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Receptor-interacting protein (RIP) has been implicated in the induction of death receptor-mediated, nonapoptotic cell death. However, the mechanisms remain to be elucidated. Here we show that tumor necrosis factor alpha induced RIP-dependent inhibition of adenine nucleotide translocase (ANT)-conducted transport of ADP into mitochondria, which resulted in reduced ATP and necrotic cell death. The inhibition of ADP/ATP exchange coincided with the loss of interaction between ANT and cyclophilin D and the inability of ANT to adopt the cytosolic conformational state, which prevented cytochrome c release. Neither overexpression of Bcl-xL nor inhibition of reactive oxygen species prevented necrosis. In contrast, the ectopic expression of ANT or cyclophilin D was effective at preventing cell death. These observations demonstrate a novel mechanism initiated through death receptor ligation and mediated by RIP that results in the suppression of ANT activity and necrosis.

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-κB activation promotes resistance to cell death in macrophages. When NF-κB activation was inhibited by a super-repressor, IκBα, the TLR4 ligand lipopolysaccharide induced the activation of caspase 8, the loss of mitochondrial transmembrane potential (ΔΨm), and apoptotic cell death in macrophages. The inhibition of caspase 8 activation suppressed DNA fragmentation but failed to protect macrophages against the loss of ΔΨm and resulted in necrotic cell death. In contrast, the reduction of receptor-interacting protein 1 suppressed the loss of ΔΨm and inhibited apoptotic cell death. Further, when caspase 8 activation was suppressed, the knock down of receptor-interacting protein inhibited the loss of ΔΨm and necrotic cell death. These observations demonstrate that following TLR4 ligation by lipopolysaccharide, NF-κB is a critical determinant of macrophage life or death, whereas caspase 8 determines the pathway employed.

We assessed mast cell influence on eosinophils, the prominent cells in late and chronic allergic reactions, by comparing the proteomic pattern of eosinophils incubated with mast cells, tumor necrosis factor alpha (TNF-alpha) or granulocyte macrophage colony stimulating factor (GM-CSF). Eosinophils were incubated with the human mast cell line HMC-1 cellular sonicate and their survival and GM-CSF production were evaluated. For proteomic studies, eosinophils were cultured with HMC-1 sonicate, TNF-alpha or GM-CSF in the presence of [(35)S]methionine, solubilized and submitted to isolelectric focusing separation and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the ISODALT system, followed by radiofluorography and computer image analysis. HMC-1-incubated eosinophils displayed increased survival partly mediated by mast cell-associated TNF-alpha, and produced GM-CSF. Metabolically labeled eosinophils incubated with either HMC-1, TNF-alpha or GM-CSF released eosinophil peroxidase. Comparison of two-dimensional gel spots from the eosinophils revealed that each of the three activating signals yielded a distinctly different proteomic pattern of labeled polypeptides. GM-CSF provided the strongest signal and the highest rate of protein synthesis (1,018 spots) followed by TNF-alpha (747 spots) and HMC-1 sonicate (611 spots). A portion of spots differed both in terms of quality and quantity. Although each stimulus induced similar functional effects, the resulting biosynthetic programs of the eosinophils greatly differed. The presented proteomic analysis is the first step in the exploration of molecular mechanisms involved in eosinophil activation.

We have previously shown that mast cells enhance eosinophil survival and activation. In this study we further characterized mast cell activity toward eosinophils. Sonicate of both rat peritoneal mast cells and the human mast cell line 1 (HMC-1) induced a concentration-dependent IL-6 and IL-8 release from human peripheral blood eosinophils (ELISA). HMC-1-induced IL-8 release was significantly reduced by the tryptase inhibitors GW-45 and GW-58 (90 and 87%, respectively, at an optimal concentration) but not by anti-stem cell factor, anti-TNF-α, or anti-IFN-γ neutralizing Abs or by the antihistamine drugs pyrilamine and cimetidine. In a manner similar to HMC-1, human recombinant tryptase induced the expression of mRNA for IL-8 (RT-PCR) and caused IL-8 release from the eosinophils. Addition of cycloheximide, actinomycin D, dexamethasone, PD 98059, curcumin, or SB 202190 completely inhibited the tryptase-induced IL-6 and IL-8 release. In contrast, cyclosporin A had no effect on tryptase-induced IL-8 release. Tryptase caused phosphorylation of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases 1 and 2, and p38 (Western blot). Tryptase also induced the translocation of c-Jun from the cytosol to the nucleus (confocal microscopy) and enhanced AP-1 binding activity to the DNA (EMSA). Eosinophils were found to express proteinase-activated receptor 2 (FACS). When eosinophils were incubated with tryptase in the presence of anti-proteinase-activated receptor 2 antagonist Abs a significant decrease in the IL-6 and IL-8 release occurred. In summary, we have demonstrated that the preformed mast cell mediator tryptase induces cytokine production and release in human peripheral blood eosinophils by the mitogen-activated protein kinase/AP-1 pathway.

