Quercetin Is More Effective than Cromolyn in Blocking Human Mast Cell Cytokine Release and Inhibits Contact Dermatitis and Photosensitivity in Humans

Abstract

Mast cells are immune cells critical in the pathogenesis of allergic, but also inflammatory and autoimmune diseases through release of many pro-inflammatory cytokines such as IL-8 and TNF. Contact dermatitis and photosensitivity are skin conditions that involve non-immune triggers such as substance P (SP), and do not respond to conventional treatment. Inhibition of mast cell cytokine release could be effective therapy for such diseases. Unfortunately, disodium cromoglycate (cromolyn), the only compound marketed as a mast cell “stabilizer”, is not particularly effective in blocking human mast cells. Instead, flavonoids are potent anti-oxidant and anti-inflammatory compounds with mast cell inhibitory actions. Here, we first compared the flavonoid quercetin (Que) and cromolyn on cultured human mast cells. Que and cromolyn (100 µM) can effectively inhibit secretion of histamine and PGD2. Que and cromolyn also inhibit histamine, leukotrienes and PGD2 from primary human cord blood-derived cultured mast cells (hCBMCs) stimulated by IgE/Anti-IgE. However, Que is more effective than cromolyn in inhibiting IL-8 and TNF release from LAD2 mast cells stimulated by SP. Moreover, Que reduces IL-6 release from hCBMCs in a dose-dependent manner. Que inhibits cytosolic calcium level increase and NF-kappa B activation. Interestingly, Que is effective prophylactically, while cromolyn must be added together with the trigger or it rapidly loses its effect. In two pilot, open-label, clinical trials, Que significantly decreased contact dermatitis and photosensitivity, skin conditions that do not respond to conventional treatment. In summary, Que is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases, especially in formulations that permit more sufficient oral absorption.

About the Authors:

Zuyi Weng

Contributed equally to this work with: Zuyi Weng, Bodi Zhang

Affiliations Molecular Immunopharmacology and Drug Discovery Laboratory, Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine, Boston, Massachusetts, United States of America, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, United States of America

Bodi Zhang

Contributed equally to this work with: Zuyi Weng, Bodi Zhang

Affiliations Molecular Immunopharmacology and Drug Discovery Laboratory, Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine, Boston, Massachusetts, United States of America, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, United States of America, Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts, United States of America

Shahrzad Asadi

Affiliations Molecular Immunopharmacology and Drug Discovery Laboratory, Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine, Boston, Massachusetts, United States of America, Department of Pharmacy, Tufts Medical Center, Boston, Massachusetts, United States of America

Nikolaos Sismanopoulos

Affiliation: Molecular Immunopharmacology and Drug Discovery Laboratory, Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine, Boston, Massachusetts, United States of America

Alan Butcher

Affiliation: Thorne Research, Inc., Sandpoint, Idaho, United States of America

Xueyan Fu

Affiliation: Vitamin K Lab, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, United States of America

Alexandra Katsarou-Katsari

Affiliation: First Department of Dermatology, A. Sygros Hospital, Athens University Medical School, Athens, Greece

Christina Antoniou

Affiliation: First Department of Dermatology, A. Sygros Hospital, Athens University Medical School, Athens, Greece

Theoharis C. Theoharides

* E-mail: [email protected]

Affiliations Molecular Immunopharmacology and Drug Discovery Laboratory, Department of Molecular Physiology and Pharmacology, Tufts University School of Medicine, Boston, Massachusetts, United States of America, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, United States of America, Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts, United States of America, Department of Internal Medicine, Tufts University School of Medicine and Tufts Medical Center, Boston, Massachusetts, United States of America

Introduction

Mast cells derive from hematopoietic progenitors and mature in tissues depending on microenvironmental conditions [1], [2]. Mast cells are important effector cells in allergic reactions [3]–[6] by secreting histamine, leukotrienes (LTs), prostaglandin D2 (PGD2), proteolytic enzymes and several multifunctional cytokines, such as interleukin-6 (IL-6), IL-8, IL-13, tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF) [7]–[9]. These mediators contribute to the late-phase reactions and to inflammation through the recruitment and activation of immune cells [10], [11]. In addition to IgE and antigen, anaphylatoxins, cytokines, hormones and neuropeptides, such as substance P (SP), can trigger mast cell secretion [12] of several mediators often selectively [13]. More importantly, SP has been implicated in many skin inflammatory disorders, including contact dermatitis [14], [15]. As a result, mast cells are also involved in innate and acquired immunity [16], as well as autoimmunity and inflammation [12], especially in the skin [17], [18]. Mast cells were also shown to be involved in contact dermatitis in mice [19], [20].

Mast cells play an essential role in contact hypersensitivity through a complex interaction with different kinds of immune cells, including antigen presenting cells, T, B, NK lymphocytes, keratinocytes, endothelium, and platelets [21]. In allergic contact dermatitis, mast cells regulate the inflammatory reactions by releasing mediators like histamine, TNF-α and IL-8, leading to local vascular activation and subsequent immune cell recruitment [22]. Contact dermatitis and photosensitivity are difficult to treat with conventional treatments [23], [24], and disodium cromoglycate (cromolyn) has failed in these diseases [25]. Several studies even reported development of contact dermatitis from cromolyn-containing eyedrops 26,27. Here we compared the ability of quercetin and cromolyn to inhibit key mediators from human cultured mast cells stimulated by SP or IgE/Anti-IgE, as well as the effect of quercetin on contact dermatitis and photosensitivity in humans.

There are no effective clinically available mast cell inhibitors. In vitro, cromolyn inhibits rodent peritoneal mast cell histamine secretion [28], but could not inhibit rat mucosal mast cells even at 1000 µM [29], [30]. Moreover, 1000 µM cromolyn was required for any inhibition of histamine release from human lung and tonsillar mast cells [31]. Even though, cromolyn was ineffective in inhibiting human lung and intestinal mast cells [32]. Cromolyn is used in the management of mastocytosis to relieve diarrhea and abdominal cramping [33]. Intestinal absorption of cromolyn is severely limited (≤1%), and several weeks of therapy may be needed before any clinical benefits are seen [34]. This finding goes against the well known phenomenon of tachyphylaxis exhibited during cromolyn pretreatment [28].

Certain naturally occurring flavonoids, such as quercetin (Fig. S1B), are polyphenolic compounds found in fruits, vegetables, nuts, seeds, herbs, spices and red wine with antioxidant properties [35]–[37]. Flavonoids have potent anti-oxidant, anti-inflammatory [38], and mast cell blocking activities [39], [40]. Several flavonoids can inhibit histamine release from murine mast cells [41], [42], as well as IL-6 and TNF release from bone marrow-derived cultured murine mast cells and rat peritoneal mast cells [43]. Quercetin and its structurally related luteolin inhibit the release of histamine, leukotrienes and PGD2 from human cultured mast cells in response to cross-linkage of high affinity surface IgE receptors (FcεRI) [44]. Quercetin also inhibits histamine, IL-6, IL-8, TNF-α and tryptase release from human mast cells [39], [40], as well as asthma development in an animal model [45]. Quercetin could inhibit stimulated rat peritoneal mast cells by 75% at 10 µM, while 1000 µM of cromolyn was required for 65% inhibition [46]. However, quercetin was never compared to cromolyn on human mast cell mediator release in response to either an allergic or non-immune trigger.

Here we show that quercetin is more effective than cromolyn in inhibiting inflammatory cytokine release from human cultured mast cells.

Results

Effect on primary hCBMC cell histamine, PGD2 and leukotriene release

Stimulation of hCBMCs with IgE/Anti-IgE results in rapid secretion of large amounts of histamine, PDG2 and LTs (Fig. 1A–C). Preincubation with Que or WSQ (100 µM) significantly reduce histamine secretion (Fig. 1A) from 4347.9 to 977.3 pg/mL (82% inhibition), and to 797.7 pg/mL (87% inhibition), respectively, compared to cromolyn (1613.6 pg/mL, 67% inhibition). Que and WSQ inhibit PGD2 release (Fig. 1B) from 3771.8 to 882.5 pg/mL (77% inhibition), and to 740.8 pg/mL (81% inhibition), respectively, compared to cromolyn (958.3 pg/mL, 75% inhibition). In addition, Que and WSQ block LT secretion (Fig. 1C) from 4628.6 to 35.3 pg/mL (99% inhibition), and to 29.7 pg/mL (99% inhibition), respectively, compared to cromolyn (530.9 pg/mL, 88% inhibition). Cells were >95% viable after 24 hr incubation with the drugs (Fig. S2A). Que was effective when added 30 min prior to the trigger. No significant inhibitory effect was observed when Que was added together with the trigger (Fig. S3A–C). However, cromolyn had to be added together with the trigger for any inhibitory effect to be evident (Fig. S4).

