Scanu, Anna (2008) Il ruolo delle HDL nel controllo dell'infiammazione sinoviale. [Ph.D. thesis]
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Recent reports suggest that apolipoproteins (apo) exert an important role in controlling inflammation. It has been demonstrated that high density lipoprotein (HDL) are able to block the contact-mediated activation of monocytes-macrophages by stimulated T lymphocytes and inhibit the production of IL-1ß and TNF?.
Aim of the thesis: To investigate the effects of HDL on MCP-1 release from monosodium urate crystals-stimulated synoviocytes and IL-1ß, TNF? and IL-1Ra release from microparticles-stimulated monocytes.
Methods: Human synoviocytes were obtained by synovial tissue explants from patients with osteoarthritis and stimulated with monosodium urate (MSU) crystals (0.01-0.25 mg/ml) in the presence or absence of human HDL (50 e 100 ?g/ml).
Microparticles (MP) were isolated by ultracentrifugation from T lymphocytes and HUT-78 cultures stimulated with phorbol myristate acetate (PMA) (5 ng/ml) and phytohemagglutinin (PHA) (1 ?g/ml) for 48 and 6 h respectively. The cellular origin of MP was determined by flow cytometry. Human monocytes were activated for 48 h by MP from T lymphocytes at concentrations of 13.3 ?g/ml and 26.6 ?g/ml. HUT-78-derived MP were used at a concentration of 1.5-6 ?g/ml in the presence or absence of human HDL (0.2 mg/ml).
HDL were isolated from peripheral blood of healthy volunteers by ultracentrifugation.
MCP-1 was determined in cultured cells by western blotting and confocal microscopy, while the production of IL-1ß, TNF? and IL-1Ra was measured in culture supernatants by ELISA.
Results: Confocal microscopy and western blotting analysis revealed that MCP-1 resides in small cytoplasmatic granules on non stimulated cells. The exposure of synoviocytes to MSU crystals leads to a decrease of intracellular levels of the protein and an increase of extracellular chemokine concentration. The treatment of synoviocytes with HDL causes a dose-dependent inhibition of the release of MCP-1 which maintains its storage in granules. The same effect was observed pre-incubating cells with HDL 1 h before crystal activation.
MP generated by stimulated T cells induce a production of IL-1ß, TNF? and IL-1Ra in monocyte cultures higher than those obtained by MP from unstimulated T lymphocytes. It has also been observed that monocytes stimulated with MP generated by activated HUT-78 release IL-1ß, TNF? and IL-1Ra in a dose-dependent manner. The treatment with HDL inhibits IL-1ß and TNF? levels, whereas the production of IL-1Ra remains unchanged.
Conclusion: The inhibitory activity of HDL highlighted by the pre-treatment of cells is probably due to a direct action of lipoproteins on synoviocytes rather than to their adsorption on the surface of the crystals. By inhibiting MCP-1 release, HDL may limit the inflammatory process.
The production of cytokines depends on the activation level of cells from which MP take origin.
The almost complete inhibition of IL-1ß and TNF? levels by HDL lead us to hypothesize that HDL control cellular contact between MP and monocytes, as already observed in the lymphocytes T - monocytes interaction.
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