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Background: Mas-related G protein-coupled receptor X2 (MRGPRX2) is regarded as a mast cell-specific receptor mediating non-IgE-dependent activation. We aimed to investigate whether human basophils and eosinophils express functional MRGPRX2., Methods: Flow cytometry, immunocytochemistry, immunofluorescence, Western blot, and RT-PCR were performed in highly purified peripheral blood basophils and eosinophils of atopic and nonatopic donors. To assess functional activity, fluorescent avidin-based degranulation assay, calcium mobilization, cytokine production in supernatants, assessment of viability/apoptosis, and tricolor granulocyte activation test were used., Results: MRGPRX2 was significantly expressed by basophils and eosinophils but not neutrophils. Functional capacity was shown by anti-MRGPRX2 mAb-induced calcium influx and concentration-dependent induction of degranulation. Sequential stimulation in the calcium mobilization assay gave no evidence for desensitization or receptor internalization. Anti-MRGPRX2 mAb significantly promoted survival. Inhibition of apoptosis could be due to released IL-3, IL-5, and GM-CSF found in supernatants. Short-term incubation with IL-3 dose-dependently upregulated MRGPRX2 expression in both, stimulation for 24 hours with anti-IgE, C5a, fMLP, and IL-3 in basophils and by IL-3, IL-5, and IL-33 in eosinophils. Among known mast cell MRGPRX2 agonists ciprofloxacin but not PMX-53 was functional on basophils and eosinophils. In basophils of allergic subjects, tricolor granulocyte activation test using grass pollen demonstrated MRGPRX2 upregulation associated with degranulation and CD63 expression., Conclusion: Unraveling the regulation and signaling mechanisms of MRGPRX2 on basophils and eosinophils might enable the development of new therapeutic strategies to prevent or inhibit allergic and nonallergic hypersensitivity. Moreover, addressing MRGPRX2 might have potential for diagnostic purposes in (drug) hypersensitivity., (© 2020 The Authors. Allergy published by John Wiley & Sons Ltd.)

Background: Pruritus is a major symptom of atopic dermatitis (AD) and is transmitted by a subpopulation of non-myelinated C-type free nerve endings in the epidermis and upper dermis. Stimulation of these nerve terminals is affected by histamine, neurotrophins and physical factors. Eosinophils of patients with AD are a source of neurotrophins, including brain-derived neurotrophic factor (BDNF), levels of which correlate with disease severity., Objective: The purpose of this study was to determine the anatomical localization of eosinophils in the skin of patients with AD with regard to peripheral nerves and to investigate whether eosinophils induce sprouting and neurite outgrowth in murine sensory neurons., Methods: Cryosections of skin derived from AD and control (NA) patients were subjected to immunofluorescence analysis with markers for eosinophils, BDNF and neuronal cells. Stimulated eosinophil supernatants were used for the treatment of cultured peripheral mouse dorsal root ganglia (DRG) neurons followed by morphometric analysis., Results: Dermal axon density and the proximity of eosinophils to nerve fibres were significantly higher in AD patients vs NA. Both neuronal projections and eosinophils expressed BDNF. Furthermore, activated eosinophil supernatants induced BDNF-dependent mouse DRG neuron branching., Conclusions and Clinical Relevance: Our results indicate that BDNF-positive eosinophils are also localized in close proximity with nerve fibres in AD, suggesting a functional relationship between BDNF-expressing eosinophils and neuronal projections. These observations suggest that eosinophils may have considerable impact on pruritus by supporting sensory nerve branching., (© 2020 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Bullous pemphigoid (BP) is characterized by substantial skin and blood eosinophilia as well as intensive pruritus. Since the pruritogenic cytokine interleukin (IL)-31 is increased in inflammatory skin diseases the aim of this study was to determine whether IL-31 plays a role in BP. Using immunofluorescence, IL-31 expression was analysed in eosinophils derived from blister fluids and skin from patients with BP and IL-31 levels in blister fluids, serum and culture supernatants were determined by enzyme-linked immunoassay (ELISA). High levels of IL-31 expression were observed in BP blister fluids, but they were only marginally elevated in BP serum compared with healthy controls. Eosinophils from either BP blister fluids or skin biopsies showed strong expression of IL-31. Furthermore, peripheral blood eosinophils from patients with BP, but not healthy controls, released high levels of IL-31, reflecting those in blister fluids. In conclusion, eosinophils are a major source of IL-31 in BP and this cytokine may contribute to itch in patients with BP.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease that is more common in elderly individuals. The aim of this study was to determine the functional activity of eosinophils in patients with BP compared with healthy donors. Blood, skin and blister-derived eosinophils were strongly activated in patients with BP, seen by increased surface expression of CD69 compared with controls. CD11b was also increased in BP blood eosinophils, which may explain the striking accumulation of eosinophils in BP (1×106 per ml blister fluid). Furthermore, CCL26 was expressed by activated eosinophils in BP skin and in blister fluid. BP eosinophils also released IL-6, IL-8 and IL-1α in BP blister fluids. Apoptosis in cultivated BP eosinophils was increased and accompanied by enhanced surface externalization of CD95. Caspase 3 positive eosinophils in lesional BP skin and blister fluid also showed the initiation of apoptosis. These results reveal novel pathophysiological aspects of BP, with a strong activation pattern and increased apoptosis of eosinophils in the peripheral blood, skin and blister fluids.

