| 1. |
- Alanazi, Sultan, et al.
(författare)
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Mast Cell β-Tryptase Is Enzymatically Stabilized by DNA
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21:14
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Tidskriftsartikel (refereegranskat)abstract
- Tryptase is a tetrameric serine protease located within the secretory granules of mast cells. In the secretory granules, tryptase is stored in complex with negatively charged heparin proteoglycans and it is known that heparin is essential for stabilizing the enzymatic activity of tryptase. However, recent findings suggest that enzymatically active tryptase also can be found in the nucleus of murine mast cells, but it is not known how the enzmatic activity of tryptase is maintained in the nuclear milieu. Here we hypothesized that tryptase, as well as being stabilized by heparin, can be stabilized by DNA, the rationale being that the anionic charge of DNA could potentially substitute for that of heparin to execute this function. Indeed, we showed that double-stranded DNA preserved the enzymatic activity of human β-tryptase with a similar efficiency as heparin. In contrast, single-stranded DNA did not have this capacity. We also demonstrated that DNA fragments down to 400 base pairs have tryptase-stabilizing effects equal to that of intact DNA. Further, we showed that DNA-stabilized tryptase was more efficient in degrading nuclear core histones than heparin-stabilized enzyme. Finally, we demonstrated that tryptase, similar to its nuclear localization in murine mast cells, is found within the nucleus of primary human skin mast cells. Altogether, these finding reveal a hitherto unknown mechanism for the stabilization of mast cell tryptase, and these findings can have an important impact on our understanding of how tryptase regulates nuclear events.
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| 2. |
- Alim, Abdul, 1983-, et al.
(författare)
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Glutamate triggers the expression of functional ionotropic and metabotropic glutamate receptors in mast cells
- 2021
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Ingår i: Cellular & Molecular Immunology. - : Springer Nature. - 1672-7681 .- 2042-0226. ; 18:10, s. 2383-2392
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Tidskriftsartikel (refereegranskat)abstract
- Mast cells are emerging as players in the communication between peripheral nerve endings and cells of the immune system. However, it is not clear the mechanism by which mast cells communicate with peripheral nerves. We previously found that mast cells located within healing tendons can express glutamate receptors, raising the possibility that mast cells may be sensitive to glutamate signaling. To evaluate this hypothesis, we stimulated primary mast cells with glutamate and showed that glutamate induced the profound upregulation of a panel of glutamate receptors of both the ionotropic type (NMDAR1, NMDAR2A, and NMDAR2B) and the metabotropic type (mGluR2 and mGluR7) at both the mRNA and protein levels. The binding of glutamate to glutamate receptors on the mast cell surface was confirmed. Further, glutamate had extensive effects on gene expression in the mast cells, including the upregulation of pro-inflammatory components such as IL-6 and CCL2. Glutamate also induced the upregulation of transcription factors, including Egr2, Egr3 and, in particular, FosB. The extensive induction of FosB was confirmed by immunofluorescence assessment. Glutamate receptor antagonists abrogated the responses of the mast cells to glutamate, supporting the supposition of a functional glutamate-glutamate receptor axis in mast cells. Finally, we provide in vivo evidence supporting a functional glutamate-glutamate receptor axis in the mast cells of injured tendons. Together, these findings establish glutamate as an effector of mast cell function, thereby introducing a novel principle for how cells in the immune system can communicate with nerve cells.
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| 3. |
- Braga, Tiago, et al.
(författare)
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Serglycin proteoglycan is required for secretory granule integrity in mucosal mast cells
- 2007
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 403:1, s. 49-57
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Tidskriftsartikel (refereegranskat)abstract
- SG (serglycin) PGs (proteoglycans) are strongly implicated in the assembly of MC (mast cell) granules. However, this notion has mainly been on the basis of studies of MCs of the connective tissue subtype, whereas the role of SG PG in mucosal MCs has not been explored. In the present study, we have addressed the latter issue by using mice with an inactivated SG gene. Bone marrow cells were differentiated in vitro into the mucosal MC phenotype, expressing the markers mMCP (mouse MC protease) -1 and -2. Biosynthetic labelling experiments performed on these cells revealed an ~80% reduction of 35SO42− incorporation into PGs recovered from SG−/− cells as compared with SG+/+ counterparts, indicating that SG is the dominating cell-associated PG of mucosal MCs. Moreover, the absence of SG led to defective metachromatic staining of mucosal MCs, both in vivo and in the in vitro-derived mucosal MCs. Ultrastructural analysis showed that granules were present in similar numbers in SG+/+ and SG−/− cells, but that their morphology was markedly affected by the absence of SG, e.g. with electron-dense core formation only seen in SG+/+ granules. Analysis of the MC-specific proteases showed that mMCP-1 and mMCP-7 were completely independent of SG for storage, whereas mMCP-2 showed a partial dependence. In contrast, mMCP-4 and -6, and carboxypeptidase A were strongly dependent on SG for storage. Together, our data indicate that SG PG is of crucial importance for assembly of mature mucosal MC granules, but that the specific dependence on SG for storage varies between individual granule constituents.
