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1.  Galectin-9 Enhances Cytokine Secretion, but Suppresses Survival and Degranulation, in Human Mast Cell Line 
PLoS ONE  2014;9(1):e86106.
Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.
doi:10.1371/journal.pone.0086106
PMCID: PMC3896437  PMID: 24465902
2.  Rationale and study design of the Japan environment and children’s study (JECS) 
BMC Public Health  2014;14:25.
Background
There is global concern over significant threats from a wide variety of environmental hazards to which children face. Large-scale and long-term birth cohort studies are needed for better environmental management based on sound science. The primary objective of the Japan Environment and Children’s Study (JECS), a nation-wide birth cohort study that started its recruitment in January 2011, is to elucidate environmental factors that affect children’s health and development.
Methods/Design
Approximately 100,000 expecting mothers who live in designated study areas will be recruited over a 3-year period from January 2011. Participating children will be followed until they reach 13 years of age. Exposure to environmental factors will be assessed by chemical analyses of bio-specimens (blood, cord blood, urine, breast milk, and hair), household environment measurements, and computational simulations using monitoring data (e.g. ambient air quality monitoring) as well as questionnaires. JECS’ priority outcomes include reproduction/pregnancy complications, congenital anomalies, neuropsychiatric disorders, immune system disorders, and metabolic/endocrine system disorders. Genetic factors, socioeconomic status, and lifestyle factors will also be examined as covariates and potential confounders. To maximize representativeness, we adopted provider-mediated community-based recruitment.
Discussion
Through JECS, chemical substances to which children are exposed during the fetal stage or early childhood will be identified. The JECS results will be translated to better risk assessment and management to provide healthy environment for next generations.
doi:10.1186/1471-2458-14-25
PMCID: PMC3893509  PMID: 24410977
Birth cohort; Children; Growth and development; Environmental chemicals; Pregnant women
3.  Epithelial Cell-derived IL-25, but not Th17 cell-derived IL-17 or IL-17F, is Crucial for Murine Asthma1 
Journal of immunology (Baltimore, Md. : 1950)  2012;189(7):10.4049/jimmunol.1200461.
IL-17A, IL-17F and IL-25 are ligands for IL-17RA. In the present study, we demonstrated that IL-25-deficient mice, but not IL-17A-, IL-17F-, IL-17A/F-, IL-23p19- and ROR-γt-deficient mice, showed significant suppression of the number of eosinophils and the levels of proinflammatory mediators in bronchoalveolar lavage fluids, airway hyperresponsiveness to methacholine, or ovalbumin-specific IgG1 and IgE levels in the serum during ovalbumin-induced Th2-type/eosinophilic airway inflammation, without any effect on lung DC migration or antigen-specific memory-Th2-cell expansion during antigen sensitization. By adoptive transfer of either T cells, mast cells or bone marrow cells from IL-25-deficient mice, we found that IL-25 produced by airway structural cells such as epithelial cells—but not by such hematopoietic stem-cell-origin immune cells as T cells and mast cells—was indispensable for induction of Th2-type/eosinophilic airway inflammation by activating lung epithelial cells and eosinophils. Therefore, airway structural-cell-derived IL-25—rather than Th17-cell-derived IL-17A and IL-17F—is responsible for induction of local inflammation by promoting activation of lung epithelial cells and eosinophils in the elicitation phase—but is not required for antigen-specific Th2 cell differentiation in the sensitization phase—of Th2-type/eosinophilic airway inflammation.
doi:10.4049/jimmunol.1200461
PMCID: PMC3812057  PMID: 22942422
4.  IL-33, but Not IL-25, Is Crucial for the Development of House Dust Mite Antigen-Induced Allergic Rhinitis 
PLoS ONE  2013;8(10):e78099.
Both interleukin (IL)-33 and IL-25 induce Th2 cytokine production by various cell types, suggesting that they contribute to development of allergic disorders. However, the precise roles of IL-33 and IL-25 in house dust mite (HDM)-induced allergic rhinitis (AR) remain unclear. Both IL-33 and IL-25 were produced mainly by nasal epithelial cells during HDM-induced AR. Eosinophil and goblet cell counts in the nose and IL-5 levels in lymph node cell culture supernatants were significantly decreased in IL-33-deficient, but not IL-25-deficient, mice compared with wild-type mice during HDM-induced AR, but the serum IgE and IgG1 levels did not differ. On the other hand, HDM-induced AR developed similarly in wild-type mice transferred with either IL-33-deficient BM cells or wild-type BM cells. IL-33, but not IL-25, produced by nasal epithelial cells was crucial for the development of murine HDM-induced AR. These observations suggest that IL-33 neutralization may be a potential approach for treatment of HDM-induced AR in humans.
doi:10.1371/journal.pone.0078099
PMCID: PMC3808342  PMID: 24205109
5.  Cholesteatoma Fibroblasts Promote Epithelial Cell Proliferation through Overexpression of Epiregulin 
PLoS ONE  2013;8(6):e66725.
