Biochemical and functional characterization of glycosaminoglycans released from degranulating rat peritoneal mast cells: Insights into the physiological role of endogenous heparin.

The properties of commercially prepared heparin as an anticoagulant and antithrombotic agent in medicine are better understood than is the physiological role of heparin in its native form, where it is uniquely found in the secretory granules of mast cells. In the present study we have isolated and characterised the glycosaminoglycans (GAGs) released from degranulating rat peritoneal mast cells. Analysis of the GAGs by NMR spectroscopy showed the presence of both heparin and the galactosaminoglycan dermatan sulphate; heparinase digestion profiles and measurements of anticoagulant activity were consistent with this finding. The rat peritoneal mast cell GAGs significantly inhibited accumulation of leukocytes in the rat peritoneal cavity in response to IL-1ß (p < 0.05, n = 6/group), and inhibited adhesion and diapedesis of leukocytes in the inflamed rat cremasteric microcirculation in response to LPS (p < 0.001, n = 4/group). FTIR spectra of human umbilical vein endothelial cells (HUVECs) were altered by treatment of the cells with heparin degrading enzymes, and restored by the addition of exogenous heparin. In conclusion, we have shown that rat peritoneal mast cells contain a mixture of GAGs that possess anticoagulant and anti-inflammatory properties.

Locally available heparin modulates inflammatory cell recruitment in a manner independent of anticoagulant activity.

Heparin is known to possess anti-inflammatory properties that in many cases appear to be separable from its anticoagulant activity. Mast cells, located in tissue, are the sole source of endogenous heparin, which may be involved in control of the inflammatory response. The majority of studies of the effects of heparin on the inflammatory response, carried out to date, have involved systemic administration and the potential influence of heparin in the site of inflammation has been less clear. In the present study, the effects of locally administered heparin and a non-anticoagulant derivative were investigated on leucocyte accumulation in the inflamed peritoneal cavity and leucocyte-endothelial interactions in the mesenteric microcirculation of the rat. Heparin and non-anticoagulant heparin inhibited the influx of neutrophils to the site of inflammation, as well as leucocyte rolling and adhesion in post-capillary venules. These effects were apparent only while heparin was present in the peritoneal cavity and plasma and may reflect, in part, an action on resident peritoneal cells, as the heparins tested were found also to inhibit the expression of endothelial adhesion molecules in response to products of activated mononuclear cells, in vitro. Our data show that locally administered heparin has anti-inflammatory effects in an in vivo model of peritoneal inflammation whilst present at the site of inflammation. These non-anticoagulant properties may involve interaction with cells in the site of inflammation, in addition to inhibiting cell adhesion at the level of the microcirculation.

Interference with heparin binding and oligomerization creates a novel anti-inflammatory strategy targeting the chemokine system.

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.