Impact of ultra-small silver nanoparticles of 2 nm (AgNP2) on neutrophil biology: AgNP2 alter the actin cytoskeleton and induce karyorrhexis by a mitogen-activated protein kinase-dependent mechanism in vitro and transitorily attract neutrophils in vivo.

Silver (Ag) is known as an antibacterial agent and there is a growing interest to use silver nanoparticles (AgNPs) in a variety of medical applications and other sectors. Some studies reported that one of the undesired effects of AgNPs is inflammation and that these NPs can alter the biology of neutrophils. Since it is commonly accepted that the more NPs are small, the more toxic they are the aim of this study was to determine the impact of ultra-small silver nanoparticles of 2 nm (AgNP2) on the biology of neutrophils, key player cells in inflammation. We report that AgNP2 are potent neutrophil activators as they rapidly induce actin polymerization and dismantling the actin network. Although AgNP2 are not necrotic for neutrophils and do not induce ROS production, kinetic studies reveal that AgNP2 are rapid inducer of apoptosis. Pyknosis (mainly 1-2 large nuclear dots) was observed after only 1h of treatment followed by karyorrhexis (several small dots) and by a complete nuclear dissolution leading to anuclear neutrophils after 6h. These observations are not associated with the release of silver ions since treatment of neutrophils with 1-50 µg/ml AgNO3 (as a source of Ag+) did not induce any apparent changes. AgNP2 induce p38 and Erk-1/2 mitogen-activated protein kinase (MAPK) and although karyorrhexis was markedly reversed by MAPK inhibitors, the cell nuclei remain with a pyknotic-like phenotype but do not return to the characteristic polylobed nucleus. Using the murine air pouch model of inflammation AgNP2 were found to induce a neutrophil influx. Our data indicate that AgNP2 are potent neutrophil activators targeting the actin cytoskeleton and the mechanism involved for inducing apoptosis is rapid, complex, and partially includes MAPK pathways. Therefore, the ultra-small AgNP2 are more potent than larger ones for inducing apoptosis and they can transitorily attract neutrophils in vivo.

Evaluating the Apoptotic Cell Death Modulatory Activity of Nanoparticles in Men and Women Neutrophils and Eosinophils.

Apoptosis is an important cell death mechanism for the resolution of inflammation. Neutrophil spontaneous apoptosis rates were reported to be slightly different in men and women and to be modulated by female sex hormones. The aim of this study was to determine whether different nanoparticles (NPs) will alter the neutrophil and eosinophil apoptotic rates differently in men and women. Using the antiapoptotic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and the proapoptotic plant lectin Viscum album agglutinin-I (VAA-I) as controls, we found that these factors respectively delay and induce apoptosis in both neutrophils and eosinophils with apoptotic rates remarkably similar in both sexes. The polyamidoamine (PAMAM) dendrimers of generation 0 (G0) and G3 slightly, but not significantly, accelerate neutrophil apoptosis regardless of sex. Zinc oxide (ZnO), titanium dioxide (TiO2), cerium dioxide (CeO2), and palladium (Pd) but not platinum (Pt) NPs were found to significantly delay neutrophil apoptosis. When results were compared between men and women, only ZnO and Pd NPs were found to significantly delay neutrophil apoptosis in men while ZnO, TiO2, CeO2, and Pt NPs inhibit apoptosis in women neutrophils. In eosinophils, G3, but not G0 NPs, significantly accelerate apoptosis in women. ZnO, Pt, and Pd NPs significantly delay eosinophil apoptosis but only in women. Unlike neutrophils, TiO2 and CeO2 NPs did not significantly delay eosinophil apoptosis. We propose that future studies aiming at determining potential effect NPs on cellular biological processes should incorporate a sex-based analysis based on the differences reported here studying the impact of NPs on human granulocyte apoptosis.

Biological activities of interleukin (IL)-21 in human monocytes and macrophages.

The biological roles of interleukin (IL)-21 in human monocytes and macrophages have been neglected. We previously demonstrated that IL-21 induce phagocytosis and established that Syk is a new molecular target of IL-21. Herein, we found that IL-21 is not chemoattractant for immature THP-1 and primary monocytes but can increase the capacity of THP-1 cells (not primary monocytes) to adhere onto a cell substratum by a Syk-dependent mechanism without altering the expression of a panel of cell surface molecules. Unlike THP- 1 and monocytes, IL-21 can increase metalloproteinase (MMP)-9 secretion and activity in monocyte-derived macrophages (HMDM), as assessed by western blot and zymography experiments, respectively. We reported that IL-21 did not increase the production of IL-6 and the chemokines MIP-1α and GRO-α in HMDM. Therefore, IL-21 can increase functions other that phagocytosis, but this cytokine does not have a large spectrum of biological activities in monocytes and macrophages.

