CCL5 was initially described in 1988 and this new protein was named for its characteristics: Regulated upon Activation expressed by Normal T cells and presumably Secreted (RANTES) (Schall et al. 1988). A relatively new technology, at the time, was used to identify RANTES, subtraction cDNA libraries, which help to distinguish genes expressed in one cell and not in another. This genetic approach has since led to the uncovering of about 50 other chemokines, and in order to clarify the nomenclature of all chemokines and their receptors, a new nomenclature has been introduced, in which RANTES has been renamed CCL5 (Zlotnik and Yoshie 2000). CCL5 was first discovered in T cell-specific cDNA, and this initial report on CCL5 placed further attention on a new family of chemotactic proteins that proved to be important in influencing a series of biologic and pathological processes (Levy 2009).
Collectively, chemokines are defined as small (8–14 kDa) proteins of cytokine family that have a broad range of activities involved in the recruitment and function of specific population of leukocytes at site of inflammation, presenting therefore important roles in the initiation and maintenance of host inflammation. Four classes of chemokines have been discovered, based on the conserved cysteine (C) residues on the mature protein CXC, CC, C, and CX3C. In the view chemokine classification, CCL5 is characterized structurally and functionally as an inducible and secreted proinflammatory chemokine of the CC subfamily (Conti and DiGioacchino 2001). It is one of the most promiscuous inflammatory CC chemokines. It is highly basic with an acidic isoelectric point of 9.8 (a feature shared by most of the chemokines) and presents a similar pharmacology and function to CCL3, however shares only 45% identity at the primary amino acid level.
CCL5 Gene and Production
Initially, this chemokine was considered a T cell-specific protein; however, it was demonstrated that CCL5 can be produced by many cells in the vertebrate body including platelets, macrophages, eosinophils, and fibroblasts, as well as endothelial, epithelial, and endometrial cells. Interestingly, while stimulation of T cells inhibits the expression of CCL5, whereas as a general rule in the other cells mentioned, synthesis of CCL5 is induced by TNF-α and IL-1α, but not by TGFβ, IFNγ, and IL-6 (Graziano et al. 1999). Also, DAMPs (Damage-associated molecular patterns) as S100A, S100B, and HMGB1; PAMPs (pathogen-associated molecular patterns) such as LPS from Gram-negative bacteria and lipoteichoic acids from Gram-positive bacteria; and virus, as H1N1, can induce the upregulation of expression of CCL5. (Bianchi et al. 2011; Ramírez-Martínez et al. 2013).
CCL5 increases the adherence of monocytes to endothelial cells. It selectively supports the migration of monocytes and T lymphocytes expressing the cell surface markers CD4 and UCHL1. These cells are thought to be prestimulated helper T cells with memory T cell functions. CCL5, although activating human basophils and causing histamine release from some donor cells, has also been found to inhibit the release of histamine from basophils induced by several cytokines (Graziano et al. 1999). In addition, CCL5 is a potent eosinophil-specific activator of oxidative metabolism, and it also changes the density of eosinophils, making the hypodense, which are thought to represent a state of generalized cell activation. These cells are most often associated with diseases such as asthma and allergic rhinitis (Graziano et al. 1999; Appay and Rowland-Jones 2001).
Analysis of chemokine receptors using radiolabeled chemokines has the presence of specific cell surface binding sites on many cells type. Interestingly, CCR5 was one of the chemokines used to characterize the first human CC chemokine receptor isolated. This receptor was initially designated CC-CKR1, but it has been renamed CCR1 to fit with the new chemokine system nomenclature (Fig. 1) (Zlotnik and Yoshie 2000; Menten et al. 2002; Neote et al. 1993).
The gene encoding human CCR1 has been mapped to chromosome 3p21, in a cluster with several other chemokine receptor genes (CCR2, CCR3, CCR4, CCR5, CCR8, CCR9, XCR1, and CX3CR1). Using polyclonal antibodies to CCR1, the receptor has been shown to be predominantly expressed on monocytes and lymphocytes. On neutrophils, CCR1 can be expressed upon induction with specific cytokines. However, mouse neutrophils express CCR1 constitutively. Furthermore, CCR1 mRNA has been detected in human dendritic cells and eosinophils. CCR1 is expressed in leukocytes from a broad range of species including rhesus, rabbit, rat, and mouse, and there is a high degree of sequence homology among all these sequences. The CCR1 seven-membrane protein consists of 355 amino acid residues and belongs to the peptide subfamily of Class A GPCR family. This chemokine receptor is thought to predominantly signal through Gi/o couple pathway to regulate calcium flux and inhibit adenyl cyclase and is thought unable to signal via Gq/11 protein. In addition, at least 11 different ligands (chemokines) interact with CCR1, including CCL3 (Cheng and Jack 2008).
