bluuebunny/crossref_metadata_2025_split huggingface dataset:
NF-κB-mediated transcriptional regulation of human β-defensin-2 gene following lipopolysaccharide stimulation
Authors: Tsutsumi-Ishii Yuko , Nagaoka Isao 
Published: September 2022

Abstractβ-Defensins are cationic peptides with broad-spectrum antimicrobialactivities that contribute to innate host defense. Among humanβ-defensins (hBDs), hBD-2 is up-regulated in epithelial tissues andmononuclear phagocytes in response to bacterial infection andproinflammatory cytokines. However, little is known about the molecularmechanism of hBD-2 gene regulation. Here, we investigatedlipopolysaccharide (LPS)-mediated transcriptional regulation of thehBD-2 gene by focusing on the roles of NF-κB, STAT, and NF-IL-6 sitesin mononuclear phagocytes using RAW264.7 cells, which are sensitive to LPS. Luciferase reporter analyses demonstrated that two NF-κB siteswere essential for full LPS responsiveness of the hBD-2 gene. Further, both NF-κB sites were also crucial for basal transcriptionalactivity. In contrast, neither the NF-IL-6 nor STAT binding site wasrequired for LPS-induced hBD-2 transcription. Electrophoretic mobilityshift assay indicated that in unstimulated cells, NF-κB p50 homodimerbound to both NF-κB sites, whereas the p65-p50 heterodimer formedcomplexes with these sites following LPS stimulation. Together, theseobservations indicate that NF-κB plays an important role in theregulation of hBD-2 gene expression in response to LPS.
AP-1 is essential for p67phoxpromoter activity
Authors: Gauss Katherine A , Bunger Peggy L , Quinn Mark T 
Published: September 2022

AbstractThe cytosolic NADPH oxidase cofactor p67phoxhas been shown to be one of the limiting factors in assembly andactivation of this multi-protein enzyme complex and, therefore, must behighly regulated at the transcriptional level. In the present studies, we have further characterized the promoter for humanp67phox. Genomic sequence upstream of thetranslational start site (TLS; 2 kb) was cloned, and RACE was used toidentify and compare the transcriptional start site (TSS) in twomyeloid cell lines, HL-60 and PLB-985. Two major TSS were identifiedwithin the first intron for both cell lines, and one transcriptisolated from PLB-985 cells started approximately 34 bp 5′ of exon 1and contained no intron 1 sequence. To identify regulatory regions ofthe promoter, a luciferase reporter was used to assay a series of promoter deletion constructs. The greatest transcriptional activity wasobserved for fragments containing at least 500 bp upstream of the TLS. Sequence analysis of the p67phox promoterrevealed consensus binding sites for previously described transcriptionfactors including AP-1 and PU.1. Site-directed mutagenesis of the AP-1site demonstrated that this site was essential for basal transcription. EMSA, competition, and super-shift assays showed that this site wasspecifically recognized by nuclear factors of the AP-1 family. EMSAanalysis and promoter-reporter assays with the PU.1 consensus sites atpositons -176, -283, and -328 demonstrate that PU.1 binds the site atposition -283 with high affinity. Mutagenesis of any one of the PU.1sites reduced the basal transcriptional activity by approximately 50%,demonstrating that, although none of these sites is singularlyresponsible for the basal transcriptional activity, all three sitesplay some role in the transcriptional activity of thep67phox promoter. In support of thisconclusion, mutagenesis of all three sites completely abrogatedtranscriptional activity.
Expression and functional characterization of CFTR in mast cells
Authors: Kulka M , Gilchrist M , Duszyk M , Befus A D 
Published: September 2022

AbstractMast cell activation requires Cl− flux, which maintains the driving force for entry of extracellular calcium and initiates release of mediators such as histamine. However, chloride channel expression in mast cells has been poorly understood. For the first time, reverse transcriptase-polymerase chain reaction shows that rat-cultured mast cells (RCMC) and peritoneal mast cells (PMC) contain mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR), an important chloride channel. Immunostaining with an anti-CFTR antibody indicates expression of CFTR in PMC and RCMC. Mast cell CFTR is a functional Cl− channel because it is capable of mediating Cl− flux in response to elevated cAMP. An inhibitor of CFTR-dependent Cl− flux, diphenylamine-2-carboxylate down-regulates mast cell mediator release. These results show that rat mast cells express a functional CFTR, which might be important in mediator release.
Mycobacterium aviuminfection of macrophages results in progressive suppression of interleukin-12 production in vitro and in vivo
Authors: Wagner Dirk , Sangari Felix J , Kim Sang , Petrofsky Mary , Bermudez Luiz E 
Published: September 2022

