Endothelial production of MCP‐1: modulation by heparin and consequences for mononuclear cell activation

Abstract

SUMMARYHeparin is a polyanionic glycosaminoglycan (GAG) that can bind with high affinity to a range of cytokines including interferon-c (IFN-c) and members of the chemokine superfamily. This GAG also possesses immunomodulatory activity in vivo and can antagonize the capacity of IFN-c to induce class II MHC antigen expression, and to up-regulate intercellular adhesion molecule-1, by cultured endothelial cells. Previous studies have shown that binding to cell-surface heparan sulphate is essential for optimal activity of IFN-c and that free heparin competitively inhibits this sequestration process. The present study was performed to increase our understanding of the immunosuppressive activity of heparin by investigation of potential antagonism of the production and function of monocyte chemotactic peptide-1 (MCP-1), a chemokine important for mononuclear leucocyte recruitment across vascular endothelium. It was found that mixture of heparin with IFN-c inhibited up-regulation of the signal transducer and activator of transcription protein, STAT-1 produced normally by treatment of endothelial cells with IFN-c. An inhibition of MCP-1 production was observed that was specifically caused by mixture of IFN-c with heparin-like, and therefore cytokine-binding, GAGs. It was also shown that mixture of heparin-like GAGs with MCP-1 inhibited the rapid tyrosine phosphorylation of phosphatidylinositol 3-kinase which is normally produced by treatment of mononuclear leucocytes with this chemokine. Blockade of this intracellular signalling event was associated with a reduction in the normal transendothelial migration response towards MCP-1. Results from this study indicate that soluble, heparin-like GAGs can block IFN-c-dependent up-regulation of MCP-1 production by cultured endothelial cells, and can also antagonize the leucocyte-activating and migration-promoting properties of pre-existing MCP-1. These activities may contribute to the immunomodulatory properties of heparin. INTRODUCTIONand phagocyte-activating properties.1 A further effect of IFN-c is to augment the potential immunogenicity of a variety of Interferon-c (IFN-c) is an important proinflammatory cells, such as those of vascular endothelium.2 Specific effects cytokine, playing a central role in immune and inflammatory on endothelial cells include up-regulation of the expression of responses. This pleiotropic molecule is produced by activated adhesion molecules, such as intercellular adhesion molecule-1 CD8+ and Th1-type CD4+ T cells, and possesses anti-viral (ICAM-1), and class I major histocompatibility complex (MHC ) antigens3 and the induction of class II MHC antigens.

Examination of MCP-1 (CCL2) partitioning and presentation during transendothelial leukocyte migration

“…24. The cells recovered from the interface were washed twice with DMEM prior to being resuspended at a concentration of 2 Â 10 6 cells/ml.…”

“…Endothelial monolayers that had not been exposed to exogenous chemokine showed no significant expression of cell surface MCP-1 (data not shown); this is consistent with a previous study that demonstrated little MCP-1 production by resting EA.hy926 cells. 24 The antibodies used for the detection of heparan sulfate are specific for epitopes known to be important in MCP-1 binding: 10E4 is specific for molecules containing N-sulfated glucosamine, 43 while HepSS-1 binds to O-sulfated, N-acetylated glucosamine residues linked to glucuronic acid. 44 Reconstructed two-color X-Z sections allowed direct visualization of the distribution of both heparan sulfate and MCP-1 within endothelial monolayers after treatment for 60 min with basally applied chemokine (Figure 3b, c).…”

“…22 Lys 58 and His 66 are known to be critical to the interaction between MCP-1 and heparan sulfate, 23 and competition with heparin attenuates its chemotactic potential. 24 Transendothelial migration of leukocytes in response to chemokines is a multistep process mediated by a variety of proteins. 25,26 Initial rolling of the leukocyte along the endothelial surface, mediated by cell surface selectins, is upregulated to firm adhesion by the leukocyte integrin family and their receptors on the endothelial surface after exposure to GAG-immobilized chemokine.…”

“…Indeed, in static in vitro experiments, a soluble gradient does elicit the vectorial migration of a variety of purified leukocytes from areas of low to high protein concentration. 20,24,29 However, recent work by Cinamon et al 30 and others 31,32 has highlighted the importance of both shear flow and apical presentation of chemokine in the direction of diapedesis in model systems.…”

Chemokines and peripheral nerve demyelination

“…Monocyte chemoattractant protein (MCP)-1 production in experimental allergic neuritis MCP-1, a well characterized b-chemokine, is produced by many cell lineages including monocytes, macrophages, lymphocytes, ®broblasts, endothelia, epithelia (Struyf et al, 1998;Douglas et al, 1997), and astrocytes (Adamus et al, 1997;Glabinski et al, 1997;Sun et al, 1997). Its target cells are monocytes, T lymphocytes and NK cells, but not neutrophils (Gu et al, 1997;Schall, 1994;Vaddi et al, 1997) and while a role for MCP-1 in EAE has been proposed (Karpus, 1995) its potential role in experimental allergic neuritis (EAN) has yet to be fully explored.…”