Verena Wilhelmi

Zinc Oxide Nanoparticles Induce Necrosis and Apoptosis in Macrophages in a p47phox- and Nrf2-Independent Manner

In view of the steadily increasing use of zinc oxide nanoparticles in various industrial and consumer applications, toxicological investigations to evaluate their safety are highly justified. We have investigated mechanisms of ZnO nanoparticle-induced apoptosis and necrosis in macrophages in relation to their important role in the clearance of inhaled particulates and the regulation of immune responses during inflammation. In the murine macrophage RAW 264.7 cell line, ZnO treatment caused a rapid induction of nuclear condensation, DNA fragmentation, and the formation of hypodiploid DNA nuclei and apoptotic bodies. The involvement of the essential effector caspase-3 in ZnO-mediated apoptosis could be demonstrated by immunocytochemical detection of activated caspase-3 in RAW 264.7 cells. ZnO specifically triggered the intrinsic apoptotic pathway, because Jurkat T lymphocytes deficient in the key mediator caspase-9 were protected against ZnO-mediated toxicity whereas reconstituted cells were not. ZnO also caused DNA strand breakage and oxidative DNA damage in the RAW 264.7 cells as well as p47phox NADPH oxidase-dependent superoxide generation in bone marrow-derived macrophages. However, ZnO-induced cell death was not affected in bone marrow-derived macrophages of mice deficient in p47phox or the oxidant responsive transcription factor Nrf2. Taken together, our data demonstrate that ZnO nanoparticles trigger p47phox NADPH oxidase-mediated ROS formation in macrophages, but that this is dispensable for caspase-9/3-mediated apoptosis. Execution of apoptotic cell death by ZnO nanoparticles appears to be NADPH oxidase and Nrf2-independent but rather triggered by alternative routes.

Evaluation of apoptosis induced by nanoparticles and fine particles in RAW 264.7 macrophages: Facts and artefacts

Current hazard characterisation of nanoparticles (NP) is predominantly based on in vitro test systems, being established for small molecules of drugs and chemicals. However, specific physicochemical properties of NP may result in interference with assay components, biomarkers, or detection systems. In the present study, six types of (nano)particles were screened in RAW 264.7 macrophages by common cytotoxicity methods (WST-1, LDH). Our specific focus was on the investigation of apoptosis (analysis of hypodiploid DNA, phosphatidylserine exposure, caspase 3/7 activation, and Cell Death Detection ELISA). Assays were validated by the well-known apoptosis inducer staurosporine. Our results show that ZnO, DQ12 quartz and amorphous silica are cytotoxic with strong indications for apoptotic effects in RAW 264.7 macrophages, whereas toxicity was absent for MgO. For fine as well as ultrafine TiO(2), no apoptotic effects could be detected except for induction of DNA fragmentation. The results of our study demonstrate the necessity to control on a case-by-case basis for assay interference to avoid misinterpretation of specific in vitro test findings. To obtain valid statements on the potential induction of apoptosis by specific NP the measurement of multiple endpoints is a prerequisite.

Neutrophil-derived ROS contribute to oxidative DNA damage induction by quartz particles

The carcinogenicity of respirable quartz is considered to be driven by reactive oxygen species (ROS) generation in association with chronic inflammation. The contribution of phagocyte-derived ROS to inflammation, oxidative stress, and DNA damage responses was investigated in the lungs of C57BL/6J wild-type and p47(phox-/-) mice, 24h after pharyngeal aspiration of DQ12 quartz (100 mg/kg bw). Bone-marrow-derived neutrophils from wild-type and p47(phox-/-) mice were used for parallel in vitro investigations in coculture with A549 human alveolar epithelial cells. Quartz induced a marked neutrophil influx in both wild-type and p47(phox-/-) mouse lungs. Significant increases in mRNA expression of the oxidative stress markers HO-1 and γ-GCS were observed only in quartz-treated wild-type animals. Oxidative DNA damage in lung tissue was not affected by quartz exposure and did not differ between p47(phox-/-) and WT mice. Differences in mRNA expression of the DNA repair genes OGG1, APE-1, DNA Polβ, and XRCC1 were also absent. Quartz treatment of cocultures containing wild-type neutrophils, but not p47(phox-/-) neutrophils, caused increased oxidative DNA damage in epithelial cells. Our study demonstrates that neutrophil-derived ROS significantly contribute to pulmonary oxidative stress responses after acute quartz exposure, yet their role in the associated induction of oxidative DNA damage could be shown only in vitro.

