Study of the interactions between neuropeptides and cytokines in atopic dermatitis and their role in mast cell activation

Atopic dermatitis is a chronic, highly pruritic skin disease, whose prevalence hasincreased considerably in industrialized countries. It is characterized by allergicinflammation in the skin, and has a strong association with other atopic disorders, suchas asthma and allergic rhinitis. The pathogenesis of atopic dermatitis is poorlyunderstood. Stress is thought to worsen skin diseases like atopic dermatitis. Mast cellsare increased in the skin of atopic dermatitis patients, accompanied by an increasednumber of mast cell-nerve interactions. Mast cells also secrete and are activated byneuropeptides and cytokines. The aim of this study was to investigate the interplaybetween neuropeptides and cytokines in the pathogenesis of atopic dermatitis and theirrole on mast cell activation. We speculate that mast cell activation, especially underconditions of stress, accentuates inflammation and angiogenesis, leading to worseningof atopic dermatitis lesions. The primary aim of this doctorate thesis was to investigate the involvement ofneuropeptides and cytokines in atopic dermatitis. For this purpose we used molecularmethods, including ELISA, Luminex and PCR to measure levels of neuropeptides andcytokines that are released by and/or activate mast cells in the serum and lesional skinof atopic dermatitis patients and controls. Our results indicate that the cytokine thymicstromal lymphopoietin (TSLP), is increased in the serum and lesional skin of atopicdermatitis patients, leading to activation of the Th2 response and aggravation of localinflammation. We also found that the neuropeptide neurotensin (NT) is increased in theserum of atopic dermatitis patients, while NT and NT receptor (NTR) gene expression isincreased in the lesional skin compared to controls. The related neuropeptide substanceP (SP) was also found to be increased in the lesional atopic dermatitis skin. NT and SPwere shown to induce corticotropin releasing hormone receptor 1 (CRHR1) expressionand lead to vascular endothelial growth factor (VEGF) release from a human mast cellline, indicating their role in mast cell activation. Gene expression of VEGF, the main proangiogenicfactor, was found to be increased in lesional atopic dermatitis skin, whichhas been previously shown to correlate with progression of local inflammation. Thesefindings may explain why a skin disease like atopic dermatitis is worsened by stress. The molecular mechanism of mast cell activation and degranulation by neuropeptideshas not been studied, but is known to require intracellular calcium and energy. Mitochondria are the primary energy-generating organelles in eukaryotic cells and alsoregulate intracellular calcium. Therefore, we investigated the role of mitochondrialdynamics in mast cell activation and atopic dermatitis. Human umbilical cord bloodderivedmast cells (hCBMCs) and LAD2 mast cells were examined by confocalmicroscopy during activation with IgE/antigen and SP. Degranulation and tumornecrosis factor (TNF) release observed 30 minutes later were accompanied bymitochondrial translocation from a perinuclear location to exocytosis sites. Extracellularcalcium depletion prevented these effects indicating calcium requirement. Mitochondrialdynamics are dependent on the fission dynamin-related protein-1 (DRP1) and itsactivator calcineurin, as inhibition of DRP1 by means of the DRP1 inhibitor MDIVI-1 andsiRNA techniques blocked mitochondrial translocation. Mitochondrial translocation wasalso evident by transmission electron microscopy in skin mast cells from atopicdermatitis biopsies, in which gene expression of calcineurin, DRP1 and SP are highercompared to normal skin. In conclusion, human mast cell activation participates in the pathogenesis of atopicdermatitis and requires mitochondrial dynamics. Atopic dermatitis patients could benefitfrom the development of new therapeutic agents targeting mast cell activation