DJ-1 regulates mast cell activation and IgE-mediated allergic responses

Abstract

Background

DJ-1 is an anti-oxidant protein, known to reduce levels of reactive oxygen species (ROS), but its presence or function in mast cells and allergic diseases is unknown.

Objectives

To determine the role and mechanism of DJ-1 in allergic responses in vitro and in vivo.

Methods

The level of ROS and DJ-1 in serum or culture medium was measured with ELISA kits. The role of DJ-1 was evaluated in mast cell cultures and passive cutaneous anaphylaxis (PCA) in normal or DJ-1 knockout (KO) mice. The mechanism of DJ-1 action was examined by immunoblotting, immunoprecipitation, RT-PCR, and other molecular biological approaches.

Results

Patients with atopic dermatitis had elevated levels of ROS and diminished levels of DJ-1. DJ-1 KO mice exhibited enhanced PCA and augmented ROS levels in serum and in bone marrow-derived mast cells (BMMCs) from these mice. Furthermore, antigen-induced degranulation as well as production of TNF-α and IL-4 were significantly amplified in DJ-1 KO and anti-DJ-1 siRNA-transfected BMMCs compared to wild type BMMCs. Studies with these and BMMCs transfected with siRNAs against the phosphatases SHP-1 or SHP-2 revealed that the DJ-1 KO phenotype could be attributed to suppression of SHP-1 activity and enhancement of SHP-2 activity leading to strengthened signaling through LAT, phospholipase Cγ, and MAP kinases.

Conclusions

A deficiency or constitutive activation of DJ-1 can have implications in mast cell-driven allergic diseases such as asthma and anaphylaxis.

Introduction

There is evidence that allergic disorders such as asthma, rhinitis, and atopic dermatitis, are associated with oxidative stress.^1,2 One regulator of intracellular and extracellular reactive oxygen species (ROS) levels is DJ-1 for which no role has been reported in mast cells and allergy. However, this protein plays a critical role in anti-oxidative stress activity by reducing ROS levels and transcriptional regulation. Loss of function of DJ-1 is associated with some types of neurodegenerative diseases and cancer.³ The anti-oxidant activity of DJ-1 is achieved through several mechanisms namely self-oxidation at cysteine residues 46, 53, and 106 (C46, C53 and C106, respectively) to eliminate ROS^4–6 and interaction with superoxide dismutase, glutathione peroxidase, and catalases to enhance their ability to remove ROS.⁷

Mast cells are key mediators of IgE-mediated allergic diseases including asthma, rhinitis, and atopic dermatitis. Activation of mast cells through cross-linking of FcεRI-bound IgE with antigen results in release of three classes of mediators; granule-associated mediators, cytokines, and inflammatory lipids.^8,9 The initiating signaling event is the activation of Src family tyrosine kinases (SFKs) including Lyn.^10,11 Lyn then phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMs) of β and γ subunits of adjacent FcεRI receptors. The subsequent recruitment and activation of the tyrosine kinase, Syk, by the phosphorylated ITAMs results in activation of signaling molecules, such as LAT and downstream signaling pathways that lead to release of various allergic mediators.^12,13 In addition, other Src family kinases including Fyn, Hck, and Fgr initiates signals required for optimal activation of mast cells.^14–16

Other, but less well understood, components of mast cell activation are ROS such as superoxide anion and hydrogen peroxide. These diffusible molecules are produced by virtually all cells including mast cells.¹⁷ Earlier reports indicated that activated mast cells have elevated intracellular levels of ROS.^18,19 Also, ROS enhances histamine release from mast cells^18,20 and hydrogen peroxide in particular can regulate tyrosine phosphorylation of PLCγ and LAT.²¹ However, the mechanisms by which ROS influence mast cell signaling is largely unclear.

The above considerations led us to study the allergic reaction in DJ-1-deficient mice as well as the reactivity of mast cells from these mice. We report for the first time that DJ-1 modulates ROS levels in mast cells and as a consequence regulates activity of Fyn, Syk, and LAT as well as responses to antigen, in vitro and in vivo.