Apoptotic, inflammatory, and fibrogenic effects of two different types of multi-walled carbon nanotubes in mouse lung

There is increasing concern about the toxicity of inhaled multi-walled carbon nanotubes (MWCNTs). Pulmonary macrophages represent the primary cell type involved in the clearance of inhaled particulate materials, and induction of apoptosis in these cells has been considered to contribute to the development of lung fibrosis. We have investigated the apoptotic, inflammogenic, and fibrogenic potential of two types of MWCNTs, characterised by a contrasting average tube length and entanglement/agglomeration. Both nanotube types triggered H2O2 formation by RAW 264.7 macrophages, but in vitro toxicity was exclusively seen with the longer MWCNT. Both types of nanotubes caused granuloma in the mouse lungs. However, the long MWCNT induced a more pronounced pro-fibrotic (mRNA expression of matrix metalloproteinase-8 and tissue inhibitor of metalloproteinase-1) and inflammatory (serum level of monocyte chemotactic protein-1) response. Masson trichrome staining also revealed epithelial cell hyperplasia for this type of MWCNT. Enhanced apoptosis was detected by cleaved caspase 3 immunohistochemistry in lungs of mice treated with the long and rigid MWCNT and, to a lesser extent, with the shorter, highly agglomerated MWCNT. However, staining was merely localised to granulomatous foci, and neither of the MWCNTs induced apoptosis in vitro, evaluated by caspase 3/7 activity in RAW 264.7 cells. In addition, our study reveals that the inflammatory and pro-fibrotic effects of MWCNTs in the mouse lung can vary considerably depending on their composition. The in vitro analysis of macrophage apoptosis appears to be a poor predictor of their pulmonary hazard.

Impact of the FcγII-receptor on quartz uptake and inflammatory response by alveolar macrophages

The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the FcgammaII-receptor (FcgammaII-R). Fluorescence immunocytochemistry, demonstrating FcgammaII-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the FcgammaII-R in DQ12 phagocytosis. FcgammaII-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the FcgammaII-R. Consistent with the uptake results, FcgammaII-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF-alpha release as observed after treatment with DQ12. In conclusion, our results indicate a major role of FcgammaII-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.

B lymphocyte compartments in the human splenic red pulp: capillary sheaths and periarteriolar regions

The microvasculature of human spleens is still incompletely understood. Two enigmatic types of red pulp microvessels, penicillar arterioles and sheathed capillaries, have already been described in the nineteenth century without gaining much attention afterwards. We performed a detailed analysis of sheathed capillaries to clarify the cellular composition of their sheaths by immunohistological double-staining experiments. Capillary sheaths comprise three different cell types, namely specialized cuboidal CD271++ inner sheath cells surrounded by CD271− macrophages and accumulations of B lymphocytes. The CD271++ inner sheath cells express the chemokine CXCL13 in a unique single dot pattern. Sheath-associated B lymphocytes consist of IgM+, IgD++, and of “switched” cells. T lymphocytes do not accumulate in pericapillary sheaths. The predominant sheath-associated macrophage population is CD163−CD68+ and thus differs from the majority of red pulp macrophages. The sheath-associated macrophages strongly express CD169 only in perifollicular sheaths, but not in sheaths located deeper in the red pulp. IgM+, IgD++, and “switched” B cells are also closely associated with red pulp arterioles characterized by the expression of smooth muscle actin in muscle cells and in branched periarteriolar stromal cells. Capillary sheaths are observed in a post-arteriolar position and appear to be of limited length. We suggest to change the term “Vagina periarteriolaris makrophagocytica” of the international histological and embryological terminologies to “Vagina pericapillaris.”

Heterogeneity of stromal cells in the human splenic white pulp. Fibroblastic reticulum cells, follicular dendritic cells and a third superficial stromal cell type.

