Activation of CD40 with Platelet Derived CD154 Promotes Reactive Oxygen Species Dependent Death of Human Hepatocytes during Hypoxia and Reoxygenation

Abstract

BackgroundHypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis.MethodsHuman hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2′,7′-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay.ResultsExposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis.ConclusionsCD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.

Hepatocyte‐induced CD4+ T cell alloresponse is associated with major histocompatibility complex class II up‐regulation on hepatocytes and suppressible by regulatory T cells

“…The levels measured might therefore indicate the induction of endogenic repair mechanisms in isolated PHHs, especially because IL22 cooperates with IL17A in inflammation control . The unexpected presence of sCD40L in the control group could be a result of hepatocyte ischemia intraoperatively or during cell culture: inflammation as well as hypoxia are known to trigger the CD40/CD40L pathway …”

“…(48) The unexpected presence of sCD40L in the control group could be a result of hepatocyte ischemia intraoperatively or during cell culture: inflammation as well as hypoxia are known to trigger the CD40/CD40L pathway. (49,50) Furthermore, high levels of anti-inflammatory IL10 following allostimulation with PHH seem interesting considering the inflammatory environment. Activated CD8 1 T cells are known to stimulate hepatocytes to produce anti-inflammatory cytokines (eg, IL4, IL10) (51) and Th2 CD4 1 T cells produce IL10.…”

Circulating membrane-derived microvesicles in redox biology

“…This response is amplified following ischemia-reperfusion injury, resulting in NADPH oxidase-dependent cell death. Inhibiting JNK and p38 signaling pathways attenuates CD154-mediated apoptosis [101]. These MV-triggered signaling pathways are mediated, in part, by reactive species [101,102].…”

Novel treatments targeting immune‐related mechanisms in primary biliary cholangitis

“…CD40 is expressed on other antigen‐presenting cells including dendritic cells and macrophages, and activation of the latter by interaction with CD40L on T cells stimulates macrophages to produce interleukin (IL)‐12 and skew T helper cell into Th1 phenotype. In cholangiocytes, CD40 signaling also augments Fas‐dependent cell death …”

“…In cholangiocytes, CD40 signaling also augments Fas-dependent cell death. 6 Administration of anti-CD40L in murine models of autoimmune cholangitis reduces liver inflammation and lowers the levels of antimitocondrial antibody (AMA). A study of the safety, tolerability, and pharmacodynamics of dacetuzumab/lucatumumab, a CD40 antagonist, in patients with PBC is currently ongoing (Clinical Trial ID: NCT02193360).…”