Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome

Activation of caspase-1 and subsequent processing and secretion of the pro-inflammatory cytokine IL-1beta is triggered upon assembly of the inflammasome complex. It is generally believed that bacterial lipopolysaccharides (LPS) are activators of the inflammasome through stimulation of Toll-like receptor 4 (TLR4). Like TLRs, NALP3/Cryopyrin, which is a key component of the inflammasome, contains Leucine-Rich-Repeats (LRRs). LRRs are frequently used to sense bacterial components, thus raising the possibility that bacteria directly activate the inflammasome. Here, we show that bacterial peptidoglycans (PGN), but surprisingly not LPS, induce NALP3-mediated activation of caspase-1 and maturation of proIL-1beta. Activation is independent of TLRs because the PGN degradation product muramyl dipeptide (MDP), which is not sensed by TLRs, is the minimal-activating structure. Macrophages from a patient with Muckle-Wells syndrome, an autoinflammatory disease associated with mutations in the NALP3/Cryopyrin gene, show increased IL-1beta secretion in the presence of MDP. The activation of the NALP3-inflammasome by MDP may be the basis of the potent adjuvant activity of MDP.     

Pannexin-1-mediated intracellular delivery of muramyl dipeptide induces caspase-1 activation via cryopyrin/NLRP3 independently of Nod2

Muramyl dipeptide (MDP), the microbial activator of nucleotide-binding oligomerization domain 2 (Nod2), induces NF-kappaB and MAPK activation, leading to the production of multiple anti-bacterial and proinflammatory molecules. In addition, MDP has been implicated in IL-1beta secretion through the regulation of caspase-1. However, the mechanisms that mediate caspase-1 activation and IL-1beta secretion in response to MDP stimulation remain poorly understood. We show here that fluorescent MDP molecules are internalized in primary macrophages and accumulate in granular structures that colocalize with markers of acidified endosomal compartments. The uptake of MDP was Nod2-independent. Upon ATP stimulation, labeled MDP was rapidly released from acidified vesicles into the cytosol, a process that required functional pannexin-1. Caspase-1 activation induced by MDP and ATP required pannexin-1 and Cryopyrin but was independent of Nod2. Conversely, induction of pro-IL-1beta mRNA by MDP stimulation was abolished in Nod2-deficient macrophages but unimpaired in macrophages lacking Cryopyrin. These studies demonstrate a Nod2-independent mechanism mediated through pore-forming pannexin-1 that is required for intracellular delivery of MDP to the cytosol and caspase-1 activation. Furthermore, the work provides evidence for distinct roles of Nod2 and Cryopyrin in the regulation of MDP-induced caspase-1 activation and IL-1beta secretion.     

Pyrin levels in human monocytes and monocyte-derived macrophages regulate IL-1beta processing and release

Macrophages and their precursors, monocytes, are key cells involved in the innate immune response. Although both monocytes and macrophages produce caspase-1, the key enzyme responsible for pro-IL-1beta processing; macrophages are limited in their ability to activate the enzyme and release functional IL-1beta. In this context, because mutations in the pyrin gene (MEFV) cause the inflammatory disorder familial Mediterranean fever, pyrin is believed to regulate IL-1beta processing. To determine whether variations in pyrin expression explain the difference between monocytes and macrophages in IL-1beta processing and release, pyrin was studied in human monocytes and monocyte-derived macrophages. Although monocytes express pyrin mRNA and protein, which is readily inducible by endotoxin, monocyte-derived macrophages express significantly less pyrin mRNA and protein. Pyrin levels directly correlated with IL-1beta processing in monocytes and macrophages; therefore, we asked whether pyrin might promote IL-1beta processing and release. HEK293 cells were transfected with pyrin, caspase-1, apoptotic speck protein with a caspase recruitment domain, and IL-1beta. Pyrin induced IL-1beta processing and release in a dose-dependent manner. Conversely, pyrin small interference RNA suppressed pro-IL-1beta processing in both THP-1 cells and fresh human monocytes. In summary, both pyrin expression and IL-1beta processing and release are diminished upon the maturation of monocytes to macrophages. When pyrin is ectopically expressed or silenced, IL-1beta processing and release parallels the level of pyrin. In conclusion, in the context of endotoxin-induced activation of mononuclear phagocytes, pyrin augments IL-1beta processing and release.     

