Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcRγ-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type-I interferon (IFN) in response to viral infection or self nucleic acids. Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8), also called CMRF-35-like molecule-6 (CLM-6), is a putative activating receptor among mouse LMIR/CLM/CD300 members; however, the expression and function of LMIR8 remain unclear. Here, we characterize mouse LMIR8 as a pDC receptor. Analysis of Flag-tagged LMIR8-transduced bone marrow (BM)-derived mast cells demonstrated that LMIR8 can transmit an activating signal by interacting with immunoreceptor tyrosine-based activating motif (ITAM)-containing FcRγ. Flow cytometric analysis using a specific antibody for LMIR8 showed that LMIR8 expression was restricted to mouse pDCs residing in BM, spleen, or lymph node. FcRγ deficiency dampened surface expression of LMIR8 in mouse pDCs. Notably, LMIR8 was detected only in pDCs, irrespective of TLR9 stimulation, suggesting that LMIR8 is a suitable marker for pDCs in mouse tissues; LMIR8 is weakly expressed in Flt3 ligand-induced BM-derived pDCs (BMpDCs). Crosslinking of transduced LMIR8 in BMpDCs with anti-LMIR8 antibody did not induce IFN-α production, but rather suppressed TLR9-mediated production of IFN-α. Taken together, these observations indicate that LMIR8 is an FcRγ-coupled receptor selectively expressed in mouse tissue pDCs, which might suppress pDC activation through the recognition of its ligands.

The CD300e molecule in mice is an immune-activating receptor.

CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.

Disrupting ceramide-CD300f interaction prevents septic peritonitis by stimulating neutrophil recruitment.

Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f -/- mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f -/- mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.

Role of PU.1 in MHC Class II Expression via CIITA Transcription in Plasmacytoid Dendritic Cells.

The cofactor CIITA is a master regulator of MHC class II expression and several transcription factors regulating the cell type-specific expression of CIITA have been identified. Although the MHC class II expression in plasmacytoid dendritic cells (pDCs) is also mediated by CIITA, the transcription factors involved in the CIITA expression in pDCs are largely unknown. In the present study, we analyzed the role of a hematopoietic lineage-specific transcription factor, PU.1, in CIITA transcription in pDCs. The introduction of PU.1 siRNA into mouse pDCs and a human pDC cell line, CAL-1, reduced the mRNA levels of MHC class II and CIITA. When the binding of PU.1 to the 3rd promoter of CIITA (pIII) in CAL-1 and mouse pDCs was analyzed by a chromatin immunoprecipitation assay, a significant amount of PU.1 binding to the pIII was detected, which was definitely decreased in PU.1 siRNA-transfected cells. Reporter assays showed that PU.1 knockdown reduced the pIII promoter activity and that three Ets-motifs in the human pIII promoter were candidates of cis-enhancing elements. By electrophoretic mobility shift assays, it was confirmed that two Ets-motifs, GGAA (-181/-178) and AGAA (-114/-111), among three candidates, were directly bound with PU.1. When mouse pDCs and CAL-1 cells were stimulated by GM-CSF, mRNA levels of PU.1, pIII-driven CIITA, total CIITA, MHC class II, and the amount of PU.1 binding to pIII were significantly increased. The GM-CSF-mediated up-regulation of these mRNAs was canceled in PU.1 siRNA-introduced cells. Taking these results together, we conclude that PU.1 transactivates the pIII through direct binding to Ets-motifs in the promoter in pDCs.

IL-33 promotes MHC class II expression in murine mast cells.

