Duffy Antigen Facilitates Movement of Across the Endothelium In Vitro and Promotes Transmigration In Vitro and In Vivo This information is current as of October 2, 2021. Janet S. Lee, Charles W. Frevert, Mark M. Wurfel, Stephen C. Peiper, Venus A. Wong, Kimberley K. Ballman, John T. Ruzinski, Johng S. Rhim, Thomas R. Martin and Richard B. Goodman

J Immunol 2003; 170:5244-5251; ; Downloaded from doi: 10.4049/jimmunol.170.10.5244 http://www.jimmunol.org/content/170/10/5244

References This article cites 28 articles, 12 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/170/10/5244.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 2, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Duffy Antigen Facilitates Movement of Chemokine Across the Endothelium In Vitro and Promotes Neutrophil Transmigration In Vitro and In Vivo1

Janet S. Lee,* Charles W. Frevert,* Mark M. Wurfel,* Stephen C. Peiper,† Venus A. Wong,* Kimberley K. Ballman,* John T. Ruzinski,* Johng S. Rhim,‡ Thomas R. Martin,* and Richard B. Goodman2*

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells Downloaded from facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-␣/CXC chemokine ligand 1 (GRO-␣/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-␣/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-␣/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We

conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines http://www.jimmunol.org/ across the endothelium. The Journal of Immunology, 2003, 170: 5244–5251.

uffy Ag is a minor blood group Ag and the erythrocyte lial Duffy Ag could have a proinflammatory role by facilitating the receptor for the malarial parasite Plasmodium vivax (1). access of chemokines to circulating leukocytes and enhancing leu- D The Duffy Ag on erythrocytes binds selective CXC and kocyte migration (7, 9). Alternatively, endothelial Duffy Ag could CC chemokines, and is proposed to function as a chemokine sink reduce the intensity of inflammation by sequestering chemokines in the circulation (2–5). Postcapillary venular endothelial cells, the at sites of inflammation (10). site of leukocyte emigration in most organs, also express the Duffy We have recently shown that there is enhanced Duffy Ag ex-

Ag, even in individuals who do not express Duffy Ag on their pression on parenchymal vascular beds and the alveolar septa of by guest on October 2, 2021 erythrocytes (6, 7). The expression of Duffy Ag is up-regulated on human lung tissue during suppurative pneumonia, a process de- endothelial cells during inflammatory states in the kidney (8, 9). Its fined by neutrophilic infiltration of the airspaces (11). The goals of high affinity binding to several CXC and CC chemokines, focal this study were to determine whether Duffy Ag enhances move- expression at the site of leukocyte emigration, up-regulation during ment of chemokines across the endothelium in vitro and whether tissue inflammation, and conserved endothelial expression even in Duffy Ag alters neutrophil migration toward chemokines in vitro individuals whose erythrocytes lack Duffy Ag all suggest a bio- and in vivo. Herein, we report that Duffy Ag, stably transfected logical role for Duffy Ag in regulating inflammatory cell recruit- into an immortalized human endothelial cell line and displaying ment to sites of inflammation. Some have postulated that endothe- high affinity binding to growth-related oncogene (GRO)3-␣/CXC chemokine ligand (CXCL) 1 (GRO-␣/CXCL1), facilitates the movement of radiolabeled 125I-labeled (125I) GRO-␣/CXCL1 *Medical Research Service, Veterans Affairs Puget Sound Health Care System and across an endothelial monolayer. We show that the presence of Division of Pulmonary and Critical Care Medicine, Department of Medicine, Uni- Duffy Ag enhances neutrophil transendothelial migration toward † versity of Washington School of Medicine, Seattle, WA 98108; Department of Pa- GRO-␣/CXCL1 and IL-8/CXCL8 in vitro. Stimulation of Duffy thology, Medical College of Georgia, Augusta, GA 30912; and ‡Department of Sur- gery, Center for Prostate Disease Research, Uniformed Services University of the transfectants with GRO-␣/CXCL1 does not result in adhesion mol- Health Sciences, Bethesda, MD 20814 ecule gene up-regulation or other alterations in the endothelial cell Received for publication October 30, 2002. Accepted for publication March 11, 2003. gene profile. Finally, we show that the absence of Duffy Ag in vivo The costs of publication of this article were defrayed in part by the payment of page markedly attenuates IL-8/CXCL8-mediated neutrophil recruitment charges. This article must therefore be hereby marked advertisement in accordance into the airspaces of mice lacking the Duffy Ag. These findings with 18 U.S.C. Section 1734 solely to indicate this fact. suggest that endothelial Duffy Ag has an active role in chemokine- 1 This work was supported in part by the Medical Research Service of the Department of Veteran’s Affairs and National Institutes of Health Grants HL70178, HL69955, and mediated neutrophil recruitment by promoting the transendothelial HL30542. movement of chemokines. 2 Address correspondence and reprint requests to Dr. Richard B. Goodman, Seattle Veterans Affairs Medical Center, Pulmonary, S-111-Pulm, 1660 South Columbian Way, Seattle, WA 98108. E-mail address: [email protected] Materials and Methods 3 Abbreviations used in this paper: GRO, growth-related oncogene; CXCL, CXC Endothelial cell culture chemokine ligand; 125I, 125I-labeled; IVEC, immortalized HUVEC; CCL, CC che- mokine ligand; MCP, monocyte chemotactic protein; MIP, inflammatory HUVECs were immortalized by transformation with human papilloma vi- protein; HSA, human serum albumin; PMN, polymorphonuclear neutrophil; fl., cal- rus-16 E6-E7 (12). The immortalized HUVEC line (IVEC), clone cein fluorescence; Duffyϩ cells, endothelial Duffy transfectants. 4-5-2G, has an indefinite lifespan in culture but is nontumorigenic. The cell

