Pyroptosis is a caspase-1-dependent inflammatory form of cell death. The adapter protein ASC binds directly to caspase-1 and is critical for caspase-1 activation in response to a broad range of stimuli. To elucidate the mechanism of activation of caspase-1 by ASC and its exact role in macrophage pyroptosis, we performed time-lapse confocal bioimaging analysis on human THP-1 macrophages stably expressing an ASC-GFP fusion protein. We show that stimulation of these cells with several proinflammatory stimuli trigger the formation of a large supramolecular assembly of ASC, termed here pyroptosome. Only one distinct pyroptosome in each stimulated cell is formed, which rapidly recruits and activates caspase-1 resulting in pyroptosis and the release of the intracellular proinflammatory cytokines. The pyroptosome is largely composed of oligomerized ASC dimers. Dimerization of ASC is driven by subphysiological concentrations of potassium as in vitro incubation of purified recombinant ASC in the presence of subphysiological concentrations of potassium induces the assembly of a functional pyroptosome. Furthermore, stimulation of potassium efflux in THP-1 cells with potassium-depleting agents induces formation of the pyroptosome, while increasing potassium concentrations in the culture medium or pharmacological inhibition of this efflux inhibits its assembly. Our results establish that macrophage pyroptosis is mediated by a unique pyroptosome, distinct from the inflammasome.

Programmed cell death, or apoptosis, is important in homeostasis of the immune system: for example, non-functional or autoreactive lymphocytes are eliminated through apoptosis. One member of the tumour necrosis factor receptor (TNFR) family, Fas (also known as CD95 or Apo-1), can trigger cell death and is essential for lymphocyte homeostasis. FADD/Mort1 is a Fas-associated protein that is thought to mediate apoptosis by recruiting the protease caspase-8. A dominant-negative mutant of FADD inhibits apoptosis initiated by Fas and other TNFR family members. Other proteins, notably Daxx, also bind Fas and presumably mediate a FADD-independent apoptotic pathway. Here we investigate the role of FADD in vivo by generating FADD-deficient mice. As homozygous mice die in utero, we generated FADD-/- embryonic stem cells and FADD-/- chimaeras in a background devoid of the recombination activating gene RAG-1, which activates rearrangement of the immunoglobulin and T-cell receptor genes. We found that thymocyte subpopulations were apparently normal in newborn chimaeras. Fas-induced apoptosis was completely blocked, indicating that there are no redundant Fas apoptotic pathways. As these mice age, their thymocytes decrease to an undetectable level, although peripheral T cells are present in all older FADD-/- chimaeras. Unexpectedly, activation-induced proliferation is impaired in these FADD-/- T cells, despite production of the cytokine interleukin (IL)-2. These results and the similarities between FADD-/- mice and mice lacking the beta-subunit of the IL-2 receptor suggest that there is an unexpected connection between cell proliferation and apoptosis.

Background: It remains a matter of debate whether autophagy contributes to apoptosis. Results: Atg5 and p62 are required for an intracellular death-inducing signaling complex (iDISC) formation on autophagosomal membranes for caspase-8 self-processing. Conclusion: Autophagosome serves as a platform for the intracellular activation of caspase-8. Significance: Induction of iDISC formation may shift cytoprotective autophagy to apoptosis for more effective cancer therapies.

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G 1 /S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G 1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G 1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G 1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.Apoptosis in immature T cells and T-cell hybridomas, which may relate to negative selection during T-cell development, can be initiated by signals through the T-cell receptor-CD3 complex (18,32,33). This process of activation-induced apoptosis (anti-CD3 apoptosis) consists of two distinct phases. The first phase is the cell cycle block at the G 1 /S transition; it is followed by the second phase, with the generation of apoptotic DNA ladders (18). The second phase requires extracellular calcium and can be inhibited by the immunosuppressive drug cyclosporin A (18). We and others have shown that Nur77 (NGFI-B) orphan steroid receptor is induced during anti-CD3 apoptosis through the calcium signals and it plays an essential role in the cell death process (17, 35). Dominant negative Nur77 can block apoptosis but not the interleukin 2 production of anti-CD3-treated T-cell hybridomas (35). Thus, Nur77 is involved in the second phase of anti-CD3 T-cell apoptosis.As alluded to above, anti-CD3 death in T-cell hybridomas is also accompanied by a G 1 cell cycle block. In all organisms studied so far, cell cycle progression is mediated by cyclindependent kinases (CDKs) that consist of a catalytic subunit CDK and a regulatory subunit cyclin. In mammalian cells, cyclin E-CDK2 together with cyclin D-CDK4 and/or cyclin D-CDK6, which are active in the G 1 phase, control the G 1 -to-S transition (23, 24, 28, 31 and references therein). There are at least three different D-type cyclins, with T cells expressing cyclins D2 and D3 and two cyclin D-associating kinases, CDK4 and CDK6. One of their substrates is the retinobl...