Alexandra Katsarou-Katsari

Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: Relevance to atopic dermatitis

BACKGROUND Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. OBJECTIVE We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. METHODS Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. RESULTS Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. CONCLUSION Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.

Quercetin is more effective than cromolyn in blocking human mast cell cytokine release and inhibits contact dermatitis and photosensitivity in humans.

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.

Alopecia areata and Affected Skin CRH Receptor Upregulation Induced by Acute Emotional Stress

Background: Recent evidence indicates that acute stress can precipitate a number of dermatological conditions, including alopecia areata. This effect may be mediated by corticotropin-releasing hormone (CRH) released locally in the skin from dorsal root ganglia or immune cells. CRH typically acts through activation of specific receptors that are either type 1 or types 2α and 2β. CRH, or related peptides such as urocortin, could have proinflammatory effects directly or through activation of mast cells leading to destruction of the hair root. Objectives: To investigate the expression of CRH receptors on the affected skin of patients who developed alopecia areata following acute emotional stress. Methods: Scalp skin biopsies were obtained from 1 normal volunteer and 3 patients after ring infiltration of the relevant site with lidocaine. The biopsies were frozen and were later processed for in situ hybridization for CRH receptors type 1 or types 2α and 2β. Sections showing positive results were photographed. Results: The skin from the normal volunteer showed weak background expression of all three receptor types. However, skin from the affected sites of all 3 patients studied showed intense expression only on the type 2β receptor around the hair follicles. Conclusion: Acute emotional stress may precipitate alopecia areata by activation of overexpressed type 2β CRH receptors around the hair follicles leading to intense local inflammation.

Clinical features and natural history of acquired cold urticaria in a tertiary referral hospital: a 10-year prospective study.

Background  Acquired cold urticaria (ACU) represents a heterogeneous group of disorders that share a common clinical feature: the development of urticaria or angioedema after cold exposure. We present epidemiological and clinical data of subjects with ACU, natural progression and we examine possible parameters that could correlate with disease severity.

Contact allergens in patients with leg ulcers

Background Contact dermatitis can complicate the treatment of leg ulcers and is an acquired phenomenon resulting from the use of topical medications.

Increased serum CRH levels with decreased skin CRHR-1 gene expression in psoriasis and atopic dermatitis

There is increased serum CRH with decreased lesional skin CRHR-1 gene expression in psoriasis and atopic dermatitis, suggesting possible involvement in stress-induced worsening of symptoms.

Increased affected skin gene expression and serum levels of thymic stromal lymphopoietin in atopic dermatitis.

