ABSTRACTcDNA clones encoding the major subunit of the Duffy blood group were isolated from a human bone marrow cDNA library using a PCR-amplifled DNA anti-Duffy murine monoclonal antibody reacted with a synthetic peptide deduced from the cDNA clone. Hydropathy analysis suggested the presence of9 membrane-spanning a-helices. In bone marrow RNA blot analysis, the gpD cDNA detected.a 1.27-kb mRNA in Duffy-positive but not in Duffynegative individuals. It also identified the same size mRNA in adult kidney, adult spleen, and fetal liver; in brain, it detected a prominent 8.5-kb and a minor 2.2-kb mRNA. In Southern blot analysis, gpD cDNA identified a single gene in Duffypositive and -negative individuals. Duffy-negative individuals, therefore, have the gpD gene, but it is not expressed in bone marrow. The same or a similar gene is active in adult kidney, adult spleen, and fetal liver of Duffy-positive individuals. Whether this is true in Duffy-negative individuals remains to be demonstrated. A GenBank sequence search yielded a significant protein sequence homology to human and rabbit interleukin-8 receptors.

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 FY is 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 homozygous Dfy ؊/؊ 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 between Dfy ؊/؊ 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 to Staphylococcus 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 coding and untranslated flanking sequences of Duffy gene (FY) in humans and simians are in a single exon. The difference between the two codominant alleles, FY*A and FY*B, is a single change at nucleotide 306: guanidine is in FY*A and adenine is in FY*B. This produces a codon change that subsequently modifies the amino acid at position 43 of gpFy, the major subunit of the Duffy blood group protein complex. The glycine at this position in antigen Fya exchanges with aspartic acid in antigen Fyb. The guanidine at nucleotide 306 creates an additional Ban I restriction site in FY*A. Ban I digestion of DNA-PCR amplified products of FY*B and FY*A yields three and four fragments, respectively. Restriction fragment length polymorphism (RFLP) studies show that Fy(a+b-) and Fy(a-b+) whites are FY homozygous, that most Fy(a-b-) blacks have FY*B, and most Fy(a+b-) blacks are FY*A/FY*B heterozygous. In the black population a silent FY*B is very common, but a silent FY*A has not been found yet. On RNA blot analysis, the gpFy cDNA clone detected mRNA in the lung, spleen, and colon but not in the bone marrow of Duffy-negative individuals. Therefore, there is no null phenotype in Fy(a-b-) blacks. The gpFy homology between human and chimpanzee is 99% with a single residue change at position 116 (valine to isoleucine), whereas a 94% homology is found in squirrel and rhesus monkeys, and there is a 93% homology in aotus monkey when compared with humans. The N-terminal exocellular domain of simian gpFy helps to identify a set of amino acids critical for antibody and malarial parasite specificities.

The nonerythroid expression of the Duffy blood group protein (gp-Fy) was confined to certain cell types. Immunocytochemistry studies of the kidney showed gp-Fy in the endothelium of glomeruli, peritubular capillaries, vasa recta, and the principal cells (epithelial) of collecting ducts. Gp-Fy was also produced in the endothelial cells of large venules and epithelial cells (type-I) of pulmonary alveoli. In the thyroid, only the endothelial cells of capillaries produced gp-Fy. In the spleen, the endothelial cells of capillaries, high endothelial venule, and sinusoids produced abundant gp-Fy. Ultrastructural studies showed that apical and basolateral plasma membrane domains, including caveolae, had gp-Fy. Immunoblot analysis showed substantially less gp-Fy in nonerythroid cells than in erythrocytes. Moreover, the analyzed nonerythroid organs of Duffy-negative individuals did not produce more gp-Fy to compensate for the lack of this protein in their erythrocytes. The nucleotide sequence and the size of kidney mRNA from a Duffy-positive individual were the same as that of bone marrow. It is assumed, therefore, that nonerythroid Duffy protein is the product of the same gene as that of bone marrow. This notion is reinforced by the fact that nonerythroid and erythroid gp-Fy have the same antigenic domains.