[Figure omitted. See PDF.]

Figure 1. Quercetin and cromolyn inhibit degranulation of hCBMCs triggered by IgE/Anti-IgE.

Cells were treated with IgE (100 ng/mL) for 2 hr and then stimulated with Anti-IgE (1 µg/mL) for 2 hr. In some experiments, cells were pre-incubated with Que or WSQ (100 µM, 30 min), or treated with cromolyn (100 µM) together with Anti-IgE. (A) Histamine; (B) PGD2; (C) Leukotriene. n = 3, * indicates p<0.05, ** indicates p<0.01. WSQ = water soluble quercetin; Que = quercetin; Crom = cromolyn.

https://doi.org/10.1371/journal.pone.0033805.g001

Effect on human mast cell histamine, IL-8, TNF and IL-6 release

LAD2 mast cells release histamine 2 hr after SP (2 µM) stimulation (1611.4 pg/mL, Fig. 2A). WSQ inhibits histamine release to 350.6 pg/mL, which is even lower than that of control (388.7 pg/mL). Quercetin and cromolyn reduce histamine release to 792.3 pg/mL and 464.6 pg/mL, respectively (Fig. 2A). LAD2 mast cells also secrete newly-synthesized IL-8 and TNF 24 hr after SP (2 µM) stimulation. Incubation with WSQ, quercetin and cromolyn (100 µM) effectively block secretion of IL-8 from 437.2 pg/mL to 115.4 pg/mL, 291.2 pg/mL and 362.9 pg/mL, respectively. Similarly, WSQ, quercetin and cromolyn (100 µM) block TNF secretion from 1917.2 pg/mL to 274.7 pg/mL, 436.9 pg/mL and 1628.8 pg/mL, respectively (Fig. 2B&C). WSQ is the most effective while cromolyn is the least effective.

[Figure omitted. See PDF.]

Figure 2. Quercetin inhibits SP-triggered mediator release from human mast cells.

LAD2 cells were stimulated with SP (2 µM). In some experiments, cells were pre-incubated with Que or WSQ (100 µM, 30 min), or treated with cromolyn (100 µM) together with SP. (A) Histamine; (B) IL-8; (C) TNF. In (D) IL-6; hCBMCs were treated with either 100 µM cromolyn together with the trigger, or pre-incubated with increasing concentrations of quercetin for 30 min, then stimulated with IgE/Anti-IgE as described in Fig. 1. Histamin release was measured at 2 hr. Cytokine release was measured at 24 hr. n = 3, * indicates p<0.05.

https://doi.org/10.1371/journal.pone.0033805.g002

Primary hCBMCs release IL-6 (223.5 pg/mL) 24 hr after stimulation with IgE/Anti-IgE, and is inhibited by quercetin in a dose-dependent manner (Fig. 2D). Quercetin preincubation at 10 and 100 µM for 30 min reduces IL-6 release to 65.8 pg/mL and 29.7 pg/mL, respectively, which is even lower than that of control (96.8 pg/mL, Fig. 2D). Cromolyn (100 µM) is able to decrease IL-6 release to 141.3 pg/mL.

It should be noted again that, when added together with the trigger, quercetin does not exhibit any significant inhibitory effect on SP-triggered mediator release from LAD2 cells (Fig. S3D–F). After 30 min preincubation, cromolyn has no effect due to rapid tachyphylaxis (Fig. S4). Instead, cromolyn had to be added together with SP for any inhibitory effect to be evident. Cells were >95% viable after 24 hr incubation with the drugs (Fig. S2B).

Effect on intracellular calcium increase in human mast cells

In order to investigate the possible mechanism of action of these agents, we studied the effect on intracellular calcium ions. Preincubation with WSQ (100 µM, 30 min) completely inhibits the intracellular calcium ion increase as compared to equimolar concentration of quercetin (Fig. 3), which is half as active. Cromolyn (100 µM) has no effect on calcium levels even added together with the trigger (data not shown).

[Figure omitted. See PDF.]

Figure 3. Quercetin inhibits intracellular calcium levels in SP-stimulated LAD mast cells.

Cells were loaded with Fura-2 AM (30 nM, 20 min) and then pre-incubated with flavonoids (100 µM, 30 min). Immediately after SP addition (2 µM), Fura-2 AM fluorescence was detected for 20 min.

https://doi.org/10.1371/journal.pone.0033805.g003

Effect on NF-κB nuclear translocation in LAD2 mast cells

Cytocolic NF-κB protein levels do not change much before and after SP stimulation (Fig. 4). SP induces NF-κB translocation into the nucleus within 60 min compared to control. This NF-κB nuclear translocation is inhibited by 30 min preincubation with WSQ, which is more effective than quercetin and cromolyn (Fig. 4).

[Figure omitted. See PDF.]

Figure 4. Quercetin inhibits nuclear translocation of NF-κB in LAD2 mast cells.

Cells were pretreated with drugs (100 µM, 30 min) and then stimulated by SP (2 µM, 60 min). Upper lane, NF-κB protein levels in nuclear fractions before and after SP stimulation; Middle lane, NF-κB protein levels in cytosolic fractions before and after SP stimulation.

https://doi.org/10.1371/journal.pone.0033805.g004

Effect on contact dermatitis and skin photosensitivity in humans

Patch tests conducted with nickel in 10 volunteers showed extensive dermatitis ranging from 1+ to 3+ at 48 hr and 120 hr after nickel contact (Fig. 5A&B). Pre-administraiton of WSQ (2 g/day for 3 days) before nickel contact on the same volunteers effectively reduced this reaction by more than 50% in 8 out of 10 patietns, and 100% in the other 2 patients (Fig. 5A&B, Wilxocon paired non-parametric test, p = 0.039 for 48 hr, and p = 0.031 for 120 hr after nickel contact). Moreover, any associated pruritus disappeared as did the generalized pruritus present in one patient with pre-exsiting atopic dermatitis.

[Figure omitted. See PDF.]

Figure 5. Inhibitory effect of WSQ on nickel patch tests.

Examination of patch tests was done at 48 hr and 120 hr after application of the patch containing nickel first without WSQ and then one week later, 3 days after having taken WSQ (2 g/day). (A&B) Individual patient data from nickel patch tests were analyzed by the Wilcoxon paired non-parametric test. Representative patch test from patient No. 8 is shown in C–F. Appearance at (C) 48 hr or (D) 120 hr without WSQ. (E) 48 hr or (F) 120 hr during which patient was administered WSQ. White arrows indicate the area of nickel patch test. It should be noted that associated pruritus (itching) was eliminated in all subjects, along with widespread itching in one patient who also had chronic atopic dermatitis.

https://doi.org/10.1371/journal.pone.0033805.g005

As shown in Fig. 6, administration of 1 g WSQ increases the MED to induce skin erythema 24 hours after irradiation in all patients (Wilcoxon paired non-parametric test, p = 0.002). MED is defined as the minimal dose that induces any visible reddening. An increase in MED demonstrates increased resistance to develop skin reddening after UVB irradiation.

[Figure omitted. See PDF.]

Figure 6. Inhibitory effect of WSQ on skin photosensitivity.

(A) Energy fluence values at MED estimated 24 hr after irradiation with and without WSQ (1 g). Data was analyzed by Wilcoxon paired non-parametric test. Photos show three difference spots on the medial aspect of the forearm, representing three different energy fluence values of UVB broad band irradiation (UVB 801, Waldmann, 3.03 mW at 21 cm). (B) Skin appearance 24 hr after irradiation with WSQ administration (1 g). (C) Skin appearance 24 hr after irradiation without WSQ administration. White arrows indicate spots of UVB irradiation.

https://doi.org/10.1371/journal.pone.0033805.g006

Discussion

Here we report on the comparative inhibitory effect of WSQ, Que and the “mast cell stabilizer” cromolyn on human mast cell secretion of inflammatory mediators. Que is equally effective to cromolyn in inhibiting histamine, PGD2 and LT release but much more effective in reducing cytokine release from human mast cells.

