Biochemical and Biophysical Research Communications
The human Duffy antigen binds selected inflammatory but not homeostatic chemokines
Section snippets
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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