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Human connective tissue mast cell proteome

ABSTRACT: Mast cells are hematopoietic cells that reside preferentially in tissues exposed to internal and external environments. Mast cells sense immunological, inflammatory and environmental stimuli, and can be activated to release granules and generate inflammatory mediators. Mast cell-derived products confer protection against snake venoms and some parasite infections. Aberrant activation of mast cells is a major contributor to human pathology, including allergy, asthma and adverse drug reactions. Their strict tissue location has largely impeded the isolation of large numbers of primary mast cell for further analysis. To better understand the biology of mast cells, we analyzed the proteome of primary human and mouse mast cells by quantitative mass spectrometry. We identified a mast cell-specific protein signature that was conserved from mouse to man. Compared to a comprehensive set of other immune cell lineages, proteome analysis identified a unique and distant mast cell cluster. The mast cell signature included proteins governing granule biosynthesis and secretion, as well as proteoglycan- and neurotransmitter metabolism. Proteome conservation across species suggests evolutionary maintenance of mast cell functions.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Connective Tissue Type Mast Cell

SUBMITTER: Thomas Plum

LAB HEAD: Hans-Reimer Rodewald

PROVIDER: PXD014978 | Pride | 2020-02-12

REPOSITORIES: Pride

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Project description:Background: Exosomes are nanovesicles of endocytic origin believed to be involved in communication between cells. Recently, it has been shown that mast cell exosomes contain RNA named "exosomal shuttle RNA". The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34 positive progenitor cells. Results: Exosomes from the human mast cell line HMC-1 contain RNA. The exosomes contain no or very little ribosomal RNA compared to their donor cells. The mRNA and microRNA content in exosomes and their donor cells was examined using microarray analyses. We found 116 microRNA in the exosomes and 134 microRNA in the cells, from which some were expressed at different level. DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. Transfer experiments revealed that exosomes and their RNA can transfer to other HMC-1 cells and to CD34 positive progenitors. Conclusions: To conclude, HMC-1 exosomes contain mRNA and microRNA that can be transferred to other mast cells and to CD34 progenitors. This shuttle of exosomal RNA may represent a powerful mode of communication between cells where cells send genetic information to other cells over a distance via exosomes. [miRNA profiling] Identification of microRNA was performed by Exiqon (www.exiqon.com). Briefly, the quality of the total RNA was verified by an Agilent 2100 Bioanalyzer. Total RNA from the exosome and the HMC-1 cell samples were labelled with Hy3 and Hy5 fluorescent stain, respectively, using the miRCURY Hy3/Hy5 power labelling kit. The Hy3-labelled exosome samples and a Hy5-labeled mast cells were mixed pair-wise and hybridized to the miRCURY? LNA array (v9.2). The hybridization was performed according to the miRCURY? LNA array manual using a Tecan HS4800 hybridization station (Tecan Systems, Inc. San Jose, CA). The miRCURY? LNA array microarray slides were scanned by a ScanArray 4000 XL scanner (Packard Biochip Technologies, Billerica, MA ,USA) and the image analysis was carried out using the ImaGene 6.1.0 software (BioDiscovery, Inc, El Segundo, CA USA). The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. MicroRNA with signals equal to or below the background signal in 2 or more of the 4 replicate measurements were identified as absent in that slide. The limit for a miRNA to be listed as detectable was set to signal intensities higher than 3 x background (3 x median Hy3 or Hy5 for the total slide). In addition, where signals were detected for <3 of the slides, they were considered unreliable and excluded from sets of detected miRNAs. The experiment was performed in triplicate samples. The signal was calculated as the mean value of the log2MeanRatio Hy3/Hy5 for the triplicates M-BM-1 SD. [mRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol . The microarray experiments were performed by SweGene (www.swegene.org/) according to Affymetrix microarray DNA chip analysis (n=4). p0739_E1, p0739_ E2, p0739_E3 and p0739_E4 for the exosomes samples and p0739_C1, p0739_C2, p0739_C3, and p0739_C4 for the HMC-1 cells. [miRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol followed by RNeasy clean-up. The microarray experiments were performed by Exiqon.

Project description:Left heart disease (LHD) frequently causes lung vascular remodelling and pulmonary hypertension (PH). Yet, pharmacological treatment for PH in LHD is lacking and its pathophysiological basis remains obscure. We aimed to identify candidate mechanisms of PH in LHD and to test their relevance and therapeutic potential. In rats, LHD was induced by supracoronary aortic banding. Whole genome microarray analyses were performed, candidate genes were confirmed by RT-PCR and Western blots and functional relevance was tested in vivo by genetic and pharmacological strategies. In lungs of LHD rats, mast cell activation was the most prominently upregulated gene ontology cluster. Mast cell gene upregulation was confirmed at RNA and protein levels and remodelled vessels showed perivascular mast cell accumulations. In LHD rats treated with the mast cell stabiliser ketotifen, or in mast cell deficient Ws/Ws rats, PH and vascular remodelling were largely attenuated. Both strategies also reduced PH and vascular remodelling in monocrotaline-induced pulmonary arterial hypertension, suggesting that the role of mast cells extends to noncardiogenic PH. In PH of different aetiologies, mast cells accumulate around pulmonary blood vessels and contribute to vascular remodelling and PH. Mast cells and mast cell-derived mediators may present promising targets for the treatment of PH. Whole rat genome microarray analyses were performed in lung homogenates of three rats with established PH following supracoronary aortic banding and in three sham-operated controls. Out of a total of 28,000 analysed genes, differential expression defined as 2-fold change with p<0.05 was evident for 120 genes. Of these, 76 were upregulated and 44 downregulated in aortic banding compared with control lungs gene. Enrichment analysis revealed regulation of mast cell specific genes - 13 out of 20 genes were significantly upregulated in aortic banding compared with control lungs. To test for a putative functional role of mast cells in lung vascular remodelling and PH in LHD, we applied a pharmacological approach by treatment of aortic banding rats with the mast cell stabiliser ketotifen. Microarray analysis then compared rats that were treated with untreated. In brief, congestive heart failure (CHF) was induced in juvenile rats by supracoronary aortic banding and rats were analyzed 9 weeks thereafter when they had established left heart failure with preserved ejection fraction and secondary pulmonary hypertension. Three heart failure rats were untreated, and 3 received the mast cell stabilizer ketotifen (1 mg/kg-1 bodyweight/day-1) with the drinking water. Sham rats underwent the same surgical procedure but without placement of a clip on the supracoronary aorta. Microarray analyses: Lungs from banded and control rats were excised and total RNA was extracted (Stratagene Absolutely RNA Miniprep Kit; Stratagene, La Jolla, USA). Three Âµg total RNA from three control and three banded rats each were processed according to the One-Cycle Target Labeling protocol (GeneChip Expression Analysis, Affymetrix, Santa Clara, USA). Before and after amplifications, the total RNA/complementary RNA concentrations were checked with NanoDrop ND-1000 (Thermo Scientific, Wilmington, USA) and quality was controlled (Experion electrophoresis station, BioRad; Hercules, USA). Samples were hybridized to GeneChipÂ® Rat Genome 230 2.0 arrays (Affymetrix; Santa Clara, USA) containing 31,000 probe sets covering 28,000 rat genes.

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Human connective tissue mast cell proteome

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