The limited number of mast cells obtained from normal human tissues has led to increasing use of human leukemic cultured LAD2 cells [47] or human umbilical cord blood-derived cultured mast cells (hCBMCs) previously reported to release cytokines, histamine and tryptase in response to anti-IgE [48], [49]. In the present study, we used IgE/Anti-IgE as a positive mast cell trigger for hCBMCs, and SP for LAD2 cells. We chose to do so because recent studies have implicated sensory neuropeptides, such as SP, in the pathogenesis of many skin diseases, including contact dermatitis [14], [15].

Cromolyn had previously been reported to inhibit histamine secretion from rodent peritoneal mast cells, but was a much weaker inhibitor of human mast cells [31], [50]. Moreover, cromolyn does not inhibit lung and mucosal mast cells [31], [32]. Cromolyn was previsouly shown to inhibit TNF release from rat peritoneal mast cells by only 20% at 100 µM [51] and 1000 µM concentration was reported for any inhibition of human lung mast cells [31]. Clinically, cromolyn has been used in the management of mastocytosis [33], [52] and other allergic diseases, including atopic dermatitis [53], [54]. Although mast cell stabilization was initially considered as its mechanism of action, modulation of sensory nerve fiber activation has also been suggested. For instance, topical cromolyn treatment effectively reduces allergen-induced pruritus but has no effect on weals, supporting that in the skin, cromolyn inhibits sensory C-fiber nerve activation rather than inhibiting mast cell degranulation [55]. Instead, Que and luteolin were shown to inhibit histamine, IL-6 and TNF production from bone-marrow derived cultured murine mast cells [43]. Quercetin had also been reported to inhibit histamine release from rat connective tissue mast cells and mucosal mast cells [56], as well as from human lung and intestinal mast cells [32].

Here we also show that WSQ is more effective than quercetin in blocking intracellular calcium increases in stimulated human mast cells, while cromolyn had no effect. WSQ and Que are appear to be more effective than cromolyn in inhibiting NF-κB nuclear translocation, which is necessary for the production of pro-inflammatory cytokines. However, our results are not quantitative because we did not perform a reporter gene assay. Previous studies had reported that activation of hCBMCs by anti-IgE increased intracellular calcium levels [48], [57], a necessary step for mediator release [58]. Fewtrell and Gomperts first showed that Que could inhibit calcium influx in rat peritoneal mast cells [59]. Previous reports showed that IgE-mediated release of histamine and leukotrienes was abolished after calcium depletion [60]. Que also inhibited ionophore-induced histamine release from rat peritoneal mast cells, suggesting it had actions other than receptor-mediated calcium influx [46]. The structure of Que has some similarity to that of cromolyn (Fig. S1), but their actions appear to differ considerably. Not only is Que more effective than cromolyn, but Que inhibits intracellular calcium increases, while cromolyn does not. An additional important difference is that Que is effective prophylactically, but not when added together with the trigger, while cromolyn shows inhibition only when added together with the trigger. The lack of cromolyn's inhibitory action when used prophylactically was reported before and was termed tachyphylaxis. In fact, cromolyn's inhibitory action and tachyphylaxis were related to the phosphorylation of moesin [61], [62], which also showed tachyphylaxis with cromolyn pre-incubation in stimulated rat peritoneal mast cells [28], [62]. Que also stimulated phosphorylation of moesin in rat peritoneal mast cells [63].

Here we report for the first time to our knowledge that Que can be beneficial in contact dermatitis and photosensitivity in humans, conditions that are typically difficult to treat [64], [65]. Cromolyn was not tried in either one of these two models. There is only one publication from 1990 where patients with nickel-positive pompholyx (skin reactions even in the absence of contact with nickel) “reacted better” after prolonged (15 days) treatment with oral cromolyn [66]. The in vitro concentration of cromolyn we used (100 µM) is approximately 10 times more than the concentration administered in humans assuming even distribution of 400 mg oral cromolyn (molecular weight 460) in a 80 kg human per day. The in vitro concentration of quercetin (molecular weight 300) we used (100 µM) is almost identical to that administered to humans (1 g/day) in these two pilot studies.

The apparent beneficial effect of quercetin in these skin conditions could be extended to atopic dermatitis/eczema and psoriasis that are also difficult to treat and involve both neuropeptides and mast cells [67], [68]. The burden of these diseases to the economy is estimated at about $ 5 billion/year. Mast cells have been implicated in allergic and inflammatory diseases of the skin [17], [18]. Contact dermatitis involves both mast cells and T cells [22]. In this context, it is interesting that mast cells were reported to stimulate activated T cells [69], [70], one effect requiring TNF and blocked by the flavone luteolin [69]. Moreover, mast cells have the ability to present antigen [71] and to regulate dendritic cell trafficking through TNF [72]. In fact, mast cell is the only cell type that stores pre-formed TNF [12].

Skin photosensitivity is a rapid reaction that is due to both keratinocyte, mast cell and sensory nerve activation [73], [74]. In this context, it is important to mention that mast cells are in close contact with sensory nerve endings in the skin [75], and can be activated by sensory neuropeptides [76]. In turn, mast cell-derived mediators such as histamine and TNF can stimulate sensory nerve endings [77], [78].

Flavonoids had been reported to improve allergic symptoms or prevent the development of allergic diseases [79], [80]. Moreover, oral administration of astragalin, which is absorbed and converted into kaempferol, suppresed the onset of dermatitis in NC/Nga mice [81]. In a phase I clinical trial of Que, a dose of 2.5 g for a 70 kg individual administered via intravenous infusion at 3-week or weekly intervals was recommended for Phase II trials [82]. The Que dose used in humans in the present study (2 g/day) is clinically relevant. A major drawback of the use of Que and cromolyn in the clinical setting is their poor water solubility. Previsous studies showed that the oral absorption of quercetin and cromolyn is only 1% [83] and 1–5% [84] respectively. WSQ is soluble in aqueous medium only, but is likely to precipitate in the acidic environment of the stomach while used in vivo. A liposomal or enteric coated formulation may offer advantage for increased bioavailability. A phytoquercetin is now under development and appears to have much higher oral absorption.

In conclusion, our results indicate that quercetin is more effective than cromolyn in inhibiting release of pro-inflammatory cytokines from human mast cells. Moreover, quercetin appears to significantly reduce contact dermatitis, photosensitivity and associated pruritus that are typically resistant to anti-histamines and cromolyn [85], [86]. Quercetin is safe [87] and could be particularly useful in formulations that increase its solubility and oral absorption.

Materials and Methods

Drugs and Reagents

Recombinant human stem cell factor (rhSCF) was kindly donated by Biobitrum AB, (Stockholm, Sweden). Cromolyn, quercetin and SP were obtained from Sigma-Aldrich (St Louis, MO). A sodium salt of quercetin soluble in water at pH 7.4 or higher (WSQ) was provided by Thorne Research, Inc. (Dover, ID). All drugs were dissolved in DMSO, except for WSQ, which was dissolved in double-distilled water forming a solution of pH≈9. Final concentration of DMSO was <0.1% and pH was 7.4. Rabbit anti-NF-κB (p65, RelA) (Ab-1) antibody was purchased from Millipore (Billerica, MA), rabbit anti-actin antibody and the secondary HRP-conjugated antibody were purchased from Cell Signaling Technology (Beverly, MA).

Human mast cell culture

LAD2 human mast cells (obtained from Dr. A Krisherbaum, NIH) were cultured in serum-free media (StemPro-34, Gibco, Grand Island, NY) supplemented with 2 mM L-glutamine, 100 IU/mL penicillin, 50 µg/mL streptomycin, and 100 ng/mL rhSCF. For optimal cell growth, LAD2 cell density was maintained between 0.5×106 and 1×106 cells/mL.

In order to obtain primary human umbilical cord blood-derived cultured mast cells (hCBMCs), human cord blood was obtained from placenta during normal deliveries at Tufts Medical Center in accordance with established institutional guidelines [88]. Briefly, mononuclear cells were isolated by layering heparin-treated cord blood onto Lymphocyte Separation Medium (INC Biomedical, Aurora, OH). CD34+ progenitor cells were isolated from mononuclear cells by positive selection of AC133 (CD133+/CD34+) cells by magnetic cell sorting (Miltenyi Biotech, Auburn, CA). hCBMCs were derived by the culture of CD34+ progenitor cells with minor modifications. For the first six weeks, CD34+ cells were cultured in AIM medium (Gibco, Grand Island, NY) supplemented with 100 ng/mL rhSCF, after six weeks of culture, 50 ng/mL IL-6 (Chemicon) was added. Cells were cultured at 37°C in 5% CO2 balanced air. Mast cell viability was determined by trypan blue (0.3%) exclusion and was better than 98%.

