Ferritin Expression in Rat Hepatocytes and Kupffer Cells after Lead Nitrate Treatment

Abstract

Lead nitrate induces hepatocyte proliferation and subsequent apoptosis in rat livers. Iron is a constituent of heme and is also required for cell proliferation. In this study, the expression of ferritin light-chain (FTL), the major iron storage protein, was investigated in rat livers after a single intravenous injection of lead nitrate. Western blotting and immunohistochemistry revealed that FTL was increased in hepatocytes around the central veins and strongly expressed in nonparenchymal cells. Some FTL-positive nonparenchymal cells were identified as Kupffer cells that were positive for CD68. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in the periportal and perivenous areas. The relationships between FTL expression and apoptosis induction or the engulfment of apoptotic cells were examined. TUNEL-positive cells were increased in the treatment group, and enhanced expression of milk fat globule EGF-like 8 was demonstrated in some Kupffer cells and hepatocytes, indicating enhanced apoptosis induction and phagocytosis of apoptotic cells. FTL-positive Kupffer cells were not detected without lead nitrate treatment or in rat livers treated with clofibrate, which induces hepatocyte proliferation but not apoptosis. These results suggest that FTL expression in Kupffer cells after lead treatment is dependent on phagocytosis of apoptotic cells.

Introduction

Lead is a multitargeted toxicant, causing effects in the gastrointestinal tract, hematopoietic system, cardiovascular system, nervous system, and other systems (Needleman and Landrigan 1981). The metal blocks heme synthesis by inhibiting activities of δ-aminolevulinic acid dehydratase and ferrochela-tase, resulting in development of anemia and impaired functions of heme-containing enzymes and proteins in many organs, as described above (Jover et al. 1996; Moore et al. 1987).

Intravenous injection of lead nitrate into rats leads to marked liver enlargement and hepatocyte proliferation (Columbano et al. 1983). Acting as a direct mitogen, the metal induces such effects without precedent liver injury. Some cytokines, including tumor necrosis factor-α, are suggested to be involved in cell proliferation (Shinozuka et al. 1996), and they are derived from Kupffer cells (Milosevic and Maier 2000; Pagliara et al. 2003). Withdrawal of lead results in the regression of liver hyperplasia resulting from the apoptosis of hepatocytes (Columbano et al. 1985); Kupffer cells are also suggested to play an important role in apoptosis induction (Pagliara et al. 2003). Apoptotic cells are removed rapidly by phagocytes or macrophages. For efficient recognition, apoptotic cells mark themselves by presenting “eat-me” signals (Savill et al. 1993). Phosphatidylserine (PS) and its receptor, milk fat globule EGF factor 8 (MFG-E8), and mannose receptor are involved in their recognition by macrophages or Kupffer cells (Callahan et al. 2000; Dini et al. 1996; Hanayama et al. 2004; Ruzittu et al. 1999; Yoshida et al. 2005).

The intracellular iron storage protein ferritin plays important roles, not only in iron metabolism, but also in inflammation (Konijn et al. 1981), oxidative damage (Cairo et al. 1995), cell proliferation (Cozzi et al. 2004; Kikyo et al. 1994), and apoptosis (Cozzi et al. 2003). Ferritin is composed of twenty-four subunits of two types, the heavy chain and light chain (Harrison and Arosio 1996), and their protein levels are largely post-transcriptionally regulated by the iron-regulatory proteins IRP1 and 2 (Ishikawa et al. 2005; Klausner and Harford 1989).

By inhibiting the heme synthesis pathway and inducing hepatocyte proliferation and subsequent apoptosis, lead may cause alterations in iron metabolism and ferritin expression in the liver. In the present study, expression of ferritin light-chain (FTL) in rat livers, the dominant subunit in the organ, was investigated after lead nitrate administration. We found that FTL was increased in hepatocytes and nonparenchymal cells (NPC), and some FTL-positive NPC were identified as Kupffer cells. We further examined the relationship between FTL expression in Kupffer cells and apoptosis induction as well as phagocytic activity.

Materials and Methods

Animal Experiments

Male Sprague-Dawley rats maintained in our department, aged six to seven weeks and weighing 200–250 g, were used in the present study. The protocol for the animal experiments was approved by the Animal Care and Use Committee, Hirosaki University, and conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology. All animals were housed in plastic cages in an air-conditioned room with a twelve-hour light/dark cycle in the Institute for Animal Experiments of Hirosaki University Graduate School of Medicine, and were allowed free access to water and laboratory chow diet. Lead nitrate (Wako Chemical Inc., Osaka, Japan) was dissolved in 0.25 M sucrose just prior to use, and was given to rats as a single injection of 200 μmol/kg body weight in a volume of 0.5 mL through the tail vein (Columbano et al. 1983). Control rats received an equivalent volume of 0.25 M sucrose. Each group contained at least four rats. Seventy-two hours after the administration of lead nitrate, animals were weighed and then euthanized by decapitation under diethyl ether anesthesia. Liver slices were fixed, and remaining livers were kept frozen at −80°C until biochemical study. Blood hemoglobin levels were measured with an automatic hematology analyzer (MEK-6450, Nihon Kohden, Tokyo, Japan).

