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Inhibition of 5-lipoxygenase induces cell growth arrest and apoptosis in rat Kupffer cells: implications for liver fibrosis¹

ESTHER TITOS, JOAN CLÀRIA², ANNA PLANAGUMÀ, MARTA LÓPEZ-PARRA, NEUS VILLAMOR, MARCELINA PÁRRIZAS^¶, ANNA CARRIÓ^||, ROSA MIQUEL, WLADIMIRO JIMÉNEZ^¶, VICENTE ARROYO^*, FRANCISCA RIVERA^¶ and JOAN RODÉS^*

DNA Unit,
^¶ Hormonal,
Hematopathology,
Pathology and
^|| Genetics Laboratories and
^* Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona 08036, Spain

²Correspondence: DNA Unit, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain. E-mail: jclaria{at}clinic.ub.es

SPECIFIC AIM

The presence of an increased number of Kupffer cells, the liver resident macrophages, together with their activation are recognized to be critical events in the initiation of the inflammatory cascade leading to liver fibrosis. In the current investigation we assessed whether inhibition of the 5-lipoxygenase (5-LO) pathway, which activity is key for cell growth and survival, reduces the excessive number of Kupffer cells and attenuates liver inflammation and fibrosis.

PRINCIPAL FINDINGS

1. Kupffer cells are the only liver cell type endowed with a metabolically active 5-LO pathway
We recently demonstrated that expression of 5-LO and production of 5-LO-derived eicosanoids are elevated in livers from rats with carbon tetrachloride (CCl₄)-induced cirrhosis. To ascertain the cellular distribution of the 5-LO pathway in this organ, rat sinusoidal liver cells were isolated by a combination of collagenase perfusion and Percoll gradient methods, and mRNA expression for key enzymes of the 5-LO pathway was examined by reverse transcription and PCR. Kupffer cells consistently expressed mRNAs for 5-LO, 5-LO-activating protein (FLAP), and leukotriene (LT) C₄ synthase. In contrast, rat hepatocytes as well as endothelial and hepatic stellate cells failed to show 5-LO mRNA expression. These results indicate that Kupffer cells are apparently the only liver sinusoidal cell type endowed with the complete enzymatic machinery necessary for the biosynthesis of 5-LO-derived eicosanoids. In fact, cultured Kupffer cells generated significant amounts of LTB₄ (3.98±0.89 pg/10⁶ cells) and cysteinyl-LTs (5.74±0.99 pg/10⁶ cells).

2. Inhibition of the 5-LO pathway reduces Kupffer cell growth
Kupffer cell growth was observed after 6 days of culture in a RPMI 1640 medium supplemented with 10% FCS. Kupffer cell proliferation was stimulated in a concentration-dependent fashion by macrophage colony-stimulating factor, a soluble factor released by adjacent hepatocytes. In contrast, Kupffer cell number was significantly reduced by AA861, a selective 5-LO inhibitor (35.8 and 38.2% inhibition at 5 and 10 µM, respectively), and by BAY-X-1005, a potent inhibitor of LT biosynthesis that blocks translocation of 5-LO by altering the active site of FLAP (42.3 and 41.8% inhibition at 30 and 40 µM, respectively). The inhibitory effect of BAY-X-1005 on cell growth was partially prevented by the addition of synthetic LTD₄. To provide further evidence for the role of the 5-LO pathway in regulating cell proliferation, concentration and time response studies were performed in THP-1 cells. In these cells, cell growth was significantly inhibited in a concentration- and time-dependent fashion by both AA861 (IC₅₀ of 10 µM at 72 h) and BAY-X-1005 (IC₅₀ of 30 µM at 72 h).

