CORSO DI DOTTORATO DI RICERCA IN PATOLOGIA E NEUROPATOLOGIA SPERIMENTALI CICLO XXIII

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1 SCUOLA DI DOTTORATO IN SCIENZE BIOMEDICHE CLINICHE E SPERIMENTALI DIPARTIMENTO DI MEDICINA TRASLAZIONALE CORSO DI DOTTORATO DI RICERCA IN PATOLOGIA E NEUROPATOLOGIA SPERIMENTALI CICLO XXIII BIOLOGICAL AND TRANSDUCTIONAL EFFECTS OF ALLOSTERIC ANTAGONISTS ON THE ACTIVITY OF CHEMOATTRACTANT RECEPTORS Settore didattico/disciplinare: MED/04 Tutore: Prof. Massimo LOCATI Coordinatore: Prof. Alberto MANTOVANI Tesi di Dottorato di: NINA PATRICIA MACHADO TORRES Matr. N. R07914 Anno Accademico

2 1.INTRODUCTION 1.1 The chemokine system Over 40 chemokines have been identified to date 1. Chemokines are produced by a variety of cell types either constituvely or in response to inflammatory stimuli, the biological activities of chemokines range from the control of leukocyte trafficking in basal and inflammatory conditions to regulation of hematopoiesis, angiogenesis, tissue architecture and organogenesis. The basis for such diversified activities rests, on one hand, upon the ubiquitous nature of chemokine production and chemokine receptor expression. Indeed virtually every cell type can produce chemokines and expresses a unique combination of chemokine receptors. On the other hand, chemokine receptors make use of a flexible and complex network of intracellular signaling machineries that can regulate a variety of cellular functions ranging from cell migration, growth, differentiation and death Structure and biological function of chemokines Chemokines are small secreted proteins with the molecular weights in the range of 8-12-KDa with 20 to 70 percent homology in amino acids sequences. They have been subdivided into families based on structural and genetic considerations; structurally they are similar, having at least three β-pleated sheets and a C-terminal α-helix, with disulfide bonds stabilizing overall topology (Figure 1) 1, 3. Most chemokines have four characteristics cysteines (cys) in highly conserved positions and depending on the motif displayed by the first two cysteins, they have been classified into CXC or alpha (α)-chemokines, CC or beta (β), C or gamma (γ), and CX3C or delta (δ) chemokines classes 1, 3. The only exception to the cysteins rule is lymphotactin, which has only two cysteins. In addition the CXC or alpha subfamily 8

3 has been divided into two groups depending on the presence of the ELR motif (glutamate-leucine-arginine) preceding the first cys: the ELR-CXC chemokines and the no-elr-cxc chemokines 4 (Figure 2). After translation, most chemokines are secreted from the cell, with the exception of CX3CL1 and CXCL16, which are tethered to the extracellular surface. In the case of CXC3CL1 can act not only as chemoattractant but also as adhesion molecule 5. Figure 1. Chemokines family. Chemokines are divided into families based on structural consideration. Structurally they are similar, having at least three β-pleated sheets (indicated as β1-3) and a C-terminal α-helix. Chemokines have at least four cysteins in conserved positions. Except lymphotactin C family. (Rollins, B.J. Blood, 1997). 9

4 Important aspects concerning chemokines include their biological activities and the chromosomal location of the genes that encode them. They are best known from mammals, but chemokine genes have also been found in chicken, zebrafish, shark and jawless fish genomes, and possible homologs of chemokine receptor have been reported in nematodes. Careful analysis of the members of the superfamily and their receptors shows a logical order to its genomic organization and function, which in turn is the result of evolutionary pressures 6. The chemokines have been divided into two major groups based on their expression patterns and functions. Those that are expressed by cells of the immune system (leukocytes) or other cells (epithelial and endothelial cells, fibroblasts and so on) only upon activation belong to the inflammatory class, whereas those that are expressed in discrete location in the absence of apparent activating stimuli have been classified as homeostatic. However, the genomic organization has allowed dividing into two alternative groups: those whose genes are located in large clusters at particular chromosomal location (the major-clusters, CC and CXC chemokines genes) and the no cluster or mini-cluster. This common evolutionary origin suggests that the cluster chemokines are a group of proteins sharing a common primary function. In the case of the chemokines encoded by CXC GRO cluster on chromosome 4, which in humans include CXCL1 - CXCL8, the primary function is the regulation of neutrophils recruitment to inflammatory sites mediated by through interaction with CXCR1 and CXCR2 receptors. Similarly, the main function of the cytokines encoded in the MIP (Macrophage Inflammatory Protein) and MCP (Monocyte Chemotactic Protein) clusters of CC chemokines in human chromosome 17, which includes CCL1-CCL16, CCL18 and CCL23 is the recruitment of monocytes, subset of T cells, and eosinophils, to sites where inflammation is developing, through their interaction with CCR1, CCR2, CCR3 and/ or CCR5 (Figure 2). 10

