Jun-ichi Kashiwakura

T cell proliferation by direct cross-talk between OX40 ligand on human mast cells and OX40 on human T cells: Comparison of gene expression profiles between human tonsillar and lung-cultured mast cells

Mast cells (MCs) are the primary effector cells in allergic reactions and have also been found to activate T cells and to reside in close physical proximity to T cells. However, the molecular mechanisms involved in the MC-T cell interaction remain unclear. We hypothesized that human tonsillar MCs, which locate in the interfollicular areas, might interact with T cells. Thus, we first established a culture system of human tonsillar MCs and then compared gene expression profiles of tonsillar MCs with that of lung MCs before and after aggregation of FcεRI by using high-density oligonucleotide probe arrays. Here we show that resting tonsillar MCs, when compared with lung MCs, revealed significantly higher expression levels for CC chemokines (CCL3 and 4), which recruit T cells, and for TNFR superfamilies (OX40 ligand and 4-1BB ligand), which induce proliferation of T cells. After aggregation of FcεRI, not only tonsillar MCs but also lung MCs up-regulated the expression of these molecules. We confirmed that T cell proliferation is induced in direct cross-talk by the MC surface molecule OX40 ligand. These results suggest that human MCs may play important roles in adaptive immunity through the T 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.

Txk, a Member of Nonreceptor Tyrosine Kinase of Tec Family, Acts as a Th1 Cell-Specific Transcription Factor and Regulates IFN-γ Gene Transcription

Precise mechanisms responsible for Th1 cell activation and differentiation are not fully elucidated. We have recently reported that Txk, a member of Tec family nonreceptor tyrosine kinase, is expressed on Th1/Th0 cells, and Txk regulates specifically IFN-γ gene expression. In this study, we found that Txk bound to IFN-γ promoter region. Txk transfection increased transcriptional activity of IFN-γ promoter plus luciferase constructs severalfold, including IFN-γ promoter −538, −208, and −53. IFN-γ promoter −39 was refractory to the Txk transfection. The actual site to which Txk bound was the element consisting of −53 and −39 bp from the transcription start site of human IFN-γ gene, a site distinct from several previously characterized binding sites. We found that the entire −53/−39 region was necessary for the binding to and function of Txk, because mutant promoter oligoDNA that contained contiguous five base substitutions dispersed throughout the −53/−39 inhibited the binding, and the mutant promoters did not respond to the Txk transfection. Similar sequences of this element are found within the 5′ flanking regions of several Th1 cell-associated protein genes. Thus, Txk is expressed on Th1/Th0 cells with the IFN-γ production and acts as a Th1 cell-specific transcription factor.

Histamine-releasing factor has a proinflammatory role in mouse models of asthma and allergy

IgE-mediated activation of mast cells and basophils underlies allergic diseases such as asthma. Histamine-releasing factor (HRF; also known as translationally controlled tumor protein [TCTP] and fortilin) has been implicated in late-phase allergic reactions (LPRs) and chronic allergic inflammation, but its functions during asthma are not well understood. Here, we identified a subset of IgE and IgG antibodies as HRF-interacting molecules in vitro. HRF was able to dimerize and bind to Igs via interactions of its N-terminal and internal regions with the Fab region of Igs. Therefore, HRF together with HRF-reactive IgE was able to activate mast cells in vitro. In mouse models of asthma and allergy, Ig-interacting HRF peptides that were shown to block HRF/Ig interactions in vitro inhibited IgE/HRF-induced mast cell activation and in vivo cutaneous anaphylaxis and airway inflammation. Intranasally administered HRF recruited inflammatory immune cells to the lung in naive mice in a mast cell- and Fc receptor-dependent manner. These results indicate that HRF has a proinflammatory role in asthma and skin immediate hypersensitivity, leading us to suggest HRF as a potential therapeutic target.

JNK1 controls mast cell degranulation and IL-1β production in inflammatory arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease marked by bone and cartilage destruction. Current biologic therapies are beneficial in only a portion of patients; hence small molecules targeting key pathogenic signaling cascades represent alternative therapeutic strategies. Here we show that c-Jun N-terminal kinase (JNK) 1, but not JNK2, is critical for joint swelling and destruction in a serum transfer model of arthritis. The proinflammatory function of JNK1 requires bone marrow-derived cells, particularly mast cells. Without JNK1, mast cells fail to degranulate efficiently and release less IL-1β after stimulation via Fcγ receptors (FcγRs). Pharmacologic JNK inhibition effectively prevents arthritis onset and abrogates joint swelling in established disease. Hence, JNK1 controls mast cell degranulation and FcγR-triggered IL-1β production, in addition to regulating cytokine and matrix metalloproteinase biosynthesis, and is an attractive therapeutic target in inflammatory arthritis.