Stem cell factor (SCF) or c-Kit ligand is a cytokine associated with the differentiation, survival, and activation of mast cells. Eosinophils have pleiotropic functions in several diseases and, together with mast cells, are key cells in allergy. Mast cell–eosinophil interactions can take place during the late and chronic phases of allergy. It was, therefore, investigated whether eosinophils can produce SCF and consequently influence mast cells. Human peripheral blood eosinophils variably expressed mRNA for the soluble and uncleaved forms of SCF (reverse transcription–polymerase chain reaction) and produced the 18.5-kd protein backbone of SCF (Western blot analysis). After overnight incubation in medium, eosinophils also produced SCF of higher molecular weight (42-45 kd) that might represent its glycosylated forms. Eosinophils expressed cytoplasmic SCF that colocalized with major basic protein (confocal laser microscopy). Freshly isolated eosinophils contained 8.9 ± 1.7 pg SCF/106 (mean ± SEM; enzyme-linked immunosorbent assay). Although overnight incubation of the eosinophils in either culture medium or in phorbol 12-myristate 13-acetate–calcium ionophore did not cause the secretion of SCF, the addition of chymase induced SCF release. In summary, it was demonstrated that human peripheral blood eosinophils are a source of SCF. These results may contribute to a better understanding of the interactions between eosinophils and mast cells in allergic inflammation.

Transcription factor NF-κB and its activating kinase IKKβ are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKKβ-NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKKβ (Ikkβ(Δ) mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikkβ(Δ) mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikkβ(Δ)Il1r1(-/-) mice, unlike Ikkβ(Δ) mice, were highly susceptible to bacterial infection, which indicated that signaling via IKKβ-NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.

Macrophages are the principal source of TNFalpha, yet they are highly resistant to TNFalpha-mediated cell death. Previously, employing in vitro differentiated human macrophages, we showed that following the inhibition of NF-kappaB, TNFalpha-induced caspase-8 activation contributes to DNA fragmentation but is not necessary for the loss of the inner mitochondrial transmembrane potential (DeltaPsim) or cell death. We here extend these observations to demonstrate that, when NF-kappaB is inhibited in macrophages, TNFalpha alters lysosomal membrane permeability (LMP). This results in the release of cathepsin B with subsequent loss of DeltaPsim and caspase-8 independent cell death. Interestingly, the cytoprotective, NF-kappaB-dependent protein A20 was rapidly induced in macrophages treated with TNFalpha. Ectopic expression of A20 in macrophages preserves LMP following treatment with TNFalpha, and as a result, mitochondrial integrity is safeguarded and macrophages are protected from cell death. These observations demonstrate that TNFalpha triggers both caspase 8-dependent and -independent cell death pathways in macrophages and identify a novel mechanism by which A20 protects these cells against both pathways.

Death receptors (DRs) are more than simple killers: they control cell growth, proliferation, and survival, thereby playing a pivotal role in immune and inflammatory responses. Some of these phenomena might be explained by aberrant reactive oxygen species (ROS) production and metabolism, which can lead to oxidative stress. A key signaling molecule of DR-initiated intracellular pathways, receptor-interacting protein 1 (RIP1), orchestrates a complex control of multiple responses and may link DR-associated signaling complexes to ROS production by mitochondria. Yet, RIP1 is also an important regulator of endogenous anti-oxidants and ROS scavenging enzymes, because it is required for nuclear factor kappaB activation that results in expression of anti-apoptotic and anti-oxidant proteins. Alteration of RIP1 function may result in ROS accumulation and abnormal c-Jun N-terminal protein kinase activation, affecting inflammatory responses, innate immunity, stress responses, and cell survival. These molecular mechanisms may be involved in neoplastic, autoimmune, neurodegenerative, inflammatory, and metabolic diseases.

