Tumor necrosis factor alpha-induced inflammation is increased but apoptosis is inhibited by common food additive carrageenan

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNF-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation and utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNF-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNF-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNF-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy.     

Delayed apoptosis and modulation of phospholipase D activity by plasmid containing mammalian cDNA in human neutrophils

Phospholipase D (PLD) has been reported to have an anti-apoptotic role in neutrophils. This study examined the effects of plasmids containing the cDNA of PLD on the apoptosis of neutrophils. The apoptotic rate of neutrophils treated with the pCDNA3.1 plasmid was similar to that of the untreated cells after 24 h culture. However, the addition of pCDNA3.1 containing the cDNA of either human PLD1 (pCDNA3.1-PLD1) or -PLD2 (pCDNA3.1-PLD2) to the culture media with or without transfection reagent significantly decreased the rate of spontaneous apoptosis but not Fas-stimulated apoptosis and the decreased apoptosis was blocked by 1-butanol. pCDNA3.1-PLD blocked the cleavage of procaspase-3 and -8. The phorbol myristate acetate stimulated the PLD activities of pCDNA3.1-PLD-treated neutrophils but did not stimulate the activities of untreated or pCDNA3.1-treated neutrophils. The level of the PLD1 protein was higher in the cultured neutrophils with pCDNA3.1-PLD than with the media or pCDNA3.1. The spontaneous apoptosis of neutrophils was inhibited and the PLD1 expression level was increased by the linearized or promoterless forms of pCDNA3.1-PLD1 and the plasmids containing the cDNA of the enhanced green fluorescent protein (pEGFP) and EGFP-PLD1. These results suggest that the plasmids containing mammalian cDNA inhibit the spontaneous apoptosis of neutrophils and modulate PLD.     

Prolonged cultures of unstimulated human neutrophils lead to the apparition and persistence of rest-in-plate structures (RIPs) recognized by professional phagocytes in vitro and in vivo

Polymorphonuclear neutrophil cells (PMNs) are known to spontaneously undergo apoptosis and then eliminated by professional phagocytes to prevent inflammation, a process called efferocytosis. However, when efferocytosis is impaired, PMNs will fall into secondary necrosis. Whether this state can persist for a certain period of time is unclear, since most of the studies investigating secondary necrosis are performed within 24 h following induction by a proapoptotic agent. In this study, freshly isolated human PMNs were incubated without addition of exogenous agents in order to force them to undergo apoptosis and then secondary necrosis, an ideal experimental condition to study the behavior of secondary necrotic PMNs in absence of efferocytosis. By monitoring PMN cell morphology over time, we observed that an increasing proportion of cells harbored a ghost-like phenotype. Because these cellular remnants persist in plates for several days, we introduce here the terminology RIPs for ‘rest-in-plate’ structure. Heating of freshly isolated PMNs for 5 min did not lead to the apparition of RIPs over time. In vivo administration of 7-days old RIPs in the murine air pouch model induced a slight inflammation resorbed within 24 h. PKH26-stained RIPs were found to be ingested by professional phagocytes in vitro and in vivo in the murine air pouch and peritonitis models. Therefore, aged-PMNs have the potential to become RIPs in absence of efficient efferocytosis. Fortunately RIPs are recognized by professional phagocytes and, therefore, the concept of resolution of inflammation based on elimination of apoptotic and secondary necrotic PMNs could also be applied to RIPs.     

Determining the 3D structure of human ASC2 protein involved in apoptosis and inflammation

ASC2 structure has been well defined by 1141 NOE experimental restraints. The model consists of five alpha helices. alpha-Helices are connected by short random structure loops. The sole exception is the loop connecting helices 2 and 3, which has a 20-residue length. Folding generally agrees with the folding of recently published death domain structures in which alpha-helix structures have been reported. In spite of structural similarity, amino acid sequence homology with the most similar protein (ASC1) is just 64%. DD, DED, and CASP protein structures present six helices along their sequences; ASC2 presents 5 well-defined helices due to long distance restraints. However, a helical fragment was observed between amino acids 38 and 42 (representing helix 3) in the death domains when constructing the model.     