Methods

Reagents

Reagents were from the following sources: antibodies detecting activating phosphorylations of Syk(Y352 or Y525/526), LAT(Y191), Gab2(Y452), PLCγ1(Y783), p85 PI3K(Y458), and SHP-2(Y542) from Upstate Biotechnology (Lake Placid, NY); antibodies detecting activated phosphorylated forms of Akt(T308), Erk1/2(T202/Y204), p38(T180/Y182), and JNK(T183/Y185) from Cell Signaling Technology, Inc (Danvers, Mass); antibodies against Lyn, Fyn, Fgr, SHP1, SHP2, and DJ-1 from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Unless otherwise stated, other reagents were from Sigma (St. Louis, MO).

Generation of passive cutaneous anaphylaxis (PCA) in mice and serums from atopic dermatitis patients

PCA was induced by injection of antigen (DNP-BSA) into mice previously primed with DNP-specific IgE in one ear.²² Evans blue dye in ear tissues was extracted in formamide for assay at 620 nm. Mast cells in the ear tissues were histologically examined as described elswhere¹⁶. The animal study was approved from the Institutional Animal Care and Use Committee (IACUC) at Konkuk University. Atopic dermatitis (AD) patients (n = 56) who visited the Seoul Allergy Clinic, Seoul, Korea participated in this study. They fulfilled the criteria of Hanifin and Rajka. ²³ The patients had been suffering from wide range of AD severity with a SCORAD index of 35.0 ± 19.8 (22 mild, 17 moderate, and 17 severe patients) at the time of study (Table E1 in this article’s Online Repository at www.jacionline.org).²⁴ All medications were discontinued for at least two weeks before the study although topical application of hydrocortisone 1% was allowed. Healthy control subjects (n = 15) had no history of specific diseases including allergic disease. The study was approved by the Institutional Review Board of Eulji University, Daejeon, Korea.

Mice, cell culture, and stimulation of BMMCs

DJ-1 KO and wild type (WT) C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). BMMCs were prepared from mouse bone marrow and by culture for 4 weeks in a 50% enriched medium (RPMI 1640 containing 2 mM L-glutamine, 0.1 mM nonessential amino acids, antibiotics, and 10% FBS) containing 10 ng/mL of IL-3.²⁵ For individual experiments, cells (2×10⁵ cells/1.5 ml tube) were primed overnight with DNP-specific IgE (50 ng/ml) and then stimulated with 25 ng/ml antigen in Tyrode-BSA buffer (20 mM HEPES, pH 7.4, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, and 0.05% BSA) or complete growth medium as indicated.

Measurement of ROS in serum and cells

ROS levels were measured with the OxiSelect™ In vitro ROS/RNS Assay Kit (Cell Biolabs, Inc., Sandiego, CA). BMMCs (2×10⁵ cells/1.5 ml) were incubated with dichlorofluorescein (DCF)-diacetate (20 μM) for 10 min at 37°C and washed before stimulation with antigen for 10 min in Tyrode-BSA buffer. DCF fluorescence (excitation 492 nm; emission 535 nm) of 100 μl lysed cells (0.5% Triton-X 100) was monitored in a GENios fluorescent plate reader (ReTiSoft, San Diego, CA).²⁶ For measurement of intracellular ROS in DCF-loaded cells by confocal microscopy, cells were fixed in 4% formaldehyde for 10 min. Confocal images were obtained in an Olympus FV-1000 confocal laser scanning microscope with an Apochromat 60X objective.

Measurement of degranulation, TNF-α, IL-4, and DJ-1

IgE-primed BMMCs were stimulated with antigen in Tyrode-BSA buffer for 10 min or as indicated. Degranulation was determined by measurement of release of the granule marker, β-hexosaminidase.²⁷ Otherwise cells were stimulated with antigen for 8 h in complete media for measurement of levels of TNF-α and IL-4 using the ELISA kits from Invitrogen-Biosource Cytokine & Signaling (Camarillo, CA) or of DJ-1 using ELISA kits from R&D Systems, Inc (Minneapolis, MN).

