Andrea Becker

Developmental regulation of intracellular and surface androgen receptors in T cells

Increasing information indicates that testosterone actions on cells are mediated not only through the classical intracellular androgen receptor (iAR), but also through membrane androgen receptors (mAR) on cell surfaces. Here, we investigate the expression pattern of mAR and iAR in thymic T cells, which is compared with that of splenic T cells. Thymic T cells are testosterone-sensitive in vivo, i.e. treatment of female C57BL/10 mice with testosterone for 3 weeks decreased the total number of thymic T cells by approximately 90%. The percentage of CD4(-) CD8(-) T cells increased, whereas that of the subsequent CD4(+) CD8(+) T cells was diminished. Flow cytometry and confocal laser scanning microscopy (CLSM) with different anti-iAR antibodies localized iAR predominantly in the cytoplasm, but not on the surface of thymic T cells. The iAR are functionally active since the iAR are induced by testosterone to translocate from cytoplasm to nucleus, and they bind the testosterone analogue 3H-R1881 with high affinity (K(d) approximately 2.2 nM) and saturable capacity (approximately 10,000 binding sites per cell) as determined by Scatchard analysis. By contrast, the impeded ligand testosterone-BSA-FITC (T-BSA-FITC) did not bind to the surface of thymic T cells. In accordance, testosterone was unable to induce any rapid rise in the intracellular free Ca(2+) concentration of Fura-2 loaded thymocytes. This indicates that thymic T cells do not express any significant amounts of mAR. Conversely, splenic T cells express functionally active mAR, whereas their expressed iAR are not functional in the genomic pathway. Our results support the view of a delicately balanced developmental regulation of iAR and mAR in T cells.

Surface-Dependent Quartz Uptake by Macrophages: Potential Role in Pulmonary Inflammation and Lung Clearance

Inhalation of quartz particles is associated with a variety of adverse lung effects. Since particle surface is considered to be crucial for particle pathogenicity, we investigated the influence of quartz surface properties on lung burden, inflammation (brochoalveolar lavage cells), and cytotoxicity (protein, lactate dehyddrogenase, β -glucuronidase) 90 days after a single intratracheal instillation of 2 mg DQ12 into rats. The role of particle surface characteristics was investigated by comparative investigation of native versus surface-modified quartz, using polyvinylpyridine N-oxide (PVNO) or aluminum lactate (AL) coating. Uptake and subcellular localization of quartz samples as well as tumor necrosis factor (TNF)-α release were determined using NR8383 rat alveolar macrophages. Surface modification of quartz particles resulted in marked in vivo and in vitro changes. Compared to native quartz, modified quartz samples showed lower lung burden at 90 days, as well as decreased inflammatory and cytotoxic responses. Coating with polyvinylpyridine N-oxide (PVNO) appeared to be more effective than aluminium lactate (AL). PVNO-coating of quartz also resulted in an enhanced particle uptake by macrophages up to 24 h, whereas AL coating caused a transient reduction of quartz uptake at 2 h. At 24 h differences with the native quartz were absent. Subcellular localization of quartz particles was not affected by surface modifications. However, surface modification resulted in a reduced release of TNF-α. In conclusion, surface properties of quartz particles appear to be crucial for rate and extent of in vitro particle uptake in macrophages. Our in vivo findings also indicate that quartz surface properties may affect clearance kinetics. Particle surface-specific interactions between quartz and macrophages may therefore play a major role in the pulmonary pathogenicity of quartz.

Mechanisms of neutrophil-induced DNA damage in respiratory tract epithelial cells

Reactive oxygen species (ROS) released by neutrophils have been suggested to play an important role in cancer development. Since the mechanisms underlying this effect in the respiratory tract are still unclear, we evaluated DNA damage induced by neutrophils in respiratory tract epithelial cells in vitro and in vivo. For in vitro studies, rat lung epithelial cells (RLE) were co-incubated with activated neutrophils, neutrophil-conditioned medium, or hydrogen peroxide. For in vivo studies, we considered the human nose as a target organ, comparing neutrophilic inflammation in the nasal lavage fluid with the oxidative DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in epithelial cells obtained by nasal brush. Our in vitro data show that human neutrophils are able to induce both 8-OHdG and strand breaks in DNA from RLE cells. Our data also suggest that DNA damage induced by neutrophils is inhibited when neutrophil-derived H2O2 is consumed by myeloperoxidase. In contrast, in the nose no association between neutrophil numbers and 8-OHdG was found. Therefore, it remains unclear whether neutrophils pose a direct genotoxic risk for the respiratory tract epithelium during inflammation, and more in vivo studies are needed to elucidate the possible association between neutrophils and genotoxicity in the lung.