Ex vivo effects of naphthoquinones on allergen-sensitized mononuclear cells in mice

Abstract

Naphthoquinone (NQ), one of the extractable chemical compounds of diesel exhaust particles, enhances allergic asthma traits in mice. However, it remains unknown whether: (1) several types of NQs have the same potential to facilitate allergies; and (2) NQs synergistically disrupt the functional phenotypes of immune cells. The aim of the present study was to investigate the effects of two types (1,2- and 1,4-) of NQs on sensitized mononuclear cells using an ex vivo assay. Male BALB/c mice were repeatedly and intraperitoneally administered ovalbumin (OVA: 20 µg) plus alum with or without two different doses of each NQ. After the final administration, splenocytes (mononuclear cells) were isolated from these mice and cultured in the presence of OVA. Helper T-related cytokines in the culture supernatants and downstream molecules were then evaluated. Protein levels of interferon-γ were higher in the supernatants from 1,2-NQ and 1,4-NQ at low dose + OVA-exposed mononuclear cells following the OVA stimulation than in those from OVA-exposed mononuclear cells. Interleukin (IL)-13 levels were higher in the supernatants from low dose NQs + OVA-exposed mononuclear cells. IL-17 levels were significantly higher in the supernatants from low dose 1,2-NQ + OVA-exposed mononuclear cells. The quantity of phosphorylated STAT6 in the nuclei of these cells was significantly greater in the low dose NQ + OVA groups than in the OVA group. These findings suggest NQs differently enhance allergen sensitization in the context of the Th response against mononuclear cells such as lymphocytes.

Introduction

Although environmental chemicals are known to have adverse health effects, their immunotoxicities have not yet been examined in detail. A relationship has been suggested between stereotypical chemicals and increases in the morbidity of allergic diseases.¹ Diesel exhaust particles (DEP), which are representative of particulate matters (PM2.5) in city areas, are widely recognized to facilitate allergic reactions;^2,3 however, the responsible components have been poorly characterized. DEP are complex particles that consist of carbonaceous nuclei and a large number of organic chemical compounds including polyaromatic hydrocarbons, aliphatic hydrocarbons, heterocycles, and quinones. Of these, organic chemical components extracted from DEP have been shown to augment allergic response in vitro⁴ and in vivo.⁵ Consistent with previous findings, we also demonstrated that extracted organic chemicals from DEP, rather than the residual carbonaceous nuclei of DEP after extraction, predominantly enhanced allergen-related airway inflammation in mice.⁶

Various quinones have been identified as components of DEP.^7,8 Quinones themselves have toxicological properties to serve as alkylating agents and interact with, for example, flavoproteins to generate reactive oxygen species, which can lead to biological injury.^9–12 Naphthoquinone (NQ; MW: 158) is one of the quinones associated with DEP.¹³ We previously demonstrated that NQ deteriorated allergen-related asthma-like phenotypes in mice.¹⁴ However, it currently remains unknown whether several types of NQ have the same potential to facilitate allergies and the point of allergic inflammation adversely affected by NQs has not yet been identified.

Therefore, the aim of the present study was to investigate the effects of two types (1,2- and 1,4-) of NQs on the sensitization phase of allergy using an ex vivo assay and a subsequent allergy recall test in vitro.

Materials and methods

Animals

Seven-week-old balb/c male mice weighing 24–27 g were purchased from SLC Japan Inc. (Shizuoka, Japan), and were given sterile distilled water and a commercial diet (Labo MR Stock; Nosan Corporation Life-Tech Department., Yokohama, Japan) ad libitum. They were housed in an animal facility that was maintained at 24–26°C with 55–75% humidity and a 12-h light/dark cycle under conventional conditions. All animal studies were approved by the Institutional Review Board of the International University of Health and Welfare.

