Contribution of Duffy antigen to chemokine function

In addition to classical G protein-coupled receptors (GPCRs), a group of alternative, “silent” chemokine receptors has recently been identified. These serpentine molecules are not coupled to G proteins and subsequent signaling cascades, but can efficiently internalize their cognate chemokine ligands, thus act as “interceptors” (internalizing receptors). Here we discuss a mechanism by which a member of this family, Duffy antigen (DARC), contributes to chemokine-induced leukocyte emigration. Cumulative experimental evidence suggests that DARC on venular endothelium mediates chemokine internalization at the abluminal surface followed by transcytosis and transfer of the chemokine cargo onto the luminal surface. DARC is also expressed on the erythrocyte surface of DARC positive individuals. Erythrocyte DARC binds plasma chemokines which results, on one hand, in impediment of the chemokines loss from the circulation and, on the other hand, in neutralization of chemokines in the blood. This leads to leukocyte protection from inadvertent “desensitization” and enhancement of leukocyte recruitment.     

Direct and indirect plaque forming cells in extrapulmonary lymphoid tissue following local vs systemic injection of soluble antigen

AKR strain mice were immunized with solubilized SRBC stroma either by direct injection into the lower respiratory tract or intravenously via the tail vein. The number of plaque forming cells (PFC) in the draining plumonary lymph node (tracheobronchial node) and spleen were determined by direct (IgM) and indirect IgG₁, IgG_2b, IgA) plaque assays.Intravenously administered antigen induced an initially strong IgM response in the spleen which was subsequently followed by antibody of the IgG₁, IgG_2b, and IgA classes of immunoglobulins. The tracheobronchial lymph node contained a minimal number PFC representing all four types of immunoglobulins studied. Conversely, following a single local injection of antigen directly into the lower respiratory tract, the tracheobronchial node responded with relatively high concentrations of PFC of all classes. The response in the spleen, although higher than background, was barely detectable. The splenic response to locally administered antigen was, however, considerably augmented as a result of a second local injection given 45 days after the initial stimulation. Under these conditions, IgG₁ IgG_2b, and IgA were represented in both tissue sites by sharp increases in the number and a decrease in the time of appearance of their respective antibody forming cells. Comparable changes were not noted for the case of IgM.Serum hemagglutination titres following a single injection by either route did not vary significantly during the time course of the experiment (28 days). The sera from locally immunized mice were treated with the reducing agent dithiothreitol and hemagglutination titres, before and after treatment, were compared. The major serum activity observed during the first 10 days following injection was affected by reduction and could therefore be assigned to high molecular weight antibody (19S, 13S). Subsequent titres (Days 13–26) were less susceptible to DTT and are considered to represent low molecular weight immunoglobulins (7S).     

A role of the Duffy antigen for the maintenance of plasma chemokine concentrations

We examined plasma chemokine concentrations and chemokine clearance rates in Duffy antigen knockout mice. The plasma concentrations of eotaxin and MCP-1 in Duffy antigen knockout mice were less than one-third of those in wild-type mice. When eotaxin or hMGSA was intravenously injected, the chemokine disappeared more rapidly from the plasma of Duffy antigen knockout mice than from the plasma of wild-type mice. The half-lives of hIP-10 and interferon-gamma, which do not have an affinity for the Duffy antigen, in plasma were indistinguishable between Duffy antigen knockout mice and wild-type mice. These results suggest that the Duffy antigen delays the disappearance of chemokines from the plasma, resulting in the maintenance of plasma chemokine concentrations.     

Duffy antigen receptor for chemokines mediates chemokine endocytosis through a macropinocytosis-like process in endothelial cells

Background

The Duffy antigen receptor for chemokines (DARC) shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.

Methodology/Principal Findings

We investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated ¹²⁵I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. ¹²⁵I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression. ¹²⁵I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF) enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.

Conclusions/Significance

These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.     

The role of the Duffy antigen-related chemokine receptor in psoriasis vulgaris

Chemokines represent a family of potent biological mediators. Within the group of receptors mediating their effects, a promiscuous receptor has been found which is able to bind and inactivate diverse chemokines of both C-C and C-X-C families. It is co-localized with blood group antigens of the Duffy system on the same glycoprotein and expressed on red blood cells as well as post-capillary blood vessels. In the present study three aspects of Duffy pathophysiology were studied: firstly the amount of IL-8 and RANTES binding to red blood cells and its correlation to disease activity of psoriatic patients, secondly the distribution of Duffy phenotype among psoriatic patients and thirdly the expression of Duffy antigen in normal vs psoriatic skin. Red blood cells from psoriatic patients (n=50) were lysed by triton X (1%) and supernatants tested in IL-8- and RANTES sandwich-ELISA. Duffy phenotype of psoriatic patients (n=50) was assessed by typing red blood cells with specific antisera in indirect Coombs technique. For immunohistochemical detection in normal and psoriatic skin (n=10 respectively) a specific monoclonal antibody (Fy6) was used. Neither IL-8- nor RANTES-levels on red blood cells correlated to disease activity and distribution of Duffy phenotype in psoriatics was not significantly altered when compared to the normal population. Furthermore, Duffy antigen was expressed in a similar pattern in normal and psoriatic skin at all parts of vasculature, albeit much more abundantly in diseased skin. Altogether, chemokine binding to red blood cells seems of minor importance in psoriasis. However, Duffy antigen together with other binding mechanisms like proteoglycans may play a role at local level by binding locally produced chemokines. Thus biological effects of chemokines are both restricted and focussed to dermal tissue.     

