Iain Comerford

The chemokine receptor CCX-CKR mediates effective scavenging of CCL19 in vitro

The chemokines CCL19, CCL21 and CCL25, by signalling through the receptors CCR7 or CCR9, play critical roles in leukocyte homing. They also bind another heptahelical surface protein, CCX‐CKR. CCX‐CKR cannot couple to typical chemokine receptor signalling pathways or mediate chemotaxis, and its function remains unclear. We have proposed that it controls chemokine bioavailability. Here, using transfected HEK293 cells, we have shown that both CCX‐CKR and CCR7 mediate rapid CCL19 internalisation upon initial chemokine exposure. However, internalised CCL19 was more efficiently retained and degraded after uptake via CCX‐CKR. More importantly, CCR7 rapidly became refractory for CCL19 uptake, but the sequestration activity of CCX‐CKR was enhanced. These properties endowed CCX‐CKR with an impressive ability to mediate progressive sequestration and degradation of large quantities of CCL19, and conversely, prevented CCR7‐expressing cells from extensively altering their chemokine environment. These differences may be linked to the routes of endocytosis used by these receptors. CCX‐CKR, unlike CCR7, was not critically dependent on β‐arrestins or clathrin‐coated pits. However, over‐expression of caveolin‐1, which stabilises caveolae, blocked CCL19 uptake by CCX‐CKR while having no impact on other chemokine receptors, including CCR7. These data predict that CCX‐CKR scavenges extracellular chemokines in vivo to modify responses through CCR7.

Inhibition of CCR6 Function Reduces the Severity of Experimental Autoimmune Encephalomyelitis via Effects on the Priming Phase of the Immune Response

Chemokines are essential for homeostasis and activation of the immune system. The chemokine ligand/receptor pairing CCL20/CCR6 is interesting because these molecules display characteristics of both homeostatic and activation functions. These dual characteristics suggest a role for CCR6 in the priming and effector phases of the immune response. However, while CCR6 has been implicated in the effector phase in several models, a role in the priming phase is less clear. Herein we analyze the role of CCR6 in these two important arms of the immune response during experimental autoimmune encephalomyelitis (EAE). Both CCR6 and its chemokine ligand CCL20 were up-regulated in the draining lymph nodes and spinal cord during EAE, and CCR6 was up-regulated on CD4+ T cells that had divided following induction of EAE. The functional role of this expression was demonstrated by impaired development of EAE in gene-targeted CCR6-deficient mice and in mice treated either with a neutralizing anti-CCR6 Ab or with a novel receptor antagonist. Inhibition of EAE was due to reduced priming of autoreactive CD4+ T cells probably as a result of impaired late-stage influx of dendritic cells into draining lymph nodes. This was accompanied by reduced egress of activated lymphocytes from the lymph nodes. These results demonstrate a novel role for CCR6 in the mechanism of autoreactive lymphocyte priming and emigration to the efferent lymphatics.

Antagonism of the chemokine receptors CXCR3 and CXCR4 reduces the pathology of experimental autoimmune encephalomyelitis.

Chemokines regulate lymphocyte trafficking under physiologic and pathologic conditions. In this study, we have investigated the role of CXCR3 and CXCR4 in the activation of T lymphocytes and their migration to the central nervous system (CNS) using novel mutant chemokines to antagonize CXCR3 and CXCR4 specifically. A series of truncation mutants of CXCL11, which has the highest affinity for CXCR3, were synthesized, and an antagonist, CXCL11(4–79), was obtained. CXCL11(4–79) strongly inhibited the migration of activated mouse T cells in response to all three high‐affinity CXCR3 ligands, CXCL9, 10 and 11. CXCL12(P2G2), while exhibiting minimal agonistic activity, potently inhibited the migration of activated mouse T cells in response to CXCL12. Interfering with the action of CXCR3 and CXCR4 with these synthetic receptor antagonists inhibited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis and reduced the accumulation of CD4+ T cells in the CNS. Further investigation demonstrated that CXCL12(P2G2) inhibited the sensitization phase, whereas CXCL11(4–79) inhibited the effector phase of the immune response. Our data suggest that simultaneous targeting of CXCR4 and CXCR3 may be of benefit in the treatment of the CNS autoimmune disease.

