Enhanced CCR2 expression by ACKR2-deficient NK cells increases tumoricidal cell therapy efficacy.

Chemokines regulate leukocyte navigation to inflamed sites and specific tissue locales and may therefore be useful for ensuring accurate homing of cell therapeutic products. We, and others, have shown that atypical chemokine receptor 2 (ACKR2), deficient mice (ACKR2-/-) are protected from metastasis development in cell line and spontaneous mouse models. We have shown that this relates to enhanced CCR2 expression on ACKR2-/- NK cells allowing them to home more effectively to CCR2 ligand expressing metastatic deposits. Here we demonstrate that the metastatic-suppression phenotype in ACKR2-/- mice is not a direct effect of the absence of ACKR2. Instead, enhanced NK cell CCR2 expression is caused by passenger-mutations that originate from creation of the ACKR2-/- mouse strain in 129 embryonic stem cells. We further demonstrate that simple selection of CCR2+ NK cells enriches for a population of cells with enhanced anti-metastatic capabilities. Given the widespread expression of CCR2 ligands by tumors, our study highlights CCR2 as a potentially important contributor to NK cell tumoricidal cell therapy.

Disease activity, burden and suffering in patients with ulcerative colitis in the UK cohort recruited into the global ICONIC study.

Objective: The Understanding the Impact of Ulcerative Colitis and Its Associated Disease Burden on Patients (ICONIC) was a 2-year, global, prospective, observational study assessing disease burden in adults recently diagnosed (≤36 months) with ulcerative colitis (UC) receiving routine outpatient care, irrespective of disease severity or treatment. A subanalysis was conducted to understand the UK perspective. Design/method: All eligible consenting patients enrolled in ICONIC from the UK were included in the subanalysis of patient-reported and physician-reported outcomes at baseline and year 2 (Y2). Results: Sixty-three UK patients were included (mean age 43.4 years, 58.7% female). At baseline and Y2, the mean (±SD) Simple Clinical Colitis Activity Index (SCCAI) scores were 3.6 (±3.3) and 1.5 (±1.5); Patient Modified Simple Clinical Colitis Activity Index (P-SSCAI) were 4.9 (±4.0) and 2.6 (±2.6), respectively. Physician-reported Pictorial Representation of Illness and Self Measure (PRISM) median scores (assessing inverse of suffering) were 3.5 (IQR 2.0-6.8) at baseline and 5.5 (IQR 3.6-6.9) at Y2; patient-reported PRISM scores were 4.7 (IQR 2.6-6.9) and 5.4 (IQR 3.2-8.0), respectively. At baseline, SCCAI and P-SCCAI were strongly correlated (r=0.86, p<0.0001), and patient-reported and physician-reported PRISM scores moderately correlated (r=0.67, p<0.0001). At Y2, moderate correlations were observed (SCCAI vs P-SCCAI: r=0.72, p<0.0001; patient-reported vs physician- reported PRISM: r=0.60, p<0.0001). Rating Form of IBD Patient Concerns scores indicated patients' greatest concerns were with energy level, having an ostomy bag and effects of medication (baseline scores >3.0). Conclusions: These findings demonstrated the multifaceted burden of disease in patients recently diagnosed with UC in the UK. Agreement between patients and physicians on disease activity/severity varied according to the instrument used.

The atypical chemokine receptor ACKR2 suppresses Th17 responses to protein autoantigens.

