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 2

Intradermal injection of fluorochrome-labeled avidin does not interfere with IgE-mediated mast cell (MC) degranulation or IgE- and MC-dependent inflammation in vivo.

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Intradermal injection of fluorochrome-labeled avidin does not interfere ...
(A and B) Sulforhodamine 101–coupled avidin (Av.SRho; 5 μg) was injected intradermally (i.d.) into the ear pinna of C57BL/6 mice. One week later mice were sensitized by i.d. injection of 20 ng of mouse anti–2,4-dinitrophenyl (anti-DNP) IgE into the ear pinna. Sixteen hours later, 250 μg of 70-kDa dextran-FITC was injected retro-orbitally and the anesthetized mice were positioned under the 2-photon microscope. Anti-DNP IgE–sensitized mice were injected i.p. with 500 μg of DNP-conjugated human serum antigen (DNP-HSA) or with vehicle (control). Image sequences were acquired in 3D at a rate of 1 picture per minute over 30 minutes using a 2-photon microscope. (A) Representative 3D time-lapse sequences of merged Av.SRho (red), dextran-FITC (green), and collagen structures (blue) fluorescence. White lines identify the magnified area in which Av.SRho fluorescence was modeled. Scale bars: 20 μm. Arrows indicate exteriorized Av.SRho+ granule structures. (B) Fold change in dextran-FITC mean fluorescence intensity (MFI) in the interstitial space following injection of DNP-HSA (green circles) or vehicle (open circles). The arrow indicates when the first exteriorized Av.SRho+ granule structures were detected. (C–F) Av.SRho (5 μg, right ear, red circles or bars) or vehicle control (left ear, black or open circles or bars) was injected i.d. into the ear pinna of C57BL/6 mice. One week later mice were sensitized by i.d. injection of 20 ng of mouse anti-DNP IgE into both ear pinnae. Sixteen hours later, mice were injected i.p. with 500 μg of DNP-HSA (Ag) or with vehicle (control) (open circles or bars). (C) Changes (Δ) in ear thickness over time after i.p. injection. (D) Representative H&E (upper panel) and toluidine blue (TB, lower panel) photomicrographs of sections of ear pinnae in mice sacrificed 360 minutes after i.p. injection. Scale bars: 200 μm; asterisks indicate areas shown at higher magnification in the insets (×60). (E) Morphological assessment of MC degranulation in TB-stained sections 360 minutes after i.p. injection of Ag. Percentage and intensity of MC degranulation, as determined by classification into 3 categories: not degranulated (None [<10% of granules altered], open bars); moderately degranulated (Mod [10%–50% of granules altered], gray bars); and extensively degranulated (Ext [>50% of granules altered], black bars). (F) Number of leukocytes/mm2 dermis 360 minutes after i.p. injection. (B and C) Mean ± SEM; (E and F) mean + SEM; circles show values for individual mice. (B) Two-way ANOVA, (C, E, and F) 2-tailed, unpaired t test (in C, red and black asterisks represent significance of differences between data depicted by red or black circles vs. open circles, respectively). Data (n = 3–12 mice per group) are pooled from the 3 independent experiments performed (each done with n = 1–4 mice per group), each of which gave similar results. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.