In situ binding assay for studying chemokine interactions with endothelial cells

The association of chemokines with endothelial cells (EC) and extracellular matrices is required for the prototypical pro-emigratory and pro-migratory in vivo activity of these molecules, respectively. In order to investigate chemokine binding to intact microanatomical structures, e.g. venular EC, we have developed an in situ binding assay. This is an autoradiographic morphological method in which the saturable binding of radiolabeled chemokines is studied in vitro in pieces of viable tissues. This article discusses the general applicability, advantages and shortcomings of the in situ binding assay in comparison with the other techniques available for visualizing chemokine receptor binding by cells in the tissues. We used this assay to demonstrate: (a) selective specific binding of CXC and CC chemokines to the EC of postcapillary venules but not capillaries or arteries; (b) selective specific binding of CC chemokines to the EC of afferent lymphatic vessels; and (c) selective specific binding of inflammatory chemokines to the EC lining high endothelial venules (HEV) in lymph nodes. The assessment of ligand cross-competition provided a fingerprint of chemokine-binding specificity of the EC in each of these microanatomical sites. This fingerprint could be paralleled with the chemokine-binding profiles of two non-signaling chemokine-binding molecules, Duffy antigen receptor for chemokines (DARC) and D6, present in venular and lymphatic EC, respectively. These observations allowed us to put forward the hypotheses regarding the involvement of EC DARC and D6 in chemokine transport and presentation by the EC.