Developmental origin of interferon-alpha-producing dendritic cells from hematopoietic precursors
ABSTRACT The aim of this study was to determine the lineage origin of interferon-alpha-producing cells (IPCs), also called plasmacytoid dendritic cells, in mice by evaluating the ability of common lymphoid (CLP) and myeloid (CMP) progenitors to give rise to IPCs.
Sublethally irradiated C57Bl/6 mice were intravenously transplanted with rigorously purified lymphoid and myeloid progenitors from a congenic mouse strain. At various time points posttransplantation mice were analyzed for donor-derived cells by flow cytometry. The developmental potential of all progenitor populations was also tested in in vitro cultures. In addition, in vitro and in vivo derived IPCs were functionally assessed for their interferon-alpha production after virus challenge.
Transplantation of 1 x 10(4) common myeloid progenitors, 1 x 10(4) common lymphoid progenitors or 2.5 x 10(4) granulocyte/macrophage progenitors all led to the generation of IPCs within 2 to 3 weeks. In general, IPC reconstitution in spleen and liver by CMPs was more efficient than by CLP. Adding Flt3L alone to in vitro cultures was sufficient to support the development of IPCs from myeloid progenitors whereas CLPs required additional survival factors provided either by stroma cells or by introduction of transgenic Bcl-2. Both myeloid- and lymphoid-derived IPC were indistinguishable by function, gene expression, and morphology.
Surprisingly, our results clearly show that murine IPCs differentiate from both lineages but are mainly of myeloid origin. These results extend to IPCs the observation made originally in classical dendritic cells that cellular expression of so called lineage markers does not correlate with lineal origin.
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ABSTRACT: Alcohol intoxication suppresses both the innate and adaptive immunities. Dendritic cells (DCs) are the major cell type bridging the innate and acquired immune responses. At the present time, the effects of alcohol on DC development in hematopoietic tissues and the functional activities of DCs are incompletely elucidated. This study investigated the impact of chronic alcohol exposure on the alteration of hematopoietic precursor cell and DC populations in the bone marrow and peripheral blood of rhesus macaques. Rhesus macaques were administered alcohol or isocaloric sucrose daily for a period of 3 months through surgically implanted gastric catheters. Peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs) were isolated for flow cytometric analysis after 3 months. Monocytes were cultured with human IL-4 (10 ng/ml) and GM-CSF (50 ng/ml) in the absence and presence of alcohol (50 mM). On day 6 of the culture, a cocktail of stimulants including IL-1beta (18 ng), IL-6 (1800 U), TNF-alpha (18 ng), and PGE(2) (1.8 microg) were added to the designated wells for transformation of immature dendritic cells (iDCs) to mature myeloid DCs. The cells were analyzed on day 8 by flow cytometry for expression of DC costimulatory molecule expression. EtOH-treated animals had significantly lower numbers of myeloid DCs (lineage-HLA-DR+CD11c+CD123-) in both the PBMCs and BMCs compared to controls (5,654 +/- 1,273/10(6) vs. 2,353 +/- 660/10(6) PBMCs and 503 +/- 34 vs. 195 +/- 44/10(6) BMCs). Under culture conditions, the number of lineage-HLA-DR+CD83+ cells was low in control wells (0.38 +/- 0.08%). Alcohol inhibited the increase in the number of lineage-HLA-DR+CD83+ cells in iDC wells (2.30 +/- 0.79% vs. 5.73 +/- 1.40%). Alcohol also inhibited the increase in the number of lineage-HLA-DR+CD83+ cells in mature DC wells (1.23 +/- 0.15% vs. 4.13 +/- 0.62%). Chronic EtOH decreases the bone marrow and circulating pools of myeloid DCs. Additionally, EtOH suppresses costimulatory molecule CD83 expression during DC transformation, which may attenuate the ability of DCs to initiate T-cell expansion.Alcoholism Clinical and Experimental Research 06/2009; 33(9):1524-31. DOI:10.1111/j.1530-0277.2009.00980.x · 3.31 Impact Factor
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ABSTRACT: Exosome production represents an alternate endocytic pathway for secretion. Multivesicular endosomes (MVE) fuse with the plasma membrane expelling internal vesicles or exosomes from cells. Exosome production has been recently described for immune cells including B cells, dendritic cells (DC), mast cells, macrophages and T cells. Exosomes derived from some DC populations stimulate T lymphocyte proliferation in vitro and have potent capacity to generate anti-tumour immune responses in vivo. These reported studies have involved in vitro grown mature DC expanded from precursors with cytokines. However, immature DC produce higher numbers of exosomes than mature DC and this is thought to be due to a reduction in endocytosis as DC mature, associated with reduced reformation of MVE and reduced exosome formation. This lab pioneered a method to generate immature DC in spleen long-term cultures (LTC). DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. LTC-DC produce exosomes and contain many MVE. This prompted a study of immunogenic potential with a view to the potential use of exosomes in vaccination and immunotherapy. DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. Exosomes were isolated by differential centrifugation from LTC-DC and shown by marker expression to arise by budding from the LAMP-1+ limiting endosomal membrane of MVE. These LTC-derived exosomes appear however to lack immunostimulatory markers like CD86, CD40, MHC-I and MHC-II. While LTC-DC can stimulate antigen-specific proliferation of CD4+ T cells, exosome preparations derived from antigen-pulsed DC were unable to stimulate purified naïve T cells in vitro. They were however found to weakly activate allogeneic CD8+ T cells in vitro. Tumour antigen-pulsed LTC-DC or their exosomes could induce a protective response in mice against growth of a transplanted tumour but could not induce a response to clear an existing tumour. Exosomes derived from immature DC can modulate immune responses, but do not function in direct T cell activation in vitro. Modulation of immune responses by exosomes produced by immature DC may be dependent on the presence of other antigen presenting DC subsets in the animal. The possible function of immature DC and their exosomes in maintenance of tolerance and in the induction of immunity is discussed.Blood Cells Molecules and Diseases 09/2005; 35(2):94-110. DOI:10.1016/j.bcmd.2005.05.002 · 2.33 Impact Factor
