Sulphated disaccharides inhibit the differentiation of U937 cells in response to phorbol myristate acetate to the adherent phenotype.

Sulphated disaccharides (SDS) are released from heparin by heparinises during inflammation (Cahalon et al., Lider et al, 1995). These molecules do not possess anti thrombotic activity, being too short to bind effectively to anti-thrombin-III. They do however have immunomodulatory activity, such as inhibiting T-cell proliferation (Lider et al., 1995, Isaacs et al., 2013), and models of arthritis (Cahalon et al 1997., Jones et al., 2008). Last year we reported that some SDS inhibit the development of TNFα synthetic capability of PMA differentiated U937 cells. Here we investigate whether this is due to the inhibition of U937 differentiation to adherent macrophages.

U937 human monocytic cells were grown to confluence in RPMI 1640 and seeded into 96 well or 24 well plates at 107 cells/ml. U937 cells were incubated with PMA at 0.8, 8.0 or 80nM for periods of time to 72 hours. Non-adherent cells were washed away, and the residual adherent cells incubated with Trypsin/EDTA, aspirated, and counted with a haemocytomoeter. Flat, granular, viable cells (assessed by Trypan Blue exclusion) were then counted. Once a concentration of PMA and time of incubation was determined, cells were incubated with either sucrose octasulphate (SOS, Chemika, Germany), diglucopyranosylamine octa-sulphate (diGaS, synthesised Dr M Burnet, Synovo GmbH), heparin disaccharide-IIIH (HDS-IIIH, Sigma Aldrich) or heparin disaccharide-IS (HDS-IS, Segma Aldrich) for two hours and PMA added for 72 hours. Differentiation into adherent cells was assayed as before.

U937 cells were differentiated with 8nM PMA for 72 hours as previously reported by us. 96 well plate data elicited erratic results. Incubation of U927 cells in 24-well plates elicited reproducible findings.

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

SOS, DOS and HDS-IIIH exhibited bell shaped inhibition curves characteristic of these chemicals, with a maximal inhibition seen at 1011M. HDS-IS elicited a different shape, with inhibition of U937 cell differentiation reaching a maximum at 100µM. This indicates that a single sulphate on each sugar is required for potent activity, as a single sulphate group as found with HDS-IS results in a great reduction in activity.

SDS with a minimum of one sulphate per sugar inhibit PMA induced differentiation of U937 cells to adherent macrophage like cells. This explains the inhibition of the development of TNFα synthetic competence we have previously reported (Isaacs et al., 2013).

Refs:

Cahalon et al. 1997 Int Immunol 9: 1517-1522;

Isaacs et al. pA2 online (2013) 1(3): 204P;

Jones MR et al, Inflammation Res 57(S2): S105, 2008;

Lider O et al, 1995 PNAS 92: 5037-5041.