CCL3 was initially described in 1988, as a partially purified 8-kDa protein doublet from conditioned medium of endotoxin-stimulated mouse macrophages. In the view of its prominent proinflammatory chemotactic role, characterized both in vivo and in vitro at that time, this protein was denominated “macrophage inflammatory protein-1 alpha” (MIP-1α). Subsequently, a high nucleotide sequence similarity (69%) was found between the murine MIP-1α cDNA and a reported human cDNA cloned from stimulated lymphocytes, initially called LD78α or GOS19, assuming to be the human counterpart to murine MIP-1α (Wolpe et al. 1988; Menten et al. 2002; Maurer and von Stebut 2004). Interestingly, human and murine MIP-1α have been independently isolated in many laboratories in relatively short time span and has been named differently by each group. Similarly, several other cytokines with chemotactic abilities were identified, leading to the introduction of a new classification and nomenclature introduction for the designation of the chemokines, in which murine MIP-1α and human MIP-1α/LD78α have been renamed CCL3 (Zlotnik and Yoshie 2000).
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 the site of inflammation, presenting therefore important roles in the initiation and maintenance of host inflammation. These chemoattractive cytokines are subdivided into four groups based on a cysteine (C) motif, CXC, CC, C, and CX3C (Menten et al. 2002; Maurer and von Stebut 2004; Rollins 1997). In the view of chemokine classification, CCL3 is characterized structurally and functionally as an inducible and secreted proinflammatory chemokine of the CC subfamily.
CCL3 Gene and Production
While some chemokines expression is characteristically constitutive, CCL3 gene expression is basically inducible in most mature hematopoietic cells, being only very low levels expressed constitutively. In fact, while monocytes and macrophages present just low levels of CCL3 mRNAi, upon stimulation with a relatively wide range of stimuli – including pathogen-associated molecular patterns (PAMPs) such as LPS from Gram-negative bacteria and lipoteichoic acids from Gram-positive bacteria; damage-associated molecular patterns (DAMPs) such as HMGB1, Heat Shock Proteins, S100, and superantigens such as the lectin phytohemagglutinin (PHA); or innate and adaptive cytokines such as IL-1β and IFN-γ – a significant CCL3 secretion was observed in human monocytes/macrophages and other cells. Neutrophils have been shown to secrete CCL3 when stimulated by microbial antigens. Not only phagocytes but also lymphocyte populations have been showed to secrete CCL3 when stimulated with PAMPs, DAMPs, or superantigens. In addition, it was reported that antigen binding to B-cell receptors (BCR) induced CCL3 production by B cells. Natural killer cells were also shown to efficiently produce CCL3 upon stimulation with IL-12 and IL-15. Also, DAMPs as S100A, S100B/RAGE dependent activation of diaphanous-1/Rac1/JNK/AP-1, Ras/Rac1/NF-κB and Src/Ras/PI3K/RhoA/diaphanous-1 and HMGB1 can induce the upregulation of expression of CCL3 and CCL5 in microglia cells. Interestingly, human regulatory T cells (Tregs) can also produce chemokines, typically inflammatory molecules. Tregs produce the chemokines CCL3 and CCL4 as a means to attract CD4+ and CD8+ T cells close to their proximity in vitro and in vivo (Patterson et al. 2016; Bianchi et al. 2011).
The costimulation provided by cytokines increases the stability of the LPS-induced CCL3 mRNA, demonstrating a convergence of proinflammatory signals toward an augmented CCL3 production. In contrast, inhibition of the LPS-mediated CCL3 production in monocytes was reported upon addition of anti-inflammatory and/or Th1-type cytokines such as IL-4, IL-10, IL-13, and also by the classic anti-inflammatory and immunosuppressant glucocorticoid dexamethasone. Indeed, IL-10 suppressed the LPS-induced release of CCL3 via an accelerated degradation of CCL3 mRNA (Menten et al. 2002; Maurer and von Stebut 2004; Cook 1996).
To discover the role of determined chemokine, researchers started by looking at the receptor usage and the biological responses of the cell types. Chemokine receptors characteristically are G protein-coupled receptors containing seven transmembrane domains that are found predominantly on the surface of leukocytes, being 19 different chemokine receptors characterized to date expressed by leukocyte subpopulations with distinct degrees of selectiveness and specificness. Indeed, although chemokine receptors share high amino acid identity in their primary sequences, they typically bind a limited number of ligands.
As a CC-family member, CCL3 chemokines mediate their biological effects by binding to cell surface CC chemokine receptors (3 × 104 to 5 × 105 receptors per cell). Receptor binding involves high affinity interactions and a subsequent cascade of intracellular events that rapidly leads to a wide range of target cell functions including chemotaxis, degranulation, phagocytosis, and mediator synthesis. Signal transduction events are initiated by G protein complex leading to its dissociation into Gα and Gβ subunits. Gα induces phosphoinositide 3-kinase (PI3K) pathway activation, and Gβγ subunits activate phospholipase C and induce Ca 2+ influx resulting in the protein kinase C isoform activation. It has also been shown that MAP kinases as well as the JAK/STAT signaling cascade are involved (Maurer and von Stebut 2004; Proudfoot et al. 2003). The overall effect of chemokine receptor signaling involves the activation of specific cellular mechanisms related to leukocyte migration (such as changes in the avidity of cell adhesion molecules [called integrins] and activation of mechanisms involved in diapedesis) and activation (such as degranulation, polarization, and effector functions) (Maurer and von Stebut 2004).
