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The Journal of Immunology, 2006, 177: 1296-1305.
Copyright © 2006 by The American Association of Immunologists

Zinc Is Required for Fc{epsilon}RI-Mediated Mast Cell Activation1

Koki Kabu*,{dagger}, Satoru Yamasaki*, Daisuke Kamimura*, Yukitaka Ito*,{dagger}, Aiko Hasegawa*,{dagger}, Emi Sato*,{dagger}, Hidemitsu Kitamura*, Keigo Nishida* and Toshio Hirano2,*,{dagger}

* Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology (RCAI), Yokohama, Kanagawa, Japan; and {dagger} Laboratories of Developmental Immunology, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Zinc (Zn) is an essential nutrient, and its deficiency causes growth retardation, immunodeficiency, and neuronal degeneration. However, the precise roles and molecular mechanism(s) of Zn function in immune response have not been clarified. Mast cells (MCs) are granulated cells that play a pivotal role in allergic reactions and inflammation. The granules of MCs contain various chemical mediators and inflammatory cytokines that are released upon Fc{epsilon}RI cross-linking. In this study, we report that Zn is essential for MC activation both in vitro and in vivo. We showed that a Zn chelator, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine, inhibited in vivo allergic reactions such as PCA and PSA. Consistent with this, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine significantly inhibited the Fc{epsilon}RI-induced degranulation and cytokine production. We found that Zn was required for Fc{epsilon}RI-induced translocation of granules to the plasma membrane, a process that we have shown to be important for MC degranulation. In addition, we showed that Zn was essential for plasma membrane translocation of protein kinase C and subsequent nuclear translocation of NF-{kappa}B, leading to cytokine production, such as IL-6 and TNF-{alpha}. These results revealed that Zn was involved in multiple steps of Fc{epsilon}RI-induced MC activation and required for degranulation and cytokine production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Zinc (Zn)3 is an essential nutrient, and its deficiency causes growth retardation, immunodeficiency, and neuronal degeneration (1, 2). There are many enzymes, signaling molecules, and transcription factors whose function is critically dependent on and/or regulated by Zn. Electron microscopic studies in the 1960s demonstrated that mast cell (MC) granules are rich in Zn (3). However, the precise roles and molecular mechanism(s) of Zn function in MCs have not been clarified.

MCs are thought to participate in a variety of immune responses, such as allergic reaction, innate immunity, and autoimmunity (4, 5, 6, 7, 8, 9, 10, 11). Engagement of the Fc{epsilon}RI on MCs initiates signaling pathways leading to the exocytosis of granules containing various cytokines and chemical mediators. These released molecules play pivotal roles in the inflammatory reactions observed in patients with allergy and autoimmune diseases.

The antigenic cross-linking of Fc{epsilon}RI induces the activation of several protein tyrosine kinases, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular calcium (12, 13, 14, 15). The degranulation process is thought to be mediated by granule-to-plasma and granule-to-granule membrane fusion (16, 17). This membrane-fusion process is regulated by the Lyn/SLP-76, LAT/PLC{gamma}1-calcium mobilization pathway. In addition to this calcium-dependent process, we recently showed that a calcium-independent process is required for microtubule-dependent granule translocation to the plasma membrane (18). This calcium-independent process is regulated by the Fyn/Gab2/RhoA signaling pathway, but not by Lyn/SLP-76 (18).

In contrast, Fc{epsilon}RI stimulation can induce the de novo synthesis of proinflmmatory cytokines such as IL-6 and TNF-{alpha} (19, 20). Especially both calcium and diacylglycerol activate conventional protein kinase C (cPKC) isoforms that mediate a critical positive signal necessary for cytokine production in MCs (21, 22). In addition, cPKCs have two common regions in the regulatory domain, C1 and C2. The C1 region has two cysteine-rich loops (Zn finger-like motifs) that interact with diacylglycerol or phorbol esters and regulate activation or translocation of PKC (23, 24, 25). The C2 region mediates calcium binding. Previous studies using PKCbeta-deficient mice revealed that bone marrow-derived MCs (BMMCs) lacking both PKCbetaI and PKCbetaIII exhibited lower levels of IL-6 release than did the wild-type cells in response to Ag (26). Collectively, these reports indicate that PKC isoforms play important roles in cytokine production.

In this study, we found that Zn was required for both degranulation and cytokine production in MCs. In fact, Zn was essential for granule translocation to the plasma membrane. Furthermore, Zn was required for Fc{epsilon}RI-mediated plasma membrane translocation of PKC and NF-{kappa}B activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Metal chelators and Abs

N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine) (TPEN) (Ka = 1015.58 M–1, 1014.61 M–1, 1010 M–1, 1010.27 M–1 for Zn2+, Fe2+, Cu2+, and Mn2+, respectively) (27, 28); ZnAF-2DA (aminofluorescein-2 diacetate (Ka = 109.15 M–1 for Zn2+) (29, 30); 2,2'-dipyridyl (Ka = 1017.2M–1 for Fe2+); desferrioxamine mesylate (Ka = 1031 M–1 for Fe3+) (31); disodium bathocuproine disulphonate (for Cu2+) (31), and p-aminosalicylic acid (for Mn2+) (32, 33). TPEN, 2,2'-dipyridyl, desferrioxamine, and bathocuproine disulphonate were all purchased from Sigma-Aldrich. Zn indicators, Newport green, and ZnAF-2DA were purchased from Molecular Probes and Dai-ichi Kagaku, respectively. Abs for Syk, p65, phospho-IKK{alpha}beta, IKK{alpha}beta, PKCbetaI, and Lyn were all purchased form Santa Cruz Biotechnology. Abs for LAT, PLC{gamma}1, Gab2, pI{kappa}B{alpha}, and I{kappa}B{alpha} were purchased from Upstate Biotechnology, and anti-beta-tubulin was from Sigma-Aldrich. Anti-Fyn mAb was a gift from K. Hattori (National Center of Neurology and Psychiatry, Tokyo, Japan).

