Synergy-inducing chemokines enhance CCR2 ligand activities on monocytes

The migration of monocytes to sites of inflammation is largely determined by their response to chemokines. Although the chemokine specificities and expression patterns of chemokine receptors are well defined, it is still a matter of debate how cells integrate the messages provided by different chemokines that are concomitantly produced in physiological or pathological situations in vivo. We present evidence for one regulatory mechanism of human monocyte trafficking. Monocytes can integrate stimuli provided by inflammatory chemokines in the presence of homeostatic chemokines. In particular, migration and cell responses could occur at much lower concentrations of the CCR2 agonists, in the presence of chemokines (CCL19 and CCL21) that per se do not act on monocytes. Binding studies on CCR2(+) cells showed that CCL19 and CCL21 do not compete with the CCR2 agonist CCL2. Furthermore, the presence of CCL19 or CCL21 could influence the degradation of CCL2 and CCL7 on cells expressing the decoy receptor D6. These findings disclose a new scenario to further comprehend the complexity of chemokine-based monocyte trafficking in a vast variety of human inflammatory disorders.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.

Inhibition of meningitis-associated neutrophil apoptosis by TNF-α depends on functional PI3-kinase in monocytes

In bacterial meningitis, neutrophils cope with bacterial infection but also lead to tissue damage. The balance of beneficial and harmful effects may depend on the lifespan of the neutrophils in the CNS. Here, we show that CSF of patients with meningococcal meningitis contains a neutrophil apoptosis-inhibiting capacity that correlates with TNF-α content. In vitro experiments show that Neisseria meningitidis as well as LPS derived from these bacteria regulated neutrophil apoptosis mainly by stimulating TNF-α production in monocytes. Whereas LPS-induced PI3K-dependent survival signals in monocytes are critical for neutrophil survival, PI3K signaling in granulocytes did not contribute to the increased lifespan of neutrophils. We conclude that LPS-driven PI3K signaling in monocytes regulates neutrophil apoptosis and thereby, may be crucial in the initiation of secondary brain damage in bacterial meningitis.

Chemistry of pyrrocorphins: C-methylations at the periphery of pyrrocorphins and related corphinoid ligand systems

Various corphinoid model systems bearing a methyl group at the position C-20 have been found to undergo regioselective chemical -methylation at the ligand periphery, mimicking enzymic -methylation occurring in vitamin-B biosynthesis.

Reactive oxygen species-dependent mechanisms of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in human carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer, and neuroblastoma. 4HPR induces apoptosis in various cancer cells and production of reactive oxygen species (ROS) has been suggested as a possible cause underlying these effects. However, the mechanisms governing these effects by 4HPR are not fully elucidated. In this study, we explored the mechanisms of 4HPR-induced ROS increase and apoptosis in human cancer cells. ^ First, we identified genes modulated by 4HPR using oligonucleotide gene expression arrays and found that they fall into specific functional canonical pathways and gene networks using Ingenuity Pathways Analysis®. Further analysis has shown that 4HPR induced up-regulation of Endoplasmic Reticulum (ER)-related genes such as Heat shock proteins 70 and 90 and the transcriptional factor, GADD153. These findings were validated using quantitative real-time PCR. ^ Second, we found that 4HPR induced extensive ER stress evidenced by dilation of the ER and endoribonuclease-mediated splicing and activation of the transcriptional factor, XBP-1. In addition, 4HPR induced the up-regulation of various ER stress-related genes and their protein products, as well as cleavage and activation of the ER specific Caspase-4. Concomitantly with XBP-1 splicing, all of these effects were dependent on ROS generation by 4HPR. Furthermore, chemical inhibition and RNA interference studies revealed a novel pro-apoptotic role for HSP70/A1A in 4HPR-mediated apoptosis. ^ Third, we observed rapid activation of the small GTPase Rac by 4HPR which was upstream of ROS generation. Inhibition of Rac activity or silencing of its expression by RNA interference inhibited ROS generation and apoptosis induction by 4HPR. siRNA targeting PAK1 and expression of a dominant negative Rac, decreased 4HPR-mediated ROS generation, while expression of a constitutive active Rac increased basal and 4HPR-induced ROS generation and PARP cleavage. Furthermore, metastatic cancer cells exhibited higher Rac activation, ROS generation, and cell growth inhibition due to 4HPR exposure compared to their primary cancer cell counterparts. ^ These findings provide novel insights into 4HPR-mediated ROS generation and apoptosis induction and support the use of ROS inducing agents such as 4HPR against metastatic cancer cells. ^

Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons

Neurotoxicity induced by overstimulation of N-methyl-d-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction

Previous studies have shown that proinflammatory cytokines, such as tumor necrosis factor (TNF), are expressed after acute hemodynamic overloading and myocardial ischemia/infarction. To define the role of TNF in the setting of ischemia/infarction, we performed a series of acute coronary artery occlusions in mice lacking one or both TNF receptors. Left ventricular infarct size was assessed at 24 h after acute coronary occlusion by triphenyltetrazolium chloride (TTC) staining in wild-type (both TNF receptors present) and mice lacking either the type 1 (TNFR1), type 2 (TNFR2), or both TNF receptors (TNFR1/TNFR2). Left ventricular infarct size as assessed by TTC staining was significantly greater (P < 0.005) in the TNFR1/TNFR2-deficient mice (77.2% ± 15.3%) when compared with either wild-type mice (46.8% ± 19.4%) or TNFR1-deficient (47.9% ± 10.6%) or TNFR2-deficient (41.6% ± 16.5%) mice. Examination of the extent of necrosis in wild-type and TNFR1/TNFR2-deficient mice by anti-myosin Ab staining demonstrated no significant difference between groups; however, the peak frequency and extent of apoptosis were accelerated in the TNFR1/TNFR2-deficient mice when compared with the wild-type mice. The increase in apoptosis in the TNFR1/TNFR2-deficient mice did not appear to be secondary to a selective up-regulation of the Fas ligand/receptor system in these mice. These data suggest that TNF signaling gives rise to one or more cytoprotective signals that prevent and/or delay the development of cardiac myocyte apoptosis after acute ischemic injury.