Macrophage phagocytosis of apoptotic neutrophils is compromised by matrix proteins modified by cigarette smoke and lipid peroxidation products

Clearance of apoptotic cells by phagocytosis plays an important role in the resolution of an inflammatory response. Macrophages interacting with extracellular matrix (ECM) proteins upregulate their phagocytic capacity. Cigarette smoke contains highly reactive carbonyls that modify proteins which directly/indirectly affects cellular function. We observed, in vitro, that human macrophages interacting with carbonyl or cigarette smoke modified ECM proteins dramatically down regulated their ability to phagocytose apoptotic neutrophils. We also show that this interaction with carbonyl-adduct modified ECM proteins led to increased macrophage adhesion in vitro. We hypothesise that changes in the ECM environment as a result of cigarette smoking affect the ability of macrophages to remove apoptotic cells. Moreover, we postulate that this decreased phagocytic activity was as a result of sequestration of receptors involved in the uptake of apoptotic cells towards that of recognition of carbonyl adducts on the modified ECM proteins leading to increased macrophage adhesion.     

Improved apoptosis detection in ovine neutrophils by annexin V and carboxyfluorescein diacetate staining

Neutrophil apoptosis is critical for final resolution of the inflammation in the tissues and for maintenance of neutrophil homeostasis under normal condition. An early hallmark of apoptotic cells is translocation of phosphatidylserine (PS) residues, normally located in the inner leaflet of cellular membrane, to the external cell surface; exposed PS is recognized by specific PS receptors on disposing cells. Here we report an improved procedure to detect neutrophil apoptosis by simultaneous staining for exposed PS with Cy3-labeled annexin V (Cy3) and for membrane integrity with the vital dye 6-carboxyfluorescein diacetate (6-CFDA) based on the APOAC apoptosis detection kit (Sigma). Spontaneous apoptosis was evaluated in ovine neutrophils cultured ex vivo for 18 h. We investigated the multiple parameters involved in the assay, i.e. the type of fixative (methanol, paraformaldehyde, or no fixation) and the type of slide (coated with Vectabond, polylysine or Parafilm^®). Results indicated that both the adhesion to the slide and the fixation can modify neutrophil functional status and morphology, which result in misleading apoptosis detection. In order to minimize these artifacts, we have developed an improved APOAC assay procedure, staining cells while in suspension and using Parafilm^® coated slides.     

The differential effect of dexamethasone on granulocyte apoptosis involves stabilization of Mcl-1L in neutrophils but not in eosinophils

In the absence of activation signals, circulating human neutrophils and eosinophils undergo spontaneous apoptosis. The glucocorticoid dexamethasone (Dex) accelerates apoptosis in inflammatory cells such as eosinophils, but uniquely delays neutrophil apoptosis. Corresponding to the opposite effects of Dex on granulocyte apoptosis, we demonstrate that in neutrophils and eosinophils Dex oppositely affects expression of the anti-apoptotic Bcl-2 family protein Mcl-1L. Mcl-1L expression declines over time in vitro; however, Dex maintains Mcl-1L expression in neutrophils. In contrast, Dex accelerates Mcl-1L protein loss in eosinophils. Neither Mcl-1S, a pro-apoptotic splice variant, nor Bax were affected. Dex treatment in the presence of a translation inhibitor stabilized existing Mcl-1L protein in neutrophils, while Mcl-1L stability in eosinophils was unaffected. Accordingly, delay of neutrophil apoptosis by Dex was prevented by antisense Mcl-1L siRNA. Our findings suggest that regulation of Mcl-1L degradation plays an important role in the opposite effects of Dex on granulocyte apoptosis.     

Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts

c-Myc is known to induce or potentiate apoptotic processes predominantly by triggering or enhancing the activity of caspases, but the activation mechanisms of caspases by c-Myc remain still poorly understood. Here we found that in MycER™ rat fibroblasts the activation of c-Myc led to an early activation and cleavage of the initiator caspase-8, and concurrent processing and activation of the effector caspases 3 and 7. Interestingly, the expression of cellular FLICE inhibitory protein (c-FLIP) mRNA and the encoded protein, c-FLIP_L, a catalytically inactive homologue of caspase-8, were down-regulated prior to or coincidently with the activation of caspase-8. Of the other known initiators, caspase-9, involved in the mitochondrial pathway, was activated/processed surprisingly late, only after the effector caspases 3/7. Further, we studied the potential involvement of the Fas- and tumor necrosis factor receptor (TNFR)-mediated signaling in the activation of caspase-8 by c-Myc. Blocking of the function of these death receptors by neutralizing antibodies against Fas ligand and TNF-α did not prevent the processing of caspase-8 or cell death. c-Myc was neither found to induce any changes in the expression of TNF-related apoptosis inducing ligand (TRAIL) or its receptor. These data suggest that caspase-8 does not become activated through an extrinsic but an “intrinsic/intracellular” apoptotic pathway unleashed by the down-regulation of c-FLIP by c-Myc. Moreover, ectopic expression of c-FLIP_L inhibited the c-Myc-induced apoptosis.     

Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events.However,death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly,bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins.Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Simultaneous activation of apoptosis and inflammation in pathogenesis of septic shock: a hypothesis

Sepsis, a widely prevalent disease with increasing morbidity and mortality, is thought to result from uncontrolled inflammatory responses to microbial infection and/or components. However, failure of several experimental anti-inflammatory therapies has necessitated re-evaluation of the paradigm underlying the pathogenesis of this complex disorder. Apoptotic cell death forms a second dominant feature of septic shock in patients and animal models. Anti-apoptotic strategies may protect animals from septic death. However, simultaneous occurrence of apoptosis and inflammation is necessary for septic death. At the cellular level, apoptosis plays a central role in the development of the lymphoid system and regulation of immune responses. Immune activation renders cells refractory to apoptosis while apoptosis of activated lymphocytes is an important immunoregulatory mechanism. Factors such as complement factor 5a, caspase-1 and mitogen-activated protein kinase, which participate in apoptosis as well as pro-inflammatory pathways, may be responsible for simultaneous activation of apoptosis and inflammation in sepsis. Further identification of other similar biochemical events capable of co-activating inflammation and apoptosis may provide new targets for therapy of this hitherto untreatable disease.     

Activation of neutrophils by Chlamydia trachomatis-infected epithelial cells is modulated by the chlamydial plasmid

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial agent of sexually transmitted disease world-wide. Chlamydia trachomatis primarily infects epithelial cells of the genital tract but the infection may be associated with ascending infection. Infection-associated inflammation can cause tissue damage resulting in female infertility and ectopic pregnancy. The precise mechanism of inflammatory tissue damage is unclear but earlier studies implicate the chlamydial cryptic plasmid as well as responding neutrophils. We here rebuilt the interaction of Chlamydia trachomatis-infected epithelial cells and neutrophils in-vitro. During infection of human (HeLa) or mouse (oviduct) epithelial cells with Chlamydia trachomatis, a soluble factor was produced that attracted neutrophils and prolonged neutrophil survival, independently of Toll-like receptor signaling but dependent on the chlamydial plasmid. A number of cytokines, but most strongly GM-CSF, were secreted at higher amounts from cells infected with plasmid-bearing, compared to plasmid-deficient, bacteria. Blocking GM-CSF removed the secreted pro-survival activity towards neutrophils. A second, neutrophil TNF-stimulatory activity was detected in supernatants, requiring MyD88 or TRIF independently of the plasmid. The results identify two pro-inflammatory activities generated during chlamydial infection of epithelial cells and suggest that the epithelial cell, partly through the chlamydial plasmid, can initiate a myeloid immune response and inflammation.     

Induction of CD69 activation molecule on human neutrophils by GM-CSF,IFN-gamma,and IFN-alpha

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes but it expression is induced in vitro on cells of most hematopoietic lineages, including neutrophils after stimulation with PMA or fMLP. In this study, we investigated whether CD69 expression on human neutrophils could be modulated by inflammatory or anti-inflammatory cytokines (IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, G-CSF, GM-CSF, TNF-alpha, TGF-beta, IFN-alpha, IFN-gamma). Resting neutrophils from healthy subjects did not express CD69 on the cell surface; moreover, a preformed intracellular pool of CD69 was not evident in these cells. CD69 was barely detectable on these cells after overnight incubation in medium while overnight incubation with GM-CSF, IFN-gamma or IFN-alpha significantly induced CD69 expression on neutrophils with GM-CSF appearing to be the most potent inducer. This induction was dependent on a new protein synthesis as it was significantly inhibited by cycloheximide (about 50% inhibition). CD69 cross-linking on GM-CSF-primed neutrophils sinergized with LPS and increased TNF-alpha production and secretion suggesting a role for CD69-positive neutrophils in the pathogenesis and maintenance of different inflammatory diseases.     