Mast cell stimulation

LAD2 cells were washed with DPBS and resuspended in complete culture medium. LAD2 cells (1×105 cells/200 µL/well) were plated in 96 well flat bottom Falcon cell culture plates from Becton Dickinson (Franklin Lakes, NJ), and then stimulated with SP (2 µM) for 24 hr at 37°C in 5% CO2 incubator. Primary hCBMCs (1×105 cells/200 µL/well) were stimulated with IgE (100 ng/mL) for 2 hr followed by Anti-IgE (1 µg/mL) for another 2 hr. Control cells were treated with equal volume of culture medium only. The supernatant fluid was collected for further assays.

Degranulation assays

Mast cell degranulation was assessed by measuring histamine, PGD2 and LTs release in the supernatant fluid 30 min after cell stimulation. Histamine levels were assayed using a SPI Bio histamine EIA kit (Bertin Pharma, France). PGD2 release was measured using a PGD2-MOX EIA kit (Cayman Chemical Co., Ann Arbor, MI). LTs levels were assayed using a Luminex cysteinyl leukotriene kit (Cayman Chemical Co.) according to the manufacturer's instructions.

TNF and IL-8 release assays

IL-8 and TNF release into the supernatant fluid 24 hrs after cell stimulation were measured by Enzyme-Linked Immunosorbent Assay (ELISA) using a commercial kit from R&D Systems (Minneapolis, MN) as per the instructions. The minimum detectable level of IL-8 and TNF was 5 pg/mL.

Cytosolic calcium measurements

Cytosolic calcium was measured at 37°C using Fura-2 as indicator. LAD2 cells were loaded in Tyrode's buffer with 30 nM Fura-2 AM (Invitrogen) for 20 min to allow Fura-2 to enter the cells. After centrifugation to remove excess dye, the cells were resuspended in Tyrode's buffer at a concentration of 106 cells/mL and incubated for another 20 min. Cells were then transferred to 96-well plates with 100 µL per well. SP (2 µM) was added to cells for the time indicated. Fura-2 fluorescence was read by MDC FlexStation II (Molecular Devices) at an excitation wavelength of 340 nm/380 nm and emission wavelength of 510 nm. Results were processed according to the Invitrogen Fura-2 protocol and reported as relative value of OD 340/380.

NF-κB nuclear translocation assay

After preincubation with different drugs (100 µM, 30 min), LAD2 mast cells (2×105 cells per well) were stimulated with SP (2 µM) for 60 min. Nuclear fractions were isolated using a NE-PER nuclear extraction kit (Thermo Scientific, Rockford, IL). Changes in NF-κB protein levels in both cytosolic and nuclear fractions were detected by Western blot analysis. The protein concentrations were determined using Bio-Rad Protein Assay reagent, and equal amounts of protein were subjected to Western blotting by using the indicated antibodies. Briefly, samples separated by SDS/PAGE were transferred to nitrocellulose membranes. After being blocked in 5% BSA (w/v) at room temperature for 1 hr, the membranes were rinsed and incubated at 4°C overnight with a variety of primary antibodies (1∶1,000 dilution). The membranes were then washed and incubated with secondary antibody (1∶2,000 dilution) at room temperature for 1 hr, developed with chemiluminescence ECL reagent (LumiGold, SignaGen Laboratories, Gaithersburg, MD), and exposed to Hyperfilm MP (GE Healthcare, Piscataway, NJ).

Nickel-contact dermatitis in human subjects

Patch tests were performed to volunteers (n = 10, all female, age 30–42 years old) sensitive to contact with nickel before and after taking 2 g/day WSQ (4×500 mg/day) orally for 3 days. The patch containing nickel was first applied without pre-treatment of WSQ. Examination of the skin site of patch tests was done and photographs obtained at 48 and 120 hr after application of the patch. After one-week wash-out period, the same subjects were given 3 days WSQ as noted above before applying the patch again to the same arm a few cm distance from the previous site. Examination of the skin site of patch tests was again done at 48 and 120 hr after application of the patch. Results were recorded as scores ranging from 1 to 3, with 1 being the least affected and 3 being the worst affected condition. Skin sites were analyzed with no identifiers. Written consents have been obtained from all participants. This protocol was approved by the Human Ethics Review Committee of A. Sygros Hospital of Athens University Medical School.

Skin photosensitivity in human subjects

Minimal erythema dose (MED) that induces visible reddening was measured in the medial aspect of the forearm in three different spots (∼2 cm2) of different power fluence values of UVB broad band irradiation (UVB 801, Waldmann, 3.03 mW at 21 cm). First, the skin was irradiated without WSQ administration. Skin erythema was evaluated and photographed at 24 hr after irradiation. After one-week wash-out period, the same subjects were irradiated at three skin spots close to the previous irradiated sites with MED and higher power fluence values of UVB broad band irradiation 2 hr after administration of 1 g WSQ (4×250 mg) orally. Skin erythema was again evaluated and photographed 24 hr after irradiation. All subjects were females, age 42.6±12.2 years. Skin erythema was evaluated with no identifiers. Written consents have been obtained from all participants. This protocol was approved by the Human Ethics Review Committee of A. Sygros Hospital of Athens University Medical School.

Statistical analysis

All in vitro conditions were performed in triplicates, and all experiments were repeated at least three times (n = 3). Results are presented as mean±SD. The results on mediator release are presented as percent inhibition to normalize baseline differences between cultures that might be due to variation among the individual batches of hCBMCs obtained from different donors, as previously reported [89], [90]. Data from stimulated and control samples were compared using the unpaired, two-tailed, Student's t-test. The in vivo patient data were analyzed using two-tailed, Wilxocon matched pairs test with representative photos showing affected and unaffected sites. Significance of comparisons is denoted by p<0.05.

Supporting Information

[Figure omitted. See PDF.]

Figure S1.

Structures of cromolyn and quercetin. Circles indicate the structural similarity between cromolyn and quercetin. Rectangle indicates the hydroxyl group critical for inhibitory activity.

https://doi.org/10.1371/journal.pone.0033805.s001

(TIF)

Figure S2.

Viability of hCBMC and LAD2 mast cells. hCBMC (A) and LAD2 (B) cells were treated with different drugs as indicated in Fig. 1 & 2 for 24 hr. Cell viability was checked using the Trypan Blue exclusion test. Numbr of viable cells are presented as a percentage of the total cell number. n = 3.

https://doi.org/10.1371/journal.pone.0033805.s002

(TIF)

Figure S3.

Quercetin loses its inhibitory effects on human mast cells when added together with the trigger. Human mast cells were stimulated as described in Fig. 1 & 2. In some experiments, cells were treated with WSQ or Que (100 µM) together with the trigger without pre-incubation. (A–C) hCBMCs; (D–F) LAD2. n = 3.

https://doi.org/10.1371/journal.pone.0033805.s003

(TIF)

Figure S4.

Cromolyn does not show significant inhibition on human mast cells when added 30 min prior to the trigger. Human mast cells were stimulated as described in Fig. 1 & 2. In some experiments, cells were treated with Crom (100 µM) 30 min prior to the trigger. (A–C) hCBMCs; (D–F) LAD2. n = 3.

https://doi.org/10.1371/journal.pone.0033805.s004

(TIF)

Acknowledgments

We thank Swedish Orphan Biovitrum AB (Stockholm, Sweden) for their kind gift of rhSCF (Stemgen), and Drs. A. S. Kirshenbaum and Dean Metcalfe (National Institutes of Health, Bethesda, MD) for the LAD2 mast cells.

Author Contributions

Conceived and designed the experiments: TCT ZW BZ. Performed the experiments: ZW BZ SA NS XF AK-K CA. Analyzed the data: TCT ZW BZ AK-K CA. Contributed reagents/materials/analysis tools: AB AK-K CA. Wrote the paper: TCT ZW BZ.

