Acute pancreatitis induces FasL gene expression and apoptosis in the liver
Presented at the 37th Annual Meeting of the Association for Academic Surgery, November 13¨C15, 2003, Sacramento, CA.
Journal of Surgical Research
Volume 122, Issue 2, December 2004, Pages 201-209
Journal of Surgical Research
Author links open overlay panelScott F.GallagherM.D.*JunYangM.D.*KathrynBakshB.S.*KristaHainesB.A.*HeatherCarpenterB.S.*P.K.Epling-BurnettePh.D.†YanhuaPengPh.D.*JamesNormanM.D.(F.A.C.S.)*Michel M.MurrM.D.(F.A.C.S.)*
https://doi.org/10.1016/j.jss.2004.05.019Get rights and content
Liver injury is an important prognostic indicator in acute pancreatitis. We previously demonstrated that Kupffer cell-derived cytokines mediate liver injury. In this work, we sought to characterize the role of Fas Ligand (FasL) in liver injury during acute pancreatitis.
Acute pancreatitis was induced in mice using cerulein; serum FasL, AST, ALT, liver FasL, p38-MAPK, and caspase-3 were measured. FasL mRNA and protein and its receptor (Fas) were determined in rat Kupffer cells treated with elastase (1 U/ml) to mimic acute pancreatitis. Apoptosis was measured by flow cytometry.
Cerulein-induced pancreatitis increased serum AST, ALT, and FasL and up-regulated liver FasL (1315 ¡À 111 versus 310 ¡À 164 pg/ml, P = 0.002 versus sham), while inducing p38-MAPK phosphorylation (P < 0.01 versus sham) and cleavage of caspase-3 (P < 0.04 versus sham); all were attenuated by pretreatment with the Kupffer cell inhibitor, gadolinium (all P < 0.003). In vitro, elastase induced a time-dependent increase in Kupffer cell FasL protein (FasL = 404 ¡À 94 versus 170 ¡À 40, P = 0.02, versus control), a 100-fold increase in FasL mRNA, and up-regulated Fas (FasL receptor). Gadolinium significantly attenuated the elastase-induced increase in FasL and FasL mRNA (FasL = 230 ¡À 20 versus 404 ¡À 94, P = 0.01, versus elastase) but had little effect on Fas. Additionally, elastase-primed Kupffer cell media induced apoptosis in hepatocytes (29 ¡À 1 versus 16% ¡À 1%; versus control, P < 0.001).
Acute pancreatitis induces liver injury and hepatocyte death while up-regulating FasL, p38-MAPK, and caspase-3. Fas is up-regulated within Kupffer cells, suggesting that FasL may autoregulate its production by inducing its originator-cell death. The ability to manipulate interactions between Kupffer cells and hepatocytes may have important therapeutic implications.
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acute pancreatitisKupffer cellsFas ligandliver injuryhepatocyte apoptosisp38-MAPKcaspase-3cell signaling
Acute pancreatitis induces FasL gene expression and apoptosis in the liver1,2 - ScienceDirect https://www.sciencedirect.com/science/article/pii/S0022480404002331
Lipopolysaccharides induced increases in Fas ligand expression by Kupffer cells via mechanisms dependent on reactive oxygen species
Keiichiro Uchikura, Tatehiko Wada, Sumito Hoshino, Yuichi Nagakawa, Takashi Aiko, ¡ Show all Authors
01 SEP 2004https://doi.org/10.1152/ajpgi.00314.2003
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Fas-Fas ligand (FasL)-dependent pathways exert a suppressive effect on inflammatory responses in immune-privileged organs. FasL expression in hepatic Kupffer cells (KC) has been implicated in hepatic immunoregulation. In this study, modulation of FasL expression of KC by endogenous gut-derived bacterial LPS and the role of reactive oxygen species (ROS) as potential mediators of FasL expression in KC were investigated. LPS stimulation of KC resulted in upstream ROS generation and, subsequently, increased FasL expression and consequent Jurkat cell (Fas-positive) apoptosis. The NADPH oxidase and xanthine oxidase enzymatic pathways appear to be major sources of this upstream ROS generation. Increased FasL expression was blocked by antioxidants and by enzymatic blocking of ROS generation. Exogenous administration of H2O2 stimulated KC FasL expression and subsequent Jurkat cell apoptosis. Intracellular endogenous ROS generation may therefore represent an important signal transduction pathway for FasL expression in KC.
endogenous gut-derived bacterial LPS have been implicated as important cofactors in the pathogenesis of liver injury (24, 25). Most of the toxicities of LPS, both intrahepatic and systemic, have been related to the release of these inflammatory cytokines and mediators (12, 37).