Retinoid-related orphan receptor γ (RORγ) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis

To identify the physiological functions of the retinoid-related orphan receptor γ (RORγ), a member of the nuclear receptor superfamily, mice deficient in RORγ function were generated by targeted disruption. RORγ−/− mice lack peripheral and mesenteric lymph nodes and Peyer's patches, indicating that RORγ expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+ and CD4+ lymphocytes is reduced 6- and 10-fold, respectively, whereas the number of circulating B cells is normal. The thymus of RORγ−/− mice contains 74.4% ± 8.9% fewer thymocytes than that of wild-type mice. Flow cytometric analysis showed a decrease in the CD4+CD8+ subpopulation. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining demonstrated a 4-fold increase in apoptotic cells in the cortex of the thymus of RORγ−/− mice. The latter was supported by the observed increase in annexin V-positive cells. RORγ−/− thymocytes placed in culture exhibit a dramatic increase in the rate of “spontaneous” apoptosis. This increase is largely associated with CD4+CD8+ thymocytes and may, at least in part, be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a 6-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from RORγ−/− mice. Our observations indicate that RORγ is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Our findings support a model in which RORγ negatively controls apoptosis in thymocytes.

Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1

We have investigated the ability of Sf-caspase-1 and two mammalian caspases, caspase-1 and caspase-3, to induce apoptosis in Spodoptera frugiperda Sf-21 insect cells. While the transient expression of the pro-Sf-caspase-1 did not induce apoptosis, expression of the pro-domain deleted form, p31, or coexpression of the two subunits of mature Sf-caspase-1, p19 and p12, induced apoptosis in Sf-21 cells. The behavior of Sf-caspase-1 resembled that of the closely related mammalian caspase, caspase-3, and contrasted with that of the mammalian caspase-1, the pro-form of which was active in inducing apoptosis in Sf-21 cells. The baculovirus caspase inhibitor P35 blocked apoptosis induced by active forms of all three caspases. In contrast, members of the baculovirus inhibitor of apoptosis (IAP) family failed to block active caspase-induced apoptosis. However, during viral infection, expression of OpIAP or CpIAP blocked the activation of pro-Sf-caspase-1 and the associated induction of apoptosis. Thus, the mechanism by which baculovirus IAPs inhibit apoptosis is distinct from the mechanism by which P35 blocks apoptosis and involves inhibition of the activation of pro-caspases like Sf-caspase-1.

SARPs: A family of secreted apoptosis-related proteins

Quiescent mouse embryonic C3H/10T½ cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T½ cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T½ cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated sarp1 (secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T½ cells. Hybridization studies with sarp1 revealed two additional family members. Cloning and sequencing of sarp2 and sarp3 revealed 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with sarp2 displayed increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of β-catenin, suggesting that SARPs interfere with the Wnt–frizzled proteins signaling pathway.

Gene transfer of Fas ligand induces tumor regression in vivo

The Fas–Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Herein, we report that gene transfer of FasL inhibits tumor cell growth in vivo. Although such inhibition is expected in Fas+ tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into the CT26 colon carcinoma that does not express Fas. Infection by an adenoviral vector encoding FasL rapidly eliminated tumor masses in the Fas+ Renca tumor by inducing cell death, whereas the elimination of Fas− CT26 cells was mediated by inflammatory cells. Analysis of human malignancies revealed Fas, but not FasL, expression in a majority of tumors and susceptibility to FasL in most Fas+ cell lines. These findings suggest that gene transfer of FasL generates apoptotic responses and induces potent inflammatory reactions that can be used to induce the regression of malignancies.

Increased mitochondrial oxidative stress in the Sod2 (+/−) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis

To determine the importance of mitochondrial reactive oxygen species toxicity in aging and senescence, we analyzed changes in mitochondrial function with age in mice with partial or complete deficiencies in the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD). Liver mitochondria from homozygous mutant mice, with a complete deficiency in MnSOD, exhibited substantial respiration inhibition and marked sensitization of the mitochondrial permeability transition pore. Mitochondria from heterozygous mice, with a partial deficiency in MnSOD, showed evidence of increased proton leak, inhibition of respiration, and early and rapid accumulation of mitochondrial oxidative damage. Furthermore, chronic oxidative stress in the heterozygous mice resulted in an increased sensitization of the mitochondrial permeability transition pore and the premature induction of apoptosis, which presumably eliminates the cells with damaged mitochondria. Mice with normal MnSOD levels show the same age-related mitochondrial decline as the heterozygotes but occurring later in life. The premature decline in mitochondrial function in the heterozygote was associated with the compensatory up-regulation of oxidative phosphorylation enzyme activity. Thus mitochondrial reactive oxygen species production, oxidative stress, functional decline, and the initiation of apoptosis appear to be central components of the aging process.