Immunoprecipitation and immunoblot analysis

IgE-primed BMMCs, stimulated with antigen in Tyrode-BSA buffer for 7 min or as indicated, were lysed with ice-cold lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet p-40, 10% glycerol, 60 mM octyl β-glucoside, 10 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM nitrophenylphosphate, 0.7 μg/ml pepstatin, and protease inhibitor cocktail tablet). Lysates were kept on ice for 30 min and then centrifuged 13,000 × g for 10 min at 4°C. Equal aliquots of protein were subjected to immunoprecipitation and immunoblotting analysis.¹⁶

Transfection of DJ-1 DNA plasmid and siRNAs against DJ-1, SHP1, SHP2 or Syk

BMMCs were transfected with DNA plasmids (10 μg/5 × 10⁶ cells, unless stated otherwise) by Amaxa nucleofector (Lonza Cologne AG, Cologne, Germany) and used within 48 h of transfection. To knockdown target proteins, BMMCs (5 × 10⁶ cells) were transfected with 100 nM siGENOME ON-TARGETplus SMARTpool against target proteins or ON-TARGETplus siCONTROLpool as controls (Dharmacon Inc, Chicago, IL) and used 48 h after transfection.

Flow cytometric analysis

IgE-primed BMMCs from DJ-1-deficient or WT mice were incubated with FITC-conjugated anti-IgE or anti-CD117 (c-Kit)-PE (BD Biosciences, San Jose, CA). Multicolor analysis was performed in a FACS Calibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ).

Measurement of activities of SHP-1, SHP-2, and tyrosine kinases in vitro

SHP-1 and SHP-2 were immunoprecipitated from lysates of BMMCs stimulated or not with antigen in Tyrode-BSA buffer for 7 min. Immunoprecipitates were assayed for phosphatase activities by the malachite green SHP-1 and SHP-2 activity kit (Duoset IC ELISA, R&D Systems). Similarly Lyn, Fyn, Fgr, and Syk immunoprecipitates prepared from 1 mg protein of whole cell lysates were assayed for kinase activity using the ELISA-based Universal Tyrosine Kinase Assay Kit (Gen Way, Sandiego, USA). One unit (U) of tyrosine kinase represents incorporation of 1 pmol of phosphate into the substrate (KVEKIGEGTYGV VYK: 6 – 20 residue of p34cdc2 )/min.

Presentation of results

All data are means ± SEM from three or more independent experiments and were analyzed statistically by the one-way ANOVA and the Dunnett test. Significance (*P < 0.05 and **P < 0.01) was determined with SigmaStat software (Systat Software, Inc, Point Richmond, CA).

Results

Diminished DJ-1 serum levels in patients with atopic dermatititis

Based on evidence that allergic disorders, such as atopic dermatitis, asthma, and rhinitis, could be mediated by oxidative stress,^1,2 we first measured ROS levels in serum from normal and allergic patients with atopic dermatitis. Significantly elevated ROS levels were observed in serum from these patients (59.9 ± 1.6 μM for milds, 57.8 ± 1.7 μM for moderates, and 57.8 ± 1.6 μM for severe patients) as compared to normal subjects (24.2 ± 1.4 μM) (P ≤ 0.00019) (Fig. 1A). Conversely, serum levels of DJ-1 were substantially diminished in patients (2.8 ± 0.3 ng/ml for milds, 2.1 ± 0.2 ng/ml for moderates, and 2.2 ± 0.2 ng/ml for severe patients) versus normal subjects (8.0 ± 0.4 ng/ml) (P ≤ 0.00114) (Fig. 1B) to suggest that DJ-1 could also be associated with AD. However, we observed no significant differences in ROS and DJ-1 levels between patients with severe (solid circles) or moderate (grey circles) and mild (open circles) AD (Fig. 1).

FIG 1.

The levels of DJ-1 are decreased in serum from allergic patients, as opposed to the elevated levels of ROS, as compared to normal volunteers. The ROS (A) and DJ-1 (B) levels in serum were measured using the ELISA kit according to the manufacturer’s instructions. Values are the means ± the SEM of values from normal volunteers (n = 15) and atopic dermatitis patients (n = 56, open circles, 22 mild AD; grey circles, 17 moderate AD; solid circles, 17 severe AD patients). n.s., not significant.

