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 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

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

Neurotensin and CRH Interactions Augment Human Mast Cell Activation

<div><p>Stress affects immunity, but the mechanism is not known. Neurotensin (NT) and corticotropin-releasing hormone (CRH) are secreted under stress in various tissues, and have immunomodulatory actions. We had previously shown that NT augments the ability of CRH to increase mast cell-dependent skin vascular permeability in rodents. Here we show that NT triggered human mast cell degranulation and significantly augmented CRH-induced vascular endothelial growth factor (VEGF) release. Investigation of various signaling molecules indicated that only NF-κB activation was involved. These effects were blocked by pretreatment with the NTR antagonist SR48692. NT induced expression of CRH receptor-1 (CRHR-1), as shown by Western blot and FACS analysis. Interestingly, CRH also induced NTR gene and protein expression. These results indicate unique interactions among NT, CRH, and mast cells that may contribute to auto-immune and inflammatory diseases that worsen with stress.</p> </div

CRH induces NT and NTR gene expression in human mast cells.

<p>(A) NT and (B) NTR gene expression in LAD2 cells following incubation with the indicated concentrations of CRH for 6 h. Relative mRNA expression was measured by quantitative qPCR, normalized to GAPDH, and expressed relatively to the untreated cells (control). (C) Western blot analysis of NTR following incubation with CRH (10 µM) for 24 h. Tubulin was used as an internal control. For all the experiments, n = 3; *p<0.05, **p<0.01, compared to control.</p

Effect of NT on NF-κB activation in human mast cells.

<p>LAD2 mast cells were pretreated with/without the NTR antagonist SR 48692 (TOCRIS Bioscience, Ellisville, MO) (10 µM) for 30 min, then treated with NT (1 µM) for 5, 10, 20 min. (A) Different molecules were measured with PathScan® Sandwich ELISA Kit (Cell Signaling Technology, Inc. Danvers, MA) and the absorbance for NF-κB was determined spectrophotometrically at 450 nm. (B) NF-κB was determined by EMSA.</p