In situ binding assay for studying chemokine interactions with endothelial cells

Antal Rot

doi:10.1016/S0022-1759(02)00502-1

In situ binding assay for studying chemokine interactions with endothelial cells

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Pages (from-to)	63-71
Number of pages	9
Journal	Journal of Immunological Methods
Volume	273
Issue number	1-2
Early online date	15 Nov 2002
DOIs	https://doi.org/10.1016/S0022-1759(02)00502-1
Publication status	Published - Feb 2003

Bibliographical note

Keywords

Animals
Antigens, Protozoan
Carrier Proteins
Chemokine CCL2
Chemokine CCL5
Chemokines, CC
Chemokines, CXC
Duffy Blood-Group System
Endothelium, Vascular
Epithelial Cells
Humans
Lymphatic System
Mice
Protozoan Proteins
Radioligand Assay
Receptors, CCR10
Receptors, Cell Surface
Receptors, Chemokine
Skin

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10.1016/S0022-1759(02)00502-1

Cite this

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title = "In situ binding assay for studying chemokine interactions with endothelial cells",

abstract = "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.",

keywords = "Animals, Antigens, Protozoan, Carrier Proteins, Chemokine CCL2, Chemokine CCL5, Chemokines, CC, Chemokines, CXC, Duffy Blood-Group System, Endothelium, Vascular, Epithelial Cells, Humans, Lymphatic System, Mice, Protozoan Proteins, Radioligand Assay, Receptors, CCR10, Receptors, Cell Surface, Receptors, Chemokine, Skin",

author = "Antal Rot",

year = "2003",

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doi = "10.1016/S0022-1759(02)00502-1",

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journal = "Journal of Immunological Methods",

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T1 - In situ binding assay for studying chemokine interactions with endothelial cells

AU - Rot, Antal

PY - 2003/2

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N2 - 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.

AB - 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.

KW - Animals

KW - Antigens, Protozoan

KW - Carrier Proteins

KW - Chemokine CCL2

KW - Chemokine CCL5

KW - Chemokines, CC

KW - Chemokines, CXC

KW - Duffy Blood-Group System

KW - Endothelium, Vascular

KW - Epithelial Cells

KW - Humans

KW - Lymphatic System

KW - Mice

KW - Protozoan Proteins

KW - Radioligand Assay

KW - Receptors, CCR10

KW - Receptors, Cell Surface

KW - Receptors, Chemokine

KW - Skin

U2 - 10.1016/S0022-1759(02)00502-1

DO - 10.1016/S0022-1759(02)00502-1

M3 - Article

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SN - 0022-1759

VL - 273

SP - 63

EP - 71

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

IS - 1-2

ER -