Isolation and characterization of hepatic mast cells from cholestatic rats

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 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.

Cellular crosstalk during cholestatic liver injury

“… 51 These studies are the first to demonstrate a synergistic interaction between MCs, cholangiocytes, and HSCs during cholestatic liver injury. 51 , 75 In addition, these studies indicate that MCs and MC-derived HA are key regulators of the biliary response in addition to the activation of HSCs during cholestatic-induced hepatic fibrosis.…”

“…The authors found that treatment with BDL saline-treated MC culture supernatants increased biliary proliferation and HSC activation, demonstrating a direct interaction between MCs, cholangiocytes, and HSCs. 75 More interestingly, when cultured cholangiocytes or HSCs were stimulated with culture supernatants of MCs collected from cromolyn sodium-treated BDL rats, biliary proliferation and HSC activation were reduced significantly. These studies support a strong relationship and regulation between MCs and HSCs during biliary damage and repair.…”

“…Hargrove et al have shown that infiltrated MCs settle in close proximity to bile ducts, but not within the hepatocyte parenchyma, demonstrating that they are in close contact to interact with proliferating cholangiocytes. 75 Furthermore, MCs have been isolated from BDL rats at day 14, which were treated with saline or the MC stabilizer cromolyn sodium. Isolated MC culture supernatants were collected and placed on cultured cholangiocytes or HSCs.…”

Mast Cells in Liver Fibrogenesis

“…The infiltration (of myeloid lineage-derived cells from the bone marrow) and accumulation (differentiation of liver resident precursor cells, which can be differentiated in the presence of SCF) of MCs in the insulted liver has been shown in several studies and is unquestionable [101]. Even more, the isolation of MCs from liver of BDL-treated animals has been established [102]. It should be noted that not only the presence of MCs itself is of vital importance, but also the type of MCs within the liver (i.e., CTMCs or MMCs), when the functional interplay of cells is considered.…”

“…A study in human liver tissue demonstrated that the amount of tryptase/chymase-positive MCs is higher than tryptase-positive cells in healthy and chronically diseased livers [104]. The cells isolated from BDL rats were chymase- and tryptase-positive, reflecting that these cells most likely represent CTMCs [102].…”