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DC Cultures (dc + culture)
Selected AbstractsGeneration of self-renewing immature dendritic cells from mouse spleen that can take up mycobacteria and present antigens to T cellsAPMIS, Issue 10 2010RUBINA PAL Pal R, Marwaha S, Pepponi I, Mann JFS, Paul MJ, Reljic R. Generation of self-renewing immature dendritic cells from mouse spleen that can take up mycobacteria and present antigens to T cells. APMIS 2010; 118: 729,38. Dendritic cells (DC) play a key role in driving the adaptive immune response to Mycobacterium tuberculosis (MTB), the causative pathogen of tuberculosis (TB). However, studying these important yet very sparse immune cells in the context of MTB pathogenesis is severely restricted by the lack of suitable cell lines and the complexity of culturing of DC progenitors, usually obtained from the bone marrow. However, significant advances have been made towards generating long-term DC cultures from various lymphoid tissues. Here, we report the evidence for generating a long-term, self-renewing DC culture from the Balb/c mouse spleen. We demonstrate that these cells, termed IDC-3, have a myeloid DC origin, i.e. they are CD11c+CD11b++CD8-,,F4/80+/, and that they also display a phenotype MHC-II+CD16/32++CD80+/,CD86+, indicating that they are immature DC. Following incubation with Mycobacterium bovis BCG (Bacillus Calmette Guerin), the IDC-3 efficiently took up bacteria and acquired the morphology of mature DC. Importantly though, when IDC-3 were pre-stimulated with a mycobacterial antigen in vitro, they were able to induce proliferation of T lymphocytes from mice immunized with the same antigen. The T-cell stimulatory potential of IDC-3 was further enhanced when the cells were co-stimulated with an anti-CD40 mAb. We therefore suggest that the IDC-3 culture system could be a useful tool for studying the interaction of DC with mycobacteria. [source] IL-33 promotes DC development in BM culture by triggering GM-CSF productionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2009Nobuyasu Mayuzumi Abstract Short-term DC cultures generated with GM-CSF and other cytokines have markedly improved our ability to study the immunobiology of DC. Here, we tested 65 cytokines individually for their potential to promote the generation of CD11c+ cells in a murine BM culture system. In addition to several cytokines known to promote DC survival and/or growth, IL-33 was found to augment DC development time- and dose-dependently. Although the resulting CD11c+ cells generated in the presence of IL-33 exhibited a typical dendritic morphology, they expressed MHC class II molecules only at modest levels, showed negligible responses to TLR ligands, produced no detectable IL-12 p70, displayed PD-L1 and PD-L2 on the surface, and failed to activate immunologically naïve T cells efficiently. IL-33-induced expansion of CD11c+ cells was completely blocked by anti-GM-CSF mAb, and GM-CSF mRNA and protein expression in BM culture was markedly elevated by added IL-33, indicating that IL-33 promotes in vitro DC generation indirectly by a GM-CSF-dependent manner. With regard to the cellular source, IL-33-dependent GM-CSF production was observed exclusively within the CD45+/Fc,RI+ BM population. Not only do our results reinforce the notion that GM-CSF serves as a primary DC growth factor, but they also reveal a previously unrecognized mechanism supporting DC development. [source] Paradoxical effects of interleukin-10 on the maturation of murine myeloid dendritic cellsIMMUNOLOGY, Issue 2 2003Dianne L. Commeren Summary The immunoregulatory cytokine, interleukin-10 (IL-10), has been shown to inhibit the maturation of human myeloid dendritic cells (DC). In the present study, we demonstrate that IL-10 has paradoxical effects on the maturation of murine myeloid bone marrow-derived DC. On the one hand, IL-10 inhibits the maturation of murine myeloid DC. The addition of IL-10 to granulocyte,macrophage colony-stimulating factor (GM-CSF)-supported murine BM-derived DC cultures reduced the frequency of major histocompatibility complex (MHC) class IIbright cells. These IL-10-pretreated DC have a reduced capacity to stimulate T cells in an allogeneic mixed leucocyte reaction. On the other hand, however, and in contrast to the effects of IL-10 