Cell culture

BMMCs were prepared as described previously (34). Briefly, 8-wk-old C57/BL6 mice were sacrificed, and their BM cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10 mU/ml penicillin, 0.1 mg/ml streptomycin, 40 µM 2-ME, and IL-3 in a 5% CO2 and 95% humidified atmosphere at 37°C. After 4–5 wk of culture, the cells were confirmed to show the cell surface expression of Fc{epsilon}RI and c-Kit and were used for experiments (< 98% MCs).

BMMC degranulation assay

Cells (1 x 106/ml) were sensitized with 1 µg/ml IgE (anti-DNP IgE clone SPE-7; Sigma-Aldrich) for 6 h at 37°C. After sensitization, the cells were washed twice with Tyrode’s buffer (10 mM HEPES (pH 7.4), 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose), then suspended in the same buffer containing 0.1% BSA and stimulated with polyvalent dinitrophenyl-human serum albumin (DNP-HSA; Sigma-Aldrich) for 30 min. For the beta-hexosaminidase reaction, 50 µl of supernatant or cell lysate and 100 µl of 1.3 mg/ml p-nitrophenyl-N-acetyl-d-glucosamide (in 0.1 M citrate, pH 4.5) were added to each well of a 96-well plate, and the color was developed for 60 min at 37°C. The enzyme reaction was then stopped by adding 150 µl of 0.2 M glycine-NaOH (pH 10.2), and the absorbance at 405 nm was measured in a microplate reader (Bio-Rad). Cells were lysed with Tyrode’s buffer containing 1% Triton X-100, and the beta-hexosaminidase activity was measured. The percentage of beta-hexosaminidase released was calculated using the following formula: percentage release = supernatant/(supernatant + cell lysate) x 100.

Measurement of cytokines and chemical mediators

Cells were activated as described above, and TNF-{alpha} and IL-6 in the cell culture supernatants were measured with an ELISA kit (BioSource International). Histamine and leukotrienes were determined using an ELISA kit (Beckman Coulter and Cayman Chemical, respectively), following the manufacturer’s recommendation.

Cell lysates and immunoblotting.

BMMCs (1 x 106/ml) were sensitized with 1 µg/ml IgE for 6 h at 37°C. Cells were either untreated or treated with 10 µM TPEN for 2 h, then stimulated with 100 ng/ml DNP-HSA for 0, 2, 5, 15, or 30 min. After stimulation, the cells were harvested and lysed with 100 µl of lysis buffer (20 mM Tris-HCl (pH 7.4) 4°C, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.1% deoxycholate, 1 mM NaVO4, 3 mM EDTA, and proteinase inhibitors, (0.5 mM PMSF), 10 µg/ml aprotinin, 5 µg/ml pepstatin, 10 µg/ml leupeptin)) for 30 min at 4°C and spun at 12,000 x g, 4°C, for 15 min. The eluted and reduced samples were resolved by SDS-PAGE using a 4–20% gradient polyacrylamide gel (Dai-ichi Kagaku) and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore). For immunoblotting, membranes were incubated with anti-p65, anti-phospho I{kappa}B{alpha}, anti-I{kappa}B{alpha}, anti-beta-tubulin, anti-phospho-IKK{alpha}beta, anti-IKK{alpha}beta, anti-PKCbetaI, and anti-Lyn, respectively. For immunoprecipitation, the cell lysates were incubated for 4 h at 4°C with 1 µg of Abs bound to protein A Sepharose. The eluted and reduced samples were separated on a 4–20% gradient SDS-polyacrylamide gel and transferred to a PVDF membrane. The membranes were incubated with anti-phosphotyrosine. Following first Ab blotting, the membranes were blotted with HRP-conjugated species-specific mouse-anti-IgG (Zymed) for 1 h at room temperature. After extensive washing of the membranes, immunoreactive proteins were visualized using the Renaissance chemiluminescence system (NEN) according to the manufacturer’s recommendation. The PVDF membranes were exposed to Fuji RX film. As for phospho-IKKbeta and PKCbetaI, densitometrical analysis was performed by LAS-1000 (Fuji).

Preparation of nuclear protein and plasma membrane protein

Nuclear protein was prepared as mentioned elsewhere. Briefly, BMMCs (1 x 106/ml) were sensitized with 1 µg/ml IgE for 6 h at 37°C. After sensitization, Ag-stimulated 5 x 106 cells were washed by ice-cold PBS – (PBS without calcium and magnesium) and suspended in 400 µl of ice-cold hypotonic buffer [10 mM HEPES-KOH (pH 7.8), 10 mM KCl, 0.1 mM EDTA (pH 8.0), 0.1% Nonidet P-40, 1 mM DTT, and protein inhibitors (0.5 mM PMSF, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 10 µg/ml leupeptin)]. To obtain the crude nuclear fractions, the suspensions were centrifuged at 2,500 x g, 4 °C for 1 min. The pellets were resuspended in hypertonic buffer [50 mM HEPES-KOH (pH 7.8), 420 mM KCl, 0.1 mM EDTA (pH 8.0), 5 mM MgCl2, 20% glycerol, 1 mM DTT, and protein inhibitors (0.5 mM PMSF, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 10 µg/ml leupeptin)] and centrifuged at 12,000 x g, 4°C for 15 min. The supernatants were applied for immunoblotting to detect p65. Plasma membrane proteins were extracted using the Plasma Membrane Protein Extraction Kit (BioVision; K268-50) according to the manufacturer’s recommendation. The plasma membrane fractions were used for immunoblot to detect PKCbetaI.