Mast cells (MCs), recognized as tissue-resident cells of hematopoietic origin, are involved in cellular and pathological manifestations of allergic disorders including atopic dermatitis. IL-33, a member of the IL-1 cytokine family, activates Th2-type immune responses, and promotes the degranulation and maturation of MCs. However, it is uncertain whether IL-33 treatment induces mature mast cells to acquire the characteristics of the monocyte-dendritic cell lineage.We investigated the effect of IL-33 on the MHC class II expression and function of murine mast cells. IL-33-treated mature murine bone marrow-derived mast cells (BMMCs) were analyzed by FACS, real-time PCR, chromatin immunoprecipitation (ChIP) assay, and Western blotting. The morphology and degranulation activity of BMMCs and T-cell activation by BMMCs were also examined. BMMCs treated with IL-33 for 10 days induced cell surface expression of the MHC class II protein, whereas the expression of FcεRI and c-kit was not affected by IL-33. The expression of CIITA, driven from pIII and pIV, was up-regulated in IL-33-treated BMMCs. The amount of PU.1 mRNA and protein significantly increased in IL-33-treated BMMCs. The ChIP assay showed PU.1 binding to CIITA pIII, and enhanced histone acetylation due to IL-33 treatment. Syngeneic T cells were activated by co-culture with IL-33-treated BMMCs, although the expression of the co-stimulatory molecules, CD40, CD80, CD86, and PDL-1, was not detected. Mast cells express MHC class II after prolonged exposure to IL-33, probably due to enhanced recruitment of PU.1 to CIITA pIII, resulting in transactivation of CIITA and MHC class II. IL-33 is an important cytokine in allergic disorders. Mast cells have the ability to express MHC class II after prolonged exposure to IL-33 in a murine model. IL-33 holds a key to understanding the etiology of atopic dermatitis.

The Transcription Factor Ehf Is Involved in TGF-ß-Induced Suppression of FcεRI and c-Kit Expression and FcεRI-Mediated Activation in Mast Cells.

FcεRI, which is composed of α, ß, and γ subunits, plays an important role in IgE-mediated allergic responses. TGF-ß1 has been reported to suppress FcεRI and stem cell factor receptor c-Kit expression on mast cell surfaces and to suppress mast cell activation induced by cross-linking of FcεRI. However, the molecular mechanism by which these expressions and activation are suppressed by TGF-ß1 remains unclear. In this study, we found that the expression of Ets homologous factor (Ehf), a member of the Ets family transcriptional factors, is upregulated by TGF-ß/Smad signaling in mouse bone marrow-derived mast cells (BMMCs). Forced expression of Ehf in BMMCs repressed the transcription of genes encoding FcεRIα, FcεRIß, and c-Kit, resulting in a reduction in cell surface FcεRI and c-Kit expression. Additionally, forced expression of Ehf suppressed FcεRI-mediated degranulation and cytokine production. Ehf inhibited the promoter activity of genes encoding FcεRIα, FcεRIß, and c-Kit by binding to these gene promoters. Furthermore, the mRNA levels of Gata1, Gata2, and Stat5b were lower in BMMCs stably expressing Ehf compared with control cells. Because GATA-1 and GATA-2 are positive regulators of FcεRI and c-Kit expression, decreased expression of GATAs may be also involved in the reduction of FcεRI and c-Kit expression. Decreased expression of Stat5 may contribute to the suppression of cytokine production by BMMCs. In part, mast cell response to TGF-ß1 was mimicked by forced expression of Ehf, suggesting that TGF-ß1 suppresses FcεRI and c-Kit expression and suppresses FcεRI-mediated activation through upregulation of Ehf.

Involvement of PU.1 in NFATc1 promoter function in osteoclast development.

BACKGROUND: The transcription factors NFATc1 and PU.1 play important roles in osteoclast development. NFATc1 and PU.1 transactivate osteoclast-specific gene expression and a deficiency in NFATc1 or PU.1 genes causes osteopetrosis due to an insufficient development of osteoclasts. However, the existence of cross-regulation between NFATc1 and PU.1 is largely unknown. In the present study, the role of PU.1 in NFATc1 expression was investigated. METHODS: Osteoclasts were generated from mouse bone marrow cells. PU.1 knockdown was performed with siRNA introduction. The mRNA levels in siRNA-introduced cells were determined by quantitative RT-PCR. The involvement of PU.1 in the NFATc1 promoter was analyzed by using a chromatin immunoprecipitation (ChIP) assay and a reporter assay. Retrovirus vector was used for enforced expression of PU.1. RESULTS: Introduction of PU.1 siRNA into bone marrow-derived osteoclasts resulted in a decrease in NFATc1 mRNA level. A ChIP assay showed that PU.1 bound to the NFATc1 promoter in osteoclasts. NFATc1 promoter activity was reduced in PU.1 knockdown cells as assessed by a reporter assay. PU.1 siRNA introduction also downregulated the expression of osteoclast-specific genes and tartrate resistant acid phosphatase (TRAP) activity. Enforced expression of PU.1 using a retrovirus vector increased NFATc1 expression and TRAP activity. When NFATc1 expression was knocked down by using siRNA, the induction of osteoclast-specific genes and TRAP-positive cells was suppressed without affecting the expression level of PU.1. CONCLUSIONS: These results indicate that PU.1 is involved in osteoclast development by transactivating NFATc1 expression via direct binding to the NFATc1 promoter.