DJ-1-deficient mice show significantly enhanced PCA reaction in mice and mast cell degranulation

Next, we investigated whether DJ-1 plays a role in mast cell-mediated allergic response using the mouse PCA model in DJ-1 KO mice which had no detectable DJ-1 (Fig. 2A). Antigen-induced PCA reaction was increased in these mice as compared to WT mice (Figs. 2B and 2C). Of note, histological examination of ear tissues¹⁶ revealed that the degranulation of mast cells was significantly increased in DJ-1 KO mice (Figs. 2D and 2E). As observed previously,^16,28 the total number of mast cells also increased during the PCA reaction (Figs. 2D and 2E), presumably through infiltration and differentiation of progenitor cells.^8,30

FIG 2.

Lack of DJ-1 significantly enhances PCA reaction and mast cell degranulation in mice. (A) Representative Western blot images of indicated tissues. Representative ear images after PCA reaction (B) and extravasated amount of Evans blue dye (C) (n = 5). (D) Representative histological images of ear skin sections: Magnification, ×1000. Arrows indicate degranulated mast cells. (E) The number and percentage of degranulated mast cells in ear skin sections. All values are the means ± SEM from three independent experiments: * P < 0.05, ** P < 0.01.

DJ-1 deficiency leads to enhancement of ROS generation, degranulation, and cytokine production in antigen-stimulated BMMCs

In view of the above results and the fact that antigen-stimulated ROS production has been functionally linked to mast cell degranulation,^18,29,31 we examined ROS levels and release of bioactive mediators in DJ-1 KO and WT BMMC cultures. The generation of ROS by antigen was significantly enhanced in DJ-1 KO BMMCs (8.8 ± 0.87 μM) as compared to WT BMMCs (5.9 ± 1.38 μM) (Fig. 3A and 3B). A similar enhancement was noted in vivo in the PCA model with no significant difference in serum ROS level before antigen challenge but substantial difference between WT (24.1 ± 1.92 μM) and DJ-1 KO (37.3 ± 1.03 μM) mice following challenge (Fig. 3C).

FIG 3.

Levels of ROS, degranulation, and cytokine production in BMMCs. (A) Representative images by confocal microscopy. ROS level by ELISA analysis in BMMCs (B) or in serum after PCA reaction (C) were measured as described in the “Materials and methods” section. (D) Representative FACS images for c-Kit and FcεRI expression in BMMCs. (E) Representative Western images of α, β, and γ subunits of FcεRI receptor. (F) Percent release of granules in BMMCs. (G) TNF-α and IL-4 levels in the culture medium by ELISA. (B, C, E, F) All values are the means ± SEM from three independent experiments: * P < 0.05.

Both types of BMMCs exhibited equivalent expression of the mature mast cell markers, c-Kit and FcεRI by flow cytometric and immunoblotting analysis (Figs. 3D and 3E). Of note, antigen-induced degranulation and the secretion of IL-4 and TNF-α were markedly increased in DJ-1 KO BMMCs (Figs. 3F and 3G).

DJ-1 regulates early FcεRI-mediated signals including phosphorylation of Syk and LAT

To determine if DJ-1 regulated responses to stimulants other than antigen, we next examined degranulation by the calcium mobilizing agents, thapsigargin and ionomycin. In contrast to antigen, degranulation induced by either agent was the same in WT and DJ-1 K/O BMMCs (Fig. 4A). This suggested that DJ-1 influences FcεRI-mediated signals upstream of calcium mobilization which is a critical signal for degranulation.³² Accordingly, we determined the effects of DJ-1 deficiency on proximal FcεRI-associated signaling molecules. Surprisingly, the tyrosine phosphorylation of Syk was suppressed whereas that of LAT was enhanced in antigen-stimulated DJ-1 KO BMMCs (Fig. 4B). This was unexpected because the tyrosine phosphorylation of LAT is dependent on Syk in mast cells.³³ To verify this paradoxical finding, we transfected WT BMMCs with anti-DJ-1 siRNAs. Consistent with DJ-1 KO results, the phosphorylation of Syk was inhibited and that of LAT was increased in DJ-1 siRNA transfected BMMCs (Fig. 4C).

