5-Lipoxygenase (5-LO) is Involved in Kupffer Cell Survival. Possible Role of 5-LO Products in the Pathogenesis of Liver Fibrosis
© Titos et al; licensee BioMed Central Ltd 2004
Published: 14 January 2004
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 , 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 . 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 , 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 CCl4 as described elsewhere .
Isolation and culture of Kupffer cells
Liver cells were isolated by in situ collagenase perfusion and purified by Percollà 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 .
RNA isolation and RT-PCR
Total RNA was obtained by the guanidinium isothiocyanate-cesium chloride method. RT was performed using an avian myeloblastoma virus reverse transcriptase cDNA synthesis kit. PCR was performed using oligonucleotides designed from published rat 5-LO, 5-LO-activating protein (FLAP), LTC4 synthase and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA sequences. PCR products were analyzed by gel electrophoresis.
Analysis of 5-LO products
LTB4 and LTC4/LTD4/LTE4 (cysteinyl-LT) levels were quantified in cell supernatans of freshly isolated rat Kupffer cells (1à2.8 à 106 cells) maintained in culture for 16 hours by specific EIA kits. 5-hydroxyeicosatetraenoic acid (5-HETE) was analyzed by RP-HPLC.
Analysis of cell proliferation
Rat Kupffer cells (1à2.8 à 106 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.
Nuclear morphology was assessed by optical microscopy visualization in Diff-Quikà-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 TACSà DNA Laddering kit and visualized by agarose gel electrophoresis.
Analysis of DNA content by flow cytometry
THP-1 cells were incubated in the presence of vehicle, AA861 (10 micromolar) or BAY-X-1005 (30 micromolar) at 37 degrees C. After 72 h, cells were stained with propidium iodide and DNA content frequency cell cycle distribution analyzed by means of fluorescence-activated cell sorting (FACS) analysis.
Generation of 5-LO-derived eicosanoids by Kupffer cells isolated from control and CCl4-treated rats. N.D., not detected. *, P < 0.05 vs control.
LTB4 (pg/106 cells)
3.98 à 0.89
7.52 à 1.66*
LTC4/LTD4/LTE4 (pg/106 cells)
5.74 à 0.99
3.82 à 0.42
5-HETE (ng/106 cells)
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 à 4.7 % inhibition) and by the FLAP inhibitor BAY-X-1005 (55.2 à 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").
This work was supported by grants SAF 00/0043 and FIS 02/0029.
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