Calcium influx measurement

BMMCs (1 x 106/ml) were sensitized with 1 µg/ml IgE for 12 h and incubated with the calcium-sensitive dye indo-1 AM (Molecular Probes) in the presence of F 127 and 0.2% FBS at 37°C for 30 min. The cells were washed twice and resuspended in 1 ml Tyrode’s buffer containing 1 mM CaCl2. Cells were analyzed for calcium mobilization in a BD-LSR flow cytometer (BD Bioscience). Values were plotted as the ratio of fluorescence at FL5 (Ca2+-bound indo-1) to that at FL4 (Ca2+-free indo-1).

Retroviral infection

Retroviral infection was performed as previously described. The CD63-GFP plasmid (a kind gift from M. Nakanishi, Nagoya City University) was inserted into the BamHI and NotI sites of the retroviral vector pMX (a gift from T. Kitamura, University of Tokyo). This construct was then used to transfect the 293T-based packaging cell line, phoenix (a gift from G. Nolan, Stanford University, Stanford, CA), with Lipofectamine 2000 (Invitrogen Life Technologies) to generate recombinant retroviruses. BM cells were infected with the retrovirus in the presence of 10 µg/ml polybrene (Sigma-Aldrich) and IL-3.

Cell surface expression of CD63 upon Ag stimulation

BMMCs (1 x 106/ml) were sensitized with 1 µg/ml IgE for 6 h at 37°C. After Ag stimulation with 100 ng/ml DNP-HSA for 10 min, the cells were fixed with 4% paraformaldehyde (pH 7.4) for 30 min at room temperature. Cells were stained with 1 µl of anti-CD63 and then with PE-labeled anti-rat IgG (Cedarlane Laboratories). The exposure of CD63 to the cell surface was analyzed by flow cytometer (BD Biosciences).

Intracellular Zn measurement

BMMCs (2 x 106 cells) were treated with 5 µM Newport Green for 10 min under three conditions: no treatment, pretreatment with 10 µM TPEN, or pretreatment with 1 µM pyrithione plus 10 µM ZnSO4. The cells were washed two times with PBS (–) before fluorescent intensity was measured by FACS.

Confocal microscopy and granule translocation

Separate aliquots of 5 x 105 cells were each sensitized with 1 µg/ml IgE for 6 h. The cells were then stimulated with 100 ng/ml DNP-HSA for 10 min at 37°C and fixed with 4% paraformaldehyde for 30 min at 37°C. The cells were spun at 12,000 x g for 5 min and permeabilized in Perm Buffer (BD Biosciences) containing 1% BSA for 15 min at room temperature. The cells were washed with 1 ml of PBS (–) twice, then resuspended in 100 µl of PBS (–) and attached to glass slides by cytospin (Thramo Shandon) at 600 rpm for 6 min. Primary and secondary stainings were performed on the slides: anti-p65 at a dilution of 1/50, anti-PKCbetaI at a dilution of 1/50, anti-beta-tubulin at a dilution of 1/50, FITC-conjugated anti-rabbit IgG, (Molecular Probes), FITC-conjugated anti-mouse IgG (Molecular Probes) at 1/50, and phalloidin-rhodamine (Molecular Probes) at 1/150. Confocal microscopy was conducted using the TCS SL system (Leica). The frequency of Ag-induced MC granule translocation was enumerated according to the following criteria: 1) the increase of yellow color fluorescence around plasma membrane, which was a result of the merge of phalloidin-rhodamine and CD63-GFP signal; and 2) the obvious decrease of the cytoplasmic area containing CD63-GFP, compared with that of nonstimulated cells. The cells that satisfied both criteria were considered to be positive for granule translocation. Two hundred independent GFP-positive cells were counted for each experiment. The data are representative of three independent experiments, and the values are the mean ± SEM.

RT-PCR and real-time quantitative RT-PCR analysis

Cells were homogenized with Sepasol RNAI (Nacalai Tesque), and total RNA was isolated according to manufacturer’s instructions. For standard RT-PCR, cDNA was synthesized from 500 ng of total RNA by incubating with reverse transcriptase (ReverTra Ace; Toyobo) and 500 ng of oligo (dT) primer for 30 min at 42 °C (Invitrogen Life Technologies). A portion of the cDNA (typically 1/20 volume) was used for standard PCR to detect IL-6, TNF-{alpha}, and GAPDH. Twenty-five cycles of PCR were performed with 0.5 U of rTaq DNA polymerase and 10 pmol of gene-specific sense and antisense primers. For real-time quantitative RT-PCR, IL-6, and TNF-{alpha} gene expression was measured relative to GAPDH using Syber Green (Applied Biosystem). Primers used in this experiments were purchased from Invitrogen Life Technologies, and sequences were as follows: IL-6, (forward) 5'-GAGGATACCACTCCCAACAGACC-3' and (reverse) 5'-AAGTGCATCATCGTTGTTCATACA-3'; TNF-{alpha}, (forward) 5'-CATCTTCTCAAAATTCGAGTGACAA-3' and (reverse) 5'-TGGGAGTAGACAAGGTACAACCC-3'; GAPDH, (forward) 5'-TTCACCACCATGGAGAAGGCCG-3' and (reverse) 5'-GGCATGGACTGTGGTCATGA-3'. PCR product was separated on an agarose gel, stained with ethidium bromide (500 ng/ml), and photographed.

Passive cutaneous anaphylaxis (PCA) and histology of the ears.