In some experiments, 0.3% w/w clofibrate (a product of Tokyo Kasei Kogyo, Tokyo, Japan; purity > 98%) in the basal diet was given to male SD rats for four weeks.

Western Blotting

Rat livers were homogenized in four volumes of 0.25 M sucrose, 15 mM Tris-HCl (pH 7.9), 15 mM NaCl, 60 mM KCl, 5 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 0.1 mM phenylmethanesulfonyl fluoride, 1.0 mM dithiothreitol, 1% pro-tease inhibitor cocktail (Sigma), and centrifuged at 15,000 × g for ten minutes. The supernatant was used as a cytoplasmic extract. Nuclear extracts were prepared from rat liver tissues, as described by Dignam et al. (1983). Proteins of these extracts were separated by 12.5% or 8% SDS-PAGE gel (Laemmli 1970) and electroblotted to PVDF membranes (Amersham Biosciences, Tokyo, Japan) according to the method of Towbin et al. (1979). These were probed with anti-FTL, IRP1, IRP2, c-Jun or glutathione S-transferase (GST)-P antibodies. Antibodies against FTL (sc-14420), IRP1 (sc-14216), IRP2 (sc-14221), and c-Jun (sc-1694) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against GST-P was raised in a rabbit, as reported previously (Satoh et al. 1985). Detected bands were quantified with an image analysis system (ChemiDoc XRS, Bio-Rad, Tokyo, Japan).

RNA Preparation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from frozen liver, as described by Ookawa et al. (2002), and RT-PCR was performed with the AccessQuick RT-PCR System (Promega, Tokyo, Japan) by using 0.5 μg RNA. PCR amplification consisted of one minute at 94°C, two minutes at 55°C to 60°C, and three minutes at 72°C for twenty-one to thirty cycles. The primers used are shown in Table 1. RT-PCR products were subjected to electrophoresis in a 2% agarose gel and visualized with ethidium bromide.

Histological Analysis and Immunohistochemistry

Liver tissues from rats were fixed in 10% formaldehyde and embedded in paraffin. Tissue sections (4–6 μm thick) were routinely passed through xylene and a graded alcohol series and stained with hematoxylin and eosin. Sections for CD68 and CD34 were incubated with Liberate Antibody Binding (L.A.B.) solution (Polysciences, Inc, Warrington, PA, USA) for ten minutes for epitope retrieval. Immunohistochemical staining for FTL, CD68, CD34, α-smooth muscle actin (α-SMA), ferritin heavy chain, hemoglobin, or MFG-E8 was performed by the avidin-biotin-peroxidase complex (ABC) method (Hsu et al. 1981) with their respective antibodies. Antibody against CD68 (MCA341R) was obtained from AbD Serotec (Oxford, UK), antibodies against CD34 (ab8158) and α-SMA (ab18147) were from Abcam (Tokyo, Japan), and antibodies against ferritin heavy chain (sc-14416), hemoglobin (sc-21005), and MFG-E8 (sc-33546) were from Santa Cruz Biotechnology. The biotinylated anti-rabbit or anti-goat IgG antibodies and Vectastain ABC kit were obtained from Vector Laboratories (Burlingame, CA, USA). The specific binding was visualized with a 3,3-diaminobenzidine tetrahydrochloride (DAB) solution. Sections were then lightly counter-stained with hematoxylin for microscopic examination. The specimens were examined and photographed using a microscope (COOLSCOPE, Nikon, Tokyo, Japan) interfaced with a computer.

For immunofluorescence analysis, tissue sections were incubated with a goat anti-FTL antibody and a mouse anti-CD68 antibody. Antibodies were stained with fluorescently labeled secondary antibodies (Alexa Fluor 546 and Alexa Fluor 488) obtained from Molecular Probes (Eugene, OR, USA). Species-matched, irrelevant antibodies were used as negative staining controls. Images were viewed using a fluorescent microscope (Olympus BX60) at wavelengths of 546 and 488 nm.