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| 4. |
- Doncheva, Atanaska, I, et al.
(författare)
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Serglycin Is Involved in Adipose Tissue Inflammation in Obesity
- 2022
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 208:1, s. 121-132
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Tidskriftsartikel (refereegranskat)abstract
- Chronic local inflammation of adipose tissue is an important feature of obesity. Serglycin is a proteoglycan highly expressed by various immune cell types known to infiltrate adipose tissue under obese conditions. To investigate if serglycin expression has an impact on diet-induced adipose tissue inflammation, we subjected Srgn(+/+) and Srgn(-/-) mice (C57BL/6J genetic background) to an 8-wk high-fat and high-sucrose diet. The total body weight was the same in Srgn(+/+) and Srgn(-/-) mice after diet treatment. Expression of white adipose tissue genes linked to inflammatory pathways were lower in Srgn(-/)- mice. We also noted reduced total macrophage abundance, a reduced proportion of proinflammatory M1 macrophages, and reduced formation of crown-like structures in adipose tissue of Srgn(-/-) compared with Srgn(+/+) mice. Further, Srgn(-/-) mice had more medium-sized adipocytes and fewer large adipocytes. Differentiation of preadipocytes into adipocytes (3T3-L1) was accompanied by reduced Srgn mRNA expression. In line with this, analysis of single-cell RNA sequencing data from mouse and human adipose tissue supports that Srgn mRNA is predominantly expressed by various immune cells, with low expression in adipocytes. Srgn mRNA expression was higher in obese compared with lean humans and mice, accompanied by an increased expression of immune cell gene markers. SRGN and inflammatory marker mRNA expression was reduced upon substantial weight loss in patients after bariatric surgery. Taken together, this study introduces a role for serglycin in the regulation of obesity-induced adipose inflammation.
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| 5. |
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| 6. |
- Faroldi, Gianni, et al.
(författare)
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Active Caspase-3 Is Stored within Secretory Compartments of Viable Mast Cells
- 2013
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 191, s. 1445-1452
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Tidskriftsartikel (refereegranskat)abstract
- Caspase-3 is a main executioner of apoptotic cell death. The general notion is that, in viable cells, caspase-3 is found as a cytosolic inactive proenzyme and that caspase-3 activation is largely confined to processes associated with cell death. In this study, we challenge this notion by showing that enzymatically active caspase-3 is stored in viable mast cells. The enzymatically active caspase-3 was undetectable in the cytosol of viable cells, but was recovered in subcellular fractions containing secretory granule-localized proteases. Moreover, active caspase-3 was rapidly released into the cytosolic compartment after permeabilization of the secretory granules. Using a cell-permeable substrate for caspase-3, the presence of active caspase-3-like activity in granule-like compartments close to the plasma membrane was demonstrated. Moreover, it was shown that mast cell activation caused release of the caspase-3 to the cell exterior. During the course of mast cell differentiation from bone marrow cells, procaspase-3 was present in cells of all stages of maturation. In contrast, active caspase-3 was undetectable in bone marrow precursor cells, but increased progressively during the process of mast cell maturation, its accumulation coinciding with that of a mast cell-specific secretory granule marker, mouse mast cell protease 6. Together, the current study suggests that active caspase-3 can be stored within secretory compartments of viable mast cells.
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| 7. |
- Garcia-Faroldi, Gianni, et al.
(författare)
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Inhibition of the BET family of epigenetic reader proteins : A novel principle for modulating gene expression in IgE-activated mast cells
- 2017
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Ingår i: Immunity, Inflammation and Disease. - : Wiley. - 2050-4527. ; 5:2, s. 141-150
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: The BET family of bromodomain-containing proteins constitute epigenetic readers that bind to acetylated lysine residues of core histones, thereby translating epigenetic histone marks to effects on gene expression. BET inhibitors are currently emerging as promising therapeutic agents for treatment of various pathological conditions. Here, we explored the potential of using BET inhibition to modulate IgE-mediated responses in mast cells.Methods: We assessed the effects of BET inhibitors PFI-1, I-BET151, and I-BET762 on responses downstream of mast cell activation through IgE receptor cross-linking.Results: BET inhibitors were neither toxic for mast cells (at doses up to 20M), nor did they prevent IgE-mediated mast cell degranulation. However, we found that BET inhibition, in particular by I-BET151, suppressed IL-6 gene expression and IL-6 protein release in response to IgE-mediated mast cell activation. This was observed in both bone marrow-derived mast cells (BMMCs) and in mature peritoneal-cell derived mast cells. Further analysis showed that BET inhibition also suppressed the expression of a number of additional genes of those that were upregulated by IgE receptor cross-linking, including IL-3, IL-7R, CCR1, and ADAMTS9. However, BET inhibition was selective, i.e., several genes that were upregulated by IgE receptor cross-linking were not affected by BET inhibitors.Conclusions: These findings suggest that BET inhibition can interfere with the upregulated expression of selected genes in mast cells activated by IgE receptor cross-linking. Further, our findings introduce the concept of utilizing epigenetic mechanisms for modulating mast cell function in the context of IgE-driven disease.
