Impaired Mast Cell-Driven Immune Responses in Mice Lacking the NFATc2

This information is current as Marc Becker, Valeska Heib, Matthias Klein, Fatma Doener, of September 24, 2021. Tobias Bopp, Christian Taube, Markus Radsak, Hansjörg Schild, Edgar Schmitt and Michael Stassen J Immunol 2009; 182:6136-6142; ; doi: 10.4049/jimmunol.0802878

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Impaired Mast Cell-Driven Immune Responses in Mice Lacking the Transcription Factor NFATc21

Marc Becker,* Valeska Heib,† Matthias Klein,* Fatma Doener,* Tobias Bopp,* Christian Taube,‡ Markus Radsak,‡ Hansjo¨rg Schild,* Edgar Schmitt,* and Michael Stassen2*

The three calcium-dependent factors NFATc1, c2, and c3 are expressed in cells of the immune system and play pivotal roles in modulating cellular activation. With regard to NFATc2, it was reported that NFATc2-deficient mice display increased immune responses in several models for infection and allergy in vivo. This led to the assumption that NFATc2 is involved in the mainte- nance of immune homeostasis. Using the synthetic TLR7 agonist imiquimod as an adjuvant in epicutaneous peptide immunization, we observed that both the inflammatory reaction and the peptide-specific CTL response are severely impaired in NFATc2-deficient mice. Detailed analyses revealed that early production of proinflammatory cytokines, lymph node hypertrophy, and migration of

Langerhans cells are strongly reduced in NFATc2-deficient animals. With the aid of mast cell-deficient mice and reconstitution Downloaded from experiments using mast cells derived from either NFATc2-deficient mice or wild-type controls, we were able to show that NFATc2 expressed in mast cells is critical for the initiation of inflammation, migration of Langerhans cells, and the development of full-blown CTL responses following epicutaneous immunization. Thus, NFATc2 is an important factor controlling mast cell accessory function at the interface of innate and adaptive immunity. The Journal of Immunology, 2009, 182: 6136–6142.