Chemokine-directed leukocyte migration is a critical component of all innate and adaptive immune responses. The atypical chemokine receptor ACKR2 is expressed by lymphatic endothelial cells and scavenges pro-inflammatory CC chemokines to indirectly subdue leukocyte migration. This contributes to the resolution of acute inflammatory responses in vivo. ACKR2 is also universally expressed by innate-like B cells, suppressing their responsiveness to the non-ACKR2 ligand CXCL13, and controlling their distribution in vivo. The role of ACKR2 in autoimmunity remains relatively unexplored, although Ackr2 deficiency reportedly lessens the clinical symptoms of experimental autoimmune encephalomyelitis induced by immunization with encephalogenic peptide (MOG(35-55)). This was attributed to poor T-cell priming stemming from the defective departure of dendritic cells from the site of immunization. However, we report here that Ackr2-deficient mice, on two separate genetic backgrounds, are not less susceptible to autoimmunity induced by immunization, and in some cases develop enhanced clinical symptoms. Moreover, ACKR2 deficiency does not suppress T-cell priming in response to encephalogenic peptide (MOG(35-55)), and responses to protein antigen (collagen or MOG(1-125)) are characterized by elevated interleukin-17 production. Interestingly, after immunization with protein, but not peptide, antigen, Ackr2 deficiency was also associated with an increase in lymph node B cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that enhances T helper type 17 (Th17) cell development and survival. Thus, Ackr2 deficiency does not suppress autoreactive T-cell priming and autoimmune pathology, but can enhance T-cell polarization toward Th17 cells and increase the abundance of GM-CSF(+) B cells in lymph nodes draining the site of immunization.

Atypical chemokine receptor ACKR2 mediates chemokine scavenging by primary human trophoblasts and can regulate fetal growth, placental structure, and neonatal mortality in mice.

Inflammatory chemokines produced in the placenta can direct the migration of placental leukocytes using chemokine receptors that decorate the surface of these cells. Fetal trophoblasts can also express receptors for inflammatory chemokines, and they are one of the few cell types that express atypical chemokine receptor 2 (ACKR2), previously known as D6. ACKR2 binds many inflammatory CC chemokines but cannot stimulate cell migration or activate signaling pathways used by conventional chemokine receptors. Existing evidence suggests that ACKR2 is a specialized chemokine scavenger, but its function in primary human trophoblasts has not been explored. In mice, ACKR2 is thought to be dispensable for the reproductive success of unchallenged females that have conceived naturally, but it can suppress inflammation-induced abortion and aid the survival of implanted allogeneic embryos. In this article, we demonstrate that cultured primary human trophoblasts express ACKR2 far more strongly than genes encoding conventional receptors for inflammatory CC chemokines. Moreover, these cells are capable of the rapid internalization and efficient scavenging of extracellular chemokine, and this is mediated by ACKR2. We also report that in unchallenged DBA/1j mice, Ackr2 deficiency increases the incidence of stillbirth and neonatal death, leads to structural defects in the placenta, and can decrease fetal weight. Loss of Ackr2 specifically from fetal cells makes a key contribution to the placental defects. Thus, primary human trophoblasts use ACKR2 to scavenge chemokines, and ACKR2 deficiency can cause abnormal placental structure and reduced neonatal survival.

Characterization of conventional and atypical receptors for the chemokine CCL2 on mouse leukocytes.

Chemokine-directed leukocyte migration is crucial for effective immune and inflammatory responses. Conventional chemokine receptors (cCKRs) directly control cell movement; atypical chemokine receptors (ACKRs) regulate coexpressed cCKRs; and both cCKRs and ACKRs internalize chemokines to limit their abundance in vivo, a process referred to as scavenging. A leukocyte's migratory and chemokine-scavenging potential is determined by which cCKRs and ACKRs it expresses, and by the ligand specificity, signaling properties, and chemokine internalization capacity of these receptors. Most chemokines can bind at least one cCKR and one ACKR. CCL2 can bind to CCR2 (a cCKR) and two ACKRs (ACKR1 and ACKR2). In this study, by using fluorescent CCL2 uptake to label cells bearing functional CCL2 receptors, we have defined the expression profile, scavenging activity, and ligand specificity of CCL2 receptors on mouse leukocytes. We show that qualitative and quantitative differences in the expression of CCR2 and ACKR2 endow individual leukocyte subsets with distinctive CCL2 receptor profiles and CCL2-scavenging capacities. We reveal that some cells, including plasmacytoid dendritic cells, can express both CCR2 and ACKR2; that Ly6C(high) monocytes have particularly strong CCL2-scavenging potential in vitro and in vivo; and that CCR2 is a much more effective CCL2 scavenger than ACKR2. We confirm the unique, overlapping, ligand specificities of CCR2 and ACKR2 and, unexpectedly, find that cell context influences the interaction of CCL7 and CCL12 with CCR2. Fluorescent chemokine uptake assays were instrumental in providing these novel insights into CCL2 receptor biology, and the sensitivity, specificity, and versatility of these assays are discussed.

