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Sterile cell death mediated inflammation is linked to several pathological disorders and involves danger recognition of intracellular molecules released by necrotic cells that activate different groups of innate pattern recognition receptors. Toll-like receptors directly interact with their extrinsic or intrinsic agonists and induce multiple proinflammatory mediators. In contrast, the NLRP3 inflammasome is rather thought to represent a downstream element integrating various indirect stimuli into proteolytic cleavage of interleukin (IL)–1β and IL-18. Here, we report that histones released from necrotic cells induce IL-1β secretion in an NLRP3–ASC-caspase-1-dependent manner. Genetic deletion of NLRP3 in mice significantly attenuated histone-induced IL-1β production and neutrophil recruitment. Furthermore, necrotic cells induced neutrophil recruitment, which was significantly reduced by histone-neutralizing antibodies or depleting extracellular histones via enzymatic degradation. These results identify cytosolic uptake of necrotic cell-derived histones as a triggering mechanism of sterile inflammation, which involves NLRP3 inflammasome activation and IL-1β secretion via oxidative stress.

As a hallmark of tuberculosis (TB), Mycobacterium tuberculosis (MTB) induces granulomatous lung lesions and systemic inflammatory responses during active disease. Molecular regulation of inflammation is associated with inflammasome assembly. We determined the extent to which MTB triggers inflammasome activation and how this impacts on the severity of TB in a mouse model. MTB stimulated release of mature IL-1β in macrophages while attenuated M. bovis BCG failed to do so. Tubercle bacilli specifically activated the NLRP3 inflammasome and this propensity was strictly controlled by the virulence-associated RD1 locus of MTB. However, Nlrp3-deficient mice controlled pulmonary TB, a feature correlated with NLRP3-independent production of IL-1β in infected lungs. Our studies demonstrate that MTB activates the NLRP3 inflammasome in macrophages in an ESX-1-dependent manner. However, during TB, MTB promotes NLRP3- and caspase-1-independent IL-1β release in myeloid cells recruited to lung parenchyma and thus overcomes NLRP3 deficiency in vivo in experimental models.

NLRP3 inflammasome-dependent inflammatory responses are triggered by a variety of signals of host danger, including infection, tissue damage and metabolic dysregulation. How these diverse activators cause inflammasome activation is poorly understood. Recent data suggest that the mitochondria integrate these distinct signals and relay this information to the NLRP3 inflammasome. Dysfunctional mitochondria generate ROS, which is required for inflammasome activation. On the contrary, the NLRP3 inflammasome is negatively regulated by autophagy, which is a catabolic process that removes damaged or otherwise dysfunctional organelles, including mitochondria. In addition to the processing and secretion of pro-inflammatory cytokines such as IL-1β, NLRP3 inflammasome activation also influences cellular metabolic pathways such as glycolysis and lipogenesis. Mapping the connections between mitochondria, metabolism and inflammation is of great interest, as malfunctioning of this network is associated with many chronic inflammatory diseases.

Recent studies have shown that in humans the germinal center reactions produce three types of V(D)J mutated B cells in similar proportions, i.e. Ig-switched, IgD-IgM+ (IgM-only) and IgD+IgM+ cells, and that together they form the CD27+ compartment of recirculating B cells. We investigated the Ig isotype switch capacity of these cells. Peripheral blood B subsets were sorted and IgG subclass secretion in presence or absence of IL-4 was compared in B cell assays which lead to Ig secretion in all (coculture with EL-4 thymoma cells) or only in CD27+ (CD40L stimulation) B cells. Already switched IgG+ B cells showed no significant sequential switch and IgM-only cells also had a low switch capacity, but IgD+CD27+ switched as much as IgD+CD27- B cells to all IgG subclasses. Thus, in switched B cells some alterations compromising further switch options occur frequently; IgM-only cells may result from aborted switch. However, IgD+CD27+ human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel cross-reactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.

