Abstracts

INTRODUCTION

Chemokines constitute a large family of small cytokines and generally have low molecular weight ranging from 7 to 15kDa. Chemokines and their receptors are able to control the migration and residence of all immune cells. Some chemokines are considered pro-inflammatory and their release can be induced during an immune response at a site of infection, while others are considered homeostatic, and are involved in the control of cells migration during tissue development or maintenance. The physiologic importance of this family of mediators is resulting from their specificity; members of the chemokine family induce recruitment of well-defined leukocyte subsets.¹1 .Oppenheim JJ, Zachariae CO, Mukaida N, Matsushima K. Properties of the novel proinflammatory supergene “intercrine” cytokine family. Annu Rev Immunol. 1991;9:617-48. Review.,²2 .Raz E, Mahabaleshwar H. Chemokine signaling in embryonic cell migration: a fisheye view. Development. 2009;136(8):1223-9. Review.

Many chemokines were initially identified by subtraction hybridization as an immediate or early response genes induced by growth factors. Based on this property, it was supposed that chemokines were involved in cellular proliferation and acted as nuclear factors. However, when complete amino acid sequences were deduced, it was clear that chemokines were secretory proteins.

There are two families of chemokines based on the first cysteine residue: the family called CC chemokines (Chart 1), also known as beta-chemokines. The genes encoding the CC chemokines are located on chromosome 17. CC chemokines stimulate mainly monocytes, but also basophils, eosinophils, T-lymphocytes, and natural killer (NK) cells. The other family CXC (Chart 2), known as alpha-chemokines, have an intervening amino acid between the first two cysteines and are located in the chromosome 4.³3 .Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, et al. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev. 2000;52(1):145-76. Review.

4 .Luster AD, Ravetch JV. Genomic characterization of a gamma-interferon-inducible gene (IP-10) and identification of an interferon-inducible hypersensitive site. Mol Cell Biol. 1987;7(10):3723-31.-⁵5 .Mukaida N, Shiroo M, Matsushima K. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8. J Immunol. 1989;143(4): 1366-71.These chemokines, which mainly stimulate neutrophil chemotaxis, contain a Glu-Leu-Arg (ELR) sequence in the N-terminus that is essential for receptor binding.⁶6 .Clark-Lewis I, Schumacher C, Baggiolini M, Moser B. Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities. J Biol Chem. 1991;266(34): 23128-34.,⁷7 .Hébert CA, Vitangcol RV, Baker JB. Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. J Biol Chem. 1991;266(28):18989-94.

Chemokine receptors are differentially expressed by all leukocytes and can be divided into two groups: G protein-coupled chemokine receptors, which is activated by pertussis toxin (PTX)-sentive G_i-type G protein; and an atypical chemokine receptors, which appear to form chemokine gradients and dampen inflammation by scavenging chemokines in a G protein independent manner.⁸8 .Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014; 32:659-702. Review.

Nowadays approximately 50 endogenous chemokines ligands and 20 G-protein-coupled receptors have been described. However, there is still a lot to be comprehended in this field, particularly regarding mechanisms during diseases development. In this review, we focused on a timeline basic knowledge in chemokines.

MONOCYTE CHEMOATTRACTANT PROTEIN-1 (CCL2/MCP-1) AND CC CHEMOKINES

Acute inflammation is a complex process characterized by the coordinated migration of effector cells to the site of inflammation, as well as displacement of immune cells from peripheral sites to draining lymphoid organs to initiate immune response. Such coordinated movement of cells requires the induced expression of inflammatory chemokines and their respective receptors on target cells. The interaction between chemoattractant and immune cells trigger a series of biochemical and cellular coordinate events.⁸8 .Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014; 32:659-702. Review.,⁹9 .Murdoch C, Finn A. Chemokine receptors and their role in inflammation and infectious diseases. Blood. 2000;95(10):3032-43. Review.

CCL2 is typically secreted in two predominant forms with similar molecular weights of 9 and 13kDa. They have the same protein core and differ by the addition of O-linked carbohydrates to the larger form.¹⁰10 .Jiang Y, Valente AJ, Williamson MJ, Zhang L, Graves DT. Post-translational modification of a monocyte-specific chemoattractant synthesized by glioma, osteosarcoma, and ascular smooth muscle cells. J Biol Chem. 1990;265(30): 18318-21. Despite this difference, they have identical activities in vitro.¹¹11 .Jiang Y, Beller DI, Frendl G, Graves DT. Monocyte chemoattractant protein-1 regulates adhesion molecule expression and cytokine production in human monocytes. J Immunol. 1992;148(8):2423-8.