To investigate whether keratinocytes proliferate in response to epiregulin produced by subepithelial fibroblasts derived from middle ear cholesteatoma. Tissue samples were obtained from patients undergoing tympanoplasty. The quantitative polymerase chain reaction and immunohistochemistry were performed to examine epiregulin expression and localization in cholesteatoma tissues and retroauricular skin tissues. Fibroblasts were cultured from cholesteatoma tissues and from normal retroauricular skin. These fibroblasts were used as feeder cells for culture with a human keratinocyte cell line (PHK16-0b). To investigate the role of epiregulin in colony formation by PHK16-0b cells, epiregulin mRNA expression was knocked down in fibroblasts by using short interfering RNA and epiregulin protein was blocked with a neutralizing antibody. Epiregulin mRNA expression was significantly elevated in cholesteatoma tissues compared with that in normal retroauricular skin. Staining for epiregulin was more intense in the epithelial cells and subepithelial fibroblasts of cholesteatoma tissues than in retroauricular skin. When PHK16-0b cells were cultured with cholesteatoma fibroblasts, their colony-forming efficiency was 50% higher than when these cells were cultured with normal skin fibroblasts. Also, knockdown of epiregulin mRNA in cholesteatoma fibroblasts led to greater suppression of colony formation than knockdown in skin fibroblasts. Furthermore, the colony-forming efficiency of PHK16-0b cells was significantly reduced after treatment with an epiregulin neutralizing antibody in co-culture with cholesteatoma fibroblasts, but not in co-culture with skin fibroblasts. These results suggest that keratinocyte hyperproliferation in cholesteatoma is promoted through overexpression of epiregulin by subepithelial fibroblasts via epithelial–mesenchymal interactions, which may play a crucial role in the pathogenesis of middle ear cholesteatoma.
doi:10.1371/journal.pone.0066725
PMCID: PMC3691191  PMID: 23826119
6.  Thymic Stromal Lymphopoietin Gene Promoter Polymorphisms Are Associated with Susceptibility to Bronchial Asthma 
Thymic stromal lymphopoietin (TSLP) triggers dendritic cell–mediated T helper (Th) 2 inflammatory responses. A single-nucleotide polymorphism (SNP), rs3806933, in the promoter region of the TSLP gene creates a binding site for the transcription factor activating protein (AP)–1. The variant enhances AP-1 binding to the regulatory element, and increases the promoter–reporter activity of TSLP in response to polyinosinic-polycytidylic acid (poly[I:C]) stimulation in normal human bronchial epithelium (NHBE). We investigated whether polymorphisms including the SNP rs3806933 could affect the susceptibility to and clinical phenotypes of bronchial asthma. We selected three representative (i.e., Tag) SNPs and conducted association studies of the TSLP gene, using two independent populations (639 patients with childhood atopic asthma and 838 control subjects, and 641 patients with adult asthma and 376 control subjects, respectively). We further examined the effects of corticosteroids and a long-acting β2-agonist (salmeterol) on the expression levels of the TSLP gene in response to poly(I:C) in NHBE. We found that the promoter polymorphisms rs3806933 and rs2289276 were significantly associated with disease susceptibility in both childhood atopic and adult asthma. The functional SNP rs3806933 was associated with asthma (meta-analysis, P = 0.000056; odds ratio, 1.29; 95% confidence interval, 1.14–1.47). A genotype of rs2289278 was correlated with pulmonary function. Moreover, the induction of TSLP mRNA and protein expression induced by poly(I:C) in NHBE was synergistically impaired by a corticosteroid and salmeterol. TSLP variants are significantly associated with bronchial asthma and pulmonary function. Thus, TSLP may serve as a therapeutic target molecule for combination therapy.
doi:10.1165/rcmb.2009-0418OC
PMCID: PMC3159073  PMID: 20656951
asthma; TSLP; bronchial epithelial cells; combination therapy; genetic polymorphisms
7.  58 Long-term IFN-γ Treatment Alters Allergic Inflammation-Associated Gene Expression in Conjunctival Fibroblasts 
The World Allergy Organization Journal  2012;5(Suppl 2):S36-S37.
Background
Interferon-γ (IFN-γ) is a T helper type 1 (Th1) cytokine which has antiviral, anti-proliferative, and immunomodulatory properties. Despite the presence of IFN-γ in the conjunctiva or tear fluid of patients with severe allergic conjunctivitis, the role of IFN-γ in allergic conjunctivitis is controversial and enigmatic. In this study, we assess the effect of long-term treatment of IFN-γ on human conjuctival fibroblasts.
