Emigration of leukocytes at sites of inflammation is initiated by the selectin family of carbohydrate-binding adhesion molecules. Molecular crossbridges initiate rolling of cells along the vascular endothelium where chemokines such as IL-8 and platelet activating factor (PAF) may be presented to their receptors on the leukocyte surface resulting in cell stimulation. Integrin activation appears to be a requirement for subsequent cell localization and diapedesis into the tissue. Several recent reports have demonstrated that ligation and cross-linking of neutrophil L-selectin results in neutrophil activation, including intracellular calcium release, superoxide production, and induction of mRNA for production of IL-8 and TNF-α. The purpose of this study was to examine whether ligation and cross-linking of L- selectin would specifically result in activation of β2-integrin-dependent adhesion. A fluorescence flow cytometric assay was developed that directly measures Mac-1-dependent cell adhesion. Fluorescent latex beads (2-μm diameter) were adsorbed with albumin or fibrinogen and added in excess to human neutrophils in a shear-stirred suspension. Following stimulation the kinetics of bead capture by neutrophils was continuously measured in real time on the flow cytometer. The onset of bead binding was detected in the presence of extremely low concentrations of PAF (10 pM) or formyl peptide (0.2 nM) stimulation. Ligation of L-selectin with whole IgG DREG200 or DREG56 Ab, but not controls (anti-CD44, -CD45, -CD11a), resulted in a significant potentiation of bead binding. Cross-linking F(ab')2 fragments of DREG200 with a goat anti-mouse F(ab')2 secondary Ab also stimulated β2-integrin- dependent adhesion in a dose-dependent fashion. A chimeric form of DREG200 expressing γ4 or γ1 isotypes of human Fc domain also stimulated cell adhesion when cross-linked. Surface expression of CD18 and an activation- dependent epitope, as detected with mAb24, also increased in response to L- selectin cross-linking. Cross-linking L-selectin induced significant adhesion and transmigration of neutrophils across human umbilical vein endothelial cells. We propose that cross-linking of L-selectin results in a cell signal that directly stimulates β2-integrin adhesive responses.
|Original language||English (US)|
|Number of pages||13|
|Journal||Journal of Immunology|
|State||Published - 1995|
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