benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Inflammation

M1 macrophages, also known as inflammatory macrophages, play an important role in the innate and adaptative immune responses against pathogens. However, the overactivation of these macrophages leads to the development and progression of various inflammatory diseases. Thus, the regulation of these macrophages is necessary to prevent such diseases. Necroptosis, a programmed form of necrosis, induces several damage-associated molecular patterns, such as high-mobility group box 1, adenosine triphosphate, and mitochondrial DNA, which activate various immune cells, thus leading to inflammation. Recent studies have shown that necroptosis in M1 macrophages is associated with inflammation in many pathological conditions. However, the molecular mechanisms underlying necroptosis in M1 macrophages are not completely understood. Thus, we examined the effects of a broad-spectrum caspase inhibitor, zVAD-fmk, on cell death in lipopolysaccharide (LPS)-induced M1 macrophages. Necrostatin-1, an inhibitor of necroptosis, partially inhibited zVAD-fmk-induced cell death and phosphorylation of mixed lineage kinase domain-like protein (MLKL) in M1 macrophages. Moreover, the inhibition of generation of reactive oxygen species (ROS) and activation of p38 mitogen-activated protein kinase (MAPK) reduced zVAD-fmk-induced necroptosis in M1 macrophages. Furthermore, the inhibition of ROS generation suppressed the activation of MLKL and p38 MAPK in zVAD-fmk-treated M1 macrophages. These results indicate that zVAD-fmk-induced cell death occurs via necroptosis through ROS-mediated activation of MLKL and p38 MAPK in M1 macrophages. Unraveling the molecular mechanisms of necroptosis in M1 macrophages might help understand their significance in inflammatory diseases.

Topics: Amino Acid Chloromethyl Ketones; Animals; Humans; Inflammation; Macrophages; Necroptosis; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinases; Reactive Oxygen Species; Toll-Like Receptor 4

Smoking can lead to airway inflammation and mucus secretion through the nucleotide-binding domain-like receptor protein 3/caspase-1 pathway. In this study, z-VaD-Ala-Asp-fluoromethyl ketone(Z-VAD), a pan-caspase inhibitor, and acetyl-Asp-Glu-Val-Asp-aldehyde(Ac-DEVD), a caspase-3 inhibitor, were used to investigate the effect of caspase inhibitors on the expression of interleukin(IL)-1β and IL-8, airway inflammation, and mucus secretion in mice exposed to cigarette smoke(CS).. Thirty-two C57BL/6J male mice were divided into a control group, Smoke group, Z-VAD group, and Ac-DEVD group. Except for the control group, the animals were all exposed to CS for three months. After the experiment, lung function was measured and hematoxylin and eosin staining and periodic acid-Schiff staining were performed. The levels of IL-1β, IL-8, and mucin 5ac(Muc5ac) in serum and bronchoalveolar lavage fluid(BALF) were determined by enzyme-linked immunosorbent assay.. Compared with the control group, the lung function of mice exposed to smoke was poorer, with a large number of inflammatory cells infiltrating around the airway, collapse of alveoli, expansion and fusion of distal alveoli, and formation of emphysema. The Z-VAD group was relieved compared with the smoke group. Airway inflammation was also reduced in the Ac-DEVD group compared with the Smoke group, but the degree of emphysema was not significantly improved. Although Z-VAD relieved airway inflammation and emphysema, Ac-DEVD only relieved inflammation. Z-VAD and Ac-DEVD decreased serum IL-1β and IL-8 levels. In BALF, IL-1β was decreased in Z-VAD group and IL-8 was highest in Smoke +Ac-DEVD group compared with control group and Ac-DEVD group. There was no significant difference in the expression of Muc5ac in serum. However, in BALF, levels of Muc5ac were higher in the smoking group and the lowest in the Ac-DEVD group.. Mice exposed to smoke had decreased lung function and significant cilia lodging, epithelial cell shedding, and inflammatory cell infiltration, with significant emphysema formation. The pan-caspase inhibitor, Z-VAD, improved airway inflammation and emphysema lesions in the mice exposed to smoke and reduced IL-1β and IL-8 levels in serum. The caspase-3 inhibitor, Ac-DEVD, reduced airway inflammation, serum IL-1β and IL-8 levels, and Muc5ac levels in BALF, but it did not improve emphysema.

Topics: Animals; Caspase 3; Cigarette Smoking; Inflammation; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Mucus; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema

