Fig 1.
Distribution of MC in mouse airways.
Lung and trachea of a pathogen-free mouse was stained to show MC (mouse mast cell protease 6, mMCP6, red) and dendritic cells (CD11c-YFP, green) imaged by fluorescence (A, B) or confocal microscopy (C). (A) Section (thickness, ~ 100 μm) of left lung without prior allergen challenge cut in a transverse plane near the hilum. Most MC are around the main stem bronchus (Br, left panel), whereas DC are more widely distributed and more abundant (middle and right panels). Bar: 1 mm. (B) Whole mount of trachea and proximal bronchi (Br) showing abundant MC between cartilage rings (horizontal black bands) and near the trachealis muscle (TM) of the posterior membrane, and some in the overlying adventitia (left panel, arrowheads). DC are more widely distributed and abundant in the trachea and in the thyroid gland (*) (middle and right panels). Bar: 1 mm. (C) Cross-section (thickness, ~ 100 μm) of trachea showing abundant MC in the adventitia over the trachealis muscle (left panel). DC are more abundant than MC and distributed around the entire tracheal circumference (middle and right panels). Bar: 100 μm.
Fig 2.
Mast cell staining and imaging methods and validation.
(A) Method for excising and mounting trachea for imaging. (B) Baseline anti-OVA Alexa 647-IgE MC staining in the trachea of a CD11c-EYFP x Actin-CFP mouse. (C) Anti-OVA Alexa 647-IgE MC staining in trachea of the Basoph8 reporter strain and the basophil deficient Basoph8 × Rosa-DTα. (D) Co-localization of mMCP6 and anti-OVA Alexa 647-IgE in fixed, permeabilized tracheal whole mount. The background red staining of non-MC was an artifact introduced by the fixation necessary for mMCP6 staining and was not present in unfixed trachea. (E) Normalized ratiometric measurement of cytosolic Ca2+ flux in FURA-2AM-loaded, IgE receptor-positive RBL-2H3 cells. Cells were incubated with anti-OVA Alexa 647-IgE, then Ca2+ flux was measured with or without addition of anti-Mouse IgE (cross-linker).
Fig 3.
Spatiotemporal interactions of anti-OVA IgE Alexa 647 MC and DC in PBS- and OVA-challenged tracheas.
(A) Spatial distribution of anti-OVA IgE Alexa 647 MC and CD11cYFP DC in saline (SAL)-challenged or OVA-challenged trachea. Tracheas were analyzed one day after the third OVA challenge (i.e., on day 24 of the OVA challenge regimen [5]). Side views show distribution in relation to the lumen. Scale bar = 50 μm (see also S1 Movie) (B). Density of DC and MC was determined by counting each cell type in images and measuring the complete volume of tissue and expressing data as cells/volume. (C) Quantified distribution of anti-OVA IgE Alexa 647 MC and CD11cYFP DC relative to the mucosal edge of tracheas after PBS or OVA challenge. Total cells per region were counted in at least 4 tracheas and expressed as a probability. (D) Mean track speed of anti-OVA IgE Alexa 647-stained mast cells and Cd11cYFP dendritic cells in PBS- and OVA-challenged tracheas. (E) Percent of MCs that had a cell-cell contact when viewed from all angles (see also S2 Movie).
Fig 4.
DC- and MC-probing in PBS- and OVA-challenged tracheas.
(A) shows stills from a time lapse movie of a DC from a CD11c-EYFP x Actin-CFP mouse in a PBS-treated trachea and YZ rendering of the same data. Dendritic cell projections are shown moving over time, with a dotted white line marking the cell border at t = 0. See also S3 Movie. (B) contains still images from a second time lapse movie of a dendritic cell from a CD11c-EYFPxActin CFP mouse in an OVA-challenged trachea. Bars: XY = 50 μm; YZ = 10 μm. See also S4 Movie. (C) Protrusions of DC dendrites in 4 tracheas measured to show whether the projection entered the epithelium. (D) Still images from a time-lapse of a MC stained with anti-OVA IgE Alexa 647 (pink) near blood vessels stained by anti-CD31 PE (red). MCs protruding into vessel walls are marked by arrows. (E) Three XYZ-slices are shown, encompassing a thickness of 5 μm and separated from the next by 5 μm, illustrating that the tip of a MC lies near the vessel lumen. The rightmost panel shows a YZ rendering of the same dataset, illustrating the Z-volume rendered in mid-section. (F) The percentage of MC with protrusions that enter the vessel as defined by z-slices was quantified. * P = 0.007.