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 5245

line demonstrates cobblestone morphology, expresses Factor VIII-related 125I-GRO-␣ transit assay ␣ ␤ ␣ ␤ Ag, takes up Dil-Ac-LDL, and expresses the integrin subunits v 3, v 5, ␤ ␣ ␣ ␤ ␣ A peptide sequence from the N terminus of human Duffy Ag 1, 2, 3, 4, and 6, consistent with its endothelial origin. IVEC were maintained in endothelial growth medium-2 containing 2% FCS (BioWhit- (STENSSQLDFEDVWNSS) was used to generate goat anti-human Duffy taker, Walkersville, MD). polyclonal Ab in collaboration with Invitrogen (Huntsville, AL). Serum from the immunized goat was affinity purified and the goat anti-Duffy Ig Cloning of human Duffy cDNA was characterized by its ability to displace 125I-GRO-␣/CXCL1 from Duffy transfectants. We determined that the inhibitory concentration of the anti- The human Duffy cDNA was cloned by the PCR using primers designed Duffy Ab required to displace 50% of 125I-GRO-␣/CXCL1 from Duffy from GenBank AF055992, flanking the open reading frame and containing binding sites (IC50) was 16 nM (see Fig. 3). Nonimmune goat IgG was used the native start site (13). A human pancreas cDNA library served as the as the control Ab (Zymed Laboratories, San Francisco, CA). PCR template. PCR was conducted for 30 cycles according to the manu- Costar 24-well plates containing polycarbonate transwell filters with a facturer’s protocol (PerkinElmer/Cetus, Norwalk, CT) using Taq polymer- pore size of 3 ␮m were used for the transit experiments (Corning Costar, ase. PCR products were blunt-end ligated into a mammalian cell expression Cambridge, MA). The transwell filters were precoated with human placen- ϩ plasmid vector, pcDNA3.1/hygromycin B (Invitrogen, Carlsbad, CA) tal type IV collagen at 10 ␮g/cm2 (Sigma-Aldrich, St. Louis, MO). Duffy containing a hygromycin resistance gene and sequenced. PCR clones were expressing endothelial cells were seeded at 2 ϫ 105/well onto the collagen- selected for the correct sequence and proper orientation in the expression coated membrane, allowed to adhere, and grown to confluence at least 7 vector. days before each experiment. On the day of the experiment, cells were pretreated with the respective Transfection and stable expression of Duffy Ag Ab (0.49 ␮M) for 30 min. Following pretreatment with Ab, medium was ϩ ␮ IVEC were transfected with the pcDNA3.1/hygromycin B vector con- changed to contain 0.1% human serum albumin (HSA), 2.5 M 10,000 ␣ 125 ␣ taining the Duffy PCR insert. The transfectants were selected by resistance m.w. Texas Red dextran, 20 nM GRO- /CXCL1, 0.2 nM I-GRO- / ␮ to hygromycin B (Boehringer Mannheim, Indianapolis, IN). Individual CXCL1, 0.49 M of either anti-Duffy Ab or control Ig in the bottom well. clones were isolated using cloning cylinders and subcloned by the limiting Cells were incubated for4hat37°Cin5%CO2. At the end of the incu- Downloaded from 125 ␣ dilution technique. Individual clones were then tested for Duffy Ag surface bation, the amounts of I-GRO- /CXCL1 and Texas Red dextran were ␮ expression by flow cytometry (FACScan; BD Biosciences, San Jose, CA) measured from a 50- l aliquot obtained from the top well using a gamma using the mAb ␣Fy6, a generous gift from Dr. P. Rubinstein of the New counter (1470 Wizard; Wallac Oy, Turku, Finland) and cytofluorometer set York Blood Center (New York, NY). Clones that were used for subsequent at excitation wavelength 590 nm and emission wavelength 645 nm (Cyto- studies showed greater than one log increase in mean fluorescence in- Fluor II; PerSeptive Biosystems, Foster City, CA), respectively. The per- 10 125 ␣ tensity as compared with mock-transfected cells incubated with ␣Fy6. This cent of I-GRO- /CXCL1 crossing the monolayer was calculated using the following equation: 125I-GRO-␣/CXCL1 crossing ϭ 100% ϫ (cpm of

shift in mean fluorescence intensity displayed by Duffy-transfected cells http://www.jimmunol.org/ Ϫ labeled with ␣Fy6 was specific, because cells incubated with an isotype sample wells cpm of medium alone)/equilibrium cpm, where equilib- ␮ mouse IgG1 control Ab did not show a significant shift. rium cpm was defined as the radioactivity in a 50- l aliquot after mixing the contents of the top and bottom wells. Receptor binding assay In all wells of each experiment, Texas Red dextran, a fluorophore-con- jugated dextran of 10,000 m.w. (Molecular Probe, Eugene, OR), was 125I-GRO-␣/CXCL1 (specific activity 2200 Ci/mmol) was obtained from placed on the same side as the chemokine and served as a marker to de- Amersham Life Science (Piscataway, NJ) and New England Nuclear (Bos- termine the diffusion of a low m.w. compound similar in size to chemo- ton, MA). Unlabeled IL-8/CXCL8 and GRO-␣/CXCL1 were purchased kines. The percent dextran crossing was used to control for any differences from PeproTech (Rocky Hill, NJ). Unlabeled RANTES/CC chemokine li- in the integrity of the monolayer from well to well and was calculated using gand (CCL) 5, monocyte chemotactic protein-1 (MCP-1/CCL2), and mac- the following equation: % dextran crossing ϭ 100% ϫ (fluorescence of ␣ ␣ rophage inflammatory protein-1 (MIP-1 /CCL3) were obtained from sample wells Ϫ fluorescence of medium alone/equilibrium fluorescence), by guest on October 2, 2021 R&D Systems (Minneapolis, MN). Stable transfectants were seeded into where equilibrium fluorescence was defined as radioactivity in a 50-␮l 96-well break-apart plates (Dynatech Laboratories, Chantilly, VA) at aliquot after manually mixing the contents of a top and bottom well. 60,000 cells/well. ␣ To determine the Kd of GRO- /CXCL1 to the Duffy Ag, each well was Neutrophil transendothelial migration assay incubated with 0.2 nM 125I-GRO-␣/CXCL1 and increasing concentrations of unlabeled GRO-␣/CXCL1 for 90 min. Nonspecific binding was defined Polymorphonuclear (PMN) were isolated from a healthy vol- as the amount of radioactivity measured in the well containing 1 ␮M un- unteer, and loaded with calcein-AM as previously reported (14). PMN at labeled ligand. The total number of cells was counted in parallel wells. All 1 ϫ 106 in a 200-␮l volume of RPMI 1640 without phenol red (BioWhit- samples were performed in triplicates or quadruplicates. Data were ana- taker, Walkersville, MD) containing 0.1% HSA (Centeon, Kankakee, IL)