Using fluorescent chemokine uptake to detect chemokine receptors by fluorescent activated cell sorting.

Fluorescent activated cell sorting (FACS) is a powerful technique that allows rapid quantitative cell-by-cell analysis of the expression of multiple distinct proteins and the isolation of specific cell types from complex cellular mixtures. FACS-based detection of chemokine receptors is typically achieved using chemokine receptor-specific antibodies. However, this approach has some limitations, and, in our experience, many anti-chemokine receptor antibodies have poor signal-to-noise ratios and thus low sensitivity and reliability in FACS. Moreover, antibodies against some chemokine receptors are not commercially available, particularly in nonhuman species, and generating good antibodies against chemokine receptors is challenging and expensive. Here, we describe a simple alternative method of detecting chemokine receptors by FACS that relies on cellular internalization of fluorescently labelled chemokines. This approach detects chemokine receptors with high sensitivity, specificity, and reliability, and consistently outperforms antibody-based detection methods when applied to the analysis of mouse cells in our experience. It can also be used to study receptor specificity on primary cells and, since chemokines from one species often bind to receptors from other species, a single fluorescently labelled chemokine should allow reliable chemokine receptor detection and analysis across a range of species.

Universal expression and dual function of the atypical chemokine receptor D6 on innate-like B cells in mice.

Mouse innate-like B cells are a heterogeneous collection of multifunctional cells that control infection, play housekeeping roles, contribute to adaptive immunity, and suppress inflammation. We show that, among leukocytes, chemokine internalization by the D6 receptor is a unique and universal feature of all known innate-like B-cell populations and, to our knowledge, the most effective unifying marker of these cells. Moreover, we identify novel D6(active) B1-cell subsets, including those we term B1d, which lack CD5 and CD11b but exhibit typical B1-cell properties, including spontaneous ex vivo production of IgM, IL-10, and anti-phosphorylcholine antibody. The unprecedented opportunity to examine D6 on primary cells has allowed us to clarify its ligand specificity and show that, consistent with a scavenging role, D6 internalizes chemokines but cannot induce Ca(2+) fluxes or chemotaxis. Unexpectedly, however, D6 can also suppress the function of CXCR5, a critical chemokine receptor in innate-like B-cell biology. This is associated with a reduction in B1 cells and circulating class-switched anti-phosphorylcholine antibody in D6-deficient mice. Therefore, in the present study, we identify a unifying marker of innate-like B cells, describe novel B1-cell subsets, reveal a dual role for D6, and provide the first evidence of defects in resting D6-deficient mice.

DARC and D6: silent partners in chemokine regulation?

Chemokine receptors adorn the surface of leukocytes and other cell types ready to translate the extracellular chemokine environment into functional cellular outcomes. However, there are several molecules that, in many respects, look like chemokine receptors, but which do not have the ability to confer chemotactic potential to cell lines. This apparent silence spurred the search for signalling-independent functions and led to the development of new paradigms of chemokine regulation. In this review, we summarise the experimental basis for these ideas focussing on DARC and D6, the most studied members of this group of molecules. We discuss data generated using in vitro systems and genetically deficient mice, include results from observational human studies, and summarise the key findings of recent research. We take a critical look at current models of in vivo function highlighting important gaps in our knowledge and demonstrating that there is still much to find out about these enigmatic molecules.

The atypical chemokine receptor D6 contributes to the development of experimental colitis.

