Immunodetection of cyclooxygenase-2 (COX-2) is restricted to tissue macrophages in normal rat liver and to recruited mononuclear phagocytes in liver injury and cholangiocarcinoma

Abstract

It has been suggested that cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis is associated with liver inflammation and carcinogenesis. The aim of this study is to identify the cellular source of COX-2 expression in different stages, from acute liver injury through liver fibrosis to cholangiocarcinoma (CC). We induced in rats acute and "chronic" liver injury (thioacetamide (TAA) or carbon tetrachloride (CCl(4))) and CC development (TAA) and assessed COX-2 gene expression in normal and damaged liver tissue by RT-PCR of total RNA. The cellular localization of COX-2 protein in liver tissue was analyzed by immunohistochemistry as well as in isolated rat liver cells by Western blotting. The findings were compared with those obtained in human cirrhotic liver tissue. The specificity of the antibodies was tested by 2-DE Western blot and mass spectrometric identification of the positive protein spots. RT-PCR analysis of total RNA revealed an increase of hepatic COX-2 gene expression in acutely as well as "chronically" damaged liver. COX-2-protein was detected in those ED1(+)/ED2(+) cells located in the non-damaged tissue (resident tissue macrophages). In addition COX-2 positivity in inflammatory mononuclear phagocytes (ED1(+)/ED2(-)), which were also present within the tumoral tissue was detected. COX-2 protein was clearly detectable in isolated Kupffer cells as well as (at lower level) in isolated "inflammatory" macrophages. Similar results were obtained in human cirrhotic liver. COX-2 protein is constitutively detectable in liver tissue macrophages. Inflammatory mononuclear phagocytes contribute to the increase of COX-2 gene expression in acute and chronic liver damage induced by different toxins and in the CC microenvironment.

Fig. 1

Changes of COX-2 gene expression in rat liver tissue are shown at various time points during (a) acute liver injury after TAA (top) or CCl₄ (bottom) administration and (b) chronic liver injury as well as in dissected CC tissue after TAA administration. The graphics represent the COX-2-specific mRNA amount normalized using UBC as housekeeping gene. Error bars represent standard deviation (control 0 h n = 4, treatment n = 4, CC n = 4). h hours, w weeks, CC dissected cholangiocarcinoma tissue, *P < 0.05 against control group according to the Mann–Whitney U test

Fig. 2

Immunolocalization of COX-2 protein by double immunofluorescence staining (CK-19, Hep Par-1, and ED1) in normal rat liver. Immunofluorescence staining of a: COX-2 (red) and CK-19 (green); b: COX-2 (red) and Hep Par-1 (green); c: COX-2 (red) and ED1 (green). Co-localization of COX-2 and ED1 is detectable. Polyclonal COX-2 antibody (ab15191) was used. The blue staining with DAPI represents the nuclei (×100/×400 original magnification). pv portal vein, bd bile duct. Bar 50 μm

Fig. 3

Immunolocalization of COX-2, ED1 and ED2 in TAA-induced acute rat liver injury (48 h). An immunofluorescence staining of COX-2- (red), ED1- (green, left side), and ED2⁻ (green, right side) positive cells is shown. Polyclonal COX-2 antibody (ab15191) was used. Staining with DAPI represents the nuclei (×100/×200 original magnification). pv portal vein. Bar 50 μm

Fig. 4

Immunolocalization of COX-2, CK-19, Hep Par-1, and ED1 in TAA-induced chronic rat liver injury (16 weeks). Immunofluorescence staining of a: COX-2 (red) and CK-19 (green); b: COX-2 (red) and Hep Par-1 (green); c: COX-2 (red) and ED1 (green). Co-expression of COX-2 and ED1 is detectable only within regenerating nodules. Polyclonal COX-2 antibody (ab15191) was used. The blue staining with DAPI represents the nuclei (×100/×400 original magnification). RN regenerating nodules, CC cholangiocarcinoma. Bar 50 μm