lyzed using the LIGAND program (Biosoft, Cambridge, U.K.). The Kd was were placed in the top well of the transwell. IL-8/CXCL8 at 1 nM or obtained by Scatchard analysis. GRO-␣/CXCL1 at 20 nM were placed in the bottom chamber in a total To determine the chemokine binding properties of the expressed Duffy volume of 1.0 ml of RPMI 1640 containing 0.1% HSA. The transwell 125 Ag, each well was incubated with 0.2 nM I-GRO-␣/CXCL1 and varying chambers were incubated for3hat37°Cin5%CO2. The transwell insert concentrations of unlabeled ligand at 37°C for 90 min. Wells were washed was removed from each well, and 1.0 ml of 2% Triton-X was added to the twice with binding buffer (RPMI 1640, 0.2% BSA), and dried. The radio- bottom chamber contents, mixed, and the fluorescence of each well was activity in each well was measured by scintillation counting (ICN Bio- measured using emission and excitation wavelengths of 485 and 530 nm, medicals, Costa Mesa, CA). Nonspecific binding was defined as the respectively. To calculate the percentage of neutrophils migrating from the amount of radioactivity measured in the well containing 1 ␮M unlabeled top to the bottom chamber, a maximal fluorescence for neutrophils was ligand. derived. At time 0, 200 ␮lof1ϫ 106 PMN were added to 800 ␮lof medium. The mixture was added to 1.0 ml of 2% Triton-X, and calcein Endothelial gene expression profile fluorescence (fl.) was measured. The percent of PMN migration was cal- culated using the following formula: Duffy transfected endothelial cells were incubated at 1 ϫ 107 in the pres- ence or absence of 20 nM GRO-␣/CXCL1 for4hat37°C. Duffy trans- % PMN migration ϭ fl. of sample/maximal fl. ϫ 100 ␣ fectants stimulated with TNF- at 10 ng/ml served as the positive control. . At the end of the incubation, cells were harvested and RNA was isolated using the Qiagen RNeasy Midi kit (Valencia, CA). Biotin-labeled cRNA mDuffy knockout and wild-type mice was generated using the Enzo BioArray HighYield RNA Transcript La- beling kit (Enzo Biochem, Farmingdale, NY) and hybridized with the Af- Mice with targeted deletion of the Duffy gene have been previously de- fymetrix U95Av2 oligonucleotide array (Affymetrix, Santa Clara, CA) then scribed (10). Briefly, a 1.1-kb neo gene eliminating a segment of the intron washed and stained per the manufacturer’s protocol. Data from each array and 90 bp of exon II was inserted into the genomic Duffy locus by ho- was analyzed using the software package Microarray Suite 5.0 (Affymetrix, mologous recombination. Embryonic stem cells containing the targeted Santa Clara, CA). After normalization by average intensity, the expression mutation were injected into C57BL/6 blastocysts. DuffyϪ/Ϫ mice and their level of each gene was compared in the presence or absence of GRO-␣/ wild-type littermates were bred and housed in specific pathogen-free con- CXCL1 or TNF-␣. The data were then filtered to remove all genes with a ditions at the Seattle Veteran’s Affairs vivarium until the day of the ex- Ͻ2-fold increase or decrease in expression at a significance level of p Ͻ periment. Equal numbers of male and female mice, age- and weight-

0.001 (Wilcoxon rank-sum test). matched, in the N2F2 generation were included in each group. 5246 DUFFY Ag NEUTROPHIL MIGRATION

Homozygous Duffy knockout and wild-type mice were generated by breed- ing Duffy heterozygote breeding pairs. Mice were weaned at 3 wk of age, at which time they were genotyped using tail snip DNA. Intratracheal instillation of IL-8 Mice were weighed and anesthetized using a ketamine/xylazine solution (70 mg/kg ketamine, 10 mg/kg xylazine). The mice were placed on a 45° incline board, a fiber optic light source was placed just above the thoracic inlet (IntraLux 6000-1; Volpi Manufacturing, Auburn, NY), and the vocal cords were directly visualized using a small catheter introducer (BD Bio- sciences, Rutherford, NJ). The trachea was cannulated with a 22-gauge gavage feeding needle (Kent Scientific, Litchfield, CT) connected to a tu- berculin syringe. The plunger was removed from the tuberculin syringe and 100 ␮l of saline was placed at the distal end before its use. The correct position of the gavage needle in the trachea was verified by movement of the saline column in the tuberculin syringe. Recombinant human IL-8/ CXCL8 (1 ϫ 10Ϫ6 M) (Peprotech, Piscataway, NJ) was diluted in PBS containing 0.1% HSA or PBS ϩ 0.1% HSA alone (Centeon) and a volume of 0.15 ml/100 g was instilled directly into the trachea through the gavage needle. At 6 h, mice were euthanized with 120 mg/kg pentobarbital. Whole blood was obtained by direct cardiac puncture and the plasma was col- Ϫ lected, aliquoted, and stored in 70°C until use. The thoracic cavity was Downloaded from opened by midline incision. The trachea was exposed and cannulated with a 24-gauge catheter, which was secured with a 2-0 silk suture. Whole lung lavage was performed using 0.9% NaCl containing 0.6 mM EDTA instilled in one aliquot of 1.2 ml, followed by three aliquots of 1.0 ml. BAL processing Total cell counts in the BAL fluid were performed using a Neubauer cham- http://www.jimmunol.org/ ber. Cell differentials were performed by counting 100 consecutive cells on cytospin preparations stained with DiffQuik. Results Cloning of human Duffy cDNA Sequence analysis confirmed that the Duffy cDNA isolated was the spliced isoform derived from two exons (GenBank AY167991). This isoform is the predominant transcript in vivo (15, 16) (Fig. 1). b