TWEAK is a recently cloned novel member of the TNF ligand family. Here we show that soluble TWEAK is sufficient to induce apoptosis in Kym-1 cells within 18 h. TWEAK-induced apoptosis is indirect and is mediated by the interaction of endogenous TNF and TNF receptor (TNFR)1, as each TNFR1-Fc, neutralizing TNF-specific antibodies and TNFR1-specific Fab fragments efficiently antagonize cell death induction. In addition to this indirect mode of action, co-stimulation of Kym-1 cells with TWEAK enhances TNFR1-mediated cell death induction. In contrast to TNF, TWEAK does only modestly activate NF-kappaB or c-jun N-terminal kinase (JNK) in Kym-1 cells. Although TWEAK binding to Kym-1 cells is easily detectable by flow cytometric analysis, we found neither evidence for expression of the recently identified TWEAK receptor Apo3/TRAMP/wsl/DR3/LARD, nor indications for direct interactions of TWEAK with TNFR. Together, these characteristics of TWEAK-induced signaling in Kym-1 cells argue for the existence of an additional, still undefined non-death domain-containing TWEAK receptor in Kym-1 cells.

Normal or perforin-deficient C57BL/6 mice are able to heal spontaneously cutaneous lesions induced by Leishmania major. In this report, we show that syngeneic gld and lpr mice, lacking a functional Fas system, fail to heal their lesions. This inability to control infection could not be attributed either to a failure to mount a protective CD4+ Th1 response or to an unresponsiveness of their macrophages to the activating signals of IFN-gamma. The observation showing that administration of exogenous recombinant Fas ligand (FasL) to FasL-deficient mice resulted in the resolution of cutaneous lesions demonstrates the importance of the Fas-FasL pathway in the elimination of parasites. Furthermore, macrophages infected with L. major in vitro up-regulate their surface expression of Fas in response to IFN-gamma and as a result become susceptible to CD4+ T cell-induced apoptotic death. These results suggest that the CD4+ T cell-induced apoptotic death of MHC class II-expressing antigen-presenting cells, observed in vitro and operating through the Fas (Apo-1/CD95) pathway, is relevant in vivo.

The expression and action of Fas/Fas ligand (FasL) in multiple sclerosis has been postulated as a major pathway leading to inflammatory demyelination. To formally test this hypothesis, C57BL/6-lpr and -gld mice, which due to gene mutation express Fas and FasL in an inactive form, were immunized with myelin oligodendrocyte glycoprotein peptide(35-55). Whereas in wild-type C57BL/6 mice, experimental autoimmune encephalomyelitis (EAE), was chronic/relapsing, EAE in lpr and gld mice was characterized by a lower incidence of disease and a monophasic course. This contrasts with C57BL/6 perforin knockout mice, which showed the most severe form of EAE of all mouse strains tested, the course being chronic relapsing. The difference noted cannot be attributed to an involvement of FasL in oligodendrocyte damage since oligodendrocytes are insensitive to FasL-mediated cytotoxicity in vitro, and since in the acute phase of EAE gld mice also show CD4+ T cell infiltrates with associated demyelination in brain and spinal cord. Unlike oligodendrocytes, astrocytes were killed by FasL in vitro. It remains to be established whether this latter finding explains the different disease course of lpr and gld mice compared to wild-type and perforin knockout mice.

Cytotoxic T lymphocytes (CTL) kill target cells by perforin-mediated pore formation, induction of apoptosis by the Fas ligand, or both. It has been demonstrated that depolarized neurons can be induced to express major histocompatibility complex (MHC) class I antigens by interferon-gamma. Evidence for antigen-dependent CTL-mediated killing was obtained by transfecting neurons with MHC class I cDNA. The present study was designed to investigate the mechanisms of killing of cerebellar granule neurons depolarized by high K+ concentrations and thereby inducible for MHC class I antigen expression. We found that neurons express only low levels of Fas (APO-1/CD95) and are resistant to Fas ligand-mediated killing even when pretreated with cytokines. However, granules extracted from CTL as well as purified perforin induce almost complete lysis of neurons. These data suggest that CTL-mediated elimination of neurons involves the perforin, but not the Fas pathway of target cell killing.