In vivo exposure protocol of NQs

Mice were divided into 11 experimental groups (shown in Figure 1). Ovalbumin (OVA: Sigma Chemical, MO, USA; 20 μg/mouse) dissolved in phosphate-buffered saline (PBS) at pH 7.4 plus alum (2 mg/mouse) or PBS were administered three times (days 0, 7, and 14). Two types of NQs (Tokyo chemical, Tokyo, Japan) with two different doses (15.8 ng and 158 ng/ mouse) were dissolved in PBS containing 0.025% Tween 20 (Nacalai Tesque, Kyoto, Japan), and these NQs or the corresponding vehicle were/was administered three times (days 1, 8, and 15). In the vehicle group, mice were treated with PBS plus vehicle. In the OVA group, mice were treated with OVA plus vehicle. The NQ or NQ + OVA groups received NQ plus PBS or NQ plus OVA on the above described schedule (Figure 1). In each group, PBS, vehicle, NQ, or OVA was dissolved in 0.1 mL aliquots, and inoculated intraperitoneally (n = 4–6 in each group). Animals were examined on day 28 for the in vitro assay.

Figure 1.

Schematic diagram for the ex vivo study. Mice were divided into seven experimental groups. OVA (20 μg/mouse) dissolved in phosphate-buffered saline (PBS) at pH 7.4 plus alum (2 mg/mouse) or PBS were intraperitoneally administered three times (days 0, 7, and 14). Two types of NQs (1,2-NQ and 1,4-NQ) with two different doses (15.8 ng and 158 ng/animal) were dissolved in PBS containing 0.025% Tween 80 and these NQs or the corresponding vehicle were/was administered totally three times (days 1, 8, and 15). In the vehicle group, mice were treated with PBS plus vehicle. In the OVA group, mice were treated with OVA plus vehicle. The NQ or NQ + OVA groups received NQ plus PBS or NQ plus OVA on the above described schedule (n = 4–6 in each group). Animals were examined on day 21 for the in vitro assay. On day 21, mice were sacrificed and the spleens were removed. After removing the spleens, splenocytes (mononuclear cells) were isolated, as described in the “Materials and methods” section. The cells were centrifuged and counted. Mononuclear cells (5 × 10⁶ cells /mL) were suspended in AIM V culture medium and then cultured in the presence or absence of OVA (1 mg/mL) at 37°C for 72 h in the CO₂ incubator. After being centrifuged, culture supernatants were harvested for ELISA and pelleted cells were prepared as cytoplasmic and nuclear extractions for western blot analysis.

Preparation of mononuclear cells (splenocytes)

On day 28, mice were anesthetized with sodium pentobarbital (Dainippon Pharmaceutical Co., Osaka, Japan) given intraperitoneally (1.5 mg/mouse), and the spleens were removed. After their removal, the spleens were pushed through a nylon mesh and red blood cells were also lysed with ammonium chloride. Cells were centrifuged at 400 × g at 20°C for 5 min. After washing with PBS, these cells were resuspended in AIM V culture medium (Gibco, CA, USA). The number of viable cells was determined by the trypan blue (Invitrogen, CA, USA) exclusion method. A total of 5 × 10⁶ cells per mL were cultured in the presence or absence of OVA (2 mg/mL) at 37°C for 72 h in the CO₂ incubator.

Cytokine analysis

Culture supernatants were harvested after a 72-h incubation. Protein levels of interferon (IFN)- γ (Thermo, Waltham, MA, USA), interleukin (IL)-5 (Thermo), IL-13, and IL-17 (R&D Systems Inc., MN, USA) in the supernatants were measured using each ELISA kit according to the manufacturer’s instructions (n = 4–6 in each group).

