From Malaria to Chemokine Receptor: The Emerging Physiologic Role of the Duffy Blood Group Antigen

Blood online

SEARCH:

Advanced

This Article

Full Text (PDF)

Alert me when this article is cited

Alert me if a correction is posted

Citation Map

Services

Email this article to a friend

Similar articles in this journal

Similar articles in PubMed

Alert me to new issues of the journal

Download to citation manager

Rights and Permissions

Citing Articles

Citing Articles via HighWire

Citing Articles via CrossRef

Citing Articles via Google Scholar

Google Scholar

Articles by Hadley, T. J.

Articles by Peiper, S. C.

Search for Related Content

PubMed

PubMed Citation

Articles by Hadley, T. J.

Articles by Peiper, S. C.

Related Collections

Review Articles

Social Bookmarking


What's this?

Next Article

Blood, Vol. 89 No. 9 (May 1), 1997: pp. 3077-3091

REVIEW ARTICLE

From Malaria to Chemokine Receptor: The Emerging Physiologic Role of the Duffy Blood Group Antigen

By Terence J. Hadley and Stephen C. Peiper
From the Departments of Medicine, Pathology, and Biochemistry, Henry Vogt Cancer Research Institute, James Graham Brown Cancer Center, University of Louisville; and the Department of Veterans Affairs Medical Center, Louisville, KY.

  INTRODUCTION

Introduction
References

ONE OF THE MOST notable achievements of biomedical research in the first half of this century was the identification of red blood cell (RBC) antigens and the recognition of their importance to transfusion medicine and hemolytic disease of the newborn.¹^,² These discoveries gave rise to an era of descriptive RBC serology during which a complex array of RBC membrane antigens were defined by their reactivity with specific alloantibodies.³^,⁴ In recent years, RBC immunohematology has progressed from descriptive serology into an era of structural and functional analysis of blood group antigens, many of which are expressed in both erythroid and nonerythroid tissue.^5-8 Here we will focus on the Duffy blood group antigen, a structure that has been of particular interest because it serves as a receptor on the RBC for the malarial parasite, Plasmodium vivax (P vivax ).^8-10 We will attempt to highlight recent advances in our understanding of the molecular basis for the interaction of the P vivax malaria parasite with the Duffy blood group antigen and indicate how this information also contributed to the identification of an important gene family for cytoadherence proteins of P falciparum. In the context of normal physiology, the Duffy blood group antigen has been shown to be receptor for chemoattractant cytokines, or chemokines, and to be expressed by endothelial cells of postcapillary venules and by Purkinje cells of the cerebellum.¹¹^,¹² We will attempt to review this important area of chemokine receptor research and discuss the potential relevance of the Duffy chemokine receptor to immunology and neurobiology.

Duffy Blood Group Serology
The Duffy blood group system first came to light in a report by Cutbush et al¹³ describing an alloantibody against an antigen denoted as Fy^a in a patient with hemophilia who had received multiple transfusions. The antithetical antigen, Fy^b, was described 1 year later.¹⁴ Three phenotypes were identified in whites using anti-Fy^a and anti-Fy^b antisera: Fy(a+b-), Fy(a-b+), and Fy(a+b+).¹⁵ These phenotypes are the products of codominant alleles comprising genotypes FY*A/FY*A, FY*B/FY*B, and FY*A/FY*B, respectively. Some whites express a weak or quantitatively reduced Fy^b, the genetic basis of which remains uncertain.¹⁶

Most West Africans and 68% of African Americans do not express Fy^a or Fy^b on their RBCs.¹⁷^,¹⁸ The absence of Fy^a and Fy^b on erythrocytes is designated the Duffy negative phenotype and denoted as Fy(a-b-). When Duffy-negative individuals of African descent develop anti-Duffy alloantibodies, they are almost always anti-Fy^a rather than anti-Fy^b.¹⁹^,²⁰ The genotype designated FY/FY, which gives rise to the Fy(a-b-) erythroid phenotype, contains a coding sequence identical to that of FY*B.²¹ In individuals of the Fy(a-b-) phenotype, this sequence remains silent in erythroid cells but is transcribed and expressed on endothelial cells of postcapillary venules.²²

Reactivity with anti-Fy^a and anti-Fy^b alloantibodies is abolished after chymotrypsin or papain treatment of intact RBCs.²³^,²⁴ Albrey et al²⁵ described an anti-Duffy alloantibody, denoted anti-Fy3, that reacts with chymotrypsin-and papain-treated RBCs. Anti-Fy3 reacts with both Fy(a+b-) and Fy(a-b+) RBCs, but not Fy(a-b-) RBCs. Adsorption and elution experiments showed that anti-Fy3 reacts with a site common to both Fy(a+b-) and Fy(a-b+) RBCs. Although initially described in the serum of a rare Fy(a-b-) white woman (AZ) with a history of pregnancy and blood transfusions, anti-Fy3 can also occur in Duffy-negative individuals of African descent, often in conjunction with anti-Fy^a.¹⁸ Other Duffy-related epitopes have been defined by rare antisera, anti-Fy4 and anti-Fy5.²⁶^,²⁷ Anti-Fy4 reacts only with Fy(a-b-) RBCs from individuals of African descent.²⁶ Anti-Fy5 reacts with all human RBCs except those that are Fy(a-b-) and Rh null or express a variant of the e antigen of the Rh system.²⁷ The rarity of these antisera has impeded progress in characterizing their epitopes.

  THE DUFFY ANTIGEN IS A GLYCOPROTEIN

Moore et al²⁸ showed that when surface-radioiodinated Fy(a+b-) RBCs were incubated with anti-Fy^a before solubilization in detergent, a protein of 35 to 43 kD was specifically immunoprecipitated. Coprecipitation of a radiolabeled protein other than the Duffy antigen was not formally excluded. More conclusive Western blotting experiments confirmed that a 35- to 43-kD protein carries the Fy^a antigenic determinant.²⁹ Desialylation of RBC membranes with Vibrio cholera neuraminidase resulted in an altered electrophoretic mobility of the 35- to 43-kD Fy^a protein, suggesting that the Duffy antigen is a glycoprotein.³⁰ Treatment of RBC membranes with N-glycanase caused a shift in the apparent molecular weight of the Duffy protein to around 30 kD.³⁰^,³¹ Although treatment of intact RBCs with trypsin did not proteolytically cleave the Duffy antigen, treatment of partially purified Duffy antigen with trypsin resulted in a 28-kD fragment.³² After incremental digestion of this 28-kD Duffy tryptic peptide with N-glycanase, Western blotting resolved multiple species, indicating that this glycoprotein has two or three asparagine-linked oligosaccharide chains.³³ Parallel digestion with O-glycanase had no effect on the electrophoretic mobility of the glycoprotein, signifying the absence of oligosaccharide chains linked to serine and threonine residues.

  MONOCLONAL ANTIBODIES (MoAbs) TO THE DUFFY ANTIGEN

In 1987, Nichols et al³⁴ reported on the first MoAb (NYBC-BG6) against the Duffy antigen. This MoAb defined a new epitope, denoted Fy6, which is common to Fy^a and Fy^b, but absent from Fy(a-b-) erythrocytes. Treatment of intact RBCs with chymotrypsin destroyed the Fy6 epitope as well as Fy^a and Fy^b.³⁵ An anti-Fy6 MoAb (i3A) similar to NYBC-BG6 was produced by Riwom et al³⁶ and a MoAb (CBC-512) with a specificity similar to that of anti-Fy3 (reactivity not destroyed by treatment of RBCs with chymotrypsin or papain) was produced by Dr M. Uchikawa at the Japanese Red Cross (see Acknowledgment) (Table 1).

View this table:
[in this window] [in a new window]
Table 1. Available Human Polyclonal and Murine Monoclonal (MoAb) Anti-Duffy Antibodies

  DUFFY IS INVOLVED IN P VIVAX PATHOPHYSIOLOGY

As a background to discussing the Duffy antigen in relation to malaria, a brief overview of the malarial life cycle will be presented. Malaria is transmitted by female anophiline mosquitos. When an infected mosquito takes a blood meal, sporozoites enter the blood and invade hepatocytes where they divide into thousands of merozoites. This part of the life cycle produces no symptoms in the host. Merozoites emerge from the liver and enter the the blood where they invade erythrocytes. This stage of the life cycle is associated with the clinical illness. Inside erythrocytes, merozoites develop sequentially into ring forms, trophozoites, and schizonts; mature schizonts give rise to many new merozoites. Infected erythrocytes ultimately lyse ("rupture") and emerging merozoites invade other erythrocytes. Some ring forms develop into gametocytes instead of schizonts. Gametocytes are responsible for the transmission of malaria back to the mosquito when the mosquito takes a blood meal.

There are four species of human malaria: P falciparum, P vivax, P ovale, and P malariae. Of these, P falciparum and P vivax are the most prevalent. P falciparum causes the most mortality; P vivax is second to P falcipaurm as a cause of malaria-related morbidity. P knowlesi, a primate malaria related to P vivax and capable of invading human RBCs, has been used as a laboratory model to investigate how malaria parasites invade RBCs.

In 1975, Miller et al reported that human erythrocytes of the Fy(a-b-), or Duffy-negative phenotype, were resistant to invasion by merozoites (the RBC invasive form) of P knowlesi.³⁷^,³⁸ Human erythrocytes of all other phenotypes were invaded. Furthermore, anti-Fy^a and anti-Fy^b specifically inhibited invasion of P knowlesi merozoites into human RBCs of the Fy(a+b-) and Fy(a-b+) phenotype, respectively.³⁷

Cytochalasin B-treated merozoites were used to study attachment independent of invasion.³⁹ Cytochalasin B-treated merozoites of P knowlesi attached equally well to both Duffy-positive and Duffy-negative erythrocytes. Ultrastructural analysis of Duffy positive erythrocytes with adherent merozoites showed an electron dense structure beneath the RBC lipid bilayer in the area of apposition between RBC and merozoite.³⁹ Previous studies had demonstrated that this structure, termed a tight junction, is of crucial importance for parasite invasion.⁴⁰ The tight junction was not observed with merozoites adherent to Duffy-negative erythrocytes.

The finding that P knowlesi requires the Duffy antigen to form a tight junction and to invade human erythrocytes suggested an explanation for two observations related to the human malaria, P vivax. First, P vivax, which is prevalent in most tropical and subtropical areas of the world, is specifically absent from West Africa, a geographic area where greater than 95% of individuals are Fy(a-b-). Second, during treatment of neurosyphilitic patients with therapeutically induced malaria, many African Americans were noted to be innately resistant to P vivax.⁴¹ Miller et al⁴² postulated that the innate resistance of some African Americans to P vivax malaria might be related to the Duffy-negative phenotype. A number of studies confirmed this hypothesis and demonstrated a correlation between the Fy(a-b-) phenotype and resistance to P vivax infection.^42-44 It was subsequently shown that Fy(a-b-) erythrocytes cannot be invaded by P vivax in vitro and that the anti-Fy6 MoAb, NYBC-BG6, can block invasion of Duffy-positive erythrocytes by P vivax.³⁴^,³⁵

One of the distinguishing features of P vivax is its preference for reticulocytes.⁴⁵ Because the Duffy antigen is expressed on mature erythrocytes as well as on reticulocytes, it has been postulated that in addition to the Duffy glycoprotein, P vivax parasites require a second RBC receptor for invasion that is specific for reticulocytes. Although the reticulocyte-specific RBC receptor for invasion by P vivax has not yet been identified, a P vivax ligand that binds specifically to reticulocytes has been identified and cloned.⁴⁶

  DUFFY BINDING LIGANDS OF P KNOWLESI AND P VIVAX

Haynes et al⁴⁷ identified a parasite protein (Duffy binding ligand) from P knowlesi that binds specifically to Duffy-positive erythrocytes but not to Duffy-negative erythrocytes. The 135-kD Duffy binding ligand of P knowlesi also binds to rhesus RBCs, which serologically react as Fy(a-b+) and which express an Fy^b-related antigen that is highly homologous, but not identical to, human Fy^b.²¹^,⁴⁷ A similar Duffy binding ligand of 140 kD was isolated by Wertheimer and Barnwell⁴⁸ from P vivax. Unlike the 135-kD Duffy binding protein of P knowlesi, the 140-kD Duffy binding protein of P vivax does not bind to rhesus RBCs, a cell type that cannot be invaded by P vivax.⁴⁸ Recent molecular analyses of the Duffy binding ligands of P knowlesi, P vivax, and the erythrocyte binding ligand of P falciparum (which does not bind to Duffy but to a sialic acid-dependent domain on glycophorin A) indicate regions of homology among these erythrocyte binding proteins.⁴⁹^,⁵⁰

Antibodies prepared against the 135-kD Duffy binding ligand of P knowlesi were used to screen expression complementary DNA (cDNA) libraries prepared from P knowlesi asexual erythrocyte-stage mRNA and cDNA encoding the 135-kD Duffy binding protein was cloned.⁵¹ The highly related 140-kD Duffy binding protein of P vivax was identified from cDNA clones obtained by cross-hybridization with the P knowlesi cDNA probe.⁵² Immunohistochemical studies localized the Duffy binding protein of P knowlesi to the micronemes, specialized organelles at the apical end of the parasite (merozoite) that appear to function in the process of invasion. This further supported the conclusion that Duffy binding proteins are indeed parasite ligands that bind to the Duffy receptor during invasion.⁵¹ How these proteins are translocated from micronemes to the apical surface of the merozoite to interact with the Duffy receptor is unknown.

