IL-9 Induces VEGF Secretion from Human Mast Cells and IL-9/IL-9 Receptor Genes Are Overexpressed in Atopic Dermatitis

Interleukin 9 (IL-9) has been implicated in mast cell-related inflammatory diseases, such as asthma, where vascular endothelial growth factor (VEGF) is involved. Here we report that IL-9 (10–20 ng/ml) induces gene expression and secretion of VEGF from human LAD2. IL-9 does not induce mast cell degranulation or the release of other mediators (IL-1, IL-8, or TNF). VEGF production in response to IL-9 involves STAT-3 activation. The effect is inhibited (about 80%) by the STAT-3 inhibitor, Stattic. Gene-expression of IL-9 and IL-9 receptor is significantly increased in lesional skin areas of atopic dermatitis (AD) patients as compared to normal control skin, while serum IL-9 is not different from controls. These results imply that functional interactions between IL-9 and mast cells leading to VEGF release contribute to the initiation/propagation of the pathogenesis of AD, a skin inflammatory disease

Rupatadine inhibits inflammatory mediator release from human laboratory of allergic diseases 2 cultured mast cells stimulated by platelet-activating factor

Background: Mast cells are involved in allergy and inflammation by the secretion of multiple mediators, including histamine, cytokines, and platelet-activating factor (PAF), in response to different triggers, including emotional stress. PAF has been associated with allergic inflammation, but there are no clinically available PAF inhibitors. Objective: To investigate whether PAF could stimulate human mast cell mediator release and whether rupatadine (RUP), a dual histamine-1 and PAF receptor antagonist, could inhibit the effect of PAF on human mast cells. Methods: Laboratory of allergic diseases 2 cultured mast cells were stimulated with PAF (0.001, 0.01, and 0.1 mu mol/L) and substance P (1 mu mol/L) with or without pretreatment with RUP (2.5 and 25 mu mol/L), which was added 10 minutes before stimulation. Release of beta-hexosaminidase was measured in supernatant fluid by spectrophotoscopy, and histamine, interleukin-8, and tumor necrosis factor were measured by enzyme-linked immunosorbent assay. Results: PAF stimulated a statistically significant release of histamine, interleukin-8, and tumor necrosis factor (0.001-0.1 mu mol/L) that was comparable to that stimulated by substance P. Pretreatment with RUP (25 mu mol/L) for 10 minutes inhibited this effect. In contrast, pretreatment of laboratory of allergic diseases 2 cells with diphenhydramine (25 mu mol/L) did not inhibit mediator release, suggesting that the effect of RUP was not due to its antihistaminic effect. Conclusion: PAF stimulates human mast cell release of proinflammatory mediators that is inhibited by RUP. This action endows RUP with additional properties in treating allergic inflammation. (C) 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved

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 Cells in Allergic and Inflammatory Diseases

Mast cells are important in the development of allergic and anaphylactic reactions, but also in acquired and innate immunity. There is also increasing evidence that mast cells participate in inflammatory diseases, where they can be activated by non-allergic triggers, such as neuropeptides and cytokines, often having synergistic effects as in the case of substance P (SP) and IL-33. Secretion of vasoactive mediators, cytokines and proteinases contribute to the development of coronary artery disease (CAD), as well as to diet-induced obesity and the metabolic syndrome. Mast cells may be able to orchestrate such different biological processes through their ability to release pro-inflammatory mediators selectively without the degranulation typical of allergic reactions. Recent evidence suggests that mitochondrial uncoupling protein 2 (UCP2) and mitochondrial translocation regulate mast cell degranulation, but not selective mediator release. Better understanding of these two processes and how mast cells exert both immunostimulatory and immunosuppressive actions could lead to the development of inhibitors of release of specific mediators with novel therapeutic applications

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

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

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

IL-9 Induces VEGF Secretion from Human Mast Cells and IL-9/IL-9 Receptor Genes Are Overexpressed in Atopic Dermatitis

Interleukin 9 (IL-9) has been implicated in mast cell-related inflammatory diseases, such as asthma, where vascular endothelial growth factor (VEGF) is involved. Here we report that IL-9 (10-20 ng/ml) induces gene expression and secretion of VEGF from human LAD2. IL-9 does not induce mast cell degranulation or the release of other mediators (IL-1, IL-8, or TNF). VEGF production in response to IL-9 involves STAT-3 activation. The effect is inhibited (about 80%) by the STAT-3 inhibitor, Stattic. Gene-expression of IL-9 and IL-9 receptor is significantly increased in lesional skin areas of atopic dermatitis (AD) patients as compared to normal control skin, while serum IL-9 is not different from controls. These results imply that functional interactions between IL-9 and mast cells leading to VEGF release contribute to the initiation/propagation of the pathogenesis of AD, a skin inflammatory disease