Preparation of nuclear and cytoplasmic extracts and western blot analysis

In another experiment, splenocytes from these experimental groups that had been incubated with or without OVA were collected with centrifugation. Cell nuclear and cytoplasmic fractions were then prepared using the Minute cytoplasmic and nuclear extraction kit of Invent Biotechnologies (Eden Prairie, MN, USA) according to the manufacturer’s direction for a western blot analysis. Protein concentrations were determined with the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of cytosolic and nuclear proteins (10–20 μg) were subjected to 8% SDS-PAGE (Bio-Rad) and blotted onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, UK). The membranes were blocked with Blocking One (Nacalai Tesque) in incubation buffer for 30 min at room temperature. Then, they were incubated with the appropriate primary antibodies (a rabbit anti-STAT3 antibody (Cell Signaling Technology, Tokyo, Japan; diluted to 1:1000 ), rabbit anti-STAT4 antibody (Cell Signaling Technology; diluted to 1:1000 ), rabbit anti-STAT6 antibody (Cell Signaling Technology; diluted to 1:1000 ), anti-phosphorylated STAT3 antibody (Ser 727, Santa Cruz Biotechnology, TX, USA; diluted to 1:1000 ), anti-phosphorylated STAT4 antibody (Tyr 693, Santa Cruz Biotechnology; diluted to 1:1000 ), or anti-phosphorylated STAT6 antibody (Tyr 641, Santa Cruz Biotechnology; diluted to 1:1000) and incubated at 4°C overnight. After washing with PBS plus Tween-20, each membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology; diluted to 1:1000) at RT for 1 h. After washing, the membrane was developed using an enhanced chemiluminescence light detecting kit (Amersham ECL-prime, GE healthcare) and visualized by an enhanced chemiluminescence reaction (GE Healthcare LAS 4000). Loading accuracy was evaluated by membrane rehybridization with anti- proliferating cell nuclear antigen (PCNA: Cell Signaling Technology; diluted to 1:1000) against nuclear proteins or anti-β-actin (Sigma-Aldrich; diluted to 1:1000) against cytosolic proteins. Semi-quantitative analyses of the immunoreactive bands were performed on digitalized films using ImageJ software.

Statistical analysis

All data are expressed as the mean ± SD. Statistical comparisons were made using unpaired Student’s t-test or analysis of variance (ANOVA) with Dunnett’s post-hoc tests.

Results

IFN-γ levels were significantly higher in the supernatants from 1,2-NQ at both 15.8 and 158 ng concentration levels per mouse plus OVA- and 1,4-NQ (at 15.8 ng) + OVA-exposed mononuclear cells in the presence of the OVA stimulation than in those from OVA-exposed mononuclear cells (Figure 2a). IL-13 protein levels were significantly higher in the supernatants from the groups treated with 1,2 - and 1,4-NQ at 15.8 ng plus OVA only (Figure 2b). For IL-5 (Figure 2c) and IL-17 (Figure 2d), both the cytokine levels were higher in both 1,2- and 1,4-NQ plus OVA-treated mice at 15.8 ng concentration level (P <0.05; 1,2 NQ + OVA vs. OVA in IL-17) when compared to 158 ng concentration level. In the current experimental protocol, there was no significant difference in levels of IL-4 between these experimental groups (2.35 pg/mL − 7.73 pg/mL). Also, no significant differences were observed in these levels between other experimental groups (NQ groups with an OVA rechallenge or NQ + OVA groups without an OVA rechallenge), and the vehicle group (Figure 2a–d). Intracellular signaling analyses revealed that the protein expression levels of phosphorylated STAT6 in the nuclei of these cells was significantly greater in the NQ at 15.8 ng + OVA groups than in the OVA group (Figure 3a), whereas (pan) STAT6 protein levels in the cytoplasm were slightly higher (Figure 3b). On the other hand, the expression levels of phosphorylated STAT3 in the nuclei were higher in the NQ at 15.8 ng + OVA group than in the OVA group (P <0.05 vs. 1,4-NQ + OVA; Figure 4a), whereas (pan) STAT3 expression levels in the cytoplasm were lower (Figure 4b). Phosphorylated and (pan) STAT4 protein expression levels in the nuclear and cytoplasmic extracts were similar among the experimental groups (Figure S1a and b).

Figure 2.

