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. (Blood. 2011; 117(7):2211-2222) IntroductionT-cell initiation requires 2 signals from antigen-presenting cells (APCs). The first signal comes from ligation of the T-cell receptor and the major histocompatibility complex (MHC)/antigen presented on the surface of APCs, and the second signal is via additional costimulatory molecules, including the interaction between the CD28 family on T cells and B7, eg, CD80 (B7-1) and CD86 (B7-2), expressed on the APC. 1-3 CD80 and CD86 are members of the immunoglobulin supergene family encoded by separate genes, and are expressed on dendritic cells (DCs) or up-regulated on activated macrophages/monocytes, B cells, and activated T cells. 1-6 CD80 or CD86 can provide costimulatory signals by engaging CD28 or CTLA4 (cytotoxic T-lymphocyte antigen 4; CD152) on T cells. The engagement of CD28 with CD80 or CD86 leads to multiple effects on the immune response, including T-cell activation and differentiation and tissue migration. [7][8][9] In contrast to CD28, the outcome of CTLA4 engagement on T cells is to suppress proliferation by transmitting an inhibitory signal. 10 In addition, a subset of T cells with potent immunoregulatory properties, CD4 ϩ CD25 ϩ regulatory T cells, constitutively expresses CTLA4. 11,12 Thus, interactions between CTLA4 and CD80 or CD86 provides an immunosuppressive function in modulating T-cell proliferation and plays a role in immune tolerance. Based on these observations, the regulated expression of costimulatory molecules is critical for immune function. Therefore, revealing the molecular mechanisms of gene expression of costimulatory molecules is essential for an understanding of the regulation of T cell-mediated immune responses.Despite the importance of CD80 and CD86, the critical transcription factor for gene expression of CD80 or CD86 is sti...

Cell-type-specific transcription of mouse high-affinity IgE receptor (Fc⑀RI) ␤-chain is positively regulated by the transcription factor GATA-1. Although GATA-1 is expressed in erythroid cells, megakaryocytes, and mast cells, the expression of mouse Fc⑀RI ␤-chain is restricted to mast cells. In the present study, we characterized the role of GATA-associated cofactor FOG-1 in the regulation of the Fc⑀RI ␤-chain promoter. The expression levels of FOG-1, GATA-1, and ␤-chain in each hematopoietic cell line were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. IntroductionThe high-affinity IgE receptor Fc⑀RI is composed of an ␣-chain, a ␤-chain, and a ␥-chain. Allergen-IgE antibody complex-induced cross-linking of Fc⑀RI results in activation of mast cells, which subsequently secrete various chemical mediators that induce the symptoms of an allergic response.In humans, Fc⑀RI is expressed as a tetramer (␣␤␥ 2 ) on mast cells and basophils and as a trimer (␣␥ 2 ) on Langerhans cells, monocytes, and dendritic cells. [1][2][3][4][5] Thus, while ␤-chain may facilitate cell-surface expression of Fc⑀RI, 6 it is not necessarily required for cell-surface expression of human Fc⑀RI. By contrast, in mice, Fc⑀RI is expressed as a tetramer (␣␤␥ 2 ) only on mast cells and basophils, and the ␤-chain is necessary for cell-surface expression of mouse Fc⑀RI 7 and acts as an amplifier for Fc⑀RI signaling by increasing phosphorylation of the ␥-chain 8 and by enhancing signaling protein recruitment. 9 Therefore, characterization of the mechanisms of mouse ␤-chain expression is critical for the understanding of mast-cell-and basophil-specific transcriptional regulatory systems.We previously reported that the transcription factor GATA-1 positively regulated cell-type-specific ␤-chain expression via 4 GATA motifs in the promoter. 10 GATA-1 mediates the maturation of various cell lineages, including erythroid cells, megakaryocytes, eosinophils, basophils, and mast cells. [11][12][13] However, the expression of mouse ␤-chain is limited to mast cells but not observed in other GATA-1-positive cell lineages. Thus, other factors may regulate cell-type-specific transcription of the ␤-chain.A zinc finger cofactor, FOG-1, interacts with GATA-1 and can either enhance or repress GATA-1-dependent gene expression. 14-17 FOG-1 is abundantly expressed in erythroid cells and in megakaryocytes, where it regulates growth and differentiation. Erythroid and megakaryocyte lineage development is arrested at proerythroblast stage in Gata-1 Ϫ/Ϫ or Fog-1 Ϫ/Ϫ mice, 18,19 and FOG-1 mutants that lack GATA-binding activity result in abnormal differentiation of megakaryotic cells. 19,20 Recent studies have implicated abnormalities in GATA-1 and FOG-1 in various human diseases, including thrombocytopenia and idiopathic myelofibrosis (IM). [21][22][23] Thus, the goal of the present study was to characterize the role of GATA-associated cofactor FOG-1 in the regulation of the Fc⑀RI ␤-chain promoter. Materials and methods Cell culture...

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The α-chain is a specific component of FcεRI, which is essential for the cell surface expression of FcεRI and the binding of IgE. Recently, two single nucleotide polymorphisms (SNPs) in the α-chain promoter, −315C>T and −66T>C, have been shown by statistic studies to associate with allergic diseases. The effect of −66 SNP on GATA-1-mediated promoter activity has been already indicated. In the present study, to investigate roles of the −315 SNP on the α-chain promoter functions, the transcription activity was evaluated by reporter assay. The α-chain promoter carrying −315T (minor allele) possessed significantly higher transcriptional activity than that of −315C (major allele). EMSA indicated that the transcription factor Sp1, but not Myc-associated zinc finger protein (MAZ), was bound to the −315C allele probe and that a transcription factor belonging to a high mobility group-family bound to the −315T allele probe. The chromatin immunoprecipitation assay suggested that high mobility group 1, 2, and Sp1 bound around −315 of FcεRIα genomic DNA in vivo in the human basophil cell line KU812 with −315C/T and in human peripheral blood basophils with −315C/C, respectively. When cell surface expression level of FcεRI on basophils was analyzed by flow cytometry, basophils from individuals carrying −315T allele expressed significantly higher amount of FcεRI compared with those of −315C/C. The findings demonstrate that a −315 SNP significantly affects human FcεRI α-chain promoter activity and expression level of FcεRI on basophils by binding different transcription factors to the SNP site.