Both apoptosis and necroptosis have been recognized to be involved in ischemia reperfusion-induced lung injury. We aimed to compare the efficacies of therapies targeting necroptosis and apoptosis and to determine if there is a synergistic effect between the two therapies in reducing lung ischemia reperfusion injury.. Forty Sprague-Dawley rats were randomized into 5 groups: sham (SM) group, ischemia reperfusion (IR) group, necrostatin-1+ischemia reperfusion (NI) group, carbobenzoxy-Val-Ala-Asp-fluoromethylketone+ischemia reperfusion (ZI) group, and necrostatin-1+carbobenzoxy-Val-Ala-Asp-fluoromethylketone+ischemia reperfusion (NZ) group. The left lung hilum was exposed without being clamped in rats from the SM group, whereas the rats were subjected to lung ischemia reperfusion by clamping the left lung hilum for 1 hour, followed by reperfusion for 3 hours in the IR group. 1 mg/kg necrostatin-1 (Nec-1: a specific necroptosis inhibitor) and 3 mg/kg carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk: a pan caspase inhibitor) were intraperitoneally administrated prior to ischemia in NI and ZI groups, respectively, and the rats received combined administration of Nec-1 and z-VAD-fmk in the NZ group. Upon reperfusion, expressions of receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), and caspase-8 were measured, and the flow cytometry analysis was used to assess the cell death patterns in the lung tissue. Moreover, inflammatory marker levels in the bronchoalveolar lavage fluid and pulmonary edema were evaluated.. Both Nec-1 and z-VAD-fmk, either alone or in combination, significantly reduced morphological damage, inflammatory markers, and edema in lung tissues following reperfusion, and cotreatment of z-VAD-fmk with Nec-1 produced the optimal effect. The rats treated with Nec-1 had lower levels of inflammatory markers in the bronchoalveolar lavage fluid than those receiving z-VAD-fmk alone (. Nec-1 synergizes the pan caspase inhibitor to attenuate lung ischemia reperfusion injury in rats. Our data support the potential use of Nec-1 in lung transplantation-related disorders.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Bronchoalveolar Lavage Fluid; Caspase 8; Caspase Inhibitors; Cell Death; Flow Cytometry; HMGB1 Protein; Imidazoles; Indoles; Inflammation; Lung Injury; Male; Necrosis; Protein Serine-Threonine Kinases; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Receptor-Interacting Protein Serine-Threonine Kinases; Reperfusion Injury

Parkin is an E3 ubiquitin ligase involved in Parkinson's disease (PD). Necroptosis is a regulated form of cell death that depends on receptor interacting protein 1 (RIP1) and 3 (RIP3). Importantly, parkin has been implicated in ubiquitination events that can alter inflammation and necroptosis. Here, we investigated how parkin influences microglial function. Incubation of BV-2 microglial cells with zVAD.fmk (zVAD) induced high levels of cell death and viability loss, while N9 microglial cells and primary microglia required further stimuli. Importantly, necrostatin-1 (Nec-1), an inhibitor of RIP1 kinase activity, abrogated cell death, thus implicating RIP1-dependent necroptosis in cell death. Cell death was characterized by necrosome assembly, as determined by sequestration of RIP1/RIP3 in insoluble fractions and by MLKL phosphorylation, which were all abolished by Nec-1. Also, necroptosis-inducing conditions led to TNF-α secretion, which may in turn contribute to autocrine necroptosis activation. Interestingly, parkin knockdown protected BV-2 cells from zVAD-induced necroptosis, which may depend on the higher RIP1 ubiquitination levels detected in siRNA-PARK2 transfected cells. This effect was independent of inflammation, since pro-inflammatory stimulation of BV-2 and primary microglia with silenced parkin resulted in stronger pro-inflammatory gene expression, an opposite observation from zVAD-exposed BV-2 cells. LPS-mediated inflammation was exacerbated by NF-κB/JNK over-activation. Finally, no alterations in mitochondrial ROS production were detected in any condition, thereby excluding the role of parkin in mitophagy. In conclusion, here, we reveal that parkin may have unsuspected roles in microglia by modulating ubiquitination. Parkin loss exacerbates inflammation and promotes survival of activated microglia, thus contributing to chronic neuroinflammation.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Brain; Caspase Inhibitors; Cell Death; Cell Line; Cell Survival; Disease Progression; Gene Knockdown Techniques; Gene Silencing; GTPase-Activating Proteins; Inflammation; Lipopolysaccharides; Mice, Inbred C57BL; Microglia; Necrosis; Phosphorylation; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Ubiquitin-Protein Ligases

Alendronate (ALN) is a nitrogen-containing bisphosphonate (NBP) that inhibits bone resorption. NBPs have inflammatory side effects, and ALN augments bacteria-induced interleukin (IL)-1β production. The present study aimed to examine whether ALN induces pyroptosis, a form of cell death associated with IL-1β release, in macrophage-like J774.1 cells incubated with lipid A, a component of gram-negative bacteria. Pretreatment of J774.1 cells with ALN increased lipid A-induced IL-1β production and cell death, but not IL-6 and TNF-α production. Ac-YVAD-CHO, a caspase-1 inhibitor, inhibited ALN-augmented IL-1β production induced by lipid A, although it did not affect ALN-induced cell death. Moreover, Ac-IETD-CHO, a caspase-8 inhibitor, and Z-VAD-FMK, a pan-caspase inhibitor, did not inhibit ALN-induced cell death, suggesting that the effects of ALN are exerted independently of caspase activation. We also demonstrate that a Smad3 inhibitor (SIS3) suppressed ALN-augmented IL-1β production. Moreover, SIS3 attenuated ALN-augmented release of LDH and caspase-1. These results suggest that ALN augments IL-1β production, cell death, and caspase-1 release in a manner dependent on Smad3. We then investigated whether ALN-augmented IL-1β production and cell death are dependent on apoptosis-associated speck-like protein containing a CARD (ASC) and NOD-like receptor pyrin domain containing-3 (NLRP3), which are associated with Smad3 activation. Both anti-ASC and anti-NLRP3 antibodies suppressed ALN-induced cell death and caspase-1 release, but only anti-ASC antibody inhibited ALN-augmented IL-1β production. Our findings suggest that ALN-augmented IL-1β production and cell death require Smad3 and ASC activation, and that SIS3 and anti-ASC antibodies may serve as palliative agents for necrotizing inflammatory diseases caused by ALN.

