In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases, leading to fodrin cleavage and apoptotic nuclear morphology. Bcl-2 acted in situ on mitochondria to prevent the release of cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2. Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome c translocation, thereby blocking caspase activation and the apoptotic process.

https://science.sciencemag.org/content/sci/275/5303/1132.full.pdf

The Release of Cytochrome c from Mitochondria: A Primary Site for Bcl-2 Regulation of Apoptosis

The nuclear factor NF-κB pathway has long been considered a prototypical proinflammatory signaling pathway, largely based on the role of NF-κB in the expression of proinflammatory genes including cytokines, chemokines, and adhesion molecules In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-κB in inflammation that suggest both pro- and anti-inflammatory roles for this pathway NF-κB has long been considered the “holy grail” as a target for new anti-inflammatory drugs; however, these recent studies suggest this pathway may prove a difficult target in the treatment of chronic disease In this article, we discuss the role of NF-κB in inflammation in light of these recent studies

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The Nuclear Factor NF-κB Pathway in Inflammation

Apoptosis in vivo is followed almost inevitably by rapid uptake into adjacent phagocytic cells, a critical process in tissue remodeling, regulation of the immune response, or resolution of inflammation. Phagocytosis of apoptotic cells by macrophages has been suggested to be a quiet process that does not lead to production of inflammatory mediators. Here we show that phagocytosis of apoptotic neutrophils (in contrast to immunoglobulin G-opsonized apoptotic cells) actively inhibited the production of interleukin (IL)-1beta, IL-8, IL-10, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-alpha, as well as leukotriene C4 and thromboxane B2, by human monocyte-derived macrophages. In contrast, production of transforming growth factor (TGF)-beta1, prostaglandin E2, and platelet-activating factor (PAF) was increased. The latter appeared to be involved in the inhibition of proinflammatory cytokine production because addition of exogenous TGF-beta1, prostaglandin E2, or PAF resulted in inhibition of lipopolysaccharide-stimulated cytokine production. Furthermore, anti-TGF-beta antibody, indomethacin, or PAF receptor antagonists restored cytokine production in lipopolysaccharide-stimulated macrophages that had phagocytosed apoptotic cells. These results suggest that binding and/or phagocytosis of apoptotic cells induces active antiinflammatory or suppressive properties in human macrophages. Therefore, it is likely that resolution of inflammation depends not only on the removal of apoptotic cells but on active suppression of inflammatory mediator production. Disorders in either could result in chronic inflammatory diseases.

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Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.

A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.

https://rupress.org/jem/article-pdf/182/5/1545/1107564/1545.pdf

Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.

■ Abstract Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli. Studies performed over the past 10 years have demonstrated that proteases play critical roles in initiation and execution of this process. The caspases, a family of cysteine-dependent aspartate-directed proteases, are prominent among the death proteases. Caspases are synthesized as relatively inactive zymogens that become activated by scaffold-mediated transactivation or by cleavage via upstream proteases in an intracellular cascade. Regulation of caspase activation and activity occurs at several different levels: ( a) Zymogen gene transcription is regulated; ( b) antiapoptotic members of the Bcl-2 family and other cellular polypeptides block proximity-induced activation of certain procaspases; and ( c) certain cellular inhibitor of apoptosis proteins (cIAPs) can bind to and inhibit active caspases. Once activated, caspases cleave a variety of intracellular polypeptides, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and a number of protein kinases. Collectively, these scissions disrupt survival pathways and disassemble important architectural components of the cell, contributing to the stereotypic morphological and biochemical changes that characterize apoptotic cell death.

Mammalian caspases: structure ,a ctivation ,s ubstrates, and functions during apoptosis

A number of murine T-cell hybridomas undergo apoptosis within a few hours of activation by specific antigens, mitogens, antibodies against the T-cell antigen receptor, or a combination of phorbol ester and calcium ionophore. This phenomenon has been extensively studied as a model for clonal deletion in the immune system, in which potentially autoreactive T cells eliminate themselves by apoptosis after activation, either in the thymus or in the periphery. Here we show that the Fas/CD95 receptor, which can transduce a potent apoptotic signal when ligand, is rapidly expressed following activation of T-cell hybridomas, as is its functional, membrane-bound ligand. Interference with the ensuing Fas/Fas-ligand interaction inhibits activation-induced apoptosis. Because T-cell receptor ligation can induce apoptosis in a single T hybridoma cell, we suggest that the Fas/Fas-ligand interaction can induce cell death in a cell-autonomous manner.

Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas

We have previously reported that activation of murine T cell hybridomas leads to expression of Fas (CD95) and Its ligand (FasL) which subsequently interact, even on the same cell, leading to apoptotlc cell death. Since the immunosuppressive drugs cyclosporin A (CsA) and FK506 block activation-Induced apoptosis in T cell hybridomas, we examined whether such compounds affect cell death by interfering with expression of Fas, FasL or both, or whether they block Fas signal transduction. We have found that CsA- and FK5O6-treated cells did not exhibit transcription of FasL mRNA after activation and were lacking functional FasL protein on their surface as determined by staining and the ability to induce apoptosis In Fas+ target cells. In contrast, no inhibition of the elevated Fas mRNA expression was observed in cells activated in the presence of CsA or FK506. Surprisingly, however, cell surface Fas levels were consistently lower on cells activated in the presence of Immunosuppres sive drugs than on activated cells, suggesting that Fas expression is regulated at several levels. Nevertheless, cells activated In the presence of CsA or FK5O6 underwent apoptosis upon treatment with anti-Fas antibody, while unactivated cells did not Furthermore, CsA and FK506 do not interfere with Fas signaling since anti-Fas induced apoptosis in Fas+ target cells was unaffected by these drugs. We therefore conclude that CsA and FK506 block activation-induced apoptosis in T cell hybridomas predominantly by interfering with activation signals leading to FasL expression and, further, that the regulation of the expression of Fas and FasL on activated T cells is differentially controlled.

/pdf/activation-induced-cell-death-in-murine-t-cell-hybridomas-1b3aqmqo61.pdf

Activation-induced cell death in murine T cell hybridomas. Differential regulation of Fas (CD95) versus Fas ligand expression by cyclosporin A and FK506

https://science.sciencemag.org/content/sci/269/5220/106.full.pdf

Drake LaFace

Papers

Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.

Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas

Activation-induced cell death in murine T cell hybridomas. Differential regulation of Fas (CD95) versus Fas ligand expression by cyclosporin A and FK506

p34cdc2 and apoptosis

A phage display system for detection of T cell receptor-antigen interactions