Kupffer cells (KC), the resident macrophage population in the liver, comprise one of major populations (20%) of the hepatic nonparenchymal cells. KC are found within the sinusoidal lumen, adhering to the liver sinusoidal endothelial cells. It has been reported that KC play a key role in clearing circulating LPS (5, 17, 19, 39), as demonstrated by experiments showing that the majority of injected 125I-labeled LPS can be localized to the KC within 30 min of injection (19). In addition to its ability to clear LPS, KC also respond to LPS with production of inflammatory cytokines (i.e., TNF-¦Á and IL-1) (18) and a variety of mediators, including reactive oxygen species (ROS) (2, 3) and Fas ligand (FasL) (9). Moreover, KC can directly interact with passenger leukocytes and, thus, may play a role in immunomodulation (21), which includes antigen presentation (27) and induction of T cell apoptosis via Fas-FasL interactions (23).
Fas (Apo-1/CD95) belongs to the TNF receptor/nerve growth factor receptor family and transduces apoptotic death via its intracellular death domains on cross-linking by its cognate ligand (FasL), a 40-kDa membrane protein. Although the Fas-FasL system has been widely studied for its role in tissue growth (1) and elimination of malignant (16) and damaged cells (10), its role is perhaps best characterized in the immune system. Interactions between Fas and FasL are functionally involved in maintaining homeostasis and self-tolerance of the immune system via cytotoxic effector mechanisms; malfunctions of the Fas system cause lymphoproliferative disorders and lymphoadenopathy and facilitate autoimmune disorders (30). Induction of antigen-specific T cell tolerance by FasL-transfected antigen-presenting cells suggests a novel strategy for modulating T cell response (20, 38).
Knowledge of the regulation of FasL expression in KC is limited. ROS have been found to be important mediators of other inflammatory signaling pathways. We previously reported that ROS generated via the NADPH oxidase- and xanthine oxidase (XO)-dependent pathways are each important in the killing of circulating pathogens (Escherichia coli) and in antigen processing by KC (36, 26). This, together with the reported increased expression of FasL genes in LPS-stimulated KC associated with augmented ROS formation, prompted us to question whether formation of ROS in KC induced by LPS is capable of modifying FasL transcription. Consequently, we evaluated the potential role of ROS, and their generation, as upstream mediators of FasL expression in KC.
The highly efficient mediation of apoptosis of activated T lymphocytes by FasL-dependent mechanisms may, in some instances, create an environment of antigen-specific accommodation. Such ¡°immune privilege¡± induced by FasL has been determined in the anterior chamber of the eye and in Sertoli cells of the testis (11). The immunologic repertoire of resident hepatic macrophages (KC) within the hepatic sinusoids may also endow the liver with similar immunologic privilege (21). Because KC can directly interact with circulating lymphocytes and also act as antigen-presenting cells for intrahepatic lymphocytes, they occupy a key position in the regulation of the hepatic immune response (21). It has been reported that IFN-¦Ã upregulates FasL expression in KC and, subsequently, induces lymphocyte death by a Fas/FasL-dependent mechanism (23). We recently reported that KC can regulate the T cell response via the Fas/FasL-dependent pathway and may play an important role in the induction of immune tolerance in liver allografts (33, 34). Expression of FasL in KC may represent an intrahepatic pathway of immunoregulation.
