(I)-induced Ca2 influx and mast cell activation - CiteSeerX

Uncorrected Version. Published on August 25, 2009 as DOI:10.1189/jlb.0609387

Article

Nitric oxide positively regulates Ag (I)-induced Ca2⫹ influx and mast cell activation: role of a nitric oxide synthase-independent pathway Toshio Inoue, Yoshihiro Suzuki,1 Tetsuro Yoshimaru,2 and Chisei Ra Division of Molecular Cell Immunology and Allergology, Nihon University Graduate School of Medical Science, Tokyo, Japan RECEIVED JUNE 5, 2009; REVISED JULY 28, 2009; ACCEPTED JULY 29, 2009. DOI: 10.1189/jlb.0609387

J. Leukoc. Biol. 86: 000 – 000; 2009.

ABSTRACT NO is generated by NOS activity and known to act as a negative regulator of mast cell activation. We reported previously that Ag (I) directly evokes mast cell degranulation and LTC4 release via Ca2⫹ influx through thiolsensitive, store-independent channels. Here, we report that NO generated independently of NOS activity mediates the store-independent Ca2⫹ influx. Exposure of mast cells to Ag (I) resulted in increased intracellular NO levels and NO2–/NO3– contents in the extracellular fluid. The NO increase was blocked by NO scavenger Hb and DTT but not by NOS inhibitors such as aminoBH4 and L-NAME. This NO production occurred independently of the Src family kinase and PI3K activities, both of which were necessary for antigen-induced, NOS-dependent NO production. Hb and DTT reduced Ag (I)-induced ␤-hexosaminidase release and LTC4 release, whereas the NO scavengers and NOS inhibitors augmented antigen-induced mediator release. Moreover, Hb and DTT, but not the NOS inhibitors, abolished the Ag (I)-induced Ca2⫹ influx, and none of the drugs blocked CRAC channel activity. Finally, Ag (I)-induced Ca2⫹ influx was distinct from LTCC activity in terms of its sensitivities to wortmannin and LTCC antagonists and the effects of Cav1.2 LTCC gene silencing. These data show that NOS-independent NO regulates mast cell activation positively via a unique store-independent Ca2⫹ influx pathway. The present findings suggest multiple sources and functions of NO in mast cell biology.

Abbreviations: 2-APB⫽2-aminoethoxydiphenyl borate, Ag (I)⫽silver ions, Ag2SO4⫽silver sulfate, amino-BH4⫽L-erythro-1⬘,2⬘-dihydroxypropyl)5,6,7,8-tetrahydropteridine, Au(III)⫽gold ions, BMMC⫽bone marrow-derived mast cell, BN⫽Brown Norway, [Ca2⫹]c⫽cytosolic Ca2⫹ concentration, Cav⫽Voltage-dependent calcium, CRAC⫽Ca2⫹ release-activated Ca2⫹, DAF-2-DA⫽diaminofluorescein-2-diacetate, ER⫽endoplasmic reticulum, Fc␧RI⫽high-affinity IgER, Fluo3/AM⫽fluo3-acetoxylmethyl ester, Hb⫽hemoglobin, Hg(II)⫽mercury ions, HMC-1⫽human mast cell line 1, KD⫽knocked down, L-NAME⫽L-NG-nitroarginine methyl ester hydrochloride, LT⫽leukotriene, LTCC⫽L-type Ca2⫹ channel, NO2–⫽nitrite, NO3–⫽ nitrate, NOS⫽NO synthase, O2•–⫽superoxide, RMCPII⫽rat mast cell protease II, ROS⫽reactive oxygen species, SFK⫽ Src family kinase, siRNA⫽small interfering RNA, SOCE⫽store-operated Ca2⫹ entry, SOD⫽superoxide dismutase, Tg⫽thapsigargin

0741-5400/09/0086-0001 © Society for Leukocyte Biology

Introduction NO is a potent radical with diverse roles in biological systems. For example, it is a mediator of vasodilatation, platelet aggregation, and neuronal transmission, and depending on the cell type and concentration, it regulates the functions, death, and survival of various cell types including many of the cells involved in immunity and inflammation [1, 2]. NO also suppresses mast cell activation and the subsequent features of inflammation as well as antigen-induced degranulation, LT secretion, and cytokine expression and release in mast cells [3–5]. An emerging view is that mast cells by themselves are potential producers of NO. Various mast cell populations, including rat peritoneal mast cells, rat mast cell line RBL-2H3 cells, BMMCs, human skin mast cells, and human mast cell lines such as HMC-1 and LAD2, produce NO upon immunological and nonimmunological stimulation [6 – 8]. Ions of heavy metals, such as mercury, gold, and silver, are capable of inducing alterations in the immune system. Several rodent models exist for mercury–, gold–, and silver-induced autoimmunity [9 –15]. Hg (II) is the best known to evoke autoimmunity in genetically susceptible animal models. It has been demonstrated that subtoxic doses of mercury induce a systemic immune system dysregulation in BN rats [9, 16]. This disorder is characterized by the production of autoantibodies, increases in serum Igs, including IgE, polyclonal activation of B and T lymphocytes, and renal immune complex deposition, resulting in glomerulonephritis [17–20]. Metal-induced autoimmunity in mice shares many similarities with disorders observed in BN rats, including the production of autoantibodies [14]. The main target of the autoantibodies is fibrillarin, a 34kDa ribonucleoprotein. Human exposure to heavy metals also results in autoimmune disorders. For example, exposure to 1.