Abnormal killer inhibitory receptor expression on natural killer cells in patients with Behçet's disease

Viral infection has been assigned some role in the pathogenesis of Behçets disease (BD). Defects in natural killer (NK) cell repertoire may be involved in impaired antiviral immunity, leading to the development of BD. We studied killer inhibitory receptor (KIR) expression in 40 patients with BD. CD94 and CD158b expression of NK cells was normal in a great majority of BD patients. NKB1 expression was reduced in eight and increased in six. Twelve of these 14 patients (86%) had severe eye disease. Some had reduced NKB1 and enhanced CD158a expression simultaneously, or enhanced NKB1 and reduced CD158a simultaneously, suggesting a skewed NK cell repertoire in BD. Collectively, KIR expression was abnormal in the BD patients with severe eye disease. This may result from genetic predisposition, or certain viruses may affect the KIR repertoire formation in BD patients. Abnormal KIR expression of NK cells may be associated with the development of BD.

Critical Role for Mast Cell Stat5 Activity in Skin Inflammation

SUMMARY Atopic dermatitis (AD) is a chronic inflammatory skin disease. Here, we show that phospholipase C-β3 (PLC-β3)-deficient mice spontaneously develop AD-like skin lesions and more severe allergen-induced dermatitis than wild-type mice. Mast cells were required for both AD models and remarkably increased in the skin of Plcb3−/− mice because of the increased Stat5 and reduced SHP-1 activities. Mast cell-specific deletion of Stat5 gene ameliorated allergen-induced dermatitis, whereas that of Shp1 gene encoding Stat5-inactivating SHP-1 exacerbated it. PLC-β3 regulates the expression of periostin in fibroblasts and TSLP in keratinocytes, two proteins critically involved in AD pathogenesis. Furthermore, polymorphisms in PLCB3, SHP1, STAT5A, and STAT5B genes were associated with human AD. Mast cell expression of PLC-β3 was inversely correlated with that of phospho-STAT5, and increased mast cells with high levels of phospho-STAT5 were found in lesional skin of some AD patients. Therefore, STAT5 regulatory mechanisms in mast cells are important for AD pathogenesis.

Polyclonal IgE Induces Mast Cell Survival and Cytokine Production

BACKGROUND Ag-dependent activation of IgE-bearing mast cells is a critical first step in immediate hypersensitivity and other allergic responses. Recent studies have revealed Ag-independent effects of monoclonal mouse IgE molecules on mast cell survival and activation. However, no studies have been performed on the effects of polyclonal IgE molecules. Here, we tested whether polyclonal mouse and human IgE molecules affect survival and cytokine production in mast cells. METHODS Mast cells were cultured in the presence of polyclonal mouse and human IgE molecules, and cell survival and cytokine production were analyzed. RESULTS Polyclonal mouse IgE molecules in sera from mice with atopic dermatitis-like allergic skin inflammation, enhanced survival and cytokine production in mast cell cultures. Similar to the effects of monoclonal IgE, the polyclonal IgE effects were mediated by the high-affinity IgE receptor, FcepsilonRI. Human polyclonal IgE molecules present in sera from atopic dermatitis patients were also capable of activating mast cells, and inducing IL-8 production in human cord blood-derived mast cells. CONCLUSIONS These results imply that polyclonal IgE in atopic dermatitis and other atopic conditions might modulate mast cell number and function, thus amplifying the allergic response.

Significantly High Levels of Anti-dsDNA Immunoglobulin E in Sera and the Ability of dsDNA to Induce the Degranulation of Basophils from Chronic Urticaria Patients

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.

Activation of human synovial mast cells from rheumatoid arthritis or osteoarthritis patients in response to aggregated IgG through Fcγ receptor I and Fcγ receptor II

OBJECTIVE Substantial evidence suggests that human synovial mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). A plausible pathway for the activation of synovial MCs is through IgG receptors, given the prevalence of circulating IgG isotype autoantibodies and synovial immune complexes in patients with RA. However, IgG receptor expression on human synovial MCs remains uncharacterized. The aim of this study was to identify which IgG receptor(s) on synovial MCs are responsible for MC activation in immune complexes. METHODS Synovial tissue specimens were obtained from patients with RA or patients with osteoarthritis (OA) who were undergoing joint replacement surgery, and synovial MCs were enzymatically dispersed. Cultured synovium-derived MCs were generated by culturing synovial cells with stem cell factor, and receptor expression was analyzed using fluorescence-activated cell sorting. Mediators released from MCs were measured using enzyme immunoassays or enzyme-linked immunosorbent assays. RESULTS Primary synovial MCs and cultured synovium-derived MCs obtained from both patients with RA and patients with OA expressed Fcε receptor I (FcεRI), FcγRI, and FcγRII but not FcγRIII. Cultured synovium-derived MCs induced degranulation and the production of prostaglandin D2 and tumor necrosis factor α (TNFα) through FcγRI. The aggregation of FcγRII caused histamine release from cultured MCs but not from primary MCs. Histamine release induced by aggregated IgG was significantly inhibited by neutralizing anti-FcγRI monoclonal antibody and anti-FcγRII monoclonal antibody. CONCLUSION With regard to the FcR expression profile, synovial MCs from patients with RA and patients with OA were similar. FcγRI was responsible for producing abundant TNFα from synovial MCs in response to aggregated IgG. Immune complexes may activate synovial MCs through FcγRI and FcγRII.