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Cited by 121Mast cells and histamineMitochondrial Function and PathologyNeutrophil, Myeloperoxidase and Oxidative Mechanisms

The DOI matches its Crossref record; 43 of the 43 references it cites were found in a registry or an index.

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

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Zhang, Bodi, Asadi, Shahrzad, Weng, Zuyi, Sismanopoulos, Nikolaos, Theoharides, Theoharis C.·2012·PLoS ONE
DOI 10.1371/journal.pone.0049767PMC PMC3524249
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Abstract

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 from LAD2 cells and, when mitochondria added to mast cells trigger degranulation and release of histamine, PGD(2), 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 in vivo, 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.

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Figure 1 Mast cell degranulation results in extracellular mitochondrial particles translocation. 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.
Figure 1 Mast cell degranulation results in extracellular mitochondrial particles translocation. 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.CC BY · PubMed Central
Figure 2 Mast cell degranulation results in extracellular mitochondrial particle secretion. (A) LAD2 cells were stained with MitoTracker and LysoTracker as described in Fig. 1. 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.
Figure 2 Mast cell degranulation results in extracellular mitochondrial particle secretion. (A) LAD2 cells were stained with MitoTracker and LysoTracker as described in Fig. 1. 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.CC BY · PubMed Central
Figure 3 Extracellular release of mtDNA from mast cells is partially stored in exosomes. 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).
Figure 3 Extracellular release of mtDNA from mast cells is partially stored in exosomes. 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).CC BY · PubMed Central
Figure 4 Sonicated mitochondria stimulate human mast cell pro-inflammatory mediators release. LAD2 cells were incubated with mitochondria isolated from mast cells for either 30 min or 24 hr. Supernatant fluids from different conditions were collected. (A) β-hex (B) Histamine and (C) PGD2 release were assayed at 30 min, while (D) TNF, (E) IL-8 and (F) IL-1beta were assayed at 24 hr (n = 3; *p<0.05, compared to control).
Figure 4 Sonicated mitochondria stimulate human mast cell pro-inflammatory mediators release. LAD2 cells were incubated with mitochondria isolated from mast cells for either 30 min or 24 hr. Supernatant fluids from different conditions were collected. (A) β-hex (B) Histamine and (C) PGD2 release were assayed at 30 min, while (D) TNF, (E) IL-8 and (F) IL-1beta were assayed at 24 hr (n = 3; *p<0.05, compared to control).CC BY · PubMed Central
Figure 5 Mitochondrial component stimulation of LAD2 cell β-Hex release is partially P2X7 receptor dependent. 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).
Figure 5 Mitochondrial component stimulation of LAD2 cell β-Hex release is partially P2X7 receptor dependent. 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).CC BY · PubMed Central
Figure 6 Sonicated mitochondria stimulate human keratinocyte and endothelial cell cytokine release. 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).
Figure 6 Sonicated mitochondria stimulate human keratinocyte and endothelial cell cytokine release. 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).CC BY · PubMed Central
Figure 7 Electron photomicrographs showing mitochondria localization in rat peritoneal mast cells and human mtDNA presence in rat serum. 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).
Figure 7 Electron photomicrographs showing mitochondria localization in rat peritoneal mast cells and human mtDNA presence in rat serum. 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).CC BY · PubMed Central

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  1. B Zhang, 2012, Preformed, but not de novo synthesized, TNF secretion from human mast cells is regulated by mitochondria, Int Arch Allergy Immunol

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Metadata

Authors
Zhang, B., Asadi, S., Weng, Z., Sismanopoulos, N., & Theoharides, T. C.
Affiliations
Tufts University (US)
Corresponding author
Theoharides, Theoharis C.
ORCID
Theoharides, Theoharis C.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-12-17
Volume
7
Issue
12
Pages
e49767
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0049767
PMC
PMC3524249
OpenAlex
W2169711067
Cited by
121
References
43
Citations by year
2026: 3 · 2025: 8 · 2024: 10 · 2023: 6 · 2022: 7 · 2021: 7 · 2020: 7 · 2019: 10 · 2018: 10 · 2017: 11 · 2016: 11 · 2015: 13 · 2014: 7 · 2013: 11
FWCI
4.27
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR047652)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Mast cells and histamine
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Keywords
mast cells, mitochondrial DNA, ATP, substance P, mast cell degranulation, LAD2 cells, histamine release, prostanoids, interleukin-8, TNF-α, IL-1β, vascular endothelial growth factor, keratinocytes, microvascular endothelial cells, compound 48/80, systemic circulation
MeSH terms
Animals, p-Methoxy-N-methylphenethylamine, DNA, Mitochondrial, Extracellular Space, Humans, Inflammation, Male, Mast Cells, Mitochondria, Cell Degranulation, Cytokines, Autocrine Communication, Paracrine Communication, Rats, Exosomes, Receptors, Purinergic P2X7, Inflammasomes

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