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Isolation and characterization of hepatic mast cells from cholestatic rats
Mast cells (MCs) are immune cells that release histamine and other mediators. MC number increases after bile duct ligation (BDL) and blocking mast cell-derived histamine decrease biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL start...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079802/ https://www.ncbi.nlm.nih.gov/pubmed/27548803 http://dx.doi.org/10.1038/labinvest.2016.89 |
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author | Hargrove, Laura Graf-Eaton, Allyson Kennedy, Lindsey Demieville, Jennifer Owens, Jennifer Hodges, Kyle Ladd, Brittany Francis, Heather |
author_facet | Hargrove, Laura Graf-Eaton, Allyson Kennedy, Lindsey Demieville, Jennifer Owens, Jennifer Hodges, Kyle Ladd, Brittany Francis, Heather |
author_sort | Hargrove, Laura |
collection | PubMed |
description | Mast cells (MCs) are immune cells that release histamine and other mediators. MC number increases after bile duct ligation (BDL) and blocking mast cell-derived histamine decrease biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL starting at 6 hrs and up to 14 days. MC infiltration was evaluated by toluidine blue. BDL rats were perfused using standard collagenase perfusion. Following enzymatic digestion, tissue was passed through a fine gauge needle. Suspensions were incubated with MAb AA4, washed and incubated with goat anti-mouse coated Dynal® beads. MCs were stained with toluidine blue, and in isolated MCs, the expression of FCεRI and MC proteases was measured. The expression of histidine decarboxylase, histamine receptors, VEGF-receptors and TIE 1 and 2 was evaluated by qPCR. Histamine and VEGF-A secretion was measured in MC supernatants. MC purity was evaluated by CK-19, CK-8, albumin, VAP-1 and α-SMA expression. In vitro, cholangiocytes and HSCs were treated with isolated MC supernatants from BDL rats treated with either NaCl or cromolyn sodium (to block MC histamine release) and biliary proliferation and hepatic fibrosis were measured. MCs infiltrate the liver and surround bile ducts starting at day 2. We isolated a virtually pure preparation of mature, functional MCs. TEM images reveal distinct secretory granules and isolated MCs secrete histamine. MCs express FCεRI, chymase, tryptase, RMCPI and RMCPII, but were virtually void of other cell markers. Biliary proliferation and fibrosis increased following treatment with MC supernatants from BDL rats + NaCl and these parameters decreased in cells treated with MC supernatants from BDL + cromolyn sodium. In conclusion, we have isolated and characterized MCs from cholestatic livers. MCs regulate cholestatic liver injury and hepatic fibrosis. This tool provides a better understanding of the paracrine influence of mast cells on biliary/liver pathologies. |
format | Online Article Text |
id | pubmed-5079802 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
record_format | MEDLINE/PubMed |
spelling | pubmed-50798022017-02-22 Isolation and characterization of hepatic mast cells from cholestatic rats Hargrove, Laura Graf-Eaton, Allyson Kennedy, Lindsey Demieville, Jennifer Owens, Jennifer Hodges, Kyle Ladd, Brittany Francis, Heather Lab Invest Article Mast cells (MCs) are immune cells that release histamine and other mediators. MC number increases after bile duct ligation (BDL) and blocking mast cell-derived histamine decrease biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL starting at 6 hrs and up to 14 days. MC infiltration was evaluated by toluidine blue. BDL rats were perfused using standard collagenase perfusion. Following enzymatic digestion, tissue was passed through a fine gauge needle. Suspensions were incubated with MAb AA4, washed and incubated with goat anti-mouse coated Dynal® beads. MCs were stained with toluidine blue, and in isolated MCs, the expression of FCεRI and MC proteases was measured. The expression of histidine decarboxylase, histamine receptors, VEGF-receptors and TIE 1 and 2 was evaluated by qPCR. Histamine and VEGF-A secretion was measured in MC supernatants. MC purity was evaluated by CK-19, CK-8, albumin, VAP-1 and α-SMA expression. In vitro, cholangiocytes and HSCs were treated with isolated MC supernatants from BDL rats treated with either NaCl or cromolyn sodium (to block MC histamine release) and biliary proliferation and hepatic fibrosis were measured. MCs infiltrate the liver and surround bile ducts starting at day 2. We isolated a virtually pure preparation of mature, functional MCs. TEM images reveal distinct secretory granules and isolated MCs secrete histamine. MCs express FCεRI, chymase, tryptase, RMCPI and RMCPII, but were virtually void of other cell markers. Biliary proliferation and fibrosis increased following treatment with MC supernatants from BDL rats + NaCl and these parameters decreased in cells treated with MC supernatants from BDL + cromolyn sodium. In conclusion, we have isolated and characterized MCs from cholestatic livers. MCs regulate cholestatic liver injury and hepatic fibrosis. This tool provides a better understanding of the paracrine influence of mast cells on biliary/liver pathologies. 2016-08-22 2016-11 /pmc/articles/PMC5079802/ /pubmed/27548803 http://dx.doi.org/10.1038/labinvest.2016.89 Text en Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms |
spellingShingle | Article Hargrove, Laura Graf-Eaton, Allyson Kennedy, Lindsey Demieville, Jennifer Owens, Jennifer Hodges, Kyle Ladd, Brittany Francis, Heather Isolation and characterization of hepatic mast cells from cholestatic rats |
title | Isolation and characterization of hepatic mast cells from cholestatic rats |
title_full | Isolation and characterization of hepatic mast cells from cholestatic rats |
title_fullStr | Isolation and characterization of hepatic mast cells from cholestatic rats |
title_full_unstemmed | Isolation and characterization of hepatic mast cells from cholestatic rats |
title_short | Isolation and characterization of hepatic mast cells from cholestatic rats |
title_sort | isolation and characterization of hepatic mast cells from cholestatic rats |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079802/ https://www.ncbi.nlm.nih.gov/pubmed/27548803 http://dx.doi.org/10.1038/labinvest.2016.89 |
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