Interference with Heparin Binding and Oligomerization Creates a Novel Anti-Inflammatory Strategy Targeting the Chemokine System

Abstract

A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents.

An engineered monomer of CCL2 has anti-inflammatory properties emphasizing the importance of oligomerization for chemokine activity in vivo

“…1B). Finally, in a third model involving OVA-induced recruitment into the BAL fluid, P8A-CCL2 inhibited recruitment by 69% at a dose of 0.5 mg/kg, whereas the 44 ANAA 47 -CCL5 variant, previously shown to have antagonistic properties [21], was less effective at the same dose, showing a reduction of 56% (Fig. 1C).…”

“…In an experiment to determine the pharmacodynamic properties of P8A-CCL2 (see Fig. 3D), a single administration of 10 mg/kg was given at various time-points before the thioglycollate stimulus, and the cells were enumerated at 48 h. In a third model, recruitment into the lungs after OVA sensitization was conducted as described previously [21], and inhibition was monitored following i.p. administration of 0.5 mg/kg P8A-CCL2 compared with 0.5 mg/kg [ 44 AANA 47 ]-CCL5.…”

“…In the case of CCL5/RANTES, we demonstrated that interference with these properties presents a novel, anti-inflammatory strategy; administration of 44 ANAA 47 -CCL5, a mutant that is crippled in its ability to bind GAGs and to form oligomeric structures, resulted in significantly reduced clinical severity in a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) [21]. Here, we investigated the potential therapeutic effect of P8A-CCL2, a point mutant that does not oligomerize but induces cell recruitment as effectively as wildtype (WT) CCL2 in vitro [22] yet is unable to recruit cells into the peritoneal cavity [20].…”

Manipulation of hydrogel assembly and growth factor delivery via the use of peptide–polysaccharide interactions

“…The stored hydrogels were equilibrated to room temperature prior to rheological characterization (typically several minutes). 1 H NMR spectra were acquired on a Bruker DRX-400 NMR spectrometer (Bruker Daltonics, Billerica, MA). DMSO-d6 or deuterium oxide was used as the NMR solvent and TMS or DSS as references, respectively.…”

“…In particular, glycosaminoglycans have been demonstrated to mediate a wide range of biological activities such as cell adhesion, cell mobility, cell proliferation and tissue morphogenesis via binding to various cell regulatory proteins such as the chemokines, growth factors, enzymes, enzyme inhibitors, and extracellular matrix proteins [1][2][3]. The well studied glycosaminoglycan, heparin, is a linear, unbranched, highly sulfated polysaccharide chain [4,5], and it is well accepted that the electrostatic interactions between the sulfates of the glycosaminoglycan and basic residues of a protein play an important role in binding [6].…”