he NFAT family of transcription factors is widely ex- were generated by three groups independently (7–9). No defects in http://www.jimmunol.org/ pressed in numerous cell types and comprises the four the development of thymocytes were observed for NFATc2-defi- T genuine members NFATc1–c4 and the distantly related cient mice but there was a slight increase of T and B cells in the TonEBP. The latter controls the osmotic stress-induced expression periphery accompanied by a modest splenomegaly. Interestingly, of some cytokines, whereas the NFATc members of this family are such mice were shown to develop enhanced immune responses in activated by a calcium signaling pathway in which calcium-calm- several models of infection and allergy (8, 10–13). odulin complexes activate the cyclosporin-sensitive phosphatase Double deficiency for NFATc2 and NFATc3 causes massive calcineurin which then binds to and dephosphorylates NFAT in the splenomegaly, lymphadenopathy, and a strong increase in serum cytosol. Dephosphorylated NFAT molecules translocate into the IgG1 and IgE levels. Such mice show a dramatic increase in ac- nucleus and cooperatively bind with their nuclear partners to DNA tivated peripheral T cells able to overproduce cytokines indepen- by guest on September 24, 2021 (1–3). NFATc1–c3 are expressed in the immune system and es- dently of costimulatory signals. Ultimately, these animals develop pecially their role in T cells, which coexpress these factors, has an autoaggressive and fatal phenotype (14, 15). These studies sug- been intensively studied. gested that NFATc2 and NFATc3 have important and partly over- Detailed analyses using mice lacking distinct NFAT factors re- lapping functions in maintaining T cell homeostasis and tolerance vealed the development of quite disparate phenotypes. NFATc1- (16). In this context, we were able to show that CD4ϩ T cells deficient mice die in utero due to defects in the generation of car- lacking both factors are able to escape suppression by CD4ϩ diac valves and septa (4). Blastocyst complementation assays CD25ϩ T regulatory cells which might, at least partly, explain the showed that NFATc1Ϫ/Ϫ/Rag-1Ϫ/Ϫ chimeric mice have reduced development of lymphoproliferative disorder (17). numbers of thymocytes, impaired proliferation of peripheral lym- We previously reported that epicutaneous application of a phocytes, and reduced synthesis of Th2 cytokines (5). Mice lack- cytotoxic T lymphocyte (CTL)3 epitope in combination with the ing NFATc3 develop normally and are phenotypically inconspic- synthetic TLR7 ligand imiquimod leads to the transport of ap- uous, yet they have defects in positive selection of T cells due to plied Ag to draining lymph nodes (LNs) and mounts a full- increased apoptosis of thymocytes. Although diminished in num- blown CTL response (18). Imiquimod acts as an adjuvant, in- ber, an increased percentage of peripheral NFATc3Ϫ/Ϫ T cells ducing both an inflammatory response and the emigration of display an activated phenotype, however, proliferation and cyto- Ag-presenting Langerhans cells (LCs) out of the epidermis. In kine production appeared normal (6). Mice deficient for NFATc2 the present study we show that inflammation, migration of LCs, development of LN hypertrophy, and the generation of a CTL response are severely impaired in NFATc2-deficient mice. De- *Institute for Immunology, University of Mainz, Mainz, Germany; †Department of Dermatology and Venereology, University of Innsbruck, Innsbruck, Austria; and tailed analyses revealed that mast cells lacking NFATc2 are the ‡Third Medical Clinic, University of Mainz, Mainz, Germany cause of the observed phenomena, identifying NFATc2 as a Received for publication September 2, 2008. Accepted for publication March 4, 2009. critical factor for the accessory function of mast cells in vivo. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 Abbreviations used in this paper: CTL, cytotoxic T lymphocyte; BMMC, Bone 1 marrow-derived mast cell; DC, dendritic cell; HGPRT, hypoxanthine-guanine phos- Supported by the Deutsche Forschungsgemeinschaft, Grants STA 984/1-1 and phoribosyl transferase; LC, Langerhans cell; LN, lymph node; PCA, passive SFB548, A10 (M.S.), A11 (C.T.), and A6 (E.S.). cutaneous anaphylaxis; TCI, transcutaneous immunization; KC, keratinocyte 2 Address correspondence and reprint requests to: Dr. Michael Stassen, Institute for chemoattractant. Immunology, Johannes Gutenberg University, Hochhaus am Augustusplatz, 55131 Mainz, Germany. E-mail: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802878 The Journal of Immunology 6137

