Characterization of human leukocyte-HUVEC adhesion: Effect of cell preparation methods.
Sample manipulation to obtain isolated granulocytes represents a key, and often necessary, step in the in vitro studies. We investigated by the means of flow cytometry and microscopic techniques (both optical microscopy [OM] and scanning electron microscopy [SEM]), the granulocyte-endothelium adhesion and the role of sample manipulation.



By means of a co-culture method, we have analysed the adhesion of human leukocytes, originated from two different blood samples (fresh venous blood [FB] and buffy coat [BC]), to the human umbilical venous endothelial cell (HUVEC) monolayer. Cultured HUVEC were analysed for adhesion molecule expression by means of flow cytometry, while the morphological changes were evaluated by means of SEM. Cell adhesion was evaluated by means of flow cytometry and both OM and SEM.



HUVEC expressed under resting conditions the adhesion molecules ICAM-1, VCAM-1 and E-selectin and their expression was upregulated by stimulation with TNF-α (0.1-10ng/ml) as well as with LPS (1μg/ml). SEM analysis showed that stimulation with both stimuli profoundly affect cell morphology. Flow cytometric evaluation of cell adhesion showed that the ability of cells to adhere to HUVEC monolayer was quite different in the two preparations, with the lowest adhesion for FB in all the cell subsets analysed. Finally, isolated granulocytes were able to adhere to HUVEC monolayer more than cells identified in FB or BC and the adhesion was increased during activation of HUVEC with 10ng/ml of TNF-α.



Our data showed that cell manipulation necessary for the isolation of specific immune cells from whole blood profoundly affect the ability of these cells to adhere to the HUVEC monolayer although their functional properties remain unchanged.
Center of Research in Medical Pharmacology, University of Insubria, Varese, Italy. Electronic address: franca.marino@uninsubria.it.
Franca
F
Marino
Center of Research in Medical Pharmacology, University of Insubria, Varese, Italy.
Laura
L
Schembri
Center of Research in Medical Pharmacology, University of Insubria, Varese, Italy.
Emanuela
E
Rasini
Center of Research in Medical Pharmacology, University of Insubria, Varese, Italy.
Monica
M
Pinoli
Center of Research in Medical Pharmacology, University of Insubria, Varese, Italy.
Angela
A
Scanzano
Center of Research in Medical Pharmacology, University of Insubria, Varese, Italy.
Alessandra
A
Luini
Department of Morphological and Surgical Sciences, University of Insubria, Varese, Italy.
Terenzio
T
Congiu
Center of Research in Medical Pharmacology, University of Insubria, Varese, Italy.
Marco
M
Cosentino
D018821:CD18 Antigens / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D048430:Cell Shape; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D018920:Coculture Techniques; D019040:E-Selectin / Q000378:metabolism; D005434:Flow Cytometry; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008855:Microscopy, Electron, Scanning; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D010641:Phenotype; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D018821:CD18 Antigens; D015815:Cell Adhesion Molecules; D019040:E-Selectin; C573420:ICAM1 protein, human; D008070:Lipopolysaccharides; C546936:SELE protein, human; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1
Flow cytometry; Human umbilical venous endothelial cells; Leukocyte adhesion; Optical microscopy; Scanning electron microscopy