The Duffy cDNA encoded the Fy epitope (A125). It also con- by guest on October 2, 2021 tained a silent mutation (G15C), and a polymorphism (G298A: Ala100Thr) that has no observable effect on surface protein ex- pression in vivo or 125I-IL-8/CXCL8-specific binding in vitro (4, 17). The cloned Duffy cDNA also contained either a new poly- morphism (A649G:Ile217Val) or a PCR-induced mutation. This Ile217Val substitution occurs within the transmembrane region of the protein and is a conservative mutation. Duffy expressing endothelial cells bind selective CXC and CC chemokines ␣ Binding analysis was undertaken to calculate the Kd of GRO- / CXCL1 using 0.2 nM 125I-GRO-␣/CXCL1 and increasing concen- trations of unlabeled GRO-␣/CXCL1 (Fig. 2). Nonspecific binding FIGURE 1. Human Duffy Ag sequence cloned by PCR (GenBank AY167991). Base sequence numbers reference the start codon. GenBank was defined as the amount of bound 125I-GRO-␣/CXCL1 in the AF055992 sequence was used to design primers (primer targets are under- ␮ ␣ 125 presence of 1 M unlabeled GRO- /CXCL1. The binding of I- lined). Predicted transmembrane amino acid sequences are highlighted. ␣ ␣ GRO- /CXCL1 was saturable as the amount of total GRO- / Three sequence variances from GenBank AF055992, which encodes the x CXCL1 increased. Scatchard analysis indicated a Kd value of 5.6 Fy mutation, are indicated with an asterisk. The Duffy Ag cDNA pos- nM, similar to what has been observed on erythrocytes and endo- sesses the following: 1) G15C, a silent mutation; 2) A125 which codes the thelial cells (3, 6, 7). Fyb epitope; 3) C265 that renders full surface expression of Fyb, in contrast The ability of various chemokines to compete with 125I-GRO- to C265T: Arg89Cys which codes the Fyx mutation; 4) G298A:Ala100Thr, ␣/CXCL1 for binding to Duffy expressing cells was tested using a previously reported polymorphism that does not alter surface expression b 0.2 nM 125I-GRO-␣/CXCL1 incubated in the presence of increas- of Fy ; 5) A649G:Ile217Val, a chemically conserved new polymorphism ing concentrations of unlabeled chemokines (Fig. 3). In addition, or PCR mutation. we tested the ability of a goat anti-human Duffy polyclonal Ab (␣hDuffy Ab) to block binding of 125I-GRO-␣/CXCL1 on the ␣ Ͻ Ͻ Duffy transfectants. The inhibitory concentration of unlabeled li- U.K.). The relative IC50 values observed were: GRO MCP-1 gand required to displace 50% of radiolabeled GRO-␣/CXCL1 ␣hDuffy Ab Ͻ RANTES Ͻ IL-8 ϽϽϽ MIP-1␣. Thus, the binding ␣ ϩ (IC50) was measured for GRO- /CXCL1, MCP-1/CCL2, RAN- profile of the endothelial Duffy transfectants (Duffy cells) was sim- TES/CCL5, ␣hDuffy Ab, IL-8/CXCL8, and MIP1-␣/CCL3 using ilar to the findings observed in mouse erythrocytes, and K562 cells Equilibrium Binding Data Analysis software (Biosoft, Cambridge, stably transfected with the Duffy cDNA (18, 19). The Journal of Immunology 5247

FIGURE 2. Saturation curve and Scatchard plot of GRO-␣/CXCL1 Downloaded from binding. Duffy transfected endothelial cells show saturable binding of 125I- ␣ ␣ GRO- /CXCL1 (top panel) and a calculated Kd for GRO- /CXCL1 of 5.6 125 nM (bottom panel). Duffy transfectants were incubated with 0.2 nM I- FIGURE 3. Competition binding studies using 125I-GRO-␣/CXCL1 and GRO-␣/CXCL1 and increasing concentrations of unlabeled GRO-␣/ various unlabeled ligands. Data are presented as the percentage of total CXCL1. Each condition was performed in triplicate or quadruplicate; n ϭ bound 125I-GRO-␣/CXCL1 as a function of increasing concentrations of 2 independent experiments. unlabeled ligand. A curve fitting program was used to determine the IC

50 http://www.jimmunol.org/ total binding of 125I-GRO-␣/CXCL1 to unlabeled GRO-␣/CXCL1, IL-8/ CXCL8, MCP-1/CCL2, ␣hDuffy Ab, RANTES/CCL5, MIP-1␣/CCL3, and are shown in the symbol legend. Each condition was performed in Interestingly, the endothelial Duffy transfectants showed a triplicate or quadruplicate. higher than anticipated IC50 of IL-8. IL-8/CXCL8 was a less ef- fective antagonist of 125I-GRO-␣/CXCL1 in the endothelial Duffy transfectants. This may be due to differences in the membrane environment on transfected cells vs erythrocytes, as previously fectants (Duffyϩ cells) by flow cytometry (data not shown). The ␣ ϩ suggested for the slightly higher Kd values observed in K562 Duffy gene expression profile in TNF- -stimulated Duffy cells showed by guest on October 2, 2021 transfectants than native erythrocytes (18). Alternatively, the lower that ICAM-1 mRNA expression was 7.5-fold higher than in un- affinity of IL-8/CXCL8 for the Duffy transfectants may be the re- stimulated Duffyϩ cells. Moreover, TNF-␣ stimulation resulted in sult of a previously unreported polymorphism (A649G:Ile217Val). up-regulation of 42 genes including VCAM-1, endothelial-selectin (E-selectin), ICAM-1, MCP-1/CCL2, IL-8/CXCL8, GRO-␤/ Endothelial gene expression following binding of ligand to the CXCL2, GRO-␥/CXCL3, and down-regulation of ϳ142 genes, Duffy Ag similar to published findings (Table I) (21). Thus, GRO-␣/CXCL1 The Duffy Ag is a member of the family of 7-transmembrane do- stimulation of Duffy transfected endothelial cells does not alter main receptors, but it lacks the DRY amino acid motif that allows gene transcription. Moreover, genes up-regulated following coupling to G proteins (1). The Duffy Ag lacks the ability to trans- TNF-␣ stimulation of Duffy transfectants help to characterize and duce a signal given the lack of the DRY motif. Direct evidence for validate the transfected cells as possessing an endothelial this is based upon the inability to show increases in intracellular phenotype. calcium ion concentrations following Duffy Ag-ligand binding in 125 ␣ transfected 293 cells (4) and the inability of Duffy Ag to stimulate I-GRO- movement across an endothelial monolayer GTPase activity (20). To determine whether the binding of GRO- expressing Duffy Ag ␣/CXCL1 to the Duffy Ag produces a signal that alters gene tran- A transwell system was used to test the hypothesis that Duffy Ag scription in endothelial cells, the gene expression pattern of Duffy participates in chemokine movement across the endothelium (Fig. transfectants was examined using oligonucleotide microarrays 4). GRO-␣/CXCL1 and tracer amounts of 125I-GRO-␣/CXCL1 (Table I). A 2-fold or higher change in gene expression was chosen were placed in the bottom well below either a Duffyϩ and DuffyϪ as significant. endothelial monolayer, in the presence or absence of blocking Ab Of the 12,000 genes examined, there were no significant against the Duffy Ag. The concentration of Ab used was 30-fold 125 ␣ changes in the gene expression profile following stimulation of greater than its IC50 required to displace I-GRO- /CXCL1 Duffyϩ cells with GRO-␣/CXCL1 as compared with unstimulated bound on Duffy transfected cells. Chemokine movement from the Duffyϩ cells (Table I). In addition, neither CXCR1 nor CXCR2 bottom to top well was determined by measuring the amount of mRNA expression were noted in Duffyϩ cells with or without 125I-GRO-␣/CXCL1 recovered from the top well following a 4-h stimulation. To show that the Duffy transfected cells could be stim- incubation at 37°C. ulated to produce changes in their gene expression profile com- The data showed that inhibition of Duffy Ag with blocking Ab pared with baseline, transfected cells were also challenged with results in a significant reduction in the translocation of 125I-GRO- TNF-␣. TNF-␣ served as the positive control stimulus after pre- ␣/CXCL1 across a Duffyϩ endothelial monolayer (Fig. 4). To liminary studies showed that incubation with TNF-␣ at 10 ng/ml demonstrate that the Duffy-specific Ab did not alter the integrity of increased the expression of ICAM-1 on the surface of Duffy trans- Duffyϩ endothelial monolayers, we also measured the movement 5248 DUFFY Ag NEUTROPHIL MIGRATION