Topics: Alendronate; Amino Acid Chloromethyl Ketones; Animals; CARD Signaling Adaptor Proteins; Caspase 1; Cell Death; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Inflammation; Interleukin-1beta; Interleukin-6; Lipid A; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Protein Domains; Smad3 Protein; Tumor Necrosis Factor-alpha

Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL).. The aim of this study is to examine in depth in vitro and ex vivo the contribution of necroptosis to intestinal inflammation.. In vitro: we used an intestinal cell line, HCT116RIP3, produced in our laboratory and overexpressing RIP3. Ex vivo: intestinal mucosal biopsies were taken from patients with inflammatory bowel disease (IBD) (20 with Crohn's disease; 20 with ulcerative colitis) and from 20 controls.. RIP3-induced necroptosis triggers MLKL activation, increases cytokine/alarmin expression (IL-8, IL-1β, IL-33, HMGB1), NF-kBp65 translocation and NALP3 inflammasome assembly. It also affects membrane permeability by altering cell-cell junctional proteins (E-cadherin, Occludin, Zonulin-1). Targeting necroptosis through Necrostatin-1 significantly reduces intestinal inflammation in vitro and in cultured intestinal explants from IBD.. We show for the first time in vitro and ex vivo that RIP3-driven necroptosis seriously affects intestinal inflammation by increasing pMLKL, activating different cytokines and alarmins, and altering epithelial permeability. The inhibition of necroptosis causes a significant decrease of all these effects. These data strongly support the view that targeting necroptosis may represent a promising new option for the treatment of inflammatory enteropathies.

Topics: Adolescent; Amino Acid Chloromethyl Ketones; Apoptosis; Cadherins; Caspase 1; Cell Adhesion; Cell Membrane Permeability; Cell Survival; Child; Child, Preschool; Colitis, Ulcerative; Crohn Disease; Epithelial Cells; HCT116 Cells; HMGB1 Protein; Humans; Imidazoles; Indoles; Inflammasomes; Inflammation; Interleukin-1beta; Interleukin-8; Intestinal Mucosa; Necrosis; NLR Family, Pyrin Domain-Containing 3 Protein; Phosphorylation; Protein Kinases; Protein Transport; Receptor-Interacting Protein Serine-Threonine Kinases; RNA, Messenger; Transcription Factor RelA; Tumor Necrosis Factor-alpha

To elucidate the role of reactive oxygen species (ROS) in arthritis and to identify targets of arthritis treatment in conditions with different levels of oxidant stress.. Through establishing an arthritis model by injecting arthritogenic serum into wild-type and NADPH oxidase 2 (NOX2)-deficient mice, we found that arthritis had a neutrophilic infiltrate and was more severe in Ncf1(-/-) mice, a mouse strain lacking the expression of the NCF1/p47(phox) component of NOX2. The levels of interleukin-1β (IL-1β) and IL-6 in inflamed joints were higher in Ncf1(-/-) than in controls. Antagonists of tumor necrosis factor-α (TNFα) and IL-1β were equally effective in suppressing arthritis in wild-type mice, while IL-1β blockade was more effective than TNFα blockade in Ncf1(-/-) mice. A treatment of caspase inhibitor and the combination treatment of a caspase inhibitor and a cathepsin inhibitor, but not a cathepsin inhibitor alone, suppressed arthritic severity in the wild-type mice, while a treatment of cathepsin inhibitor and the combination treatment of a caspase inhibitor and a cathepsin inhibitor, but not a caspase inhibitor alone, were effective in treating Ncf1(-/-) mice. Consistently, cathepsin B was found to proteolytically process pro-IL-1β to its active form and this activity was suppressed by ROS.. This novel mechanism of a redox-mediated immune regulation of arthritis through leukocyte-produced ROS is important for devising an optimal treatment for patients with different levels of tissue ROS.. Our results suggest that ROS act as a negative feedback to constrain IL-1β-mediated inflammation, accounting for the more severe arthritis in the absence of NOX2.

Topics: Amino Acid Chloromethyl Ketones; Animals; Ankle Joint; Arthritis; Caspase Inhibitors; Cathepsin B; Cell Line; Cytokines; Dipeptides; Disease Models, Animal; Fibroblasts; Humans; Inflammation; Interleukin-1beta; Ketones; Lung; Membrane Glycoproteins; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidases; Oxidation-Reduction; Reactive Oxygen Species; Wrist Joint

