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Julius Kondratyev
Julius Kondratyev

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Bone marrow-derived DCs from Tgfbr2fspKO and Tgfbr2floxE2/floxE2 littermate control mice were generated as described above. DCs were treated overnight with 1 μg/ml lipopolysaccharide (LPS), 500 U/ml tumour necrosis factor α (TNFα) or 20 μg/ml zymosan to induce maturation. A total of 1106 treated and untreated DCs from each group underwent FACS as described above for major histocompatibility complex (MHC) class II, CD11c, CD86 (BD Pharmingen) and 7-AAD. The percentage of mature DCs was determined for each group and treatment as defined by cells simultaneously staining positive for CD11c and highly positive for CD86 and MHC class II after gating out dead (7-AAD-positive) cells. Experiments were conducted in triplicate with results reported as mean percentage mature DCs (SD), and one-way analysis of variance (ANOVA) was employed to compare Tgfbr2fspKO and Tgfbr2floxE2/floxE2 DCs using SPSS 16.


Since TGFβ is known to regulate DC function and control of DC maturation is needed to maintain immune tolerance, we hypothesised that Tgfbr2fspKO DCs lack normal maturational regulation, resulting in the development of autoimmunity. To study DC maturation, bone marrow-derived DCs from Tgfbr2fspKO and Tgfbr2floxE2/floxE2 control mice were treated in vitro with molecules that induce DC maturation, including LPS, TNFα and zymosan. After 24 h, DCs were harvested and upregulation of the maturation markers MHC class II and CD86 was compared using FACS. While the number of mature DCs in untreated Tgfbr2floxE2/floxE2 and Tgfbr2fspKO cultures was similar, LPS induced maturation of Tgfbr2fspKO DC cultures more than threefold compared with wild type DC cultures (fig 8C). Similar results were seen with TNFα and zymosan treatment (data not shown).


Although TGFβ is known to modulate DC maturity,18 our studies implicate disruption of TGFβ signalling in DCs as a critical event in the development of autoimmunity. DCs normally maintain immune tolerance by remaining immature during the presentation of self-antigens to T cells, resulting in T cell anergy. Since Tgfbr2fspKO DCs have increased maturation in response to activation, we hypothesise that autoimmunity occurs in the Tgfbr2fspKO mouse due to precocious DC maturation in response to self-antigen, resulting in the activation of self-reactive T cells. Studies using targeted disruption of Tgfbr2 in helper T cells to induce autoimmunity are consistent with the idea that TGFβ affects autoimmunity by modulating interactions between DCs and T cells. By expressing a dominant-negative form of Tgfbr2 under direction of the CD4 promoter, murine models of human primary biliary cirrhosis19 and inflammatory bowel disease20 associated with autoantibody production have been generated. In the model of inflammatory bowel disease, T cells were shown to evade immune tolerance by undergoing DC-independent activation.


Thrombopoietin (TPO) is a primary and sole factor for megakaryopoiesis,13 because the genetic elimination of c-mpl or tpo leads to profound thrombocytopenia in mice due to a greatly reduced number of megakaryocytic progenitors and mature megakaryocytes.14 TPO also acts in synergy with IL-3 and SCF on hematopoietic stem cells to induce cell-cycle progression and to increase the both primitive and committed hematopoietic progenitor cells of all lineages.15-17 Furthermore, TPO cooperates with FLT3 ligand (FLT3-L) and SCF in the generation of DC precursors from human CD34+ cells,18-20 and human DCs generated from CD34+ cells following incubation with SCF, GM-CSF, and TNF-α either with or without TPO express the TPO receptor c-mpl.21 As a result, both TPO and TNF-α enhance the proliferation of CD34+ cells and differentiation toward DCs in the presence of multipotent cytokines such as SCF, FLT3-L, and IL-3.


We recently showed that nonerythroid cells were cogenerated from human CD34+ cells during erythroid differentiation in the presence of IL-3/SCF/erythropoietin with TNF-α and expressed DC phenotypes. The CD11c+ DCs physically and selectively associate with developing damaged and immature self-erythroid cells and then phagocytose them.22 This phenomenon may not be limited to the erythroid lineage. We hypothesized that TNF-α in the course of the inflammatory response by viral and microbial infection facilitates DC development during hematopoiesis, thus leading to phagocytosis of damaged self-progenitor cells by DCs. To confirm this hypothesis, we examined the effect of TNF-α and TPO on CD34+ cells.


We herein provide evidence that TNF-α inhibits the generation of megakaryocytic progenitors from CD34+ cells in the presence of TPO while inversely increasing the number of CD4+ CD11c+ CD123+ nonmegakaryocytic cells that show a DC phenotype. We also found that DCs physically associate with immature megakaryocytic cells during differentiation and then phagocytose damaged cells. Next, DCs acquire an ability to induce autologous T-cell proliferation in vitro However, they produce cytokines, which are more tolerogenic than immunogenic, and the T cells activated by them also do not produce a significant amount of immune cytokines. Interestingly, DCs with an immature phenotype in bone marrow from hemophagocytic syndromes are also found to capture CD61+ cells. These findings may suggest the possibility that the phagocytosis of damaged cells by DCs under daily life conditions with either a weak or strong inflammatory response could thus play a pivotal role in regulating the immune responses against hematopoietic progenitor cells. 041b061a72


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