Materials and Methods 3Ј; mTNF-␣.reverse – 5Ј-AGA TAG CAA ATC GGC TGA CGG TGT Ј ␤ Ј Mice GGG-3 ; mIL-1 .forward – 5 -CAA CCA ACA AGT GAT ATT CTC CAT G-3Ј; mIL-1␤.reverse – 5Ј-GAT CCA CAC TCT CCA GCT GCA-3Ј. Genetically mast cell-deficient KitW-sh/KitW-sh mice, as well as the congenic The PCR products were separated by electrophoresis on 2% agarose gels Kitϩ/Kitϩ wild-type littermates, were obtained by intercrossing heteroge- and visualized by ethidium bromide staining with UV light illumination. neous KitW-sh/Kitϩ mice provided by Marcus Maurer (Charite´-Universita¨- There were 30 HGPRT, 35 TNF-␣, and IL-1␤ PCR cycles. To quantify tsmedizin Berlin, Berlin, Germany). NFATc2-deficient mice were provided MCP-1- and KC-mRNA, real-time PCR was performed using ABSOLUTE by Edgar Serfling (University of Wu¨rzburg, Wurzburg, Germany). These QPCR SYBR Green Mix (ABgene) and the specific primers (5 pmol each): mice were generated in the laboratory of Laurie Glimcher (Harvard Med- mMCP-1.forward – 5Ј-CTC AGC CAG ATG CAG TTA ACG-3Ј; mMCP- ical School, Boston, MA). The TCR transgenic OT-I mice were obtained 1.reverse – 5Ј-TTG GGA TCA TCT TGG TGG TG-3Ј; mKC.forward – from The Jackson Laboratory and bred in our own animal facility. All mice 5Ј-CAC CCA AAC GAA GTC ATA GC-3Ј; mKC.reverse – 5Ј-AGC CAG were on a C57BL/6 genetic background and used at the age of 6–12 wk. CGT TCA CCA GAC AGG-3Ј. Animal procedures were conducted in accordance with the institutional guidelines. Generation of lymph node cell suspensions and proliferation assay of OT-I T cells Generation of bone marrow-derived mast cells (BMMCs) and local reconstitution of mast cell-deficient mice One day before immunization, spleens of killed OT-I TCR transgenic mice were isolated and processed to single-cell suspensions. The spleen cells BMMCs were generated according to standard procedures (19). For local were labeled with 2.5 ␮M of CFSE (Molecular Probes) and injected i.v. reconstitution, 5 ϫ 105 BMMC were injected into the ear pinna of 6-wk- Immunization of mice was performed on 2 consecutive days by mixing of old KitW-sh/KitW-sh mice 6–8 wk before the experiments were conducted. 100 ␮g SIINFEKL with 40 mg preformulated imiquimod cream per mouse The assessment of reconstitution efficiencies using the heparin-binding pro- per application. The mixture was applied to one ear of the anesthetized

tein avidin was done as described by Heib et al. (20). mouse. Two days after the first immunization, mice were killed and the Downloaded from auricular LN isolated. LN cell suspensions were generated by collagenase Generation of single-cell suspensions from murine ear tissue digestion as described previously (21) and immunostained with allophy- cocyanin-conjugated anti-CD8 (eBioscience). Mice were killed and the ears were divided into dorsal and ventral halves. The two halves of one ear were incubated in a solution with 0.5 mg/ml Mouse dendritic cell (DC) generation and activation Liberase (BD Pharmingen) and DNase (Sigma-Aldrich) in MEM for 90 min at 37°C. The two halves were shredded in 1 ml of MEM by a Medi- Mouse immature DCs were generated from bone marrow according to ␮ machine (BD Pharmingen). The cell suspensions of the ears were collected standard protocols (22). On day 6, DCs were stimulated with 50 g/ml http://www.jimmunol.org/ in tubes, washed twice, and immunostained with FITC-conjugated anti- TLR3 ligand poly(I:C) (Amersham Biosciences) for 18 h. The activated Gr-1 and PE-conjugated anti-MHC-II (BD Pharmingen). DCs were loaded with SIINFEKL peptide (100 nM) for1hat37°C. The peptide-loaded and activated DCs cells (106) were injected i.p. into Passive cutaneous anaphylaxis (PCA) NFATc2Ϫ/Ϫ or NFATc2ϩ/ϩ control mice. For the induction of PCA, both NFATc2-deficient and wild-type mice Transcutaneous immunization and in vivo cytotoxicity assay (C57BL/6) received intradermal injections of 1 ␮g of murine monoclonal anti-DNP IgE (clone SPE-7) in 50 ␮l of PBS in the right ear and 50 ␮lof Transcutaneous immunization was done according our standard protocol PBS in the left ear as control. Mice were challenged 24 h later by i.v. (23). In brief, mice were immunized on their shaved backs on 2 consecu- injection of 50 ␮g of DNP-human serum albumin in 100 ␮l of PBS, in- tive days with 60 mg preformulated imiquimod cream mixed with 100 ␮g ϩ