Table I. Oligonucleotide microarray analysisa

Endothelial gene expression profile of Duffy transfectants

Following GRO-␣/CXCL1 stimulation; no significant changes Following TNF-␣ stimulation Up-regulated genes: first 10 of 42 up-regulated genes 1. VCAM-1 2. Human IFN-␥ treatment inducible mRNA 3. Human JE gene encoding a monocyte secretory protein (MCP-1) 4. Diubiquitin 5. Human endothelial leukocyte adhesion molecule-1 (E- selectin) 6. ICAM-1 7. Human TNF-inducible early response gene 8. Homo sapiens mRNA for KIAA106 protein 125 ϩ Ϫ 9. Human adhesion molecule ninjurin mRNA FIGURE 4. Recovery of I-GRO-␣ across Duffy and Duffy endo- 10. Human B94 protein mRNA thelial cell monolayer in the presence of either anti-Duffy or control Ab. Down-regulated genes: first 10 of 142 down-regulated genes Duffyϩ and DuffyϪ endothelial cell monolayers were preincubated in the 1. Human signal transducing adaptor molecule 2A presence of anti-Duffy Ab or control IgG. Twenty nanomolar concentra- (STAM2) mRNA tions of unlabeled GRO-␣/CXCL1, 0.2 nM 125I-GRO-␣/CXCL1, and 2.5 2. Human SNF-1-like protein kinase mRNAb ␮M dextran (10,000 m.w. Texas Red dextran) were placed in the bottom Downloaded from 3. Human homolog of yeast mutL (hPMS1) gene wells with 0.5 ␮M Ab. The amount of 125I-GRO-␣/CXCL1 and dextran 4. H. sapiens mRNA for vascular Rab-GAP/TBC-containing recovered from the top well were measured following a 4-h incubation at proteinb 125 ␣ 5. H. sapiens RP58 gene 37°C. Recovery of I-GRO- /CXCL1 is significantly reduced across a 6. AI961743:wt66f12.x1 H. sapiens cDNA Duffy expressing endothelial monolayer in the presence of anti-Duffy Ab p ϭ 0.002 by Mann-Whitney U test, p ϭ 0.007 by ANOVA). Data are ,ء) Human mRNA for brain-derived neurotrophic factor .7 8. Human TRF-1-interacting ankyrin-related ADP-ribose mean Ϯ SEM of three independent experiments performed in triplicate or

polymerase mRNAb quadruplicate. Wells with Ͼ10% dextran crossing were excluded from http://www.jimmunol.org/ 9. Human myosin H chain 12 (MYO5A) mRNA statistical analysis. 10. Human msg1-related gene mRNA

a The gene expression profile was examined in endothelial Duffy transfectants stimulated with 20 nM GRO-␣/CXCL1 or 10 ng/mL TNF-␣ for 4 h using oligonu- cleotide microarrays. No genes were found to be significantly up-regulated or down- regulated in the Duffyϩ cells following GRO-␣ stimulation. In Duffyϩ cells stimu- lated with TNF-␣, 42 genes were up-regulated and 142 genes were down-regulated tween Duffyϩ and DuffyϪ monolayers. Although, IL-8-mediated compared with unstimulated cells (the first 10 in each group are shown for simplicity). ϩ The results were similar in two independent experiments. neutrophil migration appeared greater across Duffy monolayers, b SNF, sucrose nonfermenting protein; GAP/TBC, GTPase-activating protein/Tre/ it was not statistically significant at 2 h. However, following a 3-h by guest on October 2, 2021 Bub2/Cdc16 domain; TRF, telomeric repeat binding factor; msg, missing. incubation, the differences in IL-8-mediated neutrophil migration across Duffyϩ monolayer compared with DuffyϪ monolayers p Ͻ 0.05; Fig. 6). Although ,ء) reached statistical significance of 10,000 m.w. dextran (an inert molecule the size of GRO-␣/ Duffy Ag is not essential for neutrophil migration, the in vitro data CXCL1) across each well tested and showed that the translocation showed that Duffy Ag can facilitate both GRO-␣/CXCL1 and IL- of dextran was not significantly different across Duffyϩ endothelial 8/CXCL8-mediated neutrophil transendothelial migration. monolayers in the presence of either anti-Duffy or control Ab. As expected, anti-Duffy Ab had no effect on either GRO-␣/CXCL1 Neutrophil recruitment into the airspaces of mice lacking the and dextran translocation across DuffyϪ endothelial monolayers. Duffy Ag ϩ Notably, translocation of GRO-␣/CXCL1 across Duffy monolay- We tested whether Duffy Ag modifies neutrophil migration in vivo Ϫ ers cannot be directly compared with Duffy monolayers given the by examining neutrophil recruitment into the pulmonary airspaces Ϫ relative “leakiness” of Duffy monolayers, as evidenced by the following intratracheal instillation of human IL-8/CXCL8 in mice Ϫ greater translocation of dextran across Duffy monolayers. Nev- lacking the Duffy Ag (Fig. 7). Direct instillation of chemokine into ertheless, the blocking Ab studies support the conclusion that the distal airways provides a direct test of whether Duffy Ag has an Duffy Ag facilitates the movement of GRO-␣/CXCL1 across an effect on chemokine-mediated inflammatory cell recruitment into endothelial monolayer. local tissue beds. IL-8/CXCL8 was chosen as the stimulus because others have shown that murine Duffy Ag also binds angiogenic In vitro neutrophil migration assay CXC chemokines, including human IL-8/CXCL8 (19). Initial dose To test whether the presence of Duffy on endothelial cells modifies response studies using 0.01, 0.1, and 1 ␮M IL-8/CXCL8 yielded neutrophil migration toward either GRO-␣/CXCL1 or IL-8/ peak neutrophil recruitment into the airspaces in mice with 1 ␮M CXCL8, Duffy expressing cells (Duffyϩ) and untransfected cells IL-8/CXCL8 (data not shown); thus, 1 ␮M IL-8/CXCL8 was used (DuffyϪ) were seeded onto transwells as previously described for the in vivo studies. Compared with wild-type littermates, there (Figs. 5 and 6) (22). We determined that 1 nM IL-8/CXCL8 and 20 was significantly less neutrophil recruitment into the airspaces fol- nM of GRO-␣/CXCL1 produced equivalent chemotactic activity lowing intratracheal instillation of IL-8/CXCL8 in DuffyϪ/Ϫ mice, for neutrophils and, thus, these concentrations were selected for suggesting that Duffy Ag contributes to chemokine-mediated neu- study (23). In four separate experiments performed either in trip- trophil migration (Fig. 7). We also examined plasma levels of IL- licate or quadruplicate, we showed that GRO-␣/CXCL1-mediated 8/CXCL8 to determine whether there were measurable differences neutrophil migration is enhanced across a Duffyϩ monolayer fol- in plasma IL-8/CXCL8 levels in DuffyϪ/Ϫ vs Duffyϩ/ϩ litter- p Ͻ 0.05; Fig. 5). There were no mates. Plasma IL-8/CXCL8 levels were undetectable in all mice ,ء) lowing a 2-h incubation significant differences in random migration or dextran crossing be- studied using a standard ELISA with a detection limit of 31 pg/ml. The Journal of Immunology 5249 Downloaded from