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ABSTRACT: Dendritic cells are professional antigen presenting cells that capture antigens and migrate to lymphoid tissues where they elicit specific immune responses. Several DC subtypes have previously been characterised, depending on their function and location. Each subtype appears to have a specialised role in both immunity and tolerance. However, less clear are the processes by which these different DC subsets develop from haematopoietic precursors, and the molecular mechanisms involved in the regulation of the different stages during DC development. The research described herein had the aim to continuatively characterize a previously described DC precursor, the Flt3+ progenitor, downstream of the haematopoietic stem cells, and to assess its developmental potential in vivo. Hereby, it was found that the Flt3+ progenitor does not only develop into dendritic cells, but can also generate all other cells of the haematopoietic compartment. Furthermore, this study examined the role of the HLH transcriptional regulator Id2 on the development of different DC subtypes, especially Langerhans cells. As monocytes had been assumed to be LC precursors, Gr-1hi monocytes as well as the Flt3+ progenitor were tested for their LC precursor potential. Using an Id2 knockout mouse model, it was found that Id2 is essential for the formation of Langerhans cells in steady state. Conversely, their development from Gr-1hi monocytes in response to inflammation is independent of Id2. Id2 is known to modulate the activity of E-proteins, such as E47, by antagonising their DNA binding. Therefore, the impact of Id2 and E47 on DC development was studied and this work provides new insights into the molecular mechanisms controlling the development of distinct DC subsets. It shows that the molecular mechanism by which Id2 controls the development of different DC subtypes is based on a modulation of E-proteins. Taken together, this research identified some of the key steps in the development of DCs downstream of the early haematopoietic precursors. It also dissected the contribution of different precursors for the Langerhans cell development in both the steady-state and during inflammation. Finally it provides evidence for the importance of Id2 and E proteins, such as E47 working in concert to regulate DC development and differentiation. Dendritische Zellen gehören zu den professionellen antigenpräsentierenden Zellen, die Antigene aufnehmen und in das Lymphgewebe wandern, wo sie antigenspezifische Immunreaktionen initiieren. Verschiedene Subtypen von Dendritischen Zellen wurden bereits charakterisiert, abhängig von ihrer Funktion und Lokalisation. Jeder dieser Subtypen scheint eine besondere Rolle, sowohl in Immunität als auch in Toleranz zu haben. Hingegen ist es weniger klar, wie sich diese verschiedenen Subtypen von Dendritischen Zellen aus ihren hämatopoetischen Vorläufern entwickeln und welche molekularen Mechanismen an der Regulation der verschiedenen Entwicklungsstufen beteiligt sind. Die vorliegende Arbeit hatte das Ziel einen bereits beschriebenen Vorläufer für Dendritische Zellen, den sogenannten Flt3+ progenitor, weiterführend zu charakterisieren und sein Entwicklungspotenzial in vivo zu untersuchen. Dabei zeigte sich, dass der Flt3+ progenitor nicht nur Dendritische Zellen bildet, sondern sich auch in alle anderen Zellen des hämatopoetischen Systems entwickeln kann. Des Weiteren bearbeitet diese Studie den Einfluss des HLH Transkriptionsfaktors Id2 auf die Entwicklung von verschiedenen Subtypen Dendritischer Zellen, insbesondere Langerhans Zellen. Da bereits gezeigt wurde, dass Monozyten Vorläufer der Langerhans Zellen sein können, wurden Gr-1hi Monozyten sowie der Flt3+ progenitor auf ihre Fähigkeit Langerhans Zellen zu bilden untersucht. Dafür wurde ein Mausmodell verwendet, in dem Id2 genetisch inaktiviert war. Hierbei wurde gefunden, dass Id2 unter normalen Bedingungen essentiell für die Bildung von Langerhans Zellen ist, während deren Bildung aus Gr-1hi Monozyten in Antwort auf eine Entzündung unabhängig von Id2 verläuft. Es ist bekannt, dass Id2 die Aktivität von E-Proteinen, wie zum Beispiel E47, verändert, indem es ihre Bindung an DNS verhindert. Daher wurde der Einfluss von Id2 und E47 auf die Entwicklung von Dendritischen Zellen analysiert und diese Arbeit zeigt, dass der molekulare Mechanismus, durch den Id2 die Entwicklung von verschiedenen Subtypen Dendritischer Zellen kontrolliert, auf einer Beeinflussung von E-Proteinen basiert. Zusammen genommen identifiziert diese Arbeit einige der wesentlichen Schritte in der Entwicklung von Dendritischen Zellen aus frühen hämatopoetischen Vorläuferzellen. Weiterhin betrachtet sie den Beitrag verschiedener Vorläufer zur Entwicklung von Langerhans Zellen, sowohl im Normalzustand, als auch während der Entzündung. Abschließend zeigt sie die Bedeutung des Zusammenspiels von Id2 und E-Proteinen, wie zum Beispiel E47, in der Entwicklung und Differenzierung von Dendritischen Zellen.