CCL2 stimulates chemotaxis of monocytes and several cellular events associated with chemotaxis, including Ca²2 .Raz E, Mahabaleshwar H. Chemokine signaling in embryonic cell migration: a fisheye view. Development. 2009;136(8):1223-9. Review.flux and integrin expression. It is also a weak inducer of cytokine expression in monocytes and, at high concentrations, elicits a respiratory burst leading to generation of reactive oxygen species.¹¹11 .Jiang Y, Beller DI, Frendl G, Graves DT. Monocyte chemoattractant protein-1 regulates adhesion molecule expression and cytokine production in human monocytes. J Immunol. 1992;148(8):2423-8.,¹²12 .Rollins BJ, Morrison ED, Stiles CD. Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Proc Natl Acad Sci USA. 1988;85(11):3738-42.

Monocytes utilize different chemoattractant molecules to migrate; however, CCL2 and CCL7 are rapidly produced by stromal cells and immune cells after pattern-recognition receptors (PRR) activation or after cytokine stimulation. CCL2 dimerizes and binds to extracellular matrix glycosaminoglycans, thus establishing a stable gradient for CCR2+ inflammatory monocytes.⁸8 .Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014; 32:659-702. Review.,¹³13 .Proudfoot AE, Handel TM, Johnson Z, Lau EK, LiWang P, Clark-Lewis I, et al. Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc Natl Acad Sci USA. 2003;100(4):1885-90.

CCL2 expression has been identified upregulated in certain cell types of some diseases, such as macrophages/foam cells and smooth muscle cells in atherosclerosis; delayed-type of hypersensitivity reactions in the skin mononuclear phagocytes, endothelial cells, and keratinocytes; melanoma cells in skin cancer and synoviocites of rheumatoid arthritis.¹⁴14 .Ylä-Herttuala S, Lipton BA, Rosenfeld ME, Särkioja T, Yoshimura T, Leonard EJ, et al. Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci USA. 1991;88(12):5252-6.

15 .Graves DT, Barnhill R, Galanopoulos T, Antoniades HN. Expression of monocyte chemotactic protein-1 in human melanoma in vivo. Am J Pathol. 1992;140(1):9-14.

16 .Villiger PM, Terkeltaub R, Lotz M. Production of monocyte chemoattractant protein-1 by inflamed synovial tissue and cultured synoviocytes. J Immunol. 1992;149(2):722-7.-¹⁷17 .Yu X, Barnhill RL, Graves DT. Expression of monocyte chemoattractant protein-1 in delayed type hypersensitivity reactions in the skin. Lab Invest. 1994;71(2):226-35.

Two other chemokines structurally related to CCL2 are named CCL8 (MCP-2) and CCL7 (MCP-3). Generally, CCL2 and CCL7 have similar properties, although CCL7 may exhibit characteristics from both CCL2 and CCL5 (RANTES), suggesting that it has a broader range of chemokine binding. Others CC chemokines include macrophage inflammatory protein 1α (MlP-1α/CCL3), macrophage inflammatory protein-1β (MIP-1β/CCL4), T-cell activation gene-3 (TCA-3/CCL1), and CCL5, which support and regulate activated T-cells.¹²12 .Rollins BJ, Morrison ED, Stiles CD. Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Proc Natl Acad Sci USA. 1988;85(11):3738-42.,¹⁸18 .Opdenakker G, Froyen G, Fiten P, Proost P, Van Damme J. Human monocyte chemotactic protein-3 (MCP-3): molecular cloning of the cDNA and comparison with other chemokines. Biochem Biophys Res Commun. 1993;191(2):535-42.,¹⁹19 .Broxmeyer HE, Sherry B, Lu L, Cooper S, Carow C, Wolpe SD, et al. Myelopoietic enhancing effects of murine macrophage inflammatory proteins 1 and 2 on colony formation in vitro by murine and human bone marrow granulocyte/macrophage progenitor cells. J Exp Med. 1989;170(5):1583-94.

Eosinophil is a leukocyte present in high numbers during inflammatory allergic reactions, such as asthma and rhinitis; there is evidence that links accumulation and activation of these cells with tissue injury and lung dysfunction.²⁰20 .De Monchy JG, Kauffman HF, Venge P, Koëter GH, Jansen HM, Sluiter HJ, et al. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am Rev Respir Dis. 1985;131(3):373-6.