on human DC, we found that the addition of IL-10 from the initiation of the culture onwards induced an up-regulation of the expression of the costimulatory molecules CD40, CD80 and CD86 on murine myeloid DC, as compared to DC generated with GM-CSF only. Moreover, a subpopulation of IL-10-pretreated MHC class IIdim DC lacked the capacity to take up dextran-fluorescein isothiocyanate (FITC), a feature of DC maturation. Taken together, our data demonstrate that the generation of murine myeloid DC in the presence of IL-10 results in a population of incompletely matured MHC class IIdim CD80+ CD86+ DC. These DC lack T-cell stimulatory capacity, suggesting a role for IL-10 in conferring tolerogenic properties on murine myeloid DC. [source] Upregulation of Serotonin Transporter by Alcohol in Human Dendritic Cells: Possible Implication in Neuroimmune DeregulationALCOHOLISM, Issue 10 2009Dakshayani Kadiyala Babu Background:, Alcohol is the most widely abused substance and its chronic consumption causes neurobehavioral disorders. It has been shown that alcohol affects the function of immune cells. Dendritic cells (DC) serve as the first line of defense against infections and are known to accumulate neurotransmitters such as 5-hydroxytryptamine (5-HT). The enzyme monoamine oxidase-A (MAO-A) degrades 5-HT that is associated with clinical depression and other neurological disorders. 5-HT is selectively transported into neurons through the serotonin transporter (SERT), which is a member of the sodium- and chloride-dependent neurotransmitter transporter (SLC6) family. SERT also serves as a receptor for psychostimulant recreational drugs. It has been demonstrated that several drugs of abuse such as amphetamine and cocaine inhibit the SERT expression; however, the role of alcohol is yet to be elucidated. We hypothesize that alcohol can modulate SERT and MAO-A expression in DC, leading to reciprocal downregulation of 5-HT in extracellular medium. Methods:, Dendritic cells were treated with different concentrations (0.05% to 0.2%v/v) of alcohol for 24,72 hours and processed for SERT and MAO-A expression using Q-PCR and Western blots analysis. In addition, SERT function in DC treated with alcohol both in the presence and absence of imipramine, a SERT inhibitor was measured using 4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide uptake assay. 5-HT levels in culture supernatant and intracellular 5-hydroxy indole acetic acid (5-HIAA) and cyclic AMP were also quantitated using ELISA. Results:, Dendritic cells treated with 0.1% alcohol for 24 hours showed significant upregulation of SERT and MAO-A expression compared with untreated DC. We also observed that 0.1% alcohol enhanced the function of SERT and decreased extracellular 5-HT levels compared with untreated DC cultures, and this was associated with the elevation of intracellular 5-HIAA and cyclic AMP levels. Conclusions:, Our study suggests that alcohol upregulates SERT and MAO-A by elevating cyclic AMP, which may lead to decreased concentration of 5-HT in the extracellular medium. As 5-HT is a major neurotransmitter and an inflammatory mediator, its alcohol-mediated depletion may cause both neurological and immunological deregulation. [source] Generation of self-renewing immature dendritic cells from mouse spleen that can take up mycobacteria and present antigens to T cellsAPMIS, Issue 10 2010RUBINA PAL Pal R, Marwaha S, Pepponi I, Mann JFS, Paul MJ, Reljic R. Generation of self-renewing immature dendritic cells from mouse spleen that can take up mycobacteria and present antigens to T cells. APMIS 2010; 118: 729,38. Dendritic cells (DC) play a key role in driving the adaptive immune response to Mycobacterium tuberculosis (MTB), the causative pathogen of tuberculosis (TB). However, studying these important yet very sparse immune cells in the context of MTB pathogenesis is severely restricted by the lack of suitable cell lines and the complexity of culturing of DC progenitors, usually obtained from the bone marrow. However, significant advances have been made towards generating long-term DC cultures from various lymphoid tissues. Here, we report the evidence for generating a long-term, self-renewing DC culture from the Balb/c mouse spleen. We demonstrate that these cells, termed IDC-3, have a myeloid DC origin, i.e. they