5 By contrast, the non cluster or mini-cluster chemokines are relatively conserved between species and tend not to act on multiple receptors (Figure 2). Indeed several of these have a single ligand-receptor relationship, such as CCL25-CCR9 or CXCL13- CXCR5. These particular chemokine ligand-receptor pairs probably have a pivotal roles in the development of the organism or in the function of physiological systems necessary for the organism survival to reproductive age. For example, CXCR4 deficient and CXCL12-deficient mice both have various defects in critical organs, such as the heart, brain and bone marrow 7. Therefore, throughout evolution, several noncluster chemokines have participated in organogenesis, and their critical functions must be conserved in order for the species to survive 6, 8. Functionally, the CXC chemokines play a key role in acute inflammation 9. The prototypic CXC chemokine is CXCL8, which was purified by several groups as a monocytes-derived factor that attracts neutrophils, but no monocytes in Boyden chamber assay. Several other CXC chemokines have been described as potent neutrophil chemoattractants, and structure/activity analyses show that this property depends on the presence of a three amino acids motif ELR between the N-terminus and the first cysteine. However, these amino acids must appear in position close to the protein s N-termini. For example PBP (platelet basic protein) and two of its N- terminally truncated derivatives, CTAPIII and β thromboglobulin, have very weak neutrophil chemoattractant activity despite the presence of ELR. Only NAP-2, a further truncated product in which ELR appears close to the N-terminus, is an active PBPderived neutrophil attractant 8. CXCL8 is produced by a variety of cells types including monocytes, T lymphocytes, neutrophils, fibroblasts, endothelial cells and epithelial cells. The most abundant form of CXCL8 is a protein with 72 amino acids. There is a variant form extended at the N-terminus occasionally called endothelial IL-8 because its synthesis by these cells. In 11

6 vitro the longer protein is ~10- fold less potent than the shorter protein in attracting and activating neutrophils, but has similar potency, perhaps due to proteolytic processing to the short form. The 77 amino acid form may be involved in neutrophil adherence to the endothelium as a prelude to diapedesis. Other properties attributed to CXCL8 include chemoattraction of T lymphocytes and angiogenic activity even if the latter effect is controversial by reported absence of IL-8 receptors on endothelial cells 8. Other members belonging to this family is CXCL1 also functionally identified as a neutrophil-specific chemoattractant secreted by activated mononuclear cells along with CXCL8 and having similar potency. CXCL2 and CXCL3 are closely related proteins that are also potent neutrophil attractants. CXCL5 places into the GRO proteins and like these chemokines it specifically attracts neutrophils. Similarly, CXCL6 is a neutrophil-specific chemoattractant and activator, but has a specific activity ~ 5-10 folds lower than CXCL8. The ELR-chemokines have an apparent uniformity of function which it makes to think of them as a family of neutrophil chemoattractants and activators. In contrast, the non-elr CXC chemokines have a poor neutrophil atracttant activity. For example CXCL4 found in platelet α-granules along with PBP and its processed products, but unlike those proteins it has no ELR motif and is an extremely weak attractant for neutrophils. Another non-elr CXC chemokine that has antiangiogenic properties is CXCL10, the product of an interferon-γ (IFN-γ)-inducible gen. CXCL10 is expressed by a variety of cells types including mononuclear cells, keratinocytes, fibroblasts, endothelial cells, and T lymphocytes. In mice, IFN-γ administration induces high levels of CXCL10 in liver and kidney with lower levels in the spleen. Similar to other non-elr chemokines, CXCL10 is a poor neutrophil chemoattractant and activator. CXCL11 is another IFN-γ-inducible protein isolated from macrophages. It has chemoattractant activity in vitro for tumor-infiltrating lymphocytes. Either CXCL11 or 12

7 CXCL10 attract tumor-infiltrating lymphocytes and cross-desensitize in other measures of receptor activation, consistent with the fact that on tumor-infiltrating lymphocytes they share the same receptor CXCR3. Finally CXCL12, cloned from mouse bone marrow stromal cells. In humans CXCL12 has a low-potency as chemoattractant for T lymphocytes in vitro. However, perhaps relevant to its provenance, CXCL12 is also a potent chemoattractant for CD34+ hematopoietic progenitors. In vivo, targeted gene disruption of murine CXCL12 indicates that it s required for normal B lymphocyte development and for normal cardiac organogenesis 8. Figure 2. The chemokine and their receptors. Systematic nomenclature for each chemokine is given along with common old name or names. The select ligands are identified with old acronym and the new nomenclature, in which the first part of the name identifies the family and L stands for ligand, followed by a progressive number. Red indentifies predominantly inflammatory or inducible chemokines; green, homeostatic agonists; yellow molecules belong to both realms. Ba, basophils; Eo, eosinophils; idc, immature dendritic cells; MC, mast cells; mdc, mature dendritic cells; Mo, monocytes; MØ, macrophages, NK, natural killer cells; PMN, neutrophils; T act, activated T cells; T naïve, naïve T cells; T reg, regulatory T cells; T skin, skin homing T cells (Locati, M. Am J Clin Pathol, 2005). 13