We have used LPS-stimulated KC FasL expression as a model to investigate the relation between KC ROS generation and FasL expression. Our results indicate that KC treated with LPS express functional FasL and induce apoptosis of Fas-positive target cells. The generation of intracellular endogenous ROS may represent an important signal transduction pathway for FasL expression in this system. To determine the pathways of ROS generation in KC stimulated with LPS, antioxidants and specific oxidant enzyme inhibitors were selected as blockers for ROS generation in our model. DPI is an inhibitor of NADPH oxidase, and allopurinol is a very specific inhibitor of XO (14), whereas SOD and catalase are enzymatic scavengers of O2− and H2O2, respectively. Our results show that antioxidants (SOD and catalase) significantly suppressed FasL expression in KC stimulated with LPS in a dose-dependent fashion. DPI or allopurinol partially blocked the FasL expression in KC stimulated with LPS. However, FasL expression was almost completely blocked when KC were pretreated with DPI + allopurinol at a relatively high dose (Fig. 2E). These findings suggest that NADPH and XO play a critical role in ROS-dependent FasL expression in KC stimulated with LPS. To confirm the role of ROS in this model of FasL expression, KC were stimulated directly with H2O2. H2O2 stimulation increased KC FasL expression, and this FasL expression was blocked by catalase, a specific enzymatic scavenger of H2O2. These results support the hypothesis that ROS may act as a signal to regulate FasL expression in KC.
We previously reported that ROS generated via the NADPH oxidase- and XO-dependent pathways are each important in the killing of circulating pathogens (E. coli) by KC (15, 36). Our data show that SOD is more effective in preventing the LPS-induced increase in FasL expression and suggest that O2− is more important than H2O2 in the observed LPS-induced increase in FasL expression. However, as a single agent, the XO inhibitor (allopurinol) or NADPH oxidase inhibitor (DPI) each prevents ∼40% of the LPS-induced increase in FasL expression. Interestingly, the inhibitory effects of DPI and allopurinol were additive (Fig. 2E). Our group previously reported that DPI + allopurinol shows additive effects in reducing the generation of reactive oxygen metabolites by XO- and NADPH oxidase-dependent pathways (14). The additive effects of DPI and allopurinol in preventing an LPS-induced increase in FasL protein levels (∼75%; Fig. 2) suggest that O2− and H2O2 are each important for the LPS-induced increase in FasL expression in KC.
It is not clear how extracellularly administered SOD was able to degrade intracellular O2−. Our previous studies (26, 36) showed that SOD inhibited phagocytic killing of bacteria in phagocytes (i.e., macrophages and KC) by suppressing ROS generation without influencing phagocytosis. SOD degradation of intracellular O2− may be explained, at least in part, by phagocytosis of SOD. Phagocytosis may transfer exogenous SOD into KC. In addition, SOD can directly degrade membrane-bound and released O2− from KC. Thus SOD may degrade intracellular and extracellular O2−. This may be one reason that SOD is more effective in preventing LPS-induced increase in FasL expression.
The correlation between the H2O2- and LPS-induced increase in FasL protein levels has been demonstrated by the observations that 1) LPS stimulation increases H2O2 generation in KC (Fig. 1), 2) H2O2 alone can increase FasL protein expression in KC (Fig. 3), and 3) antioxidants (SOD and catalase) and oxidant enzyme inhibitors (DPI and allopurinol) block the increase in FasL expression of KC stimulated with LPS (Figs. 2 and 4). A high concentration of exogenous H2O2 was required to increase FasL protein levels (Fig. 3A). Our data also show that LPS at 1 ¦Ìg/ml produced <1 ¦ÌM H2O2 within 60 min (Fig. 1B). In Western blotting assay for FasL protein expression, we used 1 ¡Á 106 cells/well in 24-well plates and stimulation with H2O2 or LPS for 24 h. However, for the H2O2 generation assay, we used 2 ¡Á 105 cells/well in 96-well plates and only quantified H2O2 generation of KC within 60 min after LPS stimulation. In addition, extracellular levels of H2O2 do not necessarily represent the intracellular levels of H2O2. That may explain why a relatively high concentration of H2O2 was required to increase FasL protein level, whereas LPS at 1 ¦Ìg/ml produced only 1 ¦ÌM H2O2 in our studies.