The second CCL5 chemokine receptor cloned is named CCR3 (with lower affinity than CCL7 or CCL11), which is primarily expressed by eosinophils and appears to play an important role in regulating the migration of these cells. After the initial report of the first CCR3 clone, the same group showed that eotaxin, an eosinophil-selective CC chemokine and currently named CCL11, was a potent agonist for CCR3. Finally, in 1996, it was demonstrated that the CC chemokine CCL5 was also functional CCR3 agonist. Having noted the importance of CCR3 for eosinophil responses, subsequent studies demonstrate that this receptor and its cognate ligands such as CCL5 are directly involved in Th2-type responses and play an important role in allergy including asthma and atopic dermatitis; and also, CCL5/CCR3 signaling can promote metastasis by inducing Th2 polarization of CD4+ T cells, with implications for prognosis and immunotherapy of luminal breast cancer (Zhang et al. 2015). Furthermore, CCR3 was shown by several groups to be a HIV-1 coreceptor, and it is expressed on microglial cells of the brain, promoting disease like AIDS dementia, and also expressed in dentritic cells, play a role there in HIV-1 infection.
Another CCL5 chemokine receptor, now with low-affinity binding properties, is CCR4, and it was originally cloned from a human basophilic leukemia cell line library (Power et al. 1995; Horuk 2001). CCR4 and its ligands are important for regulating immune balance and is known to be expressed selectively on Th2 cells and Treg cells, with special regard to the relationship between CD4+ CD25+ FOXp3+ Treg subset and CCR4 (Horuk 2001; Ishida and Ueda 2006). In a subset of patients with CCR4+ T cell leukemia/lymphoma, the tumor cells themselves function as Treg cells, contributing to tumor survival in the face of host antitumor immune responses. In other types of cancer, the specific ligand chemokines for CCR4 such as CCL5 that are produced by tumor cells and the tumor environment attract Tregs cells to the tumor, where they create a favorable environment for tumor escape from host immune response (Ishida and Ueda 2006). In addition, CCR4 is known to modulate T cell migration to several sites of inflammation in the body and plays a central role in T cell migration to the thymus and T cell maturation and education (Ishida and Ueda 2006).
In 1996 was cloned the fifth CC chemokine receptor, named CC-CKR5 (Samson et al. 1996). This name has been replaced by CCR5 in the new nomenclature; however, it is also still called CD195 (Zlotnik and Yoshie 2000). The human CCR5 receptor is encoded by CCR5 gene, located on the short arm at position 21 on chromosome 3. Interestingly, CCR5 is a member of GPCR superfamily and shares 55% identical amino acids with CCR1, the firstly identified CCL5 receptor (Menten et al. 2002). CCR5 is one of the most studied chemokine receptor by the fact that, soon its discovery, CCR5 was shown to function pathologically as a key cell entry co-receptor for HIV-1. Certain population (approximately 20%) has a genetic deletion of a portion of the CCR5 gene (CCR5 Δ32), resulting in a frameshift at amino acid 185, and produces a mutant protein which is not expressed on the cell surface, which may result in distinct functional outcomes regarding inflammatory immune responsiveness (Carrington et al. 1999). In fact, CCR5 play important roles, not only in HIV infection, but also in the elaboration of a specific immune response against a series of pathogens. Lipopolysaccharide, proinflammatory cytokines, and various other stimuli can stimulate the CCR5 expression. This increase of CCR5 expression can influence in the selection of the appropriate effector T cell (i.e., Th1 or Th2); by the way, CCR5 is expressed on both Th1 and Th2 lines, although it was absent in several Th2 clones markedly influenced by interleukin 2 (Maurer and von Stebut 2004). In addition, CCR5 expression has been detected on primary and secondary lymphoid organs, neurons, capillary endothelial cells, as well as epithelium, endothelium, vascular smooth muscle, fibroblast, Langerhans cells, macrophages, dendritic cells, CD34+ progenitor cells, and thymocytes (CD4+ and CD8+ cells) (Menten et al. 2002; Horuk 2001; Maurer and von Stebut 2004).
In addition to the binding to the chemokine receptors, chemokines (including CCL5) characteristically present a carboxyl terminus stretch of positively charged residues that recognize heparan sulfate (HS) glycosaminoglycan (GAGs). Interestingly, chemokines can signal through cognate G protein coupled receptors (GPCRs) either at their soluble or immobilized (i.e., glycosaminoglycan associated) states. Recent evidences demonstrate that GAGs are indispensable for immobilization and function of major chemokines required for leukocyte adhesion to and crossing through blood and lymphatic vessels. In fact, chemokines stably immobilized on GAGs at the luminal surface of endothelium prevent their dilution by blood flow but also to facilitate localized signaling to rolling leukocytes, while GAGs at inflamed tissue contribute to establish a chemotactic gradient that guides the influx of the leukocytes within the tissue. In spite of these versatile functions of HS GAGs in different types of endothelial cells and basement membranes, it is still possible that many extravasation processes involve HS-GAG-independent mechanisms. Interestingly, the presence of HS GAGs on leukocytes does not contribute to their migratory and inflammatory properties; subsets of antigen-presenting cells may need to immobilize the chemokines they secrete within particular immune synapses, resulting in local activities essential for adhesion, motility, and survival of the cells involved (Horuk 2001).
