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Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Published on: August 23, 2013
Monosodium urate crystals promote neutrophil adhesion via a CD18-independent and selectin-independent mechanism
P H Reinhardt1, P H Naccache, P E Poubelle
1Department of Medical Physiology, University of Calgary, Canada.
Abstract:
The primary objective of this study was to determine whether monosodium urate (MSU) crystals induced neutrophil adhesion to cellular substrata and, if so, then to elucidate the molecular mechanisms involved. Human umbilical vein endothelial cells (HUVEC), as well as various other cellular substrata, were treated with various sized MSU crystals, washed, and then coincubated in the presence of neutrophils for 60 min. HUVEC exposed to MSU crystals but not to silica crystals or uric acid promoted neutrophil adhesion in a dose- and size-dependent manner, an event also observed with monolayers of rabbit synovial cells and rat intestinal epithelial cells. The increased neutrophil adhesion could not be attenuated by anti-CD18, anti-intracellular adhesion molecule-1, or various anti-selectin antibodies, despite the fact that scanning electron microscopy revealed that neutrophils were adhering primarily to the endothelial cells rather than to exposed crystals. CD18-deficient neutrophils adhered to MSU crystal-treated HUVEC as effectively as their CD18-positive counterparts. The neutrophil adhesion was temperature dependent but did not require protein synthesis. Additionally, HUVEC phagocytosis of crystals was necessary for subsequent neutrophil-endothelial cell interactions to transpire. Pretreatment of endothelial cells and neutrophils with colchicine significantly reduced the adhesive interaction. Our data demonstrate that exposure of endothelial and other cells to MSU crystals promotes neutrophil adhesion that occurs by a firm CD18-independent and selectin-independent adhesive mechanism.
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