CXCL1 inhibits airway smooth muscle cell migration through the decoy receptor Duffy antigen receptor for chemokines.

Airway smooth muscle cell (ASMC) migration is an important mechanism postulated to play a role in airway remodeling in asthma. CXCL1 chemokine has been linked to tissue growth and metastasis. In this study, we present a detailed examination of the inhibitory effect of CXCL1 on human primary ASMC migration and the role of the decoy receptor, Duffy AgR for chemokines (DARC), in this inhibition. Western blots and pathway inhibitors showed that this phenomenon was mediated by activation of the ERK-1/2 MAPK pathway, but not p38 MAPK or PI3K, suggesting a biased selection in the signaling mechanism. Despite being known as a nonsignaling receptor, small interference RNA knockdown of DARC showed that ERK-1/2 MAPK activation was significantly dependent on DARC functionality, which, in turn, was dependent on the presence of heat shock protein 90 subunit α. Interestingly, DARC- or heat shock protein 90 subunit α-deficient ASMCs responded to CXCL1 stimulation by enhancing p38 MAPK activation and ASMC migration through the CXCR2 receptor. In conclusion, we demonstrated DARC's ability to facilitate CXCL1 inhibition of ASMC migration through modulation of the ERK-1/2 MAPK-signaling pathway.

Atypical chemokine receptors in renal inflammation.

Chemokines are structurally related proteins which form a large family of chemotactic cytokines. They provide a general communication system for cells and regulate lymphocyte migration. These proteins orchestrate the formation of microenvironments in lymphoid tissue, promote lymphoid organogenesis and help foster vascular and lymphatic angiogenesis. In addition to the classical G protein-coupled chemokine receptors, many chemokines also bind to a family of nonsignaling proteins, now called interceptors (chemokine-internalizing proteins). Here we summarize recent data on the role of interceptors in chemokine biology with a focus on renal inflammation.

Effect of interleukin-8 and RANTES on the Gardos channel activity in sickle human red blood cells: role of the Duffy antigen receptor for chemokines.

We investigated the effects of the chemokines IL-8 and RANTES on the activity of the Gardos channel (GC) of sickle red blood cells (SSRBCs). SSRBCs expressing the Duffy antigen receptor for chemokines (DARC) incubated under oxygenated conditions exhibit GC activation. The deoxygenation-stimulated K(+) loss via the GC is activated by the chemokines in the Duffy-positive SSRBCs. The percentage of cells with high density is 17 times higher in the Duffy-positive group. These findings are consistent with a greater susceptibility of Duffy-positive SSRBCs to inflammatory chemokines leading to GC activation and cellular dehydration and suggest a coupling, promoted by the sickling process, between DARC and the GC.

The role of chemokine receptors in acute lung allograft rejection.

Recruitment of inflammatory cells to vascularised allografts is a hallmark of rejection, and paves the way for chronic allograft injury. Chemokines play pivotal roles in the directed movement of leukocytes. Herein, we define the distribution of chemokine receptors for the most common cell types during human lung allograft rejection as a prerequisite for therapeutic interventions. Immunohistochemistry was performed on lung allograft biopsies from 54 patients for the chemokine receptors CCR5, CXCR3 and CXCR1 and the Duffy antigen/receptor for chemokines (DARC). Perivascular infiltrates in acute lung rejection are composed of subsets of mononuclear cells expressing the chemokine receptors CXCR1, CXCR3 and CCR5. DARC-positive small vessels and capillary vessels were associated with sites of inflammation and their number was increased during episodes of acute lung rejection. DARC expression correlated with an increase in interstitial CCR5-positive T-cells and CXCR1-positive leukocytes. Leucokytic infiltrates in bronchial/bronchiolar rejection express CXCR1 and CXCR3. This is the first study that demonstrates an induction of the chemokine binding protein DARC at sites of acute human lung allograft rejection. Co-localisation with the chemokine receptors CXCR1 and CCR5 may indicate a role for DARC expression during leukocyte adhesion and interstitial infiltration.

DARC on RBC limits lung injury by balancing compartmental distribution of CXC chemokines.

