Mast cells initiate the vascular response to contact allergens by sensing cell stress
Authors: Anja Hoppe, Konstantinos Katsoulis-Dimitriou, Hanna J. Edler, Jan Dudeck, Sebastian Drube, Anne Dudeck
Journal: Journal of Allergy and Clinical Immunology (2020)

Although mast cells (MCs) are well known as key effector cells of IgE-mediated type I allergy, there is growing evidence that MCs are also essential players in innate and adaptive immunity, and in many inflammatory or autoimmune diseases.1Dudeck A. Köberle M. Goldmann O. Meyer N. Dudeck J. Lemmens S. et al.Mast cells as protectors of health.J Allergy Clin Immunol. 2019; 144: S4-S18Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar IgE crosslinking, but also IgE-independent mechanisms of MC activation, result in degranulation of MC secretory granules containing preformed proinflammatory mediators including histamine, cytokines, and proteases. By studying hapten-induced contact hypersensitivity (CHS) responses in novel transgenic mouse lines of MC deficiency or conditional gene knockout, we have previously demonstrated that MCs are of critical impact in the pathomechanism of allergic contact dermatitis. More specifically, we have shown that MCs enhance the migration of dendritic cells (DCs) to draining lymph nodes upon hapten sensitization, thereby promoting effector T-cell expansion.2Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Kohler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar,3Dudeck J. Ghouse S.M. Lehmann C.H. Hoppe A. Schubert N. Nedospasov S.A. et al.Mast-cell-derived TNF amplifies CD8(+) dendritic cell functionality and CD8(+) T cell priming.Cell Rep. 2015; 13: 399-411Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Upon challenge (second contact with the same hapten), MCs are required for efficient skin homing of T cells and thereby facilitate the T-cell–driven skin inflammation. In our previous study, longitudinal intravital imaging of the immune events after hapten contact had surprisingly revealed an immediate MC-triggered vascular response, happening irrespectively of previous sensitization and being crucial for subsequent T-cell–driven skin inflammation.2Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Kohler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar However, this IgE-independent mode of MC activation during allergic contact dermatitis is not yet clarified. In this study, we demonstrate that epicutaneous 2,4-dinitrofluorobenzene (DNFB) administration caused a pronounced vasodilatation of dermal blood vessels within the very first minutes, which was followed by a massive leakage of the intravascular dye due to increased vessel permeability (Fig 1, A). The edema formation could be measured as an early ear swelling response peaking at 2 hours post-DNFB (Fig 1, B) and was almost completely abolished in MC-depleted mice (Fig 1, A2). In sensitized mice, the early ear swelling response was followed by a second T-cell–driven edema at 24 hours after DNFB. However, the amplitude and kinetics of the early ear swelling was similar in nonsensitized mice (in contrast to the 24-hour ear swelling), indicating its innate nature. The hapten-induced innate response further comprised an MC-driven neutrophil infiltration. The early ear swelling response was impeded by treatment with the MC silencer cromolyn or with pyrilamine, a histamine receptor antagonist, before DNFB administration, suggesting a mechanism dependent on MC degranulation and histamine release (Fig 1, C). In contrast, the adaptive skin inflammation (at 24 hours post-DNFB) was significantly reduced in cromolyn-treated mice compared with pyrilamine-treated mice, indicating additional granule-embedded MC mediators beside histamine (Fig 1, C). Given the fast MC degranulation within only few minutes post-DNFB, we questioned the underlying mode of MC activation. Previous reports demonstrated that the absence of TLR signaling in DCs prevented DC-mediated sensitization, effector T-cell expansion, and subsequent CHS responses.4Martin S.F. Dudda J.C. Bachtanian E. Lembo A. Liller S. Dürr C. et al.Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity.J Exp Med. 2008; 205: 2151-2162Crossref PubMed Scopus (184) Google Scholar Therefore, we studied the innate CHS response to DNFB in TLR2/4−/− mice and found the MC-driven early vascular response to not be affected by concomitant absence of TLR2 and TLR4; although it confirmed previous findings, that the adaptive skin inflammation at 24 hours was markedly reduced (see Fig E1 in this article's Online Repository at www.jacionline.org). Previous reports demonstrated that epicutaneous hapten treatment results in a rapid release of extracellular ATP by keratinocytes that is subsequently sensed by DCs via the purinergic receptor P2X7.5Weber F.C. Esser P.R. Müller T. Ganesan J. Pellegatti P. Simon M.M. et al.Lack of the purinergic receptor P2X(7) results in resistance to contact hypersensitivity.J Exp Med. 2010; 207: 2609-2619Crossref PubMed Scopus (167) Google Scholar In addition, IL-33, an alarmin of the IL-1 family being released upon cell stress or necrotic cell death, has been shown to have an impact on DNFB-induced CHS.6Imai Y. Yasuda K. Sakaguchi Y. Futatsugi-Yumikura S. Yoshimoto T. Nakanishi K. et al.Immediate-type contact hypersensitivity is reduced in interleukin-33 knockout mice.J Dermatol Sci. 2014; 74: 159-161Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Questioning whether danger signaling might be involved in the rapid hapten-induced MC activation, we found a prominent surface expression of the ATP receptor P2X7 as well as the IL-33 receptor ST2 on a fraction of about 40% and 65%, respectively, of mouse ear skin MCs under healthy conditions (Fig 1, D). Given that expression, we assessed whether antagonizing the P2X7 ligand binding or neutralizing IL-33 affects the amplitude of the MC-mediated early ear swelling response and neutrophil infiltration. We could show that inhibiting the ATP/P2X7 signaling axis using the antagonist KN62 markedly reduced the innate ear swelling while not significantly affecting the adaptive response at 24 hours and neutrophil infiltration (Fig 1, E and F). In contrast, neutralizing IL-33 signaling by either an anti–IL-33 antibody or a soluble IL-33 receptor not only dramatically reduced the innate ear swelling but also significantly prevented the adaptive inflammation and neutrophil recruitment to the challenged skin (Fig 1, G and H). These data could be confirmed using P2X7−/− mice showing a reduced innate and adaptive edema formation upon hapten treatment (Fig 1, I). To discern P2X7 function in MCs, we used Mcpt5-Cre P2X7FL/FL mice, which lack P2X7, specifically in connective tissue–type MCs. Importantly, Mcpt5-Cre+P2X7FL/FL (referred to as MCΔP2X7) showed a marked reduction in innate and adaptive ear swelling compared with Cre−P2X7FL/FL littermates, but neutrophil infiltration to challenged ear skin was not affected (Fig 1, J and K). These findings suggest that MCs sense extracellular ATP released upon DNFB-induced cell stress or damage by secreting the vasoactive compound histamine. Because, in CHS, MCs trigger both the immediate vascular response and neutrophil infiltration,2Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Kohler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar we speculated that IL-33 signaling might play an additional role in cell stress–related MC activation. Hence, we studied CHS in ST2−/− mice lacking the IL-33 receptor, and detected a diminished innate and adaptive ear swelling (Fig 1, L). Specific IL-33 sensing by MCs via ST2 was proven using MC-deficient Mcpt5-Cre+R-DTA mice, which were reconstituted, locally in the ear skin, with in vitro expanded peritoneal MCs from either ST2−/− mice (referred to as ΔMC + MCΔST2) or wild-type (wt) mice (ΔMC + MCwt) (Fig 1, M). ΔMC + MCΔST2 mice showed decreased innate and adaptive ear swelling post-DNFB, as well as a significant reduction in neutrophil numbers infiltrating the challenged ear skin, compared with ΔMC + MCwt mice (Fig 1, N). Translation of extracellular ATP sensing by MCs into histamine-driven early vascular responses was confirmed by intradermal injection of ATP into P2X7−/− mice (Fig 2, A and B), MCΔP2X7 mice (Fig 2, C and D), and MC-deficient Mcpt5-Cre R-DTA (ΔMC) mice (Fig 2, E and F). Intradermal injection of ATP into mouse ear pinnae resulted in a short-term, self-resolving ear swelling, peaking at 1 hour