2.

Correspondence: Division of Molecular Cell Immunology and Allergology, Nihon University Graduate School of Medical Science, 30-1 Oyaguchikamicho Itabashi-ku, Tokyo 173-8610, Japan. E-mail: [email protected] Current address: Division of Genome Medicine, Institute for Genome Research, The University of Tokushima, Tokushima, Japan.

Volume 86, December 2009

Journal of Leukocyte Biology

Copyright 2009 by The Society for Leukocyte Biology.

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mercury vapors can result in elevated IgE levels and increased numbers of T lymphocytes in humans. Autoantibodies against fibrillarin appear in patients with the autoimmune disease systemic sclerosis (scleroderma). In addition, there is a correlation between the presence of antinucleolar antibodies in systemic sclerosis patients and urinary mercury levels. These observations indicate intriguing similarities between various animal models and the results of human exposure to heavy metals. Gold and silver are capable of evoking responses that are nearly identical in nature to those caused by mercury administration, except for decreased production of autoantibodies [11–13, 15, 21–27]. Mast cells play key roles in allergic and inflammatory reactions, and cross-linking of Fc␧RI on their cell surface triggers a cascade of signal transduction pathways that leads to degranulation and new synthesis and secretion of LTs, proinflammatory cytokines, and chemokines [28]. Increasing evidence has suggested that mast cells play important roles in the development of autoimmune disorders [29]. In particular, mast cells are thought to be critical for metal-induced autoimmunity, which is characterized by early tissue injury in the form of vasculitis [20, 30]. This early vasculitis is T cell-independent but accompanied by in vivo evidence for mast cell degranulation, which can be reduced by treatment with mast cell stabilizers [31, 32]. We reported previously that Hg (II) and Ag (I) can induce mast cell degranulation commonly and directly [33] and observed more recently that Au (III) exerts similar effects. We have a special interest in the mechanisms underlying the Ag (I)-induced responses, because of their unique nature. First, Ag (I) is much weaker than Hg (II) and Au (III) in evoking the production of cytokines, including IL-4, IL-13, and TNF-␣, whereas their effects on degranulation and LTC4 release are comparable with or higher than those of Hg (II) and Au (III). Second, Ag (I) is distinct from the other two metal ions in terms of its ability to stimulate tyrosine phosphorylation. As observed in T cells [34], exposure of mast cells to Hg (II) or Au (III) results in increased tyrosine phosphorylation of a set of proteins, whose pattern is similar to that observed in antigen-stimulated cells. In contrast, Ag (I) stimulates no or very little tyrosine phosphorylation of a few proteins. Consistent with these observations, Ag (I) triggers mast cell activation without affecting tyrosine phosphorylation of receptor components, Lyn, Syk, and linker for activation of T cells [35]. Third, Ag (I) is advantageous over antigen stimulation for inducing the release of ROS into extracellular spaces. Although extracellular ROS appear to mediate Ag (I)-induced Ca2⫹ influx and mast cell activation [36], their molecular entities remain unclear. Application of exogenous SOD or catalase has minimal effects on Ag (I)-induced Ca2⫹ influx and degranulation, suggesting that ROS other than O2•– and H2O2 are involved in mediating the effects of Ag (I). These observations led us to examine the possible roles of NO in Ag (I)-induced mast cell activation. As we observed essentially similar effects of antigen stimulation on NO production in RBL-2H3 cells and BMMCs previously [8], we used RBL-2H3 cells as a model. Here, we demonstrate that Ag (I) stimulates the production and release of NO into the extracellular spaces in a NOS-independent manner. Our data reveal 2 Journal of Leukocyte Biology


that NOS-independent NO acts as a positive regulator of mast cell activation by facilitating a unique store-independent Ca2⫹ influx pathway.

MATERIALS AND METHODS

Materials Ag2SO4 and Hb were obtained from Wako Pure Chemicals (Osaka, Japan). The calcium ionophore A23187 and Tg were obtained from Sigma Chemical Co. (St. Louis, MO, USA). An anti-TNP IgE mAb (clone IgE-3) was obtained from BD PharMingen Japan (Tokyo). Nifedipine, verapamil, and diltiazem were obtained from Calbiochem (San Diego, CA, USA). TNPconjugated-BSA (TNP:BSA conjugation ratio of 25:1) was purchased from Cosmo Bio (Tokyo, Japan). DTT was obtained from Nacalai Tesque (Kyoto, Japan). l-NAME and amino-BH4, a structural analog of the cofactor tetrahydrobiopterin, were obtained from BioMol (Plymouth Meeting, PA, USA). DAF-2-DA was purchased from Daiichi Pure Chemicals (Tokyo, Japan). Fluo3/AM was obtained Dojindo Laboratories (Kumamoto, Japan).