NLR family pyrin domain containing 3 (NLRP3) is a cytoplasmic pattern recognition receptor that regulates innate immune responses by forming a protein complex, the inflammasome. It leads to production of proinflammatory cytokine productions such as interleukin 1β (IL-1β). We and others demonstrated that an induction of activated NLRP3 also induced cell death. However, little is known about the characteristics and mechanisms of the cell death and its involvement in the pathogenesis of inflammatory conditions. In this study, we established cell lines in which NLRP3 was induced by doxycycline using a tetracycline-inducible expression (Tet-on) system. Using this system, the expression of NLRP3 mutants in cryopyrin-associated periodic syndrome (CAPS) patients was sufficient for the induction of necrotic cell death without lipopolysaccharide stimulation or generation of mature IL-1β. We also found that CA074-Me, a cathepsin B inhibitor, blocked cell death before oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), whereas Z-VAD-fmk, a pan-caspase inhibitor, blocked the cell death after the oligomerization. Silencing of the ASC gene (Pycard) by small hairpin RNA treatment inhibited the NLRP3 mutant-induced cell death, but silencing of the caspase-1 gene (Casp1) did not. Taken together, these results indicated that ASC was indispensable for NLRP3-mediated programmed necrotic cell death, and that this type of cell death was distinct from 'pyroptosis', which requires caspase-1. Finally, we demonstrated in an in vivo model that the programmed necrotic cell death induced by activated NLRP3 could cause neutrophil infiltration, indicating a possible role of cell death in neutrophil infiltration of skin lesions in CAPS patients.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; CARD Signaling Adaptor Proteins; Carrier Proteins; Caspase 1; Caspase Inhibitors; Cytoskeletal Proteins; Dipeptides; Doxycycline; Humans; Inflammation; Interleukin-1beta; Mice; Necrosis; Neutrophils; NLR Family, Pyrin Domain-Containing 3 Protein; RNA Interference; RNA, Small Interfering

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)-associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non-tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H2 O2 ). A population of HCEC survived H2 O2 -induced oxidative stress via JNK-dependent cell cycle arrests. Caspases, p21(WAF1) and γ-H2AX were identified as JNK-regulated proteins. Up-regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan-caspase inhibitor Z-VAD-FMK caused up-regulation of γ-H2AX, a DNA-damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G1 -phase as first response to oxidative stress and increased S-phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non-apoptotic function by promoting cells through G1 - and S-phase by overriding the G1 /S- and intra-S checkpoints despite DNA-damage. This led to the accumulation of cells in the G2 /M-phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ-H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase-dependent proteolytic degradation of the DNA-damage checkpoint protein ATM that is upstream of γ-H2AX. As a consequence, undetected DNA-damage and increased proliferation were found in repeatedly H2 O2 -exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non-apoptotic function of caspases. Overexpression of upstream p-JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Caspases; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Colitis; Colon; Comet Assay; DNA Damage; Epithelial Cells; Histones; Humans; Hydrogen Peroxide; Immunohistochemistry; Inflammation; Inflammatory Bowel Diseases; MAP Kinase Kinase 4; Oxidative Stress; Reactive Oxygen Species; Subcellular Fractions

Caspases are intracellular cysteine-class proteases with aspartate specificity that is critical for driving processes as diverse as the innate immune response and apoptosis, exemplified by caspase-1 and caspase-3, respectively. Interestingly, caspase-1 cleaves far fewer cellular substrates than caspase-3 and also shows strong positive cooperativity between the two active sites of the homodimer, unlike caspase-3. Biophysical and kinetic studies here present a molecular basis for this difference. Analytical ultracentrifugation experiments show that mature caspase-1 exists predominantly as a monomer under physiological concentrations that undergoes dimerization in the presence of substrate; specifically, substrate binding shifts the KD for dimerization by 20-fold. We have created a hemi-active site-labeled dimer of caspase-1, where one site is blocked with the covalent active site inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. This hemi-labeled enzyme is about 9-fold more active than the apo-dimer of caspase-1. These studies suggest that substrate not only drives dimerization but also, once bound to one site in the dimer, promotes an active conformation in the other monomer. Steady-state kinetic analysis and modeling independently support this model, where binding of one substrate molecule not only increases substrate binding in preformed dimers but also drives the formation of heterodimers. Thus, the cooperativity in caspase-1 is driven both by substrate-induced dimerization as well as substrate-induced activation. Substrate-induced dimerization and activation seen in caspase-1 and not in caspase-3 may reflect their biological roles. Whereas caspase-1 cleaves a dramatically smaller number of cellular substrates that need to be concentrated near inflammasomes, caspase-3 is a constitutively active dimer that cleaves many more substrates located diffusely throughout the cell.

Topics: Allosteric Site; Amino Acid Chloromethyl Ketones; Area Under Curve; Biophysics; Caspase 1; Caspase 3; Caspases; Catalytic Domain; Dimerization; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Inflammation; Kinetics; Models, Molecular; Molecular Conformation; Protein Binding; Protein Conformation; Substrate Specificity; Ultracentrifugation

Fas-mediated caspase-dependent cell apoptosis has been well investigated. However, recent studies have shown that Fas can induce nonapoptotic caspase-independent cell death (CICD) when caspase activity is inhibited. Currently, the molecular mechanism of this alternative cell death mediated by Fas remains unclear. In this study, we investigated the signaling pathway of Fas-induced CICD in mouse embryonic fibroblasts (MEFs) whose caspase function was disrupted by the pan-caspase inhibitor Z-VAD-FMK and its coupling to inflammatory responses. Our results revealed that receptor-interacting protein 1 and tumor necrosis factor receptor-associated factor 2 play important roles in FasL-induced CICD. This death is associated with intracellular reactive oxygen species (ROS) production from mitochondria, as a ROS scavenger (BHA), antioxidants (trolox, NAC), and a mitochondrial respiratory chain uncoupler (rotenone) could prevent this event. Furthermore, delayed and sustained JNK activation, mitochondrial membrane potential breakdown, and loss of intracellular GSH were observed. In addition to CICD, FasL also induces cyclooxygenase-2 and MIP-2 gene upregulation, and both responses are attributed to ROS-dependent JNK activation. Taken together, these results demonstrate alternative signaling pathways of Fas upon caspase inhibition in MEFs that are unrelated to the classical apoptotic pathway, but steer cells toward necrosis and an inflammatory response through ROS production.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Line; Chemokine CXCL2; Chromans; Cyclooxygenase 2; Fas Ligand Protein; fas Receptor; Fibroblasts; Gene Expression Regulation; Inflammation; Membrane Potential, Mitochondrial; Mice; Mitochondria; Reactive Oxygen Species; Signal Transduction