Human chemokine CCL3 can bind in two specific chemokine receptors, namely CCR5 and CCR1; it also retained the low-affinity binding properties of CCL3 for CCR4. While murine CCL3 is able to bind to the CCR1, CCR3, CCR4, CCR5, and D6 receptors (Fig. 1) (Menten et al. 2002). From a therapeutic point of view it is more interesting to identify the chemokine receptors involved in the control of recruitment of the principal cell type responsible for the initiation and resolution of inflammatory immune responses in lymphoid and peripheral tissues (Horuk 2001).
Analysis of chemokine receptors has revealed using chemokines radiolabeled to demonstrate the presence of specific cell surface-binding sites on many cell types. Not different, the first human CC chemokine receptor was isolated using several labeled chemokines, including LD78α. This receptor was designed CC-CKR1, but it has been renamed CCR1 to fit with the new nomenclature (Menten et al. 2002; Zlotnik and Yoshie 2000; 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, 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, eosinophils, and others cells. 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, 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).
In 1996, a second receptor for CCL3 was cloned and, as it was the fifth CC chemokine receptor cloned, named as CC-CKR5 (Samson et al. 1996). This name has been replaced by CCR5 in the new nomenclature; however, it is also 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 CCL3 receptor (Menten et al. 2002). CCR5 is one of the most studied chemokine receptor by the fact that, soon after 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; Maurer and von Stebut 2004; Horuk 2001).
Nowadays, current literature describes that 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 play a central role in T cell migration to the thymus, and T cell maturation and education (Ishida and Ueda 2006).
In addition to binding to the chemokine receptors, chemokines (including CCL3) 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 do 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.
The proinflammatory activities of CCL3 overlap with, but not identical to, the activities of other CC chemokines (Menten et al. 2002; Maurer and von Stebut 2004; Repeke et al. 2010). For example, CCL3 present a similar pharmacology and function compared with CCL5, however only share 45% identity at the primary amino acid level and has an acidic isoelectric point of 4.7. In addition, CCL3 and CCL4 can induce migration of monocytes and T lymphocytes, but they differ in their effects on different T cell subsets. Thus, while CCL3 is considered primarily chemotactic for B lymphocytes and activated CD8+ T cells, CCL4 is chemotactic for activated CD4+ cells (Fig. 1). Differently of CCL4, CCL3 is able to induce natural killer chemotaxis (Menten et al. 2002; Maurer and von Stebut 2004).
The wide role of CCL3 suggests that this chemokine has an important role in inflammation. In fact, CCL3 participates in the recruitment of monocytes and T cells into the inflamed synovium in rheumatoid arthritis. Furthermore, elevated levels of CCL3 are present in the synovial fluid and tissues of arthritis patients (Patel et al. 2001). High levels of CCL3 are also been detected in the cerebrospinal fluid of patients with bacterial meningitis (Lahrtz et al. 1998). Not different, the upregulation of CCL3 in glomeruli was observed in patients with glomerulonephritis and can be responsible for the part of leukocytes recruitment (Segerer et al. 2000). This ability to recruit inflammatory cells is used to overcome the limitations of radiation therapy, which is shown by the depletion of various leukocytes. Thus, the use of CCL3 as a treatment adjuvant radiation play crucial roles in tumor regression (Kanegasaki and Tsuchiya 2014).
Production of CCL3 and CCL4 by Tregs is also required for successful adoptive Treg cell therapy in murine models. Additionally, Tregs from patients with type 1 diabetes produce reduced levels of CCL3 and CCL4 compared with those from healthy controls (Patterson et al. 2016). A rare group of HIV seropositive individuals who are able to control viral replication without antiretroviral therapy presents an increased production of CCL3 and CCL4, a mechanism that drives cellular resistance to R5-tropic virus in some of these individuals (Walker et al. 2015). In addition, CCL3 has been shown to be implicated in many blood cell diseases, such as myeloid leukemia and T cell leukemia (Menten et al. 2002).
To understand the exact role of CCL3, the researchers used embryonic stem cell technology to generate mice homozygous for a disrupted CCL3 gene, called CCL3 knockout (CCL3-KO) or MIP-1KO mice. The homozygous mutant (−/−) mice were born in Mendelian proportions and had no obvious gross or histological abnormalities, indicating that CCL3 is not required for normal development (Cook 1996). The absence of CCL3 in CCL3-KO mice have reduced but not eliminated influenza virus-induced pneumonitis and presented highly resistance to CVB3-induced myocaditis (Cook 1996). Furthermore, CCL3-KO presented reduced scores of autoimmune encephalomyelitis, arthritis rheumatoid, Trypanosoma cruzi, and other inflammatory diseases (Cook 1996; Machado et al. 2005). However, the absence of CCL3 had no significant effect in the ability to decrease the effects on the inflammatory response in some chronic diseases, such as periodontal disease. Although, CCL3 is the most abundantly expressed chemokine in diseased periodontium, its absence probably is supplied by an overlap role of the homologous chemokines CCL4 and CCL5 (Repeke et al. 2010; Silva et al. 2007).
In conclusion, CCL3 plays an important role in the induction and maintenance of inflammatory immune responses, in the context of both autoimmune reactions and host defense. In this way, this interesting chemokine has shown as an important protein in the body operation, still not appears such as an essential life protein. In addition CCL3 might therefore play an important role in the pathogenesis of inflammatory diseases, such as arthritis, pneumonitis, leukemia, AIDS, and others, which would have implications for design of new therapeutic strategies.
- Repeke CE, Ferreira Jr SB, Claudino M, Silveira EM, de Assis GF, Avila-Campos MJ, et al. Evidences of the cooperative role of the chemokines CCL3, CCL4 and CCL5 and its receptors CCR1+ and CCR5+ in RANKL + cell migration throughout experimental periodontitis in mice. Bone. 2010;46(4):1122–30.CrossRefPubMedGoogle Scholar