AbstractInterleukin-12 (IL-12) has been shown to have an important role in thehost defense against Mycobacterium avium. We sought todetermine if human monocyte-derived macrophages produce IL-12 uponM. avium infection. Although IL-12 can be measured insupernatants of M. avium-infected macrophages at 24, 48,and 72 h following infection, intracellular staining showed that24 to 48 h after infection, IL-12 was synthesized chiefly byuninfected macrophages in the monolayer, suggesting that M.avium infection inhibits IL-12 production. In addition, the dataalso suggest that the longer macrophage monolayers were infected, theless IL-12 they were able to produce. Stimulation of macrophages withIFN-γ prior to infection with M. avium resulted ingreater production of IL-12 compared with unstimulated macrophages. Culture supernatant of M. avium-infected macrophagemonolayers, but not control macrophages, partially inhibited IL-12production by IFN-γ-stimulated macrophages. This partial inhibitionwas not reversed by antiinterleukin-10 (anti-IL-10) andantitransforming growth factor β1 (anti-TGFβ1)-neutralizingantibodies. M. avium infection of macrophages in vitro alsosuppressed IL-12 synthesis induced by Listeriamonocytogenes infection. Immunohistochemistry staining of spleenof infected mice showed that IL-12 production by splenic macrophageswas more pronounced in the beginning of the infection but decreasedlater. Our data indicate that M. avium infection of macrophages suppresses IL-12 production by infected cells and that thesuppression was not a result of the presence of IL-10 and TGFβ1 inthe culture supernatant.
Regulation of gelatinase B in human monocytic and endothelial cells by PECAM-1 ligation and its modulation by interferon-beta
Authors: Nelissen Inge , Ronsse Isabelle , Van Damme Jo , Opdenakker Ghislain 
Published: September 2022

AbstractPlatelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) andgelatinase B are coexpressed at sites of inflammation, where an intenseinteraction occurs between leukocytes and endothelial cells. Toinvestigate whether a functional link exists between PECAM-1 activationand gelatinase B production, the regulatory role of PECAM-1, IFN-γ,IFN-β, LPS, and PMA on the production of gelatinase B (MMP-9) wasstudied in vitro in normal human umbilical vein endothelial cells(HUVECs), human peripheral blood mononuclear cells (PBMCs), and in ahuman monocytic leukemia cell line. In THP-1 cells, progelatinase Blevels were slightly up-regulated by immobilized PECAM-1-specificmonoclonal antibody (mAb) and soluble recombinant PECAM-1 when comparedwith strong induction by LPS and PMA. IFN-β inhibited the induced andbasal gelatinase B production but had no modulating effect on theexpression of PECAM-1. HUVECs mainly produced progelatinase A(proMMP-2). Treatment with LPS and triggering of the endothelial cellswith PECAM-1 mAb or recombinant PECAM-1 had no effect on gelatinase Aor B production, whereas PMA stimulated the production of progelatinaseB. IFN-β significantly up-regulated the expression of PECAM-1 in HUVECs but did not affect gelatinase secretion. Finally, in PBMCs, progelatinase B production was increased by soluble PECAM-1 mAb, recombinant PECAM-1, LPS, and PMA, whereas IFN-β reduced gelatinase Bsecretion. IFN-β did not alter PECAM-1 expression on PBMCs. Thus, PECAM-1 and gelatinase B are differently regulated in leukocytes andendothelial cells.
Cross-linking of GPI-80, a possible regulatory molecule of cell adhesion, induces up-regulation of CD11b/CD18 expression on neutrophil surfaces and shedding of L-selectin
Authors: Yoshitake Hiroshi , Takeda Yuji , Nitto Takeaki , Sendo Fujiro 
Published: September 2022

AbstractPreviously, we described a novel glycosylphosphatidyl inositol (GPI)-anchored glycoprotein (designated GPI-80) on human neutrophils and monocytes that may regulate β2 integrin-dependent neutrophil adherence and migration. However, the mechanism regulating β2 integrin remains to be clarified. To study this, we examined changes in β2 integrin expression and function caused by cross-linking GPI-80. GPI-80 cross-linking induced up-regulation of CD11b/CD18 (Mac-1) expression on neutrophil surfaces and shedding of L-selectin, which depends on tyrosine phosphorylation and cytoskeleton remodeling. Furthermore, the cross-linking enhanced fMLP-induced human neutrophil adherence. These results suggest that GPI-80 may be a regulator of β2 integrin in neutrophils.
Bacterial metabolite interference with maturation of human monocyte-derived dendritic cells
Authors: Säemann Marcus D , Parolini Ornella , Böhmig Georg A , Kelemen Peter , Krieger Peter-Michael , Neumüller Josef , Knarr Katharina , Kammlander Willibald , Hörl Walter H , Diakos Christos , Stuhlmeier Karl , Zlabinger Gerhard J 
Published: September 2022