Methods
Primary cultured fibroblasts derived from human conjunctiva specimens were established. Cultured fibroblasts were incubated with or without IFN-γ (10 ng/mL) for up to 14 days. After IFN-γ treatment, cells were washed out and were re-stimulated with combinations of IL-4 (10 ng/mL) and TNFα (10 ng/mL) for 6 hours. Then, total mRNAs were isolated and mRNA expression levels were measured using a microarray and real time-PCR.
Results
In IFN-γ treated fibroblasts in short-term (6 hours), we confirmed the increased expression levels of well-known interferon induced genes, such as MHC class II, IRF1 and CXCL10. Increased expression of CCL11 stimulated by IL-4 + TNFα was suppressed by short-term IFN-γ treatment as described previously. In long-term (14 days) IFN-γ treated cells, the expression of CCL11 and several proinflammatory chemokines, which were associated with Th2 cell and eosinophil migration, was slightly but significantly increased without any other stimulations. Interestingly, IL-4 + TNFα stimulation greatly enhanced the expression levels of these chemokines, suggesting that long-term IFN-γ treatment alters the competency of gene expression potential on these gene loci in contrast to the situation for short term treatment. Time-course analysis of IFN-γ treatment revealed that the treatment of IFN-γ up to 24 hours suppressed the IL-4 + TNFα-induced CCL11 expression, whereas the CCL11 expression was enhanced 3 days after the treatment.
Conclusions
These results uncovered previously unsuspected contribution of IFN-γ to the fibroblasts in allergic inflammatory milieu in terms of the change in production of certain chemokines. In other words, the antagonistic function of IFN-γ to Th2 cells at the early phase may represent only a small part. The intracellular signaling and IFN-γ-dependent secondary events are needed to be explored to explain the long-term effect or the late phase phenomenon after IFN-γ administration.
doi:10.1097/01.WOX.0000411803.48668.f8
PMCID: PMC3512772
8.  171 Augmentation of cxcl10 Expression in Nasal Fibroblasts Derived from Patients With Recalcitrant Chronic Rhinosinusitis Associated With Bronchial Asthma 
Background
The prevalence of chronic rhinosinusitis (CRS) that is refractory to traditional therapy appears to be increasing, and CRS that is refractory to traditional therapy tends to be associated with bronchial asthma (BA), especially, aspirin-intolerant asthma (AIA). After viral infections, patients with CRS associated with BA usually experience exacerbations of their CRS symptoms, including nasal polyposis, in comparison with CRS patients without BA. Alternatively tissue fibroblasts as an important component of the epithelial mesenchymal trophic unit play a key role in maintaining tissue homeostasis and may also have the potential to contribute to disease pathogenesis through their contribution to inflammatory responses. On the basis of these findings, we hypothesized that CRS patients with BA are more susceptible to inflammation of the nasal and paranasal mucosa depending on the antiviral response of nasal fibroblasts.
Methods
Tissue specimens were obtained from the nasal polyps of 3 groups of CRS patients, a group that did not have BA (CRS-NA group), a group with aspirin-tolerant asthma (CRS-ATA group), and a group with AIA (CRS-AIA group). Nasal polyp fibroblasts (NPFs) were isolated from the specimens and stimulated with poly I: C. By using a DNA microarray and performing a hierarchical clustering analysis we were able to identify a cluster containing genes that were up-regulated after poly I: C stimulation. To confirm the results of the analysis data, we used quantitative real-time PCR (qRT-PCR) and an enzyme-linked immunoadsorbent assay (ELISA).
Results
Expression of IFN-inducible protein 10 (IP-10)/CXCL10 transcript was higher in the NPFs of the CRS-AIA group and CRS-ATA group than in the CRS-NA group and control group. These findings were confirmed by qRT-PCR and ELISA.
Conclusions
The results of this study suggest that the increased poly I:C-induced CXCL10 expression in NPFs derived from the CRS patients with BA is involved in susceptible to T helper (Th)1-type immune response in the nasal and paranasal mucosa by viral infection compared with CRS patients without BA.
doi:10.1097/01.WOX.0000411928.19191.11
PMCID: PMC3513039
9.  Genome-Wide Association Study Identifies HLA-DP as a Susceptibility Gene for Pediatric Asthma in Asian Populations 
PLoS Genetics  2011;7(7):e1002170.