8 1.3 Receptors and chemokine interactions Chemokines interact with 22 G protein-coupled receptors possessing a seven transmembrane domain (7TM)organization 5. Approximately 20 signaling chemokine receptors have been reported 7 CXCRs, 10 CCRs, 1 CX3CR, and 1 XCR, 10, 11 plus 3 nonsignaling receptors with high structural similarity to classic conventional signaling receptors, namely the Duffy antigen receptor for chemokines (DARC), D6, and CCX CKR. These molecules share the ability to bind chemokines with high affinity in the absence of any demonstrable signaling function and therefore are indicated as silent receptors. Silent receptors have been suggested to favor transfer of chemokines across endothelial barriers and/or to act as decoy receptors which dampen inflammatory reaction by binding, internalizing and, in the case of D6, degrading chemokines 10, 12. Chemokines receptors belong to the large family of 7TM receptors which couple to heterotrimeric GTP-binding proteins (G proteins). All chemokine receptors are single polypeptide chains with seven helical membrane- spanning regions connected by extramembranous loops, with an acidic N-terminal extracellular domain and three extracellular loops exposed outside the cell, whereas the serine-threonine- rich C- terminus and three intracellular loops face to the cytoplasm. Two disulfide bonds between the N-terminal domain and the second extracellular loop and between the first and third extracellular loop normally are required for the molecule structure 3, 10. The ability of chemokine receptors to signal upon ligand binding is due, at least in part, to the presence of a DRY motif in the second intracellular loop, which is missing in scavenger receptors. Despite a wealth of data related to GPCRs in general, many aspects of ligand binding and signaling are poorly understood at the molecular level. Structural understanding of GPCRs has benefited from a number or recent breakthrough, including the recent release of the first structure of a chemokine 14

9 receptor 13 in complex with a small- molecule antagonist and with a cyclic peptide inhibitor. Similarly to the previously determined high- resolution structures of the β 2 - adrenergic receptor (β 2 -AR) and A 2A adenosine receptor (A 2A AR), the overall structure of CXCR4 bound to the small molecule IT1t consists of the canonical bundle 7TM α- helices (Figure 3A) with some differences regarding to the disposition of the TM helices compared with β 2 -AR and A 2A AR. Substantially both intracellular and extracellular tips of helix IV in CXCR4 deviate (~5 and ~3Å, respectively)from their consensus positions in other GPCRs. The extracellular end of the helix V in CXCR4 is about one turn longer. Helix VI has a similar shape in all structures and is characterized by a sharp kink at the highly conserved residue, Proline 254. Finally, the extracellular end of helix VII in CXCR4 is two helical turns longer that in other GPCR structures. It is comes as a surprise that in CXCR4 structure, helix VII is about one turn shorter at the intracellular side, ending just after the GPCR-conserved NPxxY motif, and that the structure lack the short α helix VIII 13 (Fig 3B). Figure 3. Chemokine receptor structure. Overall fold of the CXCR4-IT1t complex and comparison with other GPCRs structures. (A) Overall fold of the CXCR4. The receptor is colored blue. The N terminus, ECL1, ECL2, and ECL3 are highlighted in brown, blue and red, respectively. The compound IT1t is shown in magenta stick representation. The disulfide bonds are yellow. Conserved water molecules are shown as red spheres. (B) Comparison of TM helices for CXCR4 (blue); B 2 AR (yellow); A 2A AR (green); and rhodopsin (pink) (Wu, B. Science, 2010 in press). 15

10 Receptor expression is a crucial determinant of the spectrum of action of chemokines. Early studies have indicate that polarized T helper type 1/T cytotoxic type 1 (Th1/Tc1) and Th2/Tc2 populations show differential receptor expression and responsiveness to chemokines, and that activation is associated with differential regulation of receptor expression 5 (Figure 4). Figure 4. Chemokines expression in polarized type 1 and type 2 responses. Chemokines in polarized type 1 and type2 T-cell responses. During type 1 (a) and type 2 (b) immune responses, master cytokines regulate chemokine production by stromal and inflammatoru cells: chemokines then support selective recruitment of polarized T cells and specific type 1 and type 2 effector cells expressing distinct panels of chemokines receptors. Eo, eosinophils; Ba, basophils; DC, dendritic cells; IFNγ, interferon-γ; MC, mast cells; NK, natular killer cells; Tc, cytotoxic cells; Th, T helper cells (Mantovani, A. Immunol Today, 1999). 16