Monocytes are the key regulators of joint inflammation and destruction in rheumatoid arthritis; hence, suppression of their recruitment into the joint may be therapeutically beneficial. Chemokines, including RANTES, are highly expressed in the joints of patient with rheumatoid arthritis, and they promote leukocyte trafficking into the synovial tissue. Because endogenous TLR4 ligands are expressed in the rheumatoid joint, the TLR4 ligand LPS was used to characterize the effects of RANTES on the TLR4-mediated induction of TNF-α and IL-6. Using peripheral blood (PB) monocytes, RANTES decreased LPS-induced IL-6 transcriptionally, whereas TNF-α was suppressed at the posttranscriptional level. RANTES signaled through p38 MAPK, and this signaling was further enhanced by LPS stimulation in PB monocytes, resulting in the earlier and increased secretion of IL-10. Inhibition of p38 by short-interfering RNA or a chemical inhibitor, as well as neutralization of IL-10, reversed the RANTES-mediated suppression of LPS-induced IL-6 and TNF-α. Further, when rheumatoid arthritis synovial fluid was added to PB monocytes, the neutralization of RANTES in fluid reduced the LPS-induced IL-10 and increased TNF-α. In conclusion, the results of this study suggest that RANTES down-regulates TLR4 ligation-induced IL-6 and TNF-α secretion by enhancing IL-10 production in PB monocytes. These observations suggest that the therapeutic neutralization of RANTES, in addition to decreasing the trafficking of leukocytes, may have a proinflammatory effect at the site of established chronic inflammation.

Objective Resistance to apoptosis may be an important mechanism contributing to the persistence of rheumatoid arthritis (RA). This study was undertaken to characterize the expression, regulation, and function of the antiapoptotic Bcl-2 family member Mcl-1 in macrophages isolated from the joints of patients with RA. Methods Mononuclear cells were isolated from the synovial fluid (SF) of patients with RA. Mcl-1 expression was documented by intracellular staining of CD14+ cells using flow cytometry, and by real-time polymerase chain reaction or immunoblot analysis of isolated macrophages. The expression of Mcl-1 was suppressed with small interfering RNA (siRNA) or chemical inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-1 and signal transducer and activator of transcription 3 (STAT-3) pathways. Apoptosis was defined by the loss of mitochondrial transmembrane potential and by DNA fragmentation. Results The expression of Mcl-1 was increased in CD14+ macrophages from the SF of patients with RA compared with normal in vitro–differentiated macrophages. Inhibition of the PI 3-kinase/Akt-1 or STAT-3 pathways significantly reduced the percentage of CD14+ cells within the SF and resulted in the reduction of Mcl-1 and the induction of apoptosis of synovial macrophages. Transfection of RA synovial macrophages with Mcl-1 siRNA resulted in apoptotic cell death. Conclusion Mcl-1 is critical for the survival of macrophages in the joints of patients with RA, and is therefore a potential therapeutic target in this disease.

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.

SummaryBackgroundTNF-alpha is an important mediator in allergy also for its effects on eosinophils.MethodsThe effect of dexamethasone on TNF-alpha induced eosinophils survival, degranulation (ECP), cytokines release (IL-8, GM-CSF) and adhesion to VCAM-1, ICAM-1 and IgG coated wells (EPO release) were evaluated.ResultsThe drug inhibited IL-8 and GM-CSF production, but not viability, degranulation or adhesion in human peripheral blood eosinophils.ConclusionThese results indicate that part of the activity of glucocorticosteroids on eosinophils may be mediated by their ability to inhibit cytokine secretion that in turn is important for the perpetuation of the allergic inflammation.

Background Heparanase and eosinophils are involved in several diseases, including inflammation, cancer, and angiogenesis. Objective We sought to determine whether eosinophils produce active heparanase. Methods Human peripheral blood eosinophils were isolated by immunoselection and tested for heparanase protein (immunocytochemistry, Western blot), mRNA (RT-PCR) and activity (Na 2 [ 35 S]O 4 -labeled extracellular matrix degradation) before and after activation. Heparanase intracellular localization (confocal laser microscopy) and ability to bind to eosinophil major basic protein (MBP) were also evaluated (immunoprecipitation). A model of allergic peritonitis resulting in eosinophilia was induced in TNF knockout and wild-type mice for in vivo studies. Results Eosinophils synthesized heparanase mRNA and contained heparanase in the active (50-kd) and latent (65-kd) forms. Heparanase partially co-localized with and was bound to MBP. No heparanase enzymatic activity was detected in eosinophils resting or activated with various agonists, including GM-CSF/C5a. Eosinophil lysates and MBP inhibited recombinant heparanase activity in a concentration-dependent manner (100%, 2×10 –7 mol/L). Eosinophil peroxidase and eosinophil cationic protein, but not myelin basic protein or compound 48/80, partially inhibited heparanase activity. Poly-l-arginine at very high concentrations caused an almost complete inhibition. In allergic peritonitis, heparanase activity in the peritoneal fluid inversely correlated with eosinophil number. Conclusions MBP is the first identified natural heparanase-inhibiting protein. Its presence in the eosinophil granules might indicate a protective function of these cells in diseases associated with inflammation and cancer progression.