postinjection, whereas a late ear swelling and neutrophil infiltration at 24 hours was not significantly induced. Importantly, the early ear swelling response was significantly reduced in MCΔP2X7 mice, but completely abolished in ΔMC mice, suggesting additional ATP sensing via other receptors (Fig 2, C and E). In contrast, intradermal injection of IL-33 resulted not only in a prominent early ear swelling, peaking at 2 hours postinjection, but also in pronounced skin inflammation at 24 hours, accompanied by massive neutrophil infiltration (Fig 2, G and H). All these events were significantly reduced in ST2−/− mice and hence, directly induced by IL-33/ST2 binding. Confirming the in vivo findings, we found that MC stimulation with ATP in vitro resulted in prominent degranulation and histamine release but not in TNF production (Fig 2, I-K), whereas in contrast, IL-33 has been demonstrated to induce TNF production in vitro and MC degranulation in vivo but not in vitro.7Drube S. Kraft F. Dudeck J. Müller A.L. Weber F. Göpfert C. et al.MK2/3 are pivotal for IL-33-induced and mast cell-dependent leukocyte recruitment and the resulting skin inflammation.J Immunol. 2016; 197: 3662-3668Crossref PubMed Scopus (35) Google Scholar Our data indicate that IL-33 signaling by MCs and amplification of stress responses by MC degranulation may be worth studying in more detail in the context of human inflammatory skin diseases. The observed mechanisms should have an impact on therapeutic decisions in cases of contact sensitivity which may be difficult to manage. Here, for instance, a local treatment with antihistamines and MC stabilizers as cromolyn glycates, or even local neutralization of IL-33 or TNF, might be a conceivable strategy. Indeed, the IL-33/ST2 signaling axis has been suggested to contribute to the pathomechanism of atopic dermatitis and currently, first clinical trials targeting IL-33 as a novel therapeutic strategy yield promising results.8Savinko T. Matikainen S. Saarialho-Kere U. Lehto M. Wang G. Lehtimäki S. et al.IL-33 and ST2 in atopic dermatitis: expression profiles and modulation by triggering factors.J Invest Dermatol. 2012; 132: 1392-1400Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,9Renert-Yuval Y. Guttman-Yassky E. New treatments for atopic dermatitis targeting beyond IL-4/IL-13 cytokines.Ann Allergy Asthma Immunol. 2020; 124: 28-35Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Collectively, we herein demonstrate that, upon hapten encounter, MCs rapidly initiate innate vascular responses including vasodilatation, vessel permeabilization, and neutrophil influx by prompt degranulation. This MC activation in the context of hapten-induced CHS is a consequence of sensing cell stress or damage by concomitant signaling via the IL-33/ST2 and ATP/P2X7 axis. Although ATP sensing by MCs is sufficient for inducing the histamine-triggered edema formation, additional IL-33 sensing is required for efficient induction of neutrophil infiltration. We cordially thank A. Roers (Dresden, Germany) for providing the Mcpt5-Cre mouse line and for his support for this project, S. Martin (Freiburg, Germany) for the TLR2/4−/− line, M. Idzko (Freiburg, Germany) for providing P2X7−/− and P2X7FL/FL mice, and D. Voehringer (Erlangen, Germany) for the R-DTA line for breedings. Expert technical assistance by C. Haase, T. Häring, and A. Karutz is gratefully acknowledged. All animal experiments were conducted in accordance with German guidelines and were approved by the relevant local authorities in Magdeburg and Dresden. Mcpt5-Cre miceE1Scholten J. Hartmann K. Gerbaulet A. Krieg T. Müller W. Testa G. et al.Mast cell-specific Cre/loxP-mediated recombination in vivo.Transgenic Res. 2008; 17: 307-315Crossref PubMed Scopus (152) Google Scholar were provided by A. Roers (Dresden, Germany), R-DTA miceE2Voehringer D. Liang H.E. Locksley R.M. Homeostasis and effector function of lymphopenia-induced "memory-like" T cells in constitutively T cell-depleted mice.J Immunol. 2008; 180: 4742-4753Crossref PubMed Scopus (168) Google Scholar by D. Voehringer (Erlangen, Germany), TLR2/4−/− miceE3Martin S.F. Dudda J.C. Bachtanian E. Lembo A. Liller S. Dürr C. et al.Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity.J Exp Med. 2008; 205: 2151-2162Crossref PubMed Scopus (185) Google Scholar by S. Martin (Freiburg, Germany), and P2X7−/− and P2X7FL/FL miceE4Stachon P. Heidenreich A. Merz J. Hilgendorf I. Wolf D. Willecke F. et al.P2X7 deficiency blocks lesional inflammasome activity and ameliorates atherosclerosis in mice.Circulation. 2017; 135: 2524-2533Crossref PubMed Scopus (57) Google Scholar by M. Idzko (Freiburg, Germany). C57BL/6 wt mice were purchased from Janvier (Le Genest Saint Isle, France). In all experiments, mice were housed under specific pathogen-free conditions and were used at the age of 8 to 16 weeks. Littermates were used as controls. DNFB, ATP, hyaluronidase, KN-62, A23187, IL-33, and propidium iodide were purchased from Sigma Aldrich (St Louis, Mo). DNAse and Liberase were obtained from Roche (Basel, Switzerland). The goat polyclonal IL-33 blocking antibody (AF3626) was purchased from R&D Systems (Wiesbaden, Germany).E5Bruhs A. Proksch E. Schwarz T. Schwarz A. Disruption of the epidermal barrier induces regulatory T cells via IL-33 in mice.J Investig Dermatol. 2018; 138: 570-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar mAbs for flow cytometry analysis directed against the following marker proteins were purchased from eBioscience (ThermoFisher Scientific, Waltham, Mass): CD45 (30-F11), F4/80 (Bm8), CD11b (M1/70), CD117 (2B8), and FcεRIα (MAR-1). Antibodies directed against Ly6G, P2X7R (1F11), and IL33R (DIH4) were purchased from BioLegend (San Diego, Calif). Recombinant Mouse ST2/IL-33R Fc Chimera Protein was obtained from R&D Systems. Longitudinal and noninvasive intravital 2-photon microscopy of mouse ear skin was performed as previously described.E6Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Köhler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, E7Dudeck J. Froebel J. Kotrba J. Lehmann C.H.K. Dudziak D. Speier S. et al.Engulfment of mast cell secretory granules on skin inflammation boosts dendritic cell migration and priming efficiency.J Allergy Clin Immunol. 2019; 143: 1849-1864Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Mice were sensitized with 100 μL 0.5% DNFB (v/v) in acetone/olive oil (4:1) and challenged 6 days later with 20 μL 0.2% DNFB at the ear (10 μL each side of the ear). Vehicle controls were sensitized and challenged with the acetone/olive oil solvent only.E6Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Köhler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar Ear thickness was measured before and at the indicated time points after challenge with an engineer's micrometer (Mitutoyo). Ear swelling was determined as the percent increase compared with prechallenge ear thickness. Twenty-four hours after challenge ear skin samples were used for flow cytometry analysis. For analysis of cell infiltration, ear skin samples were cut into small pieces and digested in medium containing 20 mM HEPES, 0.025 mg/mL Liberase, 396 U/mL DNAse I, and 0.5 mg/mL hyaluronidase for 1 hour at 37°C and 1400 rpm on a thermo mixer. Cell suspensions were passed through a 40-μm sieve, washed twice with PBS/2% BSA, and stained in 100 μL PBS/2% BSA with the respective antibodies for 30 minutes at 4°C. Neutrophil granulocytes were assessed as CD45+Ly6G+CD11b+F4/80− cells. Infiltrated cells were quantified using Miltenyi MACS Quant or BD fortessa flow cytometer and MACS Quant Analysis software or FlowJo. Pyrilamine (10 mg/g bodyweight) and cromolyn (25 mg/g bodyweight) were injected intravenously 1 hour before challenge.E6Dudeck A. Dudeck J. Scholten J. Petzold A. Surianarayanan S. Köhler A. et al.Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity. 2011; 34: 973-984Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar Twenty microliter of KN-62 at a final concentration of 178.5 μg/mL was injected intradermally (i.d.) into the ear pinna 4 hours before challenge; control mice received an injection of 20 μL of saline.E8Weber F.C. Esser P.R. Müller T. Ganesan J. Pellegatti P. Simon M.M. et al.Lack of the purinergic receptor P2X(7) results in resistance to contact hypersensitivity.J Exp Med. 2010; 207: 2609-2619Crossref PubMed Scopus (169) Google Scholar One microgram of anti–IL-33 blocking antibodyE5Bruhs A. Proksch E. Schwarz T. Schwarz A. Disruption of the epidermal barrier induces regulatory T cells via IL-33 in mice.J Investig Dermatol. 