11 Numerous chemokine receptors are highly promiscuous in their chemokine selectivity, and viceversa, numerous chemokines bind to more than one receptor. These redundancy in the chemokine system is most frequently associated with inflammation, and is in contrast with several monogamous chemokine systems involved in homeostatic leukocyte development and migration processes 11. Chemokine receptors are expressed on different types of leukocytes. Some receptors are restricted to certain cells, (e.g., CXCR1 is predominantly restricted to neutrophils), whereas others are more widely expressed (e.g., CCR2 is expressed on monocytes, T cells, natural killer cells, dendritic cells, and basophils). In addition, chemokine receptors are constituvely expressed in some cells, whereas they are inducible on others. CCR1 and CCR2 are constituvely expressed on monocytes but are expressed on lymphocytes only after stimulation by IL-2 1. In contrast, the expression of other chemokine receptors is restricted to a cell state of activation and differentiation. For example, CXCR3 is expressed on T lymphocytes of the T helper type 1 (Th1) phenotype, whereas CCR3, in addition to being expressed on eosinophils and basophils, is preferentially expressed on activated lymphocytes of the T helper type 2 (Th2) (Figure 4). In this way, transient up-regulation of chemokine receptors on leukocytes allows for the selective amplification of either a cell mediated Th1-type immune response or a Th2-type response. Some chemokine receptors are also expressed on non hematopoietic cells, including neurons, astrocytes, epithelial cells and endothelial cells, suggesting that chemokine system has other roles in addition to leukocyte chemotaxis 1. 17

12 1.4 Roles of chemokine and chemokine receptors in inflammation and immune surveillance To ensure immunity is necessary to control the homeostatic circulation of leukocytes through tissues. Different types of cells are addressed into inflamed tissue in an organized manner bringing naïve lymphocytes into the lymph nodes, where they encounter antigen and become in memory lymphocytes. This process is regulated by other bone marrow-derived cells such a macrophages, eosinophils, and mast cells all of them regulated by chemokines that ensures the movement of cells 1. The dramatic increase in the secretion of chemokines results in the selective recruitment of leukocytes into inflamed tissues. Chemokines have been detected during inflammation in most organs, including the skin, brain, joints, meninges, lungs, blood vessels, kidneys, and gastrointestinal tract. They have also been identified in many types of cells during inflammation in these organs, indicating that most, if not all cells can secrete chemokines given the appropriate stimulus 1. The role of chemokines in regulating movement of the cells into tissues began to be elucidated on the basis of studies in mice deficient in a particular chemokine. Several groups have developed models to provide insights into how chemokines work in vivo. For example CXCL12 is critical for the migration of myeloid precursor from the fetal liver to the bone marrow 14. Mice expressing CXCL8 under the control of liver-specific promoter/enhancers did not develop neutrophil infiltrates in their livers 15. Instead, they had high serum levels of IL-8 that were associated with L-selectin shedding from circulating neutrophils and an inability to induce neutrophil extravasation in response to local stimuli. This was similar to the observations that intravenous administration of CXCL8 in rabbits prevents local neutrophil accumulation 16, 17. Along the same line, mice over expressing CCL2 under the control of the MMTV-LTR had high serum levels of CCL2 and no 18

13 monocytic infiltration in expressing organs. This suggests that like, CXCL8 over expression, CCL2 over expression rendered circulating monocytes inacapable of responding to local physiological levels of CCL2 18. In contrast to these models, expression of chemokines at low levels in anatomically restricted areas can produce leukocytic infiltration. Consistent with their in vitro properties, mice expressing murine CXCL1 in the thymus or brain had neutrophil-rich infiltrates in these organs but it was not observed tissue damage associated with the acute infiltrate 19. These results all together provided at least two insights into how chemokines work in vivo. First, chemokines exert their attractant activity only when they are expressed locally at low levels; systemically administered actually antagonize the local effect. Second, chemokines appear to attract leukocytes without activating them, suggesting that chemokine function in leukocyte trafficking is restricted to attraction, and other signals are necessary for activation Role and implications in leukocyte movement Cells migrating in a tissue encounter many different signals that can potentially direct their path. Cells of the immune system require very accurate positioning within tissues to perform their biological functions. However little is known about how leukocytes navigate through the complex environments 20. Each leukocytes type has the capacity to respond to multiple different chemokines and/or classical attractants. Furthermore, many of the attractants that act on a given cell may be present together in a recruiting tissue. One classical example is a site of bacterial infection, in which a variety of neutrophil s chemoattractant are produced by different sources: host endothelial, epithelial, and stromal cells produce arrays of attractants including CXCL8, CXCL1 and others; complement deposition on pathogens releases the 19