Abstract
Several acute and chronic inflammatory diseases are driven by accumulation of activated leukocytes due to enhanced chemokine expression. In addition to specific G protein-coupled receptor-dependent signaling, chemokine–glycosaminoglycan (GAG) interactions are important for chemokine activity in vivo. Therefore, the GAG–chemokine interaction has been explored as target for inhibition of chemokine activity. It was demonstrated that CXCL9(74-103) binds with high affinity to GAGs, competed with active chemokines for GAG binding and thereby inhibited CXCL8- and monosodium urate (MSU) crystal-induced neutrophil migration to joints. To evaluate the affinity and specificity of the COOH-terminal part of CXCL9 toward different GAGs in detail, we chemically synthesized several COOH-terminal CXCL9 peptides including the shorter CXCL9(74-93). Compared to CXCL9(74-103), CXCL9(74-93) showed equally high affinity for heparin and heparan sulfate (HS), but lower affinity for binding to chondroitin sulfate (CS) and cellular GAGs. Correspondingly, both peptides competed with equal efficiency for CXCL8 binding to heparin and HS but not to cellular GAGs. In addition, differences in anti-inflammatory activity between both peptides were detected in vivo. CXCL8-induced neutrophil migration to the peritoneal cavity and to the knee joint were inhibited with similar potency by intravenous or intraperitoneal injection of CXCL9(74-103) or CXCL9(74-93), but not by CXCL9(86-103). In contrast, neutrophil extravasation in the MSU crystal-induced gout model, in which multiple chemoattractants are induced, was not affected by CXCL9(74-93). This could be explained by (1) the lower affinity of CXCL9(74-93) for CS, the most abundant GAG in joints, and (2) by reduced competition with GAG binding of CXCL1, the most abundant ELR+ CXC chemokine in this gout model. Mechanistically we showed by intravital microscopy that fluorescent CXCL9(74-103) coats the vessel wall in vivo and that CXCL9(74-103) inhibits CXCL8-induced adhesion of neutrophils to the vessel wall in the murine cremaster muscle model. Thus, both affinity and specificity of chemokines and the peptides for different GAGs and the presence of specific GAGs in different tissues will determine whether competition can occur. In summary, both CXCL9 peptides inhibited neutrophil migration in vivo through interference with GAG interactions in several animal models. Shortening CXCL9(74-103) from the COOH-terminus limited its GAG-binding spectrum.
Highlights
Several acute and chronic inflammatory diseases, such as gout and rheumatoid arthritis, are characterized by enhanced expression of chemokines and accumulation of activated leukocytes in tissues (1–6)
Directional leukocyte migration to specific primary, secondary, or tertiary lymphoid organs is important for normal functioning of our immune system
In order to prevent wash-out of chemokines from the surface of endothelial cells, chemokines can bind to negatively charged GAGs (18, 57, 58). These GAGs are linear polysaccharides which consist of repeating disaccharide subunits and dependent on the subunit they will be classified into different classes, e.g., heparin, heparan sulfate (HS), and chondroitin sulfate (CS) (59)
Summary
Several acute and chronic inflammatory diseases, such as gout and rheumatoid arthritis, are characterized by enhanced expression of chemokines and accumulation of activated leukocytes in tissues (1–6). For most chemokines an alternative biological classification can be made between homeostatic or constitutively expressed chemokines, and inflammatory or inducible chemokines The latter subclass is locally secreted by tissue cells and resident leukocytes upon infection or tissue damage, thereby creating a gradient along which leukocytes can migrate from the blood vessel to the site of inflammation (11–14). GAG-bound chemokines interact with their G protein-coupled receptors (GPCRs), expressed by specific circulating leukocyte subtypes. This results in adhesion to and extravasation of leukocytes through the endothelium (15–17). Once leukocytes enter the tissue, they can migrate to the site of inflammation through the gradient of local GAG-bound chemokines. Leukocytes of mice with disturbed heparan sulfate (HS) synthesis in endothelial cells and leukocytes showed reduced chemokine-induced migration (23)
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