Distinct roles of TLR2 and the adaptor ASC in IL-1beta/IL-18 secretion in response to Listeria monocytogenes

Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is an adaptor molecule that has recently been implicated in the activation of caspase-1. We have studied the role of ASC in the host defense against the intracellular pathogen Listeria monocytogenes. ASC was found to be essential for the secretion of IL-1beta/IL-18, but dispensable for IL-6, TNF-alpha, and IFN-beta production, in macrophages infected with Listeria. Activation of caspase-1 was abolished in ASC-deficient macrophages, whereas activation of NF-kappaB and p38 was unaffected. In contrast, secretion of IL-1beta, IL-6, and TNF-alpha was reduced in TLR2-deficient macrophages infected with Listeria; this was associated with impaired activation of NF-kappaB and p38, but normal caspase-1 processing. Analysis of Listeria mutants revealed that cytosolic invasion was required for ASC-dependent IL-1beta secretion, consistent with a critical role for cytosolic signaling in the activation of caspase-1. Secretion of IL-1beta in response to lipopeptide, a TLR2 agonist, was greatly reduced in ASC-null macrophages and was abolished in TLR2-deficient macrophages. These results demonstrate that TLR2 and ASC regulate the secretion of IL-1beta via distinct mechanisms in response to Listeria. ASC, but not TLR2, is required for caspase-1 activation independent of NF-kappaB in Listeria-infected macrophages.     

E1B 19,000-Molecular-Weight Protein Interacts with and Inhibits CED-4-Dependent, FLICE-Mediated Apoptosis

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-x_L) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-x_L have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.     

Cop, a caspase recruitment domain-containing protein and inhibitor of caspase-1 activation processing.

The production of bio-active interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves binding of the caspase-1 prodomain to a caspase recruitment domain (CARD)-containing serine/threonine kinase known as RIP2/CARDIAK/RICK. We have identified a novel protein, COP (CARD only protein), which has a high degree of sequence identity to the caspase-1 prodomain. COP binds to both RIP2 and the caspase-1 prodomain and inhibits RIP2-induced caspase-1 oligomerization. COP inhibits caspase- 1-induced IL-1beta secretion as well as lipopolysaccharide-induced IL-1beta secretion in transfected cells. Our data indicate that COP can regulate IL-1beta secretion, implying that COP may play a role in down-regulating inflammatory responses analogous to the CARD protein ICEBERG.     

Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.     

Cap-independent translation promotes C. elegans germ cell apoptosis through Apaf-1/CED-4 in a caspase-dependent mechanism

Apoptosis is a natural process during animal development for the programmed removal of superfluous cells. During apoptosis general protein synthesis is reduced, but the synthesis of cell death proteins is enhanced. Selective translation has been attributed to modification of the protein synthesis machinery to disrupt cap-dependent mRNA translation and induce a cap-independent mechanism. We have previously shown that disruption of the balance between cap-dependent and cap-independent C. elegans eIF4G isoforms (IFG-1 p170 and p130) by RNA interference promotes apoptosis in developing oocytes. Germ cell apoptosis was accompanied by the appearance of the Apaf-1 homolog, CED-4. Here we show that IFG-1 p170 is a native substrate of the worm executioner caspase, CED-3, just as mammalian eIF4GI is cleaved by caspase-3. Loss of Bcl-2 function (ced-9ts) in worms induced p170 cleavage in vivo, coincident with extensive germ cell apoptosis. Truncation of IFG-1 occurred at a single site that separates the cap-binding and ribosome-associated domains. Site-directed mutagenesis indicated that CED-3 processes IFG-1 at a non-canonical motif, TTTD(456). Coincidentally, the recognition site was located 65 amino acids downstream of the newly mapped IFG-1 p130 start site suggesting that both forms support cap-independent initiation. Genetic evidence confirmed that apoptosis induced by loss of ifg-1 p170 mRNA was caspase (ced-3) and apoptosome (ced-4/Apaf-1) dependent. These findings support a new paradigm in which modal changes in protein synthesis act as a physiological signal to initiate cell death, rather than occur merely as downstream consequences of the apoptotic event.     