RGD- and MLD-disintegrins, jarastatin and EC3, activate integrin-mediated signaling modulating the human neutrophils chemotaxis, apoptosis and IL-8 gene expression

The effects of jarastatin (JT), a monomeric RGD-disintegrin, were compared with those of the heterodimeric MLD-disintegrin, EC3, on human neutrophil activation and functions. Both disintegrins inhibited neutrophil chemotaxis induced by fMet-Leu-Phe and were also potent chemotactic agents. These effects were accompanied by an increase in actin polymerization, and both were inhibited by genistein, a tyrosine kinase inhibitor. While JT, but not other RGD-disintegrins, inhibited EC3-induced chemotaxis, EC3 was not able to inhibit JT effect. The chemotactic effect of JT was blocked by anti-alpha(M) antibody whereas anti-alpha(9)beta(1) inhibited EC3 effect. Both JT and EC3 induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Accordingly, LY294002, a PI3K inhibitor, impaired their chemotactic effect on neutrophils. JT induced Erk-2 translocation to nucleus and a delay of the spontaneous apoptosis of neutrophils in vitro. In contrast, EC3 inhibited Erk-2 activation and had a proapoptotic effect. These effects were reverted by PD98059, an MEK 1/2 inhibitor and blocked by z-VAD-FMK, a caspase inhibitor. In addition, JT, but not EC3, increased the IL-8 mRNA levels in neutrophils. The data indicate that JT and EC3 directly activate an integrin-coupled signaling and modulate the MAPK pathway in different ways, leading the neutrophils to express different functional response.     

The pro-inflammatory cytokine IL-18 is expressed in human adipose tissue and strongly upregulated by TNFalpha in human adipocytes

White adipocytes have been examined as a potential source of interleukin-18 (IL-18), the circulating levels of which are increased in obesity. IL-18 gene expression was evident in human subcutaneous and visceral adipose tissue, and expression occurred in mature adipocytes and the stromal-vascular fraction. Expression of the IL-18 receptor complex (IL-18Ralpha and IL-18Rbeta) and the IL-18 binding protein (IL-18BP) genes was also observed, mirroring that of IL-18. IL-18 mRNA level increased rapidly (within 2h) and dramatically (>900-fold) in response to TNFalpha in human adipocytes differentiated in culture. IL-18 protein was detected in lysates of cultured adipocytes, though not in the medium. There was a small increase in IL-18 in lysates of adipocytes treated with TNFalpha, but the protein was again undetectable in the medium. IL-18 may be part of the inflammatory cascade within adipose tissue; however, human adipocytes do not appear to secrete significant amounts of IL-18.     

Different signaling pathways stimulate a disintegrin and metalloprotease-17 (ADAM17) in neutrophils during apoptosis and activation

ADAM17 is a membrane-associated metalloprotease that cleaves proteins from the surface of neutrophils and modulates the density of various receptors and adhesion molecules. The protease activity of ADAM17 is highly inducible and occurs upon neutrophil activation as well as apoptosis. At this time, little is known about the signal transduction pathway that promotes ADAM17 activity in neutrophils upon the induction of apoptosis. We show that caspase-8 activation, Bid cleavage, and the release of mitochondrial reactive oxygen species are sequential transduction components of the Fas signaling cascade that induces ADAM17. This is different from ADAM17 stimulation upon overt neutrophil activation, which requires MAPK p38 or ERK, but not caspases and reactive oxygen species. ADAM17 activity in apoptotic neutrophils may serve to inactivate select effector molecules that promote the pro-inflammatory activity of recruited neutrophils. For instance, TNFα receptors TNF-RI and TNF-RII are substrates of ADAM17, and we show that they are shed during apoptosis, decreasing neutrophil sensitivity to TNFα. Altogether, our findings provide significant new insights into the signal transduction pathway that stimulates ADAM17 during induced neutrophil apoptosis. ADAM17 induction during apoptosis may rapidly diminish neutrophil sensitivity to the inflammatory environment, complementing other anti-inflammatory activities by these cells during inflammation resolution.     

Hesperidin ameliorates cisplatin induced hepatotoxicity and attenuates oxidative damage,cell apoptosis,and inflammation in rats