Cell activation RBL-2H3 cells were obtained from the National Institute of Health Sciences (Japan Collection of Research Bioresources, Tokyo, Japan; Cell Number JCRB0023) and grown in DMEM supplemented with 10% FBS (JRH Biosciences, Lenexa, KS, USA) in a 5% CO2-containing atmosphere. The cells were harvested by incubation in HBSS supplemented with bicarbonate (pH 7.4) containing 1 mM EDTA and 0.25% trypsin for 5 min at 37°C. For IgE sensitization, cells suspended in supplemented DMEM were plated on 100 mm culture dishes (1⫻106 cells/ml) or 24-well plates (2⫻105 cells/ well) and incubated with the anti-TNP IgE antibody (1 ␮g/dish or 0.1 ␮g/ well) at 37°C overnight. The IgE-sensitized cells were washed with PBS and suspended in HBSS. IgE-sensitized cells were stimulated with antigen, or IgE-untreated cells were stimulated directly with Ag2SO4. As exposure to 30 ␮M Ag2SO4 for 30 min exerted the maximum effect on degranulation with minimal cytotoxicity, we used this experimental protocol throughout the present study unless indicated otherwise.

Measurement of intracellular NO The intracellular NO levels were measured by flow cytometry using DAF-2DA, a cell-permeable, NO-sensitive fluorescent dye, as described previously [8]. For flow cytometrical analysis, IgE-sensitized or IgE-untreated cells suspended in HBSS (1⫻106 cells/ml) were incubated with 5 ␮M DAF-2-DA for 15 min at 37°C in a final volume of 450 ␮l. Next, 50 ␮l stimulant (10⫻) was added and incubated at 37°C for various times before harvesting. The cells were washed, resuspended in HBSS on ice, centrifuged at 4°C, and analyzed using a FACSCalibur (Becton Dickinson, San Jose, CA, USA) with excitation and emission at 488 and 575 nm, respectively.

Measurements of NO2–/NO3– RBL-2H3 cells were stimulated with Ag2SO4 or antigen at 37°C for 2 h, and the supernatants were collected. The NO2–/NO3– contents in the supernatants were measured using a Griess assay kit [NO2/NO3 Assay Kit-FX (Fluorometric)-2,3-Diaminonaphthalene Kit, Dojindo Laboratories], according to the manufacturer’s protocol.

Degranulation assay Degranulation was determined by ␤-hexosaminidase release. RBL-2H3 cells were stimulated at 37°C for 30 min, and the ␤-hexosaminidase activities in the supernatants were determined spectrometrically as described previously [36]. Briefly, 40 ␮l sample or cell lysate and 100 ␮l 2 mM p-nitirophenyl-Nacetyl- ␤-D-glucosaminidase [in 0.4 M citrate and 0.2 M phosphate buffer (pH 4.5; ICN Biochemicals, Eschwege, Germany] were added to each well of a 96-well plate, and color was allowed to develop for 30 min at 37°C.

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Inoue et al. NO-dependent mast cell activation by Ag (I) The enzyme reaction was terminated by adding 200 ␮l 0.2 M glycineNaOH (pH 10.7). The absorbances at 415 nm were measured using a Model 680 microplate reader (Nippon Bio-Rad Laboratories, Osaka, Japan). The cells were lysed with 0.1% Triton X-100, and the extracts were analyzed for the total ␤-hexosaminidase activities, which in untreated cells (spontaneous release, ⬍5% of the total enzyme activity) was subtracted from the enzyme activity (test sample release). The percentage of ␤-hexosaminidase released into the supernatant was calculated using the following formula: Release (%) ⫽ (test–spontaneous)/ (total–spontaneous) ⫻ 100.

LTC4 secretion assay LTC4 release was determined as described previously [36]. Briefly, cells were activated as described above, and the LTC4 contents in the supernatants were determined using an ELISA kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s protocol.

Measurement of [Ca2ⴙ]c [Ca2⫹]c was measured using the Ca2⫹-reactive fluorescent probe Fluo3/AM as described previously [36]. Briefly, a cell suspension (1⫻106 cells/ml) was incubated with 4 ␮M Fluo3/AM for 40 min at 37°C, washed with HBSS, and resuspended in HBSS supplemented with 1 mM CaCl2. To study Ca2⫹ stores and Ca2⫹ entry separately, aliquots of the Fluo3/AM-loaded cells were resuspended in HBSS supplemented with 1 mM EGTA instead of 1 mM CaCl2. Fluorescence was monitored at 5 s intervals for up to 3 min using a microplate fluorometer (Fluoroskan Ascent CF, Labsystems, Helsinki, Finland) with excitation and emission at 485 and 527 nm, respectively.

Analysis of ␣1C transcript expression The transcript expression of the ␣1C subunit of Cav1.2 LTCC was analyzed using RT-PCR as described previously [37]. Briefly, total RNA was isolated from 1 ⫻ 106 cells using Isogen (Nippon Gene, Tokyo, Japan) and reversetranscribed into cDNAs using SuperScript™ II RT (Invitrogen, Carlsbad, CA, USA), oligo dT primer (Invitrogen), and 0.5 mM dNTP mixture (Invitrogen). The ␣1C subunit and GAPDH mRNA levels were measured by RT-PCR using a Model PC-802 thermal cycler (Astec Corp., Fukuoka, Japan). The primers used were as follows: rat ␣1C, 5⬘-GACAACCTGGCTGATGCGGAGAGCCTGAC-3⬘ and 5⬘- ATGCGGTGGCACTGCAGGCGGAACCTG-3⬘; GAPDH, 5⬘-ACCACAGTCCATGCCATCAC-3⬘ and 5⬘-TCCACCACCCTGTTGCTGTA-3⬘. The PCR amplifications were performed for 30 cycles of 1 min at 95°C for denaturation, 1 min at 60°C for annealing, and 1 min at 72°C for extension.

