Differential expression of Rel/NF-kappaB and octamer factors is a hallmark of the generation and maