Valérie Lavastre

Mechanisms Involved in Spontaneous and Viscum album Agglutinin-I-Induced Human Neutrophil Apoptosis: Viscum album Agglutinin-I Accelerates the Loss of Antiapoptotic Mcl-1 Expression and the Degradation of Cytoskeletal Paxillin and Vimentin Proteins Via Caspases

Viscum album agglutinin-I (VAA-I) is a plant lectin that possesses interesting potential therapeutic properties and immunomodulatory activities. We have recently found that VAA-I is a potent inducer of human neutrophil apoptosis, but the mechanism(s) involved require further elucidation. In this study, we found that VAA-I alters mitochondrial transmembrane potential and increases intracellular levels of reactive oxygen species (ROS). Despite these observations, treatment with the mitochondrial stabilizer, bongkrekic acid, or with catalase, known to degrade H2O2, fails to reverse VAA-I-induced apoptosis. Moreover, VAA-I was found to induce apoptosis in PLB-985 cells deficient in gp91phox, indicating that the lectin acts via an ROS-independent mechanism. Pretreatment of neutrophils with brefeldin A, an inhibitor of vesicular transport, was found to reverse VAA-I-induced apoptosis. Protein expression of Mcl-1 was decreased by VAA-I. The role of caspases in the degradation of cytoskeletal proteins during both spontaneous and VAA-I-induced neutrophil apoptosis was also investigated. Paxillin and vimentin were markedly degraded by VAA-I when compared with neutrophils that undergo spontaneous apoptosis, but not vinculin or α- and β-tubulin. Caspases were involved in cytoskeletal protein degradation because preincubation with the pan-caspase inhibitor N-benzyloxycarbonyl-V-A-D-O-methylfluoromethyl ketone was found to reverse protein cleavage. We conclude that VAA-I needs to be internalized to mediate apoptosis and that its activity is not dependent on a cell surface receptor-mediated pathway. Also, we conclude that VAA-I induces apoptosis by ROS-independent and Mcl-1-dependent mechanisms and that caspases are involved in cytoskeletal protein degradation in both spontaneous and VAA-I-induced neutrophil apoptosis.

Silver nanoparticles induce degradation of the endoplasmic reticulum stress sensor activating transcription factor-6 leading to activation of the NLRP-3 inflammasome.

Background: Some nanoparticles are known to induce endoplasmic reticulum (ER) stress and lead to cell death. Results: Silver nanoparticles induce ATF-6 degradation, leading to activation of the NLRP-3 inflammasome and pyroptosis. Conclusion: ATF-6 is an important target to silver nanoparticles. Significance: Our results provide a new link between ER stress and activation of the NLRP-3 inflammasome. In the past decade, the increasing amount of nanoparticles (NP) and nanomaterials used in multiple applications led the scientific community to investigate the potential toxicity of NP. Many studies highlighted the cytotoxic effects of various NP, including titanium dioxide, zinc oxide, and silver nanoparticles (AgNP). In a few studies, endoplasmic reticulum (ER) stress was found to be associated with NP cytotoxicity leading to apoptosis in different cell types. In this study, we report for the first time that silver nanoparticles of 15 nm (AgNP15), depending on the concentration, induced different signature ER stress markers in human THP-1 monocytes leading to a rapid ER stress response with degradation of the ATF-6 sensor. Also, AgNP15 induced pyroptosis and activation of the NLRP-3 inflammasome as demonstrated by the processing and increased activity of caspase-1 and secretion of IL-1β and ASC (apoptosis-associated speck-like protein containing a CARD domain) pyroptosome formation. Transfection of THP-1 cells with siRNA targeting NLRP-3 decreased the AgNP15-induced IL-1β production. The absence of caspase-4 expression resulted in a significant reduction of pro-IL-1β. However, caspase-1 activity was significantly higher in caspase-4-deficient cells when compared with WT cells. Inhibition of AgNP15-induced ATF-6 degradation with Site-2 protease inhibitors completely blocked the effect of AgNP15 on pyroptosis and secretion of IL-1β, indicating that ATF-6 is crucial for the induction of this type of cell death. We conclude that AgNP15 induce degradation of the ER stress sensor ATF-6, leading to activation of the NLRP-3 inflammasome regulated by caspase-4 in human monocytes.

Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases are involved in induction of apoptosis

The plant lectin Viscum album agglutinin‐I (VAA‐I) was recently found to modulate protein synthesis and to induce apoptosis in various cells of immune origin. We found that VAA‐I induces de novo protein synthesis of metabolically 35S‐labeled human neutrophils when used at low concentrations (<100 ng/mL) but acts as an inhibitor at higher concentrations. Using both flow cytometry (FITC‐Annexin‐V/PI labeling) and cytology (Diff‐Quick staining) approaches, we found that VAA‐I could not modulate neutrophil apoptosis at low concentrations but could induce it in >98% of cells at 500 and 1000 ng/mL. VAA‐I was also found to reverse the delaying effect of GM‐CSF on neutrophil apoptosis and to inhibit GM‐CSF‐inducedde novo protein synthesis. In contrast to GM‐CSF, VAA‐I does not induce tyrosine phosphorylation by itself and does not alter the GM‐CSF‐induced response. Among the inhibitors used, genistein, pertussis toxin, staurosporine, H7, Calphostin C, manoalide, BpB, quinacrine HA‐1077, and z‐VAD‐FMK, only the latter (inhibitor of caspases‐1, ‐3, ‐4, and ‐7) was found to inhibit VAA‐I‐induced neutrophil apoptosis as the percentage of apoptotic cells decrease from 98 ± 1.3 to 54 ± 3.2% (n =4). Furthermore, we confirm that caspases are involved in VAA‐I‐induced neutrophil apoptosis as we have observed the fragmentation of the cytoskeletal gelsolin protein that is known to be caspase‐3‐dependent. Such degradation was reversed by the z‐VAD‐FMK inhibitor. We conclude that induction of neutrophil apoptosis by VAA‐I is a caspase‐dependent mechanism that does not involve tyrosine phosphorylation events, G‐proteins, PKCs, and PLA2. In addition, we conclude that at least caspase‐3 is involved. Correlation between VAA‐I‐induced neutrophil apoptosis and VAA‐I‐induced inhibition of de novo protein synthesis is discussed.

Tributyltin induces human neutrophil apoptosis and selective degradation of cytoskeletal proteins by caspases

Tributyltin (TBT) has frequently been used as a pesticide and in biocidal paints for marine vessels, leading to its presence in the environment. Although TBT was recently found to induce apoptosis in different immune cells, by a mechanism that is not fully established, its effect on neutrophils is not known. In this study, it was found that TBT induced apoptosis in human neutrophils as assessed by cytology, flow cytometry, and degradation of the microfilament-associated protein gelsolin. Furthermore, data showed that TBT induced neutrophil apoptosis by a caspase-dependent mechanism, since addition of z-Val-Ala-Asp(MOe)-CH 2 F (z-VAD-FMK) in the culture prevented the effect of TBT. It was also found that the cytoskeletal proteins gelsolin, paxillin, and vimentin, but not vinculin, were degraded by TBT via caspases, as assessed by immunoblotting. Data indicate that gelsolin, paxillin, and vimentin are three caspase substrates involved in both spontaneous and TBT-induced neutrophil apoptosis. Cells were not necrotic as assessed by trypan blue dye exclusion, and this is in agreement with the absence of vinculin degradation. Evidence indicates that TBT-induced fragmentation of cytoskeletal proteins via caspases is a process that is tightly regulated.

Anti-inflammatory effect of Viscum album agglutinin-I (VAA-I): induction of apoptosis in activated neutrophils and inhibition of lipopolysaccharide-induced neutrophilic inflammation in vivo

Viscum album agglutinin‐I (VAA‐I) is a plant lectin which possesses antitumoral properties. This lectin is also known for its immunostimulatory effects when used at low concentrations (1–100 ng/ml). We have demonstrated recently that VAA‐I is a potent inducer of human neutrophil apoptosis in vitro when used at higher concentrations. The role of VAA‐I on activated neutrophils has not so far been investigated and its potential proinflammatory properties in vivo are poorly documented. Herein, we demonstrated that VAA‐I (1000 ng/ml) induces apoptosis in lipopolysaccharide (LPS)‐treated human neutrophils in vitro as well as in murine neutrophils isolated from lipopolysaccharide (LPS)‐induced neutrophil influx. Using this model, we found that administration of VAA‐I (100 or 1000 ng/ml) did not induce an inflammatory response. However, when used at 1 or 10 ng/ml, VAA‐I was found to significantly induce a transitory inflammatory response, based on an increased leucocyte infiltration (>98% neutrophils). Also, we found that VAA‐I inhibits LPS‐induced neutrophil influx when administered simultaneously with LPS. In such conditions, some characteristic apoptotic neutrophils were observed in the pouch. Unlike LPS, which increased the production of some cytokines, VAA‐I (1 or 10 ng/ml) did not increase the production of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1Ra, IL‐1α, IL‐β, IL‐8, IL‐10 or IL‐12 (p70) in human neutrophils. We conclude that VAA‐I possesses the ability to induce apoptosis of preactivated neutrophils at a concentration that does not induce a proinflammatory response. Moreover, we conclude that VAA‐I can inhibit a LPS‐induced proinflammatory response in vivo. These data may provide new clinical perspectives in future mistletoe therapy and favour its potential utilization based on anti‐inflammatory activity that at first appears contradictory with its use as immunostimulant.

