Macrophage cell death plays a role in many physiological and pathophysiological conditions. Previous work has shown that macrophages can undergo caspase-independent cell death, and this process is associated with Nur77 induction, which is involved in inducing chromatin condensation and DNA fragmentation. Here we show that autophagy is a cytosolic event that controls caspase-independent macrophage cell death. Autophagy was induced in macrophages treated with lipopolysaccharides (LPSs) and the pan-caspase inhibitor benzyloxycarbonylVal-Ala-Asp (Z-VAD), and the inhibition of autophagy by either chemical inhibitors or by the RNA interference knockdown of beclin (a protein required for autophagic body formation) inhibited caspase-independent macrophage cell death. We also found an increase in poly(ADP-ribose) (PAR) polymerase (PARP) activation and reactive oxygen species (ROS) production in LPS ؉ Z-VAD-treated macrophages, and both are involved in caspase-independent macrophage cell death. We further determined that the formation of autophagic bodies in macrophages occurs downstream of PARP activation, and PARP activation occurs downstream of ROS production. Using macrophages in which receptor-interacting protein 1 (RIP1) was knocked down by small interfering RNA, and macrophages isolated from Toll/ interleukin-1 receptor-domain-containing adaptor inducing IFN-␤ (TRIF)-deficient mice, we found that TRIF and RIP1 function upstream of ROS production in LPS ؉ Z-VAD-treated macrophages. We also found that Z-VAD inhibits LPS-induced RIP1 cleavage, which may contribute to ROS over-production in macrophages. This paper reveals that TRIF, RIP1, and ROS production, as well as PARP activation, are involved in inducing autophagy, which contributes to caspase-independent macrophage cell death.

The stimulation of Toll-like receptors (TLRs) on macrophages triggers production of the cytokine tumor necrosis factor (TNF). TNF production occurs within 1 h of TLR stimulation and is sustained for 1 d. Here we document a function for the TNF family member 4-1BB ligand (4-1BBL) in sustaining TLR-induced TNF production. TLR signaling induced 4-1BBL, and 4-1BBL interacted with TLRs on the macrophage surface. The influence of 4-1BBL on TNF production was independent of its receptor (4-1BB) and did not require the adaptors MyD88 or TRIF. It did not influence TLR4-induced activation of transcription factor NF-kappaB (an early response) but was required for TLR4-induced activation of transcription factors CREB and C/EBP (a late event). Transient TLR4-MyD88 complexes appeared during the first hour after lipopolysaccharide stimulation, and TLR4-4-1BBL interactions were detected between 2 h and 8 h after lipopolysaccharide stimulation. Our results indicate that two different TLR4 complexes sequentially form and selectively control early and late TNF production.

TNF-α is a potent proinflammatory cytokine, essential for initiating innate immune responses against invading microbes and a key mediator involved in the pathogenesis of acute and chronic inflammatory diseases. To identify molecules involved in the production of TNF-α, we used a functional gene identification method using retroviral integration-mediated mutagenesis, followed by LPS-stimulated TNF-α production analysis in macrophages. We found that cathepsin B, a lysosomal cysteine proteinase, was required for optimal posttranslational processing of TNF-α in response to the bacterial cell wall component LPS. Mouse bone marrow-derived macrophages from cathepsin B-deficient mice and macrophages treated with the cathepsin B-specific chemical inhibitor CA074 methyl ester or small interfering RNA against cathepsin B secreted significantly less TNF-α than wild-type or nontreated macrophages. We further showed that the inhibition of cathepsin B caused accumulation of 26-kDa pro-TNF-containing vesicles. Ectopic expression of GFP-conjugated pro-TNF further suggests that pro-TNF failed to reach the plasma membrane without intracellular cathepsin B activity. Altogether, these data suggest that intracellular cathepsin B activity is involved in the TNF-α-containing vesicle trafficking to the plasma membrane.

Anthrax lethal toxin (LeTx) is a virulence factor secreted byBacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTxcontaining endosomes. Also, binding of protective antigen to ANXTR1 or -2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me-or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux. Anthrax lethal toxin (LeTx)2 and edema toxin are two key virulence factors secreted by Bacillus anthracis, the causative agent of anthrax (1, 2). LeTx and edema toxin are composed of lethal factor (LF) or edema factor (EF), respectively, and protective antigen (PA), which functions as a cytoplasmic transporter of LF or EF. These toxins are main contributors to the clinical manifestations of anthrax and are cytotoxic to host cells once delivered into the cytoplasm. EF is an adenylate cyclase that raises cAMP levels in cells (3). LF is a metalloproteinase that targets the N-terminal end of the mitogen-activated protein kinase kinase 1-7 (MEK1-7) (except MEK5) (4 -6) and in certain mouse cells induces pyronecrosis by activating NACHTleucine-rich repeat and pyrin domain-containing protein 1b (NALP1b) (7).Incorporation of LF or EF into the cytoplasm is initiated by the binding of PA to the host cell surface through interacting with either of two known receptors: anthrax receptor 1 (ANTXR1, also known as the tumor endothelial marker 8) (8) and ANTXR2 (also known as the capillary morphogenesis gene-2) (9). Both ANTXR1 and -2 are widely distributed in human tissues and share molecular and biochemical similarities in their extracellular PA interacting domains known as von Willebrand factor A or integrin-like inserted (I) domain (10), post-translational modifications such as palmitoylation and ubiquitination of cytoplasmic domains (11), and associations with the co-receptor lipoprotein receptor-related 6 molecule (12, 13). ANTXR1 and -2 are also distinct in that ANTXR1 is highly expressed in tumor endothelial and cancer cells, and ANTXR2 has a higher binding affinity to PA and requires a lower pH to form a transmembrane pore than ANTXR1 (10,14).After binding PA to either ANTXR, a furin-like surface protease then cleaves PA at the N-terminal end to release a 20-kDa soluble fragment, yielding membrane-associated 63-kDa PA (PA 63 ) that forms a ring-shape heptameric ...

Activation-induced cell death in macrophages has been observed, but the mechanism remains largely unknown. Activation-induced cell death in macrophages can be independent from caspases, and the death of activated macrophages can even be triggered by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD). Here, we show that this type of macrophage death can occur in the septic mouse model and that toll-like receptor (TLR)-2 or TLR4 signaling is required in this process. We conclude that Nur77 is involved in the macrophage death because Nur77 expression correlates with cell death, and cell death is reduced significantly in Nur77-deficient macrophages. The extracellular signal–regulated kinase pathway, which is downstream of TLR2 or TLR4, and myocyte-specific enhancer binding factor 2 (MEF2) transcription factor activity, which is up-regulated by zVAD, are required for Nur77 induction and macrophage death. Reporter gene analysis suggests that Nap, Ets, Rce, and Sp1 sites in the Nur77 promoter are regulated by TLR4 signaling and that MEF2 sites in the Nur77 promoter are regulated by zVAD treatment. MEF2 transcription factors are constitutively expressed and degraded in macrophages, and zVAD increases MEF2 transcription factor activity by preventing the proteolytic cleavage and degradation of MEF2 proteins. This paper delineates the dual signaling pathways that are required for Nur77 induction in macrophages and demonstrates a role of Nur77 in caspase-independent cell death.