cluding 0.5% Evans blue, into the tail vein (all reagents from Sigma-Al- SIINFEKL peptide per application. For detection of peptide-specific CD8 by guest on September 24, 2021 drich). Mice were killed 30 min after i.v. injection of Ag and the ears T cells, blood samples were collected 6 days after immunization. After a removed, weighted, and incubated in 200 ␮l of formamide at 55°C for 24 h. hypotonic lysis step, the cells were stained with PE-labeled SIINFEKL- Extravasation of Evans blue was determined by photometric analysis at H2-Kb tetramer and allophycocyanin-Cy7-conjugated anti-CD8. Pro- 620 nm. pidium iodide was used to exclude nonviable cells from analysis. Functional analysis of CTL responses induced by immunizations were Application of imiquimod and measurement of ear thickness performed by an in vivo cytotoxicity assay with differentially CFSE-la- beled syngeneic target cells (0.4 ␮M and 4 ␮M CFSE; Molecular Probes). The application of imiquimod was progressed by daily treatment of one ear Six days after immunization (immunized with imiquimod or DC immuni- with ϳ40 mg Aldara 5% creme (3M Pharmaceuticals) for up to 6 days. The zation), the CFSElow population was pulsed with 1 ␮M SIINFEKL and the contralateral ear was treated with vehicle. The resultant ear swelling after CFSEhigh population was left without peptide. Some of these target cells local application was measured with a micrometer (Mitutoyo). Aldara con- (2 ϫ 107) were adoptively transferred into the immunized mice at a ratio tains 5% imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4- 1:1 (CFSElow/CFSEhigh). Eight or 20 hours (mice immunized with imi- amine) in vehicle (isooctadecanoic acid (20–30%), ethoxylated sorbitan quimod or DC-immunized mice, respectively) after i.v. injection of the monostearate (1–5%), octadecan-1-ol (1–5%), petrolatum (1–5%), hexa- target cells, mice were killed and the spleens processed to single-cell sus- decan-1-ol (1–5%), glycerol (1–5%), benzyl alcohol (1–5%), and water pensions. Specific in vivo lysis of target cells was evaluated as follows: (40–60%)). specific lysis [%] ϭ (No. of nonpeptide-pulsed cells Ϫ No. of peptide- Preparation of epidermal sheets pulsed cells)/No. of nonpeptide-pulsed cells. The dorsal and ventral halves of mouse ears were split and the dorsal Statistical analysis halves were incubated in 0.5% dispase solution for 20 min (Dispase II; Data are expressed as mean Ϯ SD. Student’s t test was used to compare Roche). The epidermis was then removed, fixed in acetone, and stained mean values between two experimental groups where appropriate. Values with anti-MHC-II (clone M5.114.15.2) and goat anti-rat Ab labeled with of p Ͻ 0.05 were considered significant. Alexa594 (Molecular Probes). All washing steps were done in PBS/0.5% BSA. LC numbers were determined by fluorescence microscopy using Cell Imaging Software (Olympus) by counting at least three independent areas Results of 0.3 ϫ 0.4 mm each. The values shown in Figs. 2A and 4D for LC Delayed inflammation in mice lacking NFATc2 migration refer to the reduction in LC numbers compared with the un- treated contralateral ears. Topical treatment with imiquimod initiates an inflammatory skin response which can be measured as an increase in ear thickness PCR against time. In wild-type mice, ear swelling can be detected 24 h Mouse ears were frozen in liquid nitrogen, total RNA was purified (RNeasy after the initial application of imiquimod (Fig. 1A). In contrast, the kit; Qiagen), reverse transcribed into cDNA, and analyzed by PCR. PCR of inflammatory skin reaction is severely delayed in NFATc2-defi- the cDNA was performed using Advantage 2 Polymerase Mix (Clontech) cient mice, reaching on day 3 an extent comparable with that ob- with specific primers: hypoxanthine-guanine phosphoribosyl transferase (HGPRT).forward – 5Ј-GTT GGA TAC AGG CCA GAC TTT GTT G-3Ј; served in wild-type animals on day 1. HGPRT.reverse – 5Ј-GAG GGT AGG CTG GCC TAT AGG CT-3Ј; It has been reported that pronounced inflammatory changes in mTNF-␣.forward – 5Ј-TCT ACT GAA CTT CGG GGT GAT CGG TCC- the skin following topical application of imiquimod are based on a 6138 NFATc2 CONTROLS MAST CELL ACTIVITY