Asthma is a complex phenotype influenced by genetic and environmental factors. We conducted a genome-wide association study (GWAS) with 938 Japanese pediatric asthma patients and 2,376 controls. Single-nucleotide polymorphisms (SNPs) showing strong associations (P<1×10−8) in GWAS were further genotyped in an independent Japanese samples (818 cases and 1,032 controls) and in Korean samples (835 cases and 421 controls). SNP rs987870, located between HLA-DPA1 and HLA-DPB1, was consistently associated with pediatric asthma in 3 independent populations (Pcombined = 2.3×10−10, odds ratio [OR] = 1.40). HLA-DP allele analysis showed that DPA1*0201 and DPB1*0901, which were in strong linkage disequilibrium, were strongly associated with pediatric asthma (DPA1*0201: P = 5.5×10−10, OR = 1.52, and DPB1*0901: P = 2.0×10−7, OR = 1.49). Our findings show that genetic variants in the HLA-DP locus are associated with the risk of pediatric asthma in Asian populations.
Author Summary
Asthma is the most common chronic disorder in children, and asthma exacerbation is an important cause of childhood morbidity and hospitalization. Here, taking advantage of recent technological advances in human genetics, we performed a genome-wide association study and follow-up validation studies to identify genetic variants for asthma. By examining 6,428 Asians, we found rs987870 and HLA-DPA1*0201/DPB1*0901 were associated with pediatric asthma. The association signal was stretched in the region of HLA-DPB2, collagen, type XI, alpha 2 (COL11A2), and Retinoid X receptor beta (RXRB), but strong linkage disequilibrium in this region made it difficult to specifically identify causative variants. Interestingly, the SNP (or the HLA-DP allele) associated with pediatric asthma (Th-2 type immune diseases) in the present study confers protection against Th-1 type immune diseases, such as type 1 diabetes and rheumatoid arthritis. Therefore, the association results obtained in the present study could partially explain the inverse relationship between asthma and Th-1 type immune diseases and may lead to better understanding of Th-1/Th-2 immune diseases.
doi:10.1371/journal.pgen.1002170
PMCID: PMC3140987  PMID: 21814517
10.  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, FcεRI. 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.
doi:10.2332/allergolint.08-OA-0080
PMCID: PMC3123538  PMID: 19542764
allergy; antibodies; cell activation; human; mast cells/basophils
11.  Identification of a polyI:C-inducible membrane protein that participates in dendritic cell–mediated natural killer cell activation 
The Journal of Experimental Medicine  2010;207(12):2675-2687.
The novel polyI:C-inducible membrane protein INAM triggers dendritic cell–mediated natural killer cell activation.
In myeloid dendritic cells (mDCs), TLR3 is expressed in the endosomal membrane and interacts with the adaptor toll/interleukin 1 receptor homology domain–containing adaptor molecule 1 (TICAM-1; TRIF). TICAM-1 signals culminate in interferon (IFN) regulatory factor (IRF) 3 activation. Co-culture of mDC pretreated with the TLR3 ligand polyI:C and natural killer (NK) cells resulted in NK cell activation. This activation was triggered by cell-to-cell contact but not cytokines. Using expression profiling and gain/loss-of-function analyses of mDC genes, we tried to identify a TICAM-1–inducing membrane protein that participates in mDC-mediated NK activation. Of the nine candidates screened, one contained a tetraspanin-like sequence and satisfied the screening criteria. The protein, referred to as IRF-3–dependent NK-activating molecule (INAM), functioned in both the mDC and NK cell to facilitate NK activation. In the mDC, TICAM-1, IFN promoter stimulator 1, and IRF-3, but not IRF-7, were required for mDC-mediated NK activation. INAM was minimally expressed on NK cells, was up-regulated in response to polyI:C, and contributed to mDC–NK reciprocal activation via its cytoplasmic tail, which was crucial for the activation signal in NK cells. Adoptive transfer of INAM-expressing mDCs into mice implanted with NK-sensitive tumors caused NK-mediated tumor regression. We identify a new pathway for mDC–NK contact-mediated NK activation that is governed by a TLR signal-derived membrane molecule.
doi:10.1084/jem.20091573
PMCID: PMC2989763  PMID: 21059856
12.  Paracrine IL-33 Stimulation Enhances Lipopolysaccharide-Mediated Macrophage Activation 
PLoS ONE  2011;6(4):e18404.
Background
IL-33, a member of the IL-1 family of cytokines, provokes Th2-type inflammation accompanied by accumulation of eosinophils through IL-33R, which consists of ST2 and IL-1RAcP. We previously demonstrated that macrophages produce IL-33 in response to LPS. Some immune responses were shown to differ between ST2-deficient mice and soluble ST2-Fc fusion protein-treated mice. Even in anti-ST2 antibody (Ab)-treated mice, the phenotypes differed between distinct Ab clones, because the characterization of such Abs (i.e., depletion, agonistic or blocking Abs) was unclear in some cases.