A total of 0.5 µg of IgE was injected s.c. into both ears of BALB/c mice for 12 h or overnight. After sensitization, either vehicle or TPEN was injected into the mice i.p. at the concentration indicated for 30 min, and the mice were then challenged with an i.v. injection of 250 µg of polyvalent dinitrophenyl-BSA (DNP-BSA; Cosmobio) in 250 µl of saline-5 mg/ml Evans blue dye (Sigma-Aldrich). Extravasation of Evans blue in the ear was monitored for 30 min, then the mice were sacrificed, both ears were dissected, and the Evans blue dye was extracted in 700 µl of formamide at 63°C overnight. The absorbance of Evans blue-extracted formamide was then measured at 620 nm. To observe the ear MCs, 5-µm paraffin sections were fixed and stained with nuclear fast red and alcian blue. Approximately 100 alcian blue-stained cells in each sample were counted, and the ratio of morphologically degranulated MCs to total MCs was calculated. We obtained approval from the Animal Research Committee at RIKEN for all animal experiments performed.

Passive systemic anaphylaxis (PSA)

To determine the amount of histamine released in response to PSA, we sensitized mice by the i.v. injection with 0.5 µg of IgE for 12 h. After sensitization, mice were i.v. challenged with 30 µg of DNP-BSA. One minute after the Ag challenge, blood was drawn directly from the heart, and the plasma fraction was collected by centrifugation. The plasma histamine level was measured as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Zn chelator inhibits Ag-dependent PCA and PSA

To study potential roles for Zn in MC function, we assessed the efficacy of Zn chelator TPEN on in vivo allergic reactions involving normal tissue-resident MCs. We first tested the effect of TPEN in a mouse PCA model, in which Ag-specific IgE was injected into the ear. Pretreatment with the intraperitoneal administration of TPEN significantly inhibited the IgE-dependent PCA reactions (44.9, 75.7, and 87.4% inhibition by a single dose of TPEN at 10, 20, and 30 mg/kg, respectively) (Fig. 1, A and B). We then analyzed the MCs in the ear histologically. In sham-treated control mice, intact MCs densely filled with granules (blue) were easily identified (Fig. 1C, left panel), and degranulated MCs were detected as early as 30 min after Ag challenge (Fig. 1C, right panel). In contrast, the number of degranulated MCs was significantly reduced in the TPEN-treated mice (Fig. 1C, lower panel). By microscopic analysis, the total number of ear MCs per field was approximately equal in the TPEN- and sham-treated mice (data not shown). We also assessed the effect of TPEN on Ag-induced histamine release into the blood using a mouse model of PSA. The histamine release was significantly reduced, by 59.1%, in the TPEN-treated mice (Fig. 1D). These results clearly show that a Zn chelator effectively inhibited Ag-dependent MC degranulation in vivo.


Figure 1
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  		FIGURE 1. Zn chelator administration inhibits Ag-dependent PCA and PSA. A, The effects of TPEN on the PCA reaction were verified. Mice were sensitized with IgE injected into the ear, then challenged with 250 µg Ag and Evans Blue i.v. for 30 min. The injected Evans blue dye was extravasated into the ears of control mice (0 mg/kg, n = 10), but a dose-dependent inhibition was observed in TPEN-treated mice (10, 20, and 30 mg/kg, n = 5 respectively). Almost no Evans blue dye extravasation was observed in saline-challenged mice (data not shown). B, The amount of extravasated Evans Blue dye in both ears was calculated by extracting the dye and measuring the OD of the extract at 620 nm. C, Upper, Histological analysis of MCs in the ear. Ear sections were stained with nuclear fast red and alcian blue. Granules and the nucleus were stained blue and red respectively. Representative sections containing granulated MCs (upper left, arrowheads) and degranulated MCs (upper right, arrows) are shown. Lower, Approximately 100 cells in the sections were counted randomly, and the frequency of morphologically degranulated MCs in 30 mg/kg TPEN-treated (n = 3) or nontreated (n = 3) mice were calculated. D, IgE-dependent PSA was induced, and the plasma histamine concentration was measured after sham (n = 10), control (n = 10), and 30 mg/kg TPEN (n = 10) treatment. Data are representative of three independent experiments, which gave consistent results. The values are the mean ± SEM.

 
Zn is required for Fc{epsilon}RI-mediated MC activation.

Because MCs are the major effector cells in PCA and systemic anaphylaxis, we explored the effect of specific Zn chelator, TPEN on BMMC activation. To estimate the extent of Zn depletion caused by TPEN treatment, we performed FACS analysis using impermeable Zn-specific fluorescent indicator, Newport Green, As shown in Fig. 2F, TPEN treatment reduced the level of intracellular Zn in BMMCs. TPEN treatment significantly inhibited the Fc{epsilon}RI-induced beta-hexosaminidase, TNF-{alpha}, histamine, and leukotriene C4 release in a concentration-dependent manner (Fig. 2, A and B). Another Zn chelator, ZnAF-2DA <Ueno, 2002 #65> (30), also inhibited the release of beta-hexosaminidase (Fig. 2A). However, other metal chelators, such as 2,2'-dipyridyl (for Fe2+), desferrioxamine mesylate (for Fe3+), bathocuproine disulphonate (for Cu2+), and p-aminosalicylic acid (for Mn2+) (31, 32, 33) did not affect Fc{epsilon}RI-induced MC degranulation (Fig. 2, A and B). Next, we tested whether inhibitory effect of TPEN is rescued by the addition of ZnSO4. To avoid the use of very high extracellular Zn concentration, which is very cytotoxic, we used a pyrithione, which is an ionophore for Zn, in the rescue experiment (35). The combination of Zn and pyrithione more rapidly increases intracellular Zn levels than by ZnSO4 alone. This inhibitory effect of TPEN was completely rescued by the addition of ZnSO4 (Fig. 2C). Taken together, all results indicated that Zn is critically involved in MC degranulation, although we cannot completely neglect the possible involvement of other metals. We also determined the effect of TPEN on cytokine production after 6 h of stimulation. Zn depletion in BMMCs decreased the Fc{epsilon}RI-induced TNF-{alpha} and IL-6 production (Fig. 2D). Even at the highest TPEN concentration tested (20 µM), the survival and differentiation of BMMCs were not disturbed, in that the TPEN-treated BMMCs were 7-aminoactinomycin D (7AAD)-negative and expressed c-Kit and Fc{epsilon}RI on their cell surface at levels similar to those of control BMMCs (Fig. 2E).