On T cells, CCL5 can act by two quite separate signaling pathways. At nanomolar concentrations, CCL5 acts as a typical chemokine, by the GPCR-mediated pathway, as described above. And, at micromolar concentrations, CCL5 also triggers a distinct herbimycin A-sensitive protein tyrosine kinase (PTK)-mediated pathway (GPCR independent), leading to prolonged calcium influx, hyperphosphorylation, and generalized cell activation. The exact signaling pathways of CCL5 on T cells appear to be very complex and are not yet fully characterized, especially as the same kinases can be induced within both the GPCR and GPCR-independent signaling pathways (e.g., ZAP-70 and p125). The ability of CCL5 to induce the GPRC-independent pathway and activate leukocytes is a distinct and important feature of the biology of this unusual chemokine. However, because high, and perhaps nonphysiological, concentrations of CCL5 are required to demonstrate this phenomenon, its relevance remains unclear (Appay and Rowland-Jones 2001).
CCL5 is a potent chemoattractant for memory T lymphocytes, monocytes, and eosinophils (Fig. 1). It was found highly expressed in activated T lymphocytes, macrophages, fibroblasts, platelets, mesangial cells, epithelial cells, megakaryocytes, and some tumors (Graziano et al. 1999; Appay and Rowland-Jones 2001). The activity of the chemokine CCL5 is not restricted merely to chemotaxis. It is a powerful leukocytes activator, a feature potentially relevant in a range of inflammatory disorders (Conti and DiGioacchino 2001). In fact, increased CCL5 expression has been associated with a wide range of inflammatory disorders and pathologies, including allogeneic transplant rejection, atherosclerosis, arthritis, atopic dermatitis, inflammatory airway disorders such as asthma, delayed-type hypersensitivity reactions, glomerulonephritis, endometriosis, some neurological disorders (such as Alzheimer’s disease), and certain malignancies. In all these pathologies, CCL5 is thought to act by promoting leukocytes infiltration to site of inflammation (Appay and Rowland-Jones 2001).
At high concentration, CCL5 is able to induce the activation of T cells. This activation induced by CCL5 is followed by many diverse effects, including T-cell proliferation and apoptosis and the release of proinflammatory cytokines such as Interleukin 2 (IL-2), IL-5, interferon (IFN)-γ, and CCL4. CCL5-induced activation is not only restricted to T cells, but can also extend to monocytes and neutrophils, where similar dual-signaling pathways are induced (Graziano et al. 1999; Appay and Rowland-Jones 2001). Furthermore, CCL5 not only plays a key role in cells activation and inflammatory diseases but also in the immune response to viral infection. In 1995, CCL5 was shown to be the most potent member of a trio CC chemokines (CCL3–5) released by CD8+ T cells that were able to suppress the replication of nonsyncytium-inducing (NSI) HIV-1 strains in vitro. Indeed, the treatment of CD4+ T cell with CC-chemokines with antiviral function (CCL3, CCL4, and CCL5) made it resistant to macrophage-tropic HIV-1, by downregulation of CCR5. (Appay and Rowland-Jones 2001; Barrios et al. 2013; Cocchi et al. 1995).
In the view of the relative unspecificity of CCL5, a modified version of this chemokine was developed by the removal of the two N-terminal residues of CCL5 in order to create a partial agonist of chemokine receptors and consequently therapeutically interfere with chemokine-based cell migration. This compound, named Met-RANTES, was described to function as a chemotaxis inhibitor and more effectively blocks CCR5-mediated HIV 1 infection of monocytes. Met-RANTES was initially produced during the synthesis of recombinant RANTES by the extension of the product with a single methionine residue, and its treatment was extensively studied in inflammatory bone diseases, such as arthritis rheumatoid (AR) and PD (Doodes et al. 2009; Repeke et al. 2010; Proudfoot et al. 1996). Furthermore, studies demonstrated the ability of met-RANTES treatment to extenuate tissue injury in models of arthritis, renal inflammation, and experimental colitis, among others (Doodes et al. 2009). Similarly, met-RANTES was able to decrease the experimental periodontal disease scores such as alveolar bone loss and inflammatory cell influx (Repeke et al. 2010). Interference with this homologous chemokine production or activity by blocking target receptors represents a potential novel therapeutic strategy in chronic inflammatory disease (Wells et al. 1999).
In summary, CCL5 plays a key physiological role in T cell migrations and in the inflammation disorders, inducing leukocyte infiltration and activation. CCL5 might therefore play an important role in the pathogenesis of inflammatory diseases than previously thought, which would have implications for design of new therapeutic strategies.
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