*3.4. Immunohistochemistry*

The epithelial cell marker (pan-cytokeratin) was strongly expressed in the epithelium of bile ducts and, with less intensity, in acute and chronic biliary ductular reaction. This antibody was especially useful for the identification of the bile duct epithelium of damaged bile ducts that developed granulomas composed of macrophages, as well as the ectatic bile ducts surrounded by fibrous tissue in portal tracts (Figure 3A,B).

In the acute phase, there was an increase of α-SMA-positive into the parenchyma in areas of canalicular cholestasis and hepatocyte necrosis as well as within portal tracts around proliferating typical bile ductules (Figure 3C). In chronic FE these cells showed strong immunopositivity and accumulate surrounding bile ductular structures and ectatic bile ducts in fibrotic portal tracts and septa (Figure 3D). In this last case α-SMA-positively immunolabelled HSCs, appeared as spindle-shaped cells that delimited both bile ducts and atypical ductules. α-SMA was not expressed in ductular cells, cholangyocytes or hepatocytes. An increase in the number of macrophages and PMNs that stained positively for both anti-lysozyme and anti-MAC387 antibodies was observed in the areas of canalicular cholestasis and hepatocyte necrosis in acute FE liver lesions (Figure 4A). Many cells with the characteristic morphology of KCs have strong cytoplasmic labelling for lysozyme, and MAC387 immunostaining clearly defined cell aggregates, with extensive immunoreactivity, scattered in the liver parenchyma, both in zones 2 and 3 of the hepatic lobes. In addition MAC387 positivity was observed in cells having the morphology of blood monocytes within blood vessels.

**Figure 3.** Immunohistochemistry in naturally acute and chronic acquired cases of FE in sheep. (**A**) Ectatic interlobular bile duct surrounded by a thin layer of collagen fibers ('onion skin fibrosis'). HE. Bar, 50 μm. (**B**) Pancytokeratin antibody stain positively ectatic bile ducts with flattened epthelium, similar to showed in Figure 3A, and bile ductules. Bar, 50 μm. ( **C**) α-SMA-+ HSCs in areas of canalicular cholestasis in acute FE liver lesions. Bar, 50 μm. ( **D**) In chronic liver lesions observed in FE, α-SMA-+ HSCs cells accumulated and surrounded ectatic bile ducts. Bar, 50 μm.

In the chronic hepatic lesions, abundant pigmented macrophages in close association with remnants of hepatocyte lobules, were positive for lysozyme and MAC387. Also, strong lysozyme and MAC387 positively immunolabelled macrophages were found as part of the granulomatous lesions formed around degenerated bile ducts. Remarkably, a decrease in the number of cells immunostained with these antibodies were observed in the fibrous septa in relation to atypical ductular proliferation. The mannose receptor (CD206) staining was only present in some pigmented macrophages observed as scattered cells in the portal tracts (acute phase), but were more numerous in chronic lesions, in fibrotic septa with marked ductular reaction (Figure 4B). No co-localization was found with lysozyme and MAC387 markers within cells in the hepatic lobes. It is remarkable that cells positively immunostained for CD206 antibody corresponded to macrophages showing intense TGF-β

immunoreactivity. (Figure 4C). Sparse to intense intracellular staining for TGF- β was also observed in α-SMA-positive cells present in the thickened subendothelial areas and in the tunica media of hepatic arteries (Figure 4D).

In acute FE lesions, intrahepatic T CD3+-lymphocytes were scattered in sinusoids and portal tracts and occasionally were seen in intraepithelial location in bile ducts. In chronic FE lesions, there was a prominent T lymphocytic infiltrate forming aggregates intimately associated with the bile ducts, with a diffuse pattern or forming aggregates in areas of bile ductular reaction in the fibrous septa (Figure 4E). T CD3-positively immunostained lymphocytes were identified in granulomas around degenerate bile ducts and extravasated bile pigment as well as surrounding large bile ducts contain bile stones. IgG+ plasma cells were occasionally seen in portal tracts in acute FE liver lesions. In chronic cases, plasma cells were found scattered or in small amounts followed a similar distributional pattern than T lymphocytes, although they were less abundant. Like T lympohocytes, plasma cells were found in the concentric arrangemen<sup>t</sup> of fibrous tissue around bile ducts and ductules (Figure 4F).

**Figure 4.** Immunohistochemistry in naturally acute and chronic acquired cases of FE in sheep. (**A**) Intense positive MAC387 immunolabeling of a cluster of macrophages and neutrophils in an area of cholestasis (acute lesion). Bar, 50 μm. (**B**) Presence of CD206-+ macrophages in areas of fibrosis and ductular reaction (DR) (chronic lesion). Bar, 50 μm. ( **C**) Anti TGF-β antibody red stained cells morphologically compatible with macrophages in areas of DR (chronic lesion). Bar, 50 μm. (**D**) Positive immunolabeling for anti TGF-β antibody in a hepatic artery with an occlusive lesion (subintimal proliferation) in chronic FE cases. Bar, 50 μm. (**E**) Aggregates of T CD3-+ lymphocytes in areas of DR in chronic FE lesions. Bar, 50 μm. (**F**) In chronic FE lesions IgG-+ plasma cells were seen scattered around ectatic bile ducts. Bar, 20 μm.
