Imaging protective mast cells in living mice during severe contact hypersensitivity
Laurent L. Reber, Riccardo Sibilano, Philipp Starkl, Axel Roers, Michele A. Grimbaldeston, Mindy Tsai, Nicolas Gaudenzio, Stephen J. Galli
Laurent L. Reber, Riccardo Sibilano, Philipp Starkl, Axel Roers, Michele A. Grimbaldeston, Mindy Tsai, Nicolas Gaudenzio, Stephen J. Galli
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Research Article Immunology Inflammation

Imaging protective mast cells in living mice during severe contact hypersensitivity

Abstract

Contact hypersensitivity (CHS) is a common skin disease induced by epicutaneous sensitization to haptens. Conflicting results have been obtained regarding pathogenic versus protective roles of mast cells (MCs) in CHS, and this has been attributed in part to the limitations of certain models for studying MC functions in vivo. Here we describe a fluorescent imaging approach that enables in vivo selective labeling and tracking of MC secretory granules by real-time intravital 2-photon microscopy in living mice, and permits the identification of such MCs as a potential source of cytokines in different disease models. We show using this method that dermal MCs release their granules progressively into the surrounding microenvironment, but also represent an initial source of the antiinflammatory cytokine IL-10, during the early phase of severe CHS reactions. Finally, using 3 different types of MC-deficient mice, as well as mice in which IL-10 is ablated specifically in MCs, we show that IL-10 production by MCs can significantly limit the inflammation and tissue pathology observed in severe CHS reactions.

Authors

Laurent L. Reber, Riccardo Sibilano, Philipp Starkl, Axel Roers, Michele A. Grimbaldeston, Mindy Tsai, Nicolas Gaudenzio, Stephen J. Galli

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Figure 3

Longitudinal imaging of mast cell (MC) degranulation and Il10 gene activation in a model of severe CHS.

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Longitudinal imaging of mast cell (MC) degranulation and Il10 gene activ...
Sulforhodamine 101–coupled avidin (Av.SRho; 5 μg) was injected intradermally (i.d.) into the ear pinna of mice. One week later, the mice were treated as depicted in Supplemental Figure 2A to induce a severe 1-fluoro-2,4-dinitrobenzene–induced (DNFB-induced) contact hypersensitivity (CHS) reaction. (A) Longitudinal monitoring of the release of Av.SRho+ granules by dermal MCs at the site of CHS using intravital 2-photon microscopy. Representative 3D photographs of the ear pinna before DNFB challenge or at day 1, 2, or 3 after DNFB challenge. Upper panel: merged fluorescence of Av.SRho (red) and Mcpt5-EYFP (green). Lower panel: Av.SRho fluorescence (pseudocolor scale). Dashed white circles identify hair follicles. (B) Longitudinal monitoring of both the release of dermal MC Av.SRho+ granules and activation of Il10 gene transcription (IL-10-GFP, as detected by emission of GFP fluorescent signal) at the site of CHS using intravital 2-photon microscopy. Representative 3D photographs of the ear pinna before DNFB challenge or at day 1, 2, or 3 after DNFB challenge. Upper panel: merged fluorescence of Av.SRho (red) and IL-10-GFP (green). Middle panel: Av.SRho (red) fluorescence. Lower panel: IL-10-GFP (green) fluorescence. White lines identify the magnified areas and dashed white circles identify hair follicles. Scale bars: 20 μm. (C) Percentage of Mcpt5-EYFP+ cells with exteriorized Av.SRho+ structures (i.e., degranulated dermal MCs, red circles) and of Av.SRho+ IL-10-GFP+ cells (i.e., representing MCs expressing the Il10 gene, green circles) per field of view (FOV) in ear pinnae. (D) Total number of Av.SRho+ IL-10-GFP+ cells (MCs expressing the Il10 gene, green circles) per FOV in ear pinnae and total number of IL-10-GFP+ cells in ear pinnae (black circles). (E) Percentage of Av.SRho+ IL-10-GFP+ cells (i.e., representing MCs expressing the Il10 gene, green) and of Av.SRho–IL-10-GFP+ cells (i.e., representing other cell types expressing the Il10 gene, gray) among total IL-10-GFP+ cells in ear pinnae per FOV. Mean ± SEM; data (n = 3 per group) are pooled from the 3 independent experiments performed (each done with 1 mouse per group), each of which gave similar results.