Background: Recently, the pivotal role of brain-derived neurotrophic factor (BDNF) has been described in allergic asthma. However, the role of this neurotrophin in atopic dermatitis (AD) still remains unknown., Objective: The aim of this study was to investigate the functional role of BDNF on eosinophils and to assess BDNF levels in patients with AD and nonatopic control subjects. Methods p75 Neurotrophin receptor and tyrosine kinase B receptor expression was demonstrated by using FACS analysis and immunohistochemistry. BDNF levels were assessed with ELISA and FACS analysis. Chemotactic activity (modified Boyden chamber assay), eosinophil cationic protein release (fluoroenzyme immunoassay), respiratory burst (lucigenin-dependent chemiluminescence), and apoptosis (Nicoletti protocol and Annexin-V method) assays were used to assess BDNF functional activity., Results: BDNF levels were increased in serum, plasma, eosinophils, and supernatants of stimulated eosinophils from patients with AD compared with levels seen in nonatopic control subjects ( P < .05-.001). In addition, p75 neurotrophin receptor and tyrosine kinase B expression was higher on eosinophils from patients with AD compared with that seen on eosinophils from nonatopic control subjects ( P < .05-.001). Eosinophil apoptosis was inhibited by BDNF ( P < .05-.01) and chemotactic index was increased ( P < .001) in BDNF-stimulated eosinophils from patients with AD, whereas this effect was not shown in eosinophils from nonatopic control subjects. However, no response of BDNF through the release of eosinophil cationic protein or reactive oxygen species was found., Conclusion: This study provides the first evidence for a functional role of BDNF on eosinophils from patients with AD, probably mediated by an increased expression of BDNF receptors compared with that seen in nonatopic control subjects. In addition, higher intracellular, serum, and plasma BDNF levels, as well as the release of BDNF by eosinophils, underline the particular importance of BDNF in patients with AD, pointing to new pathophysiologic aspects of this chronic inflammatory skin disease.

Background: Staphylococcus aureus colonization of the skin represents a potent trigger factor of atopic dermatitis. Our previous studies demonstrated that in atopic dermatitis eosinophil apoptosis is significantly delayed., Objective: We sought to investigate the effect of staphylococcal exotoxins (SETs) on eosinophil apoptosis and functional activities., Methods: Apoptotic eosinophils were investigated by determining their hypodiploid DNA peak. Eosinophil surface-antigen expression and intracellular production of hydrogen peroxide were assessed by means of flow cytometric analysis and respiratory burst by lucigenin-dependent chemiluminescence., Results: The SETs SEA, SEB, SEC, and toxic shock syndrome toxin 1 significantly inhibited eosinophil apoptosis in a manner comparable with that of high concentrations of IL-3. The LPS inhibitor polymyxin B was found to significantly inhibit LPS-mediated, but not SET-mediated, inhibition of apoptosis. Neither SETs nor LPS was able to modulate eosinophil surface expression of CD9, CD11a, CD16, CD40, CD44, or CD63. However, 24- and 48-hour incubation with all SETs, but not with LPS, significantly upregulated expression of CD11b and CD45 in a manner similar to that of IL-3, whereas dexamethasone induced a downregulation. Moreover, all SETs resulted in a significant upregulation of CD54 after 24 hours but not after 48 hours. Interestingly, CD69 was upregulated by means of IL-3 and SEB only. Neither direct stimulation of eosinophils nor 24-hour incubation with SETs or stimulation with SETs after a 24-hour prestimulation with IL-3, IL-5, or GM-CSF resulted in a significant extracellular production of reactive oxygen species or in a significant production of intracellular hydrogen peroxide. However, SEB and toxic shock syndrome toxin 1 were able to enhance cytokine-induced respiratory burst., Conclusion: Taken together, our data demonstrate that SETs may modulate the course of the allergic inflammatory response through modulation of potent eosinophil effector functions. This may be of particular importance in atopic dermatitis, in which the skin is regularly colonized with SET-producing S aureus.