The IgA Fc receptor (FcalphaRI) has dual proinflammatory and anti-inflammatory functions that are transmitted through the immunoreceptor tyrosine-based activation motifs (ITAMs) of the associated FcRgamma subunit. Whereas the involvement of FcalphaRI in inflammation is well documented, little is known of its anti-inflammatory mechanisms. Here we show that monomeric targeting of FcalphaRI by anti-FcalphaRI Fab or serum IgA triggers apoptosis in human monocytes, monocytic cell lines, and FcalphaRI+ transfectants. However, the physiologic ligand IgA induced apoptosis only when cells were cultured in low serum conditions, indicating differences with induction of anti-inflammatory signaling. Apoptosis signaling required the FcRgamma ITAM, as cells transfected with FcalphaRI or with a chimeric FcalphaRI-FcRgamma responded to death-activating signals, whereas cells expressing a mutated FcalphaRI(R209L) unable to associate with FcRgamma, or an ITAM-mutated chimeric FcalphaRI-FcRgamma, did not respond. FcalphaRI-mediated apoptosis signals were blocked by treatment with the pan-caspase inhibitor zVAD-fmk, involved proteolysis of procaspase-3, and correlated negatively with SHP-1 concentration. Anti-FcalphaRI Fab treatment of nude mice injected subcutaneously with FcalphaRI+ mast-cell transfectants prevented tumor development and halted the growth of established tumors. These findings demonstrate that, on monomeric targeting, FcalphaRI functions as an FcRgamma ITAM-dependent apoptotic module that may be fundamental for controlling inflammation and tumor growth.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Motifs; Animals; Antigens, CD; Apoptosis; Caspase 3; Cell Line, Tumor; Cells, Cultured; Culture Media, Serum-Free; Cysteine Proteinase Inhibitors; Enzyme Activation; Female; Humans; Immunoglobulin A; Immunoglobulin Fab Fragments; Inflammation; Leukemia, Basophilic, Acute; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Neoplasms; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Rats; Receptors, Fc; Receptors, IgG; Recombinant Fusion Proteins; RNA, Small Interfering; Skin Transplantation; Transfection

The aim of this study was to investigate the effects of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketones (z-VAD-fmk) on the degree of experimental spinal cord trauma induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of a range of inflammatory mediators, tissue damage, and apoptosis. Treatment of the mice with z-VAD-fmk, a potent broad specific caspase inhibitor, significantly reduced the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation, and (4) apoptosis (TUNEL staining and Bax and Bcl-2 expression). In a separate set of experiments, z-VAD-fmk significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with z-VAD-fmk reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Humans; In Situ Nick-End Labeling; Inflammation; Male; Mice; Neuroprotective Agents; Neutrophils; Peroxidase; Proto-Oncogene Proteins c-bcl-2; Spinal Cord Injuries

Macrophages are known to be one of the initial responders to bacterial infection. While infection of macrophages with bacteria induces apoptosis, a pro-inflammatory response is also elicited. Thus, the aim of this study is to further elucidate the differential effect of infections with bacteria on the survival and function of macrophages.. THP-1 monocytic cells induced to differentiate into macrophages were infected with non-pathogenic Escherichia coli (E. coli) and analyzed for apoptosis and inflammatory response over time. Following infection with E. coli macrophages underwent apoptosis which was reduced by the general caspase inhibitor, zVAD.fmk. Inhibition of caspase activity resulted in increased DNA binding activity of NF-kappaB and enhanced production of NF-kappaB-dependent reporter gene expression following infection. Increased activity of NF-kappaB was independent of IkappaBalpha since IkappaBalpha degradation was unaffected by zVAD.fmk. Further, suppression of caspase activity reduced the proteolytic cleavage of NF-kappaB. The increased activity of NF-kappaB in the zVAD.fmk-treated macrophages was associated with a markedly enhanced production of pro-inflammatory cytokines and elimination of E. coli.. These data indicate that infection of macrophages with E. coli induces a caspase-dependent inhibition of NF-kappaB that results in a reduced production of pro-inflammatory cytokines and impaired clearance of bacteria.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspases; Cell Line; Enzyme Activation; Escherichia coli; Humans; Inflammation; Macrophages; Monocytes; NF-kappa B