AbstractDendritic cells (DC), the most potent APC, are central to antimicrobial immunity. Because of evolutionary pressure, it is reasonable that pathogens have evolved strategies to also subvert this host-defense mechanism. In the present study, we describe a novel way of bacterial interference with DC maturation. The bacterial metaboliten-butyrate, which occurs physiologically in high concentrations in the gastrointestinal tract and has well-known anti-inflammatory effects, is able to prevent LPS-induced maturation of DC resulting in a reduced capability to stimulate T cells. In particular, n-butyrate prevents homotypic DC clustering, inhibits IL-12 while sparing IL-10 production, and at the molecular level, blocks NF-κB translocation. These results demonstrate efficient targeting of DC function by a bacterial metabolite, which might explain the particular type of immune responsiveness in the presence of this bacterial agent as exemplified in the gastrointestinal tract.
Themicrophthalmiatranscription factor (MITF) contains two N-terminal domains required for transactivation of osteoclast target promoters and rescue ofmimutant osteoclasts
Authors: Mansky Kim C , Marfatia Kavita , Purdom Georgia H , Luchin Alex , Hume David A , Ostrowski Michael C 
Published: September 2022

AbstractThe microphthalmia transcription factor (MITF) regulates gene expression during differentiation of several distinct cell types, including osteoclasts. A structure/function analysis was performed to determine whether transcription activation domains were important for MITF action in osteoclasts. In addition to a previously characterized acidic activation necessary for melanocyte differentiation, the analysis defined a second potential activation domain located between amino acids 140 and 185. This second domain is required for MITF transactivation of two probable targets, the E-cadherin promoter and the tartrate-resistant acid phosphatase promoter, in transient transfection assays. An intact MITF gene rescued differentiation when introduced into osteoclasts derived from mi/mi mice using a retrovirus vector. In parallel experiments, an MITF gene lacking the acidic-activation domain rescued differentiation twofold less efficiently than wild type, and a gene lacking the region between amino acid residues 140 and 185 rescued differentiation tenfold less efficiently than wild type. The results indicate that the N-terminal region of MITF is necessary for activation of gene expression in osteoclasts and provides one mechanism by which this factor regulates distinct target genes in different cell types.
Cloning and sequencing of rabbit leukocyte NADPH oxidase genes reveals a unique p67phoxhomolog
Authors: Gauss Katherine A , Mascolo Patrice L , Siemsen Daniel W , Nelson Laura K , Bunger Peggy L , Pagano Patrick J , Quinn Mark T 
Published: September 2022

AbstractThe NADPH oxidase plays an important role in immune and nonimmune cell functions. Because rabbits represent an established model for studying a number of important disease processes that involve NADPH oxidase activity, we carried out studies to clone and sequence all five rabbit leukocyte NADPH oxidase genes. Comparison of the rabbit sequences with those of other species showed that, with the exception of p67phox, the rabbit phox proteins were highly conserved. In contrast, rabbit p67phox had a very divergent C-terminus and was 17 amino acids longer than any other known p67phox homolog. This was surprising, given the high degree of conservation among all of the phox proteins sequenced previously. To evaluate the functional consequences of this difference, wild-type rabbit p67phox and a mutated rabbit p67phox missing the C-terminal 17 amino acids were expressed and analyzed in a cell-free assay. Our results show that the full-length and truncated rabbit p67phox proteins were able to support oxidase activity, although the truncated form reproducibly supported a higher level of activity than full-length p67phox. These studies contribute to our understanding of the nature of the leukocyte NADPH oxidase in different species and will be valuable in future research using the rabbit model.
Molecular profiles of allograft rejection following inhibition of CD40 ligand costimulation differentiated by cluster analysis
Authors: Damrauer Scott M , DeFina Rachel , He Hongzhen , Haley Kathleen J , Perkins David L 
Published: September 2022

