Cytokine gene expression and production in human LPS-stimulated mononuclear cells are inhibited by sulfated heparin-like semi-synthetic derivatives

Abstract number: P1250

Gori^* A., Attanasio^* M., Gazzini^* A., Giusti^* B., Lucarini^* L., Miletti† S., Chini† J., Manoni† M., Abbate^* R., Gensini^* G. F.

†INALCO RSM SpA Research Center, Pistoia, Italy ^*University of Florence, Italy;

The K5 polysaccharide obtained from E. coli strain 010:K5:H4 is a polymer of the disaccharidic unit formed by D-glucuronic acid and N-acetylglucosamine linked by a a-1-[-4)-b Glc-(1,4)-a GlcNAc-(1-] bond. This structure is alike to N-acetylheparosan, the precursory polymer of heparin and of heparan sulfate. It has been demonstrated that both UFH and LMWHs are able to inhibit cytokine production and expression by monocytes. Aim of this study was to evaluate the effects of 4 different heparin-like semi-synthetic derivatives (HSDs) on inflammatory cytokine production and expression by human mononuclear cells. Mononuclear cells of 10 healthy male subjects were stimulated with LPS and incubated with 4 HSDs (5 or 10 µg mL^-1) at 37 °C at 0, 5 and 10 µg mL^-1 for 4 h: 1-O-sulfated K5 polysaccharide (K5OS), 2-N,O-sulfated K5 polysaccharide (K5NS, OS), 3-Epimerized N sulfated K5 polysaccharides (K5NS epi) and 4-Epimerized N and O sulfated K5 polysaccharides (K5NS,OS epi). After incubation mononuclear cells were centrifuged and in the supernatant IL-1b IL-6 and TNF-a levels were determined by using ELISA methods. Total RNA was extracted using a commercial kit. TNF-a mRNA expression was determined by real time PCR. The addition of LPS and the K5 polysaccharide, did not inhibit IL-1b, IL-6 and TNF-a production by stimulated mononuclear cells. IL-1b, IL-6 and TNF-a concentrations in supernatants of LPS-stimulated mononuclear cells were not significantly influenced by the addition of K5 NS,OS and K5 NS epi at 5 and 10 µg mL^-1. Whereas, the addition of K5 NS, OS epi (5 and 10 µg mL^-1) and K5 OS (5 and 10 µg mL^-1) to LPS-stimulated cell caused a significant dose-dependent inhibition of IL-1b (K5 NS, OS epi: 26.1% and 40.0%; K5 OS: 17.4% and 34.7%), IL-6 (K5 NS, OS epi: 21.9% and 34.7%; K5 OS: 17.8% and 28.2%) and TNF-a (K5 NS, OS epi: 21.8% and 35.2%; K5 OS: 17.8% and 31.4%). The presence of both K5 OS and K5 NS, OS epi at 5 and 10 µg mL^-1 in cell suspension decreased TNF-a mRNA expression by LPS-stimulated cells with an inhibition of 46.1% (10 µg mL^-1), 32.2% (5 µg mL^-1) and 74.1 (10 µg mL^-1), 41.3% (5 µg mL^-1), respectively. These results indicate that sulfated heparin-like semi-synthetic derivatives and in particular the O-sulfated, N-acetylated K5 and O-sulfated, N-sulfated and epimerized K5 are able to inhibit both the expression and the production of pro-inflammatory cytokines, suggesting a potential role for these products as modulators of inflammatory reactions.