Evaluation of the in vitro and in vivo proinflammatory activities of gold (+) and gold (-) nanoparticles.

OBJECTIVE AND DESIGN: The aim of this study was to determine potential effects of gold (+) and gold (-) nanoparticles, AuNP(+) and AuNP(-), on neutrophil biology. MATERIAL OR SUBJECTS: Freshly isolated human neutrophils were used for the in vitro aspects and CD-1 mice were used in the in vivo murine air pouch model of acute neutrophilic inflammation. TREATMENT: Human neutrophils were treated with the indicated concentrations of AuNP(+) or AuNP(-) in vitro and mice received 100 or 500 µg/ml AuNP(+) or AuNP(-) into air pouches. METHODS: Cellular uptake of AuNP by neutrophils was confirmed by transmission electron microscopy and the ability of the NP to modulate apoptosis, gelatinase activity, and chemokine production and chemotaxis was determined by cytology, zymography, ELISArray, antibody array, and ELISA and by a micro-chemotaxis chamber, respectively. In vivo, exudates were harvested after 6 h to determine the leukocyte infiltration to detect the production of several cytokines by an antibody array approach and ELISA. One-way analysis of variance was used for statistical analysis. RESULTS: AuNP possess proinflammatory activities in vitro and induce mainly a neutrophil influx in vivo, albeit at different degrees. CONCLUSIONS: AuNP(+) and AuNP(-) should be added as new candidates into a growing list of NP having proinflammatory activities by themselves.

Silver nanoparticles induce irremediable endoplasmic reticulum stress leading to unfolded protein response dependent apoptosis in breast cancer cells.

Nowadays, silver nanoparticles (AgNP) are widely used in the medical field mainly for their antibacterial properties. Although some studies report a cytotoxic activity of the particles, the mechanisms involved in AgNP-induced cell death remain to be determined. Herein, we report that AgNP of 2 (AgNP2) and 15 nm (AgNP15) induce apoptosis in human MCF-7 and T-47D breast cancer cells. Treatment with AgNP2 and AgNP15 led to accumulation and aggregation of misfolded proteins causing an endoplasmic reticulum (ER) stress and activating the unfolded protein response (UPR). The three main ER sensors, PERK, IRE-1α and ATF-6, were rapidly activated in response to AgNP2 and AgNP15. Although Grp78 levels remained unchanged, AgNP2 and AgNP15 induced upregulation of the transcription factors ATF-4 and GADD153/CHOP. Moreover, the initiating caspase-9 and the effector caspase-7 were activated in response to these NPs. The expression levels of the pro-apoptotic BIM and BAD proteins remained unchanged. In contrast, a downregulation of Mcl-1 and xIAP protein expression as well as a processing of PARP were observed. Pharmacological inhibition of PERK kinase and IRE-1 endonuclease activities, as well as inhibition of ER-stress, partially protected cells from AgNP2- and AgNP15-induced apoptosis. Of note, the non-cancerous MCF-10A cells were more resistant to both AgNP2 and AgNP15 when compared to MCF-7 and T-47D cell lines. Taken together, our results demonstrate that AgNP induce ER stress and can target the UPR-dependent apoptotic pathway in MCF-7 and T-47D, which highlights new potential strategies for the treatment of breast cancers.

In vivo proinflammatory activity of generations 0-3 (G0-G3) polyamidoamine (PAMAM) nanoparticles.

OBJECTIVE AND DESIGN: The aim of this study was to determine whether different generations (G) polyamidoamine (PAMAM) dendrimers possess proinflammatory activities in vivo. MATERIAL OR SUBJECTS: Several hundred female CD-1 mice were used to test four different PAMAM dendrimers using the murine air pouch model. TREATMENT: Mice received appropriate negative and positive controls or G0-G3 PAMAM nanoparticles at 100 and 500 µg/ml into air pouches. METHODS: Exudates were harvested after 3, 6, 24 and 48 h. Cell pellets and supernatants were used to determine the number of total leukocytes and neutrophils and to detect the production of several analytes by an antibody array approach, respectively. One-way analysis of variance was used for statistical analysis. RESULTS: PAMAM dendrimers rapidly increased a leukocyte influx after 3 h, the vast majority of cells being neutrophils. This was also observed after 6 and 24 h, and resolution of inflammation was noted after 48 h. In general, the increased production of a greater number of analytes detected in the exudates after 6 h correlated with the number of dendrimer generations (G3 > G2 > G1 > G0). CONCLUSIONS: PAMAM dendrimers devoid of any delivering molecules possess proinflammatory activities in vivo by themselves, probably via the production of different chemokines released by air pouch lining cells.