Neutrophils represent an important source of autoantigens for antineutrophil cytoplasmic antibody associated with vasculitis. To date, two cytoskeletal proteins, vinculin and vimentin, have been reported to be expressed on the cell surfaces of activated macrophages, platelets, and apoptotic T lymphocytes. However, such cell surface expression has never been studied in human neutrophils. As we recently demonstrated that different cytoskeletal proteins were cleaved in apoptotic neutrophils, we hypothesized that some of these were expressed on the cell surface of apoptotic neutrophils. Herein, we found that among vinculin, paxillin, gelsolin, vimentin, lamin B1, alpha-tubulin, and beta-tubulin, only the two intermediate filament (INFIL) proteins, vimentin and lamin B1, are expressed on the cell surface of 24-h aged neutrophils [spontaneous apoptosis (SA)]. By monitoring intracellular expression of vimentin and lamin B1 during SA, we found that these two proteins were cleaved and that such cleavage was reversed by the pan caspase inhibitor N-benzyloxy-carbonyl-V-A-D-O-methylfluoromethyl ketone (z-VAD-fmk). When neutrophil apoptosis was delayed or suppressed by lipopolysaccharide or the cytokines granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage (GM)-CSF, or interleukin-4, the loss of intracellular expression of vimentin and lamin B1 was prevented. The INFIL proteins were absent from the cell surface when neutrophil apoptosis was delayed. Addition of z-VAD-fmk significantly decreased the cell surface expression of vimentin and lamin B1 during SA. This study provides the first evidence that apoptotic neutrophils express cytoskeletal proteins on their surface, opening the possibility that these cells may participate in the development of autoantibodies directed against cytoskeletal proteins, a condition frequently reported in several inflammatory diseases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Autoantibodies; Autoimmune Diseases; Caspase Inhibitors; Caspases; Cell Membrane; Cells, Cultured; Down-Regulation; Enzyme Inhibitors; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Lamin Type B; Lipopolysaccharides; Membrane Proteins; Neutrophils; Vimentin

Caspases are the main executioners of apoptosis as well as interleukin (IL)-1beta and IL-18 conversion to active forms. They are activated after acute kidney injuries. In this study, we evaluated the importance of the caspase family in the pathogenesis and recovery of glycerol-induced acute renal failure in rats (Gly-ARF). Rats were treated with pan-caspase or selective caspase 1 and 3 inhibitors at the moment we injected glycerol. Renal function, renal histology (HE), transferase-mediated deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining for apoptosis, leukocytes infiltration (immunohistochemistry), renal expression of IL-1beta and IL-18 (immunohistochemistry and Western blot), tubular regeneration (5-bromo-2'-deoxyuridine (BrdU) incorporation), and P27(Kip) expression (Western blot) were evaluated at appropriate times. All inhibitors reduced the renal function impairment. Pan-caspase and caspase-3 inhibitors reduced cellular death (necrosis and apoptosis) 24 h after Gly-ARF. All caspases inhibitors reduced macrophages infiltration. The expression of total IL-1beta was enhanced in Gly-ARF, but the active IL-1beta and IL-18 forms were abolished in pan-caspase treated rats. Caspase-1 inhibitor attenuated Gly-ARF but not tubular injury suggesting glomerular hemodynamic improvement. There was striking regenerative response 48 h after Gly-ARF characterized by enhanced BrdU incorporation and reduced expression of p27(Kip). This response was not blunted by caspases inhibition. Our findings demonstrate that caspases participate in important pathogenic mechanisms in Gly-ARF such as inflammation, apoptosis, vasoconstriction, and tubular necrosis. The early inhibition of caspases attenuates these mechanisms and reduces the renal function impairment in Gly-ARF.

Topics: Acute Kidney Injury; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Western; Caspase Inhibitors; Caspases; Cell Cycle; Cell Death; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Activation; Enzyme Inhibitors; Glycerol; Immunohistochemistry; Inflammation; Interleukin-1; Interleukin-18; Male; Rats; Rats, Wistar

Statins, which inhibit 3-hydroxy-3-methylglutaryl CoA reductase, have been shown recently to promote proinflammatory responses. We show in this study that both atorvastatin and simvastatin induced proinflammatory responses in mitogen-activated PBMCs by increasing the number of T cells secreting IFN-gamma. This is abolished by the presence of mevalonate, suggesting that statins act specifically by blocking the mevalonate pathway for cholesterol synthesis to promote the proinflammatory response. Both statins at low concentrations induced a dose-dependent increase in the number of IFN-gamma-secreting T cells in mitogen-activated PBMCs, whereas at higher concentrations the effect was abolished. The proinflammatory effect of statins was not seen in purified T cells per se activated with mitogen. However, conditioned medium derived from statin-treated PBMCs enhanced the number of IFN-gamma-secreting cells in activated purified T cells. This effect was not blocked by mevalonate, but was abolished by neutralizing Abs to IL-18 and IL-12. Similarly, the up-regulation of IFN-gamma-secreting T cells in PBMCs costimulated with statins and mitogens was blocked by the neutralizing anti-IL-18 and anti-IL-12. We showed that simvastatin stimulates the secretion of IL-18 and IL-1beta in monocytes. Active caspase-1, which is required for the processing and secretion of IL-18 and IL-1beta, was activated in simvastatin-treated monocytes. This was blocked by mevalonate and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone. Taken together, the proinflammatory response mediated by statins in activated PBMCs is mediated mainly via the activation of caspase-1 and IL-18 secretion in the monocytes and to a lesser extent by IL-12.