3. The anti-proliferative properties of 5-LO inhibition are associated with induction of apoptosis
Several apoptosis assays including cell morphology studies, analysis of DNA fragmentation by agarose gel electrophoresis, and examination of DNA content by flow cytometry were performed in Kupffer and THP-1 cells. As shown in Fig. 1 A, compared with vehicle (a), propidium iodide staining of Kupffer cells incubated with 5 µM AA861 (c) or 30 µM BAY-X-1005 (d) showed an increased number of nuclei with densely compacted chromatin characteristic of apoptotic cells (denoted by arrows). Similar cell morphology changes were observed in Kupffer cells treated with 1% ethanol (Fig. 1A , b), a well-established inductor of apoptosis. Diff-Quick® staining of THP-1 cells (Fig. 1B ) also showed that compared with vehicle (a), incubation with 15 µM AA861 (c) or 30 µM BAY-X-1005 (d) induces morphological apoptotic changes, namely, cell shrinkage and condensation of nuclear chromatin (denoted by arrows). An increased cell vacuolation was also observed after the exposure of cells to AA861 or BAY-X-1005 (denoted by arrowheads in Fig. 1B ). Treatment with 3% ethanol was used as a positive control (Fig. 1B , b). DNA laddering, which is the result of the internucleosomal cleavage of DNA by specific endonucleases producing 180 bp fragments of DNA or multiples thereof, was evident in THP-1 cells treated with 10 and 15 µM AA861 and 30 and 40 µM BAY-X-1005 (Fig. 1C ). These findings were confirmed by the analysis of DNA content by flow cytometry, which revealed the presence of an increased sub-G₀/G₁ peak in cells exposed to either AA861 or BAY-X-1005. Taken together, the detection of condensed chromatin, DNA laddering, and cell cycle changes indicate that the anti-proliferative effects of 5-LO and FLAP inhibitors are secondary to the induction of programmed cell death.

View larger version (46K): [in this window] [in a new window]   Figure 1. Apoptotic effects of 5-LO and FLAP inhibitors. A) Propidium iodide-stained Kupffer cells exposed to vehicle (a), 1% ethanol (b), AA861 (5 µM) (c), or BAY-X-1005 (30 µM) (d) for 8 h at 37°C. x600. Arrows indicate representative examples of nuclei with characteristic features of apoptosis, such as reduced size and intense chromatin staining. B) Diff-Quik®-stained THP-1 cells exposed to vehicle (a), 3% ethanol (b), AA861 (15 µM) (c), or BAY-X-1005 (30 µM) (d) for 96 h at 37°C. x500. Arrows indicate apoptotic nuclei. Arrowheads indicate intense cytoplasmic vacuolations. C) detection of DNA fragmentation (DNA "ladder" pattern) by gel electrophoresis after 72 h of incubation of THP-1 cells with vehicle (0.1% DMSO), 10 and 15 µM AA861, and 30 and 40 µM BAY-X-1005. The X174 HaeIII digest DNA size standard is shown on the left (m). Results are representative of 4 separate experiments.

4. The oral FLAP inhibitor BAY-X-1005 has hepatic anti-fibrotic effects in vivo
To investigate the relevance of these in vitro findings to the intact liver, we evaluated the effects of daily oral administration of BAY-X-1005 (100 mg/kg body weight) to rats with CCl₄-induced liver fibrosis. As expected, histological sections of livers from rats treated with CCl₄ for >4 wk showed remarkable changes in liver morphology, namely, steatosis, inflammation, hepatocyte ballooning, and necrosis. At the 8th wk of CCl₄ treatment, liver architecture was extensively disorganized, as sinusoids were no longer distinguishable and few areas of healthy hepatocytes were present together with extensive collagen deposition with septa bridging portal regions (Fig. 2 A, left panels). Compared with placebo, administration of BAY-X-1005 for 8 wk significantly attenuated liver damage and fibrosis (Fig. 2A , right panels) and reduced hepatic levels of hydroxyproline (Fig. 2B ) in CCl₄-treated rats.

View larger version (54K): [in this window] [in a new window]   Figure 2. Anti-fibrotic effects of the oral FLAP inhibitor BAY-X-1005 in CCl4-treated rats. A) Histological sections of liver tissue stained with hematoxylin-eosin (H&E) (upper panels, original magnification x25) and Masson’s trichrome (lower panels, original magnification x125) from CCl4-treated rats receiving either placebo (carboxymethylcellulose, n=3) (left panels) or BAY-X-1005 (100 mg·kg–1·day–1, n=4) (right panels) for 8 wk. B) Hepatic hydroxyproline content. Rats were treated as described in panel A and hepatic hydroxyproline levels were determined by RP-HPLC. Results are expressed as mean ± SE.

