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