Cisplatin is one of the most widely used chemotherapeutic anti-cancer drugs that is associated with multiple systemic toxicities limiting its use. The present study aimed to evaluate the hepato-protective effect of hesperidin against cisplatin-induced toxicity. Thirty-two adult male albino rats were equally split into four groups, the first group served as control received normal saline, the second group (CIS) received a single intraperitoneal dose of cisplatin (7.5 mg/kg bw) on the 22nd day of the experiment, the third group (HES) treated once daily with hesperidin (200 mg/kg bw, orally) for 21 days, and the last group (HES + CIS) pretreated once daily with hesperidin followed by a single intraperitoneal dose of cisplatin. Twenty-four hours later, samples were collected for further investigations. CIS-intoxication resulted in a significant decrease in the erythrogram along with thrombocytopenia leukopenia, and lymphopenia. Furthermore, CIS administration significantly elevated serum activity of liver enzymes, total, and indirect bilirubin as well serum glucose, total cholesterol, and triglycerides levels, meanwhile serum total protein, and globulin levels were significantly reduced. The hepatic MDA was markedly elevated with a concomitant decline in the hepatic antioxidant enzymes and severe alterations in the hepatic tissue architecture in CIS-intoxicated rats. Additionally, CIS-induced overexpression of hepatic Bax, caspase-3, and TNF-α, with no effect on hepatic expression of IL-10. Interestingly, HES pretreatment improved the CIS-induced hemato-biochemical, molecular and histopathological alterations. In conclusion, hesperidin hepato-protective effects against CIS might be mediated by its antioxidant, anti-inflammatory, and anti-apoptotic properties.     

Death receptors, Fas and TRAIL receptors, are involved in human osteoclast apoptosis

Survival and apoptosis are crucial aspects of the osteoclast life cycle. Although osteoclast survival has been extensively studied, little is known about the mechanisms involved in human osteoclast apoptosis. In the present study, cord blood monocytes (CBMs) were used as the source of human osteoclast precursors. When cultured in the presence of M-CSF and RANKL, CBMs formed multinucleated cells that expressed RANK and calcitonin receptor, and were able to resorb bone. These cells expressed TRAIL receptors (R1-R4). Surprisingly, although TRAIL-receptor expression was not detectable in osteoclasts from normal bone, osteoclasts from myeloma specimens did express TRAIL receptors to a variable extent. Significantly, we have shown for the first time that this pathway is indeed functional in human osteoclasts, and that apoptosis occurred and was significantly greater in the presence of TRAIL. In addition, we have shown that a Fas-activating antibody is also able to induce osteoclast apoptosis, as did TGFbeta, whereas the survival factor M-CSF decreased apoptosis. Overall, these findings suggest that death receptors, TRAIL receptors and Fas, could be involved in osteoclast apoptosis in humans.     

Death-associated protein kinase 2: Regulator of apoptosis,autophagy and inflammation

Death-associated protein kinase 2 (DAPK2/DRP-1) belongs to a family of five related serine/threonine kinases that mediate a range of cellular processes, including membrane blebbing, apoptosis, and autophagy, and possess tumour suppressive functions. The three most conserved family members DAPK1/DAPK, DAPK2 and DAPK3/ZIPK share a high degree of homology in their catalytic domain, but differ significantly in their extra-catalytic structures and tissue-expression profiles. Hence, each orthologue binds to various unique interaction partners, localizes to different subcellular regions and controls some dissimilar cellular functions. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms that activate DAPK2 and that execute DAPK2-mediated apoptosis, autophagy and inflammation. In this “molecules in focus” review on DAPK2, the structure, modes of regulation and various cellular functions of DAPK2 will be summarized and discussed.     

Cytoglobin is a respiratory protein in connective tissue and neurons, which is up-regulated by hypoxia

Cytoglobin is a recently discovered vertebrate globin distantly related to myoglobin, and its function is unknown. Here we present the first detailed analysis of the distribution and expression of cytoglobin. Northern and Western blotting experiments show the presence of cytoglobin mRNA and protein in a broad range of tissues. Quantitative PCR demonstrates an up-regulation of cytoglobin mRNA levels in rat heart and liver under hypoxic conditions (22 and 44 h of 9% oxygen). Immunofluorescence studies with three antibodies directed against different epitopes of the protein consistently show cytoglobin in connective tissue fibroblasts as well as in hepatic stellate cells. Cytoglobin is also present in chondroblasts and osteoblasts and shows a decreased level of expression upon differentiation to chondrocytes and osteocytes. Cytoglobin is located in the cytoplasm of these cell types. Evidence against an exclusively nuclear localization of cytoglobin, as recently proposed, is also provided by transfection assays with green fluorescent protein fusion constructs, which demonstrates the absence of an active nuclear import. The differential expression of cytoglobin argues against a general respiratory function of this molecule, but rather indicates a connective tissue-specific function. We hypothesize that cytoglobin may be involved in collagen synthesis. Cytoglobin expression was also observed in some neuronal subpopulations of the central and the peripheral nervous systems. Surprisingly, cytoglobin is localized in both the cytoplasm and nucleus of neurons, indicating a possible additional role of this protein in neuronal tissues.