FIG 4.

The antigen-mediated phosphorylations of Syk and LAT were regulated by DJ-1. (A) Percent release of granules by 300 nM thapsigargin or 1 μM ionomycin in BMMCs. (B) Representative Western blot images for phosphorylation of Syk and LAT. (C) Representative Western blot images for phosphorylation of Syk and LAT (upper panel) and percent release of granules in BMMCs (lower panel) with or without anti-DJ-1 siRNAs. All values are the means ± SEM from three independent experiments: * P < 0.05.

DJ-1 exerts positive and negative effects on signaling

It is generally accepted that activation of PLCγ, Ca²⁺ mobilization, and MAP kinases are regulated by the Syk/LAT pathway whereas Gab2 and PI3K/Akt are additionally regulated by a Fyn-mediated complementary pathway in mast cells.¹¹ Therefore, we next examined the regulation of these signaling molecules by DJ-1. In DJ-1 KO BMMCs, the phosphorylation of PLCγ and the three typical MAP kinases, Erk1/2, JNK, and p38, were augmented (Fig. 5A) whereas phosphorylation of Gab2 and PI3K/Akt were suppressed (Fig. 5A). Also, transfection of DJ-1 into DJ-1 KO BMMCs reversed the positive and negative effects of DJ-1-deficiency on degranulation and phosphorylation of LAT/PLCγ and Syk (Fig. 5B). Apparently, DJ-1 positively regulates Syk and Fyn/Gab2 signals but negatively regulates LAT-dependent signaling events.

FIG 5.

DJ-1 regulates downstream signaling molecules either positively or negatively. (A) Representative Western blot images in BMMCs. (B) Representative Western blot images (upper panel) and percent release of granules (lower panel) in BMMCs with overexpression of DJ-1 or empty vector. All values are the means ± SEM from three independent experiments: * P < 0.05 (compared with WT BMMCs).

Studies in DJ-1 KO BMMCs reveal differential regulation of tyrosine kinases and SHP-1/SHP-2 phosphatases

We next investigated the effects of DJ-1 on the activation of tyrosine kinases that are required for activation of mast cells.^11,16 Lyn and Fgr were equally activated in DJ-1 KO and WT mast cells (Fig. 6A and 6B). However, in DJ-1 KO BMMCs the activation of Fyn was totally blocked (Fig. 6C) and that of Syk was significantly, albeit partially, reduced (Fig. 6D).

FIG 6.

Lack of DJ-1 significantly suppresses the activation of Fyn, and partially so Syk. Lyn (A), Fyn (B), Fgr (C) and Syk (D) were immunoprecipitated from 1 mg protein of whole cell lysates in wild type or DJ-1 KO BMMCs. The activities of immunoprecipitates were measured by ELISA-based Tyrosine Kinase Assay Kit. All values are the means ± SEM from three independent experiments: * P < 0.05 (compared with wild type BMMCs).

The apparent differential regulation of signaling molecules by DJ-1 was examined further by investigating the activation status of SHP-1 and SHP-2. SHP-1 is reported to regulate phosphorylation of LAT and MAP kinases without impacting that of Syk.^34,35 SHP-1 phosphorylation and activation was much diminished in stimulated DJ-1 KO BMMCs (Fig. 7A, upper panel) but was restored by treatment with 30 μM Tempo, a general ROS scavenger (Fig. 7A, lower panel), indicating that the SHP-1 activation was positively regulated by increases in intracellular levels of ROS.

FIG 7.

