Agonistic anti-ICAM-1 antibodies in scleroderma: activation of endothelial pro-inflammatory cascades.

BACKGROUND: Scleroderma (SSc) is a complex autoimmune disorder that can be characterised by the presence 2of circulating autoantibodies to nuclear, cytoplasmic and cell surface antigens. In particular antibodies directed against endothelial cell antigens (anti-endothelial cell antibodies; AECA) have been detected. ICAM-1 is an adhesion molecule expressed on the surface of human endothelial cells. We have previously shown that cross-linking ICAM-1 with monoclonal antibodies leads to pro-inflammatory activation of human endothelial and vascular smooth muscle cells and that cardiac transplant recipients with transplant associated vasculopathy make antibodies directed against ICAM-1. OBJECTIVES: To determine whether SSc patients make antibodies directed against ICAM-1 and whether these antibodies induce pro-inflammatory activation of human endothelial cells in vitro. METHODS: Using recombinant ICAM-1 as capture antigen, an ELISA was developed to measure ICAM-1 antibodies in sera from SSc patients. Antibodies were purified using ICAM-1 micro-affinity columns. HUVEC were incubated with purified anti-ICAM-1 antibodies and generation of reactive oxygen species, and expression of VCAM-1 was measured. RESULTS: Significantly elevated levels of anti-ICAM-1 antibodies were detected in patients with diffuse (dSSc; 10/31 32%) or limited (lSSc; 14/36 39%) scleroderma. Cross-linking of HUVEC with purified anti-ICAM-1 antibodies caused a significant increase in ROS production (2.471±0.408 fold increase above untreated after 150 min p<0.001), and significant increase in VCAM-1 expression (10.6±1.77% vs 4.12±1.33%, p<0.01). CONCLUSION: AECA from SSc patients target specific endothelial antigens including ICAM-1, and cause pro-inflammatory activation of human endothelial cells, suggesting that they are not only a marker of disease but that they contribute to its progression.

Microparticle formation after exposure of blood to activated endothelium under flow.

Increased numbers of circulating microparticles (MPs) are indicative of poor clinical outcome in a number of inflammatory disorders, including atherosclerosis. Platelets and megakaryocytes are a major source of MP and are identified by presence of CD42b on the MP surface. MP shed from activated platelets can be identified by presence of P-selectin (CD62P). Tissue factor (TF) is the principal initiator of blood coagulation and its activity has been identified in MPs derived from patient plasma, which may contribute to thrombosis. Here, we have investigated by flow cytometry the expression of TF and CD62P on MP after exposure of diluted whole blood to TNF-activated endothelial cells (EC) both under static conditions and in our newly established model of flow. MPs were significantly increased in blood subjected to flow and this was further enhanced after exposure of blood to TNF-activated EC. MP surface expression of CD62P or TF was upregulated following exposure to TNF-activated EC under flow compared with flow with nonactivated EC or after static coculture with and without prior EC activation. These data strongly suggest that interactions of blood with inflamed EC can modulate production of CD62P and TF bearing MP under flow conditions, and thus may contribute to a prothrombotic environment.

Expression of blood coagulation factors on monocytes after exposure to TNF-treated endothelium in a novel whole blood model of arterial flow.

Activated blood monocytes are a major source of tissue factor (TF), the principal initiator of blood coagulation. TF can be shed from the monocyte surface in association with microparticles (MPs) and increased numbers of circulating MPs are indicative of poor clinical outcome in a number of inflammatory disorders, including atherosclerosis. The mechanisms coupling inflammation with aberrant TF production/activity remain obscure but the protease-activated receptor (PAR) family has been implicated. We have previously shown (i) that freshly isolated human monocytes express low levels of cell surface PAR-2, (ii) that cell surface PAR-2 is rapidly upregulated from intracellular stores following mechanical stimulation, and (iii) that PAR-2 stimulation results in elevation of intracellular calcium and cytokine release. Here, we have investigated the expression of PAR-2 on monocytes exposed to TNF-activated endothelial cells both under static conditions and in our newly-established model of arterial flow, using diluted whole blood. Monocyte surface PAR-2 expression was upregulated following static exposure to activated EC and with laminar (atheroprotective) arterial flow there was a further increase in monocyte PAR-2 expression. We have also shown under arterial flow conditions that exposure to TNF-stimulated EC resulted in a significant increase in expression of TF on monocytes compared to that on cells exposed to quiescent EC. These data strongly suggest that direct or indirect interactions with inflamed EC can modulate expression of PAR-2 and TF on the monocyte cell surface.