The human Duffy antigen binds selected inflammatory but not homeostatic chemokines

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Abstract

The aim of the study was to compare the ability of the human Duffy antigen to bind homeostatic and inflammatory chemokines. Homeostatic chemokines did not bind to the Duffy antigen on erythrocytes with high affinity. In contrast, 60% of inflammatory chemokines bound strongly to Duffy, with no obvious preference for CXC or CC classes. It was investigated if this binding profile was reflected in the binding pattern of endothelial cells. Two examples of homeostatic (125I-CXCL12 and 125I-CCL21) and inflammatory (125I-CXCL8 and 125I-CCL5) chemokines were incubated with human synovia. In agreement with the erythrocyte binding data, intense specific signals for CXCL8 and CCL5 binding were found on endothelial cells, whereas CXCL12 and CCL21 showed only weak binding to these cells. Our study provides evidence that the human Duffy antigen binds selected inflammatory, but not homeostatic, chemokines and that this binding pattern is reflected by endothelial cells within inflamed and non-inflamed tissue.

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Materials and methods

Chemokines.125I-CXCL8 (specific activity 2200 Ci/mmol), 125I-CCL5 (specific activity 2200 Ci/mmol), and 125I-CCL21 (specific activity 1290 Ci/mmol) were obtained from Perkin–Elmer (Cambridge, UK). 125I-CXCL12 (specific activity 2000 Ci/mmol) was from Amersham (Little Chalfont, UK). Recombinant human chemokines were all from R&D Systems (Abingdon, UK).

Chemokine binding to human erythrocytes. Whole blood was obtained from healthy human donors and the erythrocytes were isolated by a standard protocol

Results and discussion

In order to examine the chemokine binding profile of the Duffy antigen, radioligand binding displacement experiments were carried out on human red blood cells. Initial experiments using 125I-CXCL-8 showed that the Kd was 19.5 nM (Bmax = 1.46 × 10−5 fmol/cell, 8000 binding sites per cell) and in all displacement experiments the concentration of labelled CXCL8 was 0.5 nM. The displacement of specific binding by a large excess of unlabelled CXCL8 or CCL2 (both 1 μM) showed that >86% of the total binding

Acknowledgments

We gratefully acknowledge funding from the Biotechnology and Biological Sciences, Research Council (UK), AstraZeneca (UK), the Wellcome Trust (UK), and the Droitwich Medical Trust, UK. We wish to thank the following surgeons and rheumatologists for their assistance: Dr. R. Butler, Dr. J. Dixey, Mr. C. McGeoch, Mr. D. Rees, Mr. R. Spencer-Jones, Mr. R. Wade, Mr. S. White, and the Daycase unit (RJAH Orthopaedic Hospital). Acknowledgement is given to P. Evans, N. Harness, and M. Pritchard for

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