Citation: Weng Z, Zhang B, Asadi S, Sismanopoulos N, Butcher A, Fu X, et al. (2012) Quercetin Is More Effective than Cromolyn in Blocking Human Mast Cell Cytokine Release and Inhibits Contact Dermatitis and Photosensitivity in Humans. PLoS ONE7(3): e33805. https://doi.org/10.1371/journal.pone.0033805

References

1. Rodewald HR, Dessing M, Dvorak AM, Galli SJ (1996) Identification of a committed precursor for the mast cell lineage. Science 271: 818–822.HR RodewaldM. DessingAM DvorakSJ Galli1996Identification of a committed precursor for the mast cell lineage.Science271818822

2. Chen CC, Grimbaldeston MA, Tsai M, Weissman IL, Galli SJ (2005) Identification of mast cell progenitors in adult mice. Proc Natl Acad Sci U S A 102: 11408–11413.CC ChenMA GrimbaldestonM. TsaiIL WeissmanSJ Galli2005Identification of mast cell progenitors in adult mice.Proc Natl Acad Sci U S A1021140811413

3. Galli SJ (1993) New concepts about the mast cell. N Engl J Med 328: 257–265.SJ Galli1993New concepts about the mast cell.N Engl J Med328257265

4. Siraganian RP (2003) Mast cell signal transduction from the high-affinity IgE receptor. Curr Opin Immunol 15: 639–646.RP Siraganian2003Mast cell signal transduction from the high-affinity IgE receptor.Curr Opin Immunol15639646

5. Blank U, Rivera J (2004) The ins and outs of IgE-dependent mast-cell exocytosis. Trends Immunol 25: 266–273.U. BlankJ. Rivera2004The ins and outs of IgE-dependent mast-cell exocytosis.Trends Immunol25266273

6. Kraft S, Rana S, Jouvin MH, Kinet JP (2004) The role of the FcepsilonRI beta-chain in allergic diseases. Int Arch Allergy Immunol 135: 62–72.S. KraftS. RanaMH JouvinJP Kinet2004The role of the FcepsilonRI beta-chain in allergic diseases.Int Arch Allergy Immunol1356272

7. Castells M (2006) Mast cell mediators in allergic inflammation and mastocytosis. Immunol Allergy Clin North Am 26: 465–485.M. Castells2006Mast cell mediators in allergic inflammation and mastocytosis.Immunol Allergy Clin North Am26465485

8. Galli SJ, Tsai M (2010) Mast cells in allergy and infection: versatile effector and regulatory cells in innate and adaptive immunity. Eur J Immunol 40: 1843–1851.SJ GalliM. Tsai2010Mast cells in allergy and infection: versatile effector and regulatory cells in innate and adaptive immunity.Eur J Immunol4018431851

9. Mekori YA, Metcalfe DD (2000) Mast cells in innate immunity. Immunol Rev 173: 131–140.YA MekoriDD Metcalfe2000Mast cells in innate immunity.Immunol Rev173131140

10. Wedemeyer J, Tsai M, Galli SJ (2000) Roles of mast cells and basophils in innate and acquired immunity. Curr Opin Immunol 12: 624–631.J. WedemeyerM. TsaiSJ Galli2000Roles of mast cells and basophils in innate and acquired immunity.Curr Opin Immunol12624631

11. Theoharides TC, Cochrane DE (2004) Critical role of mast cells in inflammatory diseases and the effect of acute stress. J Neuroimmunol 146: 1–12.TC TheoharidesDE Cochrane2004Critical role of mast cells in inflammatory diseases and the effect of acute stress.J Neuroimmunol146112

12. Theoharides TC, Alysandratos KD, Angelidou A, Delivanis DA, Sismanopoulos N, et al. (2010) Mast cells and inflammation. Biochim Biophys Acta. 1822. : 21–33.TC TheoharidesKD AlysandratosA. AngelidouDA DelivanisN. Sismanopoulos2010Mast cells and inflammation.Biochim Biophys Acta18222133

13. Theoharides TC, Kempuraj D, Tagen M, Conti P, Kalogeromitros D (2007) Differential release of mast cell mediators and the pathogenesis of inflammation. Immunol Rev 217: 65–78.TC TheoharidesD. KempurajM. TagenP. ContiD. Kalogeromitros2007Differential release of mast cell mediators and the pathogenesis of inflammation.Immunol Rev2176578

14. Scholzen TE, Steinhoff M, Bonaccorsi P, Klein R, Amadesi S, et al. (2001) Neutral endopeptidase terminates substance P-induced inflammation in allergic contact dermatitis. J Immunol 166: 1285–1291.TE ScholzenM. SteinhoffP. BonaccorsiR. KleinS. Amadesi2001Neutral endopeptidase terminates substance P-induced inflammation in allergic contact dermatitis.J Immunol16612851291

15. El-Nour H, Lundeberg L, Al-Tawil R, Granlund A, Lonne-Rahm SB, et al. (2006) Upregulation of the axonal growth and the expression of substance P and its NK1 receptor in human allergic contact dermatitis. Immunopharmacol Immunotoxicol 28: 621–631.H. El-NourL. LundebergR. Al-TawilA. GranlundSB Lonne-Rahm2006Upregulation of the axonal growth and the expression of substance P and its NK1 receptor in human allergic contact dermatitis.Immunopharmacol Immunotoxicol28621631

16. Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, Williams CM, et al. (2005) Mast cells as “tunable” effector and immunoregulatory cells: recent advances. Annu Rev Immunol 23: 749–786.SJ GalliJ. KalesnikoffMA GrimbaldestonAM PiliponskyCM Williams2005Mast cells as “tunable” effector and immunoregulatory cells: recent advances.Annu Rev Immunol23749786

17. Metz M, Maurer M (2009) Innate immunity and allergy in the skin. Curr Opin Immunol 21: 687–693.M. MetzM. Maurer2009Innate immunity and allergy in the skin.Curr Opin Immunol21687693

18. Paus R, Theoharides TC, Arck PC (2006) Neuroimmunoendocrine circuitry of the ‘brain-skin connection’. Trends Immunol 27: 32–39.R. PausTC TheoharidesPC Arck2006Neuroimmunoendocrine circuitry of the ‘brain-skin connection’.Trends Immunol273239

19. Askenase PW, Van Loveren H, Kops SK, Ron Y, Meade R, et al. (1983) Defective elicitation of delayed-type hypersensitivity in W/Wv and S1/S1d mast cell-deficient mice. J Immunol 131: 2687–2694.PW AskenaseH. Van LoverenSK KopsY. RonR. Meade1983Defective elicitation of delayed-type hypersensitivity in W/Wv and S1/S1d mast cell-deficient mice.J Immunol13126872694

20. Matsuda H, Ushio H, Paliwal V, Ptak W, Askenase PW (1995) Adoptive cell transfer of contact sensitivity-initiation mediated by nonimmune cells sensitized with monoclonal IgE antibodies. J Immunol 154: 5080–5092.H. MatsudaH. UshioV. PaliwalW. PtakPW Askenase1995Adoptive cell transfer of contact sensitivity-initiation mediated by nonimmune cells sensitized with monoclonal IgE antibodies.J Immunol15450805092

21. Dudeck A, Dudeck J, Scholten J, Petzold A, Surianarayanan S, et al. (2011) Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens. Immunity 34: 973–984.A. DudeckJ. DudeckJ. ScholtenA. PetzoldS. Surianarayanan2011Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens.Immunity34973984

22. Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF (2009) Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64: 1699–1714.M. VocansonA. HenninoA. RozieresG. PoyetJF Nicolas2009Effector and regulatory mechanisms in allergic contact dermatitis.Allergy6416991714

23. Usatine RP, Riojas M (2010) Diagnosis and management of contact dermatitis. Am Fam Physician 82: 249–255.RP UsatineM. Riojas2010Diagnosis and management of contact dermatitis.Am Fam Physician82249255

24. Millard TP, Hawk JL (2002) Photosensitivity disorders: cause, effect and management. Am J Clin Dermatol 3: 239–246.TP MillardJL Hawk2002Photosensitivity disorders: cause, effect and management.Am J Clin Dermatol3239246

25. Christensen OB, Christensen MB, Wall LM (1986) Does sodium cromoglycate have an effect on contact dermatitis? Contact Dermatitis 15: 183–185.OB ChristensenMB ChristensenLM Wall1986Does sodium cromoglycate have an effect on contact dermatitis?Contact Dermatitis15183185

26. Camarasa JG, Serra-Baldrich E, Monreal P, Soller J (1997) Contact dermatitis from sodium-cromoglycate-containing eyedrops. Contact Dermatitis 36: 160–161.JG CamarasaE. Serra-BaldrichP. MonrealJ. Soller1997Contact dermatitis from sodium-cromoglycate-containing eyedrops.Contact Dermatitis36160161

27. Kudo H, Tanaka T, Miyachi Y, Imamura S (1988) Contact dermatitis from sodium cromoglycate eyedrops. Contact Dermatitis 19: 312.H. KudoT. TanakaY. MiyachiS. Imamura1988Contact dermatitis from sodium cromoglycate eyedrops.Contact Dermatitis19312