The Duffy antigen receptor for chemokines (DARC) has a high affinity for CC and CXC chemokines. However, it lacks the ability to induce cell responses that are typical for classical chemokine receptors. The role of DARC in inflammatory conditions remains to be elucidated. We studied the role of DARC in a murine model of acute lung injury. We found that in Darc-gene-deficient (Darc(-/-)) mice, LPS-induced PMN migration into the alveolar space was elevated more than twofold. In contrast, PMN adhesion to endothelial cells and within the interstitial space was reduced in Darc(-/-) mice. Darc(-/-) mice also exhibited increased microvascular permeability. Elevated PMN migration in Darc(-/-) mice was associated with increased concentrations of two essential CXCR2 ligands, CXCL1 and CXCL2/3 in the alveolar space. In the blood, CXCL1 was mostly associated with RBC in WT mice and with plasma in Darc(-/-) mice. We found that DARC on RBC prevented excessive PMN migration into the alveolar space. In contrast, DARC on non-hematopoietic cells appeared to have only minor effects on leukocyte trafficking in this model. These findings show how DARC regulates lung inflammation by controlling the distribution and presentation of chemokines that bind CXCR2.

Racial differences in endotoxin-induced tissue factor-triggered coagulation.

BACKGROUND: Racial differences in coagulation are poorly understood. While some studies suggest a 'prothrombotic' coagulation profile in blacks compared with whites, others report an increased bleeding risk for blacks in various clinical settings. Moreover, preclinical data suggest a link between the Duffy antigen (=DARC, Duffy antigen receptor of chemokines) and coagulation. OBJECTIVES: Based on our previous research in Duffy antigen negative Africans, we hypothesized that Africans have an attenuated procoagulant response compared with Caucasians in a model of lipopolysaccharide (LPS)-induced, tissue factor (TF)-triggered coagulation activation. PATIENTS/METHODS: Healthy male volunteers (16 Duffy-negative Africans, 16 Duffy-positive Caucasians) received 2 ng kg(-1) LPS, and outcome parameters were measured using enzyme immunoassays and real-time polymerase chain reaction (RT-PCR, Taqman). RESULTS: LPS increased microparticle (MP)-associated TF procoagulant activity (PCA) less in Africans than Caucasians. Africans had reduced in vivo thrombin formation compared with Caucasians: they generated less thrombin-antithrombin (TAT) complexes (10.4 pg mL(-1) vs. 23.0 pg mL(-1), P<0.0001) and less prothrombin fragments (F1+2) (337 pmol mL(-1) vs. 819 pmol mL(-1), P<0.0001). Consistently, Africans also had decreased fibrin formation (D-dimer: 0.3 pg mL(-1) vs. 0.5 pg mL(-1), P=0.02). CONCLUSION: Duffy-negative subjects of African descent have a markedly reduced procoagulant response in a model of LPS-induced, TF-triggered coagulation activation compared with Duffy-positive healthy Caucasians.

Opposing roles of murine duffy antigen receptor for chemokine and murine CXC chemokine receptor-2 receptors in murine melanoma tumor growth.

The Duffy antigen receptor for chemokines (DARC) has been classified as a "silent" receptor, as it can bind CXC and CC chemokines to undergo ligand-induced receptor internalization, but is not coupled to trimeric G proteins required for the classic G protein-coupled receptor-mediated signaling. CXC chemokine receptor-2 (CXCR2) has been shown to play a major role in tumor angiogenesis. To test the hypothesis that these two chemokine receptors might play opposing roles in the growth of melanoma tumors, we developed a transgenic mouse model, where the preproendothelin promoter/enhancer (PPEP) is used to drive expression of either murine DARC (mDARC) or murine CXCR2 (mCXCR2) in endothelial cells. We show herein that the growth of melanoma tumor xenografts, established from s.c. injection of immortalized murine melanocytes overexpressing macrophage inflammatory protein-2, was inhibited or enhanced in the PPEP-mDARC and PPEP-mCXCR2 transgenic mice, respectively, compared with control mice. The early tumors formed in mDARC transgenic mice exhibited a significantly higher number of infiltrating leukocytes compared with either the control or mCXCR2 transgenic mice, suggesting a potential role for DARC expressed on endothelial cells in leukocyte migration. In addition, the tumor-associated angiogenesis in mDARC transgenic mice was reduced when compared with the control. Conversely, tumor angiogenesis was significantly increased in mCXCR2 transgenic mice. Results indicate that endothelial cell overexpression of mDARC increased leukocyte trafficking to the tumor, reduced the growth of blood vessels into the tumor, and reduced the growth rate of the tumor, whereas endothelial cell overexpression of mCXCR2 had the reverse effect on tumor angiogenesis and growth.

Duffy antigen/receptor for chemokines (DARC) attenuates angiogenesis by causing senescence in endothelial cells.

Duffy antigen/receptor for chemokines (DARC), expressed on erythrocytes and post-capillary venular endothelial cells, selectively binds both CXC and CC chemokines. DARC binds ELR + angiogenic chemokines such as IL-8 (CXCL8). We show that the DARC on endothelial cells plays a direct role in regulating angiogenesis. Matrigel(TM) in vivo plug assay showed that there was more capillary formation in DARC knockout mice compared to wild type mice indicating that DARC attenuated angiogenic activity. In vitro angiogenic assay on Matrigel coated plates using DARC expressing stable human cerebro-microvascular endothelial cells (HCEC) showed that, although capillary formation in transfected cells started early within 4-8 h; capillary formation was attenuated within 12-24 h. Contrarily, mock transfected cells continued to show vascular capillary formation during that time without demonstrating any attenuation. Preincubation of DARC-expressing HCEC with monoclonal antibody (mAb-Fy6) against the N-terminal chemokine-binding domain of DARC increased capillary formation in vitro. Moreover, addition of excess IL-8 during incubation had the similar effect. DARC-expressing transfected endothelial cells underwent senescence in conditioned medium, whereas DARC non-expressing cells remained healthy. Interestingly, after several days in the conditioned medium, DARC expressing senescent cells started to initiate capillary formation; whereas capillary formed with DARC non-expressing cells remained the same. Our data evidently demonstrated that DARC on endothelial cells attenuated the angiogenic activity by causing senescence.

The Duffy antigen receptor for chemokines in acute renal failure: A facilitator of renal chemokine presentation.

OBJECTIVE: Acute renal failure remains a major challenge in critical care medicine. Both neutrophils and chemokines have been proposed as key components in the development of acute renal failure. Although the Duffy antigen receptor for chemokines (DARC) is present in several tissues and a highly specific ligand for various chemokines, its exact role in vivo remains unclear. DESIGN: Prospective, controlled experimental study. SETTING: University-based research laboratory. SUBJECTS: C57BL/6 wild-type and DARC gene-deficient mice (DARC-/-). INTERVENTIONS: To unravel the functional relevance of DARC in vivo, we compared wild-type and DARC-/- using neutrophil-dependent models of acute renal failure, induced by either local (renal ischemia-reperfusion) or systemic (endotoxemia, lipopolysaccharide) injury. MEASUREMENTS AND MAIN RESULTS: Plasma creatinine and blood urea nitrogen concentrations served as indicators of renal function or dysfunction. Enzyme-linked immunosorbent assays were used to measure tissue and plasma chemokine concentrations. We also performed immunostaining to localize chemokine expression and flow cytometry to evaluate neutrophil recruitment into the kidney. Following renal injury, wild-type mice developed moderate renal ischemia-reperfusion(lipopolysaccharide, 300% increase in plasma creatinine concentrations) to severe acute renal failure (renal ischemia-reperfusion, 40% mortality) as well as extensive renal neutrophil recruitment. DARC-/- mice exhibited no renal dysfunction (renal ischemia-reperfusion) or only very mild renal dysfunction (lipopolysaccharide, 20% increase in serum creatinine concentrations). DARC-/- mice showed no postischemic neutrophil infiltration. Although DARC-/- and wild-type mice exhibited similar global renal neutrophil-recruitment during endotoxemia, DARC-/- mice showed significantly impaired neutrophil extravasation. Total renal concentrations of the chemokine macrophage inflammatory protein 2, which has been shown to bind to DARC and to be crucial in postischemic acute renal failure, were either identical (lipopolysaccharide) or only moderately different (renal ischemia-reperfusion) between wild-type and DARC-/- mice. Immunostaining revealed an absence of macrophage inflammatory protein-2 in renal endothelial cells of DARC-/- mice. CONCLUSIONS: We suggest that DARC predominantly exerts its effects by controlling spatial chemokine distribution, which in turn regulates neutrophil recruitment and subsequent acute renal failure.

[Interceptors:--"silent" chemokine receptors]. / Interceptory--"ciche" receptory chemokin.

The physiological effect caused by chemokines is regulated by interactions with a group of rodopsin-like G protein-coupled receptors (GPCRs). These receptors share a number of common features: the polypeptide chain is a 7-transmembrane ?-helix (7 TMD motif) and the region involved in G-protein interaction (the DRYLAIV sequence) is located in the second transmembrane loop. So far, 19 chemokine receptors have been identified. Three of them (Duffy glycoprotein, D6, and CCX-CKR proteins), although structurally related to other GPCRs, lack the ability of G-protein signal transduction. Instead, they efficiently internalize their cognate ligands, regulating chemokine levels in various body compartments. These three proteins are suggested to form a distinct chemokine receptor family, designated "interceptors" or "silent" chemokine receptors.