Eosinophils play an important role in allergic diseases such as allergic asthma, rhinoconjunctivitis and atopic dermatitis. Recruitement of eosinophils to the side of inflammation, the release of reactive oxygen species, leading to tissue damage, and the propagation of the inflammatory response are mediated by chemokines. Thus, the applicability of agents able to inhibit or antagonize chemokine–induced eosinophil activation seems to be of interest in the treatment of allergic diseases. Therefore, the effect of the CC chemokine antagonist, Met–RANTES, on its effect on human eosinophil effector functions in response to RANTES, MCP–3 and eotaxin was investigated. Met–RANTES had no intrinsic activity on [Ca[sup 2+] ][sub i] transients in eosinophils and was able to dose–dependently inhibit [Ca[sup 2+] ][sub i ] transients in eosinophils following stimulation with RANTES, MCP–3 and eotaxin. Besides its effect on [Ca[sup 2+] ][sub i] transients, Met–RANTES dose–dependently inhibited actin polymerization in eosinophils and the release of reactive oxygen species following stimulation with RANTES, MCP–3 and eotaxin. The results of this study lead to the conclusion that Met–RANTES is an effective and powerful compound to antagonize effector functions of human eosinophils following stimulation with RANTES, MCP–3 and eotaxin and is therefore a promising therapeutic approach to prevent the invasion and destructive power of eosinophils in allergic diseases. [ABSTRACT FROM AUTHOR]

Human dermal mast cells are capable of releasing cytokines, particularly preformed TNFα, upon appropriate stimulation. Mast cell activation in vivo was shown to be associated with an influx and activation of inflammatory cells, initially PMN (polymorphonuclear neutrophilic granulocytes) then eosinophils. In order to learn more about the mechanisms by which TNFα is capable of activating eosinophils, in the present study the effect of TNFα on morphology and function of highly purified normal eosinophils ( ≥ 95%) was examined. As estimated by transmission and scanning electron microscopy, TNFα-stimulated eosinophils appeared to be strictly adherent and flattened exhibiting a characteristic "hemispheric" shape. TNFα induced a dose-dependent, long-lasting production of reactive oxygen species as measured by lucigenin-dependent chemiluminescence (CL), even at a concentration of 0.001 U/mI. The maximal response upon stimulation with TNFα, however, was significantly lower than optimal effects induced by IL-5. TNFα- induced responses were completely inhibited by cytochalasin B and staurosporin, and partially blocked by pertussis toxin. Separation of eosinophils by discontinuous density gradients revealed the existence of at least two hypodense eosinophil populations with a distinct susceptibility to stimulation with TNFα. Based on functional assay systems, in contrast to a significant extracellular, only a small intracellular H[SUB2]O[SUB2] production was detected. Accordingly, H[SUB2]O[SUB2] production, detected by an ultrastructural technique, was observed only on the outer surface of the plasma membrane in the contact zones in between adjacent cells. [ABSTRACT FROM AUTHOR]

Eosinophils are predominant effector cells in allergic diseases attracted by several CC chemokines into the inflammatory tissue. According to their important role in attracting leukocytes, several kinds of chemokine receptor antagonists have been developed. Therefore, the aim of this study was to investigate the effect of aminooxypentane (AOP)-RANTES on the activation of the CC chemokine receptor 3, CCR3, exemplary on human eosinophils, because they represent the dominant CCR3+ cell type. AOP-RANTES dose-dependently induced an increase of intracellular calcium concentration ([Ca[sup 2+] ][sub i] ) and a release of reactive oxygen species, which could be inhibited by pertussis toxin, in human eosinophils from normal nonatopic donors. AOP-RANTES was as effective as RANTES but less effective than eotaxin and eotaxin-2 in the activation of the respiratory burst. Flow-cytometric analyses revealed that eosinophils constitutively expressed the CC chemokine receptors CCR1 and CCR3, whereas CCR5 was not expressed. AOP-RANTES, RANTES, eotaxin and eotaxin-2, but not Met-RANTES, induced a downregulation of CCR3 at 37°C. Reexpression of CCR3 on eosinophils was observed within 120 min. Whereas no differences of CCR3 downregulation and recycling after stimulation with AOP-RANTES, RANTES, eotaxin and eotaxin-2 were found there exists a distinct profile of activity with respect to the activation of the respiratory burst in human eosinophils.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Compares eosinophil (EOS) survival and apoptosis in nonatopic volunteers and in patients with inhalant allergy or acute exacerbation of atopic dermatitis. Cytokines that support EOS survival and delay apoptosis; Use of the ELISA technique.

