Atypical chemokine receptors: from silence to sound.

ACRs (atypical chemokine receptors) were initially referred to as 'silent' receptors on the basis of a lack of signalling and functional activities that are typically observed with conventional chemokine receptors. Although ACRs do not directly induce cell migration, they indirectly control leucocyte recruitment by shaping chemokine gradients in tissues through degradation, transcytosis or local concentration of their cognate ligands. Recent evidence also suggests that these biological activities are supported by G-protein-independent, ß-arrestin-dependent signalling events. In the present article, we review current knowledge on structural and signalling properties of ACRs that are changing our view on this entire class of receptors from silent to endogenous ß-arrestin-biased signalling receptors.

Reduced G protein signaling despite impaired internalization and ß-arrestin recruitment in patients carrying a CXCR4Leu317fsX3 mutation causing WHIM syndrome.

WHIM syndrome is an inherited immune disorder caused by an autosomal dominant heterozygous mutation in CXCR4. The disease is characterized by neutropenia/leukopenia (secondary to retention of mature neutrophils in bone marrow), recurrent bacterial infections, treatment-refractory warts, and hypogammaglobulinemia. All mutations reported in WHIM patients lead to the truncations in the C-terminal domain of CXCR4, R334X being the most frequent. This defect prevents receptor internalization and enhances both calcium mobilization and ERK phosphorylation, resulting in increased chemotaxis in response to the unique ligand CXCL12. Here, we describe 3 patients presenting neutropenia and myelokathexis, but normal lymphocyte count and immunoglobulin levels, carrying what we believe to be a novel Leu317fsX3 mutation in CXCR4, leading to a complete truncation of its intracellular tail. The analysis of the L317fsX3 mutation in cells derived from patients and in vitro cellular models reveals unique signaling features in comparison with R334X mutation. The L317fsX3 mutation impairs CXCR4 downregulation and ß-arrestin recruitment in response to CXCL12 and reduces other signaling events - including ERK1/2 phosphorylation, calcium mobilization, and chemotaxis - all processes that are typically enhanced in cells carrying the R334X mutation. Our findings suggest that, overall, the L317fsX3 mutation may be causative of a form of WHIM syndrome not associated with an augmented CXCR4 response to CXCL12.

Chemokine decoy receptors: structure-function and biological properties.

Chemokines induce cell migration through the activation of a distinct family of structurally related heterotrimeric G protein-coupled receptors (GPCR). Over the last few years, several receptors in this family that recognize chemokines but do not induce cell migration have been identified. These "atypical" chemokine receptors are unable to activate transduction events that lead directly to cell migration, but appear nonetheless to play a nonredundant role in the control of leukocyte recruitment at inflammatory sites and in tumors by shaping the chemoattractant gradient, either by removing, transporting, or concentrating their cognate ligands.

The lymphatic system controls intestinal inflammation and inflammation-associated Colon Cancer through the chemokine decoy receptor D6.

BACKGROUND AND AIMS: Inflammatory CC chemokines have long been associated with cancer, but unequivocal evidence of a role in clinically relevant models of carcinogenesis is lacking. D6, a promiscuous decoy receptor that scavenges inflammatory CC chemokines, plays a non-redundant role in reducing the inflammatory response in various organs. As inflammation is a key player in the development of inflammatory bowel disease (IBD) and IBD-associated colorectal cancer, we investigated D6 expression in human colitis and colon cancer, and its role in experimental colitis and inflammation-associated colon cancer. RESULTS: In humans, D6 was mainly expressed by lymphatic vessels and leukocytes in the mucosa of individuals with IBD and colon cancer, as well as the mucosa of control individuals. Mice lacking expression of D6 were significantly more susceptible to experimental colitis than wild-type mice and failed to resolve colitis, with significantly higher levels of several pro-inflammatory chemokines. In bone marrow chimeric mice, the ability of D6 to regulate colitis was tracked to the stromal/lymphatic compartment, with no contribution of haemopoietic cells. Finally, after administration of the carcinogen azoxymethane, D6(-/-) mice showed increased susceptibility to colitis-associated cancer in the distal segment of the colon compared with wild-type mice. CONCLUSIONS: D6 expressed on lymphatic vessels plays a key role in the control of intestinal inflammation and the development of inflammation-associated colon cancer. Our results reveal a new unexpected role for the lymphatic system in the pathogenesis of IBD and intestinal cancer, and candidate chemokines as novel players in tumour promotion and progression.

Endogenous modification of the chemoattractant CXCL5 alters receptor usage and enhances its activity toward neutrophils and monocytes.