Nod1, a CARD protein,enhances pro-interleukin-1beta processing through the interaction with pro-caspase-1

The production of bioactive interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves interaction between the caspase recruit domains (CARDs) of caspase-1 and a serine/threonine kinase RIP2. While the association of Nod1 with both caspase-1 and RIP2 is already known, the consequences of these interactions are poorly understood. Because Nod1 also binds to RIP2, we hypothesized that Nod1 plays a role in pro-caspase-1 activation and IL-1beta processing. We show here that Nod1 binds to both RIP2 and caspase-1 by CARD interactions. Nod1 enhances pro-caspase-1 oligomerization and pro-caspase-1 processing. Nod1 enhances caspase-1-induced IL-1beta secretion, as well as lipopolysaccharide (LPS)-induced IL-1beta secretion in transfected cells. Moreover, HT1080 cells stably transfected with Nod1 showed higher LPS-induced IL-1beta secretion than non-transfected cells, suggesting a role of Nod1 in LPS-induced responses. Our data indicate that Nod1 can regulate IL-1beta secretion, implying that Nod1 may play a role in inflammatory responses to bacterial LPS.     

Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1beta activation by this protease in THP-1 monocytes. ASC enhances IL-1beta secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1beta secretion but that coexpression of ASC with these proteins results in enhanced IL-1beta secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1beta secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.     

Balance between autocrine interleukin-1beta and caspases defines life versus death of polymorphonuclear cells

The role of endogenous IL-1beta in regulating spontaneous and Fas-triggered apoptosis of human PMN has been studied in relation to the activity of the IL-1beta-generating enzyme ICE (caspase-1), an enzyme also involved in the mechanism of cell death. Upon in vitro culture, PMN undergo spontaneous apoptosis and express increasing levels of IL-1beta, caspase-1- and caspase-3-like enzymes. Endogenous IL-1beta protects PMN from apoptosis, since inhibition of either IL-1beta or caspase-1 activity can accelerate PMN apoptotic death. Thus, in spontaneous PMN apoptosis caspase-1 essentially plays an anti-apoptotic role by inducing maturation of protective IL-1beta, whereas other molecules are responsible of driving apoptosis. Upon Fas triggering, PMN apoptosis is greatly accelerated, in correlation with increased caspase activity, whereas IL-1beta production is not augmented. Inhibition of IL-1beta activity can increase Fas-induced apoptosis, whereas caspase-1 inhibitors are without significant effect. It is hypothesized that in Fas-induced PMN apoptosis caspase-1 has a double role: it can protect from apoptosis through generation of protective IL-1beta, as in spontaneous apoptosis, and it can also exert pro-apoptotic activity which counterbalances the protective effect and allows accelerated apoptosis.     

YopJ-induced caspase-1 activation in Yersinia-infected macrophages: independent of apoptosis, linked to necrosis, dispensable for innate host defense

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity.     

Apoptosomes: engines for caspase activation

Activation of the caspases that initiate apoptosis typically requires cognate scaffold proteins, including CED-4 in Caenorhabditis elegans, Apaf-1 in mammals and Dark in Drosophila. Each scaffold protein oligomerizes procaspases into a complex called the apoptosome, but the regulation and biological roles of the scaffolds differ. Whereas CED-4 is restrained by the Bcl-2 homologue CED-9, Apaf-1 is inhibited by its WD40 repeat region, until it is activated by cytochrome c, derived from damaged mitochondria. Although Dark also has a WD40 region, its activation does not seem to involve cytochrome c. CED-4 is essential for apoptosis in the worm and Dark for many apoptotic responses in the fly, but the Apaf-1/caspase-9 system probably amplifies rather than initiates the mammalian caspase cascade.     

Caspase-1-deficient mice have delayed neutrophil apoptosis and a prolonged inflammatory response to lipopolysaccharide-induced acute lung injury

