Phagocytic ability is induced by ATRA differentiation.

<p>A. Effect of opsonization on interaction. Differentiated HL-60 cells were allowed to interact at 37°C with Oregon Green-labeled BMJ71 bacteria, either IgG-opsonized (1 mg/ml) or not, at a bacteria/cell ratio of 10∶1. After a synchronized presentation, the samples were incubated at 37°C as indicated. Analysis was by flow cytometry. An interaction ratio was calculated by dividing the number of cells interacting with Oregon Green-labeled bacteria with the total number of cells. Error bars show SEM, based on a total of three experiments; *  = p<0.05. B–C. Interaction and internalization during differentiation. The ability of HL-60 cells to associate with and to internalize BMJ71 bacteria was measured. Oregon Green-labeled and IgG-opsonized bacteria were allowed to interact for 5 min with the cells at a bacteria/cell ratio of 2∶1. Analysis was by fluorescence microscopy. Error bars represent SEM of two separate experiments. D. Phagocytic ability induced by ATRA-treatment. Fold increase of phagocytic ability arrived at by normalizing the phagocytic ability of ATRA-treated cells to the corresponding control sample. E. Basis for calculations. The different formulas used for the analysis of data in the figures B–D are presented. Interaction is defined as the fraction of all cells that are associated with at least one bacterium. Interacting cells were further analyzed to determine how large a fraction of the associated bacteria was intracellular. The term phagocytic ability was constructed to take interaction as well as internalization into account.</p

ATRA-induced oxidative ability.

<p>A. Induction of Nox2 activation during differentiation. Cells were allowed to phagocytose IgG-opsonized BMJ71 bacteria for 5 min (bacteria/cells, 2∶1), or were stimulated with 160 nM PMA, in the presence of 1 mg/ml NBT. Measurement of absorbance was used to quantitate the intracellular respiratory burst. Data are presented as the ratio between formazan formation in ATRA-treated cells compared to control cells. B. Localization of formazan formation. Differentiated HL-60 cells interacting with IgG-opsonized Oregon Green-labeled BMJ71 bacteria were incubated with 1 mg/ml NBT. A bacteria/cell ratio of 10∶1 was used. Respiratory burst activity was indicated by blue-black formazan precipitates, visible by light microscopy (iii, vi). Oregon Green fluorescence (i, iv) shows the total number of bacteria, and anti-streptococcal staining (ii, v) shows the location of extracellular bacteria. In the upper panel, precipitate formation on cell-adherent bacteria is illustrated. The lower panel shows that NBT precipitates may also be found on internalized bacteria. Size bar: 10 µm. C. Quantitation of respiratory burst-positive cells. The diagram shows the proportion of bacteria-interacting cells that display formazan precipitates and the effect of DPI (10 µM). At least 50 cells per sample were analyzed. Error bars show SEM, based on a total of three experiments.</p

Killing of BMJ71 bacteria does not require Nox2 activation.

<p>A. Intracellular killing with inhibited oxidase. Differentiated HL-60 cells were allowed to phagocytose BMJ71 bacteria at 37°C, bacteria/cell ratio 10∶1, in the presence or absence of 10 µM DPI. After a synchronized presentation, the samples were incubated at 37°C as indicated, before killing of extracellular bacteria by PlyC. Intracellular survival of bacteria was determined by diluting HL-60 lysates and counting the number of colonies formed after overnight growth at 37°C. Data shown are expressed as the CFU ability relative to the control value at 1 min. Error bars show SEM, based on a total of five experiments. A significant difference was found between control and DPI-treated cells at the 1 min time point, p<0.05. B. H₂O₂ susceptibility of <i>S. pyogenes</i>. BMJ71 and AP1 bacteria were incubated with 1.5% H₂O₂ at 37°C as indicated. The samples were stained using a bacterial viability kit (Viagram) and analyzed by fluorescence microscopy. As a control, catalase (1,000 U/ml) was added. At least 100 bacteria per condition were analyzed. Error bars show SEM, based on a total of three experiments.</p

Intracellular survival of <i>S. pyogenes</i> bacteria.

