-. .' A Receptor for the Malarial Parasite Plasmodium vivax: The Erythrocyte Chemokine Receptor Richard Horuk,* Chetan E. Chitnis, Walter C. Darbonne, Timothy J. Colby, Anne Rybicki, Terence J. Hadley, Louis H. Miller Plasmodium vivax and P. falciparum are the major causes of human malaria, except in sub-Saharan Africa where people lack the Duffy blood group antigen, the erythrocyte receptor for P. vivax. Duffy negative human erythrocytes are resistant to invasion by P. vivaxand the related monkey malaria, P. knowlesi. Several lines of evidence in the present study indicate that the Duffy blood group antigen is the erythrocyte receptor for the chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA). First, IL-8 binds minimally to Duffy negative erythrocytes. Second, a monoclonal antibody to the Duffy blood group antigen blocked binding of IL-8 and other chemokines to Duffy positive erythrocytes. Third, both MGSA and IL-8 blocked the binding of the parasite ligand and the invasion of human erythrocytes by P. knowlesi, suggesting the possibility of receptor blockade for anti-malarial therapy. The erythrocyte chemokine receptor binds a family of chemotactic and proinflamma- tory soluble peptides (1-3), including IL-8, MGSA, monocyte chemotactic protein 1 (MCP-1), and regulated on activation, nor- mal T expressed and secreted (RANTES) (4). We have previously shown that the erythrocyte chemokine receptor differs from the IL-8 receptors, IL-8RA and IL-8RB, on neutrophils (1-3, 5, 6). Whereas the neu- trophil IL-8 receptors bind only IL-8 and MGSA (5, 6), the erythrocyte receptor binds IL-8, MGSA, RANTES, and MCP-1 (1-3). In the course of characterizing the erythrocyte chemokine receptor, we noted that erythrocytes from the majority of Af- rican Americans studied did not bind IL-8 (7). A high percentage of African Ameri- cans have the Duffy blood group negative phenotype (Duffy negative), which is rare in Caucasians (8). In this study we found that African Americans whose erythrocytes did not bind IL-8 lacked the Duffy blood group antigen. The Duffy blood group an- tigen has previously been shown to be required for the invasion of human eryth- rocytes by the human malarial parasite Plas- modium vivax (9) and the related monkey malaria P. knowlesi (10). We now show that IL-8 and MGSA block the binding of the P. knowlesi protein that binds to the Duffy blood group antigen and also block invasion of human erythrocytes by P. knowlesi. Erythrocytes from African American and Caucasian blood donors were classified as Duffy positive or Duffy negative, and R. Horuk, W. C. Darbonne, T. J. Colby, Genentech, Inc., South San Francisco, CA 94080. C. E. Chitnis and L. H. Miller, Laboratory of Malaria Research, National Institutes of Health, Bethesda, MD 20892. A. Rybicki, Montefiore Medical Center, Bronx, NY 10467. T. J. Hadley, Division of Medical Oncology/Hematolo- gy, J. G. Brown Cancer Center, Louisville, KY 40292. *To whom correspondence should be addressed. their ability to bind 1251-labeled IL-8 was determined (Table 1). There was an abso- lute association of IL-8 binding and the Duffy positive phenotype, indicating that the erythrocyte chemokine receptor is the Duffy blood group antigen or that they are closely linked. There are a number of similarities in the biochemical properties of the erythrocyte chemokine receptor and the Duffy blood group antigen. First, both proteins have a molecular size of 35 to 45 kD (2, 3, 11). Second, both proteins aggregate to a high molecular weight complex in SDS-polyacryl- amide gels when boiled in SDS-2-mercapto- ethanol (11, 12). Third, the number of chemokine receptor binding sites detected by radiolabeled IL-8 binding is about the same as the number of Duffy blood group antigens detected by antibody (5,000 to 10,000 per cell) (1, 2, 13). Fourth, both proteins are resistant to degradation by treatment of intact erythrocytes with trypsin; both are degraded by treatment of erythrocytes with chymotryp- sin (1, 11). A monoclonal antibody to the Duffy blood group antigen, anti-Fy6 (13), which inhibits the binding of the P. knowlesi ligand (14), was tested for its ability to inhibit the binding of radioactively labeled IL-8 to Duffy positive erythrocytes. Prein- cubation of erythrocytes with anti-Fy6 in- hibited the binding of IL-8 in a dose- dependent manner, with 70% inhibition at an antibody concentration of 3 nM (Fig. 1A). An irrelevant, isotype-matched anti- body had no effect at a similar concentra- tion. The inhibition of IL-8 binding to the Duffy blood group antigen was specific be- cause anti-Fy6 had no effect on IL-8 bind- ing to cells transfected with one of the cloned IL-8 receptors, IL-8RA (Fig. 1A). Because the erythrocyte chemokine recep- tor binds MGSA, MCP-1, and RANTES in addition to IL-8 (2), we examined the ability of anti-Fy6 to inhibit the binding of these chemokines to erythrocytes. Preincu- bation of Duffy positive erythrocytes with anti-Fy6 at a final concentration of 3 nM inhibited the binding of each of these chemokines (Fig. 1B). Fig. 1. Inhibition of chemokine binding to the A erythrocyte chemokine receptor by anti-Fy6, a 120 monoclonal antibody to the human Duffy blood ioo- group antigen. (A) Inhibition of specific 1251- El labeled IL-8 binding. Erythrocytes (2 x 1 08 cells * 80- in 500 ,i) were preincubated with increasing n 60\ concentrations of anti-Fy6 for 2 hours at 40C. The cells were then incubated for a further hour with 40- hry 0.5 nM 1251-labeled IL-8 in a final volume of 600 *IL-8R 111. The binding reactions were terminated as 6 20- NonspecificAb(3nM) described in Table 1. Nonspecific binding was O 0 determined by addition of 100 nM unlabeled IL-8 0o 0.015 0.15 1.5 to the binding reactions. This value was sub- Anti-Fy6(nM) tracted from the total binding to determine the 12000 specific binding of IL-8. Parallel binding experi- a IL-8 ments to examine the effect of anti-Fy6 on the E 10000- E MGSA 1251 i% 0~~~~~~~~~~~~~ MCP-1 binding of 1251-labeled IL-8 to human kidney 2~ El RANTES cells transfected with one of the leukocyte IL-8 8000 _ receptors, IL-8RA (5), were carried out as de- X 6000- scribed for the erythrocytes. The effect of an _ isotype-matched antibody to gp120 on IL-8 C 4000- binding to erythrocytes was also determined. (B) ' 2000 Inhibition of specific binding of radiolabeled _ IL-8, MGSA, MCP-1, and RANTES. 1251-labeled 0_ IL-8 (specific activity 874 Ci/mmol) and 1251- Control +3nManti-Fy6 labeled MGSA (specific activity 322 Ci/mmol) were labeled as previously described (21); 1251-labeled MCP-1 and 1251-labeled RANTES (specific activity 1 100 Ci/mmol) were from New England Nuclear. Experiments were carried out as described above except that cells were preincubated with anti-Fy6 at a final concentration of 3 nM for 2 hours at 4°C. The cells were then incubated for a further hour with 0.5 nM radiolabeled chemokines. Data are represented as the mean ± SEM (22). SCIENCE * VOL. 261 * 27 AUGUST 1993 1182 on December 6, 2014 www.sciencemag.org Downloaded from on December 6, 2014 www.sciencemag.org Downloaded from on December 6, 2014 www.sciencemag.org Downloaded from