Caspase-1, the prototypic caspase, is known to process the cytokines IL-1beta and IL-18 to mature forms but it is unclear whether, like other caspases, it can induce apoptosis by activation of downstream protease cascades. Neutrophils are known to express caspase-1, to release IL-1beta and to undergo rapid, caspase-dependent apoptosis. We examined apoptosis and IL-1beta production in peripheral blood neutrophils of caspase-1-deficient and wild-type mice. Constitutive apoptosis of caspase-1-deficient neutrophils was delayed compared with wild-type neutrophils and LPS-mediated inhibition of apoptosis was absent, but caspase-1-deficient neutrophils were susceptible to Fas-mediated apoptosis. LPS-stimulated IL-1beta production was absent from caspase-1-deficient neutrophils. To ascertain whether these differences in apoptosis and IL-1beta production would alter the response to acute lung injury, we studied pulmonary neutrophil accumulation following intratracheal administration of LPS. Caspase-1-deficient mice showed increased, predominantly neutrophilic pulmonary inflammation, but inflammation had resolved in both wild-type and deficient animals by 72 h after LPS instillation. IL-1beta production was increased in wild-type lungs but was also detected in caspase-1-deficient mice. We conclude that caspase-1 modulates apoptosis of both peripheral blood and inflammatory neutrophils, but is not essential for IL-1beta production in the lung.     

PAN1/NALP2/PYPAF2, an inducible inflammatory mediator that regulates NF-kappaB and caspase-1 activation in macrophages

Genes encoding proteins with PYRIN/PAAD/DAPIN domains, a nucleotide binding fold (NACHT), and leucine rich repeats have recently been recognized as important mediators in autoimmune inflammatory disorders. Here we characterize the expression and function of a member of the PYRIN and NACHT domain (PAN) family, PAN1 (also known as NALP2 and PYPAF2). PAN1 protein expression is regulated by lipopolysaccharide (LPS) and interferons (IFNbeta and IFNgamma) in THP-1 macrophage cells. In gene transfection studies PAN1 manifests an inhibitory influence on NF-kappaB activation induced by various pro-inflammatory stimuli, including tumor necrosis factor TNFalpha and interleukin-1beta (IL-1beta). Gene transfer-mediated elevations in PAN1 protein also suppressed activation of IkappaB kinases induced by inflammatory cytokines. Conversely, reducing endogenous levels of PAN1 using small interfering RNA enhanced LPS-induced production of ICAM-1 (intercellular adhesion molecule 1), an NF-kappaB-dependent gene. We also show here that PAN1 binds via its PYRIN domain to ASC, an adapter protein involved in caspase-1 activation. This binding is disrupted by mutation of the alpha1 helix of ASC. In gene transfer experiments PAN1 enhances caspase-1 activation and IL-1beta secretion in collaboration with ASC. Conversely, reducing endogenous levels of PAN1 using small interfering RNA significantly reduced LPS-induced secretion of IL-1beta in monocytes. We propose that PAN1 functions as a modulator of the activation of NF-kappaB and pro-caspase-1 in macrophages.     

Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells

CED-9 blocks programmed cell death (apoptosis) in the nematode C. elegans by binding to and neutralizing CED-4, an essential activator of the aspartate-directed cysteine protease (caspase) CED-3. In mammals, the CED-9 homologs Bcl-2 and Bcl-xL also block apoptosis by interfering with the activation of CED-3-like caspases. However, it is unknown whether this occurs by binding to the CED-4 homolog Apaf-1. Whilst two groups previously detected an interaction between Bcl-xL and Apaf-1 in immunoprecipitates,1,2 another group found no interaction between Apaf-1 and any of ten individual members of the Bcl-2 family using the same experimental approach.3 In this study, we aimed to resolve this discrepancy by monitoring the binding of Apaf-1 to three Bcl-2 family members within cells. Using immunofluorescence and Western blot analysis, we show that whilst Apaf-1 is a predominantly cytoplasmic protein, Bcl-2, Bcl-xL and Bax mostly reside on nuclear/ER and mitochondrial membranes. This pattern of localization is maintained when the proteins are co-expressed in both normal and apoptotic cells, suggesting that Bcl-2, Bcl-xL or Bax do not significantly sequester cytoplasmic Apaf-1 to intracellular membranes. In addition, we confirm that Apaf-1 does not interact with Bcl-2 and Bcl-xL in immunoprecipitates. Based on these data, we propose that Apaf-1 is not a direct, physiological target of Bcl-2, Bcl-xL or Bax.     

TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages

The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO-), leading to macrophage apoptosis. Since TNF-alpha and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-alpha and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1-/- peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-alpha+Nramp1+/+ and B10R, and more IL-10+Nramp1-/- and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-alpha inhibited apoptosis, diminished NO- production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO- production. Murine rTNF-alpha induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO- production and caspase 1 activation. NO- and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO- production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-alpha- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-alpha-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival.