TNFalpha-induced IEC-6 cell apoptosis requires activation of ICE caspases whereas complete inhibition of the caspase cascade leads to necrotic cell death.

Tumor necrosis factor (TNF)alpha is considered to play a key pathogenetic role in inflammatory bowel diseases. In this study we analyzed the mechanisms by which TNFalpha induces intestinal epithelial cell apoptosis. TNFalpha alone, and more potently in combination with IFNgamma, induced a high degree of IEC-6 cell apoptosis. This effect was more than 100-fold stronger if both of the TNF-R were stimulated, compared to stimulation of the p55-TNF-R alone, indicating an important apoptosis enhancing effect of the p75-TNF-R. TNFalpha-induced apoptosis required activation of ICE caspases and was completely abolished by its inhibitor, zVAD-fmk. Specific inhibition of caspase-3 with zDEVD-fmk did not alter the effect of TNFalpha. Western blot analyses confirmed that caspase-3 was not activated in response to TNFalpha. In the presence of complete inhibition of the caspase cascade with zVAD-fmk (>/=50 microM), TNFalpha induced cell necrosis rather than apoptosis. Our data reveal that TNFalpha can trigger enterocyte cell death via apoptosis or necrosis, depending upon the activation or blockade of specific caspases.     

Molecular characterization of the surface of apoptotic neutrophils: implications for functional downregulation and recognition by phagocytes

We have used a panel of monoclonal antibodies and lectins to examine the profile of surface molecule expression on human neutrophils that have undergone spontaneous apoptosis during in vitro culture. Neutrophil apoptosis was found to be accompanied by down-regulation of the immunoglobulin superfamily members PECAM-1 (CD31), ICAM-3 (CD50), CD66acde, and CD66b and the integrin-associated proteins CD63 and urokinase plasminogen activator receptor (CD87) that may alter the potential for adhesive interactions. Cellular interactions may be further influenced by the reduction of the expression of surface carbohydrate moieties, including sialic acid. Reduced expression of FcgammaRII (CD32), complement receptor type 1 (CD35) and receptors for pro-inflammatory mediators C5a (CD88) and TNFalpha (CD120b) associated with apoptosis might limit neutrophil responsiveness to stimuli that trigger degranulation responses. Although many of the receptors we have examined are expressed at reduced levels on apoptotic neutrophils, we found that there was differential loss of certain receptors (e.g. CD16, CD15 and CD120b) and increased expression of aminopeptidase-N (CD13). Together with our previous data showing that expression of certain molecules e.g. LFA-3 (CD58) is not altered during neutrophil apoptosis, these data are suggestive of specific changes in receptor mobilisation and shedding associated with apoptosis. Although reduced expression of CD63 (azurophilic granules) and CR1 (specific granules) indicates that granule mobilisation does not accompany apoptosis, a monoclonal antibody (BOB78), that recognises a 90 kDa antigen localised in intracellular granules, defines a subpopulation of apoptotic neutrophils that exhibit nuclear degradation yet retain intact plasma membranes. BOB78 positive neutrophils were found to bind biotinylated thrombospondin, suggesting that this mAb defines surface molecular changes associated with exposure of thrombospondin binding moieties.     

Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death

Multiple cytokines are secreted in the brain during pro-inflammatory conditions and likely affect neuron survival. Previously, we demonstrated that glutamate and tumor necrosis factor alpha (TNFalpha) kill neurons via activation of the N-methyl-d-aspartate (NMDA) and TNFalpha receptors, respectively. This report continues characterizing the signaling cross-talk pathway initiated during this inflammation-related mechanism of death. Stimulation of mouse cortical neuron cultures with TNFalpha results in a transient increase in NMDA receptor-dependent calcium influx that is additive with NMDA stimulation and inhibited by pre-treatment with the NMDA receptor antagonist, DL-2-amino-5-phosphonovaleric acid, or the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione. Pre-treatment with N-type calcium channel antagonist, omega-conotoxin, or the voltage-gated sodium channel antagonist, tetrodotoxin, also prevents the TNFalpha-stimulated calcium influx. Combined TNFalpha and NMDA stimulation results in a transient increase in activity of extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs). Specific inhibition of ERKs but not JNKs is protective against TNFalpha and NMDA-dependent death. Death is mediated via the low-affinity TNFalpha receptor, TNFRII, as agonist antibodies for TNFRII but not TNFRI stimulate NMDA receptor-dependent calcium influx and death. These data demonstrate how microglial pro-inflammatory secretions including TNFalpha can acutely facilitate glutamate-dependent neuron death.     

A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils

Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.     

Actin polymerization induces shedding of FcgammaRIIIb (CD16) from human neutrophils

FcgammaRIIIb (CD16) is a glycosyl phosphatidylinositol (GPI)-anchored low-affinity IgG receptor, exclusively expressed on human neutrophils. FcgammaRIIIb associates with complement receptor 3 (CR3, Mac-1, CD11b/CD18), which may indirectly link FcgammaRIIIb to the actin cytoskeleton. Upon neutrophil activation, apoptosis, or chemotaxis, FcgammaRIIIb is shed from the cell surface. In all of these events, actin rearrangements play an important role. To establish a role for the actin cytoskeleton in the control of FcgammaRIIIb shedding, we treated human neutrophils with jasplakinolide, an actin-polymerizing peptide. We show that enhanced actin polymerization induces time- and dose-dependent shedding of FcgammaRIIIb. This effect was not restricted to FcgammaRIIIb, because the cell surface expression of CD43, CD44, and L-selectin was also downregulated after induction of actin polymerization. This actin-dependent pathway is staurosporine sensitive but does not appear to involve activation of PKC or CR3. These data show that the actin cytoskeleton can regulate protein ectodomain shedding from human neutrophils.     

Quantitative flow cytometric analysis of expression of tumor necrosis factor receptor types I and II on mononuclear cells

Abstract

Background: Tumor necrosis factor (TNF)-α is an inflammatory cytokine, the biological effects of which are mediated by the interaction with specific membrane-bound receptors. To assess TNF-α receptor (TNFR) expression, it is important to estimate both the number of cells that carry these receptors and the number of receptors per cell, because the cell fate depends on the balance between TNFRI and TNFRII signaling. Objective: The aim of the present study was to develop an optimized protocol to estimate the level of expression of membrane-bound TNFRI and TNFRII, using QuantiBRITE PE calibration beads. Materials and methods: The percentage of cells that expressed membrane-bound TNFRI and TNFRII and the mean number of receptors per cell were determined by flow cytometry using PE-labeled antibodies against TNFR. To create a calibration curve and convert cell fluorescence intensity values to absolute numbers of receptors, we used QuantiBRITE PE beads. Results: CD19⁺ B lymphocytes had the least percentage of cells expressing TNFRI and the greatest number of receptor molecules per cell, whereas CD3⁺ T lymphocytes had the greatest percentage of cells expressing TNFRII and the lowest density of these receptors. We also established that stimulation of peripheral blood mononuclear cells (PBMCs) with the lipopolysaccharide (LPS) significantly increased the number of TNFRI and TNFRII on CD14⁺ monocytes. Conclusion: Application of the protocol-identified differences in the percentage of cells that expressed TNFRs, as well as the absolute number of receptors per cell, among different subpopulations of PBMCs, and between PBMCs cultured with and without LPS.     

Tumor necrosis factor-alpha activation of the c-Jun N-terminal kinase pathway in human neutrophils. Integrin involvement in a pathway leading from cytoplasmic tyrosine kinases apoptosis

The intensity and duration of an inflammatory response depends on the balance of factors that favor perpetuation versus resolution. At sites of inflammation, neutrophils adherent to other cells or matrix components are exposed to tumor necrosis factor-alpha (TNFalpha). Although TNFalpha has been implicated in induction of pro-inflammatory responses, it may also inhibit the intensity of neutrophilic inflammation by promoting apoptosis. Since TNFalpha is not only an important activator of the stress-induced pathways leading to p38 MAPk and c-Jun N-terminal kinase (JNK) but also a potent effector of apoptosis, we investigated the effects of TNFalpha on the JNK pathway in adherent human neutrophils and the potential involvement of this pathway in neutrophil apoptosis. Stimulation with TNFalpha was found to result in beta2 integrin-mediated activation of the cytoplasmic tyrosine kinases Pyk2 and Syk, and activation of a three-part MAPk module composed of MEKK1, MKK7, and/or MKK4 and JNK1. JNK activation was attenuated by blocking antibodies to beta2 integrins, the tyrosine kinase inhibitors, genistein, and tyrphostin A9, a Pyk2-specific inhibitor, and piceatannol, a Syk-specific inhibitor. Exposure of adherent neutrophils to TNFalpha led to the rapid onset of apoptosis that was demonstrated by augmented annexin V binding and caspase-3 cleavage. TNFalpha-induced increases in annexin V binding to neutrophils were attenuated by blocking antibodies to beta2 integrins, and the caspase-3 cleavage was attenuated by tyrphostin A9. Hence, exposure of adherent neutrophils to TNFalpha leads to utilization of the JNK-signaling pathways that may contribute to diverse functional responses including induction of apoptosis and subsequent resolution of the inflammatory response.     