Viscum album agglutinin‐I induces apoptosis and degradation of cytoskeletal proteins via caspases in human leukaemia eosinophil AML14.3D10 cells: differences with purified human eosinophils

Although there are several agents that induce neutrophil apoptosis, few are known as inducers of eosinophil apoptosis. As eosinophils are potent effector cells contributing to allergic inflammation and asthma, we investigated whether the pro‐apoptotic agent Viscum album agglutinin‐I (VAA‐I) could induce eosinophil apoptosis. VAA‐I was found to induce apoptosis in eosinophilic AML14.3D10 (3D10) cells and that these cells expressed caspases‐1, ‐2, ‐3, ‐4, ‐7, ‐8, ‐9 and ‐10. VAA‐I‐induced gelsolin degradation was reversed by the pan‐caspase inhibitor N‐benzyloxycarbonyl‐V‐A‐D‐O‐methylfluoromethyl ketone (z‐VAD). Also, paxillin, vimentin and lamin B1 were cleaved by caspases in VAA‐I‐induced 3D10 cells. VAA‐I activated caspase‐3 and ‐8 in 3D10 cells but, unlike z‐VAD, treatment with a caspase‐8 inhibitor slightly reversed apoptosis. Treatment of purified human eosinophils with VAA‐I was found to induce apoptosis, degradation of gelsolin and lamin B1, but unlike 3D10 cells, cleavage of lamin B1 and cell apoptosis was not reversed by z‐VAD. We conclude that VAA‐I is a potent inducer of eosinophil apoptosis and that proteases other than those inhibited by z‐VAD in 3D10 cells are involved in VAA‐I‐induced peripheral blood eosinophil apoptosis and lamin B1 cleavage. Thus, VAA‐I represents a potential candidate for the reduction of the number of eosinophils in diseases where they play important roles.

Viscum album agglutinin‐I induces degradation of cytoskeletal proteins in leukaemia PLB‐985 cells differentiated toward neutrophils: cleavage of non‐muscle myosin heavy chain‐IIA by caspases

The role of the anti‐cancer agent Viscum album agglutinin‐I (VAA‐I) in leukaemia PLB‐985 cells differentiated toward a neutrophil‐like phenotype by dimethylsulphoxide (PLB‐985D) has never been studied. This study investigated whether or not VAA‐I can induce cytoskeletal breakdown in PLB‐985D cells, as previously observed in undifferentiated PLB‐985 cells. VAA‐I was found to induce apoptosis in PLB‐985D cells, as assessed by cytology and by degradation of gelsolin, an event known to occur via caspase‐3 activation. VAA‐I induced cytoskeletal breakdown based on the disruption of the F‐actin network and cleavage of paxillin, vimentin and lamin B1. In addition, we demonstrated, for the first time, that non‐muscle myosin heavy chain IIA (NMHC‐IIA) was cleaved by VAA‐I treatment. Degradation of NMHC‐IIA was reversed by the pan caspase inhibitor z‐VAD‐fmk in PLB‐985D cells and neutrophils. However, unlike lamin B1, no NMHC‐IIA was detected on the cell surface of apoptotic neutrophils. In conclusion, PLB‐985D cells responded in a similar manner to neutrophils regarding the degradation of the tested cytoskeletal. Therefore, PLB‐985D cells may provide a suitable substitute for neutrophils in screening experiments, preventing extensive neutrophil cell isolation.

Activation of human AML14.3D10 eosinophils by nanoparticles: Modulatory activity on apoptosis and cytokine production

Abstract Eosinophilic inflammation is frequently observed in response to nanoparticle (NP) exposure in airway rodent models of allergies where the number of eosinophils is increased in lungs. Despite this, it is surprising that the potential cytotoxic effect of NP, as well as their direct role on eosinophils is poorly documented. The present study investigated how different NP can alter the biology of the human eosinophilic cell line AML14.3D10. It was found that among NP forms of CeO2, ZnO, TiO2, and nanosilver of 20 nm (AgNP20) or 70 nm (AgNP70) diameters, only ZnO and AgNP20 induced apoptosis. Caspases-7 and -9 were not activated by the tested NP while caspase-3 was activated by AgNP20 only. However, both ZnO and AgNP20 induced cytoskeletal breakdown as evidenced by the cleavage of lamin B1. Using an ELISArray approach for the simultaneous detection of several analytes (cytokines/chemokines), it was found that only ZnO and AgNP20 increased the production of different analytes including the potent pro-inflammatory CXCL8 (IL-8) chemokine. From the data here, we conclude that toxic effects of some NP could be observed in human eosinophil-like cells and that this could be related, at least partially, by induction of apoptosis and production of cytokines and chemokines involved in inflammation. The results of this study also indicate that distinct NP do not activate similarly human eosinophils, since ZnO and AgNP20 induce apoptosis and cytokine production while others such as TiO2, CeO2, and AgNP70 do not.