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| 8. |
- Grujic, Mirjana, et al.
(författare)
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Delayed contraction of the CD8+ T cell response toward lymphocytic choriomeningitis virus infection in mice lacking serglycin
- 2008
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Ingår i: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 181:2, s. 1043-1051
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Tidskriftsartikel (refereegranskat)abstract
- We previously reported that the lack of serglycin proteoglycan affects secretory granule morphology and granzyme B (GrB) storage in in vitro generated CTLs. In this study, the role of serglycin during viral infection was studied by infecting wild-type (wt) mice and serglycin-deficient (SG(-/-)) mice with lymphocytic choriomeningitis virus (LCMV). Wt and SG(-/-) mice cleared 10(3) PFU of highly invasive LCMV with the same kinetics, and the CD8(+) T lymphocytes from wt and SG(-/-) animals did not differ in GrB, perforin, IFN-gamma, or TNF-alpha content. However, when a less invasive LCMV strain was used, SG(-/-) GrB(+) CD8(+) T cells contained approximately 30% less GrB than wt GrB(+) CD8(+) T cells. Interestingly, the contraction of the antiviral CD8(+) T cell response to highly invasive LCMV was markedly delayed in SG(-/-) mice, and a delayed contraction of the virus-specific CD8(+) T cell response was also seen after infection with vesicular stomatitis virus. BrdU labeling of cells in vivo revealed that the delayed contraction was associated with sustained proliferation of Ag-specific CD8(+) T cells in SG(-/-) mice. Moreover, wt LCMV-specific CD8(+) T cells from TCR318 transgenic mice expanded much more extensively in virus-infected SG(-/-) mice than in matched wt mice, indicating that the delayed contraction represents a T cell extrinsic phenomenon. In summary, the present report points to a novel, previously unrecognized role for serglycin proteoglycan in regulating the kinetics of antiviral CD8(+) T cell responses.
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| 9. |
- Grujic, Mirjana, et al.
(författare)
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Distorted Secretory Granule Composition in Mast Cells with Multiple Protease Deficiency
- 2013
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 191:7, s. 3931-3938
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Tidskriftsartikel (refereegranskat)abstract
- Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.
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| 10. |
- Grujic, Mirjana, et al.
(författare)
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Phenotypic Switch and Growth of Melanoma Spheroids in the Presence of Mast Cells : Potential Impact of Nutrient-starvation Effects
- 2023
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Ingår i: Anticancer Research. - : International Institute of Anticancer Research. - 0250-7005 .- 1791-7530. ; 43:4, s. 1415-1426
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Tidskriftsartikel (refereegranskat)abstract
- Background/Aim: Mast cells are abundant in melanoma tumors, and studies suggest that they can be either detrimental or protective for melanoma growth. However, the underlying mechanisms are not fully understood.Materials and Methods: Here, we adopted an established hanging-drop spheroid system to investigate how mast cells influence melanoma growth and phenotype in a 3-D context. To address the underlying mechanism, we conducted transcriptomic and pathway analyses.Results: In the presence of mast cells or mast cell-conditioned medium, growth of melanoma spheroids was profoundly reduced. Transcriptomic analysis revealed that mast cell-conditioned medium had extensive effects on the gene-expression patterns of melanoma. Pathway analyses revealed profound effects on the expression of genes related to amino acid and protein metabolism. The conditioned medium also induced up-regulation of cancer-related genes, including adhesion molecules implicated in metastatic spreading. In line with this, after transfer to a Matrigel extracellular matrix milieu, spheroids that had been developed in the presence of mast cell-conditioned medium displayed enhanced growth and adhesive properties. However, when assessing the possible impact of nutrient starvation, i.e., reduced nutrient content in mast cell-conditioned medium, we found that the observed effects on growth of melanoma spheroids could potentially be explained by such a scenario.Conclusion: Our findings suggest that the phenotypic alterations of melanoma spheroids grown in the presence of mast cells or mast cell-conditioned media are, at least partly, due to nutrient starvation rather than to the action of factors secreted by mast cells. Our findings may provide insight into the effects on gene-expression events that occur in melanoma tumors under nutrient stress.
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