2018; 138: 570-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar or soluble IL-33 receptorE9Fu A.K. Hung K.W. Yuen M.Y. Zhou X. Mak D.S. Chan I.C. et al.IL-33 ameliorates Alzheimer's disease-like pathology and cognitive decline.Proc Natl Acad Sci U S A. 2016; 113: E2705-E2713Crossref PubMed Scopus (197) Google Scholar, E10Drube S. Heink S. Walter S. Löhn T. Grusser M. Gerbaulet A. et al.The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells.Blood. 2010; 115: 3899-3906Crossref PubMed Scopus (97) Google Scholar in 20 μL of saline was injected i.d. into the ear pinna 1 hour before challenge; control mice received an injection of 20 μL saline only. Twenty microliter of ATP at a final concentration of 826.7 μg/mL (1.5 μM) was injected i.d. in the ear pinna. Ten microliter of IL-33 (500 ng) was injected i.d. into the ear pinnae. Ear swelling was measured directly after the injection at the indicated time points and flow cytometry analysis were performed at 24 hours postinjection as described above. MC-deficient Mcpt5-Cre+R-DTA mice were locally reconstituted with in vitro expanded peritoneal MCs (PMCs) isolated from wt or ST2−/− mice by i.d. injection of 1 × 106 PMCs in 20 μL sterile saline into the ear pinnae as previously proven to result in MC numbers comparable to healthy skin.E11Drube S. Kraft F. Dudeck J. Müller A.L. Weber F. Göpfert C. et al.MK2/3 are pivotal for IL-33-induced and mast cell-dependent leukocyte recruitment and the resulting skin inflammation.J Immunol. 2016; 197: 3662-3668Crossref PubMed Scopus (39) Google Scholar Following experiments were conducted at least 2 weeks after reconstitution. PMCs were isolated and cultured as previously described.E12Dudeck A. Suender C.A. Lopez Kostka S. von Stebut E. Maurer M. Mast cells promote Th1 and Th17 responses by modulating dendritic cell maturation and function.Eur J Immunol. 2011; 41: 1883-1893Crossref PubMed Scopus (108) Google Scholar In brief, PMCs were isolated by flushing the peritoneal cavity with 5 mL ice-cold PBS. Cells were pelleted for 5 minutes at 4°C and 1200 rpm; resuspended in 5 mL RPMI containing 10% FCS, 1% penicillin/streptomycin, 1% l-Ala-l-Glu, 1% sodiumpyruvate; supplemented with 10 ng/mL IL-3 and 10-30 ng/mL SCF; and cultured for 8 days at 37°C and 5% CO2. If required, PMCs were enriched using CD117 MicroBeads (Miltenyi, Bergisch-Gladbach, Germany). Purity for reconstitution or following in vitro experiments was 95% or more as determined by flow cytometry using antibodies against CD117 and FcεRIα. PMCs were plated in a 96-well plate at a density of 2 × 105 cells/200 μL/well, and stimulated with 500 μM ATP in nonconditioned RPMI. Supernatant was collected after 10 minutes and 24 hours. PMCs stimulated with the calcium ionophore A23187 (500 ng/mL) served as a positive control, and PMCs cultured in RPMI alone served as negative control. Histamine (DRG) and TNF (TNF-α ELISA Max Standard from BioLegend) release was quantified using respective ELISAs according to the manufacturer's instructions. MC degranulation was assessed by means of β-hexosaminidase assay. Twenty-five microliter cell supernatant and cell lysate, respectively, was mixed with 25 μL 1 mM p-nitrophenyl-N-acetyl β-d-glucosamine in citrate buffer (0.1 mol; pH 4.5) and incubated for 1 hour at 37°C. The reaction was stopped with 200 μL carbonate buffer (0.1 mol Na2CO3; 0.1 mol NaHCO3; pH 9) and OD was measured at 405 nm. Release of β-hexosaminidase was calculated as percent release of total content (100x amount supernatant/amount supernatant + cell lysate). Data are shown as mean ± SD. Data are pooled from 1 (Fig 1, B, D, G, H, and L), 2 (Fig 1, C, E, F, M, and N; Fig 2, A, B, F, G, and H), or 3 (Fig 1, I, J, and K; Fig 2, C, D, E, I, J, and K; Fig E1) independent experiments. Statistical analysis was performed with nonparametric 1-way ANOVA for all the graphs that contain more than 2 groups and 2-way ANOVA for the ear swelling curves. For the ear swelling curves, unless stated otherwise in the figure legend, the difference depicted is between the red and the black curves. #P < .001, ∗∗P < .01, and ∗P < .05.