FIGURE 5. Effect of endothelial Duffy Ag expression on GRO-␣/ CXCL1-mediated neutrophil migration in vitro. GRO-␣/CXCL1-mediated FIGURE 6. Effect of endothelial Duffy Ag expression on IL-8/CXCL8- ϩ mediated neutrophil migration in vitro. IL-8/CXCL8-mediated neutrophil neutrophil migration is enhanced across Duffy monolayers in vitro fol- ϩ p Ͻ 0.05 by Mann-Whitney U test). IL-8- migration is enhanced across Duffy monolayers in vitro following a 3-h ,ء) lowing a 2-h incubation Ͻ ء mediated neutrophil migration is slightly greater across Duffyϩ monolay- incubation ( , p 0.05 by Mann-Whitney U test). No significant differ- ences in the percent dextran crossing were noted between Duffyϩ and ers, but this is not statistically significant. No significant differences in the Ϫ http://www.jimmunol.org/ percent dextran crossing were noted between Duffyϩ and DuffyϪ mono- Duffy monolayers to account for differences in neutrophil migration. Data Ϯ layers to account for differences in neutrophil migration. Data are mean Ϯ are mean SEM of six independent experiments performed in either trip- SEM of four independent experiments performed in either triplicate or licate or quadruplicate. quadruplicate.