The extracellular regions of the 140-kD Duffy binding protein of P vivax and the 135-kD Duffy binding protein of P knowlesi were classified into six domains based on amino acid sequence similarities.⁵¹^,⁵³ COS 7 cells were transfected with constructs encoding region I, region II, regions III-V, and region VI, each designed to yield cell-surface expression.⁵³ Cells expressing the cysteine-rich region II specifically formed rosettes with Duffy-positive, but not Duffynegative, erythrocytes, indicating that this domain is responsible for interactions with erythrocytes mediated by the Duffy glycoprotein.⁵³

Although the 140-kD Duffy binding ligand of P vivax did not bind to rhesus RBCs in the rosetting assay, binding did occur if rhesus RBCs were first treated with N-glycanase.⁵⁴ Synthetic peptides consisting of the first 35 amino acids from the N-terminus of the human Duffy protein and the first 34 amino acids from the N-terminus of the rhesus Duffy homolog both blocked rosetting of Duffy-positive human erythrocytes with COS cells expressing the 140-kD Duffy binding protein of P vivax.⁵⁴ These data support the conclusion that the Duffy homolog on rhesus RBCs contains the peptide binding site for the P vivax Duffy binding protein. However, it appears that on the intact rhesus RBCs, this site is altered or obscured by the presence of one or more N-linked sugar side chains. Thus, it appears that differences in glycosylation may contribute to species specificity of malaria parasite-host cell interactions.

  DUFFY BINDING LIGAND MOTIFS IN THE VAR GENES OF P FALCIPARUM

Identification of the Duffy binding proteins of P knowlesi and P vivax provided a clue to the identification of another important malarial gene family, termed the var genes.^55-58 Howard et al^59-61 had shown that malaria parasites, including P falciparum, insert parasite-derived proteins into the membranes of infected RBCs. Biologic data indicated that one or more of these proteins function in cytoadherence of P falciparum-infected RBCs to endothelial cells.⁶²^,⁶³ Cytoadherence of P falciparum-infected RBCs to endothelial cells of postcapillary venules accounts for the sequestration of mature parasite-infected RBCs in the deep vasculature and their consequent absence on diagnostic blood films.⁶⁴^,⁶⁵ Ultrastructural studies of sequestered parasites showed that the points of contact between infected RBCs and endothelial cells consist of electron dense knobs on the RBC membranes.⁶⁶^,⁶⁷ Sequestration allows parasites to grow within the vasculature of the host without circulating through the spleen, an organ known to have anti-malarial properties.⁶⁷^,⁶⁸ Cytoadherence of infected RBCs to endothelial cells of cerebral vasculature also contributes to the pathogenesis of cerebral malaria.⁶⁹ David et al⁷⁰ demonstrated that treatment of P falciparum-infected monkeys with hyperimmune serum reversed sequestration in a strain-specific fashion and led to the appearance of crisis forms (morphologically deteriorating parasites within erythrocytes). Serologic and immunochemical evidence indicated that the endothelial binding proteins on the surface of parasite-infected RBCs undergo antigenic variation, a phenomenon of obvious importance to the parasite as it affords a mechanism of circumventing the effects of specific antibodies that might otherwise block cytoadherence.^71-75 Understanding the molecular basis for endothelial cytoadherence and antigenic variation was thwarted for years by difficulties purifying sufficient quantities of the malarial endothelial cytoadherence protein for microsequencing. Unexpectedly, analysis of Duffy binding ligands provided a clue to the indentification of genes encoding variant endothelial cytoadherence proteins of P falciparum.⁵⁵^,⁵⁶

While sequencing DNA from regions of parasite chromosome 7, Peterson et al⁵⁵ and Su et al⁵⁶ identified several gene sequences that contain domains similar to region II of the erythrocyte binding proteins of P knowlesi, P vivax, and P falciparum. This family of related sequences possessed properties predicted for genes encoding cytoadherence proteins: they were polymorphic, they contained large open reading frames consistent with the size of putative cytoadherence proteins, and their predicted protein products included multiple domains with regions homologous to Duffy binding proteins, which had already been shown to have cytoadhesive properties. Taking advantage of homologies within these Duffy binding-like domains, Smith et al⁵⁷ used degenerate oligonucleotide primers to study messenger RNA from these genes (designated variant or var genes) expressed in cloned parasites with serologically distinct surface antigens and cytoadherence properties. They showed that the expression of distinct cytoadhesive properties and antigenic reactivity at the surface of infected erythrocytes correlated with expression of distinct var genes.⁵⁷ Baruch et al⁵⁸ cloned cDNA from an expression library for the putative cytoadherence protein, PfEMP1 (P falciparum erythrocyte membrane protein 1), and demonstrated by immunochemical methods that peptides derived from the cDNA had antigenic properties predicted for a cytoadherence protein. These investigators, together with Su et al, showed that cDNA for PfEMP1 is a member of the var gene family.⁵⁷^,⁵⁸ The studies by Su et al,⁵⁶ Smith et al,⁵⁷ and Baruch et al⁵⁸ were published simultaneously and confirmed that var genes encode related, but antigenically variant, cytoadherence proteins.

View this table:
[in this window] [in a new window]
Table 2. Malarial Receptor/Adhesion Proteins: Potential Targets of Receptor/Adhesion Blocking Immunity

Identification of var genes of P falciparum represents a major advance in malaria research because it provides the molecular basis for understanding sequestration, antigenic variation, and the chronicity of malaria. There is evidence that each parasite of P falciparum contains a large repertoire of var genes (50 to 150 copies) scattered throughout the malarial genome.⁵⁷ These genes, which encode highly homologous but antigenically distinct cytoadherence proteins, account for 2% to 6% of the haploid parasite genome.⁵⁷ Despite antigenic differences among these variant cytoadherence proteins of P falciparum, their capacity to bind to endothelial cells is conserved.⁵⁶ The molecular basis for this conservation of function in the face of antigenic variation remains to be elucidated, although it is clear that a number of endothelial receptors, including CD36, ICAM, VCAM, E-selectin, and thrombospondin may be involved in cytoadherence.^76-78

  THE IMPORTANCE OF MALARIAL CYTOADHERENCE TO VACCINE RESEARCH

Cytoadherence of all species of malarial merozoites to host RBCs, followed by invasion, is crucial for the life cycle of these obligate intracellular parasites. Cytoadherence of P falciparum-infected RBCs to endothelial cells of postcapillary venules appears crucial to the survival of this particular parasite. The parasite proteins discussed above are part of a newly described superfamily of malarial cytoadherence molecules (Table 2). If these molecules could be used as immunogens to induce antibodies that block cytoadherence, protection against malaria might be achieved (Fig 1). Even if the protection were partial, it would potentially be beneficial because many of the pathologic sequelae of malarial infections in the partially immune host are related to the level of parasitemia.⁷⁹ Because the endothelial cytoadherence molecule of P falciparum is highly variant, it will present a major challenge to development of a malaria vaccine. The parasite ligands that bind to RBC receptors appear much less variant, although there is evidence that P falciparum can invade by more than one pathway and can switch between at least two alternative pathways for invasion.⁸⁰ Although there is some variation within the adhesion domain of the P vivax Duffy binding protein, this parasite appears to be absolutely dependent on the Duffy pathway for invasion.⁸¹ P vivax provides a model for testing the efficacy of an erythrocyte binding protein-based vaccine, especially now that inhibition of ligand-receptor interaction by immune sera can be tested in vitro using the transfected COS cell-RBC rosetting assay established by Chitnis et al.⁵³^,⁵⁴

View larger version (19K):
[in this window]
[in a new window]

View larger version (17K):
[in this window]
[in a new window]
Fig 1. (A) Two receptors for apical attachment of P vivax merozoites to RBCs have been cloned: the Duffy binding protein and the reticulocyte binding protein.⁴⁶^,⁵² One strategy for vaccine development is to use these proteins as immunogens to induce antibodies that might block invasion of the parasite into the RBC. (B) Variant cytoadherence proteins of P falciparum have also been cloned.^55-57 Antibodies against this protein can block cytoadherence of P falciparum-infected RBCs to endothelial cells, forcing the parasite to circulate through the spleen, where immunologic mechanisms (which have not been clearly defined) are brought into play against P falciparum parasites. The cytoadherence antigen is variant and antigenic variation is used by the parasite to escape antibody mediated blockade of cytoadherence. Another strategy for vaccine development is to identify domains that are shared among different variants of the cytoadherece protein of P falciparum; antibodies against shared domains could block cytoadherence in a variant-independent fashion.

  THE DUFFY ANTIGEN IS A NOVEL RECEPTOR FOR CHEMOATTRACTANT CYTOKINES

Thus far we have discussed the function of the Duffy antigen in the pathogenesis of malaria. We have attempted to describe how knowledge of the Duffy antigen and its relationship to P knowlesi and P vivax malaria led to identification of malarial Duffy binding ligands, and this in turn provided clues to the identification of endothelial binding ligands of P falciparum. But clearly the Duffy glycoprotein exists for purposes other than malarial invasion of RBCs. Unexpectedly, research on chemoattractant cytokines, abbreviated chemokines, and their receptors converged with investigation on the Duffy blood group antigen, providing insight into a potential normal physiologic role for this allelic glycoprotein.¹⁰^,¹¹ To put this research into proper perspective, it is necessary to briefly review the biologic activities of chemokines.