Fas ligand (FasL, Apo-1L) is a member of the tumor necrosis factor protein family and binding to its receptor (Fas, Apo-1, CD95) triggers cell death through apoptosis. Ligand expression is restricted to cells with known cytolytic activity and found on hematopoietic cells of the T cell and natural killer lineage. Here we provide evidence that B lymphocytes can express FasL. Flow cytometric analysis revealed that FasL is expressed on the surface of B cells upon stimulation with either lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin. FasL expression on activated B cells was confirmed by western blot and reverse transcriptase polymerase chain reaction analysis. FasL on B cells is functional since lipopolysaccharide-activated B lymphocytes derived from wild type, but not from gld mutant mice, were able to kill Fas-sensitive target cells. Our data suggest that the Fas system may contribute to the control of B cell homeostasis.

Aeromonas hydrophila is a Gram-negative pathogen that causes serious infectious disease in humans. A. hydrophila induces apoptosis in infected macrophages, but the host proinflammatory responses triggered by macrophage death are largely unknown. Here, we demonstrate that the infection of mouse macrophages with A. hydrophila triggers the activation of caspase-1 and release of IL-1β. Caspase-1 activation was abrogated in macrophages deficient in Nod-like receptor family, pyrin domain containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), but not NLR family, CARD domain containing 4 (NLRC4). The activation of the NLRP3 inflammasome was mediated by three cytotoxins (aerolysin, hemolysin and multifunctional repeat-in-toxin) produced by A. hydrophila. Our results indicated that the NLRP3 inflammasome senses A. hydrophila infection through the action of bacterial cytotoxins.

The TNF family ligand B cell-activating factor (BAFF, BLyS, TALL-1) is an essential factor for B cell development. BAFF binds to three receptors, BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA), but only BAFF-R is required for successful survival and maturation of splenic B cells. To test whether the effect of BAFF is due to the up-regulation of anti-apoptotic factors, TACI-Ig-transgenic mice, in which BAFF function is inhibited, were crossed with transgenic mice expressing FLICE-inhibitory protein (FLIP) or Bcl-2 in the B cell compartment. FLIP expression did not rescue B cells, while enforced Bcl-2 expression restored peripheral B cells and the ability to mount T-dependent antibody responses. However, many B cells retained immaturity markers and failed to express normal amounts of CD21. Marginal zone B cells were not restored and the T-independent IgG3, but not IgM, response was impaired in the TACI-IgxBcl-2 mice. These results suggest that BAFF is required not only to inhibit apoptosis of maturating B cells, but also to promote differentiation events, in particular those leading to the generation of marginal zone B cells.

Human B cell-activating factor (BAFF) induces mouse surface IgM+ B cells of the immature type from bone marrow and of the immature types 1 and 2 from spleen, as well as of the mature type from spleen to increased longevity in tissue culture. BAFF does so polyclonally and without inducing proliferation in any of these B cell subpopulations. BAFF induces phenotypic and functional maturation of immature to mature B cells so that all immature cells loose C1qRp (AA4.1, 493) expression and type 1 immature cells up-regulate IgD, CD21 and CD23. Immature B cells of types 1 and 2, upon pre-incubation with BAFF, change their reactiveness to Ig-specific antibodies so that they no longer enter apoptosis but now proliferate. However, BAFF does not seem to overcome negative selection of developing immature B cells in vitro.

The mechanisms underlying neurotoxicity mediated by cytotoxic T lymphocytes (CTL) and their secretory granule proteins perforin and granzymes remain unclear. We evaluated the possible role of the neurotransmitter glutamate in cell death observed in differentiated neurons exposed to CTL-derived granules. Excitotoxicity induced by excessive concentrations of extracellular glutamate is associated with a rise in intracellular calcium and can lead to generation of NO through the activation of glutamatergic N-methyl-D-aspartate (NMDA) receptors. Consistent with an involvement of glutamate, we found that cell death in mature cerebral granule cells was inhibited by 65-80% by two NMDA receptor blockers (MK-801 and D-2-amino-5-phosphonovaleric acid) or a NO synthase blocker (N(G)-nitro-L-arginine methylester). Furthermore, neurons treated with secretory granules responded with a biphasic rise in the intracellular calcium concentration ([Ca2+]i). Whereas MK-801 did not interfere with the immediate rise of [Ca2+]i, the second wave of calcium accumulation starting at 40 min was delayed by 20 min and reduced in amplitude in the presence of MK-801. In immature, NMDA receptor-negative neurons, MK-801 prevented neither the cytotoxicity nor the calcium influx observed 5 min after addition of cytotoxic granules. The demonstration that NMDA receptors and NO are involved in granule-mediated killing of mature neurons opens new avenues in the treatment of neuronal cell death in CTL-mediated diseases such as viral encephalitis.

