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Background: Substantial evidence suggests that human synovial mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-33 is believed to play an important role in the pathogenesis of RA. We recently reported that FcγRI is responsible for producing abundant tumor necrosis factor alpha (TNF-α) from cultured synovium-derived MCs (SyMCs) in response to aggregated immunoglobulin G (IgG). However, whether or not IL-33 affects immune complex (IC)-induced synovial MC activation remains unknown. This study sought to evaluate the effect of IL-33 on IC-induced synovial MC activation. Methods: Cultured SyMCs were generated by culturing synovial cells with stem cell factor. ST2 expression was analyzed using FACS and immunohistochemical techniques. Mediators released from the MCs were measured using EIAs or ELISAs. Results: SyMCs obtained from patients with RA or osteoarthritis (OA) expressed ST2 on their surfaces. We confirmed the expression of ST2 in MCs using immunofluorescence staining in joint tissue obtained from RA patients. IC-triggered histamine release was not enhanced by IL-33. However, IL-33 synergistically enhanced IC-induced IL-8 and TNF-α production in SyMCs. Conclusions: ICs and IL-33 may exacerbate inflammation associated with RA by abundantly producing TNF-α and IL-8 from SyMCs. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Basophils and mast cells are important initiator/effector cells capable of rapidly responding to IgE-mediated stimulation, but the precise mechanisms regulating their functions in vivo have not been fully identified. In this study, we assessed whether low levels of antigen can modulate activation of basophils and mast cells. Methods: Human basophils and cultured mast cells were pretreated with low concentrations of anti-FcεRI α-chain mAb (CRA-1 mAb), and their cell functions were assessed. Results: Basophils preincubated with CRA-1 mAb at as low as 1 ng/ml for 1 h showed significantly enhanced degranulation in response to various secretagogues such as MCP-1, FMLP, leukotriene B4 and Ca ionophore A23187. FMLP-induced leukotriene C4 production by basophils was also enhanced by CRA-1 mAb pretreatment. Degranulation was further enhanced when CRA-1 mAb-pretreated basophils were additionally treated with IL-3, IL-33 or leptin before stimulation with MCP-1. Priming by subthreshold CRA-1 mAb was a slow process, since 1 h of pretreatment was needed for maximal enhancement. Basophil priming also resulted from preincubation with subthreshold doses of an allergen, Der f 2. In parallel mAb experiments, CRA-1 mAb showed weak priming effects on human umbilical cord blood-derived cultured mast cells; a higher dose, 100 ng/ml, was necessary for this priming. Conclusion: These results indicate that subthreshold doses of CRA-1 mAb or allergens can prime basophils and induce exaggerated responses to various IgE-independent stimuli. This may be a potentially important mechanism that explains environmental allergen-induced exacerbation of IgE-mediated allergic diseases such as asthma. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Zinc (Zn) affects many aspects of immune function, including thymic development and the activities of immune cells. Zn is also involved in many steps of high-affinity IgE receptor (FcεRI)-induced mast cell (MC) activation, which is required for degranulation and cytokine production. Intracellular Zn levels increase in mouse MCs after FcεRI stimulation. We previously reported that Zn distribution in a human MC line, LAD2, changed dramatically following FcεRI aggregation with synchrotron radiation microbeams. However, the kinetics of Zn distribution and the underlying mechanisms following FcεRI cross-linking remain unknown. Methods: We used cord-blood-derived MCs and LAD2 cells. Degranulation was assessed by β-hexosaminidase (β-hex) release. Extracellular Zn levels were determined by inductively coupled plasma atomic emission spectrometry or based on the fluorescence intensity of a Zn indicator. We also used RNAi to knockdown ZnT1 expression. mRNA expression levels were determined by real-time RT-PCR. Results: Zn was rapidly released from human MCs after FcεRI aggregation. The kinetics and optimal conditions for FcεRI cross-linking for Zn release were different from those for degranulation. Treating LAD2 cells with an intracellular Ca2+ chelator significantly inhibited IgE-mediated β-hex release but not Zn release. We investigated IgE-mediated β-hex and Zn release with specific inhibitors of signaling pathways. Zn and β-hex release were partly correlated with but also partly independent of IgE. Knockdown of the Zn efflux transporter, ZnT1, significantly inhibited Zn release from human MCs. Conclusions: Our results indicate that IgE-dependent Zn release from human MCs involves signaling cascades that are distinct from those of degranulation. Thus, Zn may have a unique function as a mediator of allergic inflammation. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Chronic urticaria (CU) appears to be of autoimmune origin in about half of all patients, since several autoreactive immunoglobulin Gs (IgGs), such as anti-FcεRIα and anti-IgE, are detected in the sera of such patients. However, whether autoreactive IgE is associated with CU remains unclear. In this study, we attempted to identify autoreactive IgE antibodies in sera from patients with CU. Methods: Sera were collected from 67 normal subjects, 85 patients with CU and 28 patients with atopic dermatitis (AD). An autologous serum skin test (ASST) was performed on 27 of the CU patients. Autoreactive IgE and IgG levels against self-antigens were measured using enzyme-linked immunosorbent assays. The basophils were activated with dsDNA, and the CD63 expression level was examined using a fluorescence-activated cell sorter. Results: The anti-dsDNA IgE levels were significantly higher in patients with CU and AD than in normal subjects, but no differences in the anti-dsDNA IgG levels were seen. The levels of thioredoxin-, peroxiredoxin- and thyroglobulin-reactive IgE and IgG were not significantly higher in the CU patients than in the other 2 groups. There was no significant difference in the levels of anti-dsDNA IgE between ASST-positive and ASST-negative patients. The basophils from 2 out of 9 CU patients exhibited degranulation in response to dsDNA. Conclusions: Our data suggest that anti-dsDNA IgE is involved in the pathogenesis of some cases of CU. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A growing body of evidence indicates that neutrophils may play an important role in the pathogenesis of asthma. However, the involvement of the house dust mite (HDM) in neutrophil activation associated with the pathogenesis of asthma is not fully understood yet. To address this situation, we harvested neutrophils isolated from 15 HDM-sensitized asthmatic subjects and 18 HDM-sensitized nonasthmatic subjects and measured the amounts of neutrophil reactive oxygen species (ROS) production in response to the major HDM allergens Der-f and Der-f1. Der-f and Der-f1 significantly increased ROS production in neutrophils isolated from asthmatic subjects versus nonasthmatic subjects. To assess the involvement of Der-f-specific IgE antibodies binding to their receptors in HDM allergen-induced ROS production, we examined whether neutrophils produce ROS by cross-linking of cell-bound IgE antibodies with anti-IgE. Treatment with anti-IgE antibodies did not induce ROS production by neutrophils isolated from 6 asthmatic subjects. On the other hand, pretreatment of Der-f with E-64, a cysteine protease inhibitor, eliminated Der-f-induced ROS production. These results suggest that HDM-allergen exposure may result in greater production of ROS in asthmatic patients and may be involved in the pathogenesis of asthma. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Mast cell-released chemical mediators such as histamine, leukotriene (LT) C4 and prostaglandin (PG) D2 lead to the onset of allergic disorders. ATP provided from glycolysis is essential for histamine release and LTC4 secretion from mast cells upon FcεRI cross-linking, indicating that glucose is a primary environmental factor for mast cell activation. In this study, we investigated whether increases in concentrations of glucose in culture media affect the activation of bone marrow-derived mouse mast cells (BMMCs) upon FcεRI cross-linking. Methods: BMMCs were cultured in RPMI-1640 supplemented with varying concentrations (5.5, 11, 16.5, 22, 27.5 and 33 mM) of D-glucose for 3 h, or 1, 3 or 7 days. D-Mannitol was added to the medium containing 5.5 mMD-glucose for osmotic control. After culturing, these cells were sensitized with anti-TNP IgE and then stimulated with TNP-BSA. Results: We found that long-term culture (7 days) of BMMCs with 33 mMD-glucose increases the FcεRI-dependent release of β-hexosaminidase and LTC4 without affecting surface expression levels of FcεRI, intracellular ATP levels or calcium signaling. Biochemical analyses demonstrated that FcεRI-dependent phosphorylation of cytosolic phospholipase A2 (cPLA2) at the Ser505 residue was significantly increased by culturing with 33 mM glucose. Conclusions: Taken together, our data suggest that glucose can augment FcεRI-mediated mast cell activation, particularly the degranulation response and LTC4 secretion after prolonged culture of mast cells with high-glucose medium. Moreover, it is suggested that increased phosphorylation of cPLA2 at the Ser505 residue contributes to the enhancement of LTC4 secretion. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3-kinase (PI3K) has been recognized as an important downstream effector of high-affinity receptor for IgE (FcεRI) signaling in mast cells, but little is known about the isoform specificity of PI3Ks on the FcεRI-mediated migration toward the antigen (Ag). In the present study, we explored the role of PI3Kγ on mast cell migration. The treatment of bone marrow-derived mast cells (BMMCs) with a PI3Kγ inhibitor, AS605240, significantly repressed FcεRI-induced degranulation and migration. The culture supernatants of wild-type mast cells stimulated with IgE and Ag attracted FcεRIβ–/– mast cells which did not express FcεRI on their cell surface, indicating that the migration appears to be dependent on an autocrine/paracrine secretion of soluble factors from the mast cells. Adenosine, which is produced by mast cells, showed a strong activity to attract mast cells. Pertussis toxin (PTX) significantly inhibited the migration toward both the supernatant and adenosine. The supernatant from mast cells pretreated with wortmannin (Wort) and stimulated with IgE and Ag still exhibited the activity as chemoattractant, while the BMMCs pretreated with Wort did not migrate toward the supernatant. Although PTX significantly reduced the activation of AKT/PKB and migration, PTX had no effects on degranulation. These results suggest that PI3Kγ activation through PTX-sensitive G-protein-coupled receptor as a secondary response of FcεRI cross-linking regulates FcεRI-mediated mast cell migration toward the Ag, while simultaneously activated PI3Kγ through a PTX-insensitive pathway might have an effect on degranulation. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Surface-expressed CD69 is a recently recognized activation marker for basophils and is reported to be strongly induced in vitro by IL-3. In this study, we investigated whether IgE- and high-affinity receptor for IgE (FcεRI)-dependent stimuli can affect basophil CD69 expression. Highly purified basophils were cultured for 24 h in the presence of anti-FcεRI α-chain mAb, CRA-1 and IL-3, and surface CD69 expression was analyzed by flow cytometry. CRA-1 mAb at 1 ng/ml or lower concentrations, levels too low to provoke direct histamine release, dose-dependently enhanced surface CD69 expression in the presence of IL-3, although low-dose CRA-1 mAb failed to induce CD69 expression in the absence of IL-3. Recombinant Der f 2 at 10 to 100 pg/ml enhanced CD69 levels in the presence of IL-3 in basophils from mite-sensitive subjects. These results suggest that allergens may influence basophil CD69 expression even when the levels of the antigens are too low to trigger direct degranulation. Upregulated CD69 expression on locally accumulated basophils in bronchial asthma may be attributed at least in part to a combination of local cytokines, especially IL-3, plus exposure to low levels of IgE-crosslinking allergens. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Although the ability of basophils to release mediators, called releasability, may be an important aspect which influences the proinflammatory role of these cells, clinical approaches aiming at the depletion of the releasability have not been established. We examined whether the desensitization procedure in Ca[sup 2+] -containing physiological conditions can make basophils completely unresponsive to IgE-mediated stimulation, and whether basophil desensitization is affected by the surface IgE levels. Methods: Human peripheral blood basophils were cultured with low concentrations of anti-IgE antibody or recombinant mite allergen. Following culture, cells were stimulated and their histamine release was measured. Results: Culturing with mite allergen or anti-IgE antibody below threshold concentrations induced potent desensitization in