Topics: Amino Acid Chloromethyl Ketones; Antibodies; Atorvastatin; Caspase 1; CD28 Antigens; CD3 Complex; Cells, Cultured; Enzyme Activation; Heptanoic Acids; Humans; Inflammation; Interferon-gamma; Interleukin-1; Interleukin-12; Interleukin-18; Mevalonic Acid; Mitosis; Monocytes; Pyrroles; Simvastatin; Solubility; T-Lymphocytes

Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Azepines; Carrageenan; Caspase 3; Cyclin-Dependent Kinases; Enzyme Inhibitors; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Male; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Neutrophils; Pleurisy; Proto-Oncogene Proteins c-bcl-2; Purines; Pyrroles; Roscovitine

Inflammatory mediators delay neutrophil apoptosis, which contributes to the persistence of inflammation. The mechanisms responsible for this delay and resistance to Fas antibody-induced apoptosis are unknown but are dependent on protein synthesis. These proteins have been shown to inhibit caspase activity central to the induction of apoptosis. The inhibitors of apoptosis proteins have been shown to inhibit caspase activity and prevent apoptosis in a number of cellular systems. We hypothesize that the regulation of neutrophil apoptosis is dependent on the expression of the IAPs. c-IAP-1, c-IAP-2, and XIAP are expressed in the neutrophil at both the mRNA and protein level, but their relative protein expression is low compared with other cell types. The in vitro aging of human neutrophils results in their induction of apoptosis, which is associated with the loss of c-IAP-1 expression. The pancaspase inhibitor (zVAD-FMK) and LPS, which delay spontaneous apoptosis, also prevented this loss of c-IAP-1. Gene chip microarrays have shown that LPS increases c-IAP-1 and c-IAP-2 mRNA expression in neutrophils. However, this does not correspond to an increase in protein. Neutrophils from septic patients with delayed apoptosis show an increase in XIAP, with no change in cIAP-1 or cIAP-2 mRNA, demonstrating that different mechanisms contribute to the delay in neutrophil apoptosis. This study demonstrates that the loss of IAP expression may facilitate the induction of neutrophil apoptosis, and preventing this loss of IAP expression may represent a more significant contribution to delayed apoptosis rather than an increase in their expression.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Cells, Cultured; Cysteine Proteinase Inhibitors; Gene Expression Profiling; Humans; Inflammation; Inhibitor of Apoptosis Proteins; Lipopolysaccharides; Neutrophils; Polymerase Chain Reaction; Proteins; Reference Values; Sepsis; X-Linked Inhibitor of Apoptosis Protein

Asthma is characterized by acute and chronic airway inflammation, and the severity of the airway hyperreactivity correlates with the degree of inflammation. Many of the features of lung inflammation observed in human asthma are reproduced in OVA-sensitized/challenged mice. T lymphocytes, particularly Th2 cells, are critically involved in the genesis of the allergic response to inhaled Ag. In addition to antiapoptotic effects, broad-spectrum caspase inhibitors inhibit T cell activation in vitro. We investigated the effect of the broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), on airway inflammation in OVA-sensitized/challenged mice. OVA-sensitized mice treated with z-VAD-fmk immediately before allergen challenge showed marked reduction in inflammatory cell infiltration in the airways and pulmonary blood vessels, mucus production, and Th2 cytokine production. We hypothesized that the caspase inhibitor prevented T cell activation, resulting in the reduction of cytokine production and eosinophil infiltration. Treatment with z-VAD-fmk in vivo prevented subsequent T cell activation ex vivo. We propose that caspase inhibitors may offer a novel therapeutic approach to T cell-dependent inflammatory airway diseases.

Topics: Aerosols; Allergens; Amino Acid Chloromethyl Ketones; Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Caspase Inhibitors; Cell Movement; Cysteine Proteinase Inhibitors; Disease Models, Animal; Inflammation; Interleukin-4; Interleukin-5; Intubation, Intratracheal; Leukocytes; Lung; Lymphocyte Activation; Methacholine Chloride; Mice; Mice, Inbred BALB C; Ovalbumin; T-Lymphocytes

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.

Topics: Amino Acid Chloromethyl Ketones; Antigens, CD; Apoptosis; Butadienes; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; fas Receptor; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrogen-Ion Concentration; Imidazoles; Inflammation; Interferon-gamma; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Necrosis; NF-kappa B; Nitriles; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Pyridines; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; T-Lymphocytes; Tumor Necrosis Factor-alpha

Dendritic cells (DC) are professional antigen-presenting cells playing a pivotal role in the induction of immunological responses. There is evidence that DC survival during ongoing immune responses is finite. However, little is known about the mechanisms regulating apoptosis in these cells. Here, we have investigated the effects of the anti-inflammatory cyclopentenone prostaglandins on human monocyte-derived DC.. Phenotype of DC was determined by flow cytometry and their allostimulatory potential in mixed leukocyte reaction. Induction of apoptosis in DC was monitored by staining with annexin-V-FITC and propidium iodide, propidium iodide staining of cell nuclei, and fluorimetric assay of caspase activity. Induction of maturation in DC was obtained by stimulation with TNF-alpha, LPS, IFN-gamma, CD40-ligand, or different combinations of these stimuli. PPAR-gamma expression in DC was determined by RT-PCR.. Exposure of immature DC to cyclopentenone prostaglandins blunted their allostimulatory capacity and skewed their phenotype by downregulating CD1a and costimulatory molecules. These effects were due to activation of caspases and induction of apoptotic cell death in DC by cyclopentenone prostaglandins. Mature DC showed enhanced susceptibility to apoptosis via cyclopentenone prostaglandins as compared with immature DC. Although DC express PPAR-gamma, the corresponding receptor for some of these metabolites, PPAR-gamma activation by a synthetic high-affinity agonist failed to impair DC viability.. Cyclopentenone prostaglandins induce apoptosis of human DC by a PPAR-gamma-independent mechanism. Since these compounds are released during an inflammatory event and show anti-inflammatory properties, they may contribute to the downregulation of DC function through apoptotic cell death.

