Effects of fibronectin and collagen on cytokine production by ECFCs.

<p>ECFCs expanded on collagen produced higher levels of IL-6 and IL-8 that ECFCs expanded on fibronectin. (A) Representative curves showing that the concentration of IL-6 and IL-8 in the supernatants of ECFCs expanded on fibronectin (white circles) and collagen (gray circles) tended to increase with increasing passages. (B) ECFCs expanded on collagen (gray bars) reached higher levels of IL-6 and IL-8, but not bFGF, than ECFCs cultured on fibronectin (white bars). The mean ± SEM of the highest cytokine concentration reached during culture of the same 14 ECFC colonies reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066734#pone-0066734-g002" target="_blank">Figure 2</a> is shown. p values calculated by the Wilcoxon signed-rank test.</p

Effects of IL-6 and IL-8 on ECFC proliferation.

<p>IL-6 and IL-8 may account, at least in part, for the higher cell proliferation of ECFCs cultured on collagen as compared to fibronectin. Cell proliferation was assessed by the colorimetric CV assay, and expressed as OD. (A) Addition of IL-6 or IL-8 to ECFCs cultured on fibronectin increased cell proliferation in a dose-dependent manner. The mean ± SEM of 7 ECFC cultures is shown. *p = 0.046 and **p = 0.009 compared with untreated ECFCs cultured on fibronectin, as calculated by the Wilcoxon signed-rank test. (B) Addition of IL-6 and IL-8 together in the same culture did not further increase cell proliferation. One representative of three experiments is shown. (C) Addition of neutralizing anti-IL-6 or anti-IL-8 mAb to ECFCs cultured on collagen induced a dose-dependent reduction of cell proliferation. One representative of three experiments is shown.</p

Effects of fibronectin and collagen on isolation of ECFC colonies.

<p>Fibronectin promoted the appearance of ECFC colonies earlier than collagen. (A) PBMCs from 9 donors were seeded on fibronectin-coated plates and PBMCs from other 9 donors were seeded on collagen-coated plates; ECFC colonies from PBMCs seeded on fibronectin (white bar) appeared earlier than those seeded on collagen (gray bar). p value calculated by the Mann-Whitney U-test. (B) PBMCs from 8 donors were divided into portions, with half of them seeded on fibronectin-coated plates and the rest seeded on collagen-coated plates; also in these paired experiments ECFC colonies from PBMCs seeded on fibronectin (white circles) appeared earlier than those seeded on collagen (gray circles). p value calculated by the Wilcoxon signed-rank test.</p

Effects of fibronectin and collagen on the ability of ECFCs for in vitro tubulogenesis.

<p>ECFCs expanded on fibronectin or collagen showed a similar ability to form capillary-like structures in vitro. Independently from the substrate used for cell culture, ECFCs cultured in Matrigel gave rise within 12 hours of incubation to vascular structures. Representative phase-contrast photographs showing that the capillary-like structures formed by ECFCs cultured on fibronectin and collagen were similar. Photographs were obtained using an Olympus inverted microscope IX51, x4 magnification.</p

Effects of fibronectin and collagen on the immunophenotype of ECFCs.

<p>ECFCs expanded on fibronectin or collagen showed a similar immunophenotype. On the left are reported flow cytometric histograms showing that, independently from the substrate used for cell culture, ECFCs did not express CD45 and CD14, while they expressed at similar levels CD31, VEGFR2, CD144, CD146, CD54 and CXCR4. On the right are reported immunofluorescence photographs showing that ECFCs expanded on both substrates incorporated Dil-ac-LDL and bound lectin UEA-1. Photographs were obtained using an an Olympus Fluoview FV1000 confocal microscope, x40 magnification. One of three representative experiments is reported.</p

Effects of fibronectin and collagen on ECFC cell expansion.