TUNEL Assay

Apoptotic cell death was located in tissue sections by TUNEL analysis (Waddell et al. 2000). Paraffin tissue sections (6 μm) were dewaxed at 60°C and passed through a graded xylene series for five minutes each. Sections were hydrated through a graded series of ethanol and phosphate-buffered saline and then incubated with 5 μg/mL proteinase K in phosphate-buffered saline for ten minutes. TUNEL assay was performed using a commercial kit, following the manufacturer’s instructions (in situ apoptosis detection kit, TAKARA, Shiga, Japan). The TUNEL labels were visualized with DAB as a peroxidase substrate. Postweaning mammary tissue was included as a positive control. Apoptotic cells in liver sections were quantitated by counting the number of TUNEL-positive cells in nine random microscope fields (200X, about 250 hepatocytes/field).

Statistical Analysis

Data were expressed as mean ± SEM. Statistical differences between groups were determined using Student’s t test, taking p < .05 as the level of significance.

Results

Increase of FTL in Rat Livers by Lead Nitrate

After a single injection of lead nitrate, the livers were significantly enlarged at seventy-two hours, 6.59 ± 0.59 g/100 g body weight versus 4.31 ± 0.17 in the control group (p < .01). Blood hemoglobin levels were not different between the two groups (15.4 ± 0.7 g/100 mL in the treatment group versus 15.3 ± 0.5 g/100 mL in control). By western blotting, FTL protein was 3.5 ± 1.0-fold increased in the treatment group, as compared with that in the control group (p < .05, Figure 1A). Since the ferritin level is regulated post-transcriptionally by IRP1 and IRP2 (Leibold and Munro 1988), these proteins were also examined. However, IRP1 and 2 levels were hardly changed after the treatment (Figures 1B and 1C). The up-regulation of c-Jun protein (Figure 1D) and GST-P (Figure 1E) was confirmed in the treatment group, which is in line with the findings reported by Coni et al. (1993) and Roomi et al. (1986), respectively. To confirm post-transcriptional regulation of FTL, FTL mRNA and IRP2 mRNA levels were investigated by RT-PCR. Neither mRNA was different between the two groups (Figure 2).

Increase of FTL in Hepatocytes and Kupffer Cells by Lead Nitrate

Immunohistochemical analysis was performed to clarify cell types exhibiting enhanced FTL expression. As shown in Figure 3B, some hepatocytes around the central veins were more heavily stained by anti-FTL antibody in the treatment group than those in the control group (Figure 3A). Some NPC were very heavily stained in the treatment group (arrows in Figure 3B), whereas such cells were not detected in the control group. To identify FTL-positive NPC, expression of CD68, CD34, and α-SMA, markers for Kupffer cells, endothelial, cells and stellate cells, respectively, was examined in quasi-serial sections. The expression pattern of CD68 (Figure 3D) was similar to FTL-positive NPC in the treatment group, whereas those of CD34 and α-SMA were not (Figures 3F and 3H). Kupffer cells and hepatocytes around the central veins were also stained with anti-ferritin heavy chain antibody (Figure 3J).

To further examine the relationship between FTL-positive NPC and Kupffer cells, two-color fluorescence analysis with the respective antibodies was performed (Figure 4). Although the distinction of FTL-positive hepatocytes and NPC was not clear in the treatment group (Figure 4D), the merged image of FTL and CD68 revealed FTL expression in some CD68-positive cells (Figure 4F) and identified some FTL-positive NPC as Kupffer cells.

The distributions of CD68-positive cells and FTL-positive Kupffer cells in areas around the central veins and in the periportal areas were evaluated (Figure 5). In the control group, the number of CD68-positive cells was higher in the periportal areas than in areas around the central veins (p < .01), confirming the finding reported by Sleyster and Knook (1982). After the lead nitrate treatment, CD68-positive cells were increased in both areas (vs values in the control, p < .01), and the values in the two areas were comparable. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in both areas (56.7% in the periportal areas and 60.9% in the perivenous areas).