Figure 2
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  		FIGURE 2. (Figure legend continues)

Fc{epsilon}RI-dependent MC degranulation and cytokine production were suppressed by Zn chelator. A, The effect of Zn chelators and other metal chelators on MC degranulation was assessed. BMMCs were treated with each metal chelator (TPEN and ZnAF-2DA for Zn, 2,2'-dipyridyl and desferrioxamine mesylate for Fe, bathocuproine disulphonate for Cu, and p-aminosalicylic acid for Mn) for 2 h, then degranulation after 30 min Ag stimulation was assayed by beta-hexosaminidase release. The concentration of each chelator was depicted in the column. B, The level of TNF-{alpha}, histamine, and leukotriene C4 released into the culture supernatant after Ag stimulation for 30 min were determined by ELISA. Each chelator was pretreated for 2 h. The values represent the mean ± SEM (n = 3). C, BMMCs were untreated or treated with 10 µM TPEN for 2 h, then they were stimulated with Ag containing the indicated concentration of ZnSO4 and 1 µM pyrithione. D, Production levels of TNF-{alpha} and IL-6 in the cell culture supernatant upon Ag stimulation for 6 h were measured by ELISA. E, c-Kit and Fc{epsilon}RI expression and cell viability were detected by staining with anti-CD117 (c-Kit), anti-IgE, and 7AAD, respectively, in vehicle-, 10, and 20 µM TPEN-treated BMMCs. The term 7AAD-negative means that the cells are viable. F, Intracellular Zn contents were detected using Zn indicator, Newport Green. The cells were untreated (left), treated with 10 µM TPEN (center) and 10 µM ZnSO4 plus 1 µM pyrithione (right) as positive control. These experiments were performed at least three times independently, and representative data are shown.

 
Zn chelator inhibits Fc{epsilon}RI-induced granule translocation but not microtubule formation.

Recently, we reported that the Fyn/Gab2/RhoA signaling pathway induces granule translocation to the plasma membrane in a manner independent of calcium (18). Therefore, we tested whether Zn is involved in the Fc{epsilon}RI-induced granule translocation process. To visualize the granules, we retrovirally transduced a GFP-CD63 fusion protein expressing plasmids into BMMCs. CD63 is located on the granule membrane in resting MCs (36). Before Ag stimulation, the granule structures were retained in the cytoplasm (Fig. 3A). After Ag stimulation, the CD63 signal was observed near the plasma membrane by confocal microscopy (Fig. 3A), and detected on the cell surface by FACS (Fig. 3C, left panel), suggesting that the granules were translocated from the cytoplasm to the plasma membrane, and that granule-plasma membrane fusion had occurred. In calcium-free medium, in which degranulation is completely abolished, no increased cell surface expression of CD63 was detected by FACS (Fig. 3C, center panel), while the granule translocation to the plasma membrane was still detected by confocal microscopy as we previously showed (Fig. 3A, center panel, and B) (18). Notably, treatment with TPEN significantly suppressed the Fc{epsilon}RI-induced granule translocation to the plasma membrane detected by confocal microscopy (Fig. 3A, right panel, and B) as well as the Ag-induced cell surface expression of CD63 (Fig. 3C, right panel, and D). Furthermore, we confirmed that reloading with Zn rescued the defect in Fc{epsilon}RI-induced granule translocation in Zn-depleted BMMCs (Fig. 3E). These results show that Zn was essential for the calcium-independent process of granule translocation to the plasma membrane. Because granule translocation is dependent on microtubules (18, 37), we next investigated whether Zn-depletion inhibited Fc{epsilon}RI-induced microtubule formation. As a result, TPEN treatment had no effect on microtubule formation upon Ag stimulation, while granules did not move to the plasma membrane in TPEN-treated BMMCs. (Fig. 3F and data not shown). The above results suggested that Zn chelator could be effective for interfering with Fc{epsilon}RI-mediated granule translocation in MCs.


Figure 3
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  		FIGURE 3. Zn chelator inhibits Fc{epsilon}RI-induced granule translocation but not microtubule formation. A, BMMCs retrovirally transfected with CD63-GFP were stimulated with Ag for 10 min (0 ng/ml, upper; 100 ng/ml, lower), and the cells were stained with phalloidin-rhodamine (red) to detect F-actin. Both F-actin and CD63-GFP (green) were visualized by confocal microscopy (left, control; center, Ca2+ free; right, TPEN treatment). Scale bar, 5 µm. B, The frequency of cells showing granule translocation to the plasma membrane was enumerated as mentioned in the materials and methods. C, CD63 expression on the cell surface was detected by FACS. BMMCs were stimulated with Ag in various conditions (left, control; center, Ca2+ free; right, TPEN treatment) for 10 min and stained with anti-CD63 after fixation. Gray histograms indicate nonstimulated cells. The percentage of cells in the CD63-positive population is indicated in the figure. D, CD63 expression on the cell surface was detected by FACS. BMMCs were stimulated with Ag for 10 min at the concentration indicated under no treatment or 10 µM TPEN treatment and stained with anti-CD63 after fixation. Gray histograms indicate nonstimulated cells. The percentage of cells in the CD63-positive population is indicated in the figure. E, The effect of exogenously added Zn2+ ion on the granule translocation in TPEN-treated BMMCs. Cells were treated with 10 µM TPEN for 2 h and stimulated with Ag containing the indicated concentrations of ZnSO4, and the frequency of granule-translocated cells was calculated as done in B. F, Microtubule formation was detected in TPEN-untreated or TPEN-treated cells upon Ag stimulation. BMMCs were stained with phalloidin-rhodamine (red) and FITC-conjugated anti-{alpha}-tubulin (green) after stimulation. Scale bar, 10 µm.