28. Theoharides TC, Sieghart W, Greengard P, Douglas WW (1980) Antiallergic drug cromolyn may inhibit histamine secretion by regulating phosphorylation of a mast cell protein. Science 207: 80–82.TC TheoharidesW. SieghartP. GreengardWW Douglas1980Antiallergic drug cromolyn may inhibit histamine secretion by regulating phosphorylation of a mast cell protein.Science2078082

29. Barrett KE, Metcalfe DD (1985) The histologic and functional characterization of enzymatically dispersed intestinal mast cells of nonhuman primates: effects of secretagogues and anti-allergic drugs on histamine secretion. J Immunol 135: 2020–2026.KE BarrettDD Metcalfe1985The histologic and functional characterization of enzymatically dispersed intestinal mast cells of nonhuman primates: effects of secretagogues and anti-allergic drugs on histamine secretion.J Immunol13520202026

30. Pearce FL, Befus AD, Gauldie J, Bienenstock J (1982) Mucosal mast cells. II: Effects of anti-allergic compounds on histamine secretion by isolated intestinal mast cells. J Immunol 128: 2481–2486.FL PearceAD BefusJ. GauldieJ. Bienenstock1982Mucosal mast cells. II: Effects of anti-allergic compounds on histamine secretion by isolated intestinal mast cells.J Immunol12824812486

31. Okayama Y, Benyon RC, Rees PH, Lowman MA, Hillier K, et al. (1992) Inhibition profiles of sodium cromoglycate and nedocromil sodium on mediator release from mast cells of human skin, lung, tonsil, adenoid and intestine. Clin Exp Allergy 22: 401–409.Y. OkayamaRC BenyonPH ReesMA LowmanK. Hillier1992Inhibition profiles of sodium cromoglycate and nedocromil sodium on mediator release from mast cells of human skin, lung, tonsil, adenoid and intestine.Clin Exp Allergy22401409

32. Fox CC, Wolf EJ, Kagey-Sobotka A, Lichtenstein LM (1988) Comparison of human lung and intestinal mast cells. J Allergy Clin Immunol 81: 89–94.CC FoxEJ WolfA. Kagey-SobotkaLM Lichtenstein1988Comparison of human lung and intestinal mast cells.J Allergy Clin Immunol818994

33. Castells M, Metcalfe DD, Escribano L (2011) Diagnosis and treatment of cutaneous mastocytosis in children: practical recommendations. Am J Clin Dermatol 12: 259–270.M. CastellsDD MetcalfeL. Escribano2011Diagnosis and treatment of cutaneous mastocytosis in children: practical recommendations.Am J Clin Dermatol12259270

34. Gruchalla RS (1995) Southwestern Internal Medicine Conference: mastocytosis: developments during the past decade. Am J Med Sci 309: 328–338.RS Gruchalla1995Southwestern Internal Medicine Conference: mastocytosis: developments during the past decade.Am J Med Sci309328338

35. Huxley RR, Lean M, Crozier A, John JH, Neil HA, et al. (2004) Effect of dietary advice to increase fruit and vegetable consumption on plasma flavonol concentrations: results from a randomised controlled intervention trial. J Epidemiol Community Health 58: 288–289.RR HuxleyM. LeanA. CrozierJH JohnHA Neil2004Effect of dietary advice to increase fruit and vegetable consumption on plasma flavonol concentrations: results from a randomised controlled intervention trial.J Epidemiol Community Health58288289

36. Lako J, Trenerry C, Wahlqvist ML, Wattanapenpaiboon N, Sotheeswaran S, et al. (2004) Total antioxidant capacity and selected flavonols and carotenoids of some Australian and Fijian fruits and vegetables. Asia Pac J Clin Nutr 13: S127.J. LakoC. TrenerryML WahlqvistN. WattanapenpaiboonS. Sotheeswaran2004Total antioxidant capacity and selected flavonols and carotenoids of some Australian and Fijian fruits and vegetables.Asia Pac J Clin Nutr13S127

37. Schroeter H, Holt RR, Orozco TJ, Schmitz HH, Keen CL (2003) Nutrition: milk and absorption of dietary flavanols. Nature 426: 787–788.H. SchroeterRR HoltTJ OrozcoHH SchmitzCL Keen2003Nutrition: milk and absorption of dietary flavanols.Nature426787788

38. Middleton E Jr, Kandaswami C, Theoharides TC (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacol Rev 52: 673–751.E. Middleton JrC. KandaswamiTC Theoharides2000The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer.Pharmacol Rev52673751

39. Kempuraj D, Madhappan B, Christodoulou S, Boucher W, Cao J, et al. (2005) Flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells. Br J Pharmacol 145: 934–944.D. KempurajB. MadhappanS. ChristodoulouW. BoucherJ. Cao2005Flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells.Br J Pharmacol145934944

40. Park HH, Lee S, Son HY, Park SB, Kim MS, et al. (2008) Flavonoids inhibit histamine release and expression of proinflammatory cytokines in mast cells. Arch Pharm Res 31: 1303–1311.HH ParkS. LeeHY SonSB ParkMS Kim2008Flavonoids inhibit histamine release and expression of proinflammatory cytokines in mast cells.Arch Pharm Res3113031311

41. Foreman JC (1984) Mast cells and the actions of flavonoids. J Allergy Clin Immunol 73: 769–774.JC Foreman1984Mast cells and the actions of flavonoids.J Allergy Clin Immunol73769774

42. Middleton E Jr (1998) Effect of plant flavonoids on immune and inflammatory cell function. Adv Exp Med Biol 439: 175–182.E. Middleton Jr1998Effect of plant flavonoids on immune and inflammatory cell function.Adv Exp Med Biol439175182

43. Kimata M, Shichijo M, Miura T, Serizawa I, Inagaki N, et al. (2000) Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells. Clin Exp Allergy 30: 501–508.M. KimataM. ShichijoT. MiuraI. SerizawaN. Inagaki2000Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells.Clin Exp Allergy30501508

44. Kimata M, Shichijo M, Miura T, Serizawa I, Inagaki N, et al. (2000) Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells. Clin Exp Allergy 30: 501–508.M. KimataM. ShichijoT. MiuraI. SerizawaN. Inagaki2000Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells.Clin Exp Allergy30501508

45. Moon H, Choi HH, Lee JY, Moon HJ, Sim SS, et al. (2008) Quercetin inhalation inhibits the asthmatic responses by exposure to aerosolized-ovalbumin in conscious guinea-pigs. Arch Pharm Res 31: 771–778.H. MoonHH ChoiJY LeeHJ MoonSS Sim2008Quercetin inhalation inhibits the asthmatic responses by exposure to aerosolized-ovalbumin in conscious guinea-pigs.Arch Pharm Res31771778

46. Ennis M, Truneh A, White JR, Pearce FL (1981) Inhibition of histamine secretion from mast cells. Nature 289: 186–187.M. EnnisA. TrunehJR WhiteFL Pearce1981Inhibition of histamine secretion from mast cells.Nature289186187

47. Kirshenbaum AS, Akin C, Wu Y, Rottem M, Goff JP, et al. (2003) Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI. Leuk Res 27: 677–682.AS KirshenbaumC. AkinY. WuM. RottemJP Goff2003Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI.Leuk Res27677682

48. Kempuraj D, Huang M, Kandere-Grzybowska K, Basu S, Boucher W, et al. (2003) Azelastine inhibits secretion of IL-6, TNF-a and IL-8 as well as NF-kB activation and intracellular calcium ion levels in normal human mast cells. Int Arch Allergy Immunol 132: 231–239.D. KempurajM. HuangK. Kandere-GrzybowskaS. BasuW. Boucher2003Azelastine inhibits secretion of IL-6, TNF-a and IL-8 as well as NF-kB activation and intracellular calcium ion levels in normal human mast cells.Int Arch Allergy Immunol132231239

49. Igarashi Y, Kurosawa M, Ishikawa O, Miyachi Y, Saito H, et al. (1996) Characteristics of histamine release from cultured human mast cells. Clin Exp Allergy 26: 597–602.Y. IgarashiM. KurosawaO. IshikawaY. MiyachiH. Saito1996Characteristics of histamine release from cultured human mast cells.Clin Exp Allergy26597602

50. Theoharides TC, Patra P, Boucher W, Letourneau R, Kempuraj D, et al. (2000) Chondroitin sulfate inhibits connective tissue mast cells. Br J Pharmacol 131: 1039–1049.TC TheoharidesP. PatraW. BoucherR. LetourneauD. Kempuraj2000Chondroitin sulfate inhibits connective tissue mast cells.Br J Pharmacol13110391049