CONCLUSIONS AND SIGNIFICANCE

Blocking the inflammatory cascade leading to tissue remodeling is a major target in liver fibrosis. Kupffer cells, the predominant hepatic inflammatory cell type, exponentially increase their number during the early stages of the disease (Fig. 3 ). Here, we specifically targeted the increased Kupffer cell growth by inhibiting the 5-LO pathway. Two different pharmacological compounds, namely, AA861, an antioxidant derivative of benzoquinone that competitively inhibits 5-LO activity, and BAY-X-1005, a quinolone derivative acting not only by competition with the substrate for its binding site at FLAP but also by inhibiting 5-LO translocation from the cytosol to membranes, were used. In our experiments, both compounds consistently induced Kupffer cell growth arrest. The inhibitory actions of AA861 and BAY-X-1005 in Kupffer cells were similar to those described in human neutrophils and blood mononuclear cells in primary cultures of cerebellar granule neurons and endothelial cells and in a wide variety of human cancer cell lines exposed to AA861, nordihydroguaiaretic acid (NDGA, a nonselective LO inhibitor), or MK886, a FLAP inhibitor. The anti-proliferative properties of AA861 and BAY-X-1005 in Kupffer cells were associated with major changes in cell morphology and DNA content frequency distribution, which, together with DNA fragmentation, are characteristic features of cells undergoing apoptosis (Fig. 3 ). These apoptotic effects were similar to those previously reported in human cancer cell lines treated with AA861, NDGA, and MK886. The mechanism by which 5-LO inhibitors induce apoptosis is at present unknown, although several molecular mechanisms including vascular endothelial growth factor, up-regulation of peroxisome proliferator-activated receptors and , direct interaction of 5-LO with proteins involved in cell signaling and cytoskeletal organization, and increased levels of intracellular free arachidonate have been postulated. Finally, some FLAP inhibitors may also act independently of the 5-LO pathway because FLAP inhibition induces apoptosis in cells devoid of 5-LO activity. In our experiments, however, similar results were obtained with the FLAP inhibitor and the selective 5-LO inhibitor, suggesting that in our case apoptosis is probably linked to the inhibition of the 5-LO pathway.

View larger version (103K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the possible mechanism by which 5-LO inhibition attenuates liver fibrosis. In Kupffer cells, 5-LO translocates from the cytosol to the nucleus membrane, where it binds to FLAP and generates arachidonic acid (AA) metabolites such as LTs and hydroxyeicosatetraenoic acids (HETEs). The presence of a metabolically active 5-LO pathway in these macrophages is vital for their growth and survival. In the early stages of liver inflammation and fibrosis, Kupffer cells proliferate and produce large amounts of 5-LO-derived products. These eicosanoids in turn exert their paracrine actions on hepatic stellate cells, promoting fibrogenesis. Inhibition of the 5-LO pathway induces apoptosis and inactivates Kupffer cells, thereby reducing liver inflammation and fibrosis.

Our in vitro findings demonstrate that BAY-X-1005 does not induce massive apoptosis in Kupffer cells (Fig. 3 ), suggesting this compound apparently is able to reduce the excessive number of liver macrophages without threatening vital functions such as host defense and immune response. In fact, inhibition of the 5-LO pathway with BAY-X-1005 exerted a significant anti-fibrotic effect in vivo in CCl₄-treated rats. Similar beneficial actions of 5-LO inhibition have recently been reported in animals with experimental intimal hyperplasia. A protection against bleomycin-induced pulmonary fibrosis has been observed in 5-LO knockout mice. Taken together, these findings provide a novel and selective (only affects cells endowed with an active 5-LO pathway, i.e., Kupffer cells) mechanism for prevention of liver inflammation and fibrosis.

In summary, the current study provides the first evidence that the presence of a metabolically active 5-LO pathway is critical for the survival of Kupffer cells. In addition, this study provides the first in vivo evidence that the 5-LO pathway is involved in the pathogenesis of liver fibrosis. These data together demonstrate that by inhibiting the 5-LO pathway in Kupffer cells, the extent of fibrosis can be decreased in inflammatory liver injury.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1157fje; doi: 10.1096/fj.02-1157fje

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