Determination of neuropeptide amounts and of DC motility in mouse airways in an acute and prolonged model of allergic airway inflammation.

<p>(A) Representative confocal <i>z</i>-stack projections of mouse airways stained as whole-mounts against the pan-neuronal marker PGP9.5 (blue) and neuropeptides SP or CGRP (yellow), respectively. Airways of control-treated and asthmatic mice were analyzed (acute model: i.p. booster on days 14 and 21, challenge and analysis on day 27; prolonged protocol: i.p. booster on days 14 and 21, two additional challenges on days 27 and 28, final challenge on day 35, analysis day 36). The total area of fluorescence signals of maximum intensity projections of <i>z</i>-stack images was determined by using Imaris and ImageJ software. (B) The total area of neuropeptide fluorescence signal (SP or CGRP signal) was set into relation to the area of all nerves (PGP9.5 signal). This ratio was determined for six regions of each airway. Per group three mice donating one airway each were analyzed. Unpaired statistical <i>t</i>-test; outliers were determined by Grubb’s test; ns not significant; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. Scale: 40 µm. (C) Mean velocities of airway DC were determined both, in an acute OVA asthma model and in a model using a prolonged protocol and in their respective control groups (ctrl) as indicated. Each dot represents the mean velocity of an individual cell; black horizontal bars indicate the medians. Data of living lung slices show a pool of three (acute) to four (prolonged) independent experiments. Mann-Whitney statistical test; ns not significant; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001.</p

Characterization of CD11c⁺ cells in mouse main bronchi.

<p>Mouse airways of the CD11c-EYFP reporter mouse (CD11c in yellow) or of the CX₃CR1^+/<i>gfp</i> transgenic mouse (CX₃CR1 in yellow) were stained as whole-mounts against MHC-II (red), F4/80 (red) or CD68 (red) as indicated. Airway epithelium stained with cytokeratin (Ck) is shown in grey. Left column shows merges of all acquired channels. Other columns show according single channel images. Image projections were generated with Imaris (Bitplane) from raw confocal <i>z</i>-stacks. Scale: 40 µm.</p

Motility of CD11c⁺ cells after electrical field stimulation.

<p>Mean velocities of lung CD11c⁺ cells were determined under untreated conditions (ctrl) or after different time-points (1 h, 24 h) of EFS. Each dot represents the mean velocity of an individual cell; black horizontal bars indicate the medians. Data of living lung slices show a pool of two independent experiments (left part). Mean velocities of <i>in vitro</i>-cultured magnetically sorted CD11c⁺ total lung cells (right part) were acquired from one experiment; Brownian Motion (Brown) was determined by analyzing non-motile paraformaldehyde-fixed cells. Mann Whitney statistical test; ns not significant; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001.</p

Motility of neuropeptide-treated CD11c⁺ cells.

<p>Mean velocities of lung CD11c⁺ cells and airway DC five minutes after neuropeptide treatment. Groups as indicated. For antagonistic treatment, slices were pre-treated with CGRP_8–37 five minutes prior to EFS and analyzed 1 h after EFS. Each dot represents the mean velocity of an individual cell; black horizontal bars indicate the medians. Data of living lung slices and <i>in vitro</i>-generated BMDC show a pool of three independent experiments for each group. Mann Whitney statistical test; ns not significant; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001.</p

Phagocytosis capacity of CD11c⁺ cells after neuropeptide treatment.

<p>(A) Representative confocal images of <i>in vitro</i>-cultured BMDC after different treatments as indicated. Upper panel shows a merge of red fluorescence of phagocytosed <i>E. coli</i> pHrodo particles and transmitted light channel to visualize all cells. Lower panel shows only the red fluorescence according to images of the upper panel. Lower panel images were used to determine the total area of red fluorescence in each image with ImageJ software. Scale: 40 µm. (B) Graph of phagocytosis indices (PI) of BMDC and magnetically sorted total lung CD11c⁺ cells. Cells were neuropeptide or control treated for five minutes. <i>E. coli</i> pHrodo particles were then applied for 2 h. Beforehand a fraction of cells was triggered over night with LPS (three independent experiments). To inhibit phagocytosis, another fraction of cells was treated with Cytochalasin D (one experiment for BMDC, two experiments for whole lung CD11c⁺ cells). Neuropeptide-treated groups (0.1 nM VIP, 1 µM SP, 1 nM CGRP) were compared to a water-treated group (ctrl) (four independent experiments for BMDC and two independent experiments for whole lung CD11c⁺ cells). Each dot represents PI of one image. Black horizontal bars indicate the means. Per well two images were acquired. Unpaired statistical <i>t</i>-test; ns not significant; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001.</p

Analysis of DC motility in mouse main bronchi.

<p>Main bronchi of mouse living lung sections were analyzed by two-photon time-lapse microscopy. (A) 300 µm thick mouse living lung slice visualized with a stereomicroscope. Box indicates a typical region used for three-dimensional time-lapse microscopy. Scale: 1 mm. (B) Three-dimensional stack projection of DC in the airway epithelium. CD11c⁺ cells shown in yellow, SHG signal of airway collagen fibers in blue (two-photon excitation: 920 nm), autofluorescence of airway epithelium in grey (two-photon excitation: 820 nm). Side and bottom panel show side views of the stack in <i>yz</i> and <i>xz</i>-direction, respectively. Scale: 40 µm. (C) Example of a three-dimensional stack projection of a single analysis time point. CD11c⁺ cells of CD11c-EYFP transgenic mice are shown in yellow; SHG signal of collagen fibers of the airway in blue (two-photon excitation: 920 nm). Scale: 50 µm. (D) Color-coded tracks of DC during the total analysis time of 1 h image acquisition (resume pertains to C). Blue color code marks the start of imaging; white, the end of imaging. Scale: 50 µm.</p