Notch signaling enhances FcεRI-mediated cytokine production by mast cells through direct and indirect mechanisms.

Th2-type cytokines and TNF-α secreted by activated mast cells upon cross-linking of FcεRI contribute to the development and maintenance of Th2 immunity to parasites and allergens. We have previously shown that cytokine secretion by mouse mast cells is enhanced by signaling through Notch receptors. In this study, we investigated the molecular mechanisms by which Notch signaling enhances mast cell cytokine production induced by FcεRI cross-linking. FcεRI-mediated production of cytokines, particularly IL-4, was significantly enhanced in mouse bone marrow-derived mast cells by priming with Notch ligands. Western blot analysis showed that Notch signaling augmented and prolonged FcεRI-mediated phosphorylation of MAPKs, mainly JNK and p38 MAPK, through suppression of the expression of SHIP-1, a master negative regulator of FcεRI signaling, resulting in the enhanced production of multiple cytokines. The enhancing effect of Notch ligand priming on multiple cytokine production was abolished by knockdown of Notch2, but not Notch1, and FcεRI-mediated production of multiple cytokines was enhanced by retroviral transduction with the intracellular domain of Notch2. However, only IL-4 production was enhanced by both Notch1 and Notch2. The enhancing effect of Notch signaling on IL-4 production was lost in bone marrow-derived mast cells from mice lacking conserved noncoding sequence 2, which is located at the distal 3' element of the Il4 gene locus and contains Notch effector RBP-J binding sites. These results indicate that Notch2 signaling indirectly enhances the FcεRI-mediated production of multiple cytokines, and both Notch1 and Notch2 signaling directly enhances IL-4 production through the noncoding sequence 2 enhancer of the Il4 gene.

Critical Roles for PU.1, GATA1, and GATA2 in the expression of human FcεRI on mast cells: PU.1 and GATA1 transactivate FCER1A, and GATA2 transactivates FCER1A and MS4A2.

The high-affinity IgE receptor, FcεRI, which is composed of α-, ß-, and γ-chains, plays an important role in IgE-mediated allergic responses. In the current study, involvement of the transcription factors, PU.1, GATA1, and GATA2, in the expression of FcεRI on human mast cells was investigated. Transfection of small interfering RNAs (siRNAs) against PU.1, GATA1, and GATA2 into the human mast cell line, LAD2, caused significant downregulation of cell surface expression of FcεRI. Quantification of the mRNA levels revealed that PU.1, GATA1, and GATA2 siRNAs suppressed the α transcript, whereas the amount of ß mRNA was reduced in only GATA2 siRNA transfectants. In contrast, γ mRNA levels were not affected by any of the knockdowns. Chromatin immunoprecipitation assay showed that significant amounts of PU.1, GATA1, and GATA2 bind to the promoter region of FCER1A (encoding FcεRIα) and that GATA2 binds to the promoter of MS4A2 (encoding FcεRIß). Luciferase assay and EMSA showed that GATA2 transactivates the MS4A2 promoter via direct binding. These knockdowns of transcription factors also suppressed the IgE-mediated degranulation activity of LAD2. Similarly, all three knockdowns suppressed FcεRI expression in primary mast cells, especially PU.1 siRNA and GATA2 siRNA, which target FcεRIα and FcεRIß, respectively. From these results, we conclude that PU.1 and GATA1 are involved in FcεRIα transcription through recruitment to its promoter, whereas GATA2 positively regulates FcεRIß transcription. Suppression of these transcription factors leads to downregulation of FcεRI expression and IgE-mediated degranulation activity. Our findings will contribute to the development of new therapeutic approaches for FcεRI-mediated allergic diseases.

Role of PU.1 in MHC class II expression through transcriptional regulation of class II transactivator pI in dendritic cells.