FIGURE 1. NFATc2-deficient mice display delayed inflammatory re- sponses following topical treatment with imiquimod. A, Starting on day 0, ears of mice where treated daily with imiquimod. The indicated values re- fer to the ear thickness at the begin- ning of the experiment. Shown are the means (ϩSD) from at least six mice per group. B, Ears of mice were treated daily with imiquimod and cell suspensions were prepared on days 3 and 6 and analyzed for the presence of Gr-1pos MHC-IIneg neutrophils by FACS. Shown are the means (ϩSD) from six to eight animals per condi- tion. n.s., Not significant. C and D, Two hours following application of

either imiquimod or vehicle, RNA Downloaded from was isolated from the ears and ana- lyzed for the expression of target mRNAs using RT-PCR (TNF-␣, IL- 1␤) or quantitative RT-PCR (KC, IFN-␤) as indicated. HGPRT served as housekeeping transcript. Shown are

one representative gel and the means http://www.jimmunol.org/ p Ͻ ,ء .ϩSD) of four experiments) .p Ͻ 0.002 ,ءء ;0.001

massive increase in leukocytes in the dermis, mostly MHC-IIϩ, CD4ϩ, CD11cϩ, and Gr-1ϩ cells (24). By using FACS analy- by guest on September 24, 2021 ses, we were able to confirm these data (not shown), but in addition we also found a striking difference between wild-type and NFATc2-deficient mice with respect to the appearance of neutrophils in the inflamed tissue (Fig. 1B). In the latter, influx of neutrophils is markedly delayed, which is in accordance with data shown in Fig. 1A. Obviously, the early innate inflammatory response is delayed in mice lacking NFATc2. To investigate this phenomenon in more detail, we performed RT-PCR anal- yses using ear skin 2 h following the initial application of imi- quimod. As depicted in Fig. 1, C and D, the expression of im- portant proinflammatory mediators, TNF-␣, IL-1␤, KC, and IFN-␤, is severely impaired in NFATc2-deficient animals.

NFATc2-deficiency leads to impaired migration of LCs and reduced hypertrophy of draining LNs In combination with the initiation of an inflammatory response, it was also reported recently that imiquimod induces the mi- gration of LCs, important APCs in the epidermis (25). LCs form a dense cellular network that can be visualized by the staining of epidermal sheets with Abs to langerin (CD207), a C-type lectin selectively expressed in LCs (26). NFATc2-deficiency per se has no influence on the numbers of LCs, which is ϳ900 LCs/mm2 in both wild-type and knockout mice, but it severely FIGURE 2. NFATc2-deficiency impairs emigration of LCs and hypertro- impairs emigration of LCs out of the epidermis following top- phy of LNs following application of imiquimod. A, Mice were treated daily with imiquimod. On days 3 and 6, epidermal sheets were prepared and stained ical treatment with imiquimod (Fig. 2A). Importantly, emigra- for the presence of langerin-positive cells. The values shown refer to the re- tion of LCs is not only delayed in NFATc2-deficient mice but duction in langerin-positive cells after treatment compared with the untreated also remains reduced at later time points. Additionally, hyper- contralateral ears. B, Auricular LNs were prepared at the end of the experiment trophy of draining LNs is severely reduced in the absence of shown in A and total cell numbers were assessed. Shown are the means of eight .p Ͻ 0.001 ,ءء ;p Ͻ 0.02 ,ء .(NFATc2 as well (Fig. 2B). mice per group (ϩSD The Journal of Immunology 6139

Table I. Engraftment of mast cell-deficient mice with BMMCs derived from different donor strainsa