Surface expression of ␣1C The cell surface expression levels of ␣1C were determined by flow cytometry using a ZenonTMAlexa Fluor 488 rabbit IgG labeling kit (Invitrogen) as described previously [37]. Briefly, for ZenonTM complex formation, 1 ␮g anti-␣1C antibody and an excess amount of ZenonTM Alexa Fluor 488-conjugated IgG were mixed in 20 ␮l PBS. After incubation for 5 min, the surplus fluorochrome was blocked with ZenonTM blocking agent for 5 min. Subsequently, the Alexa Fluor 488-conjugated anti-␣1C antibody was added to RBL-2H3 cells (5⫻105 cells) and incubated for 30 min at 4°C. In parallel, negative controls were incubated with anti-␣1C antibody that had been preincubated with the antigen peptide for 1 h. The cell surface expression levels of ␣1C were evaluated in a FACSCalibur and analyzed using the CellQuest software (Becton Dickinson).

siRNA knockdown of ␣1C expression Blocking of ␣1C expression by a siRNA was performed as described previously [37]. Briefly, a target siRNA for ␣1C and a negative control siRNA with an irrelevant sequence were purchased from Ambion (Austin, TX, USA). Adherent cells in six-well plates (2.5⫻105 cells/well) or 24-well plates (5.0⫻104 cells/well) were transfected with the ␣1C siRNA (final concentra-

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tion, 5 nM) for 48 h using the siPORT™ NeoFX transfection agent (Ambion), according to the manufacturer’s instructions. The efficacy for knockdown of ␣1C gene expression was evaluated by RT-PCR analysis and flow cytometry as described above.

Statistical analysis Data were analyzed by one-way ANOVA with application of a post hoc Tukey test. Values of P ⬍ 0.05 were considered to be significant.

RESULTS

Ag (I) stimulates NO production and release in a NOS-independent manner To examine whether Ag (I) stimulates NO production in mast cells, we measured the intracellular NO levels in RBL-2H3 cells initially after exposure to Ag (I) using the NO-sensitive dye DAF-2-DA [38 – 41]. By using this technique, we demonstrated recently that antigen-stimulated mast cells produce NO in a NOS-dependent manner [8]. The intracellular NO levels were elevated within 5 min after antigen stimulation and continued to increase gradually over time (Fig. 1, A and B). Exposure to Ag (I) also resulted in rapid (within 5 min) increases in the intracellular NO levels compared with unstimulated cells (Fig. 1A), indicating the onset of NO production. Unlike antigen stimulation, however, the intracellular NO levels decreased over time thereafter. As a consequence, the intracellular NO levels at 10 min and beyond became even lower than the resting levels (Fig. 1B). These results suggest that NO is produced initially inside of the cells after Ag (I) exposure but then released rapidly into the extracellular fluid. Moreover, the NOS inhibitors amino-BH4 [42, 43] and l-NAME had minimal effects on Ag (I)-mediated NO production but inhibited antigen-induced NO production significantly (maximum of 75% inhibition; Fig. 1C). As our previous study revealed that antigen-induced NO production is also dependent on SFK and PI3K activities [8], we examined the roles of these activities in Ag (I)-induced NO production. As shown in Figure 1D, the SFK inhibitor PP1 and the PI3K inhibitor wortmannin had minimal effects on Ag (I)-induced NO production but strongly reduced antigen-induced NO production. To confirm the NO production, we measured the NO2–/NO3– contents in the extracellular fluid. Marginal increases in the NO2–/NO3– contents in the culture medium were observed at 15 min (data not shown). As shown in Figure 2A, the NO2–/NO3– contents were increased significantly in Ag (I)-stimulated cells after 2 h, whereas no significant changes were observed in antigen-stimulated cells. No increases in the NO2–/NO3– contents were observed in the absence of cells, thereby excluding the possibility that these increases were artifacts resulting from chemical reactions between Ag (I) and components of the medium. Hb is known to scavenge NO for the production of NO3– (for a review, see ref. [44]). Consistent with this report, treatment with Hb increased the NO2–/NO3– contents significantly but blocked the effects of Ag (I) (Fig. 2B). Similar effects were observed with myoglobin, another NO scavenger (data not shown). DTT, a cell-impermeable, thiol-reducing agent, also abolished the effects of Ag (I) completely, and the agent alone had minimal effects on the NO2–/NO3– contents (Fig. 2B). Volume 86, December 2009


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Figure 1. Ag (I) induces NO production and release in a NOS-independent manner. (A and B) RBL-2H3 cells that had been sensitized passively with anti-TNP IgE and IgE-untreated cells were washed and suspended in HBSS. The cells were incubated with 5 ␮M DAF-2-DA for 15 min at 37°C and washed. The DAF-2-loaded cells (5ⴛ105 cells/450 ␮l) were stimulated with 30 ng/ml TNP-BSA (antigen) or 30 ␮M Ag2SO4 for 5 min (A) or for the indicated times (B) at 37°C. After washing, the cells were resuspended immediately in HBSS on ice and measured for their DAF-2 fluorescence by flow cytometry. The data are shown as the fluorescence ratios (F/F0), where F0 is the fluorescence in unstimulated cells, and F is the fluorescence in stimulated cells and represent the means ⫾ sem of nine (A) and four (B) independent experiments. (C and D) The DAF-2-loaded cells were incubated for 5 min with or without 100 ␮M amino-BH4 and 100 ␮M l-NAME (C) and 30 ␮M PP1 and 100 nM wortmannin (D) and then stimulated with antigen or Ag2SO4 and measured for their DAF-2 fluorescence as described above. The data are shown as percentages of control cells treated with stimulus alone (set at 100%) and represent the means ⫾ sem of four independent experiments. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001, versus control cells. WT, wortmannin.

These findings support the release of NO into the extracellular fluid, as DTT exists predominantly outside of cells, owing to its cell-impermeability. Moreover, similar to the increases in the intracellular NO levels, the increases in the NO2–/NO3– contents were unaffected by treatments with amino-BH4 and l-NAME (Fig. 2C), thereby validating the NOS independence of the NO production. Collectively, these results suggest that Ag (I) stimulates intracellular NO production in a NOS-independent manner, followed by release of NO into the extracellular spaces.

Positive roles of NOS-independent NO in Ag (I)-induced degranulation and LTC4 release To clarify the roles of NO production in Ag (I)-induced mast cell activation, we examined the effects of Hb and DTT on ␤-hexosaminidase and LTC4 release. Both agents strongly inhibited Ag (I)-induced degranulation but did not suppress antigen-induced degranulation (Fig. 3A). Hb and DTT also blocked Ag (I)-induced LTC4 release, whereas both agents enhanced rather than reduced antigen-induced LTC4 release (Fig. 3B). On the other hand, amino-BH4 and l-NAME enhanced LTC4 release substantially, regardless of the stimuli used (Fig. 3C), consistent with the previous reports about antigen-induced LTC4 release [7, 45]. These results show that decreased NO blocks Ag (I)-induced mediator release but not antigen-induced release, suggesting that NOS-independent NO acts specifically as a positive regulator of Ag (I)-induced mast cell activation.

Ca2ⴙ influx is necessary for Ag (I)-induced mast cell activation Elevation of [Ca2⫹]c is believed to be an essential process for mast cell degranulation and LTC4 release. We reported 4 Journal of Leukocyte Biology

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previously that Ag (I)-induced degranulation and Ca2⫹ increase are dependent on extracellular Ca2⫹ [36]. Consistent with that report, Ag (I) failed to induce ␤-hexosaminidase release when extracellular Ca2⫹ was depleted by EGTA (Fig. 4A). Moreover, we found that Ag (I)-induced LTC4 release was also strictly dependent on extracellular Ca2⫹ (Fig. 4B). Similarly, antigen-induced ␤-hexosaminidase release and LTC4 release were dependent on extracellular Ca2⫹ (Fig. 4, A and B).

Positive roles of NOS-independent NO in Ag (I)-induced Ca2ⴙ influx To clarify the roles of NO in Ag (I)-induced Ca2⫹ influx, we examined the effects of Hb on the Ca2⫹ influx. We reported previously that Ag (I) does not induce an increase in [Ca2⫹]c in the absence of extracellular Ca2⫹ [35], indicating that the increase in Ag (I)-stimulated cells mainly results from an influx of extracellular Ca2⫹. Hb inhibited the Ca2⫹ influx substantially in a dose-dependent manner with a minimum effective dose of 3 ␮M (Fig. 5A). At low concentrations (ⱕ10 ␮M), Hb caused a considerable delay in the onset of Ca2⫹ influx, and at a high concentration (30 ␮M), it abolished the response completely for at least 3 min. Although Hb alone at concentrations of ⱖ10 ␮M caused substantial decreases in the resting [Ca2⫹]c (the maximum decrease at 30 ␮M was ⬍100 nM), Hb reduced the effects of Ag (I) more extensively (⬎1000 nM at 30 ␮M; Fig. 5, A and B). Strikingly, even when Hb alone had a minimal effect on the resting [Ca2⫹]c (e.g., 3 ␮M), a substantial (400 –1000 nM) decrease in the Ca2⫹ influx was observed, thereby excluding the possibility that the inhibitory effect of Hb results merely from artificial interference with the Ca2⫹ response. We reported previously that DTT is a

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Inoue et al. NO-dependent mast cell activation by Ag (I)

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Figure 2. Hb and DTT block the NO release. (A) RBL-2H3 cells (5ⴛ105 cells/200 ␮l) were stimulated with 30 ng/ml antigen or 30 ␮M Ag2SO4 for 2 h at 37°C. The NO2–/NO3– contents in the supernatants were measured by a Griess reagent assay. The data represent the means ⫾ sem of seven independent experiments. (B and C) RBL-2H3 cells were incubated with or without 30 ␮M Hb and 100 ␮M DTT (B) or 100 ␮M amino-BH4 and 100 ␮M l-NAME (C) and then stimulated immediately with antigen or Ag2SO4 for 2 h at 37°C. In parallel, aliquots of the cells were incubated with Hb or DTT alone. The NO2–/ NO3– contents in the supernatants were measured as described above. The data represent the mean ⫾ sem of at least three independent experiments. *, P ⬍ 0.05; ***, P ⬍ 0.001, versus control cells.

