MFI of phenotypic markers of LDGs and NDGs.

<p>LDGs and NDGs were isolated as described in materials and methods and the expression levels of phenotypic markers were determined by flow cytometry (median±SEM). The percentage increase or decrease in MFI was calculated for controls (n = 11) and HIV+ patients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048939#pone-0048939-g002" target="_blank">figures 2A–G</a>, n = 22).</p

Morphology of LDGs and NDGs.

<p>LDGs and NDGs were isolated as described in materials and methods and their morphology was compared after H&E staining. Data show the results of one representative experiment out of five independent experiments.</p

Phenotypic analysis of LDGs and NDGs.

<p>LDGs and NDGs were isolated as described in materials and methods (n = 22) and the expression levels of CD11b (A), CD15 (B), CD33 (C), CD66b (D), CD16 (E), CD13 (F), CD63 (G) and arginase 1 (H) were determined by flow cytometry. Isotype controls: <1%. Statistical significance was determined by a two-tailed Mann-Whitney test. Box = interquartile range and median; whiskers = range.</p

ARGINASE 1 Panel.

<p>LDGs and NDGs were isolated as described in materials and methods and the expression levels of phenotypic markers were determined by flow cytometry.</p><p>NP  =  Not provided by manufacturer.</p

A Role for Toll-Like Receptor Mediated Signals in Neutrophils in the Pathogenesis of the Anti-Phospholipid Syndrome

<div><p>The anti-phospholipid syndrome (APS) is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL). aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN) and Toll-like receptors (TLR) to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonist LPS monitoring neutrophil effector functions, namely the oxidative burst, phagocytosis, L-Selectin shedding and IL-8 production. aPL alone were only able to induce minor activation of PMN effector functions at high concentrations. However, in the additional presence of LPS the activation threshold was markedly lower indicating a synergistic activation pathway of aPL and TLR in PMN. In summary, our results indicate that PMN effector functions are directly activated by aPL and boosted by the additional presence of microbial products. This highlights a role for PMN as important innate immune effector cells that contribute to the pathophysiology of APS.</p> </div

Phenotype of LDGs.

<p>LDGs were isolated from the blood of HIV+ patients with CD4⁺ T cell counts >350 cells/µL (n = 11) or <350 cells/µL (n = 10) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072034#s2" target="_blank">materials and methods</a> and the expression levels of phenotypic markers were determined by flow cytometry.</p

LDGs: Correlation between CD4⁺ T cell counts and MFIs.

<p>LDGs were isolated from the blood of HIV+ patients with CD4⁺ T cell counts >350 cells/µL (n = 11) or <350 cells/µL (n = 10) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072034#s2" target="_blank">materials and methods</a> and the correlations between CD4⁺ T cell counts and phenotypic markers were determined by a Spearman's rank test.</p

LPS stimulation can be enhanced by facilitating binding to CD14.

<p>Human PMN (2×10⁵ cells per well) were incubated with LPS (100 ng/ml) with or without the addition of aPL (10 µg/ml) or LPS-binding protein (LBP, 10 ng/ml). ROS formation was measured in a fluorescence reading device. Detection of specific fluorescence index (SFI) over time was calculated by subtraction of the background fluorescence of labelled cells incubated in medium. Data from one representative experiment with two replicates is shown.</p

Phenotypes of NDGs and LDGs in CD4low and CD4high HIV+ patients.

<p>PBMCs and NDGs were isolated from the blood of HIV+ patients with CD4⁺ T cell counts >350 (n = 11) or <350 cells/µL (n = 10) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072034#s2" target="_blank">materials and methods</a> and the expression levels of phenotypic markers were determined by flow cytometry. Isotype controls: <1%. Statistical significance was determined by a two-tailed Mann-Whitney test. Box = interquartile range and median; whiskers = range.</p

Synergistic activation of PMN with aPL and TLR2 ligation.

<p>Human PMN (2×10⁵ cells per well) were incubated with the TLR2 ligand Pam₃Cys (100 ng/ml, 1 µg/ml, 10 µg/ml). A) Production of radical oxygen species was determined after 170 minutes. B) Phagocytosis rate was analysed defining percentage of PMN taken up PE-labelled microspheres. C) Shedding of L-Selectin was determined by surface staining of expressed molecules. D) Supernatants of stimulated PMN culture were harvested and an IL-8 specific ELISA was performed after 2 hours (left panel) or 6 hours (right panel) of incubation. Data shown are from one representative experiment out of 3 independent with 2 replicates per group. *Indicates significant difference of Pam₃Cys-stimulated groups compared to corresponding hIgG controls (p<0.05 in a Tukey’s multiple comparison test).</p