Mast Cells/mm Ear Cartilage

KitW-sh/W-sh ND Kitϩ/ϩ 30.9 (Ϯ4.24) NFATc2Ϫ/Ϫ 32.2 (Ϯ6,98) NFATc2Ϫ/Ϫ3 KitW-sh/W-sh 33.9 (Ϯ4.91) Kitϩ/ϩ3 KitW-sh/W-sh 36.1 (Ϯ2.51)

a Mice were reconstituted intra pinna with BMMCs from different donors, as described in the methods section. After 6 weeks, ear sections were stained with avi- din-fluorophor conjugate and mast cell numbers determined. Shown are the means (ϮSD) from four individuals per group. ND, Not detectable.

daily on 2 consecutive days. Peripheral blood was collected on day 6 and stained for the presence of SIINFEKL-specific CTLs as de- picted in Fig. 3A. In wild-type mice, expansion of Ag-specific

CTLs can easily be followed over 1% of CTLs can be stained with Downloaded from H-2KbSIINFEKL tetramer. In contrast, expansion of CTLs is se- verely reduced in the absence of NFATc2 (0.36%). It should be mentioned that we did not find significant differences between NFATc2-deficient mice and their congenic littermates on a C57BL/6 background with respect to the total numbers of periph-

eral CTLs, monitored between 7- and 23-wk of age (data not http://www.jimmunol.org/ shown). Besides phenotypic staining of Ag-specific CTLs, their cytolytic activity can also be monitored in vivo. To this end, SIINFEKL- laden CFSElow-labeled syngeneic splenocytes were mixed in a ra- tio 1:1 with CFSEhigh-labeled splenocytes without Ag and injected i.v. Thereafter, lysis of SIINFEKL-laden target cells can be fol- lowed by a decrease in CFSElow-labeled cell numbers, whereas the CFSEhigh-labeled fraction serves as internal reference (Fig. 3B). According to the expectations, the appearance of high numbers of by guest on September 24, 2021 Ag-specific CTLs in wild-type mice is accompanied by a strong FIGURE 3. NFATc2-deficient mice display an impaired CTL response cytolytic activity in vivo, while the ability to initiate a cytolytic after transcutaneous peptide immunization with imiquimod as adjuvant. A, T cell response by transcutaneous immunization is severely im- Mice were treated with peptide plus imiquimod or peptide plus vehicle paired in mice lacking NFATc2. However, the ability to present once daily on two consecutive days on their shaved backs. On day 6, Ag and to generate an efficient CTL response per se is unaf- peripheral blood was stained for peptide-specific CD8ϩ T cells using a fected in such animals. This can be concluded from data shown fluorescently labeled SIINFEKL-specific tetramer. The depicted numbers in Fig. 3C, where NFATc2Ϫ/Ϫ Ag-laden bone marrow-derived ϩ represent the percentages of Ag-specific cells within the CD8 population. dendritic cells elicit full-blown cytolytic responses in both B, Animals from the experiment described in A were injected i.v. on day 6 wild-type and NFATc2-deficient mice. with CFSE-labeled syngeneic splenocytes. CFSElow-labeled splenocytes were previously pulsed with the antigenic SIINFEKL peptide and mixed at NFATc2 expressed in mast cells is critical to initiate high a ratio 1:1 with CFSE -labeled splenocytes. Lysis of peptide-laden target inflammatory and adaptive immune responses cells in spleen cell suspensions was determined by FACS analysis 8 h after adoptive transfer. Specific lysis [%] ϭ (number of cells nonpeptide NFATc2 deficiency obviously does not impair the development of pulsed Ϫ number of peptide-pulsed cells)/number of cells nonpeptide mast cells in vivo, which can be concluded from comparable mast pulsed. C, Wild-type or NFATc2-deficient mice were immunized i.p. with cell numbers found in skin from wild-type and NFATc2-deficient peptide-laden DC generated from NFATc2-deficient mice in vitro. Six days mice (Table I). Furthermore, anaphylactic degranulation of mast after immunization, cytolytic activity against SIINFEKL-pulsed syngeneic cells following cross-linking of IgE is unaffected in mice lacking splenocytes was assessed as described in B. Shown are representative NFATc2 (Fig. 4A). FACS stainings and means (ϮSD) from eight to ten animals per group. This observation fits to our previous report showing that IgE- mediated degranulation of BMMCs derived from NFATc2-defi- cient animals is unimpaired, although the expression of some cy- Priming of CTLs by transcutaneous peptide immunization (TCI) tokines is severely reduced (27). with imiquimod as adjuvant is impaired in NFATc2-deficient Consequently, we investigated the roles of NFATc2 expressed mice in mast cells in the TCI model. BMMCs express low but detectable Our observation that both emigration of LCs and hypertrophy of levels of TLR7 mRNA (28) and expression is unaffected in the draining LNs are impaired in mice lacking NFATc2 prompted us absence of NFATc2 (data not shown). Incubation of BMMCs only to investigate whether the induction of an Ag-specific CTL re- marginally induced production of IL-1␤ mRNA, as shown in Fig. sponse is also affected in these animals. As a model we chose the 4B, but the expression of this cytokine mRNA was significantly epitope SIINFEKL from chicken OVA (OVA257–264) administered reduced in the absence of NFATc2. Under these conditions, we did in combination with imiquimod on the shaved backs of mice once not observe expression of TNF-␣ mRNA (data not shown). 6140 NFATc2 CONTROLS MAST CELL ACTIVITY

