5-Lipoxygenase (5-LO) is Involved in Kupffer Cell Survival. Possible Role of 5-LO Products in the Pathogenesis of Liver Fibrosis

A wealth of evidence indicates that inflammation plays a central role in the current paradigm of liver fibrosis. Kupffer cells, which represent the largest population of resident macrophages in the body [1], are uniquely positioned as the predominant primary inflammatory effector cells to initiate the inflammatory cascade leading to tissue remodeling and fibrosis. For this reason, the presence of an increased population of Kupffer cells together with the bulk release of inflammatory mediators by these macrophages are considered to be critical events during the early stages of liver inflammation and fibrosis [2,3]. 
 
Arachidonic acid metabolites derived from 5-lipoxygenase (5-LO) are essential regulators of cell growth and survival [4]. Given that we recently demonstrated that 5-LO expression and leukotriene (LT) formation are increased in livers from rats with carbon tetrachloride (CCl4)-induced cirrhosis [5], it is our hypothesis that 5-LO products play a role in Kupffer cell survival and in the pathogenesis of liver inflammation and fibrosis. Therefore, in the current study we examined the 5-LO pathway in sinusoidal liver cells and specifically analyzed the role of 5-LO in Kupffer cell growth and survival.


Introduction
A wealth of evidence indicates that inflammation plays a central role in the current paradigm of liver fibrosis. Kupffer cells, which represent the largest population of resident macrophages in the body [1], are uniquely positioned as the predominant primary inflammatory effector cells to initiate the inflammatory cascade leading to tissue remodeling and fibrosis. For this reason, the presence of an increased population of Kupffer cells together with the bulk release of inflammatory mediators by these macrophages are considered to be critical events during the early stages of liver inflammation and fibrosis [2,3].
Arachidonic acid metabolites derived from 5-lipoxygenase (5-LO) are essential regulators of cell growth and survival [4]. Given that we recently demonstrated that 5-LO expression and leukotriene (LT) formation are increased in livers from rats with carbon tetrachloride (CCl 4 )induced cirrhosis [5], it is our hypothesis that 5-LO products play a role in Kupffer cell survival and in the pathogenesis of liver inflammation and fibrosis. Therefore, in the current study we examined the 5-LO pathway in sinusoidal liver cells and specifically analyzed the role of 5-LO in Kupffer cell growth and survival.

Experimental model of hepatic fibrosis
Liver injury was induced in male adult Wistar rats by inhalation of CCl 4 as described elsewhere [6].

Isolation and culture of Kupffer cells
Liver cells were isolated by in situ collagenase perfusion and purified by Percoll™ density gradients as previously described [5,7]. Kupffer cells were characterized by nonspecific esterase activity staining and by immunolabeling with the monoclonal antibody RPE-ED2 and cultured in RPMI 1640 supplemented with 2 mM L-glutamine, penicillin (50 U/ml), streptomycin (50 micrograms/ml) and 10% FCS [5].

Analysis of cell proliferation
Rat Kupffer cells (1-2.8 × 10 6 cells) were cultured for up to 6 days in complete RPMI 1640 medium and cell growth was determined by the microculture MTT assay. To ascertain the effects of 5-LO inhibitors on cell survival, Kupffer cells from cirrhotic livers were grown in the presence of vehicle, AA861 (10 micromolar) and BAY-X-1005 (30 micromolar) for 8 h at 37 degrees C and the number of cells examined by direct counting using the Neuebauer chamber. The effects of 5-LO inhibitors on cell proliferation were further assessed in THP-1 cells by the MTT assay.

Apoptosis Assays
Nuclear morphology was assessed by optical microscopy visualization in Diff-Quik ® -stained THP-1 cells exposed to vehicle, AA861 (10 micromolar) or BAY-X-1005 (30 micromolar) for 96 hours at 37 degrees C. DNA fragmentation was detected using the TACS™ DNA Laddering kit and visualized by agarose gel electrophoresis.

Results
Among the different hepatic sinusoidal cell types, Kupffer cells have been historically considered to possess the capacity to produce most of liver arachidonic acid metabolites including the 5-LO products LTB 4 and cysteinyl-LT [8]. Indeed, Kupffer cells are apparently the only liver sinusoidal cells endowed with the complete enzymatic set necessary for LT formation (see Figure 1 and Reference [5]). (Table  1). 5-HETE was not detected in these incubations. Interestingly, the ability to produce LTB 4 was found to be increased in Kupffer cells from rats treated with CCl 4 .

Among the different 5-LO products, Kupffer cells generated significant amounts of LTB 4 and cysteinyl-LT
These 5-LO derived products are essential for Kupffer cell survival, because the number of Kupffer cells in culture was significantly reduced by the selective 5-LO inhibitor AA861 (42.7 ± 4.7 % inhibition) and by the FLAP inhibitor BAY-X-1005 (55.2 ± 2.5% inhibition). These findings were further characterized in THP-1 cells where AA861 and BAY-X-1005 inhibited proliferation in a dose-and time-dependent fashion. In these cells, the antiproliferative effect was associated with induction of programmed cell death, as evaluated by using different techniques for apoptosis detection (see "Methods").

Discussion
The current knowledge of the 5-LO pathway in liver sinusoidal cells is shown in figure 2. Kupffer cells, which constitutively express 5-LO, have the ability to produce LTB 4 and cysteinyl-LT. Cysteinyl-LT are also produced in hepatocytes by transcellular metabolism of LTA 4 formed by Kupffer cells [5]. Once synthesized, 5-LO-derived products may act in both paracrine and autocrine fashion modulating the contraction of nearby HSC or regulating macrophage cell growth. Interestingly, biosynthesis of 5-LO products is located in the nuclear cell membrane, where they can exert important nuclear functions. Taken together these data indicate that 5-LO plays an important role in cell proliferation and survival and set new ground for the application of 5-LO inhibitors during the inflammatory stage previous to the development of liver fibrosis.
Representative RT-PCR analysis of 5-LO mRNA expression in rat liver cells  Biosynthesis of 5-LO products in sinusoidal liver cells Figure 2 Biosynthesis of 5-LO products in sinusoidal liver cells