DJ-1 differentially regulates FcεRI-mediated activation of SHP-1 and SHP-2 and, in turn, phosphorylation of LAT and Syk, respectively. (A) Representative Western images (upper panel) and activities (lower panel) of immunoprecipitated SHP-1 or SHP-2 in BMMCs. (B and C) Representative Western images of whole cell lysates from BMMCs with transfection of control siRNAs or siRNAs against SHP-1 or SHP-2 (B), or Syk (C) in WT or DJ-1 KO BMMCs. (D) Percent release of granules in DJ-1 KO BMMCs with transfection of siRNAs against SHP-1 or SHP-2 (upper panel) or overexpression of DNA plasmid for SHP-1 or SHP-2 (lower panel). (A and D) All values are the means ± SEM from three independent experiments: * P < 0.05.

Another SHP isoform, SHP-2, has been linked to negative regulation of Syk.³⁶ We found that SHP-2 phosphorylation and activity, in contrast to SHP-1, was augmented in DJ-1 KO mast cells and that this augmentation was reversed by treating cells with 30 μM Tempo (Fig. 7A).

Syk and LAT are differentially dephosphorylated by SHP-2 and SHP-1, respectively

The roles of both SHPs were examined further by knock-down (KD) of the individual SHPs with siRNAs in WT and DJ-1 KO BMMCs. Antigen-induced phosphorylation of LAT was substantially increased in anti-SHP-1 siRNA-transfected WT or DJ-1 KO BMMCs but was not enhanced in anti-SHP-2 siRNA-transfected BMMCs (Fig. 7B). Conversely, antigen-induced phosphorylation of Syk was increased in anti-SHP-2 siRNA-transfected WT and DJ-1 KO BMMCs, but was not altered in anti-SHP-1 siRNA-transfected cells although overall Syk phosphorylation was diminished in DJ-1 KO BMMCs (Fig. 7B). LAT is thought to be the downstream target of Syk in antigen-stimulated mast cells.³² We found accordingly that transfection with anti-Syk siRNAs suppressed phosphorylation of LAT in both WT or DJ-1 KO mast cells (Fig. 7C). In addition, the KD of SHP-1 by anti-SHP-1 siRNAs further augmented the degranulation in DJ-1 KO BMMCs, presumably because of the additional increase of LAT phosphorylation (Fig. 7D, upper panel). Moreover, the overexpression of SHP-1 plasmid in DJ-1 KO BMMCs reversed the phenotype in DJ-1 KO BMMCs to the level in wild type BMMCs (Fig. 7D, lower panel). In contrast, the siRNA transfection and overexpression of SHP-2 did not affect significantly degranulation in DJ-1 KO BMMCs (Fig. 7D).

Fyn, SHP-1, and SHP-2 are regulated by H₂O₂ in vitro

As shown in Figure 7A, Tempo reversed the DJ-1 KO effects on SHP-1 and SHP-2. We, therefore, tested the direct effect of H₂O₂ on the activity of Fyn, SHP-1, and SHP-2 in vitro. Consistent with the above results, the activity of Fyn and SHP-1 were diminished by H₂O₂ in a dose dependent manner (Fig. 8A and 8B). In contrast, the activity of SHP-2 was significantly increased (Fig. 8B).

FIG 8.

Fyn, SHP-1, and SHP-2 activities are regulated directly by H₂O₂ and a proposed scheme for DJ-1 regulation of ROS and downstream signals. (A and B) Activities of immunoprecipitated Fyn, SHP-1, and SHP-2 from non-stimulated (NS) and antigen-stimulated (Ag) BMMCs were assayed in vitro with different concentrations of H₂O₂ for 15 min. Values (means ± SEM) are from three independent experiments: * P < 0.05. (C) Representative confocal images and Western blot images for co-immunoprecipitation of LAT and SHP-1 or Syk and SHP-2. (D) Proposed scheme: Absence of DJ-1 leads to excess ROS which inhibits SHP-1 and enhances phosphorylation of LAT and LAT-dependent signals while inhibiting Fyn and, via activation of SHP-2, Syk. Nevertheless, the enhancement of LAT phosphorylation is sufficient to ultimately augment mediator release in DJ-1-deficient BMMCs and PCA in DJ-1-deficient mice.