BACKGROUND: PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family. We hypothesized that PU.1 is involved in MHC class II expression in dendritic cells (DCs). OBJECTIVE: The role of PU.1 in MHC class II expression in DCs was analyzed. METHODS: Transcriptional regulation of the DC-specific pI promoter of the class II transactivator (CIITA) gene and subsequent MHC class II expression was investigated by using PU.1 small interfering RNA (siRNA) and reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays. RESULTS: PU.1 siRNA introduction suppressed MHC class II expression, allogeneic and syngeneic T-cell activation activities of bone marrow-derived DCs (BMDCs) with reduction of CIITA mRNA driven by the DC-specific promoter pI, and MHC class II mRNA. The chromatin immunoprecipitation assay showed constitutive binding of PU.1 to the pI region in BMDCs, whereas acetylation of histone H3 on pI was suppressed by LPS stimulation in parallel with shutdown of CIITA transcription. PU.1 transactivated the pI promoter through cis-elements at -47/-44 and -30/-27 in a reporter assay and to which PU.1 directly bound in an electrophoretic mobility shift assay. Acetylation of histones H3 and H4 on pI was reduced in PU.1 siRNA-introduced BMDCs. Knockdown of interferon regulatory factor 4 or 8, which is a heterodimer partner of PU.1, by siRNA did not affect pI-driven CIITA transcription or MHC class II expression. CONCLUSION: PU.1 basally transactivates the CIITA pI promoter in DCs by functioning as a monomeric transcription factor and by affecting histone modification, resulting in the subsequent expression and function of MHC class II.

Critical role of transcription factor PU.1 in the expression of CD80 and CD86 on dendritic cells.

In this study, we investigated the role of a transcription factor, PU.1, in the regulation of CD80 and CD86 expression in dendritic cells (DCs). A chromatin immunoprecipitation assay revealed that PU.1 is constitutively bound to the CD80 and CD86 promoters in bone marrow-derived DCs. In addition, co-expression of PU.1 resulted in the transactivation of the CD80 and CD86 promoters in a reporter assay. The binding of PU.1 to cis-enhancing regions was confirmed by electromobility gel-shift assay. As expected, inhibition of PU.1 expression by short interfering RNA (siRNA) in bone marrow-derived DCs resulted in marked down-regulation of CD80 and CD86 expression. Moreover, overexpression of PU.1 in murine bone marrow-derived lineage-negative cells induced the expression of CD80 and CD86 in the absence of monocyte/DC-related growth factors and/or cytokines. Based on these results, we conclude that PU.1 is a critical factor for the expression of CD80 and CD86. We also found that subcutaneous injection of PU.1 siRNA or topical application of a cream-emulsified PU.1 siRNA efficiently inhibited murine contact hypersensitivity. Our results suggest that PU.1 is a potential target for the treatment of immune-related diseases.

Suppressive effects of transcription factor GATA-1 on cell type-specific gene expression in dendritic cells.

To evaluate the effects of the transcription factor GATA-1 on determining cell fate between dendritic cell (DC) and mast cell (MC) lineages, GATA-1 was exogenously expressed in bone marrow-derived (BM) DCs. Exogenous expression of GATA-1 by a retrovirus in BMDCs inhibited expression of CD11c, CD80, CD86, and major histocompatibility complex class II with suppression of antigen-presenting ability and morphological changes toward granulocyte-like cells. Transcription of MC proteases and c-kit was markedly upregulated by GATA-1. Expression of IRF-4 and -8 was markedly suppressed, whereas PU.1 mRNA level was not affected by GATA-1. Chromatin immunoprecipitation assay showed that recruitment of PU.1 on the IRF-8 promoter was reduced in GATA-1-expressing DCs. These results indicate that GATA-1 suppresses PU.1 function but not PU.1 transcription. Thus, GATA-1 appears to determine cell fate by regulating several cell-specific transcription factors.

Opposite effects of Trichostatin A on activation of mast cells by different stimulants.

Mast cells (MCs) are activated upon stimulation via TLRs or FcepsilonRI, contributing to immune protection and/or leading to allergic diseases. In the present study, the effects of Trichostatin A (TSA) on the activation of MCs were analyzed with bone marrow-derived (BM) MCs. TSA increased the transcription and protein secretion of IL-6 in case of LPS-stimulation, in contrast to the suppressive effect on IgE-mediated activation of BMMCs. Chromatin immunoprecipitation assay showed IgE-mediated signaling-specific suppression of transcription factors recruitment to the IL-6 promoter. TSA-treatment inhibited nuclear translocation of NF-kappaB following IgE-mediated, but not LPS-induced activation in MCs.