Chemokines are members of a superfamily of small, secreted proteins ( $approx$ 8 to 13 kD), numbering over 20, that recruit leukocytes to sites of inflammation.⁸² This superfamily has two major branches, encoded in separate gene clusters, that preferentially promote acute and chronic inflammatory processes.^83-85 In addition to the functional differences between the two families, they are also distinguished by structural characteristics and at the level of genomic organization.^83-85 Chemokines of the C-X-C family, in which the two amino proximal cysteine residues are separated by one amino acid, function primarily as chemoattractants for neutrophils, whereas chemokines of the C-C family, in which these cysteines are juxtaposed, function primarily as chemoattractants for lymphocytes and monocytes. A newly discovered chemokine, lymphotactin, has a single cysteine, or C-motif rather than a C-C or C-X-C motif.⁸⁶

The effects of chemokines are mediated through high-affinity receptors that have seven hydrophobic membrane-spanning helices and signal through coupling with G-proteins.^87-92 In general, each chemokine has a specific receptor to which it binds with high affinity and another receptor to which it binds with lower affinity and with which it shares binding with another chemokine of the same family. In 1991, Darbonne et al⁹³ discovered a novel chemokine receptor on RBCs. Analysis of the repertoire of ligands bound by this novel receptor showed that it binds selected members of both the C-X-C and C-C families with high affinity (K_d $approx$ 5 nM).⁹⁴^,⁹⁵ These include interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA) from the C-X-C family and RANTES (regulated upon activation, normally T-cell expressed) and monocyte chemotactic peptide (MCP-1) from the C-C family.⁹⁴^,⁹⁵ The RBC chemokine receptor does not bind the C chemokine, lymphotactin.⁹⁶ The multispecific erythrocyte chemokine binding activity is preserved in other mammalian species, including mice, and in avian species.¹¹^,²¹^,⁹⁶

Further studies on the multispecific chemokine receptor on human RBCs led to the observation that approximately two thirds of African Americans lack this receptor activity, a frequency that was intriguingly similar to that of the Duffy-negative phenotype. Subsequent investigation led to the conclusion that the RBC chemokine receptor and the Duffy blood group antigen are identical.⁹⁷ There was an absolute correlation between chemokine binding activity and Duffy blood group antigen expression. Anti-Fy6 specifically blocked binding of chemokines to Duffy positive RBCs (Fig 2). Both the RBC chemokine receptor and the Duffy blood group antigen were destroyed by chymotrypsin treatment, but not trypsin treatment, of intact RBCs. Cross-linking experiments with ¹²⁵I-labeled MGSA indicated that the RBC chemokine receptor is a 35- 43-kD protein with a similar appearance on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as that of the Duffy antigen when detected by immunoblotting with anti-Fy6. MGSA, IL-8, and a mutant MGSA, designated MGSA-E₆A, which evinces high-affinity binding only to the RBC chemokine receptor, were shown to block invasion of human RBCs by P knowlesi, a parasite known to use the Duffy antigen for invasion.⁹⁷^,⁹⁸ MGSA was also shown to block binding of the 135-kD Duffy binding protein of P knowlesi to Duffy-positive human RBCs.⁹⁷ The conclusion drawn from this evidence that the human RBC multispecific chemokine receptor and the Duffy blood group antigen are identical was subsequently confirmed by transfection experiments. K562 and 293 cells (both of which normally lack the Duffy antigen and are devoid of a multispecific chemokine receptor) were transfected with cloned Duffy cDNA and consequently displayed concomitant Duffy antigenic and multispecific chemokine receptor activity on their surfaces.⁹⁹^,¹⁰⁰

View larger version (35K):
[in this window]
[in a new window]
Fig 2. Inhibition of chemokines binding to the DARC by the anti-Duffy MoAb, anti-Fy6. (A) Inhibition of specific ¹²⁵I-labeled IL-8 binding to Duffy-positive RBCs by increasing concentrations of anti-Fy6. The closed circles represent binding of ¹²⁵I-labeled IL-8 to cells expressing type A IL-8 receptor. Anti-Fy6 specifically inhibits binding of IL-8 to DARC in a dose-dependent fashion. (B) Inhibition of specific binding of radiolabeled IL-8, MGSA, MCP-1, and RANTES to Duffy-positive RBCs by anti-Fy6. Anti-Fy6 inhibits the binding of both C-X-C chemokines (IL-8, MGSA) and C-C chemokines (MCP-1, RANTES) to DARC. (Reprinted with permission from Science, Horuk R, Chitnis CE, Darbonne WC, Colby TJ, Rybicki A, Hadley TJ, Miller LH: A receptor for the malarial parasite Plasmodium vivax: The erythrocyte chemokine receptor. 261:1182, 1993. Copyright 1993 American Association for the Advancement of Science.)⁹⁷

  DUFFY ANTIGEN RECEPTOR FOR CHEMOKINES (DARC) IS EXPRESSED BY ENDOTHELIAL CELLS OF POSTCAPILLARY VENULES

The physiologic significance of the novel multispecific RBC chemokine receptor, designated the Duffy antigen receptor for chemokines (DARC), initially appeared questionable because no pathologic or inflammatory consequences have been associated with the Duffy-negative phenotype. However, immunohistochemical staining of human tissues with anti-Fy6 provided additional insight into a potential physiologic function for DARC.²²^,¹⁰¹ Specific and intense staining with anti-Fy6 was observed with endothelial cells lining postcapillary venules throughout the body. Staining was not observed on endothelial cells lining capillaries or larger vessels, including venules, veins, arterioles, and arteries.^101a Littoral cells, specialized endothelial cells that line the sinusoids in the red pulp of the spleen, were also strongly positive (Fig 3) as were the endothelial cells that line the bone marrow sinusoids and choroid plexus. Parallel chemokine binding experiments confirmed the presence of a high-affinity receptor having a promiscuous chemokine binding repertoire in membrane fractions from kidney and spleen.²²^,¹⁰⁰ Endothelial cells lining the hepatic sinusoids lacked immunoreactivity. Tissue from individuals of the Fy(a-b-) erythroid phenotype were also examined and found to react with anti-Fy6 in a fashion identical to tissue from Duffy-positive individuals.²² Interestingly, endothelial cells of larger vessels were observed to react with anti-Fy6 under conditions of inflammation (eg, temporal arteritis, thrombophlebitis, omphalitis), suggesting upregulation of DARC under these conditions.^101a

View larger version (155K):
[in this window]
[in a new window]
Fig 3. Immunohistochemistry of spleen using the anti-Duffy MoAb, anti-Fy6. Analysis was done on both freshly obtained and archival specimens of spleen. Anti-Fy6 reacts with specialized endothelial cells (littoral cells) lining the sinusoids in the red pulp. Endothelial cells lining arterioles, venules, arteries, and veins did not stain with anti-Fy6. Similar staining was observed with endothelial cells of postcapillary venules in every organ examined thus far. Immunohistochemical staining of endothelial cells in tissue obtained from Duffy-negative individuals (by RBC typing) was identical to that observed in tissue obtained from Duffy-positive individuals. In contrast to the sinusoids of the spleen, endothelial cells lining hepatic sinusoids did not stain.

View larger version (147K):
[in this window]
[in a new window]
Fig 6. Purkinje cells of the cerebellum express the DARC. Immunohistochemical staining of archival specimens of human cerebellum with the anti-DARC MoAb, anti-Fy6, showed high-level expression of DARC by Purkinje neurons. This staining was inhibited by a recombinant fusion protein in which the amino terminal extracellular domain of DARC was expressed in continuous translational frame with glutathione-S-transferase. Cross-linking experiments with ¹²⁵I-labeled MGSA and immunoblots with anti-Fy6 showed that MGSA and anti-Fy6 react with a protein component of cerebellar membranes with the same size and appearance on SDS-PAGE as RBC and endothelial cell DARC (data not shown).

As noted previously, DARC-like molecules are expressed on RBCs of rodents and other mammalian and avian species.¹¹^,²¹^,⁹⁶ Because many model systems for the actions of chemokines on leukocyte trafficking, angiogenesis, and microvascular leak syndromes have been developed in rodents, experiments were performed to determine whether DARC is expressed by endothelial cells of postcapillary venules in rats. Because immunologic reagents for the rodent homolog of DARC are not yet available, intravital microscopy was performed on rats infused with fluorescent microspheres covalently coated with IL-8 or MCP-1 into the cremaster vessels.^101a Monitoring of fluorescent microsphere circulation revealed attachment to endothelial cells lining small venules, but not to larger vessels. This binding of IL-8-coated microspheres was inhibited by previous infusion of IL-8, as well as MCP-1. Control microspheres did not adhere to endothelial cells. These experiments provide indirect functional data that the rodent homolog of DARC is expressed by subsets of endothelium according to a program that mimics that observed in humans.

View larger version (49K):
[in this window]
[in a new window]

View larger version (26K):
[in this window]
[in a new window]
Fig 4. Proposed seven transmembrane spanning topology of DARC. (A) Molecular modeling predicts that DARC has topologic features similar to other members of the chemokine receptor family. (Modified and reprinted with permission.¹¹³ ) The amino terminal extracellular domain contains the binding site for anti-Fy6 MoAbs and the polymorphism resulting in the Fy^a and Fy^b blood groups. This domain has also been found to contain sequences necessary for multi-specific chemokine binding. Sequences required for binding a MoAb with anti-Fy3 specificity have been tentatively localized to the third predicted extracellular loop. (B) The deduced amino acid sequence of DARC; the first seven N-terminal amino acids shown are those predicted from the exon identified by Iwamoto¹¹¹ and are thought to be present in the major DARC transcript; the N-terminal amino acids shown in parentheses and the rest of the amino acid sequence are derived from the single DARC exon as originally cloned by Chaudhuri et al.¹⁰⁵ (Modified and reprinted with permission from Horuk R: Molecular properties of the chemokine receptor family. Trends in Pharmacological Sciences, vol 15, p 159, 1994.⁸⁸ ).

It is of note that the subset of endothelial cells that express DARC is similar to the anatomic site of leukocyte trafficking and are targets for both adhesion and diapedesis.^102-104 In addition, endothelial cells in this anatomic distribution are targets for vascular leak syndromes induced by pathologic actions of IL-8. Thus, in addition to expression on erythrocytes, which is not essential, DARC is expressed by endothelial cells at a dynamic interphase of leukocyte trafficking, a process that is highly regulated by the actions of chemokines that bind to DARC. Whether or not DARC actually plays a role in leukocyte trafficking remains to be determined.

  THE MOLECULAR BIOLOGY OF DARC

Complementary DNA (cDNA) encoding DARC was molecularly cloned by Chaudhuri et al¹⁰⁵ at the New York Blood Center in 1993. Sequences of various peptides from the Duffy glycoprotein, which were purified by immunoaffinity chromatography with Fy6 MoAbs, were used to design oligonucleotide primers that were employed in DNA amplification reactions.¹⁰⁵^,¹⁰⁶ A 72-bp probe generated by reverse transcription followed by polymerase chain reaction was used to screen a cDNA library prepared from bone marrow cells of a Duffy-positive individual. Nucleotide sequence analysis of overlapping cDNA clones revealed an open reading frame (ORF ) of 1,014 bp, which predicted a protein product of 338 amino acids, with a theoretical molecular weight of 35 kD. In situ hybridization confirmed previous linkage analysis that the Duffy locus is on chromosome 1q22 $right-arrow$ q23.¹⁰⁷^,¹⁰⁸ The Duffy cDNA was noted to share significant homology with the human and rabbit type B IL-8 receptor, which also binds MGSA at high affinity.¹⁰⁵ Surprisingly, DARC also shares a similar level of homology to the multispecific receptor for endothelins, vaso-active proteins with 21 amino acid residues and two internal disulfide bonds, which are also expressed by endothelial cells and have a profound effect on vascular biology. Whereas the similarity between DARC and the IL-8RB is more prominent in the amino-terminal and carboxyl-terminal regions, its similarity to the multispecific endothelin receptor (type B) is evident in the loops (both extracellular and cytoplasmic) and transmembrane-spanning helices. Unlike virtually all other heptahelical receptors, which are almost universally linked to G-proteins, DARC lacks a highly conserved DRY motif in the second cytoplasmic loop.

View larger version (13K):
[in this window]
[in a new window]
Fig 5. Schematic representation of the DARC genomic locus. DARC is encoded by an ORF contained in two exons separated by one intron. Primer extension and 5'RACE analysis of both human DARC and mouse DARC homolog (Wang Z.X., Lu A.H., Peiper S.C., unpublished observations, September 1995) reveal the presence of an upstream exon containing a methionine translation initiation codon and codons for six additional amino acid residues. An internal initiation codon that may be used in the human gene is not conserved in the mouse gene. The polymorphism responsible for the Fy^a and Fy^b phenotypes is encoded at codon 44.

Although molecular modeling approaches described in the original cloning report predicted the presence of nine hydrophobic helices that could serve as transmembrane spanning domains, subsequent molecular modeling analyses predicted the presence of seven hydrophobic $alpha$ -helices, analogous to receptors for all of the other known chemoattractants.¹¹^,⁹⁹^,¹⁰⁹ Although DARC undergoes cotranslational addition of asparagine-linked oligosaccharide chains and posttranslational remodeling of carbohydrates in the Golgi, the predicted primary structure lacks an N-terminal series of hydrophobic amino acid residues that could function as a signal peptide. This characteristic is common to all of the cloned chemokine receptors. It is presumed that these proteins are targeted to membrane-bound polyribosomes and the Golgi apparatus via the hydrophobic helices, which are predicted to serve as transmembrane anchors.

The presence of seven hydrophobic helices suggests a topology in which there are an amino-terminal extracellular domain (approximately 62 residues), three extracellular loops, three cytoplasmic loops, and a carboxly-terminal cytoplasmic tail (28 residues) (Fig 4). Alignment with the primary structures of other known chemokine receptors shows that DARC contains cysteine residues in each extracellular loop that line up with those present in the other receptors, as well as the presence of other highly conserved residues. The N-terminal extracellular domain contains at least two, and possibly three, sites for addition of oligosaccharide chains to asparagine residues. The N-terminal extracellular domain is rich in acidic amino acid residues, as might be expected for a domain involved in binding ligands with a basic isoelectric point (pI). As characteristic of other chemokines receptors, the cytoplasmic tail is composed of approximately 50% serine and threonine residues, which are presumed to be targets for phosphorylation, as has been observed for the type B IL-8 receptor.

