Satoshi Nunomura

Positive and Negative Regulation of Mast Cell Activation by Lyn via the Fc∊RI

Aggregation of the high affinity receptor for IgE (FcεRI) induces activation of mast cells. In this study we show that upon low intensity stimulation of FcεRI with monomeric IgE, IgE plus anti-IgE, or IgE plus low Ag, Lyn (a Src family kinase) positively regulates degranulation, cytokine production, and survival, whereas Lyn works as a negative regulator of high intensity stimulation with IgE plus high Ag. Low intensity stimulation suppressed Lyn kinase activity and its association with FcεRI β subunit, whereas high intensity stimulation enhanced Lyn activity and its association with FcεRI β. The latter induced much higher levels of FcεRI β phosphorylation and Syk activity than the former. Downstream positive signaling molecules, such as Akt and p38, were positively and negatively regulated by Lyn upon low and high intensity stimulations, respectively. In contrast, the negative regulators, SHIP and Src homology 2 domain-containing protein tyrosine phosphatase-1, interacted with FcεRI β, and their phosphorylation was controlled by Lyn. Therefore, we conclude that Lyn-mediated positive vs negative regulation depends on the intensity of the stimuli. Studies of mutant FcεRI β showed that FcεRI β subunit-ITAM (ITAM motif) regulates degranulation and cytokine production positively and negatively depending on the intensity of FcεRI stimulation. Furthermore, Lyn-mediated negative regulation was shown to be exerted via the FcεRI β-ITAM.

FcεRI Signaling of Mast Cells Activates Intracellular Production of Hydrogen Peroxide: Role in the Regulation of Calcium Signals

Earlier studies, including our own, revealed that activation of mast cells is accompanied by production of reactive oxygen species (ROS) that help to mediate the release of the inflammatory mediators, including histamine and eicosanoids. However, little is known about the mechanisms of ROS production, including the species of oxidants produced. In this study we show that in both the RBL-2H3 mast cell line and bone marrow-derived mast cells, FcεRI cross-linking stimulates intracellular oxidative burst, including hydrogen peroxide (H2O2) production, as defined with the oxidant-sensitive dyes dichlorofluorescein and scopoletin and the selective scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one). The oxidative burst was observed immediately after stimulation and was most likely due to an NAD(P)H oxidase. Experiments using selective pharmacological inhibitors demonstrated that activation of tyrosine kinases and phosphatidylinositol-3-kinase is required for induction of the oxidative burst. Blockade of the oxidative burst by diphenyleneiodonium impaired the release of preformed granular mediators, such as histamine and β-hexosaminidase, and the secretion of newly synthesized leukotriene C4, whereas selective scavenging H2O2 by ebselen impaired leukotriene C4 secretion, but not degranulation. Sustained elevation of cytosolic calcium through store-operated calcium entry was totally abolished when ROS production was blocked. In contrast, selective depletion of H2O2 caused a considerable decrease and delay of the calcium response. Finally, tyrosine phosphorylation of phospholipase Cγ and the linker for activation of T cells, an event required for calcium influx, was suppressed by diphenyleneiodonium and ebselen. These studies demonstrate that activation of the intracellular oxidative burst is an important regulatory mechanism of mast cell responses.

Expression of Mas-related gene X2 on mast cells is upregulated in the skin of patients with severe chronic urticaria.

BACKGROUND Wheal reactions to intradermally injected neuropeptides, such as substance P (SP) and vasoactive intestinal peptide, are significantly larger and longer lasting in patients with chronic urticaria (CU) than in nonatopic control (NC) subjects. Mas-related gene X2 (MrgX2) has been identified as a receptor for basic neuropeptides, such as SP and vasoactive intestinal peptide. Mast cell (MC) responsiveness to eosinophil mediators contributes to the late-phase reaction of allergy. OBJECTIVE We sought to compare the frequency of MrgX2 expression in skin MCs from patients with CU and NC subjects and to identify the receptor for basic eosinophil granule proteins on human skin MCs. METHODS MrgX2 expression was investigated by using immunofluorescence in skin tissues from NC subjects and patients with severe CU and on skin-derived cultured MCs. MrgX2 expression in human MCs was reduced by using a lentiviral small hairpin RNA silencing technique. Ca(2+) influx was measured in CHO cells transfected with MrgX2 in response to eosinophil granule proteins. Histamine and prostaglandin D2 levels were measured by using enzyme immunoassays. RESULTS The number of MrgX2(+) skin MCs and the percentage of MrgX2(+) MCs in all MCs in patients with CU were significantly greater than those in NC subjects. Eosinophil infiltration in urticarial lesions was observed in 7 of 9 patients with CU. SP, major basic protein, and eosinophil peroxidase, but not eosinophil-derived neurotoxin, induced histamine release from human skin MCs through MrgX2. CONCLUSION MrgX2 might be a new target molecule for the treatment of wheal reactions in patients with severe CU.