Involvement of PU.1 in the transcriptional regulation of TNF-alpha.

PU.1 is a myeloid- and lymphoid-specific transcription factor that serves many important roles in the development and specific gene regulation of hematopoietic lineages. Mast cells (MC) and dendritic cells (DC) express PU.1 at low and high levels, respectively. Previously, we found that enforced expression of PU.1 in MC resulted in acquisition of DC-like characteristics, including repression of several IgE-mediated responses due to reduced expression of IgE-signaling related molecules. In contrast, PU.1 overexpression in MC up-regulated TNF-alpha production in response to IgE- and LPS-stimulation suggesting that PU.1 positively regulates TNF-alpha expression. However, the role of PU.1 in the expression of TNF-alpha is largely unknown. In the present study, the effects of PU.1 on the TNF-alpha promoter in mouse bone marrow-derived (BM) MC and DC were studied. Real-time PCR, ELISA, and chromatin immunoprecipitation assays indicated that the kinetics and magnitude of TNF-alpha expression levels following LPS- or IgE-stimulation are related to the amount of PU.1 binding to the promoter. In brief, higher and delayed up-regulation of TNF-alpha promoter function was observed in DC, whereas there were lower and rapid responses in MC. When PU.1-overexpressing retrovirus vector was introduced into MC, the amount of PU.1 recruited to the TNF-alpha promoter markedly increased. The knockdown of PU.1 in BMDC by siRNA resulted in a reduction of TNF-alpha protein produced from LPS-stimulated BMDC. These observations indicate that PU.1 transactivates the TNF-alpha promoter and that the amount of PU.1 binding on the promoter is associated with promoter activity.

Roles of PU.1 in monocyte- and mast cell-specific gene regulation: PU.1 transactivates CIITA pIV in cooperation with IFN-gamma.

Over-expression of PU.1, a myeloid- and lymphoid-specific transcription factor belonging to the Ets family, induces monocyte-specific gene expression in mast cells. However, the effects of PU.1 on each target gene and the involvement of cytokine signaling in PU.1-mediated gene expression are largely unknown. In the present study, PU.1 was over-expressed in two different types of bone marrow-derived cultured mast cells (BMMCs): BMMCs cultured with IL-3 plus stem cell factor (SCF) and BMMCs cultured with pokeweed mitogen-stimulated spleen-conditioned medium (PWM-SCM). PU.1 over-expression induced expression of MHC class II, CD11b, CD11c and F4/80 on PWM-SCM-cultured BMMCs, whereas IL-3/SCF-cultured BMMCs expressed CD11b and F4/80, but not MHC class II or CD11c. When IFN-gamma was added to the IL-3/SCF-based medium, PU.1 transfectant acquired MHC class II expression, which was abolished by antibody neutralization or in Ifngr(-/-) BMMCs, through the induction of expression of the MHC class II transactivator, CIITA. Real-time PCR detected CIITA mRNA driven by the fourth promoter, pIV, and chromatin immunoprecipitation indicated direct binding of PU.1 to pIV in PU.1-over-expressing BMMCs. PU.1-over-expressing cells showed a marked increase in IL-6 production in response to LPS stimulation in both IL-3/SCF and PWM-SCM cultures. These results suggest that PU.1 overproduction alone is sufficient for both expression of CD11b and F4/80 and for amplification of LPS-induced IL-6 production. However, IFN-gamma stimulation is essential for PU.1-mediated transactivation of CIITA pIV. Reduced expression of mast cell-related molecules and transcription factors GATA-1/2 and up-regulation of C/EBPalpha in PU.1 transfectants indicate that enforced PU.1 suppresses mast cell-specific gene expression through these transcription factors.

Notch signaling confers antigen-presenting cell functions on mast cells.