A myriad of functions and complex regulation of the CCR7/CCL19/CCL21 chemokine axis in the adaptive immune system

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 control a diverse array of migratory events in adaptive immune function. Most prominently, CCR7 promotes homing of T cells and DCs to T cell areas of lymphoid tissues where T cell priming occurs. However, CCR7 and its ligands also contribute to a multitude of adaptive immune functions including thymocyte development, secondary lymphoid organogenesis, high affinity antibody responses, regulatory and memory T cell function, and lymphocyte egress from tissues. In this survey, we summarise the role of CCR7 in adaptive immunity and describe recent progress in understanding how this axis is regulated. In particular we highlight CCX-CKR, which scavenges both CCR7 ligands, and discuss its emerging significance in the immune system.

An immune paradox: how can the same chemokine axis regulate both immune tolerance and activation?: CCR6/CCL20: a chemokine axis balancing immunological tolerance and inflammation in autoimmune disease.

Chemokines (chemotactic cytokines) drive and direct leukocyte traffic. New evidence suggests that the unusual CCR6/CCL20 chemokine receptor/ligand axis provides key homing signals for recently identified cells of the adaptive immune system, recruiting both pro-inflammatory and suppressive T cell subsets. Thus CCR6 and CCL20 have been recently implicated in various human pathologies, particularly in autoimmune disease. These studies have revealed that targeting CCR6/CCL20 can enhance or inhibit autoimmune disease depending on the cellular basis of pathogenesis and the cell subtype most affected through different CCR6/CCL20 manipulations. Here, we discuss the significance of this chemokine receptor/ligand axis in immune and inflammatory functions, consider the potential for targeting CCR6/CCL20 in human autoimmunity and propose that the shared evolutionary origins of pro-inflammatory and regulatory T cells may contribute to the reason why both immune activation and regulation might be controlled through the same chemokine pathway.

PI3Kδ drives the pathogenesis of experimental autoimmune encephalomyelitis by inhibiting effector T cell apoptosis and promoting Th17 differentiation

The Class IA phosphoinositide 3-kinase delta (PI3Kδ) has been implicated in multiple signaling pathways involved in leukocyte activation and hence is an attractive target in many human autoimmune diseases, including multiple sclerosis (MS). Here, using mice expressing a catalytically inactive form of the PI3Kδ subunit p110δ, we show that signaling through PI3Kδ is required for full and sustained pathology of experimental autoimmune encephalomyelitis (EAE), a Th17-driven model of MS. In p110δ-inactivated mice, T cell activation and function during EAE was markedly reduced and fewer T cells were observed in the central nervous system (CNS). The decrease in T cell activation is unlikely to be due to defects in dendritic cell (DC) function, as p110δ-inactivated DCs migrate and present antigen normally. However, significant increases in the proportion of T cells undergoing apoptosis at early stages of EAE were evident in the absence of PI3Kδ activity. Furthermore, a profound defect in Th17 cellular responses during EAE was apparent in the absence of PI3Kδ activity while Th1 responses were less affected. A highly selective PI3Kδ inhibitor, IC87114, also had greater inhibitory effects on Th17 cell generation in vitro than it did on Th1 cell generation. Thus, PI3Kδ plays an important role in Th17 responses in EAE, suggesting that small molecule inhibitors of PI3Kδ may be useful therapeutics for treatment of MS and other autoimmune diseases.

The atypical chemokine receptor CCX-CKR scavenges homeostatic chemokines in circulation and tissues and suppresses Th17 responses

