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Updated: Mar 16, 2026
Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays
Published on: July 4, 2018
Isolation and characterization of hepatic mast cells from cholestatic rats
Laura Hargrove1, Allyson Graf-Eaton2, Lindsey Kennedy2
1Digestive Disease Research Baylor Scott and White Health and Medicine, Temple, TX, USA.
Abstract:
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 decreases biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL starting at 6 h 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 FCɛRI 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 FCɛRI, chymase, tryptase, RMCP-I, and RMCP-II, 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.
Insights
Mast cells infiltrate cholestatic livers, releasing histamine that drives biliary proliferation and fibrosis. Isolating these mast cells (MCs) provides a tool to study their role in liver injury.
Area of Science:
- Immunology
- Gastroenterology
- Cell Biology
Background:
- Mast cells (MCs) are immune cells involved in inflammatory responses.
- Increased MCs are observed in cholestatic liver disease, suggesting a role in pathology.
- Histamine released by MCs has been implicated in biliary proliferation.
Purpose of the Study:
- To isolate and characterize mast cells from cholestatic rat livers.
- To investigate the role of mast cells in regulating cholestatic liver injury and fibrosis.
- To understand the paracrine mechanisms by which mast cells influence biliary pathologies.
Main Methods:
- Bile duct ligation (BDL) in rats to induce cholestasis.
- Isolation of liver cells using collagenase perfusion and magnetic bead separation.
- Characterization of isolated mast cells by microscopy, flow cytometry, and gene expression analysis (qPCR).
- In vitro assays assessing the effects of mast cell supernatants on cholangiocytes and hepatic stellate cells (HSCs).
Main Results:
- Mast cells infiltrated rat livers post-BDL, accumulating around bile ducts.
- A pure population of mature, functional mast cells was successfully isolated.
- Isolated mast cells secreted histamine and expressed key mediators like chymase and tryptase.
- Mast cell supernatants from cholestatic rats exacerbated biliary proliferation and fibrosis in vitro, an effect blocked by inhibiting histamine release.
Conclusions:
- Mast cells are key regulators of liver injury and fibrosis in cholestasis.
- Isolated mast cells serve as a valuable tool for studying their contribution to biliary pathologies.
- Targeting mast cell mediators may offer therapeutic strategies for cholestatic liver diseases.