In allergic disorders, the role of tumor necrosis factors (TNF) is not well established. We investigated the role of TNF in allergic peritonitis induced by ovalbumin (OVA) challenge in double TNF (TNF-alpha(-/-)/lymphotoxin-alpha(-/-)) knock out (TNF-KO) mice. In the peritoneal lavage of TNF-KO mice, mast cell number and histamine level (radioenzymatic assay) were similar to that in wild type (WT) mice. TNF-alpha (ELISA) and histamine were increased 1 h after challenge in WT mice. However, three days later eosinophil number and eosinophil peroxidase (EPO) levels (colorimetric-enzymatic assay) were found to be lower in TNF-KO mice. A second challenge three days after the first, increased EPO, histamine and IL-6 (ELISA) but did not alter eosinophil and mast cell numbers in both types of mice. On the other hand histamine and IL-6 were higher, while EPO was lower in TNF-KO mice. In conclusion, our findings show that TNF is involved in eosinophil accumulation and inflammatory mediators' release in a murine model of allergy.

Prolonged eosinophil survival is an essential step in the late and chronic phases of allergic inflammation and is regulated by the eosinophil survival cytokines. Our work has demonstrated that tumour necrosis factor (TNF)-alpha enhances survival (Trypan blue exclusion test) of human peripheral blood eosinophils from mildly allergic patients in a dose-dependent manner. The survival activity of TNF-alpha was inhibited by anti-TNF-RI, anti-TNF-RII antagonist antibodies and anti-granulocyte-monocyte colony-stimulating factor (GM-CSF) neutralizing antibodies but not by anti-interleukin (IL)-3 or anti-IL-5 antibodies. Furthermore, TNF-alpha-induced GM-CSF release from eosinophils. Anti-TNF-alpha antibodies also inhibited GM-CSF release from eosinophils induced by rat mast cell sonicate, which enhances eosinophil survival. To define the signal transduction pathway involved in GM-CSF production, eosinophils were incubated either with various mitogen-activated protein kinases (MAPK) inhibitors (MEK, JNK, P38), or Cyclosporin A (calcineurin inhibitor), or MG-132 (proteasome inhibitor). Only the proteasome inhibitor significantly decreased both TNF-alpha-enhanced eosinophil survival (from 38.1+/-4.1% to 13.3+/-1.4%) and GM-CSF release (from 6.2+/-0.7 pg/ml to 0.3+/-0.1 pg/ml). TNF-alpha also induced nuclear factor-kappaB (NF-kappaB) translocation to the nucleus, an essential step in GM-CSF mRNA production. All these findings provide evidence that NF-kappaB is involved in TNF-alpha-enhanced eosinophil survival through the regulation of GM-CSF production by eosinophils.

The NF-kappaB activating kinase IKKbeta suppresses early chemically induced liver tumorigenesis by inhibiting hepatocyte death and compensatory proliferation. To study IKKbeta's role in late tumor promotion and progression, we developed a transplant system that allows initiated mouse hepatocytes to form hepatocellular carcinomas (HCC) in host liver after a long latency. Deletion of IKKbeta long after initiation accelerated HCC development and enhanced proliferation of tumor initiating cells. These effects of IKKbeta/NF-kappaB were cell autonomous and correlated with increased accumulation of reactive oxygen species that led to JNK and STAT3 activation. Hepatocyte-specific STAT3 ablation prevented HCC development. The negative crosstalk between NF-kappaB and STAT3, which is also evident in human HCC, is a critical regulator of liver cancer development and progression.

Mcl-1 is a Bcl-2-family, antiapoptotic molecule that is critical for the survival of T and B lymphocytes and macrophages; however, its role in nonhemopoietic cells remains to be fully elucidated. The current study focuses on the role of Mcl-1 in rheumatoid arthritis (RA). Mcl-1 was strongly expressed in the synovial lining and was increased in the sublining fibroblasts of patients with RA, compared with control synovial tissue. The expression of Mcl-1 in sublining fibroblasts correlated with the degree of inflammation and TNF-α, and IL-1β treatment of cultured synovial fibroblasts resulted in the increased expression of Mcl-1 at the mRNA and protein levels. Mcl-1 was critical for the survival of RA synovial fibroblasts, because the forced reduction of Mcl-1 using a Mcl-1 antisense-expressing adenoviral vector induced apoptotic cell death, which was mediated through Bax, Bak, and Bim. These observations document a critical role for Mcl-1 in protecting against apoptosis in RA and suggest that Mc1–1 is a potential therapeutic target in this disease.