Antal Rot

CCR7 and its ligands: balancing immunity and tolerance

A key feature of the immune system is its ability to induce protective immunity against pathogens while maintaining tolerance towards self and innocuous environmental antigens. Recent evidence suggests that by guiding cells to and within lymphoid organs, CC-chemokine receptor 7 (CCR7) essentially contributes to both immunity and tolerance. This receptor is involved in organizing thymic architecture and function, lymph-node homing of naive and regulatory T cells via high endothelial venules, as well as steady state and inflammation-induced lymph-node-bound migration of dendritic cells via afferent lymphatics. Here, we focus on the cellular and molecular mechanisms that enable CCR7 and its two ligands, CCL19 and CCL21, to balance immunity and tolerance.

International union of pharmacology. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors

Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145–176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.

CXCR7 functions as a scavenger for CXCL12 and CXCL11.

Background CXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues. Methodology/Principal Findings We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium. Conclusions/Significance The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.

The chemokine receptor D6 limits the inflammatory response in vivo.

How the inflammatory response is initiated has been well defined but relatively little is known about how such responses are resolved. Here we show that the D6 chemokine receptor is involved in the post-inflammatory clearance of β-chemokines from cutaneous sites. After induction of inflammation by phorbol esters, wild-type mice showed a transient inflammatory response. However, in D6-deficient mice, an excess concentration of residual chemokines caused a notable inflammatory pathology with similarities to human psoriasis. These results suggest that D6 is involved in the resolution of the cutaneous inflammatory response.

The β-Chemokine Receptor D6 Is Expressed by Lymphatic Endothelium and a Subset of Vascular Tumors

The lymphatic vessels (lymphatics) play an important role in channeling fluid and leukocytes from the tissues to the secondary lymphoid organs. In addition to driving leukocyte egress from blood, chemokines have been suggested to contribute to leukocyte recirculation via the lymphatics. Previously, we have demonstrated that binding sites for several pro-inflammatory beta-chemokines are found on the endothelial cells (ECs) of lymphatics in human dermis. Here, using the MIP-1alpha isoform MIP-1alphaP, we have extended these studies to further support the contention that the in situ chemokine binding to afferent lymphatics exhibits specificity akin to that observed in vitro with the promiscuous beta-chemokine receptor D6. We have generated monoclonal antibodies to human D6 and showed D6 immunoreactivity on the ECs lining afferent lymphatics, confirmed as such by staining serial skin sections with antibodies against podoplanin, a known lymphatic EC marker. In parallel, in situ hybridization on skin with antisense D6 probes demonstrated the expression of D6 mRNA by lymphatic ECs. D6-immunoreactive lymphatics were also abundant in mucosa and submucosa of small and large intestine and appendix, but not observed in several other organs tested. In lymph nodes, D6 immunoreactivity was present on the afferent lymphatics and also in subcapsular and medullary sinuses. Tonsilar lymphatic sinuses were also D6-positive. Peripheral blood cells and the ECs of blood vessels and high endothelial venules were consistently nonreactive with anti-D6 antibodies. Additionally, we have demonstrated that D6 immunoreactivity is detectable in some malignant vascular tumors suggesting they may be derived from, or phenotypically similar to, lymphatic ECs. This is the first demonstration of chemokine receptor expression by lymphatic ECs, and suggests that D6 may influence the chemokine-driven recirculation of leukocytes through the lymphatics and modify the putative chemokine effects on the development and growth of vascular tumors.

Platelet-released supernatants increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions

Platelet-rich plasma is currently promoted to serve as an adjuvant for bone grafts to enhance quantity and quality of newly forming bone; however, the underlying cellular mechanisms are not fully understood. We show here that supernatants of leukocyte-depleted thrombin-activated platelets increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions. Using neutralizing antibodies raised against platelet-derived growth factor (PDGF), the observed effects of platelet-released supernatants were diminished. The mitogenic response was also decreased when extracellular signal-regulated protein kinase (ERK) signalling was inhibited by PD98059; however, PD98059 did not reverse the effects of platelet-released supernatants on migration and osteogenic differentiation. Consistent with an ERK-mediated mitogenic activity, incubation of serum-starved mesenchymal cell progenitors with platelet-released supernatants increased phosphorylation of the kinase. Together, these observations indicate that PDGF is a key factor released upon platelet activation that can increase migration and proliferation, and decreases osteogenic differentiation of mesenchymal progenitor cells under in vitro conditions. The results further suggest that ERK signalling is required to mediate the mitogenic response to platelet-released supernatants.

Triggering the succinate receptor GPR91 on dendritic cells enhances immunity

Succinate acts as an extracellular mediator signaling through the G protein–coupled receptor GPR91. Here we show that dendritic cells had high expression of GPR91. In these cells, succinate triggered intracellular calcium mobilization, induced migratory responses and acted in synergy with Toll-like receptor ligands for the production of proinflammatory cytokines. Succinate also enhanced antigen-specific activation of human and mouse helper T cells. GPR91-deficient mice had less migration of Langerhans cells to draining lymph nodes and impaired tetanus toxoid–specific recall T cell responses. Furthermore, GPR91-deficient allografts elicited weaker transplant rejection than did the corresponding grafts from wild-type mice. Our results suggest that the succinate receptor GPR91 is involved in sensing immunological danger, which establishes a link between immunity and a metabolite of cellular respiration.