At least three phenotypically and morphologically distinguishable types of branched stromal cells are revealed in the human splenic white pulp by subtractive immunohistological double‐staining. CD271 is expressed in fibroblastic reticulum cells of T‐cell zones and in follicular dendritic cells of follicles. In addition, there is a third CD271− and CD271+/− stromal cell population surrounding T‐cell zones and follicles. At the surface of follicles the third population consists of individually variable partially overlapping shells of stromal cells exhibiting CD90 (Thy‐1), MAdCAM‐1, CD105 (endoglin), CD141 (thrombomodulin) and smooth muscle α‐actin (SMA) with expression of CD90 characterizing the broadest shell and SMA the smallest. In addition, CXCL12, CXCL13 and CCL21 are also present in third‐population stromal cells and/or along fibres. Not only CD27+ and switched B lymphocytes, but also scattered IgD++ B lymphocytes and variable numbers of CD4+ T lymphocytes often occur close to the third stromal cell population or one of its subpopulations at the surface of the follicles. In contrast to human lymph nodes, neither podoplanin nor RANKL (CD254) were detected in adult human splenic white pulp stromal cells. The superficial stromal cells of the human splenic white pulp belong to a widespread cell type, which is also found at the surface of red pulp arterioles surrounded by a mixed T‐cell/B‐cell population. Superficial white pulp stromal cells differ from fibroblastic reticulum cells and follicular dendritic cells not only in humans, but apparently also in mice and perhaps in rats. However, the phenotype of white pulp stromal cells is species‐specific and more heterogeneous than described so far.

Feature-based multi-resolution registration of immunostained serial sections.

&NA; The form and exact function of the blood vessel network in some human organs, like spleen and bone marrow, are still open research questions in medicine. In this paper, we propose a method to register the immunohistological stainings of serial sections of spleen and bone marrow specimens to enable the visualization and visual inspection of blood vessels. As these vary much in caliber, from mesoscopic (millimeter‐range) to microscopic (few micrometers, comparable to a single erythrocyte), we need to utilize a multi‐resolution approach. Our method is fully automatic; it is based on feature detection and sparse matching. We utilize a rigid alignment and then a non‐rigid deformation, iteratively dealing with increasingly smaller features. Our tool pipeline can already deal with series of complete scans at extremely high resolution, up to 620 megapixels. The improvement presented increases the range of represented details up to smallest capillaries. This paper provides details on the multi‐resolution non‐rigid registration approach we use. Our application is novel in the way the alignment and subsequent deformations are computed (using features, i.e. “sparse”). The deformations are based on all images in the stack (“global”). We also present volume renderings and a 3D reconstruction of the vascular network in human spleen and bone marrow on a level not possible before. Our registration makes easy tracking of even smallest blood vessels possible, thus granting experts a better comprehension. A quantitative evaluation of our method and related state of the art approaches with seven different quality measures shows the efficiency of our method. We also provide z‐profiles and enlarged volume renderings from three different registrations for visual inspection. HighlightsWe non‐rigidly register immunohistological serial sections of human specimen.Using feature detection and matching we iteratively compute non‐rigid deformations.Vascular networks in spleen and bone marrow are shown on a level not possible before.A quantitative evaluation of our method shows its efficiency. Graphical abstract Figure. No caption available.

Three-Dimensional Arrangement of Human Bone Marrow Microvessels Revealed by Immunohistology in Undecalcified Sections

The arrangement of microvessels in human bone marrow is so far unknown. We combined monoclonal antibodies against CD34 and against CD141 to visualise all microvessel endothelia in 21 serial sections of about 1 cm2 size derived from a human iliac crest. The specimen was not decalcified and embedded in Technovit® 9100. In different regions of interest, the microvasculature was reconstructed in three dimensions using automatic methods. The three-dimensional models were subject to a rigid semiautomatic and manual quality control. In iliac crest bone marrow, the adipose tissue harbours irregularly distributed haematopoietic areas. These are fed by networks of large sinuses, which are loosely connected to networks of small capillaries prevailing in areas of pure adipose tissue. Our findings are compatible with the hypothesis that capillaries and sinuses in human iliac crest bone marrow are partially arranged in parallel.