BACKGROUND: Notch signaling is involved in cell fate determination along with the development of the immune system. However, very little is known about the role for Notch signaling in mast cells. OBJECTIVE: We investigated the role of Notch signaling in mast cell functions. METHODS: After mouse bone marrow-derived mast cells (BMMCs) or peritoneal mast cells (PMCs) were cocultured with mouse Notch ligand-expressing chinese hamster ovary cells for 5 days, we examined the mast cell surface expressions of MHC-II molecules and OX40 ligand (OX40L), Fc epsilon RI-mediated cytokine production, and the effects of the mast cells on proliferation and differentiation of naive CD4(+) T cells in vitro. RESULTS: We showed that BMMCs and PMCs constitutively expressed Notch1 and Notch2 proteins on the cell surface. We also found that Delta-like 1 (Dll1)/Notch signaling induced the expression of MHC-II and upregulated the expression level of OX40L on the surface of the mast cells. Dll1/Notch signaling augmented Fc epsilon RI-mediated IL-4, IL-6, IL-13, and TNF production by BMMCs. Dll1-stimulated MHC-II(+)OX40L(high) BMMCs promoted proliferation of naive CD4(+) T cells and their differentiation into T(H)2 cells producing IL-4, IL-5, IL-10, and IL-13. CONCLUSION: Dll1/Notch signaling confers the functions as an antigen-presenting cell on mast cells, which preferentially induce the differentiation of T(H)2.

Opposite effects of PU.1 on mast cell stimulation.

An Ets-family transcription factor PU.1 is involved in the development and specific gene regulation of hematopoietic cells. PU.1 also determines the commitment between several lineages via its expression level. Although enforced expression of PU.1 in mast cells (MC) induced expression of monocyte-specific markers and morphological change from MC to monocytes, especially dendritic cells (DC), in the previous report, intracellular events caused by PU.1 are largely unknown. In the present study, effect of PU.1 on IgE- and LPS-mediated stimulation degrees was analyzed. The amounts of IL-6, IL-13, and TNF-alpha produced from LPS-stimulated MC were markedly increased by overexpression of PU.1. In contrast, IL-6 and IL-13 production levels in response to IgE were reduced by PU.1, whereas that of TNF-alpha was up-regulated. beta-Hexosaminidase release as a means of degranulation was decreased in PU.1 transfectants. When eicosanoid generation in response to IgE-stimulation was analyzed, overexpression of PU.1 reduced leukotriene C(4) (LTC(4)) release, but enhanced PGD(2) production. Microarray analysis suggested that expression of FcepsilonRI signal pathway related molecules were suppressed in PU.1 overexpressing MC as well as DC. These observations indicate that up-regulation of PU.1 suppresses expression of FcepsilonRI signal transduction-related intracellular molecules, but increases the potential of transcription activity of monocyte characters.

Procyanidin C1 from apple extracts inhibits Fc epsilon RI-mediated mast cell activation.

BACKGROUND: Polyphenol-enriched fractions, which are extracted from unripe apples (Rosaceae, Malus spp.), consisting of procyanidins (polymers of catechins) are known to have an anti-allergenic effect on patients with various allergic diseases. Although it has been reported that apple extracts inhibit histamine release from mast cells, the molecular mechanisms for this anti-allergenic effect are not well understood. To elucidate the molecular mechanisms by which apple extracts induce their anti-allergenic effects, the effects of purified apple extract components on high-affinity receptors for IgE (Fc epsilon RI)-mediated mast cell activation were investigated. METHODS: The anti-allergic effect of oral administration of apple procyanidin extracts on passive cutaneous anaphylactic responses of BALB/c mice was assessed. We evaluated the effects of procyanidin C1 (PC1) [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin], a component of the procyanidin fraction, on mouse bone-marrow-derived mast cell degranulation, cytokine production, protein tyrosine phosphorylation and on the generation of intracellular reactive oxygen species (ROS) of cells stimulated by Fc epsilon RI cross-linking in vitro. RESULTS: In an in vivo study, oral administration of the procyanidin fraction suppressed the mast-cell-dependent allergic reaction. In in vitro studies, PC1 dose-dependently decreased Fc epsilon RI-mediated degranulation and cytokine production of mast cells. Furthermore, PC1 inhibited tyrosine phosphorylation of Syk and linker for activation of T cells, and the ROS generation in stimulated mast cells. CONCLUSIONS: PC1 suppresses Fc epsilon RI-mediated mast cell activation by inhibiting intracellular signaling pathways. These observations provide evidence for the anti-allergenic effects of the procyanidin-enriched apple extract.