To the Editor:
The Duffy blood group antigen has generated great interest because it is the receptor for the human malarial parasite Plasmodium vivax, simian malarial parasite Plasmodium knowlesi, and a new class of chemokine receptor for several proinflammatory cytokines.1-4 The finding that nonerythroid organs produce Duffy mRNA motivated the identification of cells that constitutively produce the Duffy protein (gp-Fy).5 Immunohistochemical studies were performed by Hadley et al,6 Peiper et al,7 and by Chaudhuri et al.8 Hadley et al6 and Peiper et al7 used only monoclonal antibody anti-Fy6. Chaudhuri et al8 used anti-Fy6 and rabbit polyclonal antibody 6615. The latter is a Duffy-specific antibody reacting with the sugar moiety of gp-Fy.8 According to Hadley et al6 and Peiper et al,7 endothelial cells of postcapillary venules of all organs and Purkinje cells of the cerebellum are the only nonerythroid cells that constitutively express gp-Fy. Chaudhuri et al8 identified the same cells; however, their studies showed gp-Fy in other cell types. Thus, in kidney, the endothelium of glomeruli, peritubular capillaries, vasa recta, and the principal cells (epithelial) of collecting ducts showed expression of gp-Fy. Duffy protein was also noticed in the endothelial cells of large venules and epithelial cells (type-I) of pulmonary alveoli. In thyroid, only the endothelial cells of capillaries produced gp-Fy. In spleen, in addition to the endothelial cells of capillaries and sinusoids, which is consistent with the observations of Peiper et al,7 endothelial cells of high endothelial venule (HEV) also produced abundant gp-Fy according to Chaudhuri et al.8 Furthermore, ultrastructural studies performed with antibody 6615 showed that apical and basolateral plasma membrane domains, including caveolae, contained gp-Fy. This indicates that the Duffy antigen is not limited to the membrane domain lining the vessels.
Hadley and Peiper9 challenged these findings in a recently published and well-documented review article. They disputed the specificity of rabbit polyclonal antibody 6615. However, Chaudhuri et al8 unequivocally demonstrated 6615 specificity by performing the following experiments: (1) Immunoblots of membrane proteins from tissues and erythrocytes yielded the same reactive bands with either monoclonal antibody Fy6 or rabbit polyclonal antibody 6615; thus, both antibodies reacted with the same protein. (2) In the immunohistochemistry of the kidney, the cell types that reacted with monoclonal antibody Fy6 reacted also with antibody 6615. (3) Neither antibody reacted with stroma and parenchymal cells of adult liver, which is an organ that does not produce Duffy mRNA. (4) Antibody 6615, immunopurified by affinity chromatography, stained the same protein and the same cells as the nonimmunopurified 6615. The carbohydrate epitope that antibody 6615 recognized is, therefore, only present in the Duffy protein. Despite these published results, Hadley and Peiper9 sustained that 6615 specificity remains to be determined. They incorrectly claimed that Chaudhuri et al8 only used antibody 6615. This is a misinterpretation of the data, because in kidney, the labeling of Duffy antigen throughout the entire collecting ducts was analyzed using anti-Fy6 as well as both nonimmunopurified and immunopurified 6615 antibodies. All of the antibodies stained the same cell types, but the labeling appears stronger in inner medulla and very weak in the cortex with monoclonal antibody anti-Fy6. Contrarily, Hadley et al6 restricted their studies to the cortex of the kidney using only anti-Fy6. This explains why the Duffy antigen was not observed in the principal cells of collecting ducts.6 In conclusion, the constitutive expression of Duffy antigen in the organs studied so far is noticed in certain epithelial cells, Purkinje cells of the cerebellum, and endothelial cells of thyroid capillaries, post-capillary venules of some organs, and large pulmonary venules.
Response
The Duffy blood group antigen is a 7-membrane spanning protein that binds chemotactic cytokines (chemokines) from both C-X-C and C-C families.1-1,1-2 Immunohistochemical studies from our laboratory using a monoclonal antibody, anti-Fy6, showed that the Duffy chemokine receptor is present on endothelial cells of postcapillary venules, sinusoids of spleen, and Purkinje neurons of the cerebellum.1-3,1-4 Chaudhuri et al1-5 recently described a rabbit polyclonal antibody, designated 6615, that reacts with carbohydrate on the Duffy antigen. Immunohistochemistry with 6615 confirmed our results that the Duffy antigen is located on endothelial cells of postcapillary venules and sinusoids of spleen. In addition, reactivity with 6615 was noted with endothelial cells of other vascular structures, including renal glomeruli, capillaries, and vasa recta. Antibody 6615 also reacted with epithelial cells of collecting tubules in kidney and type I alveolar cells of lung.
In an addendum to a recent review on the Duffy antigen, we described the findings of Chaudhuri et al1-5 and stated “Whether or not the carbohydrate epitope recognized by polyclonal antibody 6615 is also present on molecules other than the Duffy antigen remains to be determined.”6 This comment was prompted by the Western immunoblots shown by Chaudhuri et al.1-5 In addition to the expected 35-43–kD Duffy reactive band (and expected higher molecular weight oligomers), the polyclonal rabbit antibody 6615 reacted with a lower molecular weight band of 21 kD seen with kidney and other tissues. A lower molecular weight band was also seen on immunoblots with anti-Fy6, but in the figure shown this band appeared higher than 21 kD. Also, the lower molecular weight band seen on immunoblots of kidney with 6615 was not seen with anti-Fy6 in the figure shown. The investigators stated that the lower molecular weight bands were probably degradation products of the Duffy antigen; however, this conclusion was not formally substantiated.
In their Discussion, Chaudhuri et al1-5 characterized anti-Fy6 as giving an “unreliable signal in immunocytochemistry.” This has not been our experience. We have obtained consistent and strong reactivity with endothelial cells of postcapillary venules and Purkinje neurons of cerebellum using anti-Fy6. We have not seen reactivity with endothelial cells of large vessels such as arteries or veins except under conditions of inflammation (eg, temporal arteritis, thrombophlebeitis).1-7 Our data should be interpreted in light of the fact that we used a single (albeit, reliable) monoclonal antibody, anti-Fy6. It has never been our contention that endothelial cells of postcapillary venules and Purkinje neurons of the cerebellum are the only cells that express Duffy. We simply described our findings using anti-Fy6 in immunohistochemical studies supported by Western immunoblotting and chemokine cross-linking experiments.1-3 1-4
In summary, the immunohistochemical results reported by Chaudhuri et al1-5 may indeed represent true expression of the Duffy chemokine receptor by the cells observed using antibody 6615. However, the possibility that the 21-kD 6615-reactive band represents a molecule other than a Duffy degradation product has not been formally excluded by the data presented. Both our studies and those of Chaudhuri et al1-5 show that the Duffy chemokine receptor is expressed on vascular endothelial cells and other nonerythroid cells. The challenge now is to determine the function of this unique chemokine receptor. Insight into the function of the Duffy chemokine receptor will provide an important perspective from which to view the immunohistochemical data.
