Potential interaction between CCR1 and its ligand, CCL3, induced by endogenously produced interleukin-1 in human hepatomas

Abstract

Hepatoma cell lines can produce a massive amount of chemokines in response to various stimuli including hepatitis viruses and their products. However, it remains elusive on the types of chemokine receptor(s) expressed in the hepatoma tissues and its roles in hepatoma development. To clarify these points, we examined the chemokine receptor expression in six human hepatoma cell lines. All of the hepatoma cell lines constitutively and exclusively expressed CCR1 mRNA and its protein on their cell surface. CCR1 expression was also detected on hepatoma cells and to a lesser degree, on endothelial cells in hepatoma tissues but not in normal liver tissues. Furthermore, CCL3 expression was detected in hepatoma cells, endothelial cells, and to a lesser degree, fibroblast-like cells in hepatoma tissue, whereas only occasional vascular endothelial cells and inflammatory cells in normal liver tissues were weakly positive for CCL3. Moreover, the forskolin-mediated increases in intracellular cAMP concentrations were inhibited by the ligands for CCR1, CCL3, CCL4, and CCL5, suggesting that the expressed CCR1 was functional. Four hepatoma cell lines produced CCL3 only in response to interleukin (IL)-1 alpha and IL-1 beta. Finally, IL-1 alpha and IL-1 beta were detected abundantly in hepatoma tissues but not in normal liver tissues. Thus, IL-1 may enhance the local production of CCL3, which may interact with CCR1 expressed on hepatoma cells, in an autocrine and/or paracrine manner.

Figure 1.

Expression of chemokine receptor mRNAs of human hepatoma cell lines by RT-PCR. Total RNA was extracted from HuH7 (A), HepG2 (B), HLE (C), SK-Hep1 (D), HLF (E), and Hep3B (F) hepatoma cell lines and human peripheral blood mononuclear cells (G) and amplified by RT-PCR to detect chemokine receptor mRNAs as described in Materials and Methods. As a positive control, RNA from peripheral blood mononuclear cells was used. Analysis for CCR1 expression was performed without reverse transcriptase treatment and the results are shown as no-RT-PCR in the lowest panel. β-actin served as a control for sample loading.

Figure 2.

CCR1 protein expression of hepatoma cell lines. A: An immunocytochemical analysis of CCR1 protein expression in hepatoma cell lines. Localization of CCR1 in HuH7 (A), HepG2 (B), HLE (C), SK-Hep1 (D), HLF (E), and Hep3B (F) were determined immunocytochemically using specific polyclonal rabbit anti-human CCR1 antibodies as described in Materials and Methods. As a control, the first antibody was replaced by normal rabbit IgG (a, b, and c) or by primary antibodies absorbed with respective peptide antigen (d, e, and f). Scale bar, 50 μm; original magnifications, ×400. B: Flow cytometric analysis of cell-surface CCR1 expression by hepatoma cell lines. Cells (5 to 10 × 10⁵) were incubated with rabbit anti-human CCR1 or normal rabbit IgG as an isotype control. Subsequently, cells were stained with fluorescein isothiocyanate-labeled goat anti-rabbit IgG. Open heavy-lined and filled histograms represent those with the test antibody (anti-CCR1) and the control IgG, respectively. The representative results from three independent experiments are shown.

Figure 3.

Effects of ligands for CCR1 on forskolin-stimulated cAMP production in a hepatoma cell line. A: Hep3B hepatoma cells were incubated for 20 minutes without (untreated) or with 10 μmol of forskolin at the indicated concentrations of human CCL3, CCL4, and CCL5. Then, intracellular cAMP levels were measured and are expressed in fmol/well. One representative result from three independent experiments is shown. Data are expressed as mean ± SD of data in triplicates. B: Hep3B cells were incubated for 20 minutes without (untreated) or with 10 μmol of forskolin in the absence or presence of 10 ng/ml of human CCL11 and CCL3 with or without preincubation with anti-CCR1 antibodies (10 μg/ml). Then, intracellular cAMP levels were measured and are expressed in fmol/well. One representative result from three independent experiments is shown. Data are expressed as mean ± SD of data in triplicates.

Figure 4.

Immunohistochemical analysis to identify CCR1. Human hepatoma tissues (A to C) or normal liver tissues (D) were immunostained with anti-CCR1 antibodies as described in Materials and Methods. B represents the square in A at the higher magnification. Arrows in B indicate the positively stained hepatoma cells. Arrows in C indicate the positively stained vascular endothelial cells and small bile duct epithelial cells. Representative results shown in Table 2 ▶ are shown. Scale bars, 50 μm. Original magnifications: ×100 (A, C, and D); ×400 (B).

Figure 5.

Immunohistochemical analysis to identify CCL3. HuH7 (A), HepG2 hepatoma cell lines (B), human hepatoma tissues (C to E), or normal liver tissues (F) were immunostained with anti-CCL3 antibodies as described in Materials and Methods. D represents the square in C at the higher magnification. Arrows in D indicate the positively stained hepatoma cells. Arrows in E indicate the positively stained vascular endothelial cells and small bile duct epithelial cells. Representative results shown in Table 2 ▶ are shown. Scale bars, 50 μm. Original magnifications: ×400 (A, B, and D); ×100 (C, E, and F).

Figure 6.

CCL3 production of hepatoma cell lines in response to IL-1α or IL-1β. Hepatoma cell lines were incubated at a concentration of 1 × 10⁵ cells in 1 ml of DMEM with 10% fetal bovine serum after 24 hours in the absence or presence of the indicated concentration of human IL-1α or IL-1β. Then, the supernatants were collected to measure CCL3 contents as described in Materials and Methods. A representative result from three independent experiments is shown. Mean and SD are shown.

Figure 7.

Total RNAs were extracted from HuH7 or HLE cell lines, which were incubated in the presence or the absence of IL-1α (100 ng/ml) or IL-1β (100 ng/ml) for 6 hours. RT-PCR was performed on the obtained total RNAs to detect CCL3 as described in Materials and Methods. Amplification of β-actin is shown to ensure the same amount of used cDNA. A representative result from three independent experiments is shown.

Figure 8.

Immunohistochemical analysis of IL-1α. Human hepatoma tissues (A to C) and normal liver tissues (D) were immunostained with anti-IL-1α antibodies as described in Materials and Methods. B and C represent the squares in A at the higher magnification. Arrows in B indicate the positively stained hepatoma cells. Arrows in C indicate the positively stained vascular endothelial cells and small bile duct epithelial cells. Representative results are shown here. Scale bars, 50 μm. Original magnifications: ×40 (A); ×400 (B); ×200 (C); ×100 (D).