Induction of inflammasome-dependent pyroptosis by carbon black nanoparticles

Inhalation of nanoparticles has been implicated in respiratory morbidity and mortality. In particular, carbon black nanoparticles are found in many different environmental exposures. Macrophages take up inhaled nanoparticles and respond via release of inflammatory mediators and in some cases cell death. Based on new data, we propose that exposure of macrophages (both a macrophage cell line and primary human alveolar macrophages) to carbon black nanoparticles induces pyroptosis, an inflammasome-dependent form of cell death. Exposure of macrophages to carbon black nanoparticles resulted in inflammasome activation as defined by cleavage of caspase 1 to its active form and downstream IL-1β release. The cell death that occurred with carbon black nanoparticle exposure was identified as pyroptosis by the protective effect of a caspase 1 inhibitor and a pyroptosis inhibitor. These data demonstrate that carbon black nanoparticle exposure activates caspase 1, increases IL-1β release after LPS priming, and induces the proinflammatory cell death, pyroptosis. The identification of pyroptosis as a cellular response to carbon nanoparticle exposure is novel and relates to environmental and health impacts of carbon-based particulates.     

IL‐1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence‐associated secretory phenotype

Interleukin‐1 alpha (IL‐1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL‐1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL‐1α in these contexts is unknown. We show IL‐1α is activated by caspase‐5 or caspase‐11 cleavage at a conserved site. Caspase‐5 drives cleaved IL‐1α release after human macrophage inflammasome activation, while IL‐1α secretion from murine macrophages only requires caspase‐11, with IL‐1β release needing caspase‐11 and caspase‐1. Importantly, senescent human cells require caspase‐5 for the IL‐1α‐dependent senescence‐associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase‐11 for the SASP‐driven immune surveillance of senescent cells in vivo. Together, we identify IL‐1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL‐1α is activated during senescence. Thus, targeting caspase‐5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.     

Inflammatory Cytokines and Survival Factors from Serum Modulate Tweak-Induced Apoptosis in PC-3 Prostate Cancer Cells

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the tumor necrosis factor superfamily. TWEAK activates the Fn14 receptor, and may regulate cell death, survival and proliferation in tumor cells. However, there is little information on the function and regulation of this system in prostate cancer. Fn14 expression and TWEAK actions were studied in two human prostate cancer cell lines, the androgen-independent PC-3 cell line and androgen-sensitive LNCaP cells. Additionally, the expression of Fn14 was analyzed in human biopsies of prostate cancer. Fn14 expression is increased in histological sections of human prostate adenocarcinoma. Both prostate cancer cell lines express constitutively Fn14, but, the androgen-independent cell line PC-3 showed higher levels of Fn14 that the LNCaP cells. Fn14 expression was up-regulated in PC-3 human prostate cancer cells in presence of inflammatory cytokines (TNFα/IFNγ) as well as in presence of bovine fetal serum. TWEAK induced apoptotic cell death in PC-3 cells, but not in LNCaP cells. Moreover, in PC-3 cells, co-stimulation with TNFα/IFNγ/TWEAK induced a higher rate of apoptosis. However, TWEAK or TWEAK/TNFα/IFNγ did not induce apoptosis in presence of bovine fetal serum. TWEAK induced cell death through activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-3, release of mitochondrial cytochrome C and an increased Bax/BclxL ratio. TWEAK/Fn14 pathway activation promotes apoptosis in androgen-independent PC-3 cells under certain culture conditions. Further characterization of the therapeutic target potential of TWEAK/Fn14 for human prostate cancer is warranted.     

FasL‐triggered death of Jurkat cells requires caspase 8‐induced,ATP‐dependent cross‐talk between fas and the purinergic receptor P2X7

Fas ligation via the ligand FasL activates the caspase‐8/caspase‐3‐dependent extrinsic death pathway. In so‐called type II cells, an additional mechanism involving tBid‐mediated caspase‐9 activation is required to efficiently trigger cell death. Other pathways linking FasL–Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X₇ receptors (P2X₇Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase‐8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X₇Rs participate in FasL‐stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time‐ and caspase‐8‐dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL‐induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL‐induced death. Also, oxidized‐ATP or Brilliant Blue G, two P2X₇R blockers, reduced FasL‐induced caspase‐9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X₇R connect functionally via caspase‐8 and Panx1 HC‐mediated ATP release to promote caspase‐9/caspase‐3‐dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. J. Cell. Physiol. 228: 485–493, 2013. © 2012 Wiley Periodicals, Inc.     