51. Bissonnette EY, Enciso JA, Befus AD (1995) Inhibition of tumour necrosis factor-alpha (TNF-a) release from mast cells by the anti-inflammatory drugs, sodium cromoglycate and nedocromil sodium. Clin Exp Immunol 102: 78–84.EY BissonnetteJA EncisoAD Befus1995Inhibition of tumour necrosis factor-alpha (TNF-a) release from mast cells by the anti-inflammatory drugs, sodium cromoglycate and nedocromil sodium.Clin Exp Immunol1027884

52. Castells MC (2004) Mastocytosis: classification, diagnosis, and clinical presentation. Allergy Asthma Proc 25: 33–36.MC Castells2004Mastocytosis: classification, diagnosis, and clinical presentation.Allergy Asthma Proc253336

53. Moore C, Ehlayel MS, Junprasert J, Sorensen RU (1998) Topical sodium cromoglycate in the treatment of moderate-to-severe atopic dermatitis. Ann Allergy Asthma Immunol 81: 452–458.C. MooreMS EhlayelJ. JunprasertRU Sorensen1998Topical sodium cromoglycate in the treatment of moderate-to-severe atopic dermatitis.Ann Allergy Asthma Immunol81452458

54. Stainer R, Matthews S, Arshad SH, McDonald S, Robinson J, et al. (2005) Efficacy and acceptability of a new topical skin lotion of sodium cromoglicate (Altoderm) in atopic dermatitis in children aged 2–12 years: a double-blind, randomized, placebo-controlled trial. Br J Dermatol 152: 334–341.R. StainerS. MatthewsSH ArshadS. McDonaldJ. Robinson2005Efficacy and acceptability of a new topical skin lotion of sodium cromoglicate (Altoderm) in atopic dermatitis in children aged 2–12 years: a double-blind, randomized, placebo-controlled trial.Br J Dermatol152334341

55. Vieira Dos SR, Magerl M, Martus P, Zuberbier T, Church MK, et al. (2010) Topical sodium cromoglicate relieves allergen- and histamine-induced dermal pruritus. Br J Dermatol 162: 674–676.SR Vieira DosM. MagerlP. MartusT. ZuberbierMK Church2010Topical sodium cromoglicate relieves allergen- and histamine-induced dermal pruritus.Br J Dermatol162674676

56. Pearce FL, Befus AD, Bienenstock J (1984) Mucosal mast cells. III. Effect of quercetin and other flavonoids on antigen-induced histamine secretion from rat intestinal mast cells. J Allergy Clin Immunol 73: 819–823.FL PearceAD BefusJ. Bienenstock1984Mucosal mast cells. III. Effect of quercetin and other flavonoids on antigen-induced histamine secretion from rat intestinal mast cells.J Allergy Clin Immunol73819823

57. Saito H, Ebisawa M, Tachimoto H, Shichijo M, Fukagawa K, et al. (1996) Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells. J Immunol 157: 343–350.H. SaitoM. EbisawaH. TachimotoM. ShichijoK. Fukagawa1996Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells.J Immunol157343350

58. Douglas WW (1974) Exocytosis and the exocytosis-vesiculation sequence: with special reference to neurohypophysis, chromaffin and mast cells, calcium and calcium ionophores. In: Thorn NA, Petersen AH, editors. Secretory Mechanisms of Exocrine Glands. Copenhagen: Munksgaard. pp. 116–136.WW Douglas1974Exocytosis and the exocytosis-vesiculation sequence: with special reference to neurohypophysis, chromaffin and mast cells, calcium and calcium ionophores.NA ThornAH PetersenSecretory Mechanisms of Exocrine GlandsCopenhagenMunksgaard116136

59. Fewtrell CM, Gomperts BD (1977) Quercetin: a novel inhibitor of Ca++ influx and exocytosis in rat peritoneal mast cells. Biochim Biophys Acta 469: 52–60.CM FewtrellBD Gomperts1977Quercetin: a novel inhibitor of Ca++ influx and exocytosis in rat peritoneal mast cells.Biochim Biophys Acta4695260

60. Kimata M, Shichijo M, Miura T, Serizawa I, Inagaki N, et al. (1999) Ca2+ and protein kinase C signaling for histamine and sulfidoleukotrienes release from human cultured mast cells. Biochem Biophys Res Commun 257: 895–900.M. KimataM. ShichijoT. MiuraI. SerizawaN. Inagaki1999Ca2+ and protein kinase C signaling for histamine and sulfidoleukotrienes release from human cultured mast cells.Biochem Biophys Res Commun257895900

61. Theoharides TC, Wang L, Pang X, Letourneau R, Culm KE, et al. (2000) Cloning and cellular localization of the rat mast cell 78 kD protein phosphorylated in response to the mast cell “stabilizer” cromolyn. J Pharmacol Exp Ther 294: 810–821.TC TheoharidesL. WangX. PangR. LetourneauKE Culm2000Cloning and cellular localization of the rat mast cell 78 kD protein phosphorylated in response to the mast cell “stabilizer” cromolyn.J Pharmacol Exp Ther294810821

62. Wells E, Mann J (1983) Phosphorylation of a mast cell protein in response to treatment with anti-allergic compounds; implications for the mode of action of sodium cromoglycate. Biochem Pharmacol 32: 837–842.E. WellsJ. Mann1983Phosphorylation of a mast cell protein in response to treatment with anti-allergic compounds; implications for the mode of action of sodium cromoglycate.Biochem Pharmacol32837842

63. Sieghart W, Theoharides TC, Alper SL, Douglas WW, Greengard P (1978) Calcium-dependent protein phosphorylation during secretion by exocytosis in the mast cell. Nature 275: 329–331.W. SieghartTC TheoharidesSL AlperWW DouglasP. Greengard1978Calcium-dependent protein phosphorylation during secretion by exocytosis in the mast cell.Nature275329331

64. Thyssen JP, Linneberg A, Menne T, Johansen JD (2007) The epidemiology of contact allergy in the general population–prevalence and main findings. Contact Dermatitis 57: 287–299.JP ThyssenA. LinnebergT. MenneJD Johansen2007The epidemiology of contact allergy in the general population–prevalence and main findings.Contact Dermatitis57287299

65. Kasteleijn-Nolst Trenite DG (1989) Photosensitivity in epilepsy. Electrophysiological and clinical correlates. Acta Neurol Scand Suppl 125: 3–149.DG Kasteleijn-Nolst Trenite1989Photosensitivity in epilepsy. Electrophysiological and clinical correlates.Acta Neurol Scand Suppl1253149

66. Pigatto PD, Gibelli E, Fumagalli M, Bigardi A, Morelli M, et al. (1990) Disodium cromoglycate versus diet in the treatment and prevention of nickel-positive pompholyx. Contact Dermatitis 22: 27–31.PD PigattoE. GibelliM. FumagalliA. BigardiM. Morelli1990Disodium cromoglycate versus diet in the treatment and prevention of nickel-positive pompholyx.Contact Dermatitis222731

67. Zhang B, Alysandratos KD, Angelidou A, Asadi S, Sismanopoulos N, et al. (2011) Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: Relevance to atopic dermatitis. J Allergy Clin Immunol 127: 1522–1531.B. ZhangKD AlysandratosA. AngelidouS. AsadiN. Sismanopoulos2011Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: Relevance to atopic dermatitis.J Allergy Clin Immunol12715221531

68. Theoharides TC, Zhang B, Kempuraj D, Tagen M, Vasiadi M, et al. (2010) IL-33 augments substance P-induced VEGF secretion from human mast cells and is increased in psoriatic skin. Proc Natl Acad Sci U S A 107: 4448–4453.TC TheoharidesB. ZhangD. KempurajM. TagenM. Vasiadi2010IL-33 augments substance P-induced VEGF secretion from human mast cells and is increased in psoriatic skin.Proc Natl Acad Sci U S A10744484453

69. Kempuraj D, Tagen M, Iliopoulou BP, Clemons A, Vasiadi M, et al. (2008) Luteolin inhibits myelin basic protein-induced human mast cell activation and mast cell dependent stimulation of Jurkat T cells. Br J Pharmacol 155: 1076–1084.D. KempurajM. TagenBP IliopoulouA. ClemonsM. Vasiadi2008Luteolin inhibits myelin basic protein-induced human mast cell activation and mast cell dependent stimulation of Jurkat T cells.Br J Pharmacol15510761084