basophils. The desensitizing effect of anti-IgE was dose- and time-dependent; IgE-dependent releasability was completely suppressed when basophils were incubated with a near-threshold concentration of anti-IgE for ≥4 h. In the continuous presence of subthreshold doses of anti-IgE, basophils remained desensitized even after 3 days. Basophils which had undergone an increase in surface IgE levels after 24-hour culture with IgE demonstrated enhanced desensitization. Conclusions: Near-threshold stimulation in physiological medium can affect basophils, thereby inducing complete and sustained deprivation of releasability without triggering degranulation. Basophil desensitization is regulated by their surface IgE levels. Induction of full desensitization may represent a potentially important therapeutic strategy for IgE-mediated allergic diseases in which basophils play pathogenic roles.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Recently some studies have suggested that human platelets may play an important role in allergic inflammation through the high affinity IgE receptor (FcεRI), the low affinity IgE receptor (FcεRII/CD23) and the low affinity IgG receptor (FcγRIIA/CD32) expressed on the cell surface. We reported that human platelets via the FcεRI induced the release of the chemical mediator serotonin and the chemokine RANTES (regulated upon activation, normal T expressed and presumably secreted), but the biological implication of human platelets in type I allergy has not yet been understood clearly. Methods: We compared the levels of RANTES release from platelets obtained from allergic patients and healthy individuals, stimulated with monoclonal antibody (Ab) to human FcεRI α-chain, or human myeloma IgE and anti-human IgE Ab. Results: We confirmed that the level of RANTES release from platelets of allergic patients stimulated with human IgE and anti-human IgE was significantly higher than that of healthy individuals. Conclusions: We demonstrated that the surface expression levels of FcεRI on the platelets from allergic patients and healthy individuals were not significantly different, but that the platelets of allergic patients were more activated by the IgE-FcεRI pathway than those of healthy individuals. Taken together, these results suggest a novel and important role for human platelets in perpetuating allergic inflammation through the IgE and FcεRI.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A cell line, termed NCJ, was established from the bone marrow-derived mast cells (BMMCs) of NC/Nga mice that are mouse models for atopic dermatitis. NCJ cells expressed FcεRI and c-kit and showed a metachromasia of the granules with a toluidine blue-positive and safranin-negative staining pattern that is characteristic for immature-type mast cells. Interestingly, NCJ cells showed proliferation independent of IL-3, which was associated with constitutive phosphorylation of Raf-1 and Erk kinases. Although NCJ cells had several characteristics of mast cells, we failed to detect FcεRI-mediated β-hexosaminidase release and its histamine content. These findings indicated that NCJ cells represented a mast cell line with an immature phenotype and the ability to proliferate in the absence of mast cell growth factors. NCJ cells might thus be useful to study the molecular basis of mast cell proliferation.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Recent studies have demonstrated that eosinophils from allergic patients express low levels of FcεRI on their surface, but the regulatory mechanisms of eosinophil surface FcεRI expression are not fully understood. We investigated whether IL-4 and IgE, which are reported to regulate surface FcεRI expression on human mast cells, are able to affect surface FcεRI expression in normal human eosinophils. Methods: Eosinophils purified from peripheral blood were cultured with IL-5 and with or without IL-4 and/or IgE, and surface FcεRI expression was analyzed by flow cytometry using an anti-FcεRI mAb, CRA-1. Results: Apparent FcεRI expression (∼1% of mast cell FcεRI levels) was observed in eosinophils cultured with both IL-4 and IgE. A combination of IL-4 (≥1 ng/ml) and IgE (≥ 0.5 μg/ml) was necessary for the maximal induction of surface FcεRI expression. In the presence of IL-4 and IgE, eosinophils cultured for 2 days demonstrated low but statistically significant levels of surface FcεRI, which reached a plateau after 7 days of culture. However, cross-linkage of surface FcεRI molecules by CRA-1 or anti-IgE did not induce any eosinophil activation. Conclusions: IL-4 and IgE can affect the levels of surface FcεRI on normal human eosinophils. FcεRI expression on eosinophils may be regulated by a mechanism similar to that in mast cells.