G-CSF regulates hematopoietic stem cell activity, in part, through activation of Toll-like receptor signaling.

Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte colony-stimulating factor (G-CSF) is often induced with infection and has a key role in the stress granulopoiesis response. However, its effects on HSCs are less clear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect associated with induction of Toll-like receptor (TLR) expression and signaling. The G-CSF-mediated expansion of HSCs is reduced in mice lacking TLR2, TLR4 or the TLR signaling adaptor MyD88. Induction of HSC quiescence is abrogated in mice lacking MyD88 or in mice treated with antibiotics to suppress intestinal flora. Finally, loss of TLR4 or germ-free conditions mitigates the G-CSF-mediated HSC repopulating defect. These data suggest that low-level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling.

Preferential sites for stationary adhesion of neutrophils to cytokine-stimulated HUVEC under flow conditions.

Neutrophils form CD18-dependent adhesions to endothelial cells at sites of inflammation. This phenomenon was investigated under conditions of flow in vitro using isolated human neutrophils and monolayers of HUVEC. The efficiency of conversion of neutrophil rolling to stable adhesion in this model was >95%. Neither anti-CD11a nor anti-CD11b antibodies significantly altered the extent of this conversion, but a combination of both antibodies inhibited the arrest of rolling neutrophils by >95%. The efficiency of transendothelial migration of arrested neutrophils was >90%, and the site of transmigration was typically <6 microm from the site of stationary adhesion. Approximately 70% of transmigrating neutrophils migrated at tricellular corners between three adjacent endothelial cells. A model of neutrophils randomly distributed on endothelium predicted a significantly greater migration distance to these preferred sites of transmigration, but a model of neutrophils adhering to endothelial borders is consistent with observed distances. It appears that stable adhesions form very near tricellular corners.

Neutrophil CD18-dependent arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow can be activated through L-selectin.

Neutrophil emigration through endothelial cells under shear flow involves several adhesion processes including cell rolling, arrest, and transmigration. Rolling is mediated by selectins, while arrest and transmigration both require activated CD18 integrins. One mode of CD18 activation is via selectins expressed on neutrophils and endothelial cells. We have recently reported that cross-linking of L-selectin (CD62L) resulted in the rapid activation of CD18-dependent adhesion. In the current study, we examine whether binding of E-selectin (CD62E) and L-selectin can activate neutrophil CD18-dependent adhesion under shear flow. Human ICAM-1 (CD54) and E-selectin were co-transfected into L cells. Neutrophil capture, rolling, and arrest on these monolayers were quantitated in a parallel plate flow chamber at a wall shear stress of 2.0 dyne/cm2. Under these conditions, E-selectin supported cell capture and rolling on the monolayer, but did not trigger CD18-mediated cell arrest within 200 microm of rolling. However, when neutrophils were treated with anti-L-selectin mAb and cross-linked with a secondary mAb, approximately 50% of the cells arrested within 54 microm. Cell arrest was also observed in response to IL-8 stimulation. A subthreshold level of IL-8 in combination with L-selectin cross-linking potentiated the level of cell arrest due to either stimulus alone. The transition to cell arrest involved both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Blocking either subunit alone failed to reduce arrest, while blocking both molecules with mAbs reduced the number to baseline levels. These data support the conclusion that L-selectin, but not E-selectin, can signal the transition from neutrophil rolling to cell arrest under shear flow.

L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) beta 2-integrin.

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-alpha. The purpose of this study was to examine whether ligation and cross-linking of L-selectin would specifically result in activation of beta 2-integrin-dependent adhesion. A fluorescence flow cytometric assay was developed that directly measures Mac-1-dependent cell adhesion. Fluorescent latex beads (2-microns 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 beta 2-integrin-dependent adhesion in a dose-dependent fashion. A chimeric form of DREG200 expressing gamma 4 or gamma 1 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 beta 2-integrin adhesive responses.