Our previous in vitro studies led to proposals that the atypical chemokine receptor CCX-CKR is a scavenger of CCR7 ligand homeostatic chemokines. In the present study, we generated CCX-CKR(-/-) mice and confirm this scavenger function in vivo. Compared with wild-type mice, CCX-CKR(-/-) have a 5-fold increase in the level of CCL21 protein in blood, and 2- to 3-fold increases in CCL19 and CCL21 in peripheral lymph nodes. The effect of these protein increases on immunity was investigated after immunization with MOG(35-55) peptide emulsified in complete Freund adjuvant (CFA). The subsequent characteristic paralysis develops with enhanced kinetics and severity in CCX-CKR(-/-) versus wild-type mice. Despite this effect, antigen-specific immune responses in the draining lymph nodes are diminished in CCX-CKR(-/-) mice. Instead, the earlier onset of disease is associated with enhanced T-cell priming in the CCX-CKR(-/-) spleen and a skewing of CD4(+) T-cell responses toward Th17 rather than Th1. This observation correlates with increased expression of IL-23 in the CCX-CKR(-/-) spleen and increased CCL21 levels in the central nervous system postimmunization. The early onset of disease in CCX-CKR(-/-) mice is reversed by systemic administration of neutralizing anti-CCL21 antibodies. Thus, by regulating homeostatic chemokine bioavailability, CCX-CKR influences the localization, kinetics, and nature of adaptive immune responses in vivo.

Multiple Roles for the C-terminal Tail of the Chemokine Scavenger D6

D6 is a heptahelical receptor that suppresses inflammation and tumorigenesis by scavenging extracellular pro-inflammatory CC chemokines. Previous studies suggested this is dependent on constitutive trafficking of stable D6 protein to and from the cell surface via recycling endosomes. By internalizing chemokine each time it transits the cell surface, D6 can, over time, remove large quantities of these inflammatory mediators. We have investigated the role of the conserved 58-amino acid C terminus of human D6, which, unlike the rest of the protein, shows no clear homology to other heptahelical receptors. We show that, in human HEK293 cells, a serine cluster in this region controls the constitutive phosphorylation, high stability, and intracellular trafficking itinerary of the receptor and drives green fluorescent protein-tagged β-arrestins to membranes at, and near, the cell surface. Unexpectedly, however, these properties, and the last 44 amino acids of the C terminus, are dispensable for D6 internalization and effective scavenging of the chemokine CCL3. Even in the absence of the last 58 amino acids, D6 still initially internalizes CCL3 but, surprisingly, exposure to ligand inhibits subsequent CCL3 uptake by this mutant. Progressive scavenging is therefore abrogated. We conclude that the heptahelical body of D6 on its own can engage the endocytotic machinery of HEK293 cells but that the C terminus is indispensable for scavenging because it prevents initial chemokine engagement of D6 from inhibiting subsequent chemokine uptake.

Multiple functions of CXCL12 in a syngeneic model of breast cancer

BackgroundA growing body of work implicates chemokines, in particular CXCL12 and its receptors, in the progression and site-specific metastasis of various cancers, including breast cancer. Various agents have been used to block the CXCL12-CXCR4 interaction as a means of inhibiting cancer metastasis. However, as a potent chemotactic factor for leukocytes, CXCL12 also has the potential to enhance anti-cancer immunity. To further elucidate its role in breast cancer progression, CXCL12 and its antagonist CXCL12(P2G) were overexpressed in the syngeneic 4T1.2 mouse model of breast carcinoma.ResultsWhile expression of CXCL12(P2G) significantly inhibited metastasis, expression of wild-type CXCL12 potently inhibited both metastasis and primary tumor growth. The effects of wild-type CXCL12 were attributed to an immune response characterized by the induction of CD8+ T cell activity, enhanced cell-mediated cytotoxicity, increased numbers of CD11c+ cells in the tumor-draining lymph nodes and reduced accumulation of myeloid-derived suppressor cells in the spleen.ConclusionsThis study highlights the need to consider carefully therapeutic strategies that block CXCL12 signaling. Therapies that boost CXCL12 levels at the primary tumor site may prove more effective in the treatment of metastatic breast cancer.

Mini-review series: focus on chemokines

Our understanding of the significance and complexity of the chemokine superfamily has increased at an explosive pace over the last decade. Although the rapid phase of chemokine discovery and characterisation is likely to have come to an end and basic questions regarding the functional significance of most chemokines have been addressed, many questions remain in this field, several of which are highlighted in the following review series. Through interactions with their cognate serpentine G protein-coupled receptors (Figure 1a), chemokines make a critical contribution to a diverse range of physiological and pathological processes including embryogenesis, immune system development and function, inflammation, tumourigenesis and cancer metastasis.1, 2, 3 As every immunologist is by now aware, chemokines serve as critical extracellular mediators of cell migration, particularly in the immune system.4, 5, 6