Suppression of IgE responses by passive antigen inhalation: dissociation of local (mucosal) and systemic immunity

Animals from high- and low-IgE-responder rat strains were preexposed to antigen-containing aerosols of different droplet sizes, prior to parenteral antigenic challenge. Depending upon the type of aerosol employed, systemic immunological tolerance developed in high-IgE-responder animals in the IgE antibody class either with or without concomitant production of salivary IgA, indicating that the two antibody isotypes were under independent control, and further that IgA-mediated immune exclusion was not central to the development of tolerance in the IgE class. Low-IgE-responder rats exhibited biphasic salivary IgA responses during exposure, which could not be recalled by subsequent parenteral challenge, suggesting that secretory immunity in the respiratory tract may also be down regulated by repeated exposure to airborne antigens.     

Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-alpha/CXC chemokine ligand 1 (GRO-alpha/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-alpha/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-alpha/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.     

A structural model of a seven-transmembrane helix receptor: the Duffy antigen/receptor for chemokine (DARC)

The Duffy antigen/receptor for chemokine (DARC) is an erythrocyte receptor for malaria parasites (Plasmodium vivax and Plasmodium knowlesi) and for chemokines. In contrast to other chemokine receptors, DARC is a promiscuous receptor that binds chemokines of both CC and CXC classes. The four extracellular domains (ECDs) of DARC are essential for its interaction with chemokines, whilst the first (ECD1) is sufficient for the interaction with malaria erythrocyte-binding protein. In this study, we elaborate and analyze structural models of the DARC. The construction of the 3D models is based on a comparative modeling process and on the use of many procedures to predict transmembrane segments and to detect far homologous proteins with known structures. Threading, ab initio, secondary structure and Protein Blocks approaches are used to build a very large number of models. The conformational exploration of the ECDs is performed with simulated annealing. The second and fourth ECDs are strongly constrained. On the contrary, the ECD1 is highly flexible, but seems composed of three consecutive regions: a small beta-sheet, a linker region and a structured loop. The chosen structural models encompass most of the biochemical features and reflect the known experimental data. They may be used to analyze functional interaction properties.     

Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-gamma and IFN-gamma-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-gamma, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.     

Cortisol responses of pallid sturgeon and yellow perch following challenge with lipopolysaccharide

Plasma cortisol responses of pallid sturgeon Scaphirhynchus albus and yellow perch Perca flavescens following injection with equal doses of lipopolysaccharide were compared. Concentrations of cortisol in plasma from pallid sturgeon did not change following injection (6·0–11·0 v. 6·4 ng l⁻¹ pre-stress) while in yellow perch plasma they were shown to increase up to 6 h (117·0 v. 9·8 ng l⁻¹ pre-stress) after the injection. These results are consistent with other reports for pallid sturgeon that illustrate a reduced cortisol response following other applied stressors relative to teleosts and suggest differences in the expression and regulation of their inflammatory responses.     

Silicon modifies both a local response and a systemic response to mechanical stress in tobacco leaves

Both lignin and silicon (Si) are major players in the resistance of plants to mechanical stress (MS). Focusing on the phenolic metabolism, here we studied the short-term effects of a local MS on tobacco (Nicotiana rustica L. cv. Basmas) plants with Si (+Si, 1 mM Na₂SiO₃) and without Si (?Si) treatments in order to see how Si may modify local and systemic responses. One week after starting the Si treatment, a half of the plants were exposed to a mechanical pressure applying 980 Pa for 24 h on the upper side of the 3^rd leaf of each plant (+MS). The rest of the plants remained unstressed (?MS). Plants were harvested 24 h and 72 h after starting the MS and the leaves directly exposed to the mechanical stress (DMS) and those indirectly exposed to the mechanical stress (IMS) from below and above the DMS leaf were analyzed for phenolic metabolism along with the corresponding leaves from?MS plants. In the DMS leaf, the activities of polyphenol oxidase, phenylalanine ammonia lyase, and cytosolic and covalently-bound peroxidases increased by the MS, while decreased by Si. In accordance with this in the DMS leaf, the content of soluble and cell wall-bound phenolics and lignin were enhanced by the MS but decreased by Si. Interestingly, Si influenced the pattern of response to the MS depending on whether the leaves were directly treated by the MS or not. Silicon treatment augmented MS-induced lignin accumulation in the DMS leaf while rather inhibited lignin formation in the IMS leaves. These data show that Si modified MS-mediated changes in the phenolic metabolism differently in local and systemic leaves.     

