Sulphated disaccharides: Structure Activity relationship on TNFα Synthesis by Human Macrophages

Subashini Varadharajan1, Daniel Tilley1, Hilda Bellony1, Michael Burnet2, Michael Seed1. 1University of East London, London, UK, 2Synovo GmbH, Tubingen, Germany

Sulphated disaccharides SDS have been found to be released from tissue matrix by T-cell derived heparinase (1). We (2) and others (3) have suggested that they inhibit animal models of inflammatory arthritis and inhibit macrophage TNF synthesis through inhibition of differentiation (4). Incubation conditions are important to reveal effects, and responses often exhibit bell shaped curves making mechanistic studies difficult. Here we have investigated the time dependence of drug effects in relation to the induction of monocyte differentiation by phorbol myristate acetate (PMA) and assessed the need for a high degree of sulphation and explored elements of the disaccharide structure.

U937 cells were seeded (2x105cells/200μl/well) in DMEM 10%FCS, 1%Pen/Strep 95%O2/5%CO2. Drugs were given from 48 to 2 hours before PMA (8nM) addition for differentiation (PMA-U937 cells), or 2 hours prior to lps to PMA-U937 cells. Sucrose octasulphate (SOS, TC, Canada), HSDSIIIH and HDSI (Sigma) or diglucopyranosylamine octasulphate (DiGaS, Synovo GmbH) were administered to the wells in quadruplets for each condition and incubated for two hours at 37°C and 5% CO2. After differentiation, medium was replaced and PMA-U937 cells matured for 24 hours prior to the addition of lps (1μg/ml, 6hr) to induce TNFα synthesis (ELISA, eBioscience). After another 24 hours the LPS was administered to each well at 1μg/ml (20μl of 10μg/ml LPS into the 200μl in each well to give a final volume of 220μl).

PMA induced a concentration related differentiation of U937 cells to adherent cells by 72 hours (0.8nM 79.1±2.0%, 8nM 89.1±1.0%, 80nM toxic, n=3) that synthesised TNFα (0.8nM: 301±26.4, 8nM: 557.8±13.4pg/ml, n=4, 48 hours). Preincubation of PMA-U937 adherent cells with any SDS drug for 2 hours prior to LPS did not affect LPS induced TNFα synthesis.

Figure 1. Preincubation of U937 cells with SDS for 18 or 24 hours prior to PMA significantly inhibits lps induced TNFα synthesis.

SDS drugs inhibit the PMA induced lps TNFα competence of U937 cells, for SDS containing 8 (SOS, DiGaS), 2 (HDSIII-H) but not 1 sulphate (HDS-1S, NS). Activity remains after the exchange of a glucosyl with the furanyl of sucrose. Bell shaped curves are seen with DiGaS and HDS-IS. Length of preincubation has no relevance, 2 hours (data not shown & ref 4) being sufficient, except for HDSIII-S which was ineffective.

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