A hot‐water extract of Sanguisorba officinalis ameliorates endotoxin‐induced septic shock by inhibiting inflammasome activation

The inflammasome is a multiprotein signaling complex that mediates inflammatory innate immune responses through caspase 1 activation and subsequent IL‐1β secretion. However, because its aberrant activation often leads to inflammatory diseases, targeting the inflammasome holds promise for the treatment of inflammation‐related diseases. In this study, it was found that a hot‐water extract of Sanguisorba officinalis (HSO) suppresses inflammasome activation triggered by adenosine 5′‐triphosphate, nigericin, microbial pathogens, and double stranded DNA in bone marrow‐derived macrophages. HSO was found to significantly suppress IL‐1β production in a dose‐dependent manner; this effect correlated well with small amounts of caspase 1 and little ASC pyroptosome formation in HSO‐treated cells. The anti‐inflammatory activity of HSO was further confirmed in a mouse model of endotoxin‐induced septic shock. Oral administration of HSO reduced IL‐1β titers in the serum and peritoneal cavity, increasing the survival rate. Taken together, our results suggest that HSO is an inhibits inflammasome activation through nucleotide‐binding domain and leucine‐rich repeat pyrin domain 3, nucleotide‐binding domain and leucine‐rich repeat caspase recruitment domain 4 and absent in melanoma 2 pathways, and may be useful for treatment of inflammasome‐mediated diseases.     

Dexmedetomidine protects against high mobility group box 1‐induced cellular injury by inhibiting pyroptosis

Dexmedetomidine (DEX) is a widely used clinical anesthetic with proven anti‐inflammatory effects. Both high mobility group box 1 (HMGB1) and pyroptosis play an important role in the inflammatory response to infection and trauma. Thus far, there have been no studies published addressing the effect of DEX on HMGB1 and pyroptosis. In order to fill this gap in the literature, bone marrow‐derived macrophages (BMDMs) were exposed to HMGB1 (4 µg/mL) with or without DEX (50 μM) pretreatment. The production of pro‐inflammatory cytokines [such as tumor necrosis factor α (TNF‐α), interleukin 1β (IL‐1β), and IL‐18], phosphorylation of extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) and P38, and the activation of caspase‐1 were measured by enzyme immunosorbent assay, western blot analysis, confocal microscope, and flow cytometry, respectively. We found that DEX protected against HMGB1‐induced cell death of BMDMs. In addition, DEX suppressed the generation of TNF‐α, IL‐1β, and IL‐18 as well as the phosphorylation of ERK1/2 and P38. Moreover, DEX inhibited caspase‐1 activation and decreased pyroptosis. Taken together, these findings demonstrate the protective effect of DEX in mediating HMGB1‐induced cellular injury, thus indicating that DEX may be a potential therapeutic candidate for the management of infection and trauma‐derived inflammation.     

Dimer‐specific immunoprecipitation of active caspase‐2 identifies TRAF proteins as novel activators

Caspase‐2 has been shown to initiate apoptotic cell death in response to specific intracellular stressors such as DNA damage. However, the molecular mechanisms immediately upstream of its activation are still poorly understood. We combined a caspase‐2 bimolecular fluorescence complementation (BiFC) system with fluorophore‐specific immunoprecipitation to isolate and study the active caspase‐2 dimer and its interactome. Using this technique, we found that tumor necrosis factor receptor‐associated factor 2 (TRAF2), as well as TRAF1 and 3, directly binds to the active caspase‐2 dimer. TRAF2 in particular is necessary for caspase‐2 activation in response to apoptotic cell death stimuli. Furthermore, we found that dimerized caspase‐2 is ubiquitylated in a TRAF2‐dependent manner at K15, K152, and K153, which in turn stabilizes the active caspase‐2 dimer complex, promotes its association with an insoluble cellular fraction, and enhances its activity to fully commit the cell to apoptosis. Together, these data indicate that TRAF2 positively regulates caspase‐2 activation and consequent cell death by driving its activation through dimer‐stabilizing ubiquitylation.     