Fig. 5

Immunolocalization of COX-2, CK-19, Hep Par-1, and CD68 in cirrhotic human liver. Immunofluorescence staining of a: COX-2 (red) and CK-19 (green); b: COX-2 (red) and Hep Par-1 (green); c: COX-2 (red) and CD68 (green). Co-expression of COX-2 and CD68 is detectable only within regenerating nodules. Polyclonal COX-2 antibody (ab15191) was used. The blue staining with DAPI represents the nuclei (×100/×400 original magnification). pv portal vein, bd bile duct, RN regenerating nodule. Bar 50 μm

Fig. 6

Immunolocalization of COX-2 after TAA-induced acute rat liver injury (96 h). The following antibodies were used for immunohistological staining: monoclonal COX-2 antibody (33/Cox-2), monoclonal ED1 antibody (ED1) and monoclonal ED2 antibody (ED2). To visualize the antigen/antibody reaction, the classic alkaline phosphatase antialkaline phosphatase (APAAP) technique was used. The remaining tissue is made visible by counterstaining with hematoxylin. a COX-2 in the undamaged liver of a control animal (100-fold magnification). b Detail of a (400-fold magnification): a distinct COX-2 positivity is seen in cells (tissue macrophages) within the liver parenchyma (black arrows). Scattered cells with a weak positivity can be seen (white arrows) in the area of the portal field. c COX-2 after TAA-induced acute rat liver injury (96 h). d Detail of c (400-fold magnification): A weak COX-2 positivity is detectable in cells in the area of injury (white arrows). e Serial section. In the area of damage a variety of cells (recruited inflammatory macrophages) with a significant ED1 positivity can be detected. f Detail of e (400-fold magnification). g Serial section. ED2-positive cells are detectable in the area of injury. The intensity in the area of injury is lower than in the area of the undamaged parenchyma. h Detail of g (400-fold magnification). A weak ED2 positivity is detectable in cells in the area of injury (black arrows). Bar 50 µm

Fig. 7

Immunolocalization of COX-2 in TAA-induced cholangiocarcinoma. For immunostaining the monoclonal COX-2 antibody (33/Cox-2) was used. To visualize the antigen/antibody reaction, the classic alkaline phosphatase antialkaline phosphatase (APAAP) technique was used. The remaining tissue is made visible by counterstaining with hematoxylin (×100/×400 original magnification). The black arrows indicate COX-2-positive cells in the liver parenchyma. The white arrows point to COX-2-positive cells within the tumor microenvironment (lower images, 400-fold magnification). Tumor cells are COX-2 negative. Bar 50 μm

Fig. 8

a COX-2 antibody binding was tested using protein from macrophage cell line RAW 264.7 as a positive control. Total protein extracts from unstimulated cells (RAW⁻) and LPS/PMA-stimulated cells (RAW⁺) were analyzed. b COX-2 protein expression in hepatocytes (HCs), (myo)fibroblasts (MFs), and Kupffer cells (KCs) isolated from rat liver. COX-2 protein (70 kDa) is clearly visible in isolated KCs. β-actin (43 kDa) was used as an internal standard. c COX-2 mRNA expression increases in isolated Kupffer cells after LPS-administration. d Shown is the COX-2 protein amount in isolated macrophages after CCl₄-induced acute liver injury of fractions 3 (inflammatory macrophages) and 4 (resident macrophages)

Fig. 9

Shown are the results from 2-DE Western blot analysis. After ECL staining numerous spots are visible when the polyclonal antibody (ab15191) and total protein from rat liver were used (left side). A spot in the field of 70 kDa is not visible. The results of mass spectrometry are indicated in the figure and marked with arrows. Using total protein of the macrophage cell line RAW 264.7, two spots were visible (right side). While one spot (in the range of 50 kDa) could not be located in the silver-stained gel (small box), the evaluation of the second spot (in the range just below 75 kDa) resulted in COX-2 (prostaglandin synthase 2, PGH2). NI not identified