1,25-Dihydroxyvitamin D3 Is an autonomous regulator of the transcriptional changes leading to a tolerogenic dendritic cell phenotype

Activation of vitamin D receptor (VDR) by 1,25-dihydroxyvitamin D3 (1,25-vitD) reprograms dendritic cells (DC) to become tolerogenic. Previous studies suggested that 1,25-vitD could inhibit the changes brought about by differentiation and maturation of DCs. Underpinning the described phenotypic and functional alterations, there must be 1,25-vitD-coordinated transcriptional events. However, this transcriptional program has not been systematically investigated, particularly not in a developmental context. Hence, it has not been explored how 1,25-vitD-regulated genes, particularly the ones bringing about the tolerogenic phenotype, are connected to differentiation. We conducted global gene expression analysis followed by comprehensive quantitative PCR validation to clarify the interrelationship between 1,25-vitD and differentiation-driven gene expression patterns in developing human monocyte-derived and blood myeloid DCs. In this study we show that 1,25-vitD regulates a large set of genes that are not affected by differentiation. Interestingly, several genes, impacted both by the ligand and by differentiation, appear to be regulated by 1,25-vitD independently of the developmental context. We have also characterized the kinetics of generation of 1,25-vitD by using three early and robustly regulated genes, the chemokine CCL22, the inhibitory receptors CD300LF and CYP24A1. We found that monocyte-derived DCs are able to turn on 1,25-vitD sensitive genes in early phases of differentiation if the precursor is present. Our data collectively suggest that exogenous or endogenously generated 1,25-vitD regulates a large set of its targets autonomously and not via inhibition of differentiation and maturation, leading to the previously characterized tolerogenic state.

Some aspects of IL-8 pathophysiology III: chemokine interaction with endothelial cells

Chemolrines have been convincingly implicated in driving leukocyte emigration in different inflammatory reactions. However, the cellular and molecular mechanisms of chemokine involvement in leukocyte emigration are not yet clear. We and others suggested that leukocyte adhesion to the endothelium and transmigration are induced by chemokines immobilised on the endothelial cell surface. This would require the presence of specific chemokine binding sites in this microanatomical location. Using an in situ binding assay we demonstrated the presence of binding sites for interleukin‐8 (IL‐8) and RANTES, but not monocyte inflammatory protein‐1α on the endothelium of postcapillary venules and small veins in human skin. In contrast, venules and veins in various anatomical locations showed dramatically differing IL‐8 binding patterns. The subcellular distribution of IL‐8 in the venular endothelial cells following its in vivo and ex vivo injections was studied by use of electron microscopy. Our results suggest that IL‐8 was internalized by the endothelial cells, transported transcellularly via plasmalemmal vesicles, and released onto the luminal surface where it appeared located preferentially on tips of membrane protrusions. We were unable to study the endothelial IL‐8 binding or transport in vitro because all the in vitro propagated endothelial cell lines and primary endothelial cells tested lacked IL‐8 binding sites. This includes human umbilical vein endothelial cells (HUVECs), which also did not bind IL‐8 in situ. However, HUVECs provided a satisfactory in vitro system to study the secretion of IL‐8 by the endothelial cells. Two possible alternative pathways were described: secretion directly from the Golgi apparatus or following storage in Weibel‐Palade bodies.

Murine mesenchymal stem cells exhibit a restricted repertoire of functional chemokine receptors: comparison with human.

Mesenchymal stem cells (MSCs) are non-haematopoeitic, stromal cells that are capable of differentiating into mesenchymal tissues such as bone and cartilage. They are rare in bone marrow, but have the ability to expand many-fold in culture, and retain their growth and multi-lineage potential. The properties of MSCs make them ideal candidates for tissue engineering. It has been shown that MSCs, when transplanted systemically, can home to sites of injury, suggesting that MSCs possess migratory capacity; however, mechanisms underlying migration of these cells remain unclear. Chemokine receptors and their ligands play an important role in tissue-specific homing of leukocytes. Here we define the cell surface chemokine receptor repertoire of murine MSCs from bone marrow, with a view to determining their migratory activity. We also define the chemokine receptor repertoire of human MSCs from bone marrow as a comparison. We isolated murine MSCs from the long bones of Balb/c mice by density gradient centrifugation and adherent cell culture. Human MSCs were isolated from the bone marrow of patients undergoing hip replacement by density gradient centrifugation and adherent cell culture. The expression of chemokine receptors on the surface of MSCs was studied using flow cytometry. Primary murine MSCs expressed CCR6, CCR9, CXCR3 and CXCR6 on a large proportion of cells (73±11%, 44±25%, 55±18% and 96±2% respectively). Chemotaxis assays were used to verify functionality of these chemokine receptors. We have also demonstrated expression of these receptors on human MSCs, revealing some similarity in chemokine receptor expression between the two species. Consequently, these murine MSCs would be a useful model to further study the role of chemokine receptors in in vivo models of disease and injury, for example in recruitment of MSCs to inflamed tissues for repair or immunosupression.