AbstractRecent technological advances in biomedical research, such as genomesequences and DNA microarrays, have dramatically increased the size of relevant databases. A major challenge is the extraction of a limitednumber of parameters from these databases that can differentiate anddiagnose complex biological states. In a model of cardiactransplantation investigating immunosuppression by inhibition of CD40ligand costimulation, we have applied a combination of clusteralgorithms and self-organizing maps to analyze a panel of 60 candidategenes. Dendrograms generated by cluster analysis distinguisheddifferent molecular bases of rejection. Using self-organizing maps, weidentified nine genes (CD4, CCR3, CCR5, LTβ, MIP-1α, MIP-2, CD8α,IP-10, and RANTES), each with a unique profile of transcriptionalexpression, that reproduce the differentiation of states of rejectionin dendrograms. Using histology and immunohistochemistry, we correlateddifferential regulation of CD4 and CD8 at the levels of mRNA andprotein. Our strategy of data reduction successfully decreased thenumber of genes to nine, which are sufficient to differentiate distinctstates of rejection in our experimental protocol.
Activation of the RON receptor tyrosine kinase protects murine macrophages from apoptotic death induced by bacterial lipopolysaccharide
Authors: Chen Yi-Qing , Zhou Yong-Qing , Wang Ming-Hai 
Published: September 2022

AbstractRON is a receptor tyrosine kinase activated by macrophage-stimulating protein. We demonstrate here that RON activation inhibits LPS-induced apoptosis of mouse peritoneal macrophages and Raw264.7 cells expressing RON or a constitutively active RON mutant. The antiapoptotic effect of RON was accompanied with the inhibition of LPS-induced production of nitric oxide (NO), a molecule responsible for LPS-induced cell apoptosis. This conclusion is supported by experiments using a chemical NO donor GSNO, in which RON activation directly blocked GSNO-induced apoptotic death of Raw264.7 cells and inhibited LPS-induced p53 accumulation. Furthermore, we showed that treatment of cells with wortmannin, which inhibits phosphatidylinositol (PI)-3 kinase, prevents the inhibitory effect of RON on LPS-induced macrophage apoptosis. These results were confirmed further by expression of a dominant inhibitory PI-3 kinase p85 subunit. These data suggest that by activating PI-3 kinase and inhibiting p53 accumulation, RON protects macrophage from apoptosis induced by LPS and NO. The antiapoptotic effect of RON might represent a novel mechanism for the survival of activated macrophages during inflammation.
Emerging evidence that molecules expressed by mammalian tissue grafts are recognized by the innate immune system
Authors: Fox-Marsh Annette , Harrison Leonard C 
Published: September 2022

AbstractThe innate immune system existed prior to the emergence of adaptive immunity in sharks and higher vertebrates. Homologues of many mammalian innate immune-system elements such as the toll-like receptors exist in species as distant as Drosophila. Selective pressure has led to the development of highly conserved, soluble, and cell-surface receptors that recognize functionally essential molecules shared by microbial pathogens. It is thought that molecular patterns that exquisitely distinguish pathogenic cells from mammalian cells are recognized. Therefore, it would seem unlikely that innate immune-system elements should recognize mammalian tissues. However, there is increasing evidence to suggest that this is the case and that innate immunity promotes rejection of transplanted mammalian tissues, particularly those from other species (xenografts). Evidence for innate recognition of mammalian grafts, the nature of this recognition, and the bi-directional interactions between innate and adaptive immunity that contribute to graft rejection are discussed in this review, with the emphasis on nonvascular xenografts.
Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro
Authors: Soilleux Elizabeth J , Morris Lesley S , Leslie George , Chehimi Jihed , Luo Qi , Levroney Ernest , Trowsdale John , Montaner Luis J , Doms Robert W , Weissman Drew , Coleman Nicholas , Lee Benhur 
Published: September 2022

AbstractDC-SIGN is a C-type lectin, highly expressed on the surface ofimmature dendritic cells (DCs), that mediates efficient infection of Tcells in trans by its ability to bind HIV-1, HIV-2, and SIV. Inaddition, the ability of DC-SIGN to bind adhesion molecules on surfacesof naïve T cells and endothelium also suggests its involvementin T-cell activation and DC trafficking. To gain further insights intothe range of expression and potential functions of DC-SIGN, weperformed a detailed analysis of DC-SIGN expression in adult and fetaltissues and also analyzed its regulated expression on cultured DCs andmacrophages. First, we show that DC-SIGN expression is restricted tosubsets of immature DCs in tissues and on specialized macrophages inthe placenta and lung. There were no overt differences between DC-SIGNexpression in adult and fetal tissues except that DC-SIGN expression inalveolar macrophages was only present after birth. Similarly, intissues, DC-SIGN was observed primarily on immature (CD83-negative)DCs. Secondly, in the peripheral blood, we found expression of DC-SIGNon a small subset of BDCA-2+ plasmacytoid DC precursors (pDC2),concordant with our finding of large numbers of DC-SIGN-positive cellsin allergic nasal polyps (previously shown to be infiltrated by DC2). Triple-label confocal microscopy indicated that DC-SIGN was colocalizedwith BDCA-2 and CD123 on DCs in nasal polyp tissue. Consistent withthis finding is our observation that DC-SIGN can be up-regulated onmonocyte-derived macrophages upon exposure to the Th2 cytokine, IL-13. In summary, our data demonstrate the relevant populations of DC andmacrophages that express DC-SIGN in vivo where it may impact theefficiency of virus infection and indicate that DC-SIGN expression maybe involved in the Th2 axis of immunity.
IL-12 suppresses the expression of ocular immunoinflammatory lesions by effects on angiogenesis
Authors: Lee Sujin , Zheng Mei , Deshpande Shilpa , Eo Seong Kug , Hamilton Thomas A , Rouse Barry T 
Published: September 2022

