Granulocyte colony-stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neutrophils

The exact mechanism of apoptosis in neutrophils (PMNs) and the explanation for the antiapoptotic effect of granulocyte colony-stimulating factor (G-CSF) in PMNs are unclear. Using specific fluorescent mitochondrial staining, immunofluorescent confocal microscopy, Western blotting, and flow cytometry, this study found that PMNs possess an unexpectedly large number of mitochondria, which are involved in apoptosis. Spontaneous PMN apoptosis was associated with translocation of the Bcl-2-like protein Bax to the mitochondria and subsequent caspase-3 activation, but not with changes in the expression of Bax. G-CSF delayed PMN apoptosis and prevented both associated events. These G-CSF effects were inhibited by cycloheximide. The general caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) prevented caspase-3 activation and apoptosis In PMNs, but not Bax redistribution. PMN-derived cytoplasts, which lack a nucleus, granules, and mitochondria, spontaneously underwent caspase-3 activation and apoptosis (phosphatidylserine exposure), without Bax redistribution. zVAD-fmk inhibited both caspase-3 activation and phosphatidylserine exposure in cultured cytoplasts. Yet, G-CSF prevented neither caspase-3 activation nor apoptosis In cytoplasts, confirming the need for protein synthesis in the G-CSIF effects. These data demonstrate that (at least) 2 routes regulate PMN apoptosis: one via Bax-to-mitochondria translocation and a second mitochondria-independent pathway, both linked to caspase-3 activation. Moreover, G-CSF exerts its antiapoptotic effect In the first, that Is, mitochondria-dependent, route and has no Impact on the second. (Blood. 2002;99:672-679) (C) 2002 by The American Society of Hematolog

Correction: Platelet-independent adhesion of calcium-loaded erythrocytes to von Willebrand factor.

[This corrects the article DOI: 10.1371/journal.pone.0173077.]

Correction: Platelet-independent adhesion of calcium-loaded erythrocytes to von Willebrand factor

[This corrects the article DOI: 10.1371/journal.pone.0173077.]

Filamin B Mediates ICAM-1-driven Leukocyte Transendothelial Migration

During inflammation, the endothelium mediates rolling and firm adhesion of activated leukocytes. Integrin-mediated adhesion to endothelial ligands of the Ig-superfamily induces intracellular signaling in endothelial cells, which promotes leukocyte transendothelial migration. We identified the actin cross-linking molecule filamin B as a novel binding partner for intracellular adhesion molecule-1 (ICAM-1). Immune precipitation as well as laser scanning confocal microscopy confirmed the specific interaction and co-localization of endogenous filamin B with ICAM-1. Importantly, clustering of ICAM-1 promotes the ICAM-1-filamin B interaction. To investigate the functional consequences of filamin B binding to ICAM-1, we used small interfering RNA to reduce filamin B expression in ICAM-1-GFP expressing HeLa cells. We found that filamin B is required for the lateral mobility of ICAM-1 and for ICAM-1-induced transmigration of leukocytes. Reducing filamin B expression in primary human endothelial cells resulted in reduced recruitment of ICAM-1 to endothelial docking structures, reduced firm adhesion of the leukocytes to the endothelium, and inhibition of transendothelial migration. In conclusion, this study identifies filamin B as a molecular linker that mediates ICAM-1-driven transendothelial migratio

Reactive oxygen species mediate Rac-induced loss of cell-cell adhesion in primary human endothelial cells

The integrity of the endothelium is dependent on cell-cell adhesion, which is mediated by vascular-endothelial (VE)-cadherin. Proper VE-cadherin-mediated homotypic adhesion is, in turn, dependent on the connection between VE-cadherin and the cortical actin cytoskeleton. Rho-like small GTPases are key molecular switches that control cytoskeletal dynamics and cadherin function in epithelial as well as endothelial cells. We show here that a cell-penetrating, constitutively active form of Rac (Tat-RacV12) induces a rapid loss of VE-cadherin-mediated cell-cell adhesion in endothelial cells from primary human umbilical veins (pHUVEC). This effect is accompanied by the formation of actin stress fibers and is dependent on Rho activity. However, transduction of pHUVEC with Tat-RhoV14, which induces pronounced stress fiber and focal adhesion formation, did not result in a redistribution of VE-cadherin or an overall loss of cell-cell adhesion. In line with this observation, endothelial permeability was more efficiently increased by Tat-RacV12 than by Tat-RhoV14. The loss of cell-cell adhesion, which is induced by Tat-RacV12, occurred in parallel to and was dependent upon the intracellular production of reactive oxygen species (ROS). Moreover, Tat-RacV12 induced an increase in tyrosine phosphorylation of a component the VE-cadherin-catenin complex, which was identified as alpha-catenin. The functional relevance of this signaling pathway was further underscored by the observation that endothelial cell migration, which requires a transient reduction of cell-cell adhesion, was blocked when signaling through ROS was inhibited. In conclusion, Rac-mediated production of ROS represents a previously unrecognized means of regulating VE-cadherin function and may play an important role in the (patho)physiology associated with inflammation and endothelial damage as well as with endothelial cell migration and angiogenesi

Blood cell counts measured from the washed erythrocyte concentrates used in Fig 1C and 1D.

<p>Blood cell counts measured from the washed erythrocyte concentrates used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.g001" target="_blank">Fig 1C and 1D</a>.</p

Erythrocytes bind VWF through a platelet-independent mechanism.

<p>(A) Ionomycin- (1 μM) treated (1 hour) erythrocytes, mixed with platelets and perfused (10 minutes) with histamine (100 μM) over HUVECs. An example of an adherent platelet is marked by (*) and an adherent erythrocyte is marked by an arrowhead. (B) Ionomycin-treated erythrocytes labeled with anti-CD235a-FITC (green), mixed with anti-CD42b-APC-labeled platelets (magenta) and Alexa Fluor^®568-labelled anti-VWF antibody (red) were perfused with histamine over HUVECs. Adherent erythrocytes were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.g001" target="_blank">Fig 1</a>. Values are means ± SD (n = 3; Each n represents single independent experiments using cells from different donors). Statistical analysis was performed using a 1-way ANOVA in which all means were compared with 0 platelets ml^-1 followed by Bonferroni's post-hoc test. (C) Erythrocytes were purified by FACS. The erythrocyte population based on cell size (i) was positive sorted for CD235a-FITC and negative sorted for CD42b-APC (ii). Purified erythrocytes (10⁷ cells/ml) treated (1 hour) with ionomycin (1 μM) (green), mixed with Alexa Fluor^®568-labelled anti-VWF antibody (red) were perfused with histamine (100 μM) over HUVECs. (iii) The dashed box corresponds to the zoomed region. Scale bars represent 50 μm.</p