LAT and Syk associates with SHP-1 and SHP-2 respectively

Co-immunoprecipitation and co-localization studies were undertaken to verify possible functional association of LAT with SHP-1 and Syk with SHP-2. Some SHP-1 co-localized with LAT in the proximal region of plasma membrane and both molecules were co-immunoprecipitated by antigen stimulation (Fig. 8C, left panel). Some SHP-2 co-localized with Syk and both molecules were also co-immunoprecipitated by antigen-stimulation (Fig. 8C, right panel).

Discussion

DJ-1 was shown to be linked to early onset of Parkinson’s disease³⁷ and thought to act as an anti-oxidant that is capable of quenching ROS production in vivo and in vitro.^3,38 Allergic disorders are also reported to be associated with oxidative stress,¹ yet no function has been directly attributed to DJ-1 in mast cells or allergic diseases. However, we found that the level of ROS was increased in DJ-1 KO BMMCs (Fig. 3A and 3B) and in the serum from mice during PCA (Fig. 3C). Of note, abnormally low DJ-1 levels and elevated levels of ROS were observed in serum from allergic patients (Fig. 1). Furthermore, the PCA response and extent of mast cell degranulation was enhanced in DJ-1 KO mice (Fig. 2) as were degranulation and cytokine production in antigen-stimulated DJ-1 KO BMMCs (Fig. 3F and 3G). These results suggest that mast cell activation and mast cell-related allergic responses could be regulated by DJ-1.

Although much research on allergic and other immune diseases has focused on the toxic effects of ROS, there is increasing evidence that ROS at low concentrations might regulate cell signaling.^21,29 Production of ROS accompanies mast cell activation and is thought to promote this activation.^21,29,39,40 Nevertheless, the extent and mechanisms by which DJ-1 could regulate ROS in these activities is unknown. Our studies now demonstrate that elevated levels of ROS in DJ-1 KO BMMCs (Fig. 3A and 3B) are associated with increased degranulation and cytokine production (Fig. 3F and 3G). However, DJ-1’s role is surprisingly restricted to regulation of FcεRI-mediated signals as degranulation was unaltered in thapsigargin or inonomycin-stimulated DJ-1-deficient BMMCs (Fig. 4A). Our data suggest that these DJ-1/ROS regulated signals are transduced through Src-family kinases, Syk, and LAT. The phosphorylation of Syk was suppressed in DJ-1 KO or DJ-1 siRNA-transfected BMMCs (Fig. 4B and 4C) although the activity of Syk itself was only partially inhibited in DJ-1 KO BMMCs (Fig. 6D), indicating that Syk can still deliver the signals from the FcεRI receptor to the downstream signaling molecules including LAT.

Paradoxically, other downstream signaling molecules, such as PLCγ and the three typical MAP kinases, were increased by antigen in DJ-1 KO BMMCs when compared to WT BMMCs (Fig. 5A). This was in marked contrast to the substantial reduction in phosphorylation of Gab2 and PI3K/Akt (Fig. 5A), which is regulated by Fyn.¹⁴ This reduction correlated with the virtual block in activation of Fyn, but not of Lyn and Fgr, in antigen-stimulated DJ-1 KO BMMCs (Fig. 6C). These results strongly suggest that elevated ROS in DJ-1 KO BMMCs inhibits the activation of Fyn and Syk but enhances that of PLCγ and MAP kinases. These results raised further questions on the reasons for the apparent dichotomous effects of elevated ROS.

To address these questions, we focused on factors regulating Fyn, Syk, and LAT. The mechanism of activation of Fyn by antigen is not totally clear but its activation is critical for optimal activation of mast cells (Fig. 6C).¹⁴ However, the almost complete suppression of Fyn activation in antigen-stimulated DJ-1 KO BMMCs (Fig. 6C) and its direct inhibition Fyn by H₂O₂ in vitro (Fig. 8A) are consistent with the suppression of Fyn downstream molecules, Gab2 and PI3K/Akt, in DJ-1 KO BMMCs (Fig. 5A). To our knowledge, this is the first report that Fyn activity is directly influenced by the intracellular levels of ROS in antigen-stimulated mast cells. We note, however, that previous studies^42,43 have inferred, contrary to our findings, that H₂O₂ may activate Fyn in fibroblasts and Jurkat cells. However, Fyn activity was assessed indirectly in these studies through measurement of activation of downstream targets and such activation was observed at relatively high concentrations of H₂O₂ (0.2–10 mM) as compared to our study (0.1 mM). Therefore, the differences in experimental conditions and cell types could account for the apparent discrepancy in the different studies.