Methodology/Principal Findings
To elucidate the precise role of IL-33, we newly generated neutralizing monoclonal Abs for IL-33. Exogenous IL-33 potentiated LPS-mediated cytokine production by macrophages. That LPS-mediated cytokine production by macrophages was suppressed by inhibition of endogenous IL-33 by the anti-IL-33 neutralizing mAbs.
Conclusions/Significance
Our findings suggest that LPS-mediated macrophage activation is accelerated by macrophage-derived paracrine IL-33 stimulation.
doi:10.1371/journal.pone.0018404
PMCID: PMC3073971  PMID: 21494550
13.  IL-33 and Airway Inflammation 
Interleukin-33 (IL-33) is the 11th member of IL-1 cytokine family which includes IL-1 and IL-18. Unlike IL-1β and IL-18, IL-33 is suggested to function as an alarmin that is released upon endothelial or epithelial cell damage and may not enhance acquired immune responses through activation of inflammasome. ST2, a IL-33 receptor component, is preferentially expressed by T-helper type (Th) 2 cells, mast cells, eosinophils and basophils, compared to Th1 cells, Th17 cells and neutrophils. Thus, IL-33 profoundly enhances allergic inflammation through increased expression of proallergic cytokines and chemokines. Indeed, IL-33 and its receptor genes are recognized as the most susceptible genes for asthma by several recent genomewide association studies. It has also recently been shown that IL-33 plays a crucial role in innate eosinophilic airway inflammation rather than acquired immune responses such as IgE production. As such, IL-33 provides a unique therapeutic way for asthma, i.e., ameliorating innate airway inflammation.
doi:10.4168/aair.2011.3.2.81
PMCID: PMC3062800  PMID: 21461246
IL-33; ST2; host defense; allergy; autoimmunity; chronic disease; mast cell; basophil; eosinophil
14.  Intradiscal transplantation of synovial mesenchymal stem cells prevents intervertebral disc degeneration through suppression of matrix metalloproteinase-related genes in nucleus pulposus cells in rabbits 
Arthritis Research & Therapy  2010;12(6):R206.
Introduction
Synovial mesenchymal stem cells (MSCs) have high proliferative and chondrogenic potentials, and MSCs transplanted into the articular cartilage defect produce abundant extracellular matrix. Because of similarities between the articular cartilage and the intervertebral disc cartilage, synovial MSCs are a potential cell source for disc regeneration. Here, we examined the effect of intradiscal transplantation of synovial MSCs after aspiration of nucleus pulposus in rabbits.
Methods
The nucleus pulposus tissues of rabbit's intervertebral discs were aspirated to induce disc degeneration, and allogenic synovial MSCs were transplanted. At 2, 4, 6, 8, 16, 24 weeks postoperatively, we evaluated with imaging analyses such as X-ray and magnetic resonance imaging (MRI), and histological analysis. To investigate interaction between synovial MSCs and nucleus pulposus cells, human synovial MSCs and rat nucleus pulposus cells were co-cultured, and species specific microarray were performed.
Results
The existence of transplanted cells labeled with DiI or derived from green fluorescent protein (GFP)-expressing transgenic rabbits was confirmed up until 24 weeks. X-ray analyses demonstrated that intervertebral disc height in the MSC group remained higher than that in the degeneration group. T2 weighted MR imaging showed higher signal intensity of nucleus pulposus in the MSC group. Immunohistological analyses revealed higher expression of type II collagen around nucleus pulposus cells in the MSC group compared with even that of the normal group. In co-culture of rat nucleus pulposus cells and human synovial MSCs, species specific microarray revealed that gene profiles of nucleus pulposus were altered markedly with suppression of genes relating matrix degradative enzymes and inflammatory cytokines.
Conclusions
Synovial MSCs injected into the nucleus pulposus space promoted synthesis of the remaining nucleus pulposus cells to type II collagen and inhibition of expressions of degradative enzymes and inflammatory cytokines, resulting in maintaining the structure of the intervertebral disc being maintained.
