Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents

Abstract

Cholangiopathies are characterized by dysregulation of the balance between biliary growth and loss. We have shown that histamine (HA) stimulates biliary growth via autocrine mechanisms. To evaluate the paracrine effects of mast cell (MC) stabilization on biliary proliferation, sham or BDL rats were treated by IP-implanted osmotic pumps filled with saline or cromolyn sodium (24 mg/kg BW/day (inhibits MC histamine release)) for 1 week. Serum, liver blocks and cholangiocytes were collected. Histidine decarboxylase (HDC) expression was measured using real-time PCR in cholangiocytes. Intrahepatic bile duct mass (IBDM) was evaluated by IHC for CK-19. MC number was determined using toluidine blue staining and correlated to IBDM. Proliferation was evaluated by PCNA expression in liver sections and purified cholangiocytes. We assessed apoptosis using real-time PCR and IHC for BAX. Expression of MC stem factor receptor, c-kit, and the proteases chymase and tryptase were measured by real-time PCR. HA levels were measured in serum by EIA. In vitro, MCs and cholangiocytes were treated with 0.1% BSA (basal) or cromolyn (25 mM) for up to 48 h prior to assessing HDC expression, HA levels and chymase and tryptase expression. Supernatants from MCs treated with or without cromolyn were added to cholangiocytes before measuring (i) proliferation by MTT assays, (ii) HDC gene expression by real-time PCR and (iii) HA release by EIA. In vivo, cromolyn treatment decreased BDL-induced: (i) IBDM, MC number, and biliary proliferation; (ii) HDC and MC marker expression; and (iii) HA levels. Cromolyn treatment increased cholangiocyte apoptosis. In vitro, cromolyn decreased HA release and chymase and tryptase expression in MCs but not in cholangiocytes. Cromolyn-treated MC supernatants decreased biliary proliferation and HA release. These studies provide evidence that MC histamine is key to biliary proliferation and may be a therapeutic target for the treatment of cholangiopathies.

Isolation and characterization of hepatic mast cells from cholestatic rats

“…Primers and PCR reagents were obtained from Qiagen (SABiosciences, Valencia, CA) and Bio-Rad. Histamine EIA kits were obtained from Cayman Chemical (Ann Arbor, MI)(14, 15). The antibody, Anti-Rat Mast Cells, clone AR32AA4 (MAb AA4) used to isolate hepatic mast cells was purchased from BD Biosciences (San Jose, CA), this antibody has been used by other groups to isolate mature mast cells from bone marrow and the peritoneal cavity(18, 19).…”

“…Because the number of mast cells in normal tissues is relatively low(14), we used male Fisher 344 rats (Charles Rivers, 150 – 175 gm) subjected to bile duct ligation (BDL). BDL surgeries were performed as previously described by us and according to full adherence to local Institutional Animal Care and Usage Committee at Baylor Scott & White Health(14, 20).…”

“…During cholestatic liver injury cholangiocytes proliferate and secrete numerous factors including histamine and vascular endothelial growth factor(13, 14). We have shown that histamine regulates cholangiocyte proliferation via autocrine signaling, which allows for cholangiocyte homeostasis to be maintained(15).…”

“…We have shown that histamine regulates cholangiocyte proliferation via autocrine signaling, which allows for cholangiocyte homeostasis to be maintained(15). During PBC and PSC, patients have increased amounts of circulating histamine in their serum(16) and in rats subjected to bile duct ligation (to induce cholestasis), both serum and cholangiocyte histamine release is increased(14, 17). We have demonstrated that mast cells infiltrate the liver following bile duct ligation (BDL) and that blocking mast cell-derived histamine using cromolyn sodium decreases biliary mass, proliferation and histamine secretion(14).…”

“…During PBC and PSC, patients have increased amounts of circulating histamine in their serum(16) and in rats subjected to bile duct ligation (to induce cholestasis), both serum and cholangiocyte histamine release is increased(14, 17). We have demonstrated that mast cells infiltrate the liver following bile duct ligation (BDL) and that blocking mast cell-derived histamine using cromolyn sodium decreases biliary mass, proliferation and histamine secretion(14). Further, human cholangiocarcinoma tumors exhibit large numbers of mast cells and cromolyn sodium decreases tumor growth(12).…”