Influence of EDTA and heparin on lipopolysaccharide binding, internalization, and cell activation, evaluated at single-cell level in whole blood
© The Author(s) 2001
Published: 26 June 2001
The use of whole blood (WB) for studying the LPS-induced cellular activation preserves the milieu in which LPS-cell interaction occurs in vivo. However, information at single-cell level using this system is lacking. In this study we evaluated the LPS-binding, internalization, and cell activation, in WB, using flow cytometry. The influence of heparin or EDTA as anticoagulant was also addressed.
Blood samples were obtained from healthy donors in EDTA and/or heparin tubes. Biotinilated LPS (LPSb) was used to evaluate cell binding and internalization of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by the addition of streptavidin-red 670 or -APC. LPS-induced cell activation was evaluated by expression of surface activation markers and detection of intracellular TNF-α.
LPSb bound promptly to monocytes. In EDTA-treated blood membrane-bound LPSb decreased after 60 min of incubation, reaching background levels after 240 min. In contrast, membrane-bound LPSb remained detectable in heparinized blood in a high proportion of the cells. LPS induced TNF-α and enhanced the expression of HLA-DR in monocytes, and induced the expression of CD69 in Tand Blymphocytes. Induction of TNF-α in monocytes and, to a lesser degree of CD69 in lymphocytes, was more efficient in heparinized-blood.
LPS binding was not influenced by anticoagulants, while internalization seems to occur at earlier stages with EDTA. Cellular activation was better obtained with heparin.