Establishment of a Human Allergy Model Using Human IL-3/GM-CSF–Transgenic NOG Mice

The development of animal models that mimic human allergic responses is crucial to study the pathophysiology of disease and to generate new therapeutic methodologies. Humanized mice reconstituted with human immune systems are essential to study human immune reactions in vivo and are expected to be useful for studying human allergies. However, application of this technology to the study of human allergies has been limited, largely because of the poor development of human myeloid cells, especially granulocytes and mast cells, which are responsible for mediating allergic diseases, in conventional humanized mice. In this study, we developed a novel transgenic (Tg) strain, NOD/Shi-scid-IL2rγnull (NOG), bearing human IL-3 and GM-CSF genes (NOG IL-3/GM–Tg). In this strain, a large number of human myeloid cells of various lineages developed after transplantation of human CD34+ hematopoietic stem cells. Notably, mature basophils and mast cells expressing FcεRI were markedly increased. These humanized NOG IL-3/GM–Tg mice developed passive cutaneous anaphylaxis reactions when administered anti–4-hydroxy-3-nitrophenylacetyl IgE Abs and 4-hydroxy-3-nitrophenylacetyl. More importantly, a combination of serum from Japanese cedar pollinosis patients and cedar pollen extract also elicited strong passive cutaneous anaphylaxis responses in mice. Thus, to our knowledge, our NOG IL-3/GM–Tg mice are the first humanized mouse model to enable the study of human allergic responses in vivo and are excellent tools for preclinical studies of allergic diseases.

Inhibitory effects of parthenolide on antigen-induced microtubule formation and degranulation in mast cells

Pharmacological modulation of IgE-mediated mast cell activation is important to the development of anti-allergic reagents. In this study, we investigated the effects of parthenolide (PTL) on high-affinity IgE receptor (FcepsilonRI)-induced degranulation in mast cells. PTL dose-dependently inhibited degranulation induced by IgE.antigen stimulation in RBL-2H3 cells and BMMCs. Although PTL is a potent NF-kappaB inhibitor by targeting IkappaB kinase complex, NF-kappaB inhibition by other IkappaB kinase inhibitors did not inhibit degranulation in mast cells. IgE.antigen-induced microtubule formation is well known to be critical for degranulation in mast cells. Immunocytochemical study with anti-alpha-tubulin antibody revealed that PTL significantly inhibited IgE.antigen-induced microtubule formation. However, PTL, as well as nocodazol, had no significant effects on degranulation in the fyn-deficient BMMCs, suggesting that inhibitory effects of PTL in the microtubule formation are fyn dependent. We further demonstrated that in vivo administration of PTL in mice strongly inhibited passive cutaneous anaphylaxis reaction. The present study provides a possibility to develop potent reagents against mast cell activation based on an inhibition of microtubule formation.

The high-affinity immunoglobulin E receptor (FcɛRI) regulates mitochondrial calcium uptake and a dihydropyridine receptor-mediated calcium influx in mast cells: Role of the FcɛRIβ chain immunoreceptor tyrosine-based activation motif

A growing body of evidence suggests that mitochondria take up calcium upon receptor (agonist) stimulation and that this contributes to the dynamics of spatiotemporal calcium signaling. We have previously shown that engagement of the high-affinity receptor for immunoglobulin E (FcepsilonRI) stimulates mitochondrial calcium ([Ca2+]m) uptake in mast cells. The present study was undertaken to investigate the mechanisms and biological significance of FcepsilonRI regulation of [Ca2+]m. Antigen stimulated [Ca2+]m uptake in a dose-dependent manner with a minimal effective dose of 0.03-3 ng/ml. This [Ca2+]m uptake took place immediately, reaching its peak within minutes and was inhibited by the src family kinase inhibitor PP1 and phosphatidylinositol-3-kinase inhibitor wortmannin. Analyses using mast cells expressing the wild-type or the mutated type of the FcepsilonRIbeta immunoreceptor tyrosine-based activation motif (ITAM) in which all tyrosine residues were replaced by phenylalanine revealed that the FcepsilonRIbeta ITAM is essential for a sustained [Ca2+]m uptake. The FcepsilonRIbeta ITAM was essential for overall calcium response upon weak FcepsilonRI stimulation (at low antigen concentration), while upon strong stimulation (at high antigen concentration) it appeared necessary selectively to an immediate calcium response that was sensitive to the dihydropyridine receptor (DHPR) antagonist nifedipine and wortmannin but not to the store-operated calcium entry (SOCE) antagonists such as 2-aminoethoxyphenyl borate and SK&F96365. These data demonstrate that the FcepsilonRIbeta regulates [Ca2+]m uptake in mast cells via the ITAM and suggest that this plays a key role in regulating calcium influx especially that induced via a DHPR-mediated calcium channel.