Cutting edge: a dominant negative form of TNF-alpha converting enzyme inhibits proTNF and TNFRII secretion.

TNF-alpha converting enzyme (TACE) is the protease responsible for processing proTNF from the 26-kDa membrane-anchored precursor to the secreted 17-kDa TNF-alpha. We show here that a deletion mutant of TACE (dTACE), lacking the pro and catalytic domains of the protease, acts as a dominant negative for proTNF processing in transfected HEK293 cells. We used the same system to test the effect of dTACE on TNFRII processing. Overexpression of dTACE with TNFRII resulted in >80% inhibition of TNFRII shedding. Although significant inhibition of TNF-alpha and TNFRII shedding was achieved with dTACE, we could not detect a cell surface accumulation of the noncleaved substrates above that observed in the absence of dTACE. Our results suggest that TNFRII is a substrate for TACE, and that dTACE is capable of interfering with the function of endogenous TACE, either by binding and sequestering TACE substrates via the disintegrin domain, transmembrane domain, or cytoplasmic tail, or by some other mechanism that has yet to be determined.     

Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors

A series of novel L-isoserine derivatives were synthesised and evaluated for their ability to inhibit aminopeptidase N (APN)/CD13. In our preliminary biological results, some of these compounds possessed a potent inhibitory activity against the APN. Within this series, compound 14b not only showed similar enzyme inhibition (IC?? of 12.2 μM) compared with the positive control bestatin (half maximal inhibitory concentration (IC??) of 7.3 μM), but also had a potent antiproliferative activity against human cancer cell lines cells.     

Interferon gamma (IFNgamma ) and tumor necrosis factor alpha synergism in ME-180 cervical cancer cell apoptosis and necrosis. IFNgamma inhibits cytoprotective NF-kappa B through STAT1/IRF-1 pathways

We investigated the molecular mechanism of the synergism between interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) documented in a variety of biological occasions such as tumor cell death and inflammatory responses. IFNgamma/TNFalpha synergistically induced apoptosis of ME-180 cervical cancer cells. IFNgamma induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited IFNgamma/TNFalpha-induced apoptosis, whereas IRF-1 transfection induced susceptibility to TNFalpha. Dominant-negative IkappaBalpha transfection sensitized ME-180 cells to TNFalpha. IFNgamma pretreatment attenuated TNFalpha- or p65-induced NF-kappaB reporter activity, whereas it did not inhibit p65 translocation or DNA binding of NF-kappaB. IRF-1 transfection alone inhibited TNFalpha-induced NF-kappaB activity, which was reversed by coactivator p300 overexpression. Caspases were activated by IFNgamma/TNFalpha combination; however, caspase inhibition did not abrogate IFNgamma/TNFalpha-induced cell death. Instead, caspase inhibitors directed IFNgamma/TNFalpha-treated ME-180 cells to undergo necrosis, as demonstrated by Hoechst 33258/propidium iodide staining and electron microscopy. Taken together, our results indicate that IFNgamma and TNFalpha synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways initiated by IFNgamma play a critical role in IFNgamma/TNFalpha synergism by inhibiting cytoprotective NF-kappaB. IFNgamma/TNFalpha synergism appears to activate cell death machinery independently of caspase activation, and caspase activation seems to merely determine the mode of cell death.     