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Inhibition of the interaction between IgE and the α-chain of FcεRI (FcεRIα) is a straightforward strategy to develop therapeutic reagents for IgE-mediated allergic diseases. Objective: The purpose of this study is the humanization of CRA2 and/or CRA4, mouse anti-human FcεRIα monoclonal antibodies (mAbs) which recognize the IgE-binding membrane proximal immunoglobulin-like domain of FcεRIα. Methods: The two mAbs were humanized by CDR grafting onto human V region frameworks encoded by human germline V and J genes. The activities of the recombinant antibodies to bind FcεRIα and inhibit IgE binding to FcεRIα were analyzed by flow cytometry and ELISA. Human peripheral blood basophils were pretreated with the Fab fragments of the humanized CRA2 and stimulated with IgE and an anti-IgE polyclonal antibody. The released histamine was measured. Results: The humanized CRA2 had almost the same activities of binding and inhibition of IgE binding to FcεRIα as the original mouse CRA2. Although the FcεRI-binding activity was maintained following humanization of the CRA4 light chain V region, it was lost by the humanization of the CRA4 heavy chain V region. Pretreatment of human peripheral blood basophils with the Fab fragments of the humanized CRA2 inhibited their subsequent degranulation activated by cross-linking of the FcεRI. Conclusion: In the humanized CRA2, all amino acid residues except CDR are replaced with the residues encoded by human germline genes. The humanization of CRA2 might be an important step in the development of immunotherapy to manipulate the IgE network in which mast cells, basophils, and various types of FcεRIα expressing cells are involved.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The morbidity and mortality of elderly onset asthma have recently been on the increase. To evaluate the allergic status of the elderly onset asthmatics, we performed skin tests for allergens and estimated serum total IgE levels of the patients. The proportion of the skin-test-positive patients among the elderly onset asthmatics was significantly larger than that in the younger onset group, whereas no significant difference was observed in the frequency of patients with serum total IgE levels higher than the normal range (287.2 IU/ml). On the other hand, in the experiments with aged mice, isolated peritoneal mast cells (PMC) showed a considerable decrease in the FcγRIIB/III expression and higher degranulation triggered by 2.4G2, as compared to the PMC derived from young mice. Since FcγRIIB has been shown to act as a negative regulator, we speculate that in the aged mice Fc receptor-mediated mast cell function may be upregulated by a release from the negative regulation as a result of decreased FcγRIIB expression. Our results obtained from a mouse model system may help to understand pathophysiological mechanisms underlying elderly onset asthma. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) is a common pruritic disease that occurs primarily in infancy and childhood. AD is characterized by itching and the patient having an individual or family history of atopic diseases. Although AD is also frequently associated with elevated serum IgE levels and with common environmental factors contributing to its pathogenesis, the etiology of AD is still unknown. We examined NC/Nga mice (NC mice) that showed AD-like skin lesions with aging as a possible mouse model for AD. NC mice were maintained under conventional (Conv) or specific pathogen-free (SPF) conditions. Clinical symptoms, serum IgE levels and histopathology of the skin were compared between these 2 groups, and we explored their application as a model of human AD. It was found that the skin lesions of inbred NC mice were clinically and histologically very similar to human AD when the mice were raised under Conv conditions, but not under SPF conditions, and we assumed that some kinds of environmental factors might trigger AD-like signs and symptoms in NC mice. To further investigate the pathophysiology and treatment of AD, a suitable animal model is absolutely required, and NC mice are very useful for this purpose. [ABSTRACT FROM AUTHOR]