The organization of the gene encoding DARC has not yet been completely elucidated. Using primer extension analysis, Chaudhuri et al¹⁰⁵ found the RNA cap site to be approximately 175 bp upstream from the (presumed) translation initiation codon. Amplification of the ORF with primers from the 5' and 3' untranslated regions by polymerase chain reaction yielded DNA fragments of identical size when genomic DNA and cDNA reverse transcribed from mRNA served as the source of templates. Nucleotide sequence analysis confirmed that the ORF was contained within a single, uninterrupted exon. Constructs prepared from this ORF were capable of encoding a protein of the predicted size that bound anti-Duffy MoAbs and the predicted panel of chemokines at the appropriate affinity when expressed in K562 cells and human embryonic kidney cells.⁹⁹^,¹⁰⁰ However, because the amino-terminal peptide sequence of the Duffy antigen has not been determined, it has not been confirmed that the translation initiation codon proposed by Chaudhuri et al is the predominant one used in vivo.¹⁰⁵

Analysis by other groups provide evidence that for an mRNA cap site different from that initially reported. Using the method of rapid amplification of cDNA ends (5'RACE), Tournamille et al¹¹⁰ reported that the mRNA initiation site was 495 bp upstream of the translation initiation codon. Similar studies described by Iwamoto et al¹¹¹ localized the cap site to -550 bp. The latter report also described a novel upstream exon that contained an alternative ATG codon. Direct comparison showed that this exon, which encodes a methionine and six additional amino acid residues, is preferentially utilized over the downstream cap site described by Chaudhuri et al. Analysis of the mouse gene encoding the homolog of DARC performed in our laboratory supports the findings of Iwamoto et al.¹¹¹ The downstream methionine residue is not conserved in the mouse gene. 5'RACE of mRNA from mouse spleen confirmed the presence of an upstream exon encoding seven amino acids, led by a methionine residue. This exon, which is followed by a consensus splice donor sequence, is separated from the exon containing the majority of the ORF by an intron of approximately 450 bp. Together, these findings suggest that both human DARC and the mouse homolog are encoded by a gene composed of two exons and one intron (Fig 5).

The importance of DARC is evidenced by the conservation of this gene across species. We have cloned genes encoding DARC homologs from six nonhuman primates, cow, pig, rabbit, and mouse. There is over 95% conservation of primary structure between the DARC homologs in humans and great apes. There is some divergence in macaques. The nucleotide sequence of the rhesus homolog predicts six amino acid differences compared with humans and a deletion of codon 24 which encodes an aspartic acid residue in the amino-terminal extracellular domain.²¹ Rhesus RBCs react with anti-Fy^b but not with anti-Fy6. There is divergence in the predicted amino acid sequence of the murine homolog to a level of approximately 65% identity with the human receptor protein.

  PRESERVATION OF DARC EXPRESSION IN ENDOTHELIAL CELLS OF DUFFY-NEGATIVE INDIVIDUALS

Despite the conservation of DARC function and primary structure across species, the fact that a large population of individuals of African ancestry lacks expression of this receptor on their RBCs without any detectable adverse consequences represented a problem for those wishing to assign physiologic significance to DARC. Whereas Southern blotting experiments failed to show evidence of a gross rearrangement or deletion involving the DARC gene in DNA from Duffy-negative individuals, Northern blot analysis failed to detect mRNA transcripts encoding DARC in bone marrow (erythroid) cells.¹⁰³ However, Northern blot analysis indicated that mRNA transcripts that annealed to a DARC probe under high stringency conditions were present in the spleen and other tissues of Duffy-negative individuals.²¹^,²² As noted above, immunohistochemical staining revealed reactivity of anti-Fy6 with endothelial cells lining splenic sinusoids, bone marrow sinusoids, choroid plexus, and postcapillary venules in Duffy-negative as well as Duffy-positive individuals. Parallel biochemical studies demonstrated the presence of a high-affinity chemokine receptor (K_d $approx$ 5 nM) with a binding specificity that included members of both C-X-C and C-C families, identical to that of DARC from the spleen of a Duffy individual.²² Nucleotide sequence analysis of the ORF from these patients failed to show evidence of a mutation in the coding sequences of the gene. As was observed by Chaudhuri et al, the genotype of the Duffy-negative African Americans tested was FY*B/FY*B.²¹ This was consistent with previous observations that Duffy-negative individuals of African descent produce anti-Fy^a antibodies, but not anti-Fy^b.¹⁹^,²⁰

Based on the presence of an intact gene and a tissue-specific expression defect, it was postulated that the loss of expression in erythroid cells of Duffy-negative individuals could be due to a promoter/enhancer defect.²⁰^,^108-112 Tournamille et al showed that Duffy-negative individuals have a point mutation in a consensus binding site for GATA-1, a transcription factor active in erythroid cells.¹¹⁰ This point mutation, located at -46, abolishes erythroid promoter activity in reporter gene assays.¹¹⁰ Further work is required to understand the molecular basis for maintenance of expression of DARC on endothelial cells and Purkinje cells, even in individuals who are Duffy negative.

The immunohistochemical and molecular genetic evidence suggest an important physiologic role for DARC. Although the selective pressure from malaria led to loss of DARC expression on RBCs (affording protection from P vivax malaria), nature's experiment selected a mechanism whereby expression of DARC was preserved on endothelial cells. This evolutionary outcome suggests that the function of DARC on endothelial cells is less dispensable than its function on RBCs.

Recently, the Duffy genotype of a rare Fy(a-b-) white individual was reported.¹¹³ This was the same individual (AZ) who produced anti-Fy3 described by Albrey et al.²⁵ Analysis of the nucleotide sequence of amplification products from the DARC gene disclosed the presence of a 14-bp deletion that resulted in a frameshift mutation beginning at codon 98. AZ is apparently homozygous for this rare deletion. The mutation predicted the translation of a 118-amino acid residue polypeptide that would include the amino terminal extracellular domain, the first transmembrane spanning $alpha$ -helix, and the first cytoplasmic loop. However, RBCs from this individual are devoid of serologically detectable Duffy antigen and presumably DARC is missing in other tissues as well. These studies were performed on blood cells that had been previously frozen, and neither current clinical information nor immunohistochemical information from nonerythroid tissue in this individual were obtainable. However, in the report by Albrey et al describing anti-Fy3 during AZ's third pregnancy, there is no mention of ill health. This suggests that a DARC-nullizygous phenotype may not be associated with adverse biologic consequences. Of course, biologic redundancy or compensatory mechanisms could obscure a defect in such individuals. It is hoped that a mouse nullizygous for the DARC homolog can be produced by targeted gene disruption to further explore the function of DARC. This was part of our rationale for cloning the murine DARC-homolog.

  STRUCTURE-FUNCTION ANALYSIS OF DARC

Nucleotide sequence analysis of molecular clones of DARC genes from Fy(a+b-), Fy(a-b+), and Fy(a+b+) individuals in several laboratories showed that the Fy^a and Fy^b alleles differ by a single base substitution in the second position of codon 44 (Fig 4) that encodes a glycine residue in Fy^a and an aspartic acid residue in Fy^b.²¹^,¹⁰⁰^,^113-115 This polymorphism does not appear to have any biologic consequences.

Anti-Fy6 was shown to inhibit chemokine binding to DARC in a dose-dependent fashion whereas anti-Fy3 reagent did not alter binding at concentrations up to 100 µmol/L.¹¹⁶ At higher concentrations of anti-Fy3, some inhibition of chemokine binding to DARC was obtained.⁹⁶^,¹¹⁷ IL-8 did not inhibit the binding of anti-Fy6 to DARC on RBCs, indicating that the IL-8 binding site and the Fy6 epitope are distinct.¹¹⁸ The Fy6 and Fy3 epitopes were mapped using chimeric receptor proteins composed of complementary portions of DARC and IL-8RB, the chemoattractant receptor most closely related to DARC.¹¹⁶ Only chimeric receptors containing the amino terminal extracellular domain of DARC reacted with anti-Fy6 MoAbs, whether stably expressed in human cell lines or in insect cells using baculovirus. The Fy6 epitope was further localized by Wasniowska et al¹¹⁹ using a combinatorial peptide approach and site-directed mutagenesis. These investigators showed that two adjacent acidic amino acid residues, encoded by codons 25 (aspartic acid) and 26 (glutamic acid), are critical for anti-Fy6 binding, whereas the asparagine residues encoded by codons 18 and 29 are not involved (Fig 4). Furthermore, since mutation of these two residues eliminates two sites for the addition of oligosaccharide chains, it is likely that the Fy6 epitope is composed solely of peptide residues.

The epitope recognized by anti-Fy3 has also been mapped, although not to the same degree of molecular detail as Fy6.¹¹⁶ Immunofluorescent analysis of insect cells expressing chimeric receptors showed that the anti-Fy3 MoAb bound to constructs containing the three predicted extracellular loops of DARC, but not to constructs containing only the amino terminal extracellular domain.¹¹⁹ Anti-Fy3 did not bind to receptor chimeras that included the N-terminal extracellular domain and the first and second predicted extracellular loops. It was concluded that sequences necessary for the binding of anti-Fy3 are present in the third predicted extracellular loop of DARC (Fig 4).

Parallel studies localized the regions of the receptor involved in the binding of chemokines and the malarial Duffy binding ligands to this receptor.¹¹⁹ Analysis of a chimera expressing the N-terminal extracellular domain of DARC with the remainder of the heptahelical structure being that of IL-8RB (DARC^e1/IL-RB) showed that it bound not only IL-8 and MGSA at high affinity, but also RANTES, a C-C chemokine that binds to DARC, but not IL-8RB. Moreover, the DARC^e1/IL-8RB chimera bound the DARC-specific MGSA-E₆A mutant with a K_d of 12 nM, mirroring the activity of intact DARC. Preliminary studies suggest that the 140-kD Duffy binding protein of P vivax also binds to the DARC^e1/IL-8RB chimera (C. Chitnis, L.H. Miller, S. Peiper, personal communication, June 1995). Taken together, these findings highlight the significance of the amino terminal extracellular domain in the various biologic activities of DARC. Recent studies using DARC variants created by in situ mutagenesis indicate chemokine binding to DARC is dependent on the disulfide bond between a cysteine on the N-terminal extracellular domain and domain and a cysteine on the third extracellular loop.¹¹⁷

  DARC IS EXPRESSED BY PURKINJE CELLS OF THE CEREBELLUM

Emerging evidence suggests that chemokines are involved in normal homeostatic processes in the central nervous system. Experiments using in situ hybridization localized rat mRNA that hybridizes to a human IL-8 probe in specific areas of the brain, including hippocampus and cerebellum.¹²⁰ Human IL-8 has also been shown to enhance survival of rat hippocampal neurons in vitro.¹²¹ It is thought that IL-8 is synthesized and secreted by astrocytes.¹²² There are approximately 10-fold more astrocytes in the central nervous system than neurons and one of the functions of astrocytes is to "nurture" neurons by the secretion of neurotrophic factors. Until recently there were no reports on neurons expressing chemokine receptors.

Chaudhuri et al¹⁰⁵ demonstrated the presence of mRNA in human brain that specifically hybridized with DARC cDNA. Interestingly, there were two species of mRNA observed: one of the predicted size, 1.2 kb, which was also detected in many other tissues tested; and one of 8.5 kb, which thus far has only been observed in brain. Although the nature of the 8.5-kb transcript has not yet been elucidated, the findings of DARC transcripts in the brain prompted our investigation of brain by immunohistochemistry.¹¹^,¹²³ Anti-Fy6 was found to react specifically with Purkinje neurons of the cerebellum (Fig 6, see page 3083). Parallel experiments were done with an MoAb against IL-8 receptor B (IL-8RB) which reacted specifically with subsets of neurons in diverse regions of the brain and spinal cord.¹²³ These included the hippocampus, dentate nucleus, pontine nuclei, locus coeruleus, and paraventricular nucleus in the brain and the anterior horn, interomediolateral cell column, and Clarke's column of the spinal cord. Like Purkinje neurons of the cerebellum that express DARC, the neurons in other areas of the brain and spinal cord that express the IL-8RB are projection neurons, bearing long axons that connect one neuronal region with another. Interneurons, which connect neurons within a region, did not react with either anti-Fy6 or the anti-IL-8RB antibody.

