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.

No abstract

1 The e ects of unfractionated heparin (UH) and a selectively O-desulphated derivative of heparin (ODSH), lacking anticoagulant activity, on the adhesion of human peripheral blood mononuclear cells (HPBMNC) to human stimulated umbilical vein endothelial cells (HUVECs), were investigated. 2 For comparison, the e ects of poly-L-glutamic acid (PGA), a large polyanionic molecule without sulphate groups and two di erent molecular weight sulphated dextrans (DS 5 k and DS 10 k) were studied. 3 UH (50 ± 1000 u ml 71 ) signi®cantly (P50.05) inhibited the adhesion of HPBMNC to HUVECs, stimulated with IL-1b (100 u ml 71 ), TNF-a (1000 u ml 71 ) or LPS (100 mg ml 71 ), when the drugs were added together with stimuli to HUVECs and coincubated for 6 h. Such e ects on adhesion occurred with limited in¯uence on expression of relevant endothelial adhesion molecules (ICAM-1 and VCAM-1). 4 UH (100 ± 1000 u ml 71 ), when added to prestimulated HUVECs, signi®cantly (P50.05) increased adhesion of mononuclear cells to endothelium at the higher concentrations tested, without any e ect on adhesion molecule expression. In contrast, the opposite e ect was observed when human polymorphonuclear leucocyte adhesion was examined, under the same experimental conditions, suggesting that the observed potentiation of HPBMNC adhesion is cell speci®c. 5 The e ects of UH on HPBMNC adhesion were shared by the non-anticoagulant ODSH (600 ± 6000 mg ml 71 ) but not by sulphated dextrans or PGA (300 ± 6000 mg ml 71 ). 6 Heparin a ects the adhesion of HPBMNC to stimulated endothelium, in both an inhibitory and potentiating manner, e ects which are unrelated to its anticoagulant activity and not solely dependent on molecular charge characteristics.