Effects of in vivo exposure to naphthoquinones (NQs) on allergen-sensitized mononuclear cells in terms of the in vitro allergy-related cytokine recall test. Seven groups of mice were repeatedly and intraperitoneally inoculated with vehicle, NQ, ovalbumin (OVA), or a combination of NQ + OVA. After the final administration, splenocytes were removed and incubated with OVA for 72 h. Thereafter supernatants were harvested. Protein levels of helper T-related cytokines (interleukin (IL)-13 (a), interferon (IFN)-γ (b), IL-5 (c), and IL-17 (d)) in the culture supernatants were analyzed using enzyme-linked immunosorbent assays (ELISA: n = 4–6 in each group). The figure shows the data of representative experimental groups. Results are shown as the mean ± SD. *P <0.05, **P <0.01 vs. OVA.

Figure 3.

Effects of in vivo exposure to NQs on STAT6 signaling in allergen-sensitized mononuclear cells following the in vitro allergen recall test. Mice were treated with vehicle, OVA, or a combination of low dose (15.8 ng/mouse) NQ + OVA according to the experimental protocol. Splenocytes, isolated from mice in these experimental groups, were incubated with or without OVA for 72 h. Nuclear and cytoplasmic extracts of these cell compartments were prepared and subjected to 8% SDS-PAGE and immunoblot analyses with antibodies to the tyrosine 641-phosphorylated form ([p] STAT6 (a)) and its STAT6 (b). Proliferating cell nuclear antigen (PCNA) against nuclear proteins or β-actin against cytosolic proteins was probed as the loading control. The upper panel shows the representative photograph of four experiments. In the lower panel, the expression levels of [p] STAT6 and STAT6 were normalized with respect to PCNA against nuclear proteins or β-actin against cytosolic proteins and then expressed as relative fold-changes from protein levels in the vehicle group. Results are shown as the mean ± SD. *P <0.05, **P <0.01 vs. OVA.

Figure 4.

Effects of in vivo exposure to NQs on STAT3 signaling in allergen-sensitized mononuclear cells following the in vitro allergen recall test. Mice were treated with vehicle, OVA, or a combination of low dose (15.8 ng/mouse) NQ + OVA according to the experimental protocol. Splenocytes, isolated from mice in these experimental groups, were incubated with or without OVA for 72 h. Nuclear and cytoplasmic extracts of these cell compartments were prepared and subjected to 8% SDS-PAGE and immunoblot analyses with antibodies to the serine 727-phosphorylated form ([p] STAT3 (a)) and its STAT3 (b). The upper panel shows a representative photograph of four experiments. In the lower panel, the levels of [p] STAT3 and STAT3 were normalized with respect to PCNA against nuclear proteins or β-actin against cytosolic proteins and then expressed as relative fold-changes from protein levels in the vehicle group. Results are shown as the mean ± SD. *P <0.05 vs. OVA.

Discussion

In the present study, we examined the in vivo effects of NQs on the OVA recall response and showed that splenic mononuclear cells isolated from 1,2-NQ-administered allergen-sensitized mice produced more IFN-γ following the in vitro OVA stimulation than that by control (treated with the vehicle) mice, whereas IL-13 levels were significantly higher in the supernatants from low dose NQs + OVA-exposed mononuclear cells. We previously demonstrated that 1,4-NQ drives allergic asthma-like traits such as lung inflammation and airway hyper-responsiveness in mice.^14,15 Moreover, local expression levels of Th2 cytokines such as IL-4, IL-5, and IL-13 (in lung homogenate samples) were shown to be greater in the NQ (158 ng /mouse) + OVA group than in the OVA group.^14,15 Therefore, the results of the present study, not only provided additional evidence to support the proallergic potential of NQ in at least two different strains of mice (ICR and BALB/c), but also illustrated their ability to disrupt the relatively early phase of the reaction toward the complex Th milieu.

