CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions

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.

The chemokine fragment CXCL9(74–103) diminishes neutrophil recruitment and joint inflammation in antigen-induced arthritis

“…Neutrophils play an important role in the pathogenesis of experimental antigen‐induced arthritis and their presence is related with articular damage and hypernociception . Additionally, we previously demonstrated that systemic treatment with CXCL9(74–103) decreased the recruitment of neutrophils in two different murine models of joint inflammation: local injection of CXCL8 and gout . In order to check if CXCL9(74‐103) also reduces neutrophil recruitment in an antigen‐induced arthritis, we immunized mice with mBSA and CFA as described before .…”

“…CXCL9 can recruit activated Th1 lymphocytes and NK cells and is produced by a variety of cells following stimulation with IFN‐γ . We previously demonstrated that a fragment of CXCL9 consisting of its 30 C‐terminal amino acids [CXCL9(74–103)] competes with chemokines for the binding to GAGs, inhibits CXCL8‐induced neutrophil migration, and reduces the recruitment of neutrophils to the joint in a murine model of gout …”

Anti‐inflammatory effects of the GAG‐binding CXCL9(74‐103) peptide in dinitrofluorobenzene‐induced contact hypersensitivity in mice

“…41,[44][45][46][47][48][49][50] In the CellJammer and NOX A12 approaches, chemokine mutants with increased GAGbinding affinity and abrogated GPCR signalling or aptamers that mask the GAG-binding site of a chemokine are used, respectively. 54 This indicates a certain degree of specificity for different GAGs due to a reduced spectrum of GAG binding. A, B, C Representative photomicrographs of CD11b and Ly6G stained ear sections of control (A) and DNFB-challenged mice treated with buffer (B) or CXCL9(74-103) (C) were shown (×20).…”

Impact of chemotactic factors and receptors on the cancer immune infiltrate: a bioinformatics study revealing homogeneity and heterogeneity among patient cohorts

“…Indeed, a recent work demonstrating an inhibitory activity of CXCL9 peptides on neutrophil migration in murine models supporting such a hypothesis. 74 …”