Superantigens are bacterial or viral products that polyclonally activate T cells bearing certain TCR beta chain variable elements. For instance, Vbeta8+ T cells proliferate in response to staphylococcal enterotoxin B (SEB) in vivo and then undergo Fas- and/or TNF-mediated apoptosis. We have recently shown that apoptotic SEB-reactive T cells express the B cell marker B220. Here we report the identification of a novel subset of CD4+ B220+ T cell blasts that are the precursors of these apoptotic cells in SEB-immunized mice. Moreover, we show that the CD4- CD8- B220+ T cells that accumulate in the lymphoid organs of Fas ligand-defective gld mice stably express a form of the B220 molecule which exhibits biochemical similarities to that expressed by activated wild-type T cells, but is distinct from that displayed on the surface of B cells. Surprisingly, we also find a population of CD4+ B220+ pre-apoptotic T cells in FasL-defective gld mice, arguing that these cells can be generated in a Fas-independent fashion. Collectively, our data support a general model whereby upon activation, T cells up-regulate B220 before undergoing apoptosis. When the apoptotic mechanisms are defective, T cells presumably down-regulate their coreceptor molecules but retain expression of B220 as they accumulate in lymphoid organs.

The aspartase granzyme B is one of the major components of the granules involved in cell killing by cytotoxic T lymphocytes. Granzyme B has been shown to activate the apoptotic death pathway in the target cell, and this involves activation of members of the caspase (CASP) protein family. Therefore, activational cleavage of mouse (m) CASP proforms by granzyme B was examined in vitro. CASP can be subdivided in the CASP-1 (interleukin-1 beta-converting enzyme; ICE) subfamily, the CASP-2 (Ich1) subfamily, and the CASP-3 (CPP32) subfamily. Our results reveal that the proforms of the CASP-3 subfamily members mCASP-3 and mCASP-7 are hydrolyzed by granzyme B, while proforms of CASP-2 and CASP-1 subfamily members are not directly cleaved. Only one CASP-3 subfamily member, pro-mCASP-6, was not proteolytically cleaved by granzyme B. These results indicate that two members of the CASP-3 subfamily, but no others, become activated by granzyme B.

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.

Fas was recently demonstrated to be the major target molecule engaged by CD4+ cytolytic T lymphocytes (CTL). We examined Fas expression on various cloned T cell subpopulations and their susceptibility to lysis by CD4+ or CD8+ CTL. A reciprocal relationship in Fas and Fas-ligand expression was observed in CD4+ T helper (Th)1- and Th2-type clones, and Fas mRNA was predominantly detected in Th2 clones, whereas Fas-ligand mRNA was principally found in Th1 clones. The two Th0 clones tested expressed both Fas and Fas-ligand, but only one exhibited cytolytic activity, whereas both were sensitive to CD4-mediated lysis. A functional consequence of the inverse Fas-Fas-ligand expression pattern was that Th2 and Th0 cells were sensitive to lysis by both Th1 CD4+ CTL and a CD8+ CTL clone in a Fas-dependent manner. These results suggest that cytolytic CD4+ Th1 cells may play an immunomodulatory role, regulating a Th2/Th0 response by Fas-mediated lysis.

Homologous restriction factor (HRF) is a membrane protein of erythrocytes and leukocytes that inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. HRF has also been reported to inhibit perforin-mediated cytolysis and postulated to play a role in cytotoxic T lymphocyte (CTL) self-protection. We show that paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes, lacking HRF, are no more sensitive to Ca2+-dependent human CTL-mediated lysis than normal erythrocytes. Furthermore, mouse and normal human erythrocytes, as well as PNH erythrocytes, are similarly lysed by isolated murine perforin-containing granules. We conclude that HRF does not inhibit perforin-mediated lysis and therefore is not likely to play a role in CTL self-protection.