Heat shock inhibits caspase‐1 activity while also preventing its inflammasome‐mediated activation by anthrax lethal toxin

Anthrax lethal toxin (LT) rapidly kills macrophages from certain mouse strains in a mechanism dependent on the breakdown of unknown protein(s) by the proteasome, formation of the Nalp1b (NLRP1b) inflammasome and subsequent activation of caspase‐1. We report that heat‐shocking LT‐sensitive macrophages rapidly protects them against cytolysis by inhibiting caspase‐1 activation without upstream effects on LT endocytosis or cleavage of the toxin's known cytosolic substrates (mitogen‐activated protein kinases). Heat shock protection against LT occurred through a mechanism independent of de novo protein synthesis, HSP90 activity, p38 activation or proteasome inhibition and was downstream of mitogen‐activated protein kinase cleavage and degradation of an unknown substrate by the proteasome. The heat shock inhibition of LT‐mediated caspase‐1 activation was not specific to the Nalp1b (NLRP1b) inflammasome, as heat shock also inhibited Nalp3 (NLRP3) inflammasome‐mediated caspase‐1 activation in macrophages. We found that heat shock induced pro‐caspase‐1 association with a large cellular complex that could prevent its activation. Additionally, while heat‐shocking recombinant caspase‐1 did not affect its activity in vitro, lysates from heat‐shocked cells completely inhibited recombinant active caspase‐1 activity. Our results suggest that heat shock inhibition of active caspase‐1 can occur independently of an inflammasome platform, through a titratable factor present within intact, functioning heat‐shocked cells.     

Functional ABCG1 expression induces apoptosis in macrophages and other cell types

The expression of the ATP-binding cassette transporter ABCG1 is greatly increased in macrophages by cholesterol loading via the activation of the nuclear receptor LXR. Several recent studies demonstrated that ABCG1 expression is associated with increased cholesterol efflux from macrophages to high-density lipoprotein, suggesting an atheroprotective role for this protein. Our present study uncovers an as yet not described cellular function of ABCG1. Here we demonstrate that elevated expression of human ABCG1 is associated with apoptotic cell death in macrophages and also in other cell types. We found that overexpression of the wild type protein results in phosphatidyl serine (PS) translocation, caspase 3 activation, and subsequent cell death, whereas neither the inactive mutant variant of ABCG1 (ABCG1_K124M) nor the ABCG2 multidrug transporter had such effect. Induction of ABCG1 expression by LXR activation in Thp1 cells and in human monocyte-derived macrophages was accompanied by a significant increase in the number of apoptotic cells. Thyroxin and benzamil, previously identified inhibitors of ABCG1 function, selectively prevented ABCG1-promoted apoptosis in transfected cells as well as in LXR-induced macrophages. Collectively, our results suggest a causative relationship between ABCG1 function and apoptotic cell death, and may offer new insights into the role of ABCG1 in atherogenesis.     

Induction of caspase 3 activation by multiple Legionella pneumophila Dot/Icm substrates

The intracellular pathogen Legionella pneumophila is able to strike a balance between the death and survival of the host cell during infection. Despite the presence of high level of active caspase 3, the executioner caspase of apoptotic cell death, infected permissive macrophages are markedly resistant to exogenous apoptotic stimuli. Several bacterial molecules capable of promoting the cell survival pathways have been identified, but proteins involved in the activation of caspase 3 remain unknown. To study the mechanism of L. pneumophila‐mediated caspase 3 activation, we tested all known Dot/Icm substrates for their ability to activate caspase 3. Five effectors capable of causing caspase 3 activation upon transient expression were identified. Among these, by using its ability to activate caspase 3 by inducing the release of cytochrome c from the mitochondria, we demonstrated that VipD is a phospholipase A2, which hydrolyses phosphatidylethanolamine (PE) and phosphocholine (PC) on the mitochondrial membrane in a manner that appears to require host cofactor(s). The lipase activity leads to the production of free fatty acids and 2‐lysophospholipids, which destabilize the mitochondrial membrane and may contribute to the release of cytochrome c and the subsequent caspase 3 activation. Furthermore, we found that whereas it is not detectably defectively in caspase 3 activation in permissive cells, amutant lacking all of these five genes is less potent in inducing apoptosis in dendritic cells. Our results reveal that activation of host cell death pathways by L. pneumophila is a result of the effects of multiple bacterial proteins with diverse biochemical functions.     

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi‐protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill‐characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N‐terminal fragment of caspase‐1‐cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N‐terminal fragment of caspase‐1‐cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome‐inserted GSDMD at nanometer resolution by cryo‐electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.     