Lung homogenates showed no significant differences in IL-8/ Ϫ/Ϫ ϩ/ϩ is important in facilitating chemokine-mediated neutrophil migra- CXCL8 concentrations between Duffy and Duffy animals, tion. The in vitro and in vivo approaches are complementary and indicating that the amounts instilled were similar in both groups of support the conclusion that Duffy Ag has an important biological by guest on October 2, 2021 animals. To address the possibility that the deletion of the Duffy function in tissue sites of inflammation. Ag in the knockout mice resulted in reduced peripheral neutrophil To gain a better understanding of how Duffy Ag contributes to counts or functional defects in neutrophil chemotaxis, we mea- neutrophil migration, we examined whether GRO-␣/CXCL1- sured total peripheral neutrophil counts and in vitro neutrophil Duffy Ag interactions change the endothelial gene expression pro- chemotaxis in DuffyϪ/Ϫ animals and found no differences as ϩ/ϩ file. We showed that stimulation of Duffy transfectants with GRO- compared with Duffy animals. ␣/CXCL1 had no observable effect in altering endothelial gene transcription. This is the first direct evidence that GRO-␣/CXCL1 Discussion binding to the Duffy Ag does not produce intracellular signaling We have previously shown that Duffy Ag expression is enhanced in human lungs with a histologic diagnosis of suppurative pneu- monia, a process defined by neutrophilic infiltration of the air- spaces (11). The enhanced Duffy Ag expression occurs along mi- crovessels and alveolar septa, particularly in regions of neutrophil accumulation (11). This is in contrast to normal lungs and lungs with acute lung injury, which showed only low level Duffy Ag expression. These observations suggest that expression of Duffy Ag is regulated in the lung microvasculature by the inflammatory process, and that Duffy Ag may have a functional role in the lung parenchyma during inflammation. Because of the up-regulation of Duffy Ag at tissue sites of inflammation, its colocalization to areas of inflammatory cell accumulation, and its multiple chemokine binding properties (9, 11), the main focus of this study was to test whether Duffy Ag plays a role in modifying chemokine-mediated FIGURE 7. Total neutrophil counts in the BAL of Duffyϩ/ϩ and neutrophil recruitment in vitro and in vivo. We showed enhanced Ϫ/Ϫ ϩ Duffy mice 6 h following intratracheal instillation of 1 ␮M IL-8/ neutrophil transmigration across a Duffy monolayer toward two ␣ CXCL8. There was minimal neutrophil recruitment into the pulmonary different chemokines, GRO- /CXCL1 and IL-8/CXCL8. In addi- airspaces following instillation of PBS. Alveolar neutrophil recruitment to tion, we found that Duffy knockout mice have markedly reduced IL-8/CXCL8 was impaired in DuffyϪ/Ϫ mice. Data are mean Ϯ SEM com- neutrophil counts in the bronchoalveolar lavage following intra- bined from two independent experiments performed with n ϭ 8or9in tracheal instillation of IL-8/CXCL8 as compared with wild-type each of the PBS groups and n ϭ 16 in each of the IL-8/CXCL8 groups. .(p Ͻ 0.0001 by Mann-Whitney U test ,ء) littermates. These findings support the interpretation that Duffy Ag 5250 DUFFY Ag NEUTROPHIL MIGRATION events resulting in changes in gene transcription. Despite the abil- on endothelial cells of the postcapillary venules where leukocytes ity of the Duffy endothelial transfectants to express molecules that emigrate (6). Duffy Ag is also up-regulated during inflammation might affect neutrophil transmigration when stimulated with and binds CXC and CC chemokines (4, 6, 8, 9). The data presented TNF-␣ (e.g., VCAM-1, E-selectin, ICAM-1), this is not a mech- herein are the first direct evidence that Duffy Ag can promote anism that would explain the differences we observed in the neu- neutrophil recruitment into tissue such as the lungs, when IL-8/ trophil transmigration experiments with GRO-␣/CXCL1, because CXCL8 is the stimulus. The in vitro data suggest a potential mech- GRO-␣/CXCL1 binding in these cells does not up-regulate the anism whereby Duffy Ag on endothelial cells contributes to trans- expression of adhesion molecules or other genes that might affect endothelial movement of chemokine. We conclude that endothelial neutrophil migration. Duffy Ag is a biologically relevant molecule that participates in It has been previously postulated that the Duffy Ag can partic- regulating inflammatory cell recruitment to tissue sites of ipate in chemokine transport across endothelial cells (7, 24). We inflammation. determined whether Duffy Ag contributes to chemokine movement across the endothelium in vitro, and showed that the presence of Duffy Ag facilitates movement of 125I-GRO-␣/CXCL1 across a Acknowledgments ϩ Duffy monolayer. Thus, our in vitro data are in keeping with the We gratefully acknowledge Amy Selk for excellent technical assistance. hypothesis that Duffy Ag on endothelial cells can function as a shuttling molecule for chemokines. Others also have shown lines of evidence to suggest that the References function of the Duffy Ag is different in endothelial cells than in 1. Hadley, T. J., and S. C. Peiper. 1997. From malaria to : the erythrocytes (7, 24, 25). In situ binding studies performed in rabbit emerging physiologic role of the Duffy blood group antigen. Blood 89:3077. Downloaded from 125 2. Darbonne, W. C., G. C. Rice, M. A. Mohler, T. Apple, C. A. Hebert, skin show that I-IL-8 originating from the abluminal side is A. J. Valente, and J. B. Baker. 1991. Red blood cells are a sink for 8, internalized by venular endothelial cells, localizes to caveolae, and a leukocyte chemotaxin. J. Clin. Invest. 88:1362. is subsequently presented on the luminal surface (24). The mole- 3. Neote, K., W. Darbonne, J. Ogez, R. Horuk, and T. J. Schall. 1993. Identification cule participating in the transport and presentation of IL-8 in en- of a promiscuous inflammatory peptide receptor on the surface of red blood cells. J. Biol. Chem. 268:12247. dothelial cells is unknown. However, it binds IL-8 and RANTES 4. Neote, K., J. Y. Mak, L. F. Kolakowski, and T. J. Schall. 1994. Functional and but not MIP-1␣ (24), similar to the binding profile of the Duffy Ag biochemical analysis of the cloned Duffy antigen: identity with the red blood cell http://www.jimmunol.org/ (24, 26). In addition, K562 erythroleukemic cells stably transfected chemokine receptor. Blood 84:44. with Duffy cDNA rapidly internalize radiolabeled GRO-␣/ 5. Horuk, R., C. E. Chitnis, W. C. Darbonne, T. J. Colby, A. Rybicki, T. J. Hadley, and L. H. Miller. 1993. A receptor for the malarial parasite Plasmodium vivax: the CXCL1, but this GRO-␣/CXCR1 is not degraded following inter- erythrocyte chemokine receptor. Science 261:1182. nalization (7). Furthermore, immunoelectron microscopy and im- 6. Hadley, T. J., Z. H. Lu, K. Wasniowska, A. W. Martin, S. C. Peiper, J. Hessel- munohistochemistry studies have localized Duffy Ag to both apical gesser, and R. Horuk. 1994. Postcapillary venule endothelial cells in kidney ex- press a multispecific chemokine receptor that is structurally and functionally and basal membrane domains of endothelial cells as well as within identical to the erythroid isoform, which is the Duffy blood group antigen. J. Clin. caveolae (25). These independent findings support the concept that Invest. 94:985. the potential biological role of Duffy includes internalization and 7. Peiper, S. C., Z. X. Wang, K. Neote, A. W. Martin, H. J. Showell, M. J. Conklyn, K. Ogborne, T. J. Hadley, Z. H. Lu, J. Hesselgesser, et al. 1995. The Duffy by guest on October 2, 2021 transport of chemokines across endothelial cells (7). antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Animal studies have produced discrepant results about the func- Duffy negative individuals who lack the erythrocyte receptor. J. Exp. Med. 181: tion of Duffy Ag in vivo (10, 27); however, differences in dose and 1311. 8. Liu, X. H., T. J. Hadley, L. Xu, S. C. Peiper, and P. E. Ray. 1999. Up-regulation time intervals make direct comparisons of the studies difficult. Luo of Duffy antigen receptor expression in children with renal disease. Kidney Int. Ϫ/Ϫ et al. (27) found that Duffy mice had reduced neutrophil ac- 55:1491. cumulation in the lungs and intestines 24 h following i.p. injection 9. Segerer, S., H. Regele, K. M. Mac, R. Kain, J. P. Cartron, Y. Colin, of 10 mg/kg LPS or 3% thioglycolate. These findings suggest that D. Kerjaschki, and D. Schlondorff. 2000. The Duffy antigen receptor for chemo- kines is up-regulated during acute renal transplant rejection and crescentic glo- Duffy Ag may facilitate neutrophil accumulation in tissue follow- merulonephritis. Kidney Int. 58:1546. ing specificinflammatory stimuli (27). Our in vitro and in vivo 10. Dawson, T. C., A. B. Lentsch, Z. Wang, J. E. Cowhig, A. Rot, N. Maeda, and data are in keeping with these findings and provide direct support S. C. Peiper. 2000. Exaggerated response to endotoxin in mice lacking the Duffy antigen/receptor for chemokines (DARC). Blood 96:1681. for the role of the Duffy Ag in chemokine-mediated neutrophil 11. Lee, J. S., C. W. Frevert, D. R. Thorning, S. Segerer, C. E. Alpers, J. P. Cartron, recruitment into the lungs. Y. Colin, V. A. Wong, T. R. Martin, and R. B. Goodman. 2003. Enhanced In contrast, Dawson et al. (10) found increased neutrophil ac- expression of Duffy antigen in the lungs during suppurative pneumonia. J. His- cumulation in the lungs and livers of DuffyϪ/Ϫ mice 2 h following tochem. Cytochem. 51:159. 12. Rhim, J. S., W. P. Tsai, Z. Q. Chen, Z. Chen, C. Van Waes, A. M. Burger, and i.p. injection of 30 mg/kg LPS, and they concluded that Duffy Ag J. A. Lautenberger. 1998. A human vascular endothelial cell model to study functions as a chemokine sink. However, with an overwhelming angiogenesis and tumorigenesis. 19:673. dose of LPS in circulation at the early time, the exaggerated sys- 13. Kozak, M. 1987. An analysis of 5Ј-noncoding sequences from 699 vertebrate Ϫ Ϫ messenger RNAs. Nucleic Acids Res. 15:8125. temic inflammatory response observed in the Duffy / mice could 14. Frevert, C. W., V. A. Wong, R. B. Goodman, R. Goodwin, and T. R. Martin. be explained by the absence of Duffy Ag on erythrocytes that nor- 1998. Rapid fluorescence-based measurement of neutrophil migration in vitro. mally bind excess chemokines in circulation. This interpretation is J. Immunol. Methods 213:41. supported by the findings of Olszyna et al. (28), who showed that 15. Iwamoto, S., J. Li, T. Omi, S. Ikemoto, and E. Kajii. 1996. Identification of a novel exon and spliced form of Duffy mRNA that is the predominant transcript in humans with experimental endotoxemia, erythrocyte-associated in both erythroid and postcapillary venule endothelium. Blood 87:378. IL-8 peaked 2 h following LPS injection, then fell as neutrophil- 16. Rios, M., A. Chaudhuri, G. Mallinson, L. Sausais, A. E. Gomensoro-Garcia, associated IL-8/CXCL8 began to rise at 4 and 6 h. The findings of J. Hannon, S. Rosenberger, J. Poole, G. Burgess, O. Pogo, and M. Reid. 2000. New genotypes in Fy(aϪbϪ) individuals: nonsense mutations (Trp to stop) in the Dawson et al. (10) and those of Luo et al. (27) highlight the com- coding sequence of either FY A or FY B. Br. J. Haematol. 108:448. plex roles of the Duffy Ag in two different locations: one expressed 17. Yazdanbakhsh, K., M. Rios, J. R. Storry, N. Kosower, N. Parasol, A. Chaudhuri, on erythrocytes in the circulation and the other expressed predom- and M. E. Reid. 2000. Molecular mechanisms that lead to reduced expression of inantly on endothelial cells in tissue. Duffy antigens. Transfusion 40:310. 18. Chaudhuri, A., V. Zbrzezna, J. Polyakova, A. O. Pogo, J. Hesselgesser, and Endothelial Duffy Ag is genetically conserved, even in individ- R. Horuk. 1994. Expression of the Duffy antigen in K562 cells: evidence that it uals who do not express Duffy on their RBCs (6, 7). It is expressed is the human erythrocyte chemokine receptor. J. Biol. Chem. 269:7835. The Journal of Immunology 5251