 
Zn chelator does not affect Fc{epsilon}RI-induced global tyrosine phosphorylation or calcium mobilization.

Fc{epsilon}RI-induced signaling in MCs involves the tyrosine phosphorylation of various signaling molecules and calcium influx, both of which are critical for MC degranulation. We next monitored the effect of TPEN on these Fc{epsilon}RI-induced signaling events. In TPEN-treated BMMCs the total cellular, Syk, Fyn, LAT, Gab2, and PLC{gamma}1 tyrosine phosphorylation was apparently normal, although the phosphorylation of LAT and PLC{gamma}1 appeared to be more transient and the intensity of phosphorylation of the protein bands above the 91-kDa marker appear to be intensified (Fig. 4, A–C). The Fc{epsilon}RI-induced calcium influx was also apparently normal in TPEN-treated BMMCs (Fig. 4D). These results suggest that there is an action point of TPEN besides tyrosine phosphorylation and calcium-elicited signaling.


Figure 4
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  		FIGURE 4. Zn chelator does not affect Fc{epsilon}RI-induced global tyrosine phosphorylation or calcium mobilization. A, The cells were stimulated with Ag for indicated times with or without TPEN treatment and cell lysates were blotted with anti-phosphotyrosine. B and C, The effect of TPEN on the activation of molecules involved in Fc{epsilon}RI signal transduction was assessed. Syk, LAT, PLC{gamma}1 and Gab2 were immunoprecipitated with its polyclonal Ab and blotted with phosphotyrosine. For Fyn, immunoprecipitation was performed using anti-Fyn mAb and autotyrosine phosphorylation was detected as mentioned in material and methods. The protein loading was normalized by reprobing the membranes with anti-Syk, anti-LAT, anti- PLC{gamma}1, and anti-Gab2, respectively. D, Calcium influx was measured using a calcium-sensitive indicator, indo 1-AM. After incubation with indo 1-AM, cells were stimulated with Ag (100 ng/ml) for 512 s at 37°C. Calcium mobilization is represented as the fluorescence ratio of FL5 (Ca2+-bound indo 1) to FL4 (Ca2+-free indo 1). Bars, 1 min.

 
Zn chelator inhibits Fc{epsilon}RI-induced NF-{kappa}B activation.

To define the molecular mechanism(s) responsible for the defective cytokine production in Zn-depleted BMMCs, we analyzed cytokine mRNA levels using RT-PCR. Fc{epsilon}RI stimulation of BMMCs led to an increase in IL-6 and TNF-{alpha} mRNA level in a time-dependent manner. However, Zn-depleted BMMCs decreased IL-6 and TNF-{alpha} mRNA level (Fig. 5, A and B). This inhibitory effect of TPEN was rescued by the addition of ZnSO4 (Fig. 5A). Furthermore, other metal chelators did not affect IL-6 and TNF-{alpha} mRNA level (Fig. 5C). Thus, Zn is involved in Fc{epsilon}RI-induced IL-6 and TNF-{alpha} gene transcription, in particular IL-6. In general, the transcription of IL-6 and TNF-{alpha} was regulated by a transcription factor, NF-{kappa}B. NF-{kappa}B consists of p65 and p50 heterodimer and retained in cytoplasm by binding with I{kappa}B{alpha} in resting state. NF-{kappa}B nuclear translocation was occurred after I{kappa}B{alpha} serine phosphorylation by I{kappa}B kinase {alpha}beta (IKK{alpha}beta), leading to the degradation of I{kappa}B{alpha} (38). We analyzed whether TPEN inhibited Fc{epsilon}RI-induced nuclear translocation of p65. The cells were stained with anti-p65 after stimulation under the TPEN treatment. As a result, Zn-depleted BMMCs significantly reduced Fc{epsilon}RI-induced translocation of p65 to the nucleus (Fig. 6A). Furthermore, we confirmed that this inhibitory effect of TPEN was rescued by the addition of ZnSO4 (Fig. 6A), indicating that Zn is essential for nuclear translocation of NF-{kappa}B in MCs. Consistent with these results, Fc{epsilon}RI-mediated I{kappa}B{alpha} phosphorylation and its subsequent degradation were impaired in Zn-depleted BMMCs (Fig. 6B). Finally, we examined the effect of Zn depletion on the activation of IKK{alpha} and IKKbeta, which regulate I{kappa}B{alpha} phosphorylation. As shown in Fig. 6C, TPEN inhibited Fc{epsilon}RI-induced IKKbeta phosphorylation. Together, these results showed that Zn-dependent mechanism is required for Fc{epsilon}RI-mediated NF-{kappa}B activation pathway.


Figure 5
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  		FIGURE 5. IL-6 and TNF-{alpha} gene expression induced by Ag stimulation was suppressed by Zn chelator, but not with other metal chelators. A, The cells were stimulate for 0, 1, and 3 h under various conditions, then IL-6 and TNF-{alpha} gene expression levels were analyzed for RT-PCR. Lanes 1–3; no treatment, lanes 4–6; 10 µM TPEN, lanes 7–9; 10 µM TPEN plus 0.1 µM pyrithione, lanes 10–12; 10 µM TPEN plus 10 µM ZnSO4, lanes 13–15; 10 µM TPEN plus 0.1 µM pyrithione plus 10 µM ZnSO4. B, IL-6 and TNF-{alpha} expressions at mRNA level were assessed by real-time quantitative RT-PCR. BMMCs were untreated or treated with 10 µM TPEN for 2 h and stimulated with Ag for indicated times. C, IL-6 and TNF-{alpha} expressions at mRNA level were assessed by RT-PCR as in (A). The cells were pretreated with indicated concentration of metal chelators as used in Fig. 2A. These experiments were performed at least three times independently, and representative data are shown.