SOD or catalase completely prevents the LPS-induced increase in apoptosis (Fig. 5A), whereas SOD or catalase caused only partial inhibition of the LPS-induced increase in FasL mRNA (Fig. 4A) and FasL protein (Fig. 2, A and B). It should be recognized that KC constitutively express FasL, and, indeed, the coculture of untreated KC with Jurkat cells results in a baseline level of measurable apoptosis. In our study, coculture of Jurkat cells with untreated KC at a ratio of 1 to 10 did not significantly increase Jurkat cell apoptosis compared with coculture of Jurkat cells without KC (Fig. 5A). Thus the discrepancy between the effect of antioxidants on LPS-induced apoptosis and FasL expression could be explained by FasL dose-dependent induction of Jurkat cell apoptosis. The decreased FasL expression in KC by SOD or catalase insufficiently induces Fas-positive Jurkat T cell apoptosis. On the other hand, LPS-stimulated activation of KC not only induces increased FasL expression but also stimulates production of cytokines, such as TNF-¦Á (31). These cytokines may also cause apoptosis of Jurkat T cells in our model. However, our observations that KC-induced T cell apoptosis can be blocked by addition of neutralizing anti-FasL antibody (34) further support the importance of FasL expression in LPS-induced apoptosis. We focused on FasL expression, although SOD or catalase may also inhibit cytokine production in LPS-stimulated KC. Catalase prevents up to 50% of H2O2-induced FasL expression in KC and similar levels of T cell apoptosis when cocultured with KC stimulated with H2O2 (Fig. 5B), suggesting a correlation between H2O2 and functional FasL expression in KC.
Although the mechanism of regulation of FasL expression in KC is unclear, it is likely that gut-derived LPS may act as a mediator to induce FasL expression of KC. LPS activates KC to release active substances such as ROS. Imbalance in oxidant production and elimination can lead to excessive exposure to ROS and subsequent oxidative damage of nucleic acids, proteins, and membrane lipids. A role for KC-dependent oxidative injury in hepatic ischemia-reperfusion is well supported (6). In contrast, the endogenous production of ROS may act as a signal to effect gene transcription, rather than cellular injury. ROS have been found to be important mediators of other inflammatory signaling pathways (35). Specifically, ROS have been shown to affect gene expression, including Fas and FasL gene expression in endothelial cells, microglia, and astrocytoma cells (8, 32). A number of signal transduction pathways has been implicated in FasL expression, including NF-¦ÊB, AP-1, p38 MAPK, and JNK (13, 28). LPS can activate the NF-¦ÊB signaling pathway mediated by ROS and MAPK signal transduction pathways such as JNK and p38 MAPK in KC (4, 29). The effect of ¡°oxidant stress¡± on the activation of these transcription factors is cell-type specific, and the molecular mechanism by which ROS modulates these processes is unclear. However, these signals in KC might be mediated by the endogenous generation of ROS and, thereby, induce FasL expression. We observed that exogenous H2O2 induced upregulation of KC FasL and that the increased expression of FasL in LPS-stimulated KC was blocked by addition of antioxidants. These data suggest that LPS-induced upregulation of FasL expression in KC may be dependent, at least in part, on ROS generation. More extensive studies are needed to demonstrate which factors are key modulators of FasL expression via the generation of endogenous ROS in KC.
To our knowledge, this is the first study showing that LPS has the ability to upregulate FasL mRNA and protein expression in KC in vitro. Intracellular endogenous ROS generation may represent an important signal transduction pathway for FasL expression in KC. The NADPH oxidase and the XO enzymatic pathways appear to be major sources of this upstream ROS generation. We would propose that stimulation of KC FasL expression via ROS-dependent mechanisms may provide a novel strategy for modulating the T cell response.
Lipopolysaccharides induced increases in Fas ligand expression by Kupffer cells via mechanisms dependent on reactive oxygen species | American Journal of Physiology-Gastrointestinal and Liver Physiology https://www.physiology.org/doi/full/10.1152/ajpgi.00314.2003