<p>A. Interaction. Differentiated HL-60 cells were allowed to interact at 37°C with Oregon Green-labeled IgG-opsonized (1 mg/ml) AP1 and BMJ71, at a bacteria/cell ratio of 10∶1. After a synchronized presentation, the samples were incubated at 37°C as indicated. Analysis was by flow cytometry. An interaction ratio was calculated by dividing the number of cells interacting with Oregon Green-labeled bacteria with the total number of cells. Error bars show SEM, based on a total of three experiments. B. Internalization. After phagocytosis as in A, the internalization of bacteria was determined by immunofluorescence microscopy using non-permeabilized and permeabilized conditions and anti-<i>S. pyogenes</i> antibodies. For each condition, at least 100 cells were counted. A representative experiment is shown. C. Intracellular survival. Differentiated HL-60 cells were allowed to phagocytose AP1 and BMJ71 bacteria at 37°C, bacteria/cell ratio 10∶1. After a synchronized presentation, the samples were incubated at 37°C as indicated, before killing of extracellular bacteria by PlyC. Intracellular survival of bacteria was determined by diluting HL-60 lysates and counting the number of colonies formed after overnight growth at 37°C. Data shown are expressed as the CFU ability relative to the values at 1 min. Error bars show SEM, based on a total of three experiments.</p

Fusion of azurophilic granules with phagosomes containing <i>S. pyogenes</i> bacteria.

<p>A. Western blot of isolated phagosomes and cell lysate. Differentiated HL-60 cells were allowed to phagocytose IgG-opsonized, heat-killed and magnetically labeled <i>S. pyogenes</i> bacteria for 20 min at a bacteria/cell ratio of 5∶1. Following washes, the cells were lysed by nitrogen cavitation and phagosomes were retrieved by magnetic selection. Equal amounts of phagosomes were loaded and probed with antibodies against cathepsin D (azurophilic granule content marker) and GM130 (Golgi marker) with anti-<i>S. pyogenes</i> as loading control. Increasing amounts of cell lysate (relative protein content.05×, 0.5× and 1.0×) was used as a control. B. Azurophilic granule–phagosome fusion. Differentiated HL-60 cells were allowed to phagocytose opsonized Oregon Green-labeled <i>S. pyogenes</i> bacteria, either the BMJ71 (i-iii) or the AP1 (iv–v) strains, at a bacteria/cell ratio of 10∶1. After a synchronized presentation, the samples were incubated at 37°C for 5 min, fixed and incubated with antibodies directed against CD63, subsequently detected using Alexa 594 F(ab')₂ fragments. Single deconvolved focal planes from serial z-stacks were taken from the mid-part of the HL-60 cells. Oregon Green staining shows the localization of bacteria (ii, v). Magenta staining shows the localization of the azurophilic granule membrane marker CD63 (i, iv). The images are also presented as merged (iii, vi). Size bar: 5 µm.</p

Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level

Infrared spectroscopic imaging is widely used for the visualization of biomolecule structures, and techniques such as optical photothermal infrared (OPTIR) microspectroscopy can achieve <500 nm spatial resolution. However, these approaches lack specificity for particular cell types and cell components and thus cannot be used as a stand-alone technique to assess their properties. Here, we have developed a novel tool, fluorescently guided optical photothermal infrared microspectroscopy, that simultaneously exploits epifluorescence imaging and OPTIR to perform fluorescently guided IR spectroscopic analysis. This novel approach exceeds the diffraction limit of infrared microscopy and allows structural analysis of specific proteins directly in tissue and single cells. Experiments described herein used epifluorescence to rapidly locate amyloid proteins in tissues or neuronal cultures, thus guiding OPTIR measurements to assess amyloid structures at the subcellular level. We believe that this new approach will be a valuable addition to infrared spectroscopy providing cellular specificity of measurements in complex systems for studies of structurally altered protein aggregates