Atopic dermatitis (AD) is a chronic highly pruritic inflammatory skin disease, which has increased considerably in industrialized countries.1 AD is characterized by allergic inflammation in the skin,2 and has a strong association with other atopic disorders such as asthma and allergic rhinitis.1 Thymic stromal lymphopoietin (TSLP) is a cytokine closely related to IL-7,3 mostly produced by epithelial cells and keratinocytes in response to infection, trauma and inflammation. TSLP mediates Th2 responses through activation of dendritic cells, and has emerged as an important regulator of allergic inflammation. TSLP also activates mast cells to release several cytokines, such as IL-5, IL-6 and IL-13, but only in the presence of IL-1 and TNF.4 One paper reported increased TSLP protein expression in lesional skin of AD patients.3 We investigated gene expression of TSLP in lesional skin biopsies and serum levels of AD patients (n=9, mean age ± 1 SD=41.33 ± 21.40; three males, six females) compared to controls (n=9, mean age ± 1 SD=42.8 ± 18.1; three males, six females). All 10 mm punch skin biopsies were obtained (abdomen, arm, back, hand, and neck; there was no apparent correlation between any of these sites and TSLP levels) from patients who had mild, localized, chronic AD, and from controls (back). No subject had received any anti-inflammatory medication for 15 days prior to the biopsy. Contact dermatitis, asteatotic eczema or other types of dermatitis were excluded. The Medical Ethics Committee of University of Athens Medical School approved this protocol. All participants gave their written informed consent according to the Declaration of Helsinki Principles. Patients were free of other major medical conditions except allergic rhinitis as noted. All biopsies were placed in RNAlater (Ambion Inc., Austin, TX) and stored at −20 °C. Total RNA from skin biopsies was extracted by TRIzol isolation method (Invitrogen, Carlsbad, CA). cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Quantitative PCR was performed using Taqman probes (Applied Biosystems, Foster City, CA) on a 7300 Real Time PCR System (Applied Biosystems). For qPCR, the cDNA was reverse-transcribed from 300 ng RNA from each sample. Relative mRNA levels were normalized to the GAPDH endogenous control. Data were analyzed with the non-parametric Mann-Whitney U test, since values did not conform to a normal distribution. Serum measurements are reported in pg/ml. Relative TSLP gene expression was significantly increased in the lesional AD skin samples (median=4.93; range=2.27–23.99) as compared to controls (median=0.84; range=0.49–1.85) (Fig. 1a, p<0.0001). Figure 1 Scattergrams of (a) TSLP gene expression determined in skin by qPCR and (b) TSLP levels in serum measured by ELISA from controls and atopic dermatitis patients. The value for TSLP gene expression from one patient was not included because the quality of ... Blood was obtained the same day prior to the biopsy and the serum was isolated, aliquoted and stored at −20°C until use. TSLP levels were determined with ELISA (RD range=0–2,246), in contrast to the controls (median=0; range=0–62.5) (Fig. 1b, p=0.0056). The 3 female patients with the highest serum levels (2246.6, 859.4, and 768.2 pg/ml) also had allergic rhinitis confirmed by skin prick testing. There was neither a positive nor a negative correlation between skin TSLP gene expression and serum levels in any of the subjects. Local skin TSLP gene expression and production may be one of the earliest steps in the inflammatory process, preceding systemic increases in TSLP levels. Our results contrast with another human study reporting that TSLP serum levels were not increased in AD (n=46; average age=26.5 years),5 but are in agreement with increased TSLP serum levels in children with atopic eczema (n=75) and non-atopic eczema (n=70), as compared to normal controls (n=87).6 All of our patients had mild, localized chronic AD and, therefore, do not permit an attempt to correlate with severity of disease. Besides, this study was not powered for such analysis. The highest serum values corresponded to AD patients with concomitant allergic rhinitis confirmed with skin prick testing. This finding supports the hypothesis that increase of serum TSLP levels may also precede other atopic diseases, such as allergic rhinitis and asthma. In fact, a study showed that skin-derived TSLP contributed to the aggravation of asthma in mice.7 The role of TSLP in human asthma is supported by another study, which showed that bronchial TSLP expression was increased in asthmatics (n=20) compared to controls (n=15) and correlated with disease severity.8 Based on the apparent association between increased serum TSLP levels and allergic rhinitis, investigation of atopic subjects without AD could provide further insight into the relationship between TSLP and allergic disease.

Serum neurotensin (NT) is increased in psoriasis and NT induces vascular endothelial growth factor release from human mast cells

Summary Background  Psoriasis involves skin inflammation that often worsens with stress, but the mechanism of this effect remains obscure. We have shown that corticotropin‐releasing hormone (CRH) is increased in the serum of patients with psoriasis. A peptide, neurotensin (NT), can trigger skin histamine release and augment the ability of CRH to increase skin vascular permeability.

Urticaria pigmentosa associated with acute stress and lesional skin mast-cell expression of CRF-R1

A 38‐year‐old woman presented with a pronounced increase in symptoms and proliferation of urticaria pigmentosa (UP) after acute psychological stress, which was quantified using the Spielberger’s State–Trait Anxiety Inventory. Immunohistochemical examination of a skin biopsy from a new UP lesion showed a large number of activated mast cells expressing corticotrophin‐releasing factor receptor‐1 (CRF‐R1) and there was high serum CRF. This is the first documented report to our knowledge of UP worsening associated with acute stress, possibly through activation of skin mast‐cell CRF‐R1.

Theophylline as "add-on" therapy in patients with delayed pressure urticaria: a prospective self-controlled study.

Delayed pressure urticaria (DPU) is a skin condition that involves the gradual development of wheals and edema at sites of physical pressure. Its pathogenesis is not clear and histamine-1 receptor (H-1R) antagonists provide only partial relief. In this prospective, clinical study, we investigated the effect of theophylline, which has a long history of benefit in allergic asthma, added to cetirizine in patients with DPU. Twenty three patients received during period #1 cetirizine (10 mg po QD) and theophylline (200 mg po BID) for 6 months, followed by period #2 of 1 month washout with only rescue medication as needed, and then by period #3 with cetirizine (10 mg QD plus placebo (BID) for 5 more months. The addition of theophylline resulted in statistically significant improvement over cetirizine alone by 2 months and continued for the duration of treatment. Treatment of cultured human mast cells with theophylline (10 μM) did not inhibit allergic histamine release, but the in vivo beneficial effect of theophylline may require significant pretreatment period to manifest itself, or may involve inhibition of other mast cell dependent mediators. A double-blind study, accompanied by serum histamine and tryptase levels, should be in order.