Identification of mouse Duffy antigen receptor for chemokines (Darc) as a BMD QTL gene.

It is now well known that bone mineral density (BMD) variance is determined by both genetic and environmental factors. Accordingly, studies in human and animal models have revealed evidence for the presence of several quantitative trait loci (QTL) that contribute to BMD variations. However, the identification of BMD QTL genes remains a big challenge. In the current study, we focused our efforts to identify the BMD candidate gene in chromosome 1 (Chr 1) QTL that was detected from a cross involving high BMD CAST/EiJ (CAST) and low BMD C57BL/6J (B6) mice. To this end, we have combined several approaches including: (1) fine mapping the BMD QTL in Chr 1 of the B6.CAST F2 female mice using a large number of polymorphic markers; (2) the generation of congenic sublines of mice by repeated backcrossing of CAST with B6 mice and phenotype characterization; (3) expression profiling genes in the QTL region; and (4) SNP analyses to identify the mouse Duffy Antigen Receptor for Chemokines (Darc) as a candidate gene for Chr 1 BMD QTL2. We verified the involvement of the Darc protein in BMD variation by evaluating the skeletal phenotype of Darc-knockout mice and congenic sublines of mice carrying small chromosomal segments from CAST BMD QTL. Based on the findings that Darc-antibody blocked formation of multinucleated osteoclasts in vitro and that Darc from CAST binds chemokines, known to regulate osteoclast formation, with reduced affinity compared with Darc from B6 mice, we conclude that Darc regulates BMD negatively by increasing osteoclast formation, and that the genetic association between Darc gene polymorphism and BMD variations in humans merits investigation.

Coy decoy with a new ploy: interceptor controls the levels of homeostatic chemokines.

A new subfamily of chemokine receptors is emerging that do not signal along classical G-protein-mediated pathways. Instead, these "silent" receptors efficiently internalize their cognate chemokine ligands, hence their suggested name, "chemokine interceptors", for internalizing receptors. Two of these interceptors, DARC and D6, possess intriguing patterns of tissue expression and are believed to be involved in controlling the local levels of proinflammatory chemokines. In this issue of the European Journal of Immunology, the biochemical properties of a third silent chemokine receptor, CCX-CKR, have been characterized and it is suggested that it may act as a scavenger for homeostatic chemokines, pointing to a broad and significant role for this group of chemokine binding molecules in chemokine biology.

Red cell membrane CO2 permeability in normal human blood and in blood deficient in various blood groups, and effect of DIDS.

The red cell membrane has an exceptionally high permeability for CO2, PCO2 approximately 0.15 cm/s, which is two to three orders of magnitude greater than that of some epithelial membranes and similarly greater than the permeability of the red cell membrane for HCO3-. As shown previously, this high PCO2 can be drastically inhibited by 10 microM 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate (DIDS), indicating that membrane proteins may be involved in this high gas permeability. Here, we have studied the possible contribution of several blood group proteins to CO2 permeation across the red cell membrane by comparing PCO2 of red cells deficient in specific blood group proteins with that of normal red cells. While PCO2 of normal red cells is approximately 0.15 cm/s and that of Fy(null) and Jk(null) red cells is similar, PCO2's of Colton null (deficient in aquaporin-1) and Rh(null) cells (deficient in Rh/RhAG) are both reduced to about 0.07 cm/s, i.e. to about one half. In addition, the inhibitory effect of DIDS is about half as great in Rh(null) and in Colton null red cells as it is in normal red cells. We conclude that aquaporin-1 and Rh/RhAG proteins contribute substantially to the high permeability of the human red cell membrane for CO2. Together these proteins are responsible for 50% or more of the CO2 permeability of red cell membranes. The CO2 pathways of both proteins can be partly inhibited by DIDS, which is why this compound very effectively reduces membrane CO2 permeability.

Host receptors in malaria merozoite invasion.

The clinical manifestations of Plasmodium falciparum malaria are directly linked to the blood stage of the parasite life cycle. At the blood stage, the circulating merozoites invade erythrocytes via a specific invasion pathway often identified with its dependence or independence on sialic acid residues of the host receptor. The invasion process involves multiple receptor-ligand interactions that mediate a complex series of events in a period of approximately 1 min. Although the mechanism by which merozoites invade erythrocytes is not fully understood, recent advances have put a new perspective on the importance of developing a multivalent blood stage-malaria vaccine. In this review, we highlight the role of currently identified host invasion receptors in blood-stage malaria.

