The sulfation level of glycosaminoglycans in monocytes and macrophages

Sulfation of glycosaminoglycans (GAGs) of components of extracellular matrix and cell surface is an important regulatory mechanism in inflammatory immune response. All sulfotransferases use 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a common sulfate donor that is produced by PAPS synthase.

In order to detect the sulfation level of monocytes/macrophages we established a CD44-hyaluronan binding assay based on flow cytometry. Monocytes were isolated from human peripheral blood of healthy volunteers. Macrophages were cultivated from monocytes at 37 °C in the presence of 100 U/ml granulocytes/macrophages – colony stimulating factor (GM-CSF). CD44 is a cell-surface receptor that binds via heparan sulfate side chains hyaluronan, a glycosaminoglycan that is released from immune cells at inflammatory sites. Using flow cytometry approaches, the expression of CD44 was followed by antibodies against CD44, while the ability of CD44 to bind hyaluronan was assessed with FITC-labelled hyaluronan.

The additional use of a Blyscan^TM dye binding assay was used to detect the sulfation level of glycosaminoglycans on the whole cell surface. To reduce stimulation of the cells by adherence, teflon-coated cell culture dishes were used. In order to simulate pro-inflammatory conditions monocytes were stimulated with TNF-α.

TNF-α increased the expression of CD44 on cells similar to macrophages, but shows no significant alterations of CD44 expression in monocytes. TNF-α has no significant influence concerning hyaluronan binding on monocytes. The inhibitor of sulfation reactions chlorate diminished the binding of hyaluronan in a concentration-dependent manner in both TNF-α stimulated and non-stimulated cells. This effect occurs in adherent and in non-adherent cells.

This assay is a convenient tool to investigate effects on sulfation after the treatment with anti-inflammatory agents.