FIGURE 4. NFATc2 expressed in mast cells promotes early inflammation, migration of LCs, and expansion of Ag- specific CTLs. A, In vivo mast cell acti- vation was measured by IgE-mediated PCA. Thirty minutes after application of Ag, Evans blue was extracted and mea- sured by spectrophotometric analysis. Shown are means (ϩSD) from four ani- mals per group. B, BMMCs were left un- stimulated or were treated with 10 ␮g/ml imiquimod for 2 h. Quantitative RT- PCR was performed for the presence of IL-1␤ mRNA. HGPRT served as housekeeping transcript. Shown are means (ϩSD) of p Ͻ 0.03. C, Mast ,ء ,three experiments cell-deficient sash mice were reconsti- tuted locally with NFATc2-competent or NFATc2-deficient BMMCs, indicated by

the arrows. Reconstituted mice as well as Downloaded from wild-type and sash mice were treated daily with imiquimod and ear thickness was measured. Shown are means (ϩSD) p Ͻ ,ء .from six to eight mice per group p Ͻ 0.03. D, Ears of mice were ,ءء ;0.001 treated daily with imiquimod and epider- mal sheets were prepared on days 3 and 6 http://www.jimmunol.org/ and stained for the presence of LCs. Ar- rows indicate the local reconstitution of mast cell-deficient KitW-sh/W-sh mice with BMMCs derived from either wild-type or NFATc2-deficient mice. Shown are means (ϩSD) from three to four individ- p Ͻ ,ءء ;p Ͻ 0.02 ,ء .uals per condition 0.002, #, p Ͻ 0.001. E, Mice of the indi- cated genotypes were topically treated with either vehicle or imiquimod. Two by guest on September 24, 2021 hours later, mice were killed and RNA was isolated from the ear tissue and ana- lyzed for the expression of target mRNAs TNF-␣ and IL-1␤. HGPRT served as housekeeping transcript. Shown are rep- resentatives from at least three animals per group. F, CFSE-labeled SIINFEKL- specific CTL from OT-I mice were adop- tively transferred i.v. on day Ϫ1. Ears were then either sham-treated or immu- nized with imiquimod as adjuvants and peptide Ag SIINFEKL once daily on day 0 and 1. On day 2, cell suspensions were pre- pared from draining auricular LNs and an- alyzed for the presence of CFSE-stained cells. Representatives of at least three indi- viduals per condition are shown. G, Mean fluorescence intensities from the experi- p Ͻ ,ءء ;p Ͻ 0.05 ,ء .ments described in F 0.03. n.s., Not significant.