Sistema de grupo sangüíneo Duffy: biologia e prática transfusional / Duffy blood group system: biology and transfusion practice

Após a introducão da técnica de antiglobulina indireta por Coombs em meados da década de 40, vários anticorpos antieritrocitários foram descobertos. O grupo sanguíneo Duffy foi descoberto quando Cutbush e Ikin detectaram, no início da década de 50, os primeiros anticorpos desse sistema. Os anticorpos Duffy são clinicamente significantes na prática transfusional, pois mostraram ser causadores de reacão hemolítica transfusional e de doenca hemolítica do recém-nascido, sendo de ocorrência mundial. O gene FY é constituído por dois exons e seu lócus foi mapeado no cromossomo 1q22-q23. Os antígenos Fyª e Fy b são codificados pelos alelos FYA e FYB e são responsáveis pelos fenótipos Fy(a+b-), Fy(a-b+) e Fy(a+b+). São carreados por uma glicoproteína de 336 aminoácidos também chamada DARC (Duffy Antigen/Receptor for Chemokines), que tem alta afinidade a quimiocinas, sendo também os receptores para Plasmodium vivax. Os polimorfismos relacionados aos seus alelos permitiram o desenvolvimento da técnica de genotipagem por PCR, que é de grande utilidade para a seguranca transfusional e incompatibilidade feto-materna. Na última década, inúmeras pesquisas têm sido feitas quanto ao papel biológico dos antígenos de grupos sangüíneos. Nesse artigo iremos revisar o sistema de grupo sangüíneo Duffy, em especial quanto à prática transfusional e suas funcões biológicas.

Conserved residues in the Plasmodium vivax Duffy-binding protein ligand domain are critical for erythrocyte receptor recognition.

Malaria merozoite invasion of human erythrocytes depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium vivax merozoite invasion is totally dependent on the recognition of the Duffy blood group antigen by the parasite ligand Duffy-binding protein (DBP). Receptor recognition by P. vivax relies on a cysteine-rich domain, the DBL domain or region II, at the N terminus of the extracellular portion of DBP. The minimal region of the DBP implicated for receptor recognition lies between cysteines 4 and 8 of the DBL domain, which is a region that also has the highest rate of allelic polymorphisms among parasite isolates. We previously found that allelic polymorphisms in this region altered the P. vivax DBL domain antigenic character, which contrasts with changes in receptor specificity attributed to polymorphisms in some homologous ligands of Plasmodium falciparum. To further investigate the relative importance of conserved and polymorphic residues within this DBL central region, we identified residues critical for receptor recognition by site-directed mutagenesis. Seventy-seven surface-predicted residues of the Sal-1 DBL domain were substituted with alanine and assayed for erythrocyte binding activity by expression of the mutant proteins on the surface of transiently transfected COS cells. The functional effect of alanine substitution varied from nil to complete loss of DBL erythrocyte-binding activity. Mutations that caused loss of ligand function mostly occurred in discontinuous clusters of conserved residues, whereas nearly all mutations in polymorphic residues did not affect erythrocyte binding. These data delineate DBL domain residues essential for receptor recognition.

Review: the Kell, Duffy, and Kidd blood group systems.

After the discovery (over 50 years ago) that the IAT could be applied to the detection of antibodies to blood group antigens, there was a rapid increase in the identification of alloantibodies that caused transfusion reactions or HDN. After Rh, antibodies in the Kell, Duffy, and Kidd blood group systems were the next in clinically significant antibodies to be revealed. Much of what has been learned about these blood groups since the journal Immunohematology issued its first edition has to do with the proteins, the genes, and the molecular basis for the antigens. What has not changed is that, after ABO and Rh, antibodies to antigens in these three systems are still the most clinically significant. They will form the basis of this review.

Duffy antigen is important for the lethal effect of the lethal strain of Plasmodium yoelii 17XL.

We studied the potential role of the Duffy antigen and glycophorin A as receptors for rodent malaria parasite invasion of erythrocytes. Parasitemia increased exponentially after infection with Plasmodium berghei NK65, P. chabaudi, and P. vinckei in Duffy antigen knockout, glycophorin A knockout, and wild-type mice, indicating that the Duffy antigen and glycophorin A are not essential for these malaria parasites. However, parasitemia of the Duffy antigen knockout mice infected with P. yoelii 17XL remained constant from day 5 to 14 after infection, and then decreased, resulting in autotherapy. The treatment of P. yoelii 17XL-infected Duffy antigen knockout mice with anti-CD4 antibody increased the parasitemia 15 days after infection and the mice eventually died, indicating that CD-4-positive cells play an important role in the clearance of P. yoelii 17XL at the late stage of the infection.