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potent inhibitor of Ag (I)-induced Ca2⫹ influx but exerted a minimal effect on antigen-induced Ca2⫹ influx. Moreover, DTT can reverse Ag (I)-induced Ca2⫹ influx. Specifically, even when DTT was added after exposure to Ag (I), the [Ca2⫹]c increase was reduced substantially compared with that observed in the absence of DTT [36]. Therefore, we examined whether Hb exhibited a similar reverse effect. As shown in Figure 5C, even when Hb was added at 3 min after exposure to Ag (I), the [Ca2⫹]c increase was abolished completely. Therefore, Hb and DTT exhibit similar reverse effects on Ag (I)induced Ca2⫹ influx in parallel with their abilities to scavenge extracellular NO. In contrast, exogenously added SOD and catalase, which scavenge selectively O2•– and H2O2, respectively, had minimal inhibitory effects on the Ag (I)-induced Ca2⫹ influx (Fig. 5D). These results suggest that NOS-independent NO is a principal mediator of Ag (I)-induced Ca2⫹ influx.

As Ag (I) appears to stimulate store-dependent or -independent Ca2⫹ influx [35, 36], we clarified the roles of NO in these two Ca2⫹ influxes. To activate store-operated Ca2⫹channels specifically, we used the classical Ca2⫹ readdition protocol. Briefly, cells were treated with Tg in Ca2⫹-free medium to deplete the ER Ca2⫹ stores, and Ca2⫹ was then re-added, which resulted in a robust Ca2⫹ influx blocked completely by pretreatment with the SOCE antagonists 2-APB and SK&F96365 (Fig. 6, A and C), thereby validating activation of CRAC channels. Hb and DTT had no significant inhibitory effects on this Ca2⫹ influx (Fig. 6, A and C). Similarly, amino-BH4 and l-NAME had minimal effects on the CRAC channel activity (Fig. 6, B and D). Consistent with our previous study [36], Ag (I), but not Tg, induced a robust Ca2⫹ influx into the cells that had been depleted of intracellular Ca2⫹ stores (Fig. 7A), indicating activation of a non-SOCE pathway. As shown in Figure 7B, Hb and DTT abolished this non-SOCE completely. On the contrary, this non-SOCE was quite resistant to treatments with amino-BH4, l-NAME, SOD, and catalase (Fig. 7, C and D). Collectively, these results suggest that NOS-independent NO is necessary for non-SOCE but not SOCE.

Ag (I)-induced non-SOCE is mediated by Ca2ⴙ channels other than LTCCs Recently, we reported that antigen, but not Tg, induces a non-SOCE pathway [46] and that this non-SOCE is mediated mainly by Cav1.2 LTCC [37]. One important characteristic of this kind of non-SOCE is its high sensitivities to classical LTCC antagonists and wortmannin and resistance to 2-APB and SK&F96365 [46]. Therefore, we examined the effects of LTCC antagonists and wortmannin on the Ag (I)induced non-SOCE. In accordance with our previous observations that Ag (I) induces Ca2⫹ influx in a distinct manner from antigen [35, 36], the LTCC antagonist nifedipine and wortmannin had minimal effects on Ag (I)-induced nonSOCE (Fig. 8, A and B). Similar results were obtained with Volume 86, December 2009


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other LTCC antagonists verapamil and diltiazem (data not shown). To clarify the independence of LTCCs more directly, we examined the effects of abolishing the expression of ␣1C, the ␣1 subunit of Cav1.2 LTCC. The siRNA gene-

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silencing technique enabled us to obtain ␣1C-KD cells, in which ␣1C gene expression was reduced substantially at the mRNA and protein levels [37]. Consistent with a pivotal role of Cav1.2 LTCC, robust non-SOCE, which was blocked and augmented by nifedipine and (S)-BayK8644, respectively, was observed in control cells, but not in ␣1C-KD cells after antigen stimulation (Fig. 8, C and D). On the con-

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6 Journal of Leukocyte Biology

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4 Figure 3. Positive roles of NOS-independent NO in Ag (I)-induced degranulation and LTC4 release. (A–C) RBL-2H3 cells (5ⴛ105 cells/ 200 ␮l) were incubated with or without 30 ␮M Hb and 100 ␮M DTT (A and B) or 100 ␮M amino-BH4 and 100 ␮M l-NAME (C) and then stimulated immediately with 30 ng/ml antigen or 30 ␮M Ag2SO4 for 30 min at 37°C. The ␤-hexosaminidase activities (A) and LTC4 contents (B and C) in the supernatants were determined enzymatically and by ELISA, respectively. The data are expressed as percentages of the ␤-hexosaminidase activity and LTC4 content in control cells treated with stimulus alone (set at 100%) and represent the means ⫾ sem of nine independent experiments. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001, versus control cells.

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NO and that NO facilitates non-SOCE, thereby positively regulating mast cell degranulation and LTC4 release. Our results seem to argue against the conventional view that NO is a negative regulator of mast cell activation and subsequent features of inflammation [2, 3, 5]. Endogenously produced or exogenously applied NO suppresses calcium ionophore- and IgEmediated degranulation, LT secretion, and cytokine expression and production in rodent and human mast cells. NO mediates the suppressive effects of IFN-␥ on mast cell responses. Activated mast cells are able to bind to extracellular matrix proteins, including fibronectin, and IFN-␥ and NO donors

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Figure 5. Positive role of NOS-independent NO in Ag (I)-induced Ca2ⴙ influx. RBL-2H3 cells (1⫻106 cells/ml) were incubated with 4 ␮M Fluo3/AM for 40 min at 37°C, washed, and resuspended in a solution supplemented with 1 mM CaCl2. The Fluo3/AM-loaded cells were incubated with Hb at the indicated concentrations and stimulated immediately with 30 ␮M Ag2SO4 (A) or HBSS (B) and measured for their Fluo3 fluorescence at 5 s intervals for up to 3 min in a microplate fluorometer. (C) Hb was added to the cells at 3 min after stimulation with Ag2SO4 (indicated by the bold arrow). (D) The Fluo3/AM-loaded cells were incubated with or without 10 U/ml SOD and 10 U/ml catalase and then stimulated immediately with Ag2SO4. The data shown as the calculated [Ca2⫹]c are representative of four independent experiments.

