*3.6. The Histamine*/*Histamin Receptor Axis*

In the setting of cholangiocarcinoma (CCA), MCs within the tumor environment release histamine, which increases CCA progression and angiogenesis. Conversely, cholangiocytes secrete SCF, which binds and activates the MC growth factor receptor KIT. Cholangiocytes express histidine decarboxylase and its inhibition reduces CCA growth [157]. In the study by Johnson et al., MCs were detected in human CCA biopsies and their recruitment is mediated via SCF in the tumor microenvironment stimulating CCA growth. In xenograft tumor mice treated with the MC stabilizer cromolyn sodium, MC infiltration and tumor growth decreased. Inhibition of SCF in CCA blocked MC migration and MC/EMT/ECM in CCA [157]. MCs migrate into the CCA tumor microenvironment via KIT/SCF and increase tumor progression, angiogenesis, and ECM degradation [157]. In a further study analyzing histamine function, *Mdr2*−/<sup>−</sup> mice (PSC model) were treated with cromolyn. This treatment reduced MC protease mMCPT-1, as well as serum histamine. In agreement, human PSC samples also showed a robust expression of MC markers. Necrosis, lobular damage, and bile duct reactions, such as intrahepatic biliary mass and cholangiocyte proliferation, were also reduced via MC silencing using cromolyn in *Mdr2*−/<sup>−</sup> mice.

As a confirmation of the in vivo analysis, co-culture experiments of cholangiocytes with MCs induced cholangiocyte proliferation, α-SMA, fibronectin-1, and TGF-β1 production, functions that are blocked in MC depleted using an siRNA approach targeting histidine decarboxylase. In a similar experiment, MCs induced PCNA, α-SMA, and fibronectin-1 in human HSC, and these effects were also blocked by the knockdown of HDC in MCs. These in vitro results imply that cholangiocyte and HSC responses to MCs are dependent on the histamine secreted by MCs [158]. Nevertheless, the in vivo results using cromolyn have to be handled with caution because this substance directly affects HSCs and hepatocytes. Cromolyn was originally developed as an MC stabilizer. However, the activation and collagen accumulation for the HSC cell lines LX2 and HSC-T6 were reduced by 50% after cromolyn treatment at a low concentration without signs of apoptosis. Furthermore, cromolyn treatment compromised the TGF-β-induced epithelial to mesenchymal transition and replicative senescence rate of hepatocytes, which are generally associated with fibrogenesis. Taken together, cromolyn may be the basis for an effective cure for fibrosis and cirrhosis because it targets both HSCs and hepatocytes [159].

In a more reliable/specific setting to analyze the function of MC histamine, Kennedy et al. used histamine receptor antagonists. These reduced the lobular damage, necrosis, and inflammation in *Mdr2*−/<sup>−</sup> mice. The histamine receptors H1 (H1R) and H2 (H2HR) are upregulated in cholangiocytes of *Mdr2*−/<sup>−</sup> mice, as well as in human PSC and CCA. Blocking of the histamine receptor leads to decreased activation of MCs as evaluated via the detection of MC markers in *Mdr2*−/<sup>−</sup> mice and in the CCA model [160]. Histamine serum levels decrease in *Mdr2*−/<sup>−</sup> and CCA mice in the presence of respective blockers. Bile duct reaction, i.e., biliary proliferation and intrahepatic bile duct mass, were reduced. Treated *Mdr2*−/<sup>−</sup> mice had a lower collagen deposition and lower expression of the HSC activation marker synaptophysin-9 (Syp-9). Histamine receptor blockers do not affect activation of HSCs directly [160]. The tumor growth is blocked in the CCA model and Ki67-positive proliferating cholangiocytes were reduced. In a co-culture/conditioned medium model, MCs increased the proliferation of cholangiocytes and progression of CCA, and this effect was reduced in the presence of inhibitors. In summary, histamine receptor blockers reduce PSC and CCA progression [160]. As a general feature, MCs stimulate fibroblast proliferation [161,162] and collagen synthesis [163,164] via histamine secretion. Moreover, it has been shown that MCs themselves are competent at expressing basement membrane components, including collagen IV and laminin [165].

An early study by Akiyoshi et al. reported that MCs, portal myofibroblasts, and cholinergic nerve terminals work synergistically to promote liver fibrosis [137], demonstrating that the paracrine influence from MCs is not limited to altered HSCs and cholangiocyte function.
