Human bronchial epithelial cells abundantly express SPHK1, SPHK2 and Spns2.

<p><b>A,</b> SPHK1 (red). <b>B</b>, SPHK2 (red). <b>C</b>, Spns2 (red, LifeSpan BioSciences rabbit polyclonal antibody). Green in <b>A-C</b> was beta-actin. <b>D, E</b>, Spns2 (green, Santa Cruz goat polyclonal antibody) was expressed in cilia, and colocalized with SPHK1 (red, yellow being merged color of red and green) in the cytoplasm. <b>F</b>, a negative staining control incubated with conjugated antibodies alone. Blue in <b>A-F</b> was DAPI. Arrows in <b>C, D, E</b> indicate Spns2 expression in cilia. Scale bars are in micrometers. Images are representative of bronchial epithelial cells obtained from 3 different non-smoking donors.</p

Spns2 expression in human macrophages.

<p><b>A</b>, Expression and subcellular localization of Spns2 in alveolar macrophages. Spns2 (rabbit antibody) was labeled in red, beta-actin in green, blue was staining of nuclei. Spns2 was localized to plasma membrane (arrowhead), nucleus (arrow), and cytoplasm. <b>B</b>, Representative confocal images of Spns2 (goat antibody, green) in COPD vs. non-COPD alveolar macrophages, captured for quantitative analysis. For the highly intense Spns2 immunofluorescence in the given COPD sample not become saturated, the laser settings had to be set as low as the immunofluorescence in the non-COPD sample was nearly invisible for naked eyes. Scale bars in <b>A</b> and <b>B</b> are in micrometers. <b>C</b>, Significant increase of Spns2 immunofluorescence in alveolar macrophages of smokers/COPD patients (n = 5) compared to healthy control (C, n = 8, p<0.01) and non-COPD non-smoker transplant patients (Tx, n = 4, p<0.01). <b>D</b>, Cigarette smoke extract induced significant increase (**, p<0.01) of Spns2 immunofluorescence in primary alveolar macrophages obtained from non-smokers (n = 6). <b>E</b>, Cigarette smoke extract induced significant increase (***, p<0.001, 5 experiments) of Spns2 immunofluorescence in THP-1 macrophages. <b>F</b>, Cigarette smoke extract induced significant increase (*, p<0.05, 3 experiments) of Spns2 mRNAs in THP-1 macrophages.</p

Defective phagocytosis in lung macrophages of cigarette smoked mice was associated with a down-regulated expression of Spns2 in bronchial epithelia.

<p><b>A,</b> Representative confocal images of Spns2 in lung tissue of control and cigarette smoke (CS)-exposed mice, and a negative control staining (NEG). Spns2 was stained in red, nuclei in blue. Scale bars are in micrometers. <b>B,</b> Significant decrease of Spns2 (MFI, mean fluorescence intensity) in bronchial epithelia in CS-exposed vs. control mice (p<0.001, pooled data from 6 animals per group). <b>C,</b> Cigarette smoke exposure induced a significant decrease of efferocytosis (p<0.05, n = 6 per group) in lung macrophages of CS-exposed vs. control mice, and a nearly significant trend of reduced NTHi phagocytosis (p = 0.063). <b>D,</b> Correlation between NTHi phagocytic activity of pulmonary macrophages in individual mice and expression of Spns2 in their bronchial epithelia (pooled data from 12 mice, r = 0.685; p = 0.014 by Spearman’s rho test).</p

Immunolocalization of ZIP1 and ZIP2 transporters in human macrophages.

<p><b>A:</b> Plasma membrane-like localization of ZIP1 (red) in PMA-differentiated THP-1 macrophages. <b>B</b>–<b>C:</b> Staining of human lung sections (non-tumour area) for ZIP1 (red, B) and negative control (C). <b>D</b>: Sparse and predominantly nucleolar localization of ZIP2 in PMA-differentiated THP-1 macrophages. <b>E</b>–<b>F:</b> Staining of human lung sections (non-tumour area) for ZIP2. Macrophages were identified in separate experiments using CD68 marker (not shown). Panels E and F are representative microphotos of ZIP2 staining (red) of macrophages in two different patient biopsies, showing surface and cytoplasmic fluorescence. Blue = DAPI staining of nuclei. Scale bar represents 50 µm for all microphotos, and 16.7 µm in the insets.</p

Zinc and Zinc Transporters in Macrophages and Their Roles in Efferocytosis in COPD

<div><p>Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn <i>in vitro</i> by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in <i>ZIP1</i> mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter <i>ZIP2,</i> consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.</p></div

Zn transporter mRNA expression in THP-1 monocytes and macrophages in response to intracellular Zn depletion.

<p>THP-1 monocytes and PMA differentiated THP-1 macrophages were treated with 16 µM TPEN for 4 h. RNA was isolated and target genes were detected using Taqman probes, with HPRT-1 and 18S RNA used as endogenous controls for 3 separate experiments (n = 3). Data is presented as mean fold change (±95% CI) compared to control (untreated) cells. * p: <0.05, ** p: <0.01, *** p: <0.001.</p

Effect of ZIP1 XO on cytosolic Zn and efferocytosis.

<p>Peritoneal macrophages were harvested from WT mice (n = 25) and Zip1 KO mice (n = 14). <b>A:</b> Macrophages were stained with FluoZin-3 and the resulting fluorescence was measured by flow cytometry. Positive fluorescence was determined by gating relative to an unstained control. Error bars represent the SEM. <b>B:</b> Macrophages were assayed for efferocytosis. Results are expressed as a percentage of macrophages ingesting apoptotic cells. Error bars represent the SEM. * p: <0.05, ** p: <0.005.</p