Involvement of Yes‐associated protein 1 (YAP1) in doxorubicin‐induced cytotoxicity in H9c2 cardiac cells

Cardiac cell death is one of the major events implicated in doxorubicin‐induced cardiotoxicity, which leads to heart failure. We recently reported that Yes‐associated protein 1 (YAP1) regulates cell survival and apoptosis. However, it is unclear whether YAP1 regulates doxorubicin‐induced cell death in cardiomyocytes. We investigated whether YAP1 is involved in doxorubicin‐induced cell death using H9c2 cardiac cells and mouse heart. In an in vivo study, YAP1 protein expression was significantly decreased in hearts of doxorubicin‐treated mice with increased caspase‐3 activation. Doxorubicin also caused cell death by increasing caspase‐3 activation in H9c2 cells. Doxorubicin reduced YAP1 protein expression and messenger RNA expression accompanied by increased phosphorylation of YAP1 at Ser127. Doxorubicin further increased cell death with increased caspase‐3/7 activation in the absence of YAP1 when compared with doxorubicin or siYAP1 treatment alone. Overexpression of constitutively active YAP1 (YAP1–5SA) using an adenovirus gene transfer technique significantly reversed doxorubicin‐induced cell death by decreasing caspase‐3/7 activation in H9c2 cells. Akt, a potential prosurvival factor, decreased in doxorubicin‐ and YAP1 short interfering RNA (siRNA)‐treated cells. Doxorubicin further significantly decreased Akt protein expression when YAP1 was silenced. Overexpression of YAP1 canceled decreased Akt protein expression induced by doxorubicin treatment in H9c2 cells. In conclusion, these results suggest that doxorubicin‐induced cardiac cell death is mediated in part by down‐regulation of YAP1 and YAP1‐targeted gene, Akt. Modulating YAP1 and its related Hippo pathway on local cardiomyocytes may be a promising therapeutic approach for doxorubicin‐induced cardiotoxicity.     

Execution of macrophage apoptosis by Mycobacterium avium through apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase signaling and caspase 8 activation

Macrophage apoptosis is an important component of the innate immune defense machinery (against pathogenic mycobacteria) responsible for limiting bacillary viability. However, little is known about the mechanism of how apoptosis is executed in mycobacteria-infected macrophages. Apoptosis signal-regulating kinase 1 (ASK1) was activated in Mycobacterium avium-treated macrophages and in turn activated p38 mitogen-activated protein (MAP) kinase. M. avium-induced macrophage cell death could be blocked in cells transfected with a catalytically inactive mutant of ASK1 or with dominant negative p38 MAP kinase arguing in favor of a central role of ASK1/p38 MAP kinase signaling in apoptosis of macrophages challenged with M. avium. ASK1/p38 MAP kinase signaling was linked to the activation of caspase 8. At the same time, M. avium triggered caspase 8 activation, and cell death occurred in a Fas-associated death domain (FADD)-dependent manner. The death signal induced upon caspase 8 activation linked to mitochondrial death signaling through the formation of truncated Bid (t-Bid), its translocation to the mitochondria and release of cytochrome c. Caspase 8 inhibitor (z-IETD-FMK) could block the release of cytochrome c as well as the activation of caspases 9 and 3. The final steps of apoptosis probably involved caspases 9 and 3, since inhibitors of both caspases could block cell death. Of foremost interest in the present study was the finding that ASK1/p38 signaling was essential for caspase 8 activation linked to M. avium-induced death signaling. This work provides the first elucidation of a signaling pathway in which ASK1 plays a central role in innate immunity.     

Bacterial infection of macrophages induces decrease in refractive index

Infection of cells by pathogens leads to both biochemical and structural modifications of the host cell. To study the structural modifications in a label‐free manner, we use digital holographic microscopy, DHM, to obtain the integral refractive index distribution of cells. Primary murine bone marrow derived macrophages (BMDM) infected with Salmonella enterica serovar Typhimurium, undergo highly significant reduction in refractive index, RI, compared to uninfected cells. Infected BMDM cells from genetically modified mice lacking an inflammatory protein that causes cell death, caspase 1, also exhibit similar decrease in RI. These data suggest that any reduction in RI of Salmonella ‐infected BMDMs is pathogen induced and independent of caspase 1‐induced inflammation or cell death. This finding suggests DHM may be useful for general real time monitoring of host cell interactions with infectious pathogens. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

NF-kappaB protects macrophages from lipopolysaccharide-induced cell death: the role of caspase 8 and receptor-interacting protein

Macrophages play a pivotal role in the pathogenesis of a variety of diseases. These studies were performed to characterize the mechanisms by which Toll-like receptor 4 (TLR4)-mediated NF-kappaB activation promotes resistance to cell death in macrophages. When NF-kappaB activation was inhibited by a super-repressor, IkappaBalpha, the TLR4 ligand lipopolysaccharide induced the activation of caspase 8, the loss of mitochondrial transmembrane potential (DeltaPsim), and apoptotic cell death in macrophages. The inhibition of caspase 8 activation suppressed DNA fragmentation but failed to protect macrophages against the loss of DeltaPsim and resulted in necrotic cell death. In contrast, the reduction of receptor-interacting protein 1 suppressed the loss of DeltaPsim and inhibited apoptotic cell death. Further, when caspase 8 activation was suppressed, the knock down of receptor-interacting protein inhibited the loss of DeltaPsim and necrotic cell death. These observations demonstrate that following TLR4 ligation by lipopolysaccharide, NF-kappaB is a critical determinant of macrophage life or death, whereas caspase 8 determines the pathway employed.     

