A. Oscar Pogo

Deletion of the Murine Duffy Gene (Dfy) Reveals that the Duffy Receptor Is Functionally Redundant

ABSTRACT All of the antigenic determinants of the Duffy blood group system are in a glycoprotein (gp-Fy), which is encoded by a single-copy gene (FY) located on chromosome 1. gp-Fy is also produced in several cell types, including endothelial cells of capillary and postcapillary venules, the epithelial cell of kidney collecting ducts, lung alveoli, and the Purkinje cells of the cerebellum. This protein, which spans the cell membrane seven times, is a member of the superfamily of chemokine receptors and a malarial parasite receptor. The mouse Duffy gene (Dfy) homolog of human FYis also a single-copy gene, which maps in a region of conserved synteny with FY and produces a glycoprotein with 60% homology to the human protein. The mouse Duffy-like protein also binds chemokines. To study the biological role of gp-Fy, we generated a mouse strain in which Dfy was deleted. These homozygousDfy −/− mice were indistinguishable in size, development, and health from wild-type and heterozygous littermates. We also examined components of the immune system and found no differences in lymph nodes or peripheral blood leukocyte levels between knockout and wild-type mice. The gross and histological anatomy of the thymus, spleen, lung, and brain showed no significant differences between mutants and wild-type mice. There was no indication of an overall difference between the knockout and wild-type mice in systematic neurological examinations. The only significant difference betweenDfy −/− and Dfy +/+mice that we found was in neutrophil migration in peritoneal inflammations induced by lipopolysaccharide and thioglycolate. In mice homozygous for the deletion, there was less neutrophil recruitment into the peritoneal cavity and neutrophil influx in the intestines and lungs than in wild-type mice. Despite this, the susceptibility toStaphylococcus aureus infection was the same in the absence and in the presence of gp-Fy. Our results indicate that gp-Fy is functionally a redundant protein that may participate in the neutrophil migratory process.

The Duffy protein: a malarial and chemokine receptor.

A major advance towards understanding the Duffy blood group system has been achieved with the cloning of FY, a single-copy gene located in the 1q22->q23 region of chromosome 1. The product of FY Is an acidic glycoprotein (gp-Fy), which spans the plasma membrane seven times and has an exocellular N-terminal domain and an endocellular C-terminal domain. The system consists of four alleles, five phenotypes, and five antigens. FYA, FYB, FYB(ES), and FYB(WK) are the alleles; Fy(a+b-), Fy(a-b+), Fy(a+b+), Fy(a-b+(wK)), and Fy(a-b-), are the phenotypes, and Fy(a), Fy(b), Fy3, Fy5, and Fy6 are the antigens. Fy(a-b-), or Duffy-negative individuals, lack the Duffy protein on erythrocytes and are predominantly African and American blacks. They have the FYB(Es) allele with a mutation in the promoter region, which abolishes the expression of the protein in erythrocytes only. In the few cases of non-black Fy(a-b-) individuals, a nonsense mutation prevents the synthesis of gp-Fy. In Fy(a-b+(wk)) erythrocytes, the Fy(b) antigen is weakly expressed due to a reduced amount of the protein. The Fy5 antigen includes the Rh protein, and the Fy6 antigen is defined by a murine monoclonal antibody. Gp-Fy is produced in several cell types, including endothellal cells of capillary and postcapillary venules, epithelial cells of kidney collecting ducts, and lung alveoli, as well as PurkinJe cells of the cerebellum. The Duffy protein plays a role in inflammation and in malaria Infection. The protein is a member of the superfamily of chemokine receptors and is the receptor to which certain malarial parasites bind to invade red blood cells. The parasite-specific binding site, the binding site of chemokines, and the major antigenic domains are located in overlapping regions at the exocellular N terminus of the Duffy protein.

Induction of Duffy gene (FY) in human endothelial cells and in mouse.

Duffy Blood Group protein is a glycoprotein with seven transmembrane domains that binds to C-X-C and C-C chemokines. The antigen is constitutively expressed in endothelial and epithelial cells of several nonerythroid tissues and in Purkinje cells of the cerebellum. We studied the effect of proinflammatory cytokines on Duffy gene expression in endothelial cells from human umbilical vein (HUVEC) and human pulmonary arteries (HPAEC). Also, we studied the effect of inflammatory agents like bacterial lipopolysaccharide (LPS) on Duffy gene induction in mouse. Reverse transcription-PCR and mRNA blot analyses showed that Duffy mRNA was present in these cells in negligible amounts. However, treatment with tumor necrosis factor-alpha for 6-24h resulted in a 5 to 8-fold increase in Duffy mRNA. On the other hand, treatment with interleukin-1 (IL-1), IL-6 or LPS did not have any effect. Fluorescence microscopy and fluorescence activated cell sorting showed greater expression of Duffy protein in treated cells correlating the increase in mRNA synthesis with an increase in antigen production. In mice, Duffy gene was induced in lungs and brain with LPS treatment indicating that the induction is a physiological event. Vascular endothelial cells may induce Duffy protein to regulate leukocytes and/or chemokine trafficking.

The Duffy Blood Group System and Malaria

Cutbush et al. (1950) reported the presence of a new antibody in a hemophiliac man who had many blood transfusions during the previous 20 years. “Duffy” was the patient’s last name and the authors used this name for the new blood group system (Cutbush and Mollison, 1950). Fy u was designated as the gene responsible for the antigen, anti-Fy a the antibody, and Fy b the allele. They did not discover the anti-Fy b antibody but predicted that it would be found soon. The antibody was found a year later, in the serum of a Berlin woman who had three pregnancies but no transfusions (Ikin et al., 1951). Chown et al.(1965) reported a variant of Fy b having a weak expression of Fy b that they called Fy x Several Duffy antigens were found and subsequently termed Fy3 (Albrey et al., 1971), Fy4 (Behzad et al., 1973), and Fy5 (Colledge et al., 1973). More recently the murine monoclonal antibody anti-Fy6 identified the Fy6 antigen (Nichols et al., 1987).