Mast cell degranulation results in extracellular mitochondrial particles translocation.

<p>hCBMCs were stained with MitoTracker Deep Red (20 nM) for 20 min and LysoTracker DND green (50 nM) for 30 min, then seeded in glass bottom culture dishes and observed under Leica TCS SP2 Confocal microscope. Mitochondrial distribution was observed in resting (upper panels) and degranulated (bottom panels) mast cells stimulated as shown. The left panels depict secretory granules in green and the middle panels represent mitochondria fluorescence in red. The right panels represent images merged from the two previous panels.</p

Stimulated Human Mast Cells Secrete Mitochondrial Components That Have Autocrine and Paracrine Inflammatory Actions

<div><p>Mast cells are hematopoietically-derived tissue immune cells that participate in acquired and innate immunity, as well as in inflammation through release of many chemokines and cytokines, especially in response to the pro-inflammatory peptide substance P (SP). Inflammation is critical in the pathogenesis of many diseases, but the trigger(s) is often unknown. We investigated if mast cell stimulation leads to secretion of mitochondrial components and whether these could elicit autocrine and/or paracrine inflammatory effects. Here we show that human LAD2 mast cells stimulated by IgE/anti-IgE or by the SP led to secretion of mitochondrial particles, mitochondrial (mt) mtDNA and ATP without cell death. Mitochondria purified fromLAD2 cells and, when mitochondria added to mast cells trigger degranulation and release of histamine, PGD₂, IL-8, TNF, and IL-1β. This stimulatory effect is partially inhibited by an ATP receptor antagonist and by DNAse. These results suggest that the mitochondrial protein fraction may also contribute. Purified mitochondria also stimulate IL-8 and vascular endothelial growth factor (VEGF) release from cultured human keratinocytes, and VEGF release from primary human microvascular endothelial cells. In order to investigate if mitochondrial components could be secreted <em>in vivo</em>, we injected rats intraperiotoneally (ip) with compound 48/80, which mimicks the action of SP. Peritoneal mast cells degranulated and mitochondrial particles were documented by transimission electron microscopy outside the cells. We also wished to investigate if mitochondrial components secreted locally could reach the systemic circulation. Administration ip of mtDNA isolated from LAD2 cells in rats was detected in their serum within 4 hr, indicating that extravascular mtDNA could enter the systemic circulation. Secretion of mitochondrial components from stimulated live mast cells may act as “autopathogens” contributing to the pathogenesis of inflammatory diseases and may be used as targets for novel treatments.</p> </div

Mast cell degranulation results in extracellular mitochondrial particle secretion.

<p>(A) LAD2 cells were stained with MitoTracker and LysoTracker as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049767#pone-0049767-g001" target="_blank">Fig. 1</a>. mitochondria distribution was observed in resting (upper panels) and degranulated (bottom panels) mast cells stimulated as shown. The left panels depict secretory granules in green and the middle panels represent mitochondria fluorescence in red. The right panels represent images merged from the two previous panels. The lower set of three panels show cells in lower magnification as indicated. White rectangles in the middle panels indicate extracellular mitochondriaL particles stained by MitoTracker. Supernatant fluids from both stimulated and control LAD2 cells were collected and assayed for (B, C) mt-7s and mt-CytB, as well as (D) ATP (n = 3; *p<0.05, **p<0.01 compared to control). Sup = Supernatant fluid.</p

Sonicated mitochondria stimulate human keratinocyte and endothelial cell cytokine release.

<p>HaCaT and HMVEC cells were incubated with mitochondria isolated from LAD2 cells for 24 hr. Supernatant fluids from different conditions were collected. Cytokine release from HaCat cells (A) IL-8 and (B) VEGF, as well as from HMVECs (C) VEGF and (D) TNF were measured at 24 hr (n = 3; *p<0.05, compared to control).</p

Extracellular release of mtDNA from mast cells is partially stored in exosomes.

<p>LAD2 cells were stimulated with SP (2 µM) for 30 min. Supernatant fluids from both stimulated and control LAD2 cells were collected and were treated by DNAase. Quantitative PCR (A) was performed to measure mtDNA level in supernatants with or without DNase treatment (B). Exosomes were isolated from supernatant fluids by Differential Ultracentrifugation followed by (C) mtDNA isolation from exosomes and measured by quantitative PCR. (D) Exosome-containing mtDNA level compared to mtDNA isolated from uncentrifuged supernatant fluids (n = 3; *p<0.05, **p<0.01 compared to control).</p

Mitochondrial component stimulation of LAD2 cell β-Hex release is partially P2X7 receptor dependent.

<p>LAD2 cells were (A) Stimulated with different concentrations of ATP, or (B) pre-treated with the P2X7 receptor inhibitor for 30 min and then stimulated with mitochondrial components. β-Hex release was measured 30 min later. (n = 3; *p<0.05, compared to control).</p

Electron photomicrographs showing mitochondria localization in rat peritoneal mast cells and human mtDNA presence in rat serum.

<p>Male rat peritoneal mast cells (A) control with intact electron dense granules and mitochondria inside the cell. (B, C) after C48/80 stimulation showing (B) intense degranulation (*) with most mitochondria at the cell surface close to sites of exocytosis (Magnification: 13,800×), and (C) extracellular mitochondria outside the cell perimeter (Magnification: 4,500×). Mitochondria is shown within red rectangles. (D) Presence of human mitochondria in rat serum following ip injection in male rats (n = 4).</p

(A) IL-9 gene expression in the skin of AD affected (n = 16) and normal healthy controls (n = 12).

<p>(B) IL-9 receptor (IL-9r) gene expression in skin of AD affected (n = 19) skin and normal healthy controls (n = 20). Relative quantities of mRNA expression were measured by quantitative RT-PCR and normalized to GAPDH. TaqMan was performed with cDNA reverse transcribed from 100 ng RNA from each sample. *p<0.05.</p

IL-9 stimulates VEGF production in human mast cells.

<p>(A) Gene expression. LAD2 cells were stimulated with IL-9 for 6 hrs, RNA was extracted and relative VEGF mRNA levels were determined by real-time PCR. (B) Protein release. LAD2 cells were stimulated with the indicated concentration of IL-9 (10–20 ng/ml) for 48 hrs. VEGF was measured in the supernatant fluid by ELISA. Data are the mean ± SD of 3 separate experiments performed in triplicate (*<i>P</i><0.05 versus unstimulated cells).</p

(A) IL-9 induces STAT3 phosphorylation in LAD2 cells.

<p>Cells were stimulated with IL-9 for up to 20 min. Phospho-STAT3 levels in the cell lysates were determined by ELISA. (B) STAT3 inhibitor Stattic inhibits IL-9-induced VEGF release from LAD2 cells. LAD2 cells were pre-incubated for 30 min with the indicated concentrations of Stattic. Cells were stimulated with IL-9 (10–20 ng/ml) for 24 h and supernatant VEGF was measured by ELISA. Data are representative of similar experiments.</p