Common and distinct signalling cascades in the production of tumour necrosis factor-α and interleukin-13 induced by lipopolysaccharide in RBL-2H3 cells

Background Activation of mast cells by lipopolysaccharide (LPS) results in the production of TNF‐α and IL‐13. TNF‐α and IL‐13 are key mediators in the development of neutrophilic and allergic inflammation, respectively. LPS‐induced TNF‐α and IL‐13 production in mast cells has been reported to be mediated by Toll‐like receptor 4 (TLR4) signalling, but differences in signal transduction mechanisms leading to the production of these cytokines are not clearly defined.

Abrogation of High-Affinity IgE Receptor-Mediated Mast Cell Activation at the Effector Phase Prevents Contact Hypersensitivity to Oxazolone

Inflammatory mediators released from mast cells (MCs) through engagement of the high-affinity receptor for IgE (FcepsilonRI) have pivotal roles in chemical allergen-induced contact hypersensitivity (CHS) reactions, which suggests that the blockade of MC activation through FcepsilonRI stimulation may attenuate allergic contact dermatitis (CD). To address this possibility, we employed the following two approaches: (i) modulation of FcepsilonRI-mediated MC activation by introducing mutations in tyrosine residues of the FcepsilonRI beta-chain immunoreceptor tyrosine-based activation motif (ITAM) and (ii) blockade of FcepsilonRI-mediated MC activation employing a recombinant soluble ecto-domain of the human FcepsilonRIalpha-chain (rsFcepsilonRIalpha). In this study, we show that optimal MC activation through the FcepsilonRI beta-chain ITAM has essential roles in the onset of CHS to oxazolone (Oxa), a well-characterized chemical allergen. In addition, we demonstrate that administration of the rsFcepsilonRIalpha after sensitization successfully prevents murine CHS to Oxa. In a chronic CD model elicited by multiple challenges with low-dose Oxa, application of the rsFcepsilonRIalpha during the course of the challenges showed suppressive effects on CHS to Oxa. Taken together, our data indicate that inhibition of FcepsilonRI-dependent MC activation can suppress allergic CD.

Requirement of transcription factor AML1 in proliferation of developing thymocytes

Although the transcription factor AML1/Runx1 is known to be essential for definitive hematopoiesis, its role in T cell differentiation is not well understood. In this study, we investigated the functions of AML1 in the early stage of thymocyte differentiation. For this, we crossed AML1 dominant interfering form (Runt)-transgenic mice with TCR-transgenic mice, and demonstrated the decrease of CD4+8+ (DP) thymocyte cell number although their proportion was not reduced. Reaggregation culture system for thymocytes of (RuntxTCR) double transgenic mice, in which the rate of de novo transition from DN cells to the DP stage can be estimated, showed that the cell division during the DN-to-DP transition is impaired without significant cell death. These results indicate that AML1 is involved in thymocyte differentiation by controlling cell proliferation.

Oxysterol represses high-affinity IgE receptor-stimulated mast cell activation in Liver X receptor-dependent and -independent manners

Oxysterols activating liver X receptors (LXRs) repress expression of pro‐inflammatory genes and have anti‐inflammatory effects. Here, we show for the first time that bone marrow‐derived murine mast cells (BMMCs) predominantly express LXRβ. 25‐hydroxycholesterol, a representative LXR activating oxysterol, suppressed IL‐6 production and degranulation response in BMMCs following engagement of high‐affinity IgE receptor (FcεRI). Interestingly, 25‐hydroxycholesterol reduced cell‐surface FcεRI expression by inhibiting assembly of FcεRIα and FcεRIβ. We demonstrate that LXR activation was involved in the suppression of IL‐6 production in BMMCs, but that reduced FcεRI expression and degranulation response was mediated in an LXR‐independent manner.