Signals for local and systemic responses of plants to pathogen attack

Activation of plant defences following recognition of pathogen attack involves complex reiterative signal networks with extensive signal amplification and cross-talk. The results of two approaches that have been taken to analyse signalling in plant-microbe interactions are discussed here. Activation tagging with T-DNA harbouring multiple 35S enhancer elements was employed as a gain-of-function approach to dissect signalling related to bacterial pathogen resistance in Arabidopsis thaliana. From a screen of approximately 5000 activation tagged lines, one line was identified as harbouring a T-DNA leading to over-expression of an apoplastic aspartic protease (CDR-1), that resulted in resistance to normally virulent Pseudomonas syringae. The second approach was to screen for loss-of-function mutants in T-DNA tagged populations. From a screen of 11 000 lines, one line, defective in induced resistance-1 (dir-1) lost resistance to normally avirulent P. syringae. Models for action of the products of the CDR-1 and DIR-1 genes suggest involvement of peptide and lipid signals in systemic disease resistance responses in A. thaliana.     

Integration of local and systemic signaling pathways for plant N responses

Nitrogen (N) is an essential macronutrient and a signal that has profound impacts on plant growth and development. In order to cope with changing N regimes in the soil, plants have developed complex regulatory mechanisms that involve short-range and long-range signaling pathways. These pathways act at the cellular and whole plant scale to coordinate plant N metabolism, growth and development according to external and internal N status. Although molecular components of local and systemic N signaling have been identified and characterized, an integrated view of how plants coordinate and organize the N response is still lacking. In this review, we discuss recent advances toward understanding the mechanisms of local and systemic N responses and provide an integrated model for how these responses are orchestrated.     

The mutation G298A-->Ala100Thr on the coding sequence of the Duffy antigen/chemokine receptor gene in non-caucasian Brazilians

Ala100Thr has been suggested to be a Caucasian genetic marker on the FY*B allele. As the Brazilian population has arisen from miscegenation among Portuguese, Africans, and Indians, this mutation could possibly be found in Euro- and Afro-Brazilians, or in Brazilian Indians. Fifty-three related individuals and a random sample of 100 subjects from the Brazilian population were investigated using the polymerase chain reaction and four restriction fragment length polymorphisms. Confirming the working hypothesis, among the related individuals three Afro-Brazilians (two of them a mother and daughter) and a woman of Amerindian descent had the Ala100Thr mutation on the FY*B allele. Five non-related Euro-Brazilians also carried the mutation. All nine individuals presented the Fy(a-b+) phenotype. We conclude that the Ala100Thr mutation can occur in populations other than Caucasians and that this mutation does not affect Duffy expression on red blood cells. Gene frequencies for this allele in the non-related individuals were in agreement with those of other populations. The Duffy frequencies of two Amerindian tribes were also investigated.     

Duration of antigen expression in vivo following DNA immunization modifies the magnitude, contraction, and secondary responses of CD8+ T lymphocytes

The duration of Ag expression in vivo has been reported to have a minimal impact on both the magnitude and kinetics of contraction of a pathogen-induced CD8(+) T cell response. In this study, we controlled the duration of Ag expression by excising the ear pinnae following intradermal ear pinnae DNA immunization. This resulted in decreased magnitude, accelerated contraction and differentiation, and surprisingly greater secondary CD8(+) T cell responses. Furthermore, we found delayed and prolonged Ag presentation in the immunized mice; however, this presentation was considerably decreased when the depot Ag was eliminated. These findings suggest that the magnitude and the contraction phase of the CD8(+) T cell response following intradermal DNA immunization is regulated by the duration rather than the initial exposure to Ag.     

Membrane dielectric responses of human T-lymphocytes following mitogenic stimulation

Human peripheral blood T-lymphocytes, normally resting at the G0 phase, were stimulated with phytohemagglutinin (PHA) and interleukin-2 (IL-2) to induce the cell division cycle. The cells were examined at 24-h intervals for up to 96 h by flow cytometry to determine cell cycle distributions and by electrorotation to determine dielectric properties. The average membrane specific capacitance was found to vary from 12 (+/-1.5) mF/m2 prior to stimulation to 10 (+/-1.5) and 16 (+/-3.5) mF/m2 at 24 and 48 h after stimulation, respectively, and to remain unchanged up to 96 h after stimulation. Scanning electron microscopy studies of the cells revealed an increased complexity in cell membrane morphology following stimulation, suggesting that the observed change in the membrane capacitance was dominated by the alteration of cell surface structures. The average electrical conductivity of the cell interior decreased from approximately 1.1 S/m prior to stimulation to approximately 0.8 S/m at 24 h after stimulation and showed little change thereafter. The average dielectric permittivity of the cell interior remained almost unchanged throughout the course of the cell stimulation. The percentage of T-lymphocytes in the S and G2/M phases increased from approximately 4% prior to stimulation to approximately 11 and approximately 34% at 24 and 48 h after stimulation, respectively. The large change in membrane specific capacitance between the 24 and 48 h time period coincided with the large alteration in the cell cycle distribution where the S and G2/M populations increased by approximately 23%. These data, together with an analysis of the variation of the membrane capacitance during the cell cycle based on the cell cycle-dependent membrane lipid accumulation, show that there is a correlation between membrane capacitance and cell cycle phases that reflects alterations in the cell plasma membrane.