AbstractTopical application of plasmid DNA encoding IL-12 to the cornea of mice prior to ocular infection with Herpes simplex virus type 1 (HSV) results in diminished corneal immunoinflammatory lesions. Such herpetic stromal keratitis (HSK) reactions in humans represent an important cause of blindness. The effect of IL-12 pretreatment acted via inhibitory effects on corneal neovascularization rather than by inhibiting viral replication or the function of CD4+ T cells that mediate HSK. The antiangiogenesis induced by IL-12 DNA application was mediated indirectly via the cytokine IFN-γ and one or both of two chemokine molecules, IP-10 and MIG. Thus IL-12 DNA administration lacked modulatory effects on HSK in GKO mice, indicating the necessary involvement of IFN-γ induction for antiangiogenesis. In contrast, exposure of GKO mice to IP-10 DNA did suppress the severity of HSK. Furthermore, treatment with specific antisera to IP-10 and MIG in HSV-infected mice abrogated the IL-12-induced inhibitory effect on lesion severity. Taken together, our data indicate that the HSV-induced ocular immunoinflammatory lesions can be modulated by IL-12 and that this effect results from chemokine inhibition of angiogenesis. The use of antiangiogenesis therapy might represent a useful control measure against HSK.
Beryllium fluoride-induced cell proliferation: a process requiring P21ras-dependent activated signal transduction and NF-κB-dependent gene regulation
Authors: Misra Uma Kant , Gawdi Govind , Pizzo Salvatore Vincent 
Published: September 2022

AbstractWe studied the effect of beryllium fluoride on murine peritoneal macrophages and determined its effects on signal transduction and genetic regulation. At low concentration (1–5 nM), BeF2 caused an approximate twofold increase in [3H]thymidine uptake and cell number, but above 5 nM, it showed cytotoxic effects. BeF2 increased cellular inositol (1,4,5)trisphosphate (IP3) and [Ca2+]i about twofold. The rise in [Ca2+]i occurred consequent to release from IP3-sensitive Ca2+ stores and from influx, mainly via L-type channels. A significant increase in the levels of MEK1, ERK1, p38 MAPK, and JNK phosphorylation was observed in BeF2-exposed macrophages. The levels of NF-κB and CREB transcription factors and the proto-oncogenesc-fos and c-myc were also elevated significantly. Intracellular Ca2+ chelation blocked the effect of BeF2. We conclude that BeF2 at low concentration exerts its mitogenic effects in peritoneal macrophages by elevating [Ca2+]i, which triggers the activation of p21ras-dependent MAPK signaling cascades.
Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced STAT5 activation and target-gene expression during human monocyte/macrophage differentiation
Authors: Lehtonen Anne , Matikainen Sampsa , Miettinen Minja , Julkunen Ilkka 
Published: September 2022

AbstractGM-CSF signals through JAK2 and STAT5 and stimulates the expression of STAT5 target genes, such as pim-1 and CIS. Analyzed by EMSA, GM-CSF stimulation led to much stronger STAT5 DNA-binding to pim-1 or CIS GAS elements in primary human monocytes compared with mature macrophages. Similarly, GM-CSF-induced expression of pim-1 and CIS mRNAs was much stronger in monocytes. These differencies were not a result of downregulation of the GM-CSF receptor system or STAT5 expression, because monocytes and macrophages readily expressed GM-CSF receptor, JAK2, STAT5A, and STAT5B mRNAs and proteins. Monocytes expressed significant amounts of truncated STAT5 forms that took part in STAT5-DNA complex formation in GM-CSF-stimulated monocytes. This resulted in faster moving STAT5 complexes compared with macrophages in EMSA. Our results demonstrate that STAT5 isoform expression, GM-CSF-induced STAT5 activation, and STAT5 target-gene expression are altered significantly during monocyte/macrophage differentiation.