Further experiments were performed to confirm that anti-Fy6 reactivity with Purkinje cells was indeed caused by the presence of DARC. Membranes were isolated from cerebellar tissue at postmortem examination and these were analyzed for the presence of DARC by chemokine binding assays, chemokine cross-linking experiments, and immunblotting. Results obtained by each of these methods supported the immunohistochemical data and indicated that DARC is indeed expressed on Purkinje neurons of the cerebellum. What function DARC serves on Purkinje neurons and how this relates to its expression on RBCs and on endothelial cells remains to be determined.

  THE RIDDLE OF DARC FUNCTION

Despite knowledge of structure-function relationships and tissue localization of DARC, the precise role of this receptor in normal and pathologic physiology remains uncertain. DARC does not appear to present chemokines in an active form to leukocyte receptors. Once bound to DARC expressed on the surface of erythrocytes, IL-8 did not induce effects in neutrophils associated with chemokine stimulation (K. Neote, S. Peiper, unpublished observations, September 1994).⁹³ Human erythroleukemia (HEL) cells express DARC, but its function on these cells has not been elucidated.¹²⁴ Although DARC on RBCs does not internalize ligand, there is evidence that DARC-transfectants can internalize ligands.²² It is possible that endothelial DARC internalizes ligands, thereby generating the chemotactic gradient essential for leukocyte attraction.

Based on what is known of other heptahelical membrane receptors, it seems likely that DARC transmits a signal across the membrane upon chemokine binding. However, DARC-dependent signal transduction has not yet been demonstrated. DARC does not appear to be coupled to a guanosine triphosphate binding protein (G-protein). It does not stimulate GTPase activity nor mediate calcium flux upon ligand binding; furthermore, DARC lacks a DRY motif in the second cytoplasmic loop that is characteristic of G-protein-coupled seven-membrane-spanning receptors.

To date, only a few other heptahelical receptors that lack DRY motifs have been cloned. They include the cAMP receptor in Dictostelium, bride of Sevenless (bos ) in drosophila, and the two seven-transmembrane-spanning proteins that harbor mutations in familial cases of Alzheimer's disease.^125-128 The yeast homolog of the heptahelical protein mutated in Alzheimer's disease has been shown to be involved in signaling via the notch pathway.¹²⁹ Thus, there is precedent for heptahelical receptors that signal through pathways independent of G-coupled proteins. It is possible that DARC interacts or "cross-talks" with other membrane components (which could differ depending on cell type) and thereby elicit tissue specific responses to ligand binding.

  SUMMARY

The glycoprotein expressed on surface of erythrocytes initially known as the Duffy blood group antigen has been shown to be a receptor for the invasion of these cells by P vivax parasites. The parasite ligand that binds to the Duffy antigen has now been cloned and characterized. The region on the Duffy binding ligand of the parasite responsible for interaction with the Duffy antigen has also been identified. It is hoped that this molecule will useful as an immunogen to induce antibodies capable of blocking invasion of the parasite into the erythrocyte. Research on the Duffy binding ligand also provided a clue (a shared motif ) to the identification of the long-sought family of variant endothelial binding ligands that mediate attachment of P falciparum-infected erythrocytes to endothelial cells of postcapillary venules. If this attachment could be disrupted during the course of a malarial infection, it might lead to spleen-mediated parasite death. It might also lead to amelioration of cerebral malaria, a condition which in part is due to the blockade of cerebral venules by sequestered adherent parasites.

Recently, research on the Duffy antigen has taken on a new dimension. The Duffy antigen has been shown to be a multispecific heptahelical receptor for chemokines, expressed on RBCs, endothelial cells of postcapillary venules, and Purkinje cells of the cerebellum. The challenge now is to determine its function, both in immunobiology and neurobiology. Its capacity to bind chemoattractant cytokines and its expression on endothelial cells lining postcapillary venules are highly conserved across species, suggesting that this receptor subserves a critical function. This is supported by nature's experiment, the Duffy blood group negative phenotype, in which the genetic mechanism selected to remove expression on erythroid cells to protect against malarial infection, preserved expression on endothelial cells of postcapillary venules and splenic sinusoids. Although we have significant insight into structure-function relationships for the Duffy antigen/receptor for chemokines, its mechanism of signaling and its biologic function remain to be elucidated.

  NOTE ADDED IN PROOF

Chaudhuri et al (Blood 89:701, 1997) recently described results of immunohistochemical staining with a polyclonal rabbit antibody (6615) that reacts with carbohydrate on the Duffy antigen. Reactivity was observed with endothelial cells of glomeruli, capillaries, vasa recta, and epithelial cells of collecting tubules in the kidney; reactivity was also observed with capillaries in the thyroid, and capillaries, large venules, and type I alveolar squamous cells of lung. Whether or not the carbohydrate epitope recognized by polyclonal rabbit antibody 6615 is also present on molecules other than the Duffy antigen remains to be determined.

  FOOTNOTES

   Submitted July 17, 1996; accepted December 13, 1996.
   Supported by a Merit Review Grant from the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, by the Agnes Brown Duggan Endowment for Oncologic Research, and the the Humana Endowment for Excellence.
   Address reprint requests to Terence J. Hadley, MD, James Graham Brown Cancer Center, University of Louisville, 529 S Jackson St, Louisville, KY 40292.

  ACKNOWLEDGMENT

We acknowledge the investigators in our laboratories including Zi-xuan Wang, Zhaohai Lu, and Haihong Guo. Collaborators at the University of Louisville include Alvin Martin and Victor Fingar and Stephen Slone. We also acknowledge Richard Horuk (Berlex, Richmond, CA), who has been a major collaborator, and Domanique Blanchard (Centre Regional de Transfusion Sanguine, Nantes, France) and Makoto Uchikawa (Japanese Red Cross Central Blood Center, Tokyo, Japan) for providing anti-Fy6 and anti-Fy3, respectively. Anti-Fy3 was obtained through Peter Byrne at the National Reference Laboratory (Rockville, MD). We also thank Beverly Kirkpatrick and Abby Carden for assistance in preparing the manuscript for this review.