In the present study, we have elucidated the roles of NO in Ag (I)-induced mast cell activation. The results demonstrated clearly that Ag (I) stimulates the production of intracellular

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gether, NO acts as critical down-regulators of mast cell functions in vitro and in vivo. NO also negatively regulated IgEmediated mast cell activation in our systems. Therefore, the positive regulatory role of NO is specific for Ag (I) stimulation. This finding suggests that the mechanisms and sources of Ag (I)-induced NO production may be unique. Our previous study revealed that Ag (I) is advantageous over antigen stimulation for inducing the release of ROS into extra-

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Figure 7. NOS-independent NO is essential for Ag (I)-induced non-SOCE. (A and B) The Fluo3/AM-loaded cells (1ⴛ106 cells/ml) were resuspended in Ca2⫹-free medium (HBSS supplemented with 1 mM EGTA) and treated with 2 ␮M Tg at 37°C for 10 min to deplete the ER Ca2⫹ stores. After extensive washing, the store-depleted cells were resuspended in Ca2⫹-containing medium (HBSS supplemented with 1 mM CaCl2), stimulated with 30 ␮M Ag2SO4 or 1 ␮M Tg, and measured for their Fluo3 fluorescence using a microplate fluorometer. (B–D) Store-depleted cells were resuspended in Ca2⫹-containing medium, treated with 30 ␮M Hb and 100 ␮M DTT (B), 100 ␮M amino-BH4 and 100 ␮M l-NAME (C), or 10 U/ml SOD and 10 U/ml catalase (D), and stimulated immediately with Ag2SO4. The data shown as the calculated [Ca2⫹]c are representative of four independent experiments.

down-regulate the adhesion of RBL-2H3 and HMC-1, respectively [47, 48]. In addition, NO down-regulates the biological effects of mast cells on other cell types. Rat mast cell has been shown to release RMCPII, thereby contributing to increased intestinal epithelial permeability. Kanwar and colleagues [49] have demonstrated that l-NAME enhances the RMCPII release greatly, and this effect is attenuated by adding stabilizer, suggesting that NO regulates this protease release. Taken to-

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Figure 8. Ag (I)-induced non-SOCE is mediated by Ca2ⴙ channels other than LTCCs. (A and B) After Fluo3/AM loading, ER Ca2⫹ store-depleted cells were prepared as described in the legend for Figure 7. The store-depleted cells were incubated with or without 1 ␮M nifedipine (A) and 100 nM wortmannin (B), immediately stimulated with 30 ␮M Ag2SO4, and measured for their Fluo3 fluorescence using a microplate fluorometer. (C–F) ER Ca2⫹ store-depleted cells were prepared from control cells and ␣1C-KD cells. Ca2⫹ store-depleted cells (C and E) and Ca2⫹ store-depleted ␣1C-KD cells (D and F) were incubated with or without 1 ␮M nifedipine and 10 ␮M (S)-BayK8644, stimulated immediately with 30 ng/ml antigen (C and D) or 30 ␮M Ag2SO4 (E and F), and measured for their Fluo3 fluorescence as described above. The data shown as the calculated [Ca2⫹]c are representative of four (A and B) and three (C–F) independent experiments.



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cellular spaces [36]. Consistent with these findings, we found that Ag (I)-mediated, but not IgE-mediated, activation was concomitant with a robust NO release into the extracellular fluid, although both stimuli induced substantial intracellular NO production. This difference suggests that the two stimuli evoke NO production through separate mechanisms and sources. In fact, our data indicated that Ag (I)-induced NO production was quite resistant to pharmacological inhibitors of NOS, SFK, and PI3K activities. These properties are quite different from those of IgE-mediated NO production, which is mediated by a PI3K-Akt-endothelial NOS pathway [8]. We showed previously that mast cells can produce NO by NOS-dependent and -independent pathways. Specifically, Fc␧RI-mediated signaling events, such as receptor component phosphorylation, are necessary for the NOS-dependent pathway, whereas chemicals that stimulate mast cell activation independently of Fc␧RI-mediated events, such as Tg, induce the NOS-independent pathway. These two distinct pathways of NO production are also distinct in terms of their PI3K dependences, as the NOS-dependent pathway is PI3K-dependent, and the NOS-independent pathway is PI3K-independent. Given that Ag (I) induces mast cell activation independently of Fc␧RI signaling pathways [35], the present data expand this view. The enzymatic NOS-dependent pathway is generally accepted as a source of NO. However, generation of NO that is independent of NOS activity has been reported in several cell types, in which two different sources have been implicated as playing roles. One is direct reduction of NO2– to NO under acidic and highly reducing conditions, including ischemia [50, 51]. The other is NO release from S-nitrosothiols, endogenous metabolites of NO [52]. Further studies clarifying the potential roles of these two NOS-independent NO formation pathways in Ag (I)-induced NO production are under way. As Ca2⫹ influx is necessary for Ag (I)-induced mast cell activation, it is important to identify the molecular entities of the Ca2⫹ channels activated by Ag (I). Our previous studies suggested that store-dependent and -independent Ca2⫹ channels are involved in Ca2⫹ influx in Ag (I)-stimulated cells [35, 36]. Therefore, we investigated the roles of NO in the store-dependent and -independent Ca2⫹ influx pathways. Although CRAC channels are the major Ca2⫹ channels involved in SOCE, our data indicated that regardless of whether NO existed inside or outside of cells, NO played a minor role in CRAC channel activation. Instead, we found that a non-SOCE pathway was regulated strictly by NOS-independent NO. We reported recently that antigen stimulation of mast cells induces a substantial Ca2⫹ influx in a store-independent manner, although the effects varied considerably depending on the conditions used and the sublines used. Moreover, our previous study revealed that the Fc␧RI␤ ITAM was essential for this IgE-mediated non-SOCE [46]. Given that Ag (I) has been shown to activate mast cells independently of Fc␧RI␤ [35], Ag (I)-induced nonSOCE may be distinct from IgE-mediated non-SOCE. In fact, the data obtained in the present study revealed some differences in the pharmacological and biochemical proper-

