secondary lymphoid tissues. In the setting of alloantigen introduction, dendritic cells undergo maturation and migrate to secondary lymphoid tissues such as spleen and lymph nodes where they selectively activate T and B cells. CCR1, CCR2, CCR5, and CXCR1 expressed by immature dendritic cells contribute to the ability of these cells to migrate into non-inflamed as well as inflamed tissue [2, 74, 75]. Therefore, it is clear that the central role of dendritic cells in transplantation results from their chemokine receptor repertoire.

Dermatological Diseases

Given the close interaction of chemokines in the inflammatory process and immune response, it is not surprising that a number of dermatological diseases are subject to their dysregulation. In psoriasis lesions, one finds neutrophils and activated T cells, and the neutrophil chemoattractants CXCL8 (IL-8) and CXCL1 (GRO-α) [2]. CXCL10 (IP-10) and CCL2 (MCP-1) are found in psoriatic plaques but not in normal skin [76, 77]. Indeed, decreased IP-10 in diseased skin is seen after treatment [77]. More recently investigators also found that calcipotriene-induced improvement in psoriasis is associated with reduced CCL8 (IL-8) and increased IL-10 levels within lesions of thirty randomized patients [78]. Further study on human keratinocytes and peripheral blood mononuclear cells demonstrated dose-dependent inhibition of CXCL1 (GRO-α), CXCL8 (IL-8), CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC) by the antipsoriatic drug dimethylfumarate [79]. There is also an abundance of data linking IL-8 receptors (CXCR1 and CXCR2) and CXCL8 (IL-8) to psoriatic skin [80].
The expression of chemokine genes during the induction phase of contact hypersensitivity has been well studied [81-84]. Abe et al [85] have shown that during the T cell mediated elicitation phase, CXCL10 (IP-10) expression is mediated by CD8⁺ T cells and is regulated by CD4⁺ T cells during the elicitation of contact hypersensitivity.
CC chemokine receptors are expressed on hematopoietic cells. CCR3 is the major chemokine receptor on eosinophils and is also expressed on other inflammatory cells suggesting an important role for this receptor in allergic diseases such as atopic dermatitis and bronchial asthma. Human keratinocytes have also been found to possess autocrine and paracrine mechanisms for CC chemokine secretion and CC chemokine receptor expression [80], similar to the established CXC chemokine receptors on fibroblasts, melanocytes and keratinocytes [86-88]. Petering and colleagues identified CCR3 on epidermal keratinocytes, suggesting that CCR3 and its ligands may play an important role in skin physiology and pathophysiology [80].
Chemokines are also implicated in the diseases scleroderma and systemic sclerosis. Silica, which is capable of causing scleroderma, was found to induce mRNA and protein of the chemokines CCL5 (RANTES) and CCL2 (MCP-1) in endothelial cells [89]. They also found abundant CCL5 (RANTES) mRNA expression in the skin of systemic sclerosis patients, but none in control skin. The potential contribution of systemic sclerosis fibroblasts to chemokine production and its relevance to pathogenesis has been examined [90]. They found that these fibroblasts display a specific pattern of chemokine expression characterized by constitutively increased and abnormally regulated expression of CCL2 (MCP-1) in vitro. CCL2 (MCP-1) was also expressed in lesional skin. It is interesting to note that CCL2 (MCP-1) expression has been noted in a variety of other fibrotic diseases such as hepatic cirrhosis, pulmonary fibrosis and glomerular sclerosis [91-93]. Recent results also document for the first time that CCL2 (MCP-1) induces an inflammatory response in human tubular epithelial cells [94]. The distribution of novel polymorphisms of the CXCL8 (IL-8), CXCR1 and CXCR2 genes in systemic sclerosis patients has been studied [95]. An association between systemic sclerosis and 2 polymorphisms occurring in the vicinity of each other in the CXCR2 gene has been described. In particular, a significant increase in the frequency of CXCR2 + 785 CC and CXCR2 + 1208 TT homozygotes was found in the systemic sclerosis patients compared with control subjects. Interestingly, a recent report describes a murine model of human scleroderma [96]. They found that early elevated cutaneous mRNA expression of TGF β-1 but not 2 or 3, and elevated CC chemokines CCL2 (MCP-1), CCL3 (MIP 1-α) and CCL5 (RANTES) precede skin and lung fibrosis. They suggest that this model may be useful in testing new interventions in early fibrosing diseases and that chemokines may be new potential targets for scleroderma.

Future implications

One of the first chemokines to be identified was by Yoshimura and Tanaka who reported the characterization of “human monocyte-derived neutrophil chemotactic factor” (IL-8) more than a decade ago [97]. Over the past 16 years, investigators from many scientific disciplines have demonstrated that the scope of chemokine function extends far beyond their chemoattractant moieties. Indeed, it is now well established that chemokines are involved in the most fundamental parts of the immune system, including hematopoiesis, organogenesis, angiogenesis, lymphocyte homing, and dendritic cell maturation and movement. Consequently it is not surprising that a growing number of reports indicate their crucial roles in a variety of diseases [22, 98]. Investigators are using the strategy of selectively blocking leukocyte recruitment to the site of inflammation as an approach to treat many diseases. The use of small molecule inhibitors of chemokine receptors may soon become an attractive way to accomplish these treatment goals. n

Dr. Abdi is supported by the Juvenile Diabetes Research Foundation (JDRF) Career Development Award. We thank Joan M. Sechler of NIAID for editorial assistance. There is no conflict of interest between the contents of this review and the products of Solvay Pharmaceuticals, Inc.

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