Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single-cell level in whole blood.
BACKGROUND
The use of whole blood (WB) in studying lipopolysaccharide (LPS)-induced cellular activation preserves the milieu in which LPS-cell interaction occurs in vivo. However, little information is available on using such a system at a single-cell level. We evaluated LPS binding and cell activation in WB by using flow cytometry. The influence of heparin or EDTA as anticoagulants was also addressed.


METHODS
Blood was obtained from healthy donors in EDTA and/or heparin tubes. Biotinylated LPS (LPSb) was used to evaluate cell binding of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by adding streptavidin-allophycocyanin (APC). LPS-induced activation was evaluated by the expression of surface activation markers and by the detection of intracellular tumor necrosis factor-alpha (TNF-alpha).


RESULTS
LPSb bound promptly to monocytes in EDTA- and heparin-treated blood. In EDTA-treated blood, membrane-bound LPSb decreased after 60 min of incubation, whereas it remained detectable in heparinized blood during the 6 h of incubation. LPS induced TNF-alpha and enhanced the expression of HLA-DR in monocytes, as well as the expression of CD69 in T and B lymphocytes. Induction of both TNF-alpha in monocytes and CD69 in lymphocytes was more efficient in heparinized blood.


CONCLUSIONS
Detection of membrane-bound LPSb on monocytes differed in EDTA or heparin-treated blood, and cell activation was better obtained in heparinized blood.
Brunialti
Milena Karina Coló
MK
Division of Infectious Diseases, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
Kallás
Esper Georges
EG
Freudenberg
Marina
M
Galanos
Chris
C
Salomao
Reinaldo
R
D000925:Anticoagulants / Q000494:pharmacology; D015703:Antigens, CD / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D004306:Dose-Response Relationship, Immunologic; D004492:Edetic Acid / Q000494:pharmacology*; D005434:Flow Cytometry; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D037181:Lectins, C-Type; D008070:Lipopolysaccharides / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000187:drug effects*; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D012475:Salmonella / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D000099192:CD69 Antigens
D000925:Anticoagulants; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D004492:Edetic Acid; D006493:Heparin; D037181:Lectins, C-Type; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D000099192:CD69 Antigens