doi:10.1186/ar3182
PMCID: PMC3046513  PMID: 21054867
15.  Dexamethasone and FK506 Inhibit Expression of Distinct Subsets of Chemokines in Human Mast Cells1 
Mast cells produce a large amount of several chemokines after cross-linking of FcεRI and participate in the pathogenesis of allergic diseases. The objective of this study was to comprehensively investigate FcεRI-mediated chemokine induction in human mast cells and the effect of a corticosteroid (dexamethasone) and a calcineurin inhibitor (FK506). Human peripheral blood-derived mast cells were stimulated with anti-IgE Ab in the presence of dexamethasone or FK506. Gene expression profiles were evaluated using GeneChip and confirmed by real-time PCR, and chemokine concentrations were measured by cytometric bead arrays and ELISA. Expression of eight chemokines was significantly induced in mast cells by anti-IgE stimulation. Induction of CCL2, CCL7, CXCL3, and CXCL8 by anti-IgE was significantly inhibited by dexamethasone but was enhanced by FK506. In contrast, induction of CCL1, CCL3, CCL4, and CCL18 was significantly inhibited by FK506 but, with the exception of CCL1, was enhanced by dexamethasone. Combination of dexamethasone and FK506 suppressed production of all chemokines by anti-IgE stimulation. Studies using protease inhibitors indicate that mast cell proteases may degrade several of the chemokines. These results suggest that corticosteroids and calcineurin inhibitors inhibit expression of distinct subsets of chemokines, and a combination of these drugs almost completely suppresses the induction of all chemokine genes in human mast cells in response to FcεRI-dependent stimulation. This implies that a combination of a corticosteroid and a calcineurin inhibitor may be more effective than each single agent for the treatment of allergic diseases in which mast cell-derived chemokines play a major role.
doi:10.4049/jimmunol.0801375
PMCID: PMC2824683  PMID: 19454720
16.  Zinc transporter Znt5/Slc30a5 is required for the mast cell–mediated delayed-type allergic reaction but not the immediate-type reaction 
The Journal of Experimental Medicine  2009;206(6):1351-1364.
Zinc (Zn) is an essential nutrient and its deficiency causes immunodeficiency. However, it remains unknown how Zn homeostasis is regulated in mast cells and if Zn transporters are involved in allergic reactions. We show that Znt5/Slc30a5 is required for contact hypersensitivity and mast cell–mediated delayed-type allergic response but not for immediate passive cutaneous anaphylaxis. In mast cells from Znt5−/− mice, Fcϵ receptor I (FcϵRI)–induced cytokine production was diminished, but degranulation was intact. Znt5 was involved in FcϵRI-induced translocation of protein kinase C (PKC) to the plasma membrane and the nuclear translocation of nuclear factor κB. In addition, the Zn finger–like motif of PKC was required for its plasma membrane translocation and binding to diacylglycerol. Thus, Znt5 is selectively required for the mast cell–mediated delayed-type allergic response, and it is a novel player in mast cell activation.
doi:10.1084/jem.20082533
PMCID: PMC2715059  PMID: 19451265
17.  Large scale genotyping study for asthma in the Japanese population 
BMC Research Notes  2009;2:54.
Background
Asthma is a complex phenotype that is influenced by both genetic and environmental factors. Genome-wide linkage and association studies have been performed to identify susceptibility genes for asthma. These studies identified new genes and pathways implicated in this disease, many of which were previously unknown.
Objective
To perform a large-scale genotyping study to identify asthma-susceptibility genes in the Japanese population.
Methods
We performed a large-scale, three-stage association study on 288 atopic asthmatics and 1032 controls, by using multiplex PCR-Invader assay methods at 82,935 single nucleotide polymorphisms (SNPs) (1st stage). SNPs that were strongly associated with asthma were further genotyped in samples from asthmatic families (216 families, 762 members, 2nd stage), 541 independent patients, and 744 controls (3rd stage).
Results
SNPs located in the 5' region of PEX19 (rs2820421) were significantly associated with P < 0.05 through the 1st to the 3rd stage analyses; however, the P values did not reach statistically significant levels (combined, P = 3.8 × 10-5; statistically significant levels with Bonferroni correction, P = 6.57 × 10-7). SNPs on HPCAL1 (rs3771140) and on IL18R1 (rs3213733) were associated with asthma in the 1st and 2nd stage analyses, but the associations were not observed in the 3rd stage analysis.
Conclusion
No association attained genome-wide significance, but several loci for possible association emerged. Future studies are required to validate these results for the prevention and treatment of asthma.
doi:10.1186/1756-0500-2-54
PMCID: PMC2674055  PMID: 19335888
18.  Human Sclera Maintains Common Characteristics with Cartilage throughout Evolution 
PLoS ONE  2008;3(11):e3709.
Background
The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm.
Methodology/Principal Findings
We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-βs and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human ‘sclera’-derived cells can be considered chondrocytes when cultured ex vivo.
Conclusions/Significance
Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia.
doi:10.1371/journal.pone.0003709
PMCID: PMC2579486  PMID: 19002264
19.  Interleukin-3 Does Not Affect the Differentiation of Mast Cells Derived from Human Bone Marrow Progenitors 
Immunological Investigations  2008;37(1):1-17.