  REFERENCES

Introduction
References

1. Landsteiner K: Individual differences in human blood. Science 73:405, 1931
2. Levine P, Burnham L, Katzin EM, Vogel P: Role of iso-immunization in pathogenesis of erythroblastosis fetalis. Am J Obstet Gynecol 42:925, 1941
3. Anstee DJ: Blood group-active surface molecules of the human red blood cell. Vox Sang 58:1, 1990[Medline] [Order article via Infotrieve]
4. Issitt PD, Issitt CH: Applied Blood Group Serology. Miami, FL, Montgomery Scientific, 1985
5. Anstee DJ: Blood group antigens defined by the amino acid sequences of red cell surface proteins. Transf Med 5:1, 1995[Medline] [Order article via Infotrieve]
6. Agre PC, Catron J-P (eds): Blood Group Antigens of the Human Red Cell. Structure, Function, and Clinical Significance. Baltimore, MD, Johns Hopkins University Press, 1992
7. Cartron, J-P, Rouger P (eds): Molecular Basis of Major Blood Group Antigens, Blood Cell Biochemistry, vol 8. New York, NY, Plenum, 1995
8. Telen MJ: Erythrocyte blood group antigens: Not so simple after all. Blood 85:299, 1995[Free Full Text]
9. Barnwell JW, Galinski MR: Plasmodium vivax: A glimpse into the unique and shared biology of the merozoite. Ann Trop Med Parasitol 89:113, 1995[Medline] [Order article via Infotrieve]
10. Pogo AO, Chaudhuri A: Duffy and receptors for P. vivax and chemokine peptides. Transf Clin Biol 2:269, 1995[Medline] [Order article via Infotrieve]
11. Horuk R, Peiper SC: The Duffy antigen receptor for chemokines, in Horuk R (ed): Chemoattractant Ligands and Their Receptors. New York, NY, CRC, 1996
12. Horuk R, Martin A, Hesselgesser J, Hadley T, Lu ZH, Wang ZX, Peiper SC: The Duffy antigen receptor for chemokines: Structural analysis and expression in the brain. J Leukoc Biol 59:29, 1996[Abstract]
13. Cutbush M, Mollison PL, Parkin DM: A new human blood group. Nature 165:188, 1950
14. Ikin EW, Mourant AE, Pettenkoffer JH, Blumenthal G: Discovery of the expected haemagglutinin anti-Fy^b. Nature 168:1077, 1951
15. Chown B, Lewis M, Kaita H: The Duffy blood group in caucasians: Evidence for a new allele. Am J Hum Genet 17:384, 1965[Medline] [Order article via Infotrieve]
16. Lewis M, Kaita H, Chown B: The Duffy blood group system in caucasians: A further population sample. Vox Sang 23:523, 1972[Medline] [Order article via Infotrieve]
17. Mourant AE, Kopec AC, Domaniewska-Sobczak K: The Distribution of Human Blood Groups and Other Polymorphisms (ed 2). London, UK, Oxford University Press, 1976
18. Sanger R, Race RR, Jack J: The Duffy blood groups of New York Negroes: The phenotype Fy(a-b-). Br J Haematol 1:370, 1955
19. Vengelen-Tyler V: Anti-Fy^a preceding anti-Fy3 or -Fy5: A study of five cases. Transfusion Abstracts S150, 1985 (suppl)
20. Le Pennec PY, Rouger P, Klein MT, Robert N, Salmon C: Study of anti-Fy^a in five black Fy(a-b-) patients. Vox Sang 52:246, 1987[Medline] [Order article via Infotrieve]
21. Chaudhuri A, Polyakova J, Zbrzezna V, Pogo O: The coding sequence of Duffy blood group gene in humans and simians: Restriction fragment length polymorphism, antibody and malarial parasite specificities and expression in nonerythroid tissues in Duffy negative individuals. Blood 85:615, 1995[Abstract/Free Full Text]
22. Peiper SC, Wang ZX, Neote K, Martin AW, Showell HJ, Conklyn MJ, Ogborne K, Hadley TJ, Lu ZH, Hesselgesser J, Horuk R: The Duffy antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Duffy negative individuals who lack the erythrocyte receptor. J Exp Med 181:1311, 1995[Abstract/Free Full Text]
23. Morton JA: Some observations on the action of blood-group antibodies on red cells treated with proteolytic enzymes. Br J Haematol 8:134, 1962
24. Judson PA, Anstee DJ: Comparative effect of trypsin and chymotrypsin on blood group antigens. Med Lab Sci 34:1, 1977[Medline] [Order article via Infotrieve]
25. Albrey JA, Vincent EE Jr, Hutchinson J, Marsh WL, Allen FH, Gavin J, Sanger R: A new antibody, anti-Fy3, in the Duffy blood group system. Vox Sang 20:29, 1971[Medline] [Order article via Infotrieve]
26. Behzad O, Lee CL, Gavin J, Marsh WL: A new anti-erythrocyte antibody in the Duffy system: Anti-Fy4. Vox Sang 24:337, 1973[Medline] [Order article via Infotrieve]
27. Colledge KI, Pezzulich M, Marsh WL: Anti-Fy5: An antibody disclosing a probable association between the Rhesus and Duffy glood group genes. Vox Sang 24:193, 1973[Medline] [Order article via Infotrieve]
28. Moore S, Woodrow CF, McClelland DB: Isolation of membrane components associated with human red cell antigens Rh(D), (c), (E) and Fy. Nature 295:529, 1982[Medline] [Order article via Infotrieve]
29. Hadley TJ, David PH, McGinniss MH, Miller LH: Identification of an erythrocyte component carrying the Duffy blood group Fy^a antigen. Science 223:597, 1984[Abstract/Free Full Text]
30. Tanner MJ, Anstee DJ, Mallison G, Ridgewell K, Martin PG, Avent ND, Parsons SF: Effect of endoglycosidase F-peptidyl N-glycosidase F preparations on the surface components of the human erythrocyte. Carbohydrate Res 178:203, 1988[Medline] [Order article via Infotrieve]
31. Chaudhuri A, Pogo OA: The Duffy is a glycoprotein, in Cartron J-P, Rouger P (eds): Molecular Basis of Major Human Blood Group Antigens, Blood Cell Biochemistry, vol 8. New York, NY, Plenum, 1995
32. Wasniowska K, Eichenberger P, Kugele F, Hadley TJ: Purification of a 28 kD non-aggregating tryptic peptide of the Duffy blood group protein. Biochem Biophys Res Commun 192:366, 1993[Medline] [Order article via Infotrieve]
33. Wasniowska K, Hadley TJ: The Duffy blood group antigen: An update. Transf Med Rev 8:281, 1994[Medline] [Order article via Infotrieve]
34. Nichols ME, Rubinstein P, Barnwell J, Rodriguez de Cordoba S, Rosenfield RE: A new human Duffy blood group specificity defined by a murine monoclonal antibody. Immunogenetics and association with susceptibility to Plasmodium vivax. J Exp Med 166:776, 1987[Abstract/Free Full Text]
35. Barnwell JW, Nichols ME, Rubenstein P: In vitro evaluation of the role of the Duffy blood group in erythrocyte invasion by Plasmodium vivax. J Exp Med 169:162, 1989
36. Riwom S, Janvier D, Navenot JM, Benbunan M, Muller JY, Blanchard D: Production of a new murine monoclonal antibody with Fy6 spcificity and characterization of the immunopurified N-glycosylated Duffy-active molecule. Vox Sang 66:61, 1994[Medline] [Order article via Infotrieve]
37. Miller LH, Mason SJ, Dvorak JA, McGinniss MH, Rothman IK: Erythrocyte receptors for (Plasmodium knowlesi ) malaria: Duffy blood group determinants. Science 189:561, 1975[Abstract/Free Full Text]
38. Mason SJ, Miller LH, Shiroishi T, Dvorak JA, McGinniss MH: The Duffy blood group determinants: Their role in the susceptibility of human and animal erythrocytes to Plasmodium knowlesi malaria. Br J Haematol 36:327, 1977[Medline] [Order article via Infotrieve]
39. Miller LH, Aikawa M, Johnson JG, Shiroishi T: Interaction between cytochalasin B-treated malarial parasites and erythrocytes. Attachment and junction formation. J Exp Med 149:172, 1979[Abstract/Free Full Text]
40. Aikawa M, Miller LH, Johnson J, Rabbege J: Erythrocyte entry by malarial parasites: A moving junction between erythrocyte and parasite. J Cell Biol 77:72, 1978[Abstract/Free Full Text]
41. Young MD, Eyles DE, Burgess RW, Jeffrey GM: Experimental testing of the immunity of Negroes to Plasmodium vivax. J Parasitol 41:315, 1955
42. Miller LH, Mason SJ, Clyde DF, McGinniss MH: The resistance factor to Plsmodium vivax in blacks: The Duffy blood group genotype, FyFy. N Engl J Med 295:302, 1976[Abstract]
43. Miller LH, McGinniss MH, Holland PV, Sigmon P: The Duffy blood group phenotype in American blacks with Plasmodium vivax in Vietnam. Am J Trop Med Hyg 27:1069, 1978
44. Spencer HC, Miller LH, Collins WE, Knud-Hansen C, McGinnis MH, Shiroishi T, Lobos RA, Feldman RA: The Duffy blood group and resistance to Plasmodium vivax in Honduras. Am J Trop Med Hyg 27:664, 1978
45. Kitchen SF: The infection of reticulocytes by Plasmodium vivax. Am J Trop Med 18:347, 1938
46. Galinski MR, Medina CC, Ingravallo P, Barnwell JW: A reticulocyte-binding protein complex of Plasmodium vivax merozoites. Cell 69:1213, 1992[Medline] [Order article via Infotrieve]
47. Haynes JD, Dalton JP, Klotz FW, McGinnis MH, Hadley TJ, Hudson DE, Miller LH: Receptor-like specificity of a Plasmodium knowlesi malarial protein that binds to Duffy antigen ligands on erythrocytes. J Exp Med 167:1873, 1988[Abstract/Free Full Text]
48. Wertheimer SP, Barnwell JW: Plasmodium vivax interaction with the human Duffy blood group glycoprotein: Identification of a parasite receptor-like protein. Exp Parasitol 69:340, 1989[Medline] [Order article via Infotrieve]
49. Sim BKL, Chitnis CE, Wasniowska K, Hadley TJ, Miller LH: Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum malaria. Science 264:1941, 1994[Abstract/Free Full Text]
50. Adams JH, Sim BK, Dolan SA, Fang X, Kaslow DC, Miller LH: A family of erythrocyte binding proteins of malaria parasites. Proc Natl Acad Sci USA 89:7085, 1992[Abstract/Free Full Text]
51. Adams JH, Hudson DE, Torii M, Ward GE, Wellems TE, Aikawa M, Miller LH: The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive Malaria merozoites. Cell 63:141, 1990[Medline] [Order article via Infotrieve]
52. Fang XD, Kaslow DC, Adams JH, Miller LH: Cloning of the Plasmodium vivax Duffy receptor. Mol Biochem Parasitol 44:125, 1992
53. Chitnis CE, Miller LH: Identification of the erythrocyte binding domains of Plasmodium vivax and Plasmodium knowlesi proteins involved in erythrocyte invasion. J Exp Med 180:497, 1994[Abstract/Free Full Text]
54. Chitnis CE, Chaudhuri A, Horuk R, Pogo O, Miller LH: The domain on the Duffy blood group antigen for binding Plasmodium vivax and P. knowlesi malarial parasites to erythroytes. J Exp Med 184:1531, 1996[Abstract/Free Full Text]
55. Peterson DS, Miller LH, Wellems TE: Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins. Proc Natl Acad Sci USA 92:7100, 1995[Abstract/Free Full Text]
56. Su XZ, Heatwole VM, Wertheimer SP, Guinet F, Herrfeldt JA, Peterson DS, Ravetch JA, Wellems TE. The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell 82:89, 1995
57. Smith JD, Chitnis CE, Craig AG, Roberts DJ, Hudson-Taylor DE, Peterson DS, Pinches R, Newbold CI, Miller LH: Switches in expression of Plamodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82:101, 1995[Medline] [Order article via Infotrieve]
58. Baruch DI, Pasloske BL, Singh HB, Bi X, Ma XC, Feldman M, Taraschi TF, Howard RJ: Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82:77, 1995[Medline] [Order article via Infotrieve]
59. Leech JH, Barnwell JW, Miller LH, Howard RJ: Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium falciparum-infected erythrocytes. J Exp Med 159:1567, 1984[Abstract/Free Full Text]
60. Howard RJ, Barnwell JW, Rock EP, Neequaye J, Ofori-Adjei D, Maloy WL, Lyon JA, Saul A: Two approximately 300 kilodalton Plasmodium falciparum proteins at the surface membrane of infected erythrocytes. Mol Biochem Parasitol 27:207, 1988[Medline] [Order article via Infotrieve]
61. van Shravendijk MR, Rock EP, Marsh K, Ito Y, Aikawa M, Neequaye J, Ofori-Adjei D, Rodriquez R, Patarroyo ME, Howard RJ: Characterization and localization of Plasmodium falciparum surface antigens on inficated erythrocytes from West Africa. Blood 78:226, 1991[Abstract/Free Full Text]
62. Udeinya IJ, Schmidt JA, Aikawa M, Miller LH, Green I: Falciparum malaria-infected erythrocytes specifically bind to cultured human endothelial cells Science 213:555, 1981
63. Pasloske BL, Howard RJ: Malaria, the red cell and the endothelium. Annu Rev Med 45:283, 1994[Medline] [Order article via Infotrieve]
64. Raventos-Suarez C, Kaul DK, Macaluso F, Nagel RL: Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium falciparum-infected erythrocytes. Proc Natl Acad Sci USA 82:3829, 1985[Abstract/Free Full Text]
65. Pongponratn E, Riganti M, Punpoowong B, Aikawa M: Microvascular sequestration of parasitized erythrocytes in human falciparum malaria: A pathologic study. Am J Trop Med Hyg 44:168, 1991
66. Luse SA, Miller LH: Plasmodium falciparum malaria: Ultrastructure of parasitized erythrocytes in cardiac vessels. Am J Trop Med Hyg 20:655, 1971
67. Langreth SG, Peterson E: Pathogenicity, stability and immunogenicity of a knobless clone of Plasmodium falciparum in Columbian owl monkeys. Infect Immun 47:760, 1985[Abstract/Free Full Text]
68. Oster CN, Koontz LC, Wyler DJ: Malaria in asplenic mice: Effects of splenectomy, congenital asplenia, and splenic reconstitution on the course of infection. Am J Trop Med Hyg 29:1138, 1980
69. Howard RJ, Gilladoga AD: Molecular studies related to the pathogenesis of cerebral malaria. Blood 74:2603, 1989[Free Full Text]
70. David PH, Hommel M, Miller LH, Udeinya IJ, Oligino LD: Parasite sequestration in Plasmodium falciparum malaria: Spleen and antibody modulation of cytoadherence of infected erythrocytes. Proc Natl Acad Sci USA 80:5075, 1983[Abstract/Free Full Text]
71. Udeinya IJ, Miller LH, McGregor IA, Jensen JB: Plasmodium falciparum strain-specific antibody blocks binding of infected erythrocytes to amelanotic melanoma cells. Nature 303:429, 1983[Medline] [Order article via Infotrieve]