To investigate the role of mast cells in vivo we used mast cell- to imiquimod, whereas reconstitution with NFATc2Ϫ/Ϫ mast cells deficient KitW-sh/W-sh (“sash”) mice, which can be reconstituted closely resembles the delayed inflammation seen in sash mice. with BMMCs derived from either wild-type or NFATc2-deficient Furthermore, emigration of LCs cannot be restored following mice (29). Six weeks following local reconstitution of the ear skin, transfer of NFATc2-deficient mast cells (Fig. 4D). Using this comparable mast cell numbers can be found in sash mice engrafted model we recently reported (20) that mast cell-derived cytokines with either wild-type or NFATc2Ϫ/Ϫ mast cells. In addition, there and TLR7 expressed in mast cells are critical for the early onset of is no difference in mast cell numbers between wild-type and inflammation (TNF-␣) and emigration of LCs (IL-1␤). Production NFATc2-deficient mice (Table I). of these cytokines is severely impaired in sash mice and can be As depicted in Fig. 4C, reconstitution of sash mice with restored following transfer of BMMCs derived from wild-type but BMMCs from wild-type mice restores the inflammatory response not from NFATc2-deficient mice (Fig. 4E). Reconstitution of sash The Journal of Immunology 6141 mice with wild-type BMMCs is critical to restore the production of This can also be concluded from experiments in which transfer of early cytokines (IL-1␤, TNF-␣) following topical application of wild-type BMMCs, but not of NFATc2-deficient BMMCs, in sash imiquimod. However, BMMCs only marginally produce IL-1␤ mice restores production of TNF-␣ and IL-1␤ mRNAs following mRNA and no TNF-␣ mRNA upon treatment with imiquimod in topical application of imiquimod. With regard to the ability of mast vitro. To explain this discrepancy, it is reasonable to assume that cells to promote CTL responses in our model, it is reasonable to mast cells are able to change their characteristics following trans- assume that mast cells act at the level of the APC. We previously fer in vivo, determined by the local microenvironment (30). reported that mast cell-derived IL-1␤ is crucial for the migration of The most outstanding consequence of NFATc2-deficiency LCs to the draining LNs (20). In line with this, selective ablation solely in mast cells is the inability of such animals to develop of LCs in the presence of mast cells before TCI severely impairs adequate cytolytic responses as depicted in Fig. 4, F and G.In the development of a CTL response in vivo, demonstrating that these experiments, the ears of sash mice were locally reconstituted LCs act as important APCs (P. Lopez and M. P. Radsak, in prep- with BMMCs derived from the indicated strains and imiquimod aration). plus peptide was applied to the ears of the animals. TCI via the ears Obviously, NFATc2 is an important factor controlling the acti- was chosen because systematic reconstitution of sash mice by i.v. vation of mast cells and appears to be an attractive target for ther- injection of BMMCs does not lead to the development of dermal apeutic intervention in mast cell-mediated diseases. mast cells (29). Immunization was followed by measuring the expansion of Disclosures adoptively transferred CFSE-labeled T cells from OT-I mice ex- The authors have no financial conflict of interest. pressing a SIINFEKL-specific TCR. As detailed in Fig. 4, F and G, Downloaded from proliferation of labeled T cells after TCI is severely impaired in References mast cell-deficient sash mice and can only be restored following 1. Macian, F. 2005. NFAT : key regulators of T-cell development and func- Ϫ/Ϫ transfer of wild-type, but not of NFATc2 , mast cells. tion. Nat. Rev. Immunol. 5: 472–484. Thus, NFATc2 expressed in mast cells plays important roles in 2. Serfling, E., F. Berberich-Siebelt, A. Avots, S. Chuvpilo, S. Klein-Hessling, M. K. Jha, E. Kondo, P. Pagel, J. Schulze-Luehrmann, and A. Palmetshofer. various aspects of mast cell-mediated immunity in vivo. 2004. NFAT and NF-␬B factors-the distant relatives. Int. J. Biochem. Cell Biol.

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