Proinflammatory CC chemokines control leukocyte recruitment and function during inflammation by engaging chemokine receptors expressed on circulating leukocytes. The D6 chemokine receptor can bind several of these chemokines, but appears unable to couple to signal transduction pathways or direct cell migration. Instead, D6 has been proposed to act as a chemokine scavenger, removing proinflammatory chemokines to dampen leukocyte responses. In this study, we have examined the role of D6 in the colon using the dextran sodium sulfate-induced model of colitis. We show that D6 is expressed in the resting colon, predominantly by stromal cells and B cells, and is up-regulated during colitis. Unexpectedly, D6-deficient mice showed reduced susceptibility to colitis and had less pronounced clinical symptoms associated with this model. D6 deletion had no impact on the level of proinflammatory CC chemokines released from cultured colon explants, or on the balance of leukocyte subsets recruited to the inflamed colon. However, late in colitis, inflamed D6-deficient colons showed enhanced production of several proinflammatory cytokines, including IFN-gamma and IL-17A, and there was a marked increase in IL-17A-secreting gammadelta T cells in the lamina propria. Moreover, Ab-mediated neutralization of IL-17A worsened the clinical symptoms of colitis at these later stages of the response in D6-deficient, but not wild-type, mice. Thus, D6 can contribute to the development of colitis by regulating IL-17A secretion by gammadelta T cells in the inflamed colon.

Hemopoietic cell expression of the chemokine decoy receptor D6 is dynamic and regulated by GATA1.

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.

Hemopoietic cell expression of the chemokine decoy receptor D6 is dynamic and regulated by GATA1.

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.

Unique features and distribution of the chicken CD83+ cell.

The central importance of dendritic cells (DC) in both innate and acquired immunity is well recognized in the mammalian immune system. By contrast DC have yet to be characterized in avian species despite the fact that avian species such as the chicken have a well-developed immune system. CD83 has proven to be an excellent marker for DC in human and murine immune systems. In this study we identify chicken CD83 (chCD83) as the avian equivalent of the human and murine DC marker CD83. We demonstrate for the first time that unlike human and murine CD83, chCD83 is uniquely expressed in the B cell areas of secondary lymphoid organs and in organs with no human or murine equivalent such as the bursa and Harderian gland. Furthermore through multicolor immunofluorescence, we identify chCD83(+) populations that have unique attributes akin to both DC and follicular DC. These attributes include colocalization with B cell microenvironments, MHC class II expression, dendritic morphology, and distribution throughout peripheral and lymphoid tissues.

Professional and part-time chemokine decoys in the resolution of inflammation.

Inflammation is essential for protection from infection and for the repair of damaged tissue. Much is now known about how inflammation is induced and maintained, but the processes underlying the resolution of inflammation are often overlooked. However, resolution is an essential component of all successful inflammatory responses because it ensures the restoration of tissue homeostasis and prevents immunopathology of the type seen in chronic inflammatory diseases and autoimmunity. Small secreted proteins called chemokines, acting through chemokine receptors, are known to be critical regulators of leukocyte recruitment and function during the initiation and maintenance of inflammation. Thus, their efficient removal would seem to be a prerequisite for successful resolution. In recent years, it has emerged that specialized chemokine "decoy" receptors exist that actively participate in this process. Moreover, other chemokine receptors have been proposed to lead a double life and perform opposing roles during inflammation: leukocyte recruitment (by signaling) and resolution (by chemokine sequestration). A recent study provides further support for this theory by showing that apoptotic inflammatory leukocytes increase the number of surface chemokine receptors and that these receptors can remove chemokines from inflamed tissue. Leukocyte apoptosis is already known to aid resolution, not just because it eliminates leukocytes from inflamed tissues, but also because their consumption by macrophages leads to the production of anti-inflammatory cytokines. The new work indicates that chemokine sequestration may be another mechanism exploited by dying cells to assist in the resolution of inflammation.