 

Figure 6
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  		FIGURE 6. NF-{kappa}B activation pathway induced by Ag stimulation is inhibited by Zn chelator pretreatment. BMMCs were untreated or treated with 10 µM TPEN for 2 h and stimulated for indicated times. A, NF-{kappa}B nuclear translocation was visualized by confocal microscopy. After Ag or Ag with 1 µM pyrithione plus 10 µM ZnSO4 stimulation, cells were stained with anti-p65, FITC-conjugated anti-rabbit Ab and 4', 6-diamidino-2-phenylindole dihydrochloride. Green indicates p65, and blue indicates nucleus. Scale bar, 5 µm. A total of 50 cells were counted randomly in each sample, and the frequency of NF-{kappa}B nuclear-translocated cells was calculated. The values are the mean ± SEM. (Bottom) Western blot analysis was performed on nuclear extracts to assess the effect of TPEN of NF-{kappa}B nuclear translocation. Nuclear extraction was blotted with anti-p65 Ab. Protein loading was normalized by reprobing the membrane with anti-beta tubulin. B and C, I{kappa}B{alpha} phosphorylation, degradation, and IKK{alpha}beta phosphorylation were determined. After Ag stimulation, cells were lysed and cytosol faraction was immunoblotted with anti-phospho-IkB{alpha} and anti-phospho-IKK{alpha}beta, respectively. Protein loading was normalized by reprobing the membrane with anti-IkB{alpha} and anti-IKK{alpha}beta, respectively. As for IKK{alpha}beta, the extent of serine phosphorylation was densitometrically enumerated normalized by densitometry of IKKbeta.

 
Zn is involved in Fc{epsilon}RI-induced plasma membrane translocation of PKCbeta I.

Because I{kappa}B{alpha} phosphorylation through IKKbeta was decreased in Zn-depleted BMMCs as described in Fig. 6, we next investigated the upstream target of Zn involved in NF-{kappa}B activation. Because recent studies demonstrated a role for PKC-mediated signals in the control of NF-{kappa}B activation (39, 40, 41), we examined whether PKC is involved in Fc{epsilon}RI-mediated NF-{kappa}B activation in MCs. As shown in Fig. 7, A and B, PKC inhibitor, Ro31–8220 inhibited Fc{epsilon}RI-induced up-regulation of IL-6 and TNF-{alpha} mRNA. Consistent with this result, Fc{epsilon}RI-induced NF-{kappa}B nuclear translocation, I{kappa}B{alpha} phosphorylation, and I{kappa}B{alpha} degradation were partially blocked by Ro31-8220 treatment (Fig. 7C). Based on known function of PKC, we hypothesized that Zn is required for NF-{kappa}B activation because it is required for PKC activation in MCs. To test this hypothesis, we examined the effect of TPEN plasma membrane translocation of PKC, a process important for PKC function (42, 43). As shown in Fig. 7, D and E, Fc{epsilon}RI-induced plasma membrane translocation of PKCbetaI was inhibited in Zn-depleted BMMCs.


Figure 7
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  		FIGURE 7. PKC is involved in Ag-induced IL-6 and TNF-{alpha} mRNA expression, and plasma membrane translocation of PKC was inhibited by Zn chelator. To assess the involvement of PKC on Ag-induced IL-6 and TNF-{alpha} gene expression, cells were treated with 2 µM PKC family inhibitor, Ro31-8220, at the same time as Ag stimulation and total RNA was extracted after Ag stimulation for 1 and 3 h. Total RNA (1 µg) was used for reverse transcription. mRNA (50 ng) was analyzed for IL-6 and TNF-{alpha} expression level by RT-PCR (A) and by real-time quantitative RT-PCR (B). C, NF-{kappa}B nuclear translocation was visualized by confocal microscopy. After Ag stimulation, cells were stained with anti-p65, FITC-conjugated anti-rabbit Ab and 6-diamidino-2-phenylindole dihydrochloride. Green indicates p65 and blue indicated nucleus. Scale bar, 5 µm. The frequency of NF-{kappa}B nuclear-translocated cells was calculated as in Fig. 6A. The values are the mean ± SEM. Phosphorylation of I{kappa}B{alpha} and degradation of I{kappa}B{alpha} in the cytosol fraction were detected by immunobloting after Ag stimulation under the treatment with or without 2 µM Ro31-8220. D, After Ag stimulation for indicated time in the presence or absence of 10 µM TPEN, cells were lysed, plasma membrane fraction was extracted, and PKCbetaI was detected by immunoblot. The protein loading was normalized by reprobing the membranes with anti-Lyn Ab. PKC plasma membrane translocation was densitometrycally enumerated in a bottom panel. E, PKCbetaI plasma membrane translocation was visualized by confocal microscopy. Cells were either untreated or treated with 10 µM TPEN for 2 h and stimulated with Ag for 10 min. After Ag stimulation, cells were fixed and stained with polyclonal anti-PKCbetaI Ab. Arrows indicate PKCbetaI. These experiments were performed at least three times independently, and representative data are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we showed that the Zn chelator TPEN inhibited the IgE-dependent PCA and systemic anaphylaxis reactions. Consistent with this in vivo effect, the Zn chelator strongly inhibited the Fc{epsilon}RI-induced degranulation (i.e., histamine and beta-hexosaminidase release) in vitro. Furthermore, it inhibited the Fc{epsilon}RI-mediated cytokine production and leukotriene release, indicating that Zn-dependent mechanisms are involved in MC activation. We also showed that the reloading of Zn could rescue the degranulation defect in Zn-depleted BMMCs, indicating that the effect of the Zn chelator is a reversible reaction. In the 1960s, electron microscopic studies demonstrated that MCs are rich in heavy metals such as Zn and Fe, but not Cu (3). Because TPEN has affinities for other heavy metals, we checked other metal chelators such as 2,2'-dipyridyl (for Fe2+), desferrioxamine mesylate (for Fe3+), bathocuproine disulphonate (for Cu2+), and p-aminosalicylic acid (for Mn2+) on degranulation and cytokine induction. These metal chelators treatment did not affect Fc{epsilon}RI-induced MC degranulation and cytokine induction. Because the inhibitory effect of TPEN was suppressed by the treatment with Zn and ionophore, all results are consistent with the notion that Zn is involved in MC degranulation process and cytokine production, although we cannot dismiss the possible involvement of other metals.