Essential role of STAT1 in caspase-independent cell death of activated macrophages through the p38 mitogen-activated protein kinase/STAT1/reactive oxygen species pathway

Unlike other immune cells, activation of macrophages by stimulating agents, such as lipopolysaccharide (LPS), confers significant resistance to many apoptotic stimuli, but the underlying mechanism of this phenomenon remains largely unknown. Here, we demonstrate that LPS-induced early caspase activation is essential for macrophage survival because blocking caspase activation with a pancaspase inhibitor (zVAD [benzyloxycarbonyl-Val-Ala-Asp]) rapidly induced death of activated macrophages. This type of death process by zVAD/LPS was principally mediated by intracellular generation of superoxide. STAT1 knockout macrophages demonstrated profoundly decreased superoxide production and were resistant to treatment with zVAD/LPS, indicating the crucial involvement of STAT1 in macrophage death by zVAD/LPS. STAT1 level and activity were reciprocally regulated by caspase activation and were associated with cell death. Activation of STAT1 was critically dependent upon serine phosphorylation induced by p38 mitogen-activated protein kinase (MAPK) because a p38 MAPK inhibitor nullified STAT1 serine phosphorylation, reactive oxygen species (ROS) production, and macrophage death by zVAD/LPS. Conversely, p38 MAPK activation was dependent upon superoxide and was also nullified in STAT1 knockout macrophages, probably due to impaired generation of superoxide. Our findings collectively indicate that STAT1 signaling modulates intracellular oxidative stress in activated macrophages through a positive-feedback mechanism involving the p38 MAPK/STAT1/ROS pathway, which is interrupted by caspase activation. Furthermore, our study may provide significant insights in regards to the unanticipated critical role of STAT1 in the caspase-independent death pathway.     

Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis

NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival. Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD. fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB. These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.     

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase‐2

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase‐2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase‐2 also regulates autophagy, genomic stability and ageing. Caspase‐2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase‐2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase‐2 and impedes dimerization and activation of caspase‐2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase‐2. Depletion of endogenous API5 leads to an increase in caspase‐2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase‐2‐dependent apoptotic cell death. These results establish API5/AAC‐11 as a direct inhibitor of caspase‐2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.     

The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram-negative rod periodontal pathogen. NO could not be induced by heat-killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.     

Induction of lymphocyte blast transformation by purified fibronectin in vitro

Purified rat plasma fibronectin (Fn) induces a dose-dependent, nonspecific proliferation of lymphoid cells isolated from spleen and lymph nodes, but has no effect on thymocytes. Proliferation of cells occurred after 4 to 5 days of incubation and was generally 5- to 10-fold greater than control cells cultured without Fn or in the presence of the same concentrations of rat serum albumin. The responding cell appears to be the T cell and requires the presence of adherent accessory cells (macrophages). Although purified T and B cells failed to demonstrate a significant increase in blastogenesis in the presence of Fn, reconstitution of T, but not B, cells with irradiated peritoneal exudate macrophages restored the stimulatory effect of Fn. Furthermore, comparison of the effect of various concentrations of macrophages on the restored T cell response shows that proliferative activity in the presence of Fn is dependent on a critical number of macrophages. Adding higher numbers of macrophages beyond the optimal concentration results in a sharp decline in lymphocyte responsiveness to Fn, although the proliferation of control cells continues to increase at these macrophage concentrations. Macrophages pulsed with Fn for 1 hr failed to evoke an increase in T cell responsiveness after 3 or 5 days of incubation. In addition, incubation of Fn-pulsed T cells with macrophages that had been precultured with or without Fn also failed to result in T cell activation. The effect of Fn on T lymphocyte transformation appears to be mediated indirectly through its interaction with the macrophage, because 125I-Fn, which shows significant binding to peritoneal exudate macrophages, fails to interact with purified T cells. Radiolabeled Fn also demonstrated little or no binding activity for unseparated lymph node cells.