Induction of Apoptosis and Phagocytosis of Apoptotic Cells

Some Kupffer cells engulfing apoptotic cells were positive for FTL (Figure 5, Insert). Since Kupffer cells have phagocytic activity (Yoshida et al. 2005), high FTL expression may be the result of the engulfment of apoptotic cells. To examine this possibility, first we performed the TUNEL assay to evaluate apoptotic cells (Figures 6A and 6B). TUNEL-positive cells were 2.5 ± 1.4% of hepatocytes in the treatment group versus 0.31 ± 0.31% in the control group (Figure 6E, p < .01). Furthermore, expression of MFG-E8 was examined to investigate phagocytic processes. Some Kupffer cells and hepatocytes were positive for MFG-E8 in the treatment group (Figure 6D), but were rarely stained in controls (Figure 6C). MFG-E8-positive Kupffer cells were 18.4 ± 7.1% of total Kupffer cells in the treated livers versus 3.4 ± 2.9% in control (Figure 6F, p < .01). Among receptors for phagocytosis, mRNA levels for PS receptor (Figure 7A), mannose receptor (Figure 7B), and MFG-E8 (Figure 7C) were examined by RT-PCR. MFG-E8 mRNA was increased in the treatment group, whereas the others were not changed. A protein pumping out iron, ferroportin, plays a crucial role in macrophages and hepatocytes to decrease the intracellular iron level (Nemeth et al. 2004), and their protein amount is regulated by hepcidin (Pigeon et al. 2001). To examine whether enhanced FTL expression in Kupffer cells and hepatocytes is a result of iron level alteration by lead nitrate, ferroportin and hepcidin mRNAs were investigated by RT-PCR; there were no differences between the two groups (Figures 7D and 7E), suggesting no change in iron export.

Loss of FTL-Positive Kupffer Cells in Clofibrate-Treated Rat Livers

To study the relationship between FTL-positive Kupffer cells and apoptosis, the appearance of such cells was immunohistochemically examined in clofibrate-administered rat livers, because the drug is known to induce hepatocyte proliferation but not apoptotic changes (Columbano and Shinozuka 1996). Although FTL expression was increased in hepatocytes around the central veins, FTL-positive Kupffer cells were not detected (Figure 8).

Discussion

In the present study, FTL protein was increased in hepatocytes around the central veins and NPC after lead nitrate treatment. Some FTL-positive NPC were identified as Kupffer cells by two-color fluorescence analysis with anti-CD68 and anti-FTL antibodies; FTL-positive Kupffer cells occupied about 60% of CD68-positive cells. Although CD68-positive cells were detected, FTL-positive Kupffer cells were not detected in controls, indicating that FTL is not expressed in Kupffer cells under basal conditions. In normal rat livers, Kupffer cells are more frequently distributed in the periportal areas than in the perivenous areas, and the periportal Kupffer cells are larger and have higher phagocytic activities than the perivenous Kupffer cells, demonstrating the presence of two different types of Kupffer cells (Sleyster and Knook 1982). In the present study, CD68-positive cells were more prominently increased after lead treatment in the perivenous areas than the periportal areas. This finding supports the proliferation of Kupffer cells by lead nitrate (Shinozuka et al. 1996) and also suggests that the perivenous Kupffer cells may be more sensitive to mitotic signals from the metal. Because FTL-positive hepatocytes were located mainly in the perivenous areas, we anticipated that FTL-positive Kupffer cells would be preferentially distributed in the same areas. However, positive Kupffer cells were evenly distributed between the periportal areas and perivenous areas, which may reflect the even distribution of TUNEL-positive cells in both areas (data not shown).

Both lead nitrate treatment and clofibrate administration induced FTL expression in hepatocytes around the central veins, whereas FTL-positive Kupffer cells were not induced by clofibrate treatment. The staining intensity of hepatocytes with anti-FTL antibody was much lower than that of Kupffer cells, and FTL expression in hepatocytes may be dependent on cell proliferation rather than apoptotic changes.

Although many hepatocytes become apoptotic after lead treatment (Columbano et al. 1985), such apoptotic cells are rapidly engulfed by Kupffer cells, and their constituents are promptly degraded (Dini et al. 2002). In fact, the number of apoptotic cells detected by TUNEL assay was increased by lead treatment, but the value is rather small and the number of Kupffer cells actively engulfing apoptotic cells is also low. MFG-E8 or mannose receptor is a marker for cells actively engulfing apoptotic cells but not for cells that have engulfed them. Phagocytosis of apoptotic cells containing iron will result in high iron content and FTL expression in Kupffer cells, when iron is not exported. We chose seventy-two hours as a time for studying FTL expression, and there were no alterations in ferroportin or hepcidin mRNA levels, suggesting that iron export is not altered during the experiment period.

Kupffer cells are reported to engulf oxidatively damaged erythrocytes (Otogawa et al. 2007). Because lead has a destabilizing effect on erythrocyte membranes and induces hemolysis by reactive oxygen species–generated lipid peroxidation (Lawton and Donaldson 1991), we also considered a possibility that erythrophagocytosis by Kupffer cells may be involved in their FTL expression. However, this is unlikely because blood hemoglobin level was not decreased and the engulfment of erythrocytes was not demonstrated on immunohistochemistry with anti-hemoglobin antibody (data not shown).

In conclusion, the results of the present study suggest that FTL expression in Kupffer cells after lead treatment seemed to be dependent on phagocytosis of apoptotic cells, and FTL may be used as a marker for cells that have phagocytosed them.