72. Marsh K, Howard RJ: Antigens induced on erythrocytes by P. falciparum: Expression of diverse and conserved determinants. Science 231:150 1986
73. Magowan C, Wollish W, Anderson L, Leech J: Cytoadherence by Plasmodium falciparum-infected erythrocytes is correlated with the expression of a family of variable proteins on infected erythrocytes. J Exp Med 168:1307, 1988[Abstract/Free Full Text]
74. Roberts DJ, Craig AG, Berendt AR, Pinches R, Nash G, Marsh K, Newbold CI: Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature 357:689, 1992[Medline] [Order article via Infotrieve]
75. Biggs BP, Anders RF, Dillon HE, Davern KM, Martin M, Peterson C, Brown GV: Adherence of infected erythrocytes to venular endothelium selects for antigenic variants of Plasmodium falciparum. J Immunol 149:2047, 1992[Abstract]
76. Ockenhouse CF, Ho M, Tandon NN, Van Seventer GA, Shaw S, White NJ, Jamieson GA, Chulay JD, Webster HK: Molecular basis of sequestration in severe and uncomplicated Plasmodium falciparum malaria: Differential adhesion of infected erythrocytes to CD36 and ICAM-1. J Infect Dis 164:163, 1991[Medline] [Order article via Infotrieve]
77. Ockenhouse CF, Tegoshi T, Maeno Y, Benjamin C, Ho M, Kan KE, Thway Y, Win K Aikawa M, Lobb RR: Human vascular endothelial cell adhesion receptors for Plasmodium-infected erythrocytes: Roles for endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1. J Exp Med 176:1183, 1992[Abstract/Free Full Text]
78. Roberts DD, Sherwood JA, Spitalnik SL, Panton LJ, Howard RJ, Dixit VM, Frazier WA, Miller LH, Ginsburg V: Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence. Nature 318:64, 1985[Medline] [Order article via Infotrieve]
79. Miller LH, Good MF, Milon G: Malaria pathogenesis. Science 264:1878, 1994[Abstract/Free Full Text]
80. Dolan SA, Miller LH, Wellems TE: Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum. J Clin Invest 86:618, 1990
81. Tsuboi T, Kappe SH, al-Yaman F, Prickett MD, Alpers M, Adams JH: Natural variation within the principal adhesion domain of the Plasmodium vivax Duffy binding protein. Infect Immun 62:5581, 1994[Abstract/Free Full Text]
82. Oppenheim JJ, Zachariae CO, Mukaida N, Matsushima K: Properties of the novel proinflammatory supergene "intercrine" cytokine family. Annu Rev Immunol 9:617, 1991[Medline] [Order article via Infotrieve]
83. Baggiolini M, Dewald B, Moser B: Interleukin-8 and related chemotactic cytokines $---$ CXC and CC chemokines. Adv Immunol 55:97, 1994[Medline] [Order article via Infotrieve]
84. Schall T: The chemokines, in Thomson AW (ed): The Cytokine Handbook (ed 2). New York, NY, Academic, 1994, p 419
85. Schall TJ: Biology of the RANTES/SIS cytokine family. Cytokine 3:165, 1991[Medline] [Order article via Infotrieve]
86. Kelner GS, Kennedy J, Bacon KB, Kleyensteuber S, Largaespada DA, Jenkins NA, Copeland NG, Bazan JF, Moore KW, Schall TJ, Zlotnik A: Lymphotactin: A cytokine that represents a new class of chemokine. Science 266:1395, 1994[Abstract/Free Full Text]
87. Murphy PM: The molecular biology of leukocyte chemoattractant receptors. Annu Rev Immunol 12:593, 1994[Medline] [Order article via Infotrieve]
88. Horuk R: Molecular properties of the chemokine receptor family. Trends Pharmacol Sci 15:159, 1994[Medline] [Order article via Infotrieve]
89. Horuk R: Cytokine receptors, in Peroutka SJ (ed): Handbook of Receptors and Channels: G Protein-Coupled Receptors. Boca Raton, FL, CRC, 1993, p 87
90. Neote K, DiGregorio D, Mak JY, Horuk R, Schall TJ: Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor. Cell 72:415, 1993[Medline] [Order article via Infotrieve]
91. Holmes WE, Lee J, Kuang WJ, Rice GC, Wood WI: Structure and functional expression of a human interleukin-8 receptor. Science 253:1278, 1991[Abstract/Free Full Text]
92. Hebert CA, Chuntharapai A, Smith M, Colby T, Kim J, Horuk R: Partial functional mapping of the human interleukin-8 type A receptor. Identification of a major ligand binding domain. J Biol Chem 268:18549, 1993[Abstract/Free Full Text]
93. Darbonne WC, Rice GC, Mohler MA, Apple T, Hegert CA, Valente AJ, Baker JB: Red blood cells are a sink for interleukin 8, a leukocyte chemotaxin. J Clin Invest 88:1362, 1991
94. Neote K, Darbonne W, Ogez J, Horuk R, Schall TJ: Identification of a promiscuous inflammatory peptide receptor on the surface of red blood cells. J Biol Chem 268:12247, 1993[Abstract/Free Full Text]
95. Horuk R, Colby TJ, Darbonne WC, Schall TJ, Neote K: The human erythrocyte inflammatory peptide (chemokine) receptor. Biochemical characterization, solubilization, and development of a binding assay for the soluble receptor. Biochemistry 32:5733, 1993[Medline] [Order article via Infotrieve]
96. Szabo MC, Soo KS, Zlotnik A, Schall TJ: Chemokine class differences in binding to the Duffy antigen/erythrocyte receptor. J Biol Chem 270:25348, 1995[Abstract/Free Full Text]
97. Horuk R, Chitnis CE, Darbonne WC, Colby TJ, Rybicki A, Hadley TJ, Miller LH: A receptor for the malarial parasite Plasmodium vivax: The erythrocyte chemokine receptor. Science 261:1182, 1993[Abstract/Free Full Text]
98. Hesselgesser J, Chitnis CE, Miller LH, Yansura DG, Simmons LC, Fairbrother WJ, Kotts C, Wirth C, Gillece-Castro BL, Horuk R: A mutant of melanoma growth stimulating activity does not activate neutrophils but blocks erythrocyte invasion by malaria. J Biol Chem 270:11472, 1995[Abstract/Free Full Text]
99. Chaudhuri A, Zbrzezna V, Polykova J, Pogo AO, Hesselgesser J, Horuk R: Expression of the Duffy antigen in K562 cells. Evidence that it is the human erythrocyte chemokine receptor. J Biol Chem 269:7835, 1994[Abstract/Free Full Text]
100. Neote K, Mak JY, Kolakowski LF, Schall TJ: Functional and biochemical analysis of the cloned Duffy antigen: Identity with the red blood cell chemokine receptor. Blood 84:44, 1994[Abstract/Free Full Text]
101. Hadley TJ, Lu Z-h, Wasniowska K, Martin AW, Peiper SC, Hesselgesser J, Horuk R: Postcapillary venule endothelial cells in kidney express a multispecfic chemokine receptor that is structurally and functionally identical to the erythroid isoform which is the Duffy blood group antigen. J Clin Invest 94:985, 1994
101a. Guo H, Slone S, Fingar V, Blanchard D, Peiper S, Hadley T: Tissue distribution and upregulation by inflammation. Blood 88:513a, 1996 (abstr, suppl 1)
102. Rot A: Endothelial cell binding of NAP-1/IL-8: Role in neutrophil emigration. Immunol Today 13:291, 1992[Medline] [Order article via Infotrieve]
103. Hechtman DH, Cybulsky MI, Fuchs HJ, Baker JB, Gimbrone MA: Intravascular IL-8: Inhibitor of polymorphonuclear leukocyte accumulation at sites of acute inflammation. J Immunol 147:883, 1991[Abstract]
104. Lawrence LB, Springer TA: Leukocytes roll on a selectin at physiologic flow rates: Distinction from and prerequisite for adhesion through integrins. Cell 65:859, 1991[Medline] [Order article via Infotrieve]
105. Chaudhuri A, Polyakova J, Zbrezna V, Williams K, Gulati S, Pogo AO: Cloning of glycoprotein D cDNA which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite. Proc Natl Acad Sci USA 90:10793, 1993[Abstract/Free Full Text]
106. Chaudhuri A, Zbrzezna V, Johnson C, Nichols M, Rubinstein P, Marsh WL, Pogo AO: Purification and characterization of an erythrocyte membrane protein complex carrying Duffy blood group antigenicity. Possible receptor for plasmodium vivax and plasmodium Knowlesi malaria parasite. J Biol Chem 264:13770, 1989[Abstract/Free Full Text]
107. Jacobs PA, Brunton M, Fackiewicz A, Newton M, Cook PJ, Robson EB: Studies on a family with three cytogenetic markers. Ann Hum Genet 33:325, 1970
108. Mathew S, Chaudhuri A, Murty VV, Pogo AO: Confirmation of Duffy blood group antigen locus (FY) at 1q22 $right-arrow$ q23 by flourescence in situ hybridization. Cytogenet Cell Genet 67:68, 1994[Medline] [Order article via Infotrieve]
109. Eisenberg D: Three-dimensional structure of membrane and surface proteins. Annu Rev Biochem 53:595, 1984[Medline] [Order article via Infotrieve]
110. Tournamille C, Colin Y, Cartron JP, Le Van Kim C: Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals. Nature Genet 10:224, 1995[Medline] [Order article via Infotrieve]
111. Iwamoto S, Li J, Omi T, Ikemoto S, Kajii E: Identification of a novel exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary endothelium. Blood 87:378, 1996[Abstract/Free Full Text]
112. Iwamoto S, Li J, Sugimoto N, Okuda H, Kajii E: Characterization of the Duffy gene promoter: Evidence for tissue specific abolishment of expression in Fy(a-b-) of black individuals. Biochem Biophys Res Commun 222:852, 1996[Medline] [Order article via Infotrieve]
113. Mallinson G, Soo KS, Schall TJ, Pisacka M, Anstee DJ: Mutations in the erythrocyte chemokine receptor (Duffy) gene: The molecular basis of the Fy^a/Fy^b antigens and identification of a deletion in the Duffy gene of an apparently healthy individual with the Fy(a-b-) phenotype. Br J Haematol 90:823, 1995[Medline] [Order article via Infotrieve]
114. Iwamoto S, Omi T, Kajii E, Ikemoto S: Genomic organization of the glycoprotein D gene: Duffy blood group Fy^a/Fy^b alloantigen system is associated with a polymorphism at the 44-amino acid residue. Blood 85:662, 1995
115. Tournamille C, Le Van Kim C, Gane P, Cartron JP, Colin Y: Molecular basis and PCR-DNA typing of the Fya/Fyb blood group polymorphism. Hum Genet 95:407, 1995[Medline] [Order article via Infotrieve]
116. Lu Z-h, Wang Z-x, Horuk R, Hesselgesser J, Lou YC, Hadley TJ, Peiper SC: The promiscuous chemokine binding profile of the Duffy antigen/recptor for chemokines (DARC) is primarily localized to sequences in the amino terminal domain. J Biol Chem 270:26239, 1995[Abstract/Free Full Text]
117. Tournamille C, Le Van Kim C, Gane P, Proudfoot A, Cartron JP, Colin Y: Epitopes of the erythrocyte receptor for chemokines (Duffy glycoprotein) involved in IL8 binding (abstr). Trans Clin Biol 3:54s, 1996 (suppl)
118. Hausman E, Dzik W, Blanchard D: The red cell chemokine receptor is distinct from the Fy6 epitope. Transfusion 36:421, 1996[Medline] [Order article via Infotrieve]
119. Wasniowska K, Blanchard D, Jauvier D, Wang ZX, Peiper SC, Hadley TJ, Lisowska E: Identification of the Fy6 epitope recognized by two monoclonal antibodies in the N-terminal extracellular portion of the Duffy antigen receptor for chemokines. Mol Immunol 33:917, 1996[Medline] [Order article via Infotrieve]
120. Rothwell NJ, Hardwick AJ, Lindley I: Central actions of interleukin-8 in the rat are independent of prostaglandins. Horm Metab Res 22:595, 1990[Medline] [Order article via Infotrieve]
121. Araujo DM, Cotman CW: Trophic effects of interleukin-4, -7 and -8 on hippocampal neuronal cultures: Potential involvement of glial-derived factors. Brain Res 600:49, 1993[Medline] [Order article via Infotrieve]
122. Van Meir E, Ceska M, Effenberger F, Walz A, Grouzmann E, Desbaillets I, Frei K, Fontana A, de Tribolet N: Interleukin-8 is produced in neoplastic and infectious diseases of the human central nervous system. Cancer Res 52:4297, 1992[Abstract/Free Full Text]
123. Horuk R, Martin AW, Wang ZY, Schweitzer L, Gerassimides A, Guo H, Lu ZH, Hesselgesser J, Perez HD, Kim J, Parker J, Hadley TJ, Peiper SC: Expression of chemokine receptors by subset of neurons in the central nervous system. J Immunol 158:2882, 1997[Abstract]
124. Horuk R, Wang ZX, Peiper SC, Hesselgesser J: Identification and characterization of a promiscuous chemokine binding protein in a human erythroleukemia cell line. J Biol Chem 269:17730, 1994[Abstract/Free Full Text]
125. Klein PS, Sun TJ, Saxe CL, Kimmel AR, Johnson RL, Devreotes PN: A chemoattractant receptor controls development in Dictyostelium discoideum. Science 241:1467, 1988[Abstract/Free Full Text]
126. Hart AC, Kramer H, Van Vactor DL, Paidhungat M, Zipursky SL: Induction of cell fate in the Drosophila retina: The bride of sevenless protein is predicted to contain a large extracellular domain and seven transmembrane segments. Genes Dev 4:1835, 1990[Abstract/Free Full Text]
127. Rogaev EI, Sherrington R, Rogaeva EA, Levesque G, Ikeda M, Liang Y, Chi H, Lin C, Holman K, Tsuda T, Mar L, Sorbi S, Nacmias B, Placentini S, Amaducci L, Chumakov I, Cohen D, Lannfelt L, Fraser PE, Rommens JM, St George-Hyslop PH: Familial Alheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzhelmer's disease type 3 gene. Nature 376:775, 1995[Medline] [Order article via Infotrieve]
128. Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K, Tsuda T, Mar L, Foncin J-F, Bruni AC, Montesi M.P., Sorbi S, Rainero I, Pinessi L, Nee L, Chumakov I, Pollen D, Brookes A, Sanseau P, Pollinsky R J, Wasco W, Da Silva HAR, Haines JL, Pericak-Vance MA, Tanzi RE, Roses AD, Fraser PE, Rommens JM, St George-Hyslop PH: Cloning of a gene bearing missense mutations in early onset familial Alzheimer's disease. Nature 375:754, 1995[Medline] [Order article via Infotrieve]
129. Levitan D, Greenwald I: Facilitation of lin-12-mediated signalling be sel-12, a Caenorhabditis elegans S182 Alzheimer's disease gene. Nature 377:351, 1995[Medline] [Order article via Infotrieve]