How might the Zn chelator exert its inhibitory effect? We confirmed that it had no effect on the early Fc{epsilon}RI-induced tyrosine phosphrylation of a variety of signaling molecules or on calcium mobilization, but it strongly suppressed the Fc{epsilon}RI-induced granule translocation. Vesicle transport generally depends on cytoskeletal proteins such as tubulin and actin (44). In MCs, several reports including ours have shown that microtubules are important for Fc{epsilon}RI-induced degranulation and granule translocation to the plasma membrane (18, 37, 45). However, in this study, we showed that TPEN had no effect on Fc{epsilon}RI-induced microtubule formation. This result may suggest the existence of a molecule linking microtubules and granules. Recently, kinesin receptors, or linker-cargo proteins, were identified (46). These molecules have a kinesin-interacting region and link kinesin to vesicles. One target of TPEN may be such a linker molecule-one with a Zn finger or Zn-binding domain. In addition to the granule translocation, TPEN might control the process of granule-membrane fusion through Rab-effector molecules because several groups have reported that Rab-effector molecules such as Rim, Granulophilin, and Rabphilin regulate fusion of synaptic vesicle and have Zn-finger domain (47, 48). In addition, PKC may be involved in granule translocation process as discussed later.

In this study, we also showed that Zn chelator suppressed Fc{epsilon}RI-mediated cytokine production and transcription of IL-6 and TNF-{alpha} mRNA. Importantly, this inhibitory effect could be rescued by addition of ZnSO4, suggesting that Zn is essential for Fc{epsilon}RI-mediated cytokine gene expression. Previously, it was reported that PKC is activated with Ag stimulation and involved in cytokine production in MCs and that Zn-binding domain of PKC is required for plasma membrane translocation after stimulation (24, 25). These observations may suggest Zn plays a role in the regulation of PKC plasma membrane translocation. In fact, in this study, we showed that TPEN inhibited plasma membrane translocation of PKCbetaI using biochemical and microscopic approaches. These results suggested that one of targets of TPEN involved in cytokine production was PKC. In addition to PKC, TPEN treatment strongly inhibited I{kappa}B{alpha} phosphorylation and its degradation. Accordingly, p65 nuclear translocation was significantly inhibited by TPEN treatment. These results indicate that modification of Zn level by TPEN can affect NF-{kappa}B activation pathway. Recently, it was reported that PKC was involved in NF-{kappa}B activation pathway in MCs by using Ro31-8220, a pharmaceutical inhibitor of PKC (49). Consistent with this reports, Ro31-8220 partially inhibited I{kappa}B{alpha} phosphorylation and its degradation. These results suggest that suppression of cytokine production by TPEN is partly due to inactivation of PKCbetaI/NF-{kappa}B activation pathway. Furthermore, PKC may contribute to Fc{epsilon}RI-mediated granule translocation, because PKC family molecules have multiple effects for regulation of exocytosis and endocytosis (50). Thus, PKC might be one of target molecules of TPEN in MCs. However, we also consider that there exist some other targets of TPEN. TPEN might control the process of protein degradation through inhibiting ubiquitination. Because E3 ligases which play a pivotal role in ubiquitination have the catalytic domain containing a Zn-binding RING finger motif (51). TPEN might suppress E3 ligase activity to inhibit I{kappa}B{alpha} degradation. Mutational analyses of these candidates molecules, such as PKC and E3 ligases will be required to clarify these points.

In summary, Zn is essential for Fc{epsilon}RI-mediated MC activation (i.e., degranulation and cytokine production). Our data indicate the presence of multiple Zn-dependent mechanisms in MC activation. These findings strongly suggest that Zn chelator is a promising new drug that acts differently from other antiallergic drugs such as histamine antagonists. We postulate that controlling the concentration of Zn in MCs may become a useful strategy for developing novel anti-allergy treatments.


   Acknowledgments
 
We thank Drs. M. Nakanishi, K. Hattori, G. Nolan, and T. Kitamura for various reagents; and Drs. E. Morii and K. Oboki for technical advice and suggestions. We also thank M. Kikuchi for excellent technical assistance and M. Shimura for secretarial assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology in Japan. K.K is a Junior Research Associate, Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan. Back

2 Address correspondence and reprint requests to Dr. Toshio Hirano, Laboratory of Developmental Immunology, Graduate School of Medicine C-7, Osaka University, 2-2, Yamada-oka, Suita, Osaka, 565-0871, Japan. E-mail address: hirano{at}molonc.med.osaka-u.ac.jp Back

3 Abbreviations used in this paper: Zn, zinc; MC, mast cell; BMMC, bone marrow-derived MC; TPEN, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine; ZnAF-2DA, Zn aminofluorescein-2 diacetate; PCA, passive cutaneous anaphylaxis; PSA, passive systemic anaphylaxis; 7AAD, 7-aminoactinomycin D; PKC, protein kinase C; cPKC, conventional PKC; IKK{alpha}, beta-I{kappa}B kinase {alpha}beta; PVDF, polyvinylidene difluoride. Back

Received for publication December 29, 2005. Accepted for publication April 21, 2006.


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 Introduction
 Materials and Methods
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