21 .Bousquet J, Chanez P, Lacoste JY, Barnéon G, Ghavanian N, Enander I, et al. Eosinophilic inflammation in asthma. N Engl J Med. 1990;323(15):1033-9.-²²22 .Bradley BL, Azzawi M, Jacobson M, Assoufi B, Collins JV, Irani AM, et al. Eosinophils, T-lymphocytes, mast cells, neutrophils, and macrophages in bronchial biopsy specimens from atopic subjects with asthma: comparison with biopsy specimens from atopic subjects without asthma and normal control subjects and relationship to bronchial hyperresponsiveness. J Allergy Clin Immunol. 1991;88(4):661-74. Humbles et al. purified and sequenced a CC-chemokine from bronchoalveolar lavage fluid of allergen-challenged sensitized animal model. They also identified CCL11 (Eotaxin-1) as a local chemoattractant responsible for eosinophil recruitment, via CCR3, a chemokine receptor highly expressed on eosinophils.²³23 .Humbles AA, Conroy DM, Marleau S, Rankin SM, Palframan RT, Proudfoot AE, et al. Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo. J Exp Med. 1997;186(4):601-12. These leukocytes express CCR1 and CCR3 and can respond to a variety of chemokines including CCL11, CCL24 (Eotaxin-2) and CCL26 (Eotaxin-3).²⁴24 .Kalomenidis I, Stathopoulos GT, Barnette R, Guo Y, Peebles RS, Blackwell TS, et al. Eotaxin-3 and interleukin-5 pleural fluid levels are associated with pleural fluid eosinophilia in post-coronary artery bypass grafting pleural effusions. Chest. 2005;127(6):2094-100.,²⁵25 .Pope SM, Zimmermann N, Stringer KF, Karow ML, Rothenberg ME. The eotaxin chemokines and CCR3 are fundamental regulators of allergen-induced pulmonary eosinophilia. J Immunol. 2005;175(8):5341-50.

CCL19 (MIP-3β) and CCL21 (SLC) are expressed mainly by stromal cells in T cell-rich zones of lymph nodes. They are mediators of homeostatic trafficking of naïve T cells, but also play a role on T cell priming and activation, as well as on lymphocytes recruitment to inflamed tissue. The pathway signaling of both proteins take place via CCR7, which is expressed on T cells and mature dendritic cells. Although CCL19 and CCL21 exhibit the same affinity for CCR7, only CCL19 has ability to desensitize and internalize the receptor.⁸8 .Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014; 32:659-702. Review.

T cells and progenitor cells enter the thymus in response to CCL21/SLC and CCL25/TECK, which are produced by thymic epithelium and surrounding structures. CCR9 guides these cells to the subcapsular zone. Subsequent thymic maturation from double-positive through single-positive thymocytes occurs in concert with their migration into the medulla, which is dependent on CCR7 and perhaps another chemokine yet to be defined.²⁶26 .Love PE, Bhandoola A. Signal integration and crosstalk during thymocyte migration and emigration. Nat Rev Immunol. 2011;11(7):469-77. Review.

In general, chemokines CCL2, CCL3, CCL4, CCL5, CCL11 and CCL13 are classified as inflammatory; CCL18, CCL19, CCL21, CCL25 and CCL27 as homeostatic. CCL1, CCL17, CCL20 and CCL22 with dual function inflammatory and homeostatic while CCL14, CCL15, CCL16 and CCL23 are related with plasma or platelet.²⁷27 .Le Y, Zhou Y, Iribarren P, Wang J. Chemokines and chemokine receptors: their manifold roles in homeostasis and disease. Cell Mol Immunol. 2004;1(2):95-104. Review.

INTERLEUKIN-8 AND CXC CHEMOKINES

lnterleukin-8 (IL-8/CXCL8) is a prototypical member of CXC chemokine family. The IL-8 gene encodes a 99-aminoacid translation product. It undergoes proteolytic cleavage of the amino terminal yielding several different products. IL-8 in solution is thought to exist as non-covalently linked dimer, and, in vitro, it is a potent chemoattractant for neutrophils. It has been reported to be also a chemoattractant for a subset of T-lymphocytes.²⁸28 .Huber AR, Kunkel SL, Todd RF 3rd, Weiss SJ. Regulation of transendothelial neutrophil migration by endogenous interleukin-8. Science. 1991;254(5028): 99-102. Erratum in: Science. 1991;254(5032):631. Science. 1991;254(5037): 1435.

IL-8 has also been named neutrophil activating protein-1 (NAP-1), since it stimulates release of neutrophil granules. Like many other chemoattractants, IL-8 induces re-arrangement of cytoskeleton, changes in intracellular Ca²2 .Raz E, Mahabaleshwar H. Chemokine signaling in embryonic cell migration: a fisheye view. Development. 2009;136(8):1223-9. Review. levels, activation of integrins, exocytosis of granule proteins, and respiratory burst.²⁹29 .Paccaud JP, Schifferli JA, Baggiolini M. NAP-1/IL-8 induces up-regulation of CR1 receptors in human neutrophil leukocytes. Biochem Biophys Res Commun. 1990;166(1):187-92.There are some chronic inflammatory diseases, such as psoriasis, rheumatoid arthritis, as well as pulmonary diseases, in which IL-8 is overexpressed, and high levels of IL-8 are also seen after septic shock or systemic administration of endotoxin.³⁰30 .Camp R, Bacon K, Fincham N, Mistry K, Ross J, Lawlor F, et al. Chemotactic cytokines in inflammatory skin disease. Adv Exp Med Biol. 1991;305:109-18. Review.