70. Archer KR (1980) The mast cell. J R Soc Med 73: 318–319.KR Archer1980The mast cell.J R Soc Med73318319

71. Stelekati E, Bahri R, D'Orlando O, Orinska Z, Mittrucker HW, et al. (2009) Mast cell-mediated antigen presentation regulates CD8+ T cell effector functions. Immunity 31: 665–676.E. StelekatiR. BahriO. D'OrlandoZ. OrinskaHW Mittrucker2009Mast cell-mediated antigen presentation regulates CD8+ T cell effector functions.Immunity31665676

72. Shelburne CP, Nakano H, St John AL, Chan C, McLachlan JB, et al. (2009) Mast cells augment adaptive immunity by orchestrating dendritic cell trafficking through infected tissues. Cell Host Microbe 6: 331–342.CP ShelburneH. NakanoAL St JohnC. ChanJB McLachlan2009Mast cells augment adaptive immunity by orchestrating dendritic cell trafficking through infected tissues.Cell Host Microbe6331342

73. Hart PH, Townley SL, Grimbaldeston MA, Khalil Z, Finlay-Jones JJ (2002) Mast cells, neuropeptides, histamine, and prostaglandins in UV-induced systemic immunosuppression. Methods 28: 79–89.PH HartSL TownleyMA GrimbaldestonZ. KhalilJJ Finlay-Jones2002Mast cells, neuropeptides, histamine, and prostaglandins in UV-induced systemic immunosuppression.Methods287989

74. Ashton GH, McLean WH, South AP, Oyama N, Smith FJ, et al. (2004) Recurrent mutations in kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin fragility and photosensitivity disorder, Kindler syndrome. J Invest Dermatol 122: 78–83.GH AshtonWH McLeanAP SouthN. OyamaFJ Smith2004Recurrent mutations in kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin fragility and photosensitivity disorder, Kindler syndrome.J Invest Dermatol1227883

75. Siebenhaar F, Magerl M, Peters EM, Hendrix S, Metz M, et al. (2008) Mast cell-driven skin inflammation is impaired in the absence of sensory nerves. J Allergy Clin Immunol 121: 955–961.F. SiebenhaarM. MagerlEM PetersS. HendrixM. Metz2008Mast cell-driven skin inflammation is impaired in the absence of sensory nerves.J Allergy Clin Immunol121955961

76. Donelan J, Boucher W, Papadopoulou N, Lytinas M, Papaliodis D, et al. (2006) Corticotropin-releasing hormone induces skin vascular permeability through a neurotensin-dependent process. Proc Natl Acad Sci USA 103: 7759–7764.J. DonelanW. BoucherN. PapadopoulouM. LytinasD. Papaliodis2006Corticotropin-releasing hormone induces skin vascular permeability through a neurotensin-dependent process.Proc Natl Acad Sci USA10377597764

77. Kovacs P, Hernadi I, Wilhelm M (2006) Mast cells modulate maintained neuronal activity in the thalamus in vivo. J Neuroimmunol 171: 1–7.P. KovacsI. HernadiM. Wilhelm2006Mast cells modulate maintained neuronal activity in the thalamus in vivo.J Neuroimmunol17117

78. Kakurai M, Monteforte R, Suto H, Tsai M, Nakae S, et al. (2006) Mast cell-derived tumor necrosis factor can promote nerve fiber elongation in the skin during contact hypersensitivity in mice. Am J Pathol 169: 1713–1721.M. KakuraiR. MonteforteH. SutoM. TsaiS. Nakae2006Mast cell-derived tumor necrosis factor can promote nerve fiber elongation in the skin during contact hypersensitivity in mice.Am J Pathol16917131721

79. Miller AL (2001) The etiologies, pathophysiology, and alternative/complementary treatment of asthma. Altern Med Rev 6: 20–47.AL Miller2001The etiologies, pathophysiology, and alternative/complementary treatment of asthma.Altern Med Rev62047

80. Hirano T, Higa S, Arimitsu J, Naka T, Shima Y, et al. (2004) Flavonoids such as luteolin, fisetin and apigenin are inhibitors of interleukin-4 and interleukin-13 production by activated human basophils. Int Arch Allergy Immunol 134: 135–140.T. HiranoS. HigaJ. ArimitsuT. NakaY. Shima2004Flavonoids such as luteolin, fisetin and apigenin are inhibitors of interleukin-4 and interleukin-13 production by activated human basophils.Int Arch Allergy Immunol134135140

81. Kotani M, Matsumoto M, Fujita A, Higa S, Wang W, et al. (2000) Persimmon leaf extract and astragalin inhibit development of dermatitis and IgE elevation in NC/Nga mice. J Allergy Clin Immunol 106: 159–166.M. KotaniM. MatsumotoA. FujitaS. HigaW. Wang2000Persimmon leaf extract and astragalin inhibit development of dermatitis and IgE elevation in NC/Nga mice.J Allergy Clin Immunol106159166

82. Ferry DR, Smith A, Malkhandi J, Fyfe DW, deTakats PG, et al. (1996) Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition. Clin Cancer Res 2: 659–668.DR FerryA. SmithJ. MalkhandiDW FyfePG deTakats1996Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in vivo tyrosine kinase inhibition.Clin Cancer Res2659668

83. Gugler R, Leschik M, Dengler HJ (1975) Disposition of quercetin in man after single oral and intravenous doses. Eur J Clin Pharmacol 9: 229–234.R. GuglerM. LeschikHJ Dengler1975Disposition of quercetin in man after single oral and intravenous doses.Eur J Clin Pharmacol9229234

84. Ashton MJ, Clark B, Jones KM, Moss GF, Neale MG, et al. (1973) The absorption, metabolism and excretion of disodium cromoglycate in nine animal species. Toxicol Appl Pharmacol 26: 319–328.MJ AshtonB. ClarkKM JonesGF MossMG Neale1973The absorption, metabolism and excretion of disodium cromoglycate in nine animal species.Toxicol Appl Pharmacol26319328

85. Formica JV, Regelson W (1995) Review of the biology of quercetin and related bioflavonoids. Food & Chemical Toxicology 33: 1061–1080.JV FormicaW. Regelson1995Review of the biology of quercetin and related bioflavonoids.Food & Chemical Toxicology3310611080

86. Katsarou A, Davoy E, Xenos K, Armenaka M, Theoharides TC (2000) Effect of an antioxidant (quercetin) on sodium-lauryl-sulfate-induced skin irritation. Contact Dermatitis 42: 85–89.A. KatsarouE. DavoyK. XenosM. ArmenakaTC Theoharides2000Effect of an antioxidant (quercetin) on sodium-lauryl-sulfate-induced skin irritation.Contact Dermatitis428589

87. Harwood M, nielewska-Nikiel B, Borzelleca JF, Flamm GW, Williams GM, et al. (2007) A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties. Food Chem Toxicol 45: 2179–2205.M. HarwoodB. nielewska-NikielJF BorzellecaGW FlammGM Williams2007A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties.Food Chem Toxicol4521792205

88. Kempuraj D, Papadopoulou NG, Lytinas M, Huang M, Kandere-Grzybowska K, et al. (2004) Corticotropin-releasing hormone and its structurally related urocortin are synthesized and secreted by human mast cells. Endocrinology 145: 43–48.D. KempurajNG PapadopoulouM. LytinasM. HuangK. Kandere-Grzybowska2004Corticotropin-releasing hormone and its structurally related urocortin are synthesized and secreted by human mast cells.Endocrinology1454348

89. Yamaguchi M, Sayama K, Yano K, Lantz CS, Noben-Trauth N, et al. (1999) IgE enhances Fce receptor I expression and IgE-dependent release of Histamine and lipid mediators from human umbilical cord blood-derived mast cells: Synergistic effect of IL-4 and IgE on human mast cell Fce receptor I expression and mediator release. J Immunol 162: 5455–5465.M. YamaguchiK. SayamaK. YanoCS LantzN. Noben-Trauth1999IgE enhances Fce receptor I expression and IgE-dependent release of Histamine and lipid mediators from human umbilical cord blood-derived mast cells: Synergistic effect of IL-4 and IgE on human mast cell Fce receptor I expression and mediator release.J Immunol16254555465

90. Tachimoto H, Ebisawa M, Tomohide H, Kashiwabara T, Ra C, et al. (2000) Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-g. J Allergy Clin Immunol 106: 141–149.H. TachimotoM. EbisawaH. TomohideT. KashiwabaraC. Ra2000Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-g.J Allergy Clin Immunol106141149