Gliotoxin causes apoptosis and necrosis of rat Kupffer cells in vitro and in vivo in the absence of oxidative stress: Exacerbation by caspase and serine protease inhibition☆
Introduction
Gliotoxin, a fungal metabolite, belongs to the epipolythiodioxopiperazine class of compounds. Gliotoxin (0.3–7.5 μM) was reported to cause apoptosis of activated rat and human hepatic stellate cells (HSCs) by inducing mitochondrial permeability, cytochrome c release and caspase-3 activation [1], [2]; higher concentrations (>7.5 μM) caused necrosis of activated human HSCs in association with increased oxidative stress [2]. These were important observations as activated HSCs play a central role in liver fibrosis and sinusoidal component of portal hypertension [3]. HSCs produce several growth factors, cytokines and chemokines [4], [5] responsible for initiation and progression of liver pathology. Therefore, strategies to remove selectively activated HSCs from the fibrotic liver are intensely investigated.
Gliotoxin possesses immunomodulating activity: it stimulates lymphocyte proliferation, and induces T cell cytotoxicity and lymphokine release by T cells [6], [7], [8], [9]. Gliotoxin (10–100 nM) was found to inhibit phagocytic activity of peritoneal and circulating macrophages [6], [7], [9] at concentrations lower than those required to cause apoptosis of HSCs. High concentration of gliotoxin (>1 μM) induces reactive oxygen species (ROS)-mediated apoptosis of peritoneal macrophages, independent of the inhibition of phagocytosis [8]. The role of hepatic macrophages Kupffer cells extends from defense against invading microbes and clearance of toxic substances to liver growth and immune regulation [10], [11], [12], [13]. These functions of Kupffer cells are even more critical in disease states as their removal can significantly augment pathological development [14]. Since gliotoxin-induced elimination of activated HSCs could be therapeutic strategy for liver fibrosis, it is important to rule out its adverse effects on other hepatic cell types. Therefore, we investigated the effects of gliotoxin on rat Kupffer cells in vitro, and in control and CCl4-induced cirrhotic rats in vivo.
Section snippets
Materials and methods
Gliotoxin, TPCK, MG132 (Sigma, St. Louis, MO); PD98059, SB203580, SP600125 and PDTC (Calbiochem, La Jolla, CA); Z-DEVD-fmk (R&D, Minneapolis, MN); Mn-TBAP (Calbiochem, La Jolla, CA); anti-rabbit NFκB-p65, ERK1/2 and caspase-3, mouse anti-rat desmin antibodies and CY-3-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-rabbit monoclonal P-ERK1/2, P-p38, p38, P-JNK and JNK antibodies (Cell Signaling, Danvers, MA); anti-rat ED2 antibody (Serotech,
Gliotoxin decreases Kupffer cell viability
Viability of control cells did not change over 24 h (Fig. 1A). Gliotoxin (0.03 μM) caused significant reduction in viability only at 24 h. At 0.3 and 3.0 μM, gliotoxin caused time-dependent loss of viability that was significant at 3 and 1 h, respectively (Fig. 1A); incubation beyond 3 h at both concentrations caused detachment of the majority of cells. Gliotoxin caused equivalent loss of viability of Kupffer cells even in the presence of serum (Fig. 1B). Pancreatic elastase (apoptotic agent for
Discussion
Here we report that gliotoxin causes death of cultured Kupffer cells via apoptosis and secondary necrosis. Previously, activated rat and human HSCs were found to undergo apoptosis and exclude trypan blue at 0.3–7.5 μM gliotoxin [1], [2] but necrosis was observed at concentration above 7.5 μM [2]. Hepatocytes are resistant to lower gliotoxin concentration but undergo necrosis at high concentration (50 μM). Apoptosis of Kupffer cells by lower concentration of gliotoxin (0.03–0.3 μM) indicates their
Acknowledgements
This work was supported by two grants from National Institutes of Health: RO1-DK54401 to CRG and CA76541 to DBS. We thank Dr. C. Thirunavukkarasu for assistance with apoptotic assays and Mr. Mark Ross for immunostaining of liver sections.
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2019, CytokineCitation Excerpt :Selective induction of apoptosis in HSCs can be another therapeutic route for curing liver fibrosis. Pharmacological agents such as gliotoxin [50], curcumin [184] and bortezomib [185] induce apoptosis in HSCs thereby inhibiting hepatic fibrogenesis. Mesenchymal stem cells (MSCs) regulate the functioning of activated HSCs via paracrine mechanisms, thereby mediating protection in liver inflammation and fibrosis.
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2014, Journal of HepatologyCitation Excerpt :A fungal metabolite gliotoxin was found to cause apoptosis of activated rat and human HSCs in vitro, and of rat HSCs in vivo resulting in resolution of fibrosis [18,19]. However, gliotoxin also induces apoptosis of KCs and endothelial cells in the fibrotic liver [20,21]. Ebrahimkhani et al. [22] administered gliotoxin into bile duct-ligated mice in conjugation with the single-chain antibody C1–3, which recognizes synaptophysin expressed by activated HSCs [23]; C1–3-gliotoxin caused resolution of fibrosis by selectively depleting HSCs.
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The authors who have taken part in this study declared that they have no relationship with the manufacturers of the drugs involved either in the past or present and did not receive funding from the manufacturers to carry out their research. The authors received funding from the National Institutes of Health, USA, which enabled them to carry out their study.