are CD11c+CD11b++CD8-,,F4/80+/, and that they also display a phenotype MHC-II+CD16/32++CD80+/,CD86+, indicating that they are immature DC. Following incubation with Mycobacterium bovis BCG (Bacillus Calmette Guerin), the IDC-3 efficiently took up bacteria and acquired the morphology of mature DC. Importantly though, when IDC-3 were pre-stimulated with a mycobacterial antigen in vitro, they were able to induce proliferation of T lymphocytes from mice immunized with the same antigen. The T-cell stimulatory potential of IDC-3 was further enhanced when the cells were co-stimulated with an anti-CD40 mAb. We therefore suggest that the IDC-3 culture system could be a useful tool for studying the interaction of DC with mycobacteria. [source] Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocyte-derived immature dendritic cellsBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2002Ulrich Grigoleit Summary. The hypothesis that productive infection of monocyte-derived immature dendritic cells (DCs) by the human cytomegalovirus (HCMV) is associated with decreased immunostimulatory capacity was tested in this study. DCs were infected with 60,80% efficiency by HCMV strain TB40/E. Infected versus uninfected cells were analysed by fluorescence-activated cell sorting and by immunocytochemistry for surface expression of major histocompatibility complex (MHC) and co-stimulatory molecules as well as cytokine secretion during the 3 d after infection. The immunostimulatory capacity of these cells was measured by mixed leucocyte reaction. In spite of the fact that HCMV infection of DCs induced an increased release of tumour necrosis factor-, (TNF-,) and a decreased interleukin 10 (IL-10) production, expression of MHC class I and II, as well as CD40 and CD80 molecules, were downregulated on infected DCs. The mixed leucocyte reaction showed significantly reduced immunostimulatory capacity of infected DC cultures. Simultaneous detection of MHC antigens and virus antigens by double immunofluorescence revealed that downregulation occurred only on infected cells, but not on uninfected bystander cells. These findings demonstrate on a single cell level, together with the marked downregulation of MHC and co-stimulatory molecules in the presence of high TNF-, and low IL-10 levels, a direct inhibitory effect of HCMV on antigen presentation by immature DCs independent of soluble mediators. [source] Altered phenotype and function of dendritic cells in children with type 1 diabetesCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2005F. Angelini Summary The importance of dendritic cells (DC) in the activation of T cells and in the maintenance of self-tolerance is well known. We investigated whether alterations in phenotype and function of DC may contribute to the pathogenesis of Type 1 diabetes (T1DM). Mature DC (mDC) from 18 children with T1DM and 10 age-matched healthy children were tested. mDC, derived from peripheral blood monocytes cultured for 6 days in presence of interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor (GM-CSF) and stimulated with lipopolysaccharide (LPS) for the last 24 h, were phenotyped for the expression of the co-stimulatory molecules B7·1 and B7·2. In six patients and six controls allogenic mixed leucocyte reaction (AMLR) was performed using mDC and cord blood-derived naive T cells at a DC/T naive ratio of 1 : 200. Proliferation was assessed on day 7 by [3H]-thymidine incorporation assay. Mature DC derived from patients showed, compared with controls, a reduced expression of B7·1 [mean of fluorescence intensity (MFI): 36·2 ± 14·3 versus 72·9 ± 34·5; P = 0·004] and B7·2 (MFI: 122·7 ± 67·5 versus 259·6 ± 154·1; P = 0·02). We did not find differences in the HLA-DR expression (P = 0·07). Moreover, proliferative response of allogenic naive T cells cultured with mDC was impaired in the patients (13471 ± 9917·2 versus 40976 ± 24527·2 cpm, P = 0·04). We also measured IL-10 and IL-12 concentration in the supernatant of DC cultures. Interestingly, we observed in the patients a sevenfold higher level of IL-10 (P = 0·07) and a ninefold lower level of IL-12 (P = 0·01). Our data show a defect in the expression of the co-stimulatory molecules and an impairment of DC priming function, events that might contribute to T1DM pathogenesis. [source] | ||||||||||