<p>Collagen sustained cell expansion of ECFCs more efficiently than fibronectin. 14 ECFC colonies were divided into portions, with half of them seeded on fibronectin-coated plates and the rest seeded on collagen-coated plates. (A) Representative phase-contrast photographs showing that ECFCs expanded on fibronectin and collagen formed similar cobblestone-like monolayers with endothelial-like morphology. Photographs were obtained using an Olympus inverted microscope IX51, x10 magnification. ECFCs cultured on collagen (gray bar) compared with fibronectin (white bar) achieved (B) higher number of passages, (C) longer lifespan and (D) higher cell yields. p values calculated by the Wilcoxon signed-rank test.</p

<i>Zymosan</i>-induced production of TNFα and IL-1α:masking of Siglec-7.

<p>Statistical histogram bar graph showing the percentages of CD14^pos monocyte producing TNF-α (left) and IL-1α (right) either in the absence (gray bars) or in the presence (white bars) of <i>Zymosan</i> and in presence of <i>Zymosan</i> cultured with blocking anti-Siglec-7 Abs (black bars). The intracellular production of TNF-α and IL-1α were evaluated by flow cytometric analysis. Data are representative of 5 independent experiments performed in triplicates (± SD).</p

Engagement of Siglec-7 Receptor Induces a Pro-Inflammatory Response Selectively in Monocytes

<div><p>Sialic acid binding immunoglobulin-like lectin-7 (Siglec-7) is a trans-membrane receptor carrying immunoreceptor tyrosine based inhibitory motifs (ITIMs) and delivering inhibitory signals upon ligation with sialylated glycans. This inhibitory function can be also targeted by several pathogens that have evolved to express sialic acids on their surface to escape host immune responses. Here, we demonstrate that cross-linking of Siglec-7 by a specific monoclonal antibody (mAb) induces a remarkably high production of IL-6, IL-1α, CCL4/MIP-1β, IL-8 and TNF-α. Among the three immune cell subsets known to constitutively express Siglec-7, the production of these pro-inflammatory cytokines and chemokines selectively occurs in monocytes and not in Natural Killer or T lymphocytes. This Siglec-7-mediated activating function is associated with the phosphorylation of the extracellular signal-regulated kinase (ERK) pathway. The present study also shows that sialic acid-free <em>Zymosan</em> yeast particles are able to bind Siglec-7 on monocytes and that this interaction mimics the ability of the anti Siglec-7 mAb to induce the production of pro-inflammatory mediators. Indeed, blocking or silencing Siglec-7 in primary monocytes greatly reduced the production of inflammatory cytokines and chemokines in response to <em>Zymosan</em>, thus confirming that Siglec-7 participates in generating a monocyte-mediated inflammatory outcome following pathogen recognition. The presence of an activating form of Siglec-7 in monocytes provides the host with a new and alternative mechanism to encounter pathogens not expressing sialylated glycans.</p> </div

Intracellular production of TNF-α and IL-1α in monocytes upon engagement of Siglec-7 and Siglec-9 and modulation of adhesion molecules in monocytes upon engagement of Siglec-7.

<p>(A) Statistical histogram bar graph showing the percentages of CD14^pos monocyte producing TNF-α (left) and IL-1α (right) in the presence of mAbs cross-linking either Siglec-7 and Siglec-9 or their relative IgG2b and IgG1 isotype controls. Data are representative of 5 independent experiments performed in triplicates (± SD). (B) Representative flow cytometry histogram graphs showing the mean fluorescence intensity (MFI) of ICAM-1 (left) and CD49e (right) on CD14^pos monocyte after incubation with either the anti-Siglec-7 mAb (blue lines) or with the matched IgG2b isotype control (red lines). (C) Statistical summary graphs of box plots with medians and standard deviation showing the MFI of ICAM-1 (left) and CD49e (right) on CD14^pos monocyte after incubation with either the anti-Siglec-7 mAb (blue boxes) or with the matched IgG2b isotype control (red boxes). Data are representative of 5 independent experiments (± SD).</p

Binding of pathogens to Siglec-7 Fc chimera.

<p>Representative flow cytometric dot plot graphs showing the binding of goat anti human (GAH) Fc Ab (upper line) and of Siglec-7 Fc chimera (lower line) to <i>Escherichia coli</i> (strains K1 and K12) and <i>Candida albicans</i>. Data are representative of 3 independent experiments.</p