With respect to LAT, its phosphorylation is increased in SHP-1-deficient BMMCs³⁴ and T cells³⁹ as well as in DJ-1 KO BMMCs (Fig. 4B). Therefore, we hypothesized that the increase in phosphorylation of LAT in DJ-1 KO and DJ-1 siRNA-transfected BMMCs (Fig. 4B and 4C) was attributable to inhibition of SHP-1 by the elevated ROS in these cells. This was confirmed by the suppressed activation of SHP-1 was inhibited in DJ-1 KO BMMCs (Fig. 7A) and restoration of activation with a low concentration of Tempo (Fig. 7A, lower panel). Furthermore, SHP-1 activity was significantly inhibited by H₂O₂ in vitro (Fig. 8B), indicating that the intracellular ROS level could inhibit SHP-1 activity and thus modulate the phosphorylation of LAT.

In regard to the diminished phosphorylation of Syk in DJ-1 KO BMMCs, we considered the role of SHP-2 because this phosphatase is known to dephosphorylate Syk.³⁶ Indeed, the activation of SHP-2 was increased (Fig. 7A) and that of Syk was decreased (Fig. 4B and 6D) in DJ-1 KO BMMCs. In addition, the phosphorylation of Syk was increased in the SHP-2 siRNA-transfected WT and DJ-1 KO BMMCs (Fig. 7B). These results suggest that the activation of Syk is negatively regulated by DJ-1 via SHP-2.

In conclusion, FcεRI signaling events including activation of Src-family kinases, protein tyrosine phosphatases, and other critical molecules¹¹ are highly impacted by excess ROS as summarized schematically in Figure 8D. These include the inhibition of activation of Fyn and Syk and the unexpected enhancement of phosphorylation of LAT in DJ-1 KO BMMCs. Such effects are associated with inhibitory actions of ROS on Fyn and SHP-1 and positive actions on SHP-2. These multiple effects of ROS are associated with enhanced degranulation and cytokine production and thus deficiency or constitutive activation of DJ-1 could have implications in mast cell-driven inflammatory allergic and autoimmune diseases (Fig. 8D). As reported here, a distinguishing feature of AD patients was the markedly diminished levels of DJ-1 with corresponding increases in ROS levels when compared normal subjects (Fig. 1), We note, however, that although no discernable differences were noted with severity of disease in the patients studied, the onset of AD occurred early in life (newborn, childhood, or early teens) before this study in almost all of our patients. Therefore, we suggest only that abnormal DJ-1/ROS levels may be associated with the onset of AD that relapses through adulthood. Whether the extent of DJ-1/ROS abnormality correlates with the onset of disease will require further study of samples examined retrospectively from pediatric subjects before the onset of disease. It would also be of interest to determine whether DJ-1/ROS dysfunction underlies other mast cell-driven inflammatory diseases or is unique to AD.

Key messages.

• The serum levels of DJ-1 were significantly decreased in atopic dermatitis patients (P ≤ 0.00114) and was associated with elevated levels of ROS.

• DJ-1 regulates antigen-induced mast cell activation and allergic responses in vitro and in vivo.

• DJ-1 differentially regulates activation of Fyn, Syk, and LAT in mast cells.

• The dysregulation of DJ-1 has implications in mast cell-driven allergic diseases.

List of Abbreviations

BMMC

Bone marrow-derived mast cells

DCF

Dichlorofluorescein

ELISA

Enzyme-linked immunosorbent assay

ITAM

Immunoreceptor tyrosine-based activation motif

KO

Knockout

LAT

Linker for activation of T cells

PCA

Passive cutaneous anaphylaxis

pY

Phosphotyrosine

ROS

Reactive oxygen species

SFK

Src-family kinase

WT

Wild type