The inflammatory human chemokine CXCL5 interacts with the G protein-coupled receptor CXCR2 to induce chemotaxis and activation of neutrophils. CXCL5 also has weak agonist activity toward CXCR1. The N-terminus of CXCL5 can be modified by proteolytic cleavage or deimination of Arg9 to citrulline (Cit), and these modifications can occur separately or together. Here, we chemically synthesized native CXCL5(1-78), truncated CXCL5 [CXCL5(9-78)], and the citrullinated (Cit9) versions and characterized their functions in vitro and in vivo. Compared with full-length CXCL5, N-terminal truncation resulted in enhanced potency to induce G protein signaling and ß-arrestin recruitment through CXCR2, increased CXCL5-initiated internalization of CXCR2, and greater Ca2+ signaling downstream of not only CXCR2 but also CXCR1. Citrullination did not affect the capacity of CXCL5 to activate classical or alternative signaling pathways. Administering the various CXCL5 forms to mice revealed that in addition to neutrophils, CXCL5 exerted chemotactic activity toward monocytes and that this activity was increased by N-terminal truncation. These findings were confirmed by in vitro chemotaxis and Ca2+ signaling assays with primary human CD14+ monocytes and human THP-1 monocytes. In vitro and in vivo analyses suggested that CXCL5 targeted monocytes through CXCR1 and CXCR2. Thus, truncation of the N-terminus makes CXCL5 a more potent chemoattractant for both neutrophils and monocytes that acts through CXCR1 and CXCR2.

Regulation of D6 chemokine scavenging activity by ligand- and Rab11-dependent surface up-regulation.

The decoy receptor D6 plays a nonredundant role in the control of inflammatory processes through scavenging of inflammatory chemokines. However it remains unclear how it is regulated. Here we show that D6 scavenging activity relies on unique trafficking properties. Under resting conditions, D6 constitutively recycled through both a rapid wortmannin (WM)-sensitive and a slower brefeldin A (BFA)-sensitive pathway, maintaining low levels of surface expression that required both Rab4 and Rab11 activities. In contrast to "conventional" chemokine receptors that are down-regulated by cognate ligands, chemokine engagement induced a dose-dependent BFA-sensitive Rab11-dependent D6 re-distribution to the cell membrane and a corresponding increase in chemokine degradation rate. Thus, the energy-expensive constitutive D6 cycling through Rab11 vesicles allows a rapid, ligand concentration-dependent increase of chemokine scavenging activity by receptor redistribution to the plasma membrane. D6 is not regulated at a transcriptional level in a variety of cellular contexts, thus ligand-dependent optimization of its scavenger performance represents a rapid and unique mechanism allowing D6 to control inflammation.

Male age: negative impact on sperm DNA fragmentation.

The main goal of semen processing in Assisted Reproductive Techniques (ART) is to select sperm with good viability and, at the same time, remove Reactive Oxygen Species (ROS) sources (such as leukocytes) and reduce the percentage of morphologically abnormal sperm for fertility treatment. We performed a comparative analysis on sperm DNA fragmentation after Density Gradient Centrifugation (DGC) using products sold by two competing companies. Our results showed comparable DNA Fragmentation Index (DFI) after treatment with both DGC products. However, in both cases, a comparable number of samples do not benefit from the treatment. Interestingly, increasing evidences indicated that male age has a negative impact on sperm DNA fragmentation, but the mechanisms underlying age-dependent patterns of sperm decline have not yet been fully understood. Thus, we performed a comparative analysis of DFI before and after treatment with DGC products in age-stratified sample populations. Our results showed a worsening of the baseline DFI in the eldest group and the benefits of DGC on sperm DNA were compromised. In conclusion, our work consolidates the current evidences suggesting that both paternal and maternal aging, critically affects reproductive success.

Non-signaling chemokine receptors: mechanism of action and role in vivo.

Cell migration is fundamental for numerous biological processes and is critical for the pathogenesis of several diseases. Chemokines represent the main class of mediators providing cell directional migration and several levels of regulation of their function have been identified. A subfamily of chemokine receptors not able to transduce chemotactic signals plays an important role in the control of chemokine concentrations through binding, internalization and degradation of chemotactic factors. Here we review in vitro and in vivo evidences indicating that these 'silent' chemokine receptors represent a strategy to regulate innate and adaptive immunity.

Review: Structure-function and biological properties of the atypical chemokine receptor D6.

The atypical chemokine receptor D6 was initially called "silent" on the basis of lack of conventional signaling events that lead to directional cell migration. It has emerged that D6 is able to bind and drive to degradative compartments most inflammatory CC chemokines and that is able to convey G-protein independent signaling events to optimize its scavenging activity. We here summarize the knowledge available today on D6 structural and signaling properties and its essential role for the control of inflammatory cells traffic and proper development of the adaptive immune response.

Expression of the atypical chemokine receptor D6 in human alveolar macrophages in COPD.