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ties of the non-SOCE pathways induced by Ag (I) and antigen. Unlike the pathway induced by antigen, the Ag (I)induced non-SOCE was quite resistant to the LTCC antagonists nifedipine, verapamil, and diltiazem. The non-SOCE was also independent of SFK and PI3K activities, both of which are necessary for IgE-mediated non-SOCE [46]. More recently, we reported that IgE-mediated non-SOCE is mediated mainly by Cav1.2 LTCC. In accordance with the resistance to the LTCC antagonist, siRNA knockdown of ␣1C, the ␣1-subunit of Cav1.2 LTCC, had minimal effects on the Ag (I)-induced non-SOCE, thereby supporting the view that this non-SOCE is mediated by Ca2⫹ channels other than LTCCs. Strikingly, NOS-dependent NO does not seem to play a positive role in SOCE. However, our preliminary experiments showed that this enzymatic NO generation positively regulated IgE-mediated non-SOCE, i.e., Cav1.2 LTCC. If this is the case, NO may facilitate two pharmacologically distinct non-SOCE pathways depending on the NO source. Specifically, NOS-dependent NO may facilitate Cav1.2 LTCC, whereas NOS-independent NO may mediate non-SOCE via other Ca2⫹ channels. However, further investigations are necessary to establish this hypothesis. Our present data suggested that NOS-dependent NO facilitated Ag (I)-induced mast cell activation. Consistent with several previous studies demonstrating that NO acts as a negative regulator of IgE-mediated mast cell activation [2, 3, 5], we observed that scavenging of NO by Hb and DTT augmented rather than suppressed IgE-mediated degranulation and LTC4 release; meanwhile, scavenging of NO blocked Ag (I)-induced degranulation and LTC4 release specifically. Given that Ag (I), but not antigen, activated NOS-independent NO production, rapid NO release, and a unique non-SOCE pathway, it is possible that the extracellular NO produced independently of NOS activity positively regulates the unique non-SOCE pathway and mast cell activation. Further studies attempting to identify the molecular entities involved in the unique non-SOCE are under way. Given that LTCCs play a pivotal role in mast cell survival after cell activation [53], the strong ability of Ag (I) to evoke

Ag (I) stimulation NOS-independent NO generation Intracellular NO Extracellular NO

Hb, DTT

Store-independent Ca2+ influx β-hexosaminidase release and LTC4 release Figure 9. Model for Ag (I)-induced mast cell activation. Stimulation of mast cells with Ag (I) results in the activation of a NOS-independent NO generation, leading to increased intracellular and extracellular NO levels. The extracellular NO in turn stimulates Ca2⫹ influx through thiol-sensitive, store-independent Ca2⫹ channel(s), thereby evoking Ca2⫹-dependent effector functions such as degranulation and LTC4 release.



9

necrosis and apoptosis in a Ca2⫹-dependent manner [54] may result from the inactivation of LTCC activity in Ag (I)-stimulated cells. In conclusion, the present study has demonstrated that NOS-independent NO positively regulates a non-SOCE pathway through unique Ca2⫹ channels, thereby playing a critical role in Ag (I)-induced mast cell activation (Fig. 9). To the best of our knowledge, this is the first report that NO can act as a positive regulator of mast cell activation. Our findings suggest multiple sources and functions of NO in mast cell biology. The NO-dependent non-SOCE pathway may be a potential target for the development of therapeutic approaches toward metal-induced allergy and autoimmunity.

AUTHORSHIP T. I. performed experiments, analyzed the results, made the figures, and wrote the paper. Y. S. designed the research, analyzed the results, and wrote the paper. T. Y. performed experiments. C. R. checked the manuscript and figures.

ACKNOWLEDGMENTS This work was partially supported by a Grant-in-Aid from the “High-Tech Research Center” Project (2002–2006) for Private Universities, matching fund subsidy from MEXT, and by Grants-in-Aid from Nihon University. We thank the National Institute of Health Sciences (Japanese Collection of Research Bioresources) for providing the RBL-2H3 cells (Cell No. JCRB0023).

REFERENCES

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KEY WORDS: silver 䡠 Ca2⫹ channel



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