Oxidation negated the stimulatory effects of pdMBL on BAL-derived alveolar macrophage surface expression of SRA detected by flow cytometry.

<p><b>A–D</b>. Representative flow cytometry plots showing SRA expression following treatment with pdMBL or oxMBL. Debris, lymphocytes and cell fragments were firstly excluded based on forward- and side-scatter characteristics then macrophages identified by positive staining for CD13 and high autofluorescence (not shown). <b>A</b>. Control tube of alveolar macrophages stained with IgG control was included and the staining patterns used to set quadrant markers for flow cytometric analysis of <b>B</b>. SRA expression in untreated macrophages (13.9%), <b>C</b>. SRA expression in macrophages treated with plasma-derived MBL (22.6%), <b>D</b>. SRA expression in macrophages treated with oxidized MBL (9.4%). <b>E</b>. There was a significant increase (*, p<0.05; Wilcoxon; data pooled from 7 experiments using BAL-derived alveolar macrophages) in % expression of SRA in the presence of pdMBL. Oxidation of MBL abrogated this stimulation effect.</p

pdMBL but not oxMBL enhanced phagocytosis by human alveolar macrophages.

<p>There was a significant increase in phagocytosis of <b>A</b>. apoptotic bronchial epithelial cells (*, p<0.05; Wilcoxon; data pooled from 9 experiments.) and <b>B</b>. Phagocytosis of NTHi (*, p<0.05; Wilcoxon; data pooled from 12 experiments) in the presence of pdMBL. Oxidation of MBL significantly reduced the phagocytic ability for both apoptotic cells and bacteria vs both control and pdMBL treatment.</p

Loss of higher-order quaternary structures in oxMBL vs pdMBL.

<p>To mimic high levels of oxidative stress, pdMBL was exposed to 74′-azobis(2-methylpropionamidine)dihydrochloride (AAPH). Polyacrylamide gel electrophoresis was then applied to measure changes to MBL by separating the different MW. Protein samples were loaded 10 ug MBL per lane for blue native PAGE and visualized with silver stain. Figure shows a loss of mid-higher order oligomeric structures in oxMBL vs pdMBL.</p

Tissue/tumor patient demographics.

<p>Values shown as mean [SEM]. COPD, chronic obstructive pulmonary disease; yrs, years; FEV₁, forced expiratory volume in 1 min; FVC, forced vital capacity; BD, bronchodilator; LLN, lower limit of normality; Cur, current-smoker; ex, ex-smoker; n, never smoker; adeno, adenocarcinoma; squam, squamous; * significantly (p<0.05) different compared to control.</p

Prostaglandin inhibition of efferocytosis involves COX-2.

<p>U937 cells were incubated in normal RPMI media or SBC-1 supernatant that had been treated with indomethacin. Following 24 hrs incubation, an efferocytosis assay was performed. (n = 5 experiments performed in triplicate). Data presented as box plots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061573#pone-0061573-g001" target="_blank">Figure 1</a>. Values are presented as percentage of macrophages ingesting apoptotic cells *, p<0.05 compared with RPMI media treatment (one-way ANOVA, Dunnett’s test).</p

Efferocytosis ability of alveolar and lung tissue macrophages.

<p><b>A.</b> Efferocytosis of BAL-derived alveolar macrophages was assessed for controls (‘C’), smokers, current- and ex- smokers with COPD (‘COPD Cur’ and ‘COPD Ex’), COPD subjects with lung cancer (‘COPD Cancer’) and patients with lung cancer and no COPD (‘Cancer’); <b>B.</b> Tissue from Controls (‘C Non-Tumor’) (non-cancer area from patients with cancer/no COPD), ‘COPD Non-Tumor’ (non-cancer area from patients with cancer+COPD), ‘COPD Tumor’ (cancer site from patients with cancer+COPD) and ‘Control Tumor’ (cancer site from patients with cancer/no COPD). *significantly (p<0.05) lower expression vs. controls (non-parametric Kruskal-Wallis test)Box plots present median±25th and 75th percentiles (solid box) with the 10th and 90th percentiles shown by whiskers outside the box.</p

Effect of cancer cell line supernatants on efferocytosis.

<p><b>A.</b> Effect of cancer cell line supernatants on the phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages. Macrophages from (A) control subjects or (B) subjects with lung cancer were incubated in normal RPMI media or cancer cell line supernatants (H2009, H1466, SBC1) for 24 hrs prior to phagocytosis assay. Values are presented as percentage of macrophages ingesting apoptotic cells *, p< 0.05 compared with RPMI media treatment (n = 5 experiments performed in triplicate; one-way ANOVA, Dunnett’s test). Data presented as box plots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061573#pone-0061573-g001" target="_blank">Figure 1</a>.</p

BAL patient demographics.

<p>BAL patient demographics.</p