Eosinophils represent major effector cells in the allergic inflammatory response. Following activation, these cells are capable of mediating tissue damage, particularly by the release of reactive oxygen species. In this study, the role of extracellular and intracellular calcium in the induction of the respiratory burst of human eosinophils was investigated in healthy non-atopic individuals. Pre-incubation of Fura-2-loaded eosinophils with the intracellular calcium chelator 2-bis(o-aminophenoxy)ethane-N,N,N',N',- tetraacetic acid prevented the increase of the [Ca++]i following stimulation by RANTES, C5a and PAF, in concentration-dependent fashion, whereas depletion of extracellular calcium in the test medium by ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid was ineffective. To investigate the potential role of extracellular and intracellular calcium on the production of reactive oxygen species, flow-cytometric measurement of H2O2 production by dihydrorhodamine 123 and lucigenin-dependent chemiluminescence were carried out. Chelation of both intracellular and extracellular calcium prevented production of reactive oxygen species after stimulation with C5a, PAF, or RANTES. However, production of reactive oxygen species after stimulation by phorbol myristate acetate, which bypasses post-receptor events by direct activation of protein kinase C, was prevented only after chelation of intracellular but not extracellular calcium. This suggested a Ca++-sensitive form of protein kinase C in the activation process of the respiratory burst. These data demonstrate that intracellular and extracellular calcium represent a prerequisite of chemotaxin-induced activation of the respiratory burst in human eosinophils. Thus, intracellular calcium seems to play a central role in the modulation of the respiratory burst in eosinophils and might therefore be an interesting target for drugs that interface with calcium homeostasis and reduce the tissue destructive power of eosinophils. [ABSTRACT FROM AUTHOR]

As many new biologically active chemokines have been cloned exploring the genomic DNA sequence database in the vicinity of already known chemokine sequences without demonstrating their natural origin, it is important to transfer findings from in vitro experiments with chemokines into the in vivo situation. With respect to eosinophils and fibroblasts that play an important part in the pathogenesis of allergic and autoimmune diseases, the role of the recently discovered members of the eotaxin family, eotaxin-2 and eotaxin-3, is not really understood. In order to elucidate the origin and biologic potency of the eotaxin family this study was performed. Conventional reverse transcription–polymerase chain reaction analysis was suitable to detect mRNA for eotaxin and eotaxin-3 but not for eotaxin-2 in dermal fibroblasts. In contrast to conventional reverse transcription–polymerase chain reaction, LightCycler analysis revealed that dermal fibroblasts constitutively expressed mRNA not only for eotaxin and eotaxin-3 but also for eotaxin-2. Moreover, with this technique we investigated mRNA expression levels after stimulation of fibroblasts with interleukin-4 and interleukin-4 plus tumor necrosis factor-α: the rank order of expression levels within the eotaxin family was eotaxin > eotaxin-3 > eotaxin-2. To address the question of the efficacy of eotaxin-3, we compared its activity with eotaxin, eotaxin-2, monocyte chemotactic protein-3, monocyte chemotactic protein-4, and RANTES in different test systems for eosinophils. The efficacy of the CC chemokines at equimolar concentrations with respect to the chemotactic response of human eosinophils was eotaxin-3 = eotaxin = eotaxin-2 > RANTES > monocyte chemotactic protein-4. The rank order of activity with respect to actin polymerization and release of toxic reactive oxygen species was eotaxin-3 = eotaxin = eotaxin-2 and eotaxin = eotaxin-2 > eotaxin-3 = monocyte chemotactic protein-3 = monocyte chemotactic... [ABSTRACT FROM AUTHOR]