Leptin inhibits neutrophil apoptosis in children via ERK/NF-κB-dependent pathways.

Previous studies have shown that delayed neutrophil apoptosis is associated with chronic airway diseases. Leptin is an adipocyte-derived hormone that acts as a regulator of energy homeostasis and food intake. Emerging evidence suggests that leptin can regulate immune responses including the release of proinflammatory cytokines and protection of inflammatory cells from apoptosis. Serum leptin is increased during allergic reactions in the airways. However, the expression and function of leptin receptor in neutrophils isolated from children is not known.Flow cytometry was used to detect leptin receptor expression in neutrophils isolated from allergic asthmatic (n = 14), allergic non asthmatic (n = 21), non allergic asthmatic (n = 7) and healthy children (n = 23); confocal laser scanning microscopy combined with immunofluorescence was performed to detect intracellular pool of leptin receptor; Annexin-V/PI staining and caspase 3 activity was used to determine neutrophil survival. Pharmacological inhibitors were utilized to understand the role of MAPK and NF-κB pathway in leptin-induced neutrophil survival.A heterogeneous leptin receptor expression was observed on neutrophils isolated from children. Neutrophils isolated from healthy children expressed more leptin receptor than those from allergic asthmatic (P0.05) or non-allergic asthmatic children (n = 7, P>0.05). Neutrophils isolated from children express an intracellular pool of leptin receptor that was mobilized to the cell surface upon GM-CSF stimulation. Finally, leptin exhibited anti-apoptotic properties on neutrophils via NF-κB and MEK1/2 MAPK pathway. Collectively, our data suggest that leptin may enhance airway inflammation by promoting neutrophil survival

Comparative expression of leptin receptor in neutrophils of different groups of children.

<p>Summary of cumulative leptin receptor expression is shown. X axis represents different groups of children, classified as normal healthy, allergic asthmatic, non-allergic asthmatic, and non-asthmatic allergic children. Y axis shows percentage positive cells compared to isotype control. * p<0.05, unpaired <i>t</i> test.</p

GM-CSF mobilizes the intracellular pool of leptin receptor to the cell surface.

<p><i>A</i>.(a <i>and b</i>). Representative FACS data showing absence of surface leptin receptor expression in freshly isolated neutrophil. <i>b</i>. Immunofluorescence of leptin receptor on neutrophil (inset shows isotype control), also stained with PI for nuclei (red color). Intracellular pool of leptin receptor (green color) is observed from freshly purified neutrophils in which no expression of leptin receptor was observed by FACS. Isotype control antibodies were used as negative controls. <i>B–E</i>. Normalized neutrophil leptin receptor expression in fresh cells at 2 h in medium, GM-CSF, Cycloheximide (10 ng/ml), and GM-CSF+Cycloheximide, quantitated in <i>F</i>. *p<0.05, ***p<0.001 compared to unstimulated control, unpaired <i>t</i> test.</p

Leptin inhibits neutrophil apoptosis via NFκB involvement.

<p><b><i>A.</i></b> Representative Annexin-V/PI staining assay of neutrophil apoptosis after 20 h incubation. <b><i>B.</i></b> Quantitative analysis of non-apoptotic cells in neutrophil from 5 children after 20 h incubation with untreated (medium), 10 ng/ml GM-CSF, 10 µg/ml Leptin, Leptin+PDTC (5 µM) and PDTC alone.n = 5 ** p<0.01 compared to medium alone, ††p<0.01 compared with leptin-stimulated group, one-way ANOVA.</p

Cell surface expression of leptin receptor on peripheral blood PMNs.

<p>Highly purified peripheral blood children PMNs from allergic asthmatics, non-asthmatic allergic, non-allergic asthmatic, and normal controls were analyzed by FACS with mouse anti-human leptin mAb(red lines) and mouse IgG2a isotype-matched control Ab (black lines).</p

Pharmacological inhibition of MEK1/2 MAPK and NFκB blocks anti-death effects of leptin in neutrophils from children.

<p>Neutrophils were cultured in the presence and absence of leptin (10 µg/ml) for 20 h (n = 3). U0126 (5 µM) significantly prevented the leptin-mediated anti-death effect while PDTC (5 µM) completely blocked anti-death effect. SB203580 (p38 inhibitor, 25 µM) and Wortmannin (PI3K inhibitor, 10 µM). **p<0.01 compared to medium control, ††p<0.01 compared to leptin-stimulated group without any inhibitor; one-way ANOVA.</p

GM-CSF up regulates surface leptin receptor expression in peripheral neutrophils isolated from children.

<p>A. Dose response effect of GM-CSF (0.1, 1, 10, 25 ng/ml) on leptin receptor expression on neutrophils from children (n = 3, P<0.01). B. Neutrophils incubated with GM-CSF (10 ng/ml), leptin (10 µg/ml), combination, or alone for 2 h were analyzed by FACS with mouse anti-human leptin mAb and mouse IgG2a isotype-matched control Ab. Mean fluorescence intensity (MFI) of anti-leptin receptor is plotted as fold increase compared to isotype control. (n = 7, data shown as mean ± SD) **p<0.01, *p<0.05 compared to freshly isolated group, one-way ANOVA; †p<0.05 compared to medium control, unpaired <i>t</i> test.</p

Clinical characteristics of the children.

<p>All children were between the ages of 10–11years. FEV1; Forced expiratory volume in 1 second, PC20; provocative concentration causing a 20% fall in FEV1, CRP; C-reactive protein.</p