Contrasting effects of myeloid dendritic cells transduced with an adenoviral vector encoding interleukin-10 on organ allograft and tumour rejection.
(1/1974)
Mouse bone marrow-derived myeloid dendritic cells (DC) propagated in granulocyte-macrophage colony-stimulating factor and transforming growth factor-beta1 (TGF-beta1) (so-called 'TGF-beta DC') are phenotypically immature, and prolong allograft survival. Interleukin-10 (IL-10) has been shown to inhibit the maturation of DC by down-regulating surface major histocompatibility complex (MHC) class II, co-stimulatory and adhesion molecule expression. Genetic engineering of TGF-beta DC to overexpress IL-10 might enhance their tolerogenic potential. In this study, adenoviral (Ad) vectors encoding the mouse IL-10 gene were transduced into B10 (H2b) TGF-beta DC. Transduction with Ad-IL-10 at a multiplicity of infection (MOI) of 50-100 resulted in a modest reduction in the incidence of DC expressing surface MHC class II, CD40, CD80 and CD86. Paradoxically, Ad-IL-10 transduction enhanced the allostimulatory activity of DC in mixed leucocyte reactions and cytotoxic T lymphocyte assays, and increased their natural killer cell stimulatory activity. Systemic injection of normal C3H recipients with Ad-IL-10-transduced B10-DC 7 days before organ transplantation, exacerbated heart graft rejection and augmented circulating anti-donor alloantibody titres. Contrasting effects were observed in relation to tumour growth. All mice preimmunized with Ad-IL-10-transduced, tumour antigen (B16F10)-pulsed DC developed palpable tumours, associated with significant inhibition of splenic anti-tumour cytotoxic T lymphocyte generation. Animals pretreated with control Ad-LacZ-transduced, B16F10-pulsed DC however, remained tumour free. These findings are consistent with the multifunctional immunomodulatory properties of mammalian IL-10. (+info)
Antagonism between C/EBPbeta and FOG in eosinophil lineage commitment of multipotent hematopoietic progenitors.
(2/1974)
The commitment of multipotent cells to particular developmental pathways requires specific changes in their transcription factor complement to generate the patterns of gene expression characteristic of specialized cell types. We have studied the role of the GATA cofactor Friend of GATA (FOG) in the differentiation of avian multipotent hematopoietic progenitors. We found that multipotent cells express high levels of FOG mRNA, which were rapidly down-regulated upon their C/EBPbeta-mediated commitment to the eosinophil lineage. Expression of FOG in eosinophils led to a loss of eosinophil markers and the acquisition of a multipotent phenotype, and constitutive expression of FOG in multipotent progenitors blocked activation of eosinophil-specific gene expression by C/EBPbeta. Our results show that FOG is a repressor of the eosinophil lineage, and that C/EBP-mediated down-regulation of FOG is a critical step in eosinophil lineage commitment. Furthermore, our results indicate that maintenance of a multipotent state in hematopoiesis is achieved through cooperation between FOG and GATA-1. We present a model in which C/EBPbeta induces eosinophil differentiation by the coordinate direct activation of eosinophil-specific promoters and the removal of FOG, a promoter of multipotency as well as a repressor of eosinophil gene expression. (+info)
The role of the DAP12 signal in mouse myeloid differentiation.
(3/1974)
DAP12 is a recently cloned, immunoreceptor tyrosine-based activation motif-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. Previous reports showed that the DAP12 message could be detected not only in NK cells but also in granulocytes, monocytes, dendritic cells, and macrophages. In this study we found a significant level of DAP12 protein expression in macrophage-related cell lines and organs. Additionally, we observed increased expression of DAP12 after LPS-induced differentiation of M1 cells into macrophages. To examine the role of DAP12 in the myeloid cell lineage, we established M1 FLAG-DAP12 transfectants (FDAP-M1) and demonstrated the marked morphological changes in FDAP-M1 cells caused by signaling through DAP12. Cell surface phenotypic analysis showed up-regulation of macrophage markers CD11b, 2.4G2, and adhesion molecule B7-2. Additionally, after stimulation through DAP12, phosphorylated FLAG -DAP12 could be immunoprecipitated using anti-phosphotyrosine mAbs. Collectively, these findings indicate that direct DAP12 signaling has an important role in macrophage differentiation. (+info)
Macrophage-derived dendritic cells have strong Th1-polarizing potential mediated by beta-chemokines rather than IL-12.