1,2-NQ also appeared to potently affect allergen-specific Th1 polarization against these allergen-sensitized mononuclear cells, suggesting its impact on the Th1/Th2 paradigm. The role of IFN-γ in asthma development/exacerbation has not yet been elucidated in detail. DEP-mediated IFN-γ decreases were previously reported in human monocytes, natural killer cells, and natural killer T cells.^16–18 On the other hand, allergen-provoked IFN-γ has been positively correlated with airway hyper-responsiveness in allergic subjects.¹⁹ Santos et al. recently demonstrated that inhaled exposure to 1,2-NQ potentiated allergic lung inflammation in neonate mice, which was concomitant with the enhanced expression of IFN-γ in the lungs.²⁰ Furthermore, the number of CD11/IFN-γ positive splenic monocytes from the 1,2-NQ-exposed mice was significantly increased following in vitro stimulation with PMA than that of vehicle group, which was consistent with the results of the present study. Therefore, the activation of Th2 only or both Th1 and Th2 under the immunological machinery may depend on asthmatic “phenotypes;” however, this remains controversial. We^6,21 and another group²² previously reported that lung expression of IFN-γ was significantly amplified in parallel with the augmentation of airway pathophysiology in murine PM-exposed asthmatic models, implying its causal role in the PM-mediated exacerbation of allergy beyond the Th1/2 theory. Alternatively, the different impacts of 1,2- and 1,4-NQ on helper T reactions convolutely resulted in the synergistic exacerbation of allergic pathobiology. In any case, it is plausible that NQs in the air can differentially promote the malfunctional activation of T cell biology in the context of the Th1 and Th2 theory.

Concerning the promotion of Th2 lateralization, such as IL-13 hyperproduction by both types of NQs, it is interesting that the low dose (15.8 ng/mouse) chemical-exposed groups showed prominent effects. This curious type of toxicity, referred to as the “low dose effect” or “non-monotonic response,” is a commonly observed phenomenon in cell cultures, animals, and human populations, especially in the endocrine and reproductive systems, following exposures to endocrine-disrupting chemicals such as bisphenol A.²³ We previously reported that 1.58 ng of 1,4-NQ significantly elevated OVA-specific IgG₁ titer in mice, whereas the chemical at the dose of 158 ng did not, although the instillation route of NQ was intratracheal in the previous study.¹⁴ Nevertheless, in vivo exposure to 158 ng/mouse of 1,4-NQ had marked effects on lung inflammation in the experiment. On the other hand, 1,2-NQ and 1,4-NQ reportedly have the potential to induce electrophile-mediated covalent modifications of sensor proteins with thiolate ions, resulting in the activation of cellular signal transduction pathways for cellular protection such as oxidative stress.^24,25 Taking these studies into consideration, we can imagine that optimal dose of NQ against promotion for Th2 response with adjuvanticity perhaps through activation of downstream STAT6 signaling pathway is lower than that against exacerbation of inflammatory (oxidative) tissue (ex. lung) injury.

Immunodisruption caused by DEP has been suggested to involve an amplified Th17 response. Previous studies reported that DEP induced IL-17 hyperproduction in vitro²⁶ and amplified Th17⁺ cell proliferation in the lungs in parallel with the exacerbation of allergic pulmonary inflammation in vivo.²⁷ DEP have also been suggested to facilitate Th17-related autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (Inoue et al., in preparation).^26,27 In the present study, IL-17 levels in mononuclear cells were higher in the (low dose of) NQ + OVA-exposed mice than in the OVA group. Compatible with this results, the nuclear translocation of phosphorylated STAT3, an important intracellular molecule for Th17 signaling,²⁸ was greater in the NQ + OVA groups than that in the OVA group, suggesting that NQs partially/functionally activated STAT3/(retinoid related orphan receptor γt) signaling in these cells. However, since these relationships were not significant, except for IL-17 levels in 15.8 ng/mouse of 1,2-NQ + OVA vs. OVA groups, further studies are warranted in order to determine the contribution of the cytokine and its related signals to the NQ plus allergic model.

In conclusion, NQs differentially enhanced allergen sensitization in the context of the Th response such as Th1/2/17 against mononuclear cells, e.g. lymphocytes, and this may, at least in part, contribute to the immunoenhancement of DEP in allergic diseases.