BACKGROUND: D6 is an atypical chemokine receptor involved in chemokine degradation and resolution of acute inflammatory responses in mice. Emerging evidence suggests that D6 might behave differently in human chronic inflammatory conditions. We, therefore, investigated the involvement of D6 in the immune responses in COPD, a chronic inflammatory condition of the lung. METHODS: D6 expression was quantified by immunohistochemistry in surgical resected lung specimens from 16 patients with COPD (FEV(1), 57% ± 6% predicted) and 18 control subjects with normal lung function (nine smokers and nine nonsmokers). BAL was also obtained and analyzed by flow cytometry, immunofluorescence, and molecular analysis for further assessment of D6 involvement. RESULTS: D6 expression in the lung was mainly detected in alveolar macrophages (AMs). The percentage of D6(+) AMs was markedly increased in patients with COPD as compared with both smoker and nonsmoker control subjects (P < .0005 for both). D6 expression was detected at both transcript and protein level in AMs but not in monocyte-derived macrophages. Finally, D6 expression was positively correlated with markers of immune activation (CD8(+) T lymphocytes, IL-32, tumor necrosis factor-α, B-cell activating factor of the tumor necrosis factor family, phospho-p38 mitogen-activated protein kinase) and negatively with lung function (FEV(1), FEV(1)/FVC). CONCLUSIONS: D6 is expressed in AMs from patients with COPD, and its expression correlates with the degree of functional impairment and markers of immune activation. Upregulation of D6 in AMs could indicate that, besides its known scavenger activity in acute inflammation, D6 may have additional roles in chronic inflammatory conditions possibly promoting immune activation.

ß-arrestin-dependent activation of the cofilin pathway is required for the scavenging activity of the atypical chemokine receptor D6.

Chemokines promote the recruitment of leukocytes to sites of infection and inflammation by activating conventional heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). Chemokines are also recognized by a set of atypical chemokine receptors (ACRs), which cannot induce directional cell migration but are required for the generation of chemokine gradients in tissues. ACRs are presently considered "silent receptors" because no G protein-dependent signaling activity is observed after their engagement by cognate ligands. We report that engagement of the ACR D6 by its ligands activates a ß-arrestin1-dependent, G protein-independent signaling pathway that results in the phosphorylation of the actin-binding protein cofilin through the Rac1-p21-activated kinase 1 (PAK1)-LIM kinase 1 (LIMK1) cascade. This signaling pathway is required for the increased abundance of D6 protein at the cell surface and for its chemokine-scavenging activity. We conclude that D6 is a signaling receptor that exerts its regulatory function on chemokine-mediated responses in inflammation and immunity through a distinct signaling pathway.

Chemokine receptors intracellular trafficking.

Chemokines coordinate leukocyte recruitment during inflammatory and immune responses through the interaction with a distinct subfamily of G protein-coupled receptors. The magnitude of the cellular response elicited by chemokines is dictated by the level of receptor expression at the plasma membrane, which is the balance of finely tuned endocytic and recycling pathways. Recent data have revealed that receptor trafficking properties can drive chemokine receptors to lysosomal degradation or recycling pathways, producing opposite effects on the strength of the intracellular signaling cascade. This review will cover recent advances on the molecular mechanisms underlying chemokine receptor internalization, recycling and degradation pathways, with particular attention to structural motifs present in receptor intracellular domains and their interacting adaptor proteins that modulate receptor trafficking and dictate proper biological response.

Role of the chemokine scavenger receptor D6 in balancing inflammation and immune activation.

Chemokines play a major role in the induction of inflammatory reactions and development of an appropriate immune response by coordinating leukocyte recruitment. The appropriate control of the chemokine system involves several chemokine decoy receptors, with distinct specificity and tissue distribution, defined as nonactivating chemokine receptors able to bind the ligands and target them to degradation. The best-characterized representative of these receptors is D6, which is located on lymphatic endothelium and controls most inflammatory CC chemokines. Here we will discuss the expression and regulation of D6 during challenge with the pathogen, and its role in dampening inflammation in tissues and draining lymph nodes and in the organization of a protective immune response.

Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6.

Fetal loss in animals and humans is frequently associated with inflammatory conditions. D6 is a promiscuous chemokine receptor with decoy function, expressed in lymphatic endothelium, that recognizes and targets to degradation most inflammatory CC chemokines. Here, we report that D6 is expressed in placenta on invading extravillous trophoblasts and on the apical side of syncytiotrophoblast cells, at the very interface between maternal blood and fetus. Exposure of D6-/- pregnant mice to LPS or antiphospholipid autoantibodies results in higher levels of inflammatory CC chemokines and increased leukocyte infiltrate in placenta, causing an increased rate of fetal loss, which is prevented by blocking inflammatory chemokines. Thus, the promiscuous decoy receptor for inflammatory CC chemokines D6 plays a nonredundant role in the protection against fetal loss caused by systemic inflammation and antiphospholipid antibodies.