CCL4 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 (MIP-1). Further biochemical experimentation resulted in the separation and characterization of two distinct but highly related proteins, then renamed as MIP-1α and MIP-1β. Also in 1988, the human equivalent of MIP-1β (Act-2, AT 744) was independently cloned by different groups, and similarly, several other cytokines with prominent chemotactic properties were identified, leading to the introduction of a new classification and nomenclature introduction for the designation of the chemokines, in which, murine MIP-1β and human MIP-1β have been renamed CCL4 (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 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 (Rollins 1997; Menten et al. 2002; Maurer and von Stebut 2004). In the view of chemokine classification, CCL4 is characterized structurally and functionally as an inducible and secreted proinflammatory chemokine of the CC subfamily.
CCL4 Gene and Production
Murine CCL4 is encoded by a single gene on chromosome 11, which consists of three exons and two introns (Widmer et al. 1993). In addition, murine CCL4 gene also codes for a pre-protein of 92 amino acids with a theoretical M r of 7826.9 Da and has a sequence homology of 60% with murine CCL3, a homologous CC family chemokine.
The three-dimensional structure of human CCL4 has been defined by heteronuclear magnetic resonance analysis. It has shown that CCL4 exists as a symmetrical homodimer. Within each human CCL4 subunit (monodimer), the main secondary structure elements comprise a triple-stranded antiparallel β sheet arranged in a Greek key structure on top of which lies an α-helix. The NH 2-terminus consists of an irregular strand and a series of nonclassical turns that form a long loop. This loop is followed by a four-residue helical turn, which leads into strand β 1. The two disulfide bridges, present in CCL4, have a left-handed spiral conformation (Menten et al. 2002; Lodi et al. 1994).
About CCL4 cell production, it has been demonstrated that monocytes produce high amounts of CCL4 when stimulated with LPS or IL-7 (Menten et al. 2002; Lukacs et al. 1995). Also, DAMPs (damage-associated molecular patterns) such as HMGB1, heat shock proteins, and S100 can induce the up-regulation of expression of CCL4 (Bianchi et al. 2011). 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).
This CCL4 production is counteracted by addition of IL-4, which reduces level of CCL4 mRNA expression. Activated T and B cells which have been triggered by antigen binding also secrete CCL4. Furthermore, NK cells are a good source of CCL4 by physiological activation signals, such as cross-linking of their Fc receptors or lysis of target cells (Menten et al. 2002; Oliva et al. 1998). In addition, brain microvessel endothelial cells were found to release CCL4 following stimulation with LPS, TNF-α, IFN-γ, or IL-1β and pulmonary vascular smooth muscle cells–secreted CCL4 after addition of IL-1β, TNF-α IL-4, and IFN-γ (Menten et al. 2002; Lukacs et al. 1995; Shukaliak and Dorovini-Zis 2000). Finally, dendritic cells have also been shown to produce CCL4 in response to LPS, TNF-α, or CD40 ligand (Menten et al. 2002).
In 1996, the first receptor for CCL4 was identified as CC-CKR5, the fifth CC chemokine receptor cloned (Samson et al. 1996). This name has been replaced by CCR5 in the new chemokine system 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 CCL3 receptor (Menten et al. 2002). CCR5 is one of the most studied chemokine receptor by the fact that, as soon as 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 produce 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 plays 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/Th17 or Th2) by the way CCR5 is expressed on Th1, Th17, and Th2 lines (Griffith et al. 2014). Although, it was absent in several Th2 clones markedly influenced by IL-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, neutrophils, monocytes, macrophages, dendritic cells, natural killers, Th1, Th17, and Tregs cells (Menten et al. 2002; Maurer and von Stebut 2004; Horuk 2001). 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 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, and 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.
When analyzed in vitro in a microchamber chemotaxis assay, CCL4 was shown to be a potent lymphocyte chemoattractant. Lymphocyte migration from blood into tissues depends on the integrin-mediated adhesion of lymphocytes to the endothelium. Adhesion requires not only the presence of integrins on the surface of leukocytes but also the activation of these molecules by chemokines. CCL4 was shown to be effective in augmentation adhesion of T lymphocytes to the integrin vascular cell adhesion molecule 1 (VCAM-1 or CD106) (Menten et al. 2002; Tanaka et al. 1993). In addition, CCL4 is a potent chemoattractant for immature dendritic cells, but appears to be totally inactive on basophils (Fig. 1).
A series of studies demonstrate that CCL4 has an important role in inflammation. In fact, CCL4 participates in the recruitment of monocytes and T-cells into the inflamed synovium in rheumatoid arthritis. Furthermore, elevated levels of CCL4 are presented in the synovial fluid and tissues of arthritis patients (Patel et al. 2001). High levels of CCL4 have also been detected in the cerebrospinal fluid of patients with bacterial meningitis (Lahrtz et al. 1998). No difference in the CCL4 levels was observed in patients with glomerulonephritis and can be responsible for the part of leukocytes recruitment (Segerer et al. 2000). Furthermore, CCL4 has been detected during rejection of liver transplant, and CCL4 has been associated with chronic hepatitis C infection (Adams et al. 1996). 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 CCL4 and CCL3, a mechanism that drives cellular resistance to R5-tropic virus in some of these individuals (Walker et al. 2015).
Interestingly, since CCL4 shares the binding to CCR5 with the analogous chemokines CCL3 and CCL5, these factors are thought to operate in a complex network that regulates cell traffic in a given tissue. Interestingly, the redundancy (more than one chemokine can ding the same receptor) and the promiscuity (one chemokine can bind to more than one receptor) are characteristics that confer a robustness to the chemokines/chemokine receptors system. Indeed, the robustness is a common feature of many cytokine and growth factor networks that assure their proper performance, since the outputs of these cytokine networks may be retained to a sufficient extent, even if genetic or epigenetic alterations affect qualitatively or quantitatively individual network components. Indeed, robustness, redundancy, and promiscuity difficult the study of individual chemokines roles within these systems, being the role of chemokine receptors usually more easily unraveled in details than its chemokine ligands.
In conclusion, CCL4 plays an important role in the induction and maintenance of inflammatory immune responses, in the context of both autoimmune reactions and host defense. In addition, CCL4 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.
- Lodi PJ, Garrett DS, Kuszewski J, Tsang ML, Weatherbee JA, Leonard WJ, et al. High-resolution solution structure of the beta chemokine hMIP-1 beta by multidimensional. NMR Sci. 1994;263(5154):1762–7.Google Scholar
- Lukacs NW, Kunkel SL, Allen R, Evanoff HL, Shaklee CL, Sherman JS, et al. Stimulus and cell-specific expression of C-X-C and C-C chemokines by pulmonary stromal cell populations. Am J Phys. 1995;268(5 Pt 1):L856–61.Google Scholar
- Oliva A, Kinter AL, Vaccarezza M, Rubbert A, Catanzaro A, Moir S, et al. Natural killer cells from human immunodeficiency virus (HIV)-infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro. J Clin Investig. 1998;102(1):223–31.CrossRefPubMedPubMedCentralGoogle Scholar