Inhibition of tumor necrosis factor alpha-mediated NFkappaB activation and leukocyte adhesion,with enhanced endothelial apoptosis,by G protein-linked receptor (TP) ligands

Tumor necrosis factor (TNF) alpha is a critical mediator of inflammation; however, TNFalpha is rarely released alone and the "cross-talk" between different classes of inflammatory mediators is largely unexplored. Thromboxane A(2) (TXA(2)) is released during I/R injury and exerts its effects via a G protein-linked receptor (TP). In this study, we found that TXA(2) mimetics stimulate leukocyte adhesion molecule (LAM) expression on endothelium via TPbeta. The potential interaction between TXA(2) and TNFalpha in altering endothelial survival and LAM expression was examined. IBOP, a TXA(2) mimetic, attenuated TNFalpha-induced LAM expression in vitro, in a concentration-dependent manner, by preventing TNFalpha-enhanced gene expression, and also reduced TNFalpha-induced leukocyte adhesion to endothelium both in vitro and in vivo. IBOP abrogated TNFalpha-induced NFkappaB activation in endothelial cells, as determined by reduced IkappaB phosphorylation and NFkappaB nuclear translocation, by inhibiting the assembly of signaling intermediates with the intracellular domain of TNF receptors 1 and 2 in response to TNFalpha. This inhibition resulted from the Galpha(q)-mediated enhancement of STAT1 activation and was reversed by anti-STAT1 antisense oligonucleotides. TNFalpha-mediated TNFR1-FADD association and caspase 8 activation were not inhibited by IBOP co-stimulation, however, resulting in a 2.6-fold increase in endothelial cell apoptosis. By stimulating the vessel wall and inducing endothelial cell apoptosis, TXA(2), in combination with TNFalpha, may hamper the angiogenic response during inflammation or ischemia, thus reducing revascularization and tissue viability.     

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.     

Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors

A series of novel L-isoserine derivatives were synthesised and evaluated for their ability to inhibit aminopeptidase N (APN)/CD13. In our preliminary biological results, some of these compounds possessed a potent inhibitory activity against the APN. Within this series, compound 14b not only showed similar enzyme inhibition (IC₅₀ of 12.2?μM) compared with the positive control bestatin (half maximal inhibitory concentration (IC₅₀) of 7.3?μM), but also had a potent antiproliferative activity against human cancer cell lines cells.     

Loss and shedding of surface markers from the leukemic myeloid monocytic line THP-1 induced to undergo apoptosis

Previous studies have established that a relationship exists between apoptosis and cell surface (ecto-) peptidase activity. Thus dose-dependent increases were found both in ectopeptidase activities and in the proportion of cells undergoing apoptosis in HeLa cell monolayers after exposure to UV and other perturbants causing arrest of DNA synthesis (indirectly or directly as a result of DNA damage). The nature of the correlation made no distinction as to whether an increase in peptidase activity was causal of, or consequential to apoptosis, nor whether the increase was a general response by all cells. As a wider approach to understanding the possible role played by ectopeptidases in apoptosis, we report the effect on expression of a known ectopeptidase, aminopeptidase N (CD13), by a myelomonocytic cell line induced to undergo apoptosis. Using THP-1 cultures exposed to low concentrations of ethanol, we used FACS technology to sort for early apoptotic cells that have an increased ability to sequester the vital dye Hoechst 33342 while excluding nonvital dyes. Apoptosis was verified by light, fluorescence, and transmission electron microscopy, and the presence of DNA fragmentation. These early apoptotic cells showed a significant loss in CD13 labeling. Another surface marker, CD33, behaved similarly, whereas CD14 was lost globally, and not just by the apoptotic cells. Peptidase assays confirmed that an aminopeptidase was shed into the bathing media and that this activity was inhibitable both by bestatin and by a CD13 neutralizing monoclonal antibody. In treated cells, there was no evidence for an increase in cell surface protease activity directed toward a highly aliphatic nonapeptide substrate used as a model for TGF-α scission from its precursor form. However, other cell surface proteases of different specificity are presumably responsible for the observed shedding of CD13. © 1996 Wiley-Liss, Inc.     

Novel aminopeptidase N inhibitors derived from antineoplaston AS2–5 (Part II)

As our ongoing work, a series of peptidomimetic l-iso-glutamine derivatives derived from antineoplaston AS2–5 scaffold were prepared and their APN/CD13 and MMP-2 inhibitory activities were evaluated hereby. The results displayed that these compounds exhibited selective inhibition against APN as compared with MMP-2, with IC₅₀ values in micromole range. Compounds A1 and A2 showed comparable APN inhibitory activities than the positive control bestatin.