19. Luo, H., A. Chaudhuri, K. R. Johnson, K. Neote, V. Zbrzezna, Y. He, and 24. Middleton, J., S. Neil, J. Wintle, I. Clark-Lewis, H. Moore, C. Lam, M. Auer, A. O. Pogo. 1997. Cloning, characterization, and mapping of a murine promiscuous E. Hub, and A. Rot. 1997. Transcytosis and surface presentation of IL-8 by chemokine receptor gene: homolog of the human Duffy gene. Genome Res. 7:932. venular endothelial cells. Cell 91:385. 20. Horuk, R., T. J. Colby, W. C. Darbonne, T. J. Schall, and K. Neote. 1993. The 25. Chaudhuri, A., S. Nielsen, M. L. Elkjaer, V. Zbrzezna, F. Fang, and human erythrocyte inflammatory peptide (chemokine) receptor: biochemical A. O. Pogo. 1997. Detection of Duffy antigen in the plasma membranes and characterization, solubilization, and development of a binding assay for the sol- caveolae of vascular endothelial and epithelial cells of nonerythroid organs. uble receptor. Biochemistry 32:5733. Blood 89:701. 21. Zhou, J., Y. Jin, Y. Gao, H. Wang, G. Hu, Y. Huang, Q. Chen, M. Feng, and 26. Rot, A., E. Hub, J. Middleton, F. Pons, C. Rabeck, K. Thierer, J. Wintle, B. Wolff, C. Wu. 2002. Genomic-scale analysis of gene expression profiles in TNF-␣ M. Zsak, and P. Dukor. 1996. Some aspects of IL-8 pathophysiology. III. Che- treated human umbilical vein endothelial cells. Inflamm. Res. 51:332. mokine interaction with endothelial cells. J. Leukocyte Biol. 59:39. 22. Smart, S. J., and T. B. Casale. 1994. TNF-␣-induced transendothelial neutrophil 27. Luo, H., A. Chaudhuri, V. Zbrzezna, Y. He, and A. O. Pogo. 2000. Deletion of migration is IL-8 dependent. Am. J. Physiol. 266:L238. the murine Duffy gene (Dfy) reveals that the Duffy receptor is functionally re- 23. Goodman, R. B., R. M. Strieter, C. W. Frevert, C. J. Cummings, dundant. Mol. Cell. Biol. 20:3097. P. Tekamp-Olson, S. L. Kunkel, A. Walz, and T. R. Martin. 1998. Quantitative 28. Olszyna, D. P., E. De Jonge, P. E. Dekkers, S. J. van Deventer, and comparison of C-X-C chemokines produced by endotoxin-stimulated human al- T. van Der Poll. 2001. Induction of cell-associated chemokines after endotoxin veolar . Am. J. Physiol. 275:L87. administration to healthy humans. Infect. Immun. 69:2736. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021