Cellular responses after exposure of lung cell cultures to secondary organic aerosol particles

The scope of this work was to examine in vitro responses of lung cells to secondary organic aerosol (SOA) particles, under realistic ambient air and physiological conditions occurring when particles are inhaled by mammals, using a novel particle deposition chamber. The cell cultures included cell types that are representative for the inner surface of airways and alveoli and are the target cells for inhaled particles. The results demonstrate that an exposure to SOA at ambient-air concentrations of about 10(4) particles/cm(3) for 2 h leads to only moderate cellular responses. There is evidence for (i) cell type specific effects and for (ii) different effects of SOA originating from anthropogenic and biogenic precursors, i.e. 1,3,5-trimethylbenzene (TMB) and alpha-pinene, respectively. There was no indication for cytotoxic effects but for subtle changes in cellular functions that are essential for lung homeostasis. Decreased phagocytic activity was found in human macrophages exposed to SOA from alpha-pinene. Alveolar epithelial wound repair was affected by TMB-SOA exposure, mainly because of altered cell spreading and migration at the edge of the wound. In addition, cellular responses were found to correlate with particle number concentration, as interleukin-8 production was increased in pig explants exposed to TMB-SOA with high particle numbers.

Macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall

The human airway epithelium serves as structural and functional barrier against inhaled particulate antigen. Previously, we demonstrated in an in vitro epithelial barrier model that monocyte derived dendritic cells (MDDC) and monocyte derived macrophages (MDM) take up particulate antigen by building a trans-epithelial interacting network. Although the epithelial tight junction (TJ) belt was penetrated by processes of MDDC and MDM, the integrity of the epithelium was not affected. These results brought up two main questions: (1) Do MDM and MDDC exchange particles? (2) Are those cells expressing TJ proteins, which are believed to interact with the TJ belt of the epithelium to preserve the epithelial integrity? The expression of TJ and adherens junction (AJ) mRNA and proteins in MDM and MDDC monocultures was determined by RT-PCR, and immunofluorescence, respectively. Particle uptake and exchange was quantified by flow cytometry and laser scanning microscopy in co-cultures of MDM and MDDC exposed to polystyrene particles (1 μm in diameter). MDM and MDDC constantly expressed TJ and AJ mRNA and proteins. Flow cytometry analysis of MDM and MDDC co-cultures showed increased particle uptake in MDDC while MDM lost particles over time. Quantitative analysis revealed significantly higher particle uptake by MDDC in co-cultures of epithelial cells with MDM and MDDC present, compared to co-cultures containing only epithelial cells and MDDC. We conclude from these findings that MDM and MDDC express TJ and AJ proteins which could help to preserve the epithelial integrity during particle uptake and exchange across the lung epithelium.

Fas-activated serine/threonine phosphoprotein promotes immune-mediated pulmonary inflammation

We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST(-/-)) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST(-/-) mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST(-/-) mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-alpha and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST(-/-) neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

Early alveolar bone grafting has a negative effect on maxillary dental arch dimensions of pre-school children with complete unilateral cleft lip and palate

To evaluate maxillary dental arch dimensions in pre-school children with a complete unilateral cleft lip and palate (CUCLP) after early alveolar bone grafting.

Local strain distribution in real three-dimensional alveolar geometries

Mechanical ventilation is not only a life saving treatment but can also cause negative side effects. One of the main complications is inflammation caused by overstretching of the alveolar tissue. Previously, studies investigated either global strains or looked into which states lead to inflammatory reactions in cell cultures. However, the connection between the global deformation, of a tissue strip or the whole organ, and the strains reaching the single cells lining the alveolar walls is unknown and respective studies are still missing. The main reason for this is most likely the complex, sponge-like alveolar geometry, whose three-dimensional details have been unknown until recently. Utilizing synchrotron-based X-ray tomographic microscopy, we were able to generate real and detailed three-dimensional alveolar geometries on which we have performed finite-element simulations. This allowed us to determine, for the first time, a three-dimensional strain state within the alveolar wall. Briefly, precision-cut lung slices, prepared from isolated rat lungs, were scanned and segmented to provide a three-dimensional geometry. This was then discretized using newly developed tetrahedral elements. The main conclusions of this study are that the local strain in the alveolar wall can reach a multiple of the value of the global strain, for our simulations up to four times as high and that thin structures obviously cause hotspots that are especially at risk of overstretching.

Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium

We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).

Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

Calcium-modulated chloride pathways contribute to chloride flux in murine cystic fibrosis-affected macrophages

Cystic fibrosis (CF), a common lethal inherited disorder defined by ion transport abnormalities, chronic infection, and robust inflammation, is the result of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a cAMP-activated chloride (Cl-) channel. Macrophages are reported to have impaired activity in CF. Previous studies suggest that Cl- transport is important for macrophage function; therefore, impaired Cl- secretion may underlie CF macrophage dysfunction. To determine whether alterations in Cl- transport exist in CF macrophages, Cl- efflux was measured using N-[ethoxycarbonylmethyl]- 6-methoxy-quinolinium bromide (MQAE), a fluorescent indicator dye. The contribution of CFTR was assessed by calculating Cl- flux in the presence and absence of cftr(inh)-172. The contribution of calcium (Ca(2+))-modulated Cl- pathways was assessed by examining Cl- flux with varied extracellular Ca(2+) concentrations or after treatment with carbachol or thapsigargin, agents that increase intracellular Ca(2+) levels. Our data demonstrate that CFTR contributed to Cl- efflux only in WT macrophages, while Ca(2+)-mediated pathways contributed to Cl- transport in CF and WT macrophages. Furthermore, CF macrophages demonstrated augmented Cl- efflux with increases in extracellular Ca(2+). Taken together, this suggests that Ca(2+)-mediated Cl- pathways are enhanced in CF macrophages compared with WT macrophages.

Disruption of SIRPα signaling in macrophages eliminates human acute myeloid leukemia stem cells in xenografts

Although tumor surveillance by T and B lymphocytes is well studied, the role of innate immune cells, in particular macrophages, is less clear. Moreover, the existence of subclonal genetic and functional diversity in some human cancers such as leukemia underscores the importance of defining tumor surveillance mechanisms that effectively target the disease-sustaining cancer stem cells in addition to bulk cells. In this study, we report that leukemia stem cell function in xenotransplant models of acute myeloid leukemia (AML) depends on SIRPα-mediated inhibition of macrophages through engagement with its ligand CD47. We generated mice expressing SIRPα variants with differential ability to bind human CD47 and demonstrated that macrophage-mediated phagocytosis and clearance of AML stem cells depend on absent SIRPα signaling. We obtained independent confirmation of the genetic restriction observed in our mouse models by using SIRPα-Fc fusion protein to disrupt SIRPα-CD47 engagement. Treatment with SIRPα-Fc enhanced phagocytosis of AML cells by both mouse and human macrophages and impaired leukemic engraftment in mice. Importantly, SIRPα-Fc treatment did not significantly enhance phagocytosis of normal hematopoietic targets. These findings support the development of therapeutics that antagonize SIRPα signaling to enhance macrophage-mediated elimination of AML.