© 1997 by The American Society of Hematology.

Add to CiteULike CiteULike    Add to Connotea Connotea    Add to Del.icio.us Del.icio.us    Add to Digg Digg    Add to Reddit Reddit    Add to Technorati Technorati    What's this?

This article has been cited by other articles:

H. Kulkarni, V. C. Marconi, W. He, M. L. Landrum, J. F. Okulicz, J. Delmar, D. Kazandjian, J. Castiblanco, S. S. Ahuja, E. J. Wright, et al.
The Duffy-null state is associated with a survival advantage in leukopenic HIV-infected persons of African ancestry
Blood, September 24, 2009; 114(13): 2783 - 2792.
[Abstract] [Full Text] [PDF]

C. Vergara, Y. J. Tsai, A. V. Grant, N. Rafaels, L. Gao, T. Hand, M. Stockton, M. Campbell, D. Mercado, M. Faruque, et al.
Gene Encoding Duffy Antigen/Receptor for Chemokines Is Associated with Asthma and IgE in Three Populations
Am. J. Respir. Crit. Care Med., November 15, 2008; 178(10): 1017 - 1022.
[Abstract] [Full Text] [PDF]

B. A. Zabel, S. Nakae, L. Zuniga, J.-Y. Kim, T. Ohyama, C. Alt, J. Pan, H. Suto, D. Soler, S. J. Allen, et al.
Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis
J. Exp. Med., September 29, 2008; 205(10): 2207 - 2220.
[Abstract] [Full Text] [PDF]

L. W. Horton, Y. Yu, S. Zaja-Milatovic, R. M. Strieter, and A. Richmond
Opposing Roles of Murine Duffy Antigen Receptor for Chemokine and Murine CXC Chemokine Receptor-2 Receptors in Murine Melanoma Tumor Growth
Cancer Res., October 15, 2007; 67(20): 9791 - 9799.
[Abstract] [Full Text] [PDF]

H. Rompler, C. Staubert, D. Thor, A. Schulz, M. Hofreiter, and T. Schoneberg
G Protein-Coupled Time Travel: Evolutionary Aspects of GPCR Research
Mol. Interv., February 1, 2007; 7(1): 17 - 25.
[Abstract] [Full Text] [PDF]

K. C. Barnes, A. V. Grant, N. N. Hansel, P. Gao, and G. M. Dunston
African Americans with Asthma: Genetic Insights
Proceedings of the ATS, January 1, 2007; 4(1): 58 - 68.
[Abstract] [Full Text] [PDF]

J. S. Lee, M. M. Wurfel, G. Matute-Bello, C. W. Frevert, M. R. Rosengart, M. Ranganathan, V. W. Wong, T. Holden, S. Sutlief, A. Richmond, et al.
The Duffy Antigen Modifies Systemic and Local Tissue Chemokine Responses following Lipopolysaccharide Stimulation
J. Immunol., December 1, 2006; 177(11): 8086 - 8094.
[Abstract] [Full Text] [PDF]

H. Shen, R. Schuster, K. F. Stringer, S. E. Waltz, and A. B. Lentsch
The Duffy antigen/receptor for chemokines (DARC) regulates prostate tumor growth
FASEB J, January 1, 2006; 20(1): 59 - 64.
[Abstract] [Full Text] [PDF]

K. C. Barnes
Genetic Determinants and Ethnic Disparities in Sepsis-associated Acute Lung Injury
Proceedings of the ATS, October 1, 2005; 2(3): 195 - 201.
[Abstract] [Full Text] [PDF]

H. Rompler, A. Schulz, C. Pitra, G. Coop, M. Przeworski, S. Paabo, and T. Schoneberg
The Rise and Fall of the Chemoattractant Receptor GPR33
J. Biol. Chem., September 2, 2005; 280(35): 31068 - 31075.
[Abstract] [Full Text] [PDF]

B Moser and K Willimann
Chemokines: role in inflammation and immune surveillance
Ann Rheum Dis, November 1, 2004; 63(suppl_2): ii84 - ii89.
[Abstract] [Full Text] [PDF]

H. Shen and A. B. Lentsch
Progressive dysregulation of transcription factors NF-{kappa}B and STAT1 in prostate cancer cells causes proangiogenic production of CXC chemokines
Am J Physiol Cell Physiol, April 1, 2004; 286(4): C840 - C847.
[Abstract] [Full Text] [PDF]

M. Kashiwazaki, T. Tanaka, H. Kanda, Y. Ebisuno, D. Izawa, N. Fukuma, N. Akimitsu, K. Sekimizu, M. Monden, and M. Miyasaka
A high endothelial venule-expressing promiscuous chemokine receptor DARC can bind inflammatory, but not lymphoid, chemokines and is dispensable for lymphocyte homing under physiological conditions
Int. Immunol., October 1, 2003; 15(10): 1219 - 1227.
[Abstract] [Full Text] [PDF]

J. S. Lee, C. W. Frevert, M. M. Wurfel, S. C. Peiper, V. A. Wong, K. K. Ballman, J. T. Ruzinski, J. S. Rhim, T. R. Martin, and R. B. Goodman
Duffy Antigen Facilitates Movement of Chemokine Across the Endothelium In Vitro and Promotes Neutrophil Transmigration In Vitro and In Vivo
J. Immunol., May 15, 2003; 170(10): 5244 - 5251.
[Abstract] [Full Text] [PDF]

M. GOTTE
Syndecans in inflammation
FASEB J, April 1, 2003; 17(6): 575 - 591.
[Abstract] [Full Text] [PDF]

J. Middleton, A. M. Patterson, L. Gardner, C. Schmutz, and B. A. Ashton
Leukocyte extravasation: chemokine transport and presentation by the endothelium
Blood, December 1, 2002; 100(12): 3853 - 3860.
[Abstract] [Full Text] [PDF]

S. Seixas, N. Ferrand, and J. Rocha
Microsatellite Variation and Evolution of the Human Duffy Blood Group Polymorphism
Mol. Biol. Evol., October 1, 2002; 19(10): 1802 - 1806.
[Full Text] [PDF]

T. Horikawa, T. Nakayama, I. Hikita, H. Yamada, R. Fujisawa, T. Bito, S. Harada, A. Fukunaga, D. Chantry, P. W. Gray, et al.
IFN-{gamma}-inducible expression of thymus and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in epidermal keratinocytes and their roles in atopic dermatitis
Int. Immunol., July 1, 2002; 14(7): 767 - 773.
[Abstract] [Full Text] [PDF]

A. B. LENTSCH
The Duffy antigen/receptor for chemokines (DARC) and prostate cancer. A role as clear as black and white?
FASEB J, July 1, 2002; 16(9): 1093 - 1095.
[Abstract] [Full Text] [PDF]

S. A. Bryan, P. J. Jose, J. R. Topping, R. Wilhelm, C. Soderberg, D. Kertesz, P. J. Barnes, T. J. Williams, T. T. Hansel, and I. Sabroe
Responses of Leukocytes to Chemokines in Whole Blood and Their Antagonism by Novel CC-Chemokine Receptor 3 Antagonists
Am. J. Respir. Crit. Care Med., June 15, 2002; 165(12): 1602 - 1609.
[Abstract] [Full Text] [PDF]

A. Fraile-Ramos, T. N. Kledal, A. Pelchen-Matthews, K. Bowers, T. W. Schwartz, and M. Marsh
The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling
Mol. Biol. Cell, June 1, 2001; 12(6): 1737 - 1749.
[Abstract] [Full Text] [PDF]

R. J. B. Nibbs, E. Kriehuber, P. D. Ponath, D. Parent, S. Qin, J. D. M. Campbell, A. Henderson, D. Kerjaschki, D. Maurer, G. J. Graham, et al.
The {beta}-Chemokine Receptor D6 Is Expressed by Lymphatic Endothelium and a Subset of Vascular Tumors
Am. J. Pathol., March 1, 2001; 158(3): 867 - 877.
[Abstract] [Full Text] [PDF]

A. N. Gubin, J. M. Njoroge, U. Wojda, S. D. Pack, M. Rios, M. E. Reid, and J. L. Miller
Identification of the Dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family
Blood, October 1, 2000; 96(7): 2621 - 2627.
[Abstract] [Full Text] [PDF]

C. Murdoch and A. Finn
Chemokine receptors and their role in inflammation and infectious diseases
Blood, May 15, 2000; 95(10): 3032 - 3043.
[Abstract] [Full Text] [PDF]

J.-P. Girard, E. S. Baekkevold, T. Yamanaka, G. Haraldsen, P. Brandtzaeg, and F. Amalric
Heterogeneity of Endothelial Cells : The Specialized Phenotype of Human High Endothelial VenulesCharacterized by Suppression Subtractive Hybridization
Am. J. Pathol., December 1, 1999; 155(6): 2043 - 2055.
[Abstract] [Full Text] [PDF]

H. H. Ho, D. Du, and M. C. Gershengorn
The N Terminus of Kaposi's Sarcoma-associated Herpesvirus G Protein-coupled Receptor Is Necessary for High Affinity Chemokine Binding but Not for Constitutive Activity
J. Biol. Chem., October 29, 1999; 274(44): 31327 - 31332.
[Abstract] [Full Text] [PDF]

M. J.G. Southcott, M. J.A. Tanner, and D. J. Anstee
The Expression of Human Blood Group Antigens During Erythropoiesis in a Cell Culture System
Blood, June 15, 1999; 93(12): 4425 - 4435.
[Abstract] [Full Text] [PDF]

G. Gentilini, N. E. Kirschbaum, J. A. Augustine, R. H. Aster, and G. P. Visentin
Inhibition of Human Umbilical Vein Endothelial Cell Proliferation by the CXC Chemokine, Platelet Factor 4�(PF4), Is Associated With Impaired Downregulation of p21Cip1/WAF1
Blood, January 1, 1999; 93(1): 25 - 33.
[Abstract] [Full Text] [PDF]

N. Parasol, M. Reid, M. Rios, L. Castilho, I. Harari, and N. S. Kosower
A Novel Mutation in the Coding Sequence of the FY*B Allele of the Duffy Chemokine Receptor Gene Is Associated With an Altered Erythrocyte Phenotype
Blood, October 1, 1998; 92(7): 2237 - 2243.
[Abstract] [Full Text] [PDF]

R. T. Palframan, P. D. Collins, T. J. Williams, and S. M. Rankin
Eotaxin Induces a Rapid Release of Eosinophils and Their Progenitors From the Bone Marrow
Blood, April 1, 1998; 91(7): 2240 - 2248.
[Abstract] [Full Text] [PDF]

S. Nielsen, A. Chaudhuri, A. O. Pogo;, T. J. Hadley, and S. C. Peiper
Which Are the Nonerythroid Cells That Constitutively Express the Duffy Antigen?
Blood, October 15, 1997; 90(8): 3231 - 3232.
[Full Text] [PDF]

C. Le Van Kim, C. Tournamille, Y. Kroviarski, J. P. Cartron, and Y. Colin
The 1.35-kb and 7.5-kb Duffy mRNA Isoforms Are Differently Regulated in Various Regions of Brain, Differ by the Length of Their 5' Untranslated Sequence, but Encode the Same Polypeptide
Blood, October 1, 1997; 90(7): 2851 - 2853.
[Full Text] [PDF]

This Article

Full Text (PDF)

Alert me when this article is cited

Alert me if a correction is posted

Citation Map

Services

Email this article to a friend

Similar articles in this journal

Similar articles in PubMed

Alert me to new issues of the journal

Download to citation manager

Rights and Permissions

Citing Articles

Citing Articles via HighWire

Citing Articles via CrossRef

Citing Articles via Google Scholar

Google Scholar

Articles by Hadley, T. J.

Articles by Peiper, S. C.

Search for Related Content

PubMed

PubMed Citation

Articles by Hadley, T. J.

Articles by Peiper, S. C.

Related Collections

Review Articles

Social Bookmarking


What's this?

  Copyright © 1997 by American Society of Hematology         Online ISSN: 1528-0020





	Copyright © 1997 by American Society of Hematology Online ISSN: 1528-0020

Blood, Vol. 89 No. 9 (May 1), 1997: pp. 3077-3091

From Malaria to Chemokine Receptor: The Emerging Physiologic Role of the Duffy Blood Group Antigen

© 1997 by The American Society of Hematology.