Topics: Amino Acid Chloromethyl Ketones; Antigens, CD1; Apoptosis; Caspases; CD40 Ligand; Cell Differentiation; Cells, Cultured; Cyclooxygenase 2; Cysteine Proteinase Inhibitors; Dendritic Cells; Dinoprostone; Drug Resistance; Enzyme Activation; Enzyme Inhibitors; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Interferon-gamma; Interleukin-4; Isoenzymes; Lipopolysaccharides; Membrane Proteins; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Prostaglandins A; Receptors, Cytoplasmic and Nuclear; T-Lymphocytes; Transcription Factors; Tumor Escape; Tumor Necrosis Factor-alpha

African swine fever (ASF) is an asymptomatic infection of warthogs and bushpigs, which has become an emergent disease of domestic pigs, characterized by hemorrhage, lymphopenia, and disseminated intravascular coagulation. It is caused by a large icosohedral double-stranded DNA virus, African swine fever virus (ASFV), with infection of macrophages well characterized in vitro and in vivo. This study shows that virulent isolates of ASFV also infect primary cultures of porcine aortic endothelial cells and bushpig endothelial cells (BPECs) in vitro. Kinetics of early and late gene expression, viral factory formation, replication, and secretion were similar in endothelial cells and macrophages. However, ASFV-infected endothelial cells died by apoptosis, detected morphologically by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and nuclear condensation and biochemically by poly(ADP-ribose) polymerase (PARP) cleavage at 4 h postinfection (hpi). Immediate-early proinflammatory responses were inhibited, characterized by a lack of E-selectin surface expression and interleukin 6 (IL-6) and IL-8 mRNA synthesis. Moreover, ASFV actively downregulated interferon-induced major histocompatibility complex class I surface expression, a strategy by which viruses evade the immune system. Significantly, Western blot analysis showed that the 65-kDa subunit of the transcription factor NF-kappaB, a central regulator of the early response to viral infection, decreased by 8 hpi and disappeared by 18 hpi. Both disappearance of NF-kappaB p65 and cleavage of PARP were reversed by the caspase inhibitor z-VAD-fmk. Interestingly, surface expression and mRNA transcription of tissue factor, an important initiator of the coagulation cascade, increased 4 h after ASFV infection. These data suggest a central role for vascular endothelial cells in the hemorrhagic pathogenesis of the disease. Since BPECs infected with ASFV also undergo apoptosis, resistance of the natural host must involve complex pathological factors other than viral tropism.

Topics: African Swine Fever Virus; Amino Acid Chloromethyl Ketones; Animals; Aorta; Apoptosis; Cell Line; Cytokines; E-Selectin; Endothelium, Vascular; Histocompatibility Antigens Class I; Inflammation; Interferon-alpha; NF-kappa B; Swine; Thrombosis

The effect of apoptosis inhibitors on experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, was investigated by intraperitoneal or intracisternal administration of apoptosis inhibitors Ac-YVAD-cmk and zVAD-fmk. After onset of the disease, these agents had no suppressive effect on EAE and resulted in impaired recovery or earlier relapse. Histological examination revealed that administration of zVAD-fmk suppressed the apoptotic death of inflammatory cells in the central nervous system (CNS) of mice with EAE. The results indicated that the apoptotic elimination of infiltrated cells in the CNS might be one of the recovery mechanisms in EAE.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Encephalomyelitis, Autoimmune, Experimental; Female; Inflammation; Injections, Intraperitoneal; Injections, Spinal; Mice; Mice, Inbred Strains; Neuroprotective Agents; Spinal Cord

Inflammatory processes may play an important role in the degeneration of basal forebrain cholinergic cells Alzheimer's disease. We infused the proinflammagen lipopolysaccharide into the basal forebrain of young rats and determined whether the chronic administration of two novel non-steroidal anti-inflammatory drugs or a pan-caspase synthesis inhibitor, z-Val-Ala-Asp(OMe)-fluoromethyl ketone (zVAD), could provide neuroprotection from the cytotoxic effects of the neuroinflammation. Chronic lipopolysaccharide infusions decreased choline acetyltransferase activity and increased the number of activated microglia within the basal forebrain region. The level of caspases 3, 8 and 9 was increased in ventral caudate/putamen. Non-steroidal anti-inflammatory drug therapy attenuated the toxicity of the inflammation upon cholinergic cells and reduced caspases 3, 8 and 9 activity in the caudate/putamen. zVAD treatment significantly decreased the levels of caspases 3, 8 and 9 but did not provide neuroprotection for the cholinergic neurons. These results suggest that prostaglandins contribute to the degeneration of forebrain cholinergic neurons in Alzheimer's disease.

Topics: Amino Acid Chloromethyl Ketones; Animals; Anti-Inflammatory Agents, Non-Steroidal; Body Weight; Caspase Inhibitors; Choline O-Acetyltransferase; Enzyme Inhibitors; Flurbiprofen; Immunohistochemistry; Inflammation; Lipopolysaccharides; Male; Neurons; Parasympathetic Nervous System; Prosencephalon; Rats; Rats, Inbred F344