31 .Kunkel SL, Standiford T, Kasahara K, Strieter RM. Interleukin-8 (IL-8): the major neutrophil chemotactic factor in the lung. Exp Lung Res. 1991;17(1):17-23.-³²32 .Martich GD, Danner RL, Ceska M, Suffredini AF. Detection of interleukin 8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effect of antiinflammatory agents. J Exp Med. 1991;173(4):1021-4.

Regarding other CXC-chemokines members, neutrophils are the main responsive cell type, since they express receptors CXCR1 and CXCR2, which are able to bind to CXCL1, CXCL2, CXCL5, CXCL6, CXCL7 and CXCL8. In acute inflammation initiated by bacterial invasion, neutrophils are the first cells that infiltrate into the tissue. Neutrophils play a critical role during infection control, first by their capacity of microorganism phagocytosis, and second by releasing other chemotactic mediators, such as CXCL1, CXCL8, CXCL9 and CXCL10 that recruit other leucocytes to the affected tissues.³³33 .De Filippo K, Dudeck A, Hasenberg M, Nye E, van Rooijen N, Hartmann K, et al. Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation. Blood. 2013; 121(24):4930-7.

Chemokines control innate immune cell trafficking between the bone marrow, blood, and peripheral tissues during inflammation. At the same time, systemic levels of G-CSF lead to a reduction on CXCL12 production by bone marrow and decreased CXCR4 expression by neutrophils. Release of neutrophil from bone marrow into the blood is triggered by the switch of activated receptor, from CXCR4 to CXCR2.⁸8 .Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014; 32:659-702. Review.

Mast cells also play an important role in acute inflammation because they express a wide variety of PRR and contain large granules of preformed inflammatory mediators. LPS stimulated murine peritoneal mast cells leads to immediate release of CXCL1- and CXCL2-containing granules, but not histamine-containing granules, as well as transcriptional activation of CXCL1 and CXCL2. This promotes early neutrophil recruitment that is abolished in mast cell-depleted mice, but not macrophage-depleted mice.⁸8 .Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014; 32:659-702. Review.

CX3CL1 (or fractalkine) is an atypical chemokine with a documented role in the development of numerous inflammatory diseases, including atherosclerosis. It represents the only known member of the CX3C family and was first characterized by Bazan et al.³⁴34 .Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, et al. A new class of membrane-bound chemokine with a CX3C motif. Nature. 1997; 385(6617):640-4. It is a unique chemotactic factor existing in both membrane-bound and soluble form. The human CX3CL1 molecule consists of 373 amino acids and it is functionally divided into four domains: an extracellular domain of 76 amino acids connected to an extended 241 amino acid mucin-like stalk, followed by transmembrane and intracellular domains of 21 and 35 amino acids, respectively. CX3CL1 is synthesized as a membrane-bound molecule with the chemokine domain presented on a mucin-like stalk, which mediates the direct capture of circulating leukocytes. Cleavage at the base of this stalk by a protease, namely the tumor necrosis alpha-converting enzyme, generates a soluble chemokine, which function as a potent chemoattractant of target cells.³⁴34 .Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, et al. A new class of membrane-bound chemokine with a CX3C motif. Nature. 1997; 385(6617):640-4.,³⁵35 .Apostolakis S, Spandidos D. Chemokines and atherosclerosis: focus on the CX3CL1/CX3CR1 pathway. Acta Pharmacol Sin. 2013;34(10):1251-6. Review.

The fractalkine receptor CX3CR1 is a well-established marker of anti-inflammatory or patrolling monocytes known by providing pro-survival signal to anti-inflammatory monocytes, but is also present in NK cells, T cells, and smooth muscle cells, where it mediates migration, adhesion and proliferation. CXCR3 is also present on pro-inflammatory monocytes and may play a role on normal function of inflammatory monocytes.⁸8 .Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014; 32:659-702. Review.

In general, chemokines CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and CXCL8 are classified as inflammatory; CXCL12 and CXCL13 as homeostatic. CXCL9, CXCL10, CXCL11 and CXCL16 with dual function inflammatory and homeostatic, while CXCL4 is plasmatic or platelet related.²⁷27 .Le Y, Zhou Y, Iribarren P, Wang J. Chemokines and chemokine receptors: their manifold roles in homeostasis and disease. Cell Mol Immunol. 2004;1(2):95-104. Review.

ACKNOWLEDGMENTS

We are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant number 2014/15504-7 and to Sociedade Beneficente Israelita Brasileira Hospital Albert Einstein for their support.

REFERENCES

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