Interleukin-12 (IL-12), a heterodimeric cytokine (p35/p40) produced mainly from macrophages and dendritic cells, is an important regulator of T-helper 1 cell responses and for host defense. We found that interferon (IFN) consensus sequence binding protein (ICSBP), which is a transcription factor essential for the expression of p40, was expressed in mouse bone marrow-derived mast cells (BMMCs). The transcription levels of p35 and p40 were increased by stimulation of BMMCs with IFN-␥/lipopolysaccharide (LPS). IL-12 was secreted from IntroductionInterleukin-12 (IL-12) is a cytokine that governs production of interferon-gamma (IFN-␥) in natural killer (NK) cells and CD4 ϩ T cells 1,2 and that is involved in the induction and maintenance of T-helper 1 (Th1) cells. [3][4][5] IL-12 also plays important roles in resistance against various infectious agents including viruses, bacteria, and parasites. 6,7 The deficiency of IL-12 in knock-out mice causes a severely depressed Th1 response, supporting the role of IL-12 in the Th1 response and resistance to infections. 3,8,9 IL-12 (p70) is a heterodimer composed of 2 subunits, p40 and p35, which are encoded by 2 separate genes. 10 In many cell types, p35 expression is induced by stimulation with pathogens or their components, such as Gram-negative bacterial lipopolysaccharide (LPS). 11 p40 expression is also induced by stimulation of LPS, but is limited to macrophages, dendritic cells, B cells, and neutrophils, among which the first 2 cells are the primary sources of [12][13][14] The p40 protein also forms a heterodimer with a p35-related protein p19. 15 The heterodimer p19/p40, known as IL-23, a member cytokine of the IL-12 family, also regulates Th1-cell responses. [16][17][18] Thus, the p40 protein is considered to be an IL-12-specific subunit required for the functional expression of IL-12.IFN consensus sequence binding protein (ICSBP), also designated IFN regulatory factor 8 (IRF-8), is a nuclear transcription factor belonging to the IRF family. ICSBP Ϫ/Ϫ mice cannot show Th1-mediated responses because of a deficiency in IL-12 production. [19][20][21] Several previous studies indicate that ICSBP binds to the Ets site of the p40 promoter to up-regulate promoter activity in response to IFN-␥ and LPS stimulation. [22][23][24] We have analyzed the regulation mechanisms of hematopoietic cell development by transcription factors and found (1) cooperation between transcription factors PU.1 and GATA-1, and (2) repression of GATA-1-dependent transactivation by cofactor FOG-1, which causes mast cell-specific gene expression. [25][26][27][28] In addition, the expression level of PU.1 determines the fate of cell differentiation between mast cells and monocytes. [29][30][31] Although ICSBP is one of the partners of PU.1, the regulatory mechanism for the expression of ICSBP in mast cells and the involvement of ICSBP in the cell fate determination between mast cells and monocytes are unclear.Mast cells play roles not only as effector cells in allergic disease but also as initiator cel...

Two promoter polymorphisms of the high-affinity IgE receptor alpha-subunit (FcepsilonRIalpha) gene (FCER1A), -66T>C (rs2251746) and -315C>T (rs2427827), were analysed in Japanese atopic dermatitis subjects. Patients with the -315CT/TT genotype tended to have higher total serum IgE levels, while the proportion of -315CT/TT genotype or the -315T allele was significantly higher in those with highly elevated total serum IgE concentrations.

The ATP2A2 gene encodes Ca2+-dependent ATPase, the dysfunction of which causes Darier disease. In this study, we analyzed the promoter structure of the human ATP2A2 gene using primary normal human keratinocytes (NHK). Reporter assays showed that deletion of -550/-529, -488/-472, -390/-362, or -42/-21 resulted in a significant decrease in human ATP2A2 promoter activity. Electrophoretic mobility shift assay (EMSA) showed that Sp1 is a transcription factor that binds to the -550/-529 and -488/-472 regions of the promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that Sp1, but not Sp3, binds to the promoter region of the ATP2A2 gene in NHK cells in vivo. Knockdown of Sp1 expression by small interfering RNA resulted in a marked reduction in ATP2A2 promoter activity and ATP2A2 mRNA levels in NHK, suggesting that Sp1 positively transactivates the ATP2A2 promoter in NHK. This is early evidence demonstrating that Sp1 plays an important and positive role in ATP2A2 gene expression in NHK in vivo and in vitro.

Cannabinoids have emerged as powerful drug candidates for the treatment of inflammatory and autoimmune diseases due to their immunosuppressive properties. While significant clinical and experimental data on the use of cannabinoids as anti-inflammatory agents exist in many autoimmune disease settings, virtually no studies have been performed on their potential role in transplant rejection. Here we suggest a theoretical role for the use of cannabinoids in preventing allograft rejection. While the psychotropic properties of CB1 agonists limit their clinical use, CB2 agonists may offer a new avenue to selectively target immune cells involved in allograft rejection. Moreover, development of mixed CB1/CB2 agonists that cannot cross the blood-brain barrier may help prevent their undesired psychotropic properties. In addition, manipulation of endocannabinoids in vivo by activating their biosynthesis and inhibiting cellular uptake and metabolism may offer yet another pathway to regulate immune response during allograft rejection.