(4/1974)
Monocyte-derived dendritic cells (MDDCs) activate naive T lymphocytes to induce adaptive immunity, effecting Th1 polarization through IL-12. However, little is known about other potential DC Th1 polarizing mechanisms, or how T cell polarization may be affected by DCs differentiating in, or exposed to, a proinflammatory environment. Macrophages (MPhis) are DC precursors abundant in inflamed tissues, lymph nodes, and tumors. Thus we studied the T cell-activating and -polarizing properties of MPhi-derived DCs (PhiDCs). Monocytes were cultured in MPhi-CSF (M-CSF) to produce MPhis, which were then differentiated into DCs following culture with GM-CSF plus IL-4. PhiDCs activated a significant allogeneic MLR and were significantly better than MDDCs in activating T cells with superantigen. Most strikingly, PhiDCs elicited up to 9-fold more IFN-gamma from naive or Ag-specific T cells compared with MDDCs (with equivalent IL-4 secretion), despite producing up to 9-fold less IL-12. Neutralization of MDDC, but not PhiDC IL-12 significantly inhibited T cell IFN-gamma induction. PhiDCs produced up to 12-fold more beta-chemokines (macrophage-inflammatory protein-1alpha, -1beta, and RANTES) than MDDCs. Ab blockade of CCR5, but not CXC chemokine receptor 4, inhibited T cell IFN-gamma induction by PhiDCs significantly greater than by MDDCs. Thus DCs differentiating from MPhis induce T cell IFN-gamma through beta-chemokines with little or no requirement for IL-12. Myeloid DCs arising from distinct precursor cells may have differing properties, including different mechanisms of Th1 polarization. These data are the first reports of IFN-gamma induction through chemokines by DCs. (+info)
In vivo roles of integrins during leukocyte development and traffic: insights from the analysis of mice chimeric for alpha 5, alpha v, and alpha 4 integrins.
(5/1974)
Mice chimeric for integrins alpha(5), alpha(V), or alpha(4) were used to dissect the in vivo roles of these adhesion receptors during leukocyte development and traffic. No major defects were observed in the development of lymphocytes, monocytes, or granulocytes or in the traffic of lymphocytes to different lymphoid organs in the absence of alpha(5) or alpha(V) integrins. However, in agreement with previous reports, the absence of alpha(4) integrins produced major defects in development of lymphoid and myeloid lineages and a specific defect in homing of lymphocytes to Peyer's patches. In contrast, the alpha(4) integrin subunit is not essential for localization of T lymphocytes into intraepithelial and lamina propria compartments in the gut, whereas one of the partners of alpha(4), the beta(7) chain, has been shown to be essential. However, alpha(4)-deficient T lymphocytes cannot migrate properly during the inflammatory response induced by thioglycolate injection into the peritoneum. Finally, in vitro proliferation and activation of lymphocytes deficient for alpha(5), alpha(V), or alpha(4) integrins upon stimulation with different stimuli were similar to those seen in controls. These results show that integrins play distinct roles during in vivo leukocyte development and traffic. (+info)
p21 is a transcriptional target of HOXA10 in differentiating myelomonocytic cells.
(6/1974)
The myeolomonocytic cell line U937 differentiates into macrophages in response to a variety of agents. Several genes including the cyclin-dependent kinase inhibitor p21(waf1/cip1) and the homeobox gene transcription factor HOXA10 are induced at the onset of differentiation. Ectopic expression of either gene results in U937 differentiation. In this paper, we describe a mechanism by which p21 and HOXA10 may act in concert, where HOXA10 can bind directly to the p21 promoter and, together with its trimeric partners PBX1 and MEIS1, activate p21 transcription, resulting in cell cycle arrest and differentiation. These experiments for the first time identify p21 as a selective target for a HOX protein and link the differentiative properties of a transcription factor and a cell cycle inhibitor. (+info)
Inhibition of IL-6 and IL-10 signaling and Stat activation by inflammatory and stress pathways.
(7/1974)
The development and resolution of an inflammatory process are regulated by a complex interplay among cytokines that have pro- and anti-inflammatory effects. Effective and sustained action of a proinflammatory cytokine depends on synergy with other inflammatory cytokines and antagonism of opposing cytokines that are often highly expressed at inflammatory sites. We analyzed the effects of the inflammatory and stress agents, IL-1, TNF-alpha, LPS, sorbitol, and H(2)O(2), on signaling by IL-6 and IL-10, pleiotropic cytokines that activate the Jak-Stat signaling pathway and have both pro- and anti-inflammatory actions. IL-1, TNF-alpha, and LPS blocked the activation of Stat DNA binding and tyrosine phosphorylation by IL-6 and IL-10, but not by IFN-gamma, in primary macrophages. Inhibition of Stat activation correlated with inhibition of expression of IL-6-inducible genes. The inhibition was rapid and independent of de novo gene induction and occurred when the expression of suppressor of cytokine synthesis-3 was blocked. Inhibition of IL-6 signaling was mediated by the p38 subfamily of stress-activated protein kinases. Jak1 was inhibited at the level of tyrosine phosphorylation, indicating that inhibition occurred at least in part upstream of Stats in the Jak-Stat pathway. Experiments using Stat3 mutated at serine 727 and using truncated IL-6Rs suggested that the target of inhibition is contained within the membrane-proximal region of the cytoplasmic domain of the gp130 subunit of the IL-6 receptor and is different from the SH2 domain-containing protein-tyrosine phosphatase/suppressor of cytokine synthesis-3 docking site. These results identify a new level at which IL-1 and TNF-alpha modulate signaling by pleiotropic cytokines such as IL-6 and IL-10 and provide a molecular basis for the previously described antagonism of certain IL-6 actions by IL-1. (+info)
Age-associated characteristics of murine hematopoietic stem cells.
(8/1974)
Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34(-/low)c-Kit(+)Sca-1(+) lineage marker-negative (CD34(-)KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34(-)KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34(-)KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34(-)KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs. (+info)