Although IL-3 is commonly used for culture of human progenitor-derived mast cells together with Stem cell factor (SCF) and IL-6, the effect of IL-3 on human mast cell differentiation has not been well elucidated. Human bone marrow CD34+ progenitors were cultured for up to 12 weeks in the presence of rhSCF and rhIL-6 either with rhIL-3 (IL-3 (+)) or without rhIL-3 (IL-3 (−)) for the initial 1-week of culture. Total cell number increased at 2 weeks in IL-3 (+), as compared to IL-3 (−), but changes in the appearance of mast cells were delayed. When IL-3 was present for the initial 1-week culture, granules looked more mature with IL-3 than without IL-3. However, tryptase and chymase contents, and surface antigen expression (CD18, CD51, CD54, and CD117) were not altered by IL-3. Surface expression and mRNA level of FcεRIα and histamine release by crosslinking of FcεRIα did not differ from one preparation to the next. GeneChip analysis revealed that no significant differences were observed between IL-3 (+) and IL-3 (−) cells either when inactivated or activated by aggregation of FcεRIα. These findings indicate that initial incubation of human bone marrow CD34+ progenitors with IL-3 does not affect the differentiation of mast cells.
doi:10.1080/08820130701741742
PMCID: PMC2430175  PMID: 18214796
Interleukin-3; Human mast cells; Bone marrow; Stem cell factor; Differentiation
20.  Gremlin Enhances the Determined Path to Cardiomyogenesis 
PLoS ONE  2008;3(6):e2407.
Background
The critical event in heart formation is commitment of mesodermal cells to a cardiomyogenic fate, and cardiac fate determination is regulated by a series of cytokines. Bone morphogenetic proteins (BMPs) and fibroblast growth factors have been shown to be involved in this process, however additional factors needs to be identified for the fate determination, especially at the early stage of cardiomyogenic development.
Methodology/Principal Findings
Global gene expression analysis using a series of human cells with a cardiomyogenic potential suggested Gremlin (Grem1) is a candidate gene responsible for in vitro cardiomyogenic differentiation. Grem1, a known BMP antagonist, enhanced DMSO-induced cardiomyogenesis of P19CL6 embryonal carcinoma cells (CL6 cells) 10–35 fold in an area of beating differentiated cardiomyocytes. The Grem1 action was most effective at the early differentiation stage when CL6 cells were destined to cardiomyogenesis, and was mediated through inhibition of BMP2. Furthermore, BMP2 inhibited Wnt/β-catenin signaling that promoted CL6 cardiomyogenesis.
Conclusions/Significance
Grem1 enhances the determined path to cardiomyogenesis in a stage-specific manner, and inhibition of the BMP signaling pathway is involved in initial determination of Grem1-promoted cardiomyogenesis. Our results shed new light on renewal of the cardiovascular system using Grem1 in human.
doi:10.1371/journal.pone.0002407
PMCID: PMC2398777  PMID: 18545679
21.  Role of Regulatory and Proinflammatory T-Cell Populations in Allergic Diseases 
Regulatory T (Treg) cells are considered to inhibit the development of both type 1 (Th1) and type 2 helper T (Th2) cells. However, it is recently reported that there are reduced numbers of Treg cells in patients with allergic diseases as compared with individuals who have high levels of serum immunoglobulin E and blood eosinophils but are asymptomatic. Therefore, Treg cells may suppress the onset of allergic disease by down-regulating other types of immune cells besides Th1 and Th2 cells. The newly discovered interleukin 17-producing helper T cells that are responsible for autoimmune inflammatory diseases may counteract Treg cells even in allergic diseases. The Th2 cells that are capable of producing of high levels of tumor necrosis factor-α may also be involved in inflammation in allergic diseases. In this review, we further discuss the role of Th1, Th2, interleukin 17-producing helper T cells, and Treg cells in allergic diseases by using the balancing square model and the factors differentiating between patients with clinical manifestations of allergic symptomatic and atopic individuals who are sensitized but asymptomatic.
doi:10.1097/wox.0b013e3181629ae3
PMCID: PMC3651097  PMID: 23283296
helper T cells; regulatory T cells; interleukin 17; mast cells; thymic stromal lymphopoietin
22.  Combination of hTERT and bmi-1, E6, or E7 Induces Prolongation of the Life Span of Bone Marrow Stromal Cells from an Elderly Donor without Affecting Their Neurogenic Potential†  
Molecular and Cellular Biology  2005;25(12):5183-5195.
Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.
doi:10.1128/MCB.25.12.5183-5195.2005
PMCID: PMC1140572  PMID: 15923633
23.  Thrombospondin 1 Is an Autocrine Negative Regulator of Human Dendritic Cell Activation 
The Journal of Experimental Medicine  2003;198(8):1277-1283.
Thrombospondin 1 (TSP) elicits potent antiinflammatory activities in vivo, as evidenced by persistent, multiorgan inflammation in TSP null mice. Herein, we report that DCs represent an abundant source of TSP at steady state and during activation. Human monocyte-derived immature dendritic cells (iDCs) spontaneously produce TSP, which is strongly enhanced by PGE2 and to a lesser extent by transforming growth factor (TGF) β, two soluble mediators secreted by macrophages after engulfment of damaged tissues. Shortly after activation via danger signals, DCs transiently produce interleukin (IL) 12 and tumor necrosis factor (TNF) α, thereby eliciting protective and inflammatory immune responses. Microbial stimuli increase TSP production, which is further enhanced by IL-10 or TGF-β. The endogenous TSP produced during early DC activation negatively regulates IL-12, TNF-α, and IL-10 release through its interactions with CD47 and CD36. After prolonged activation, DCs extinguish their cytokine synthesis and become refractory to subsequent stimulation, thereby favoring the return to steady state. Such “exhausted” DCs continue to release TSP but not IL-10. Disrupting TSP–CD47 interactions during their restimulation restores their cytokine production. We conclude that DC-derived TSP serves as a previously unappreciated negative regulator contributing to arrest of cytokine production, further supporting its fundamental role in vivo in the active resolution of inflammation and maintenance of steady state.
doi:10.1084/jem.20030705
PMCID: PMC2194231  PMID: 14568985
PGE2; TGF-β; IL-12; CD47; CD36
24.  Interferon-α/β receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006 
BMC Immunology  2003;4:8.
Background
Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported.
Results
This study was designed to identify a CpG-inducible gene cluster that potentially predicts for the molecular mechanisms of clinical efficacy of CpG ODN, by determining mRNA expression in human PBMC after stimulation with CpG ODN. PBMCs were obtained from the peripheral blood of healthy volunteers and cultured in the presence or absence of CpG ODN 2006 for up to 24 hours. The mRNA expression profile was evaluated using a high-density oligonucleotide probe array, GeneChip®. Using hierarchical clustering-analysis, out of a total of 10,000 genes we identified a cluster containing 77 genes as having been up-regulated by CpG ODN. This cluster was further divided into two sub-clusters by means of time-kinetics. (1) Inflammatory cytokines such as IL-6 and GM-CSF were up-regulated predominantly 3 to 6 hours after stimulation with CpG ODN, presumably through activation of a transcription factor, NF-κB. (2) Interferon (IFN)-inducible anti-viral proteins, including IFIT1, OAS1 and Mx1, and Th1 chemoattractant IP-10, were up-regulated predominantly 6 to 24 hours after stimulation. Blocking with mAb against IFN-α/β receptor strongly inhibited the induction of these IFN-inducible genes by CpG ODN.
Conclusion
This study provides new information regarding the possible immunomodulatory effects of CpG ODN in vivo via an IFN-α/β receptor-mediated paracrine pathway.
doi:10.1186/1471-2172-4-8
PMCID: PMC183869  PMID: 12887736
25.  Transplantation of Achilles Tendon Treated With Bone Morphogenetic Protein 7 Promotes Meniscus Regeneration in a Rat Model of Massive Meniscal Defect 
Arthritis and Rheumatism  2013;65(11):2876-2886.
Objective
This study was undertaken to examine whether bone morphogenetic protein 7 (BMP-7) induces ectopic cartilage formation in the rat tendon, and whether transplantation of tendon treated with BMP-7 promotes meniscal regeneration. Additionally, we analyzed the relative contributions of host and donor cells on the healing process after tendon transplantation in a rat model.
Methods
BMP-7 was injected in situ into the Achilles tendon of rats, and the histologic findings and gene profile were evaluated. Achilles tendon injected with 1 μg of BMP-7 was transplanted into a meniscal defect in rats. The regenerated meniscus and articular cartilage were evaluated at 4, 8, and 12 weeks. Achilles tendon from LacZ-transgenic rats was transplanted into the meniscal defect in wild-type rats, and vice versa.
Results
Injection of BMP-7 into the rat Achilles tendon induced the fibrochondrocyte differentiation of tendon cells and changed the collagen gene profile of tendon tissue to more closely approximate meniscal tissue. Transplantation of the rat Achilles tendon into a meniscal defect increased meniscal size. The rats that received the tendon treated with BMP-7 had a meniscus matrix that exhibited increased Safranin O and type II collagen staining, and showed a delay in articular cartilage degradation. Using LacZ-transgenic rats, we determined that the regeneration of the meniscus resulted from contribution from both donor and host cells.
Conclusion
Our findings indicate that BMP-7 induces ectopic cartilage formation in rat tendons. Transplantation of Achilles tendon treated with BMP-7 promotes meniscus regeneration and prevents cartilage degeneration in a rat model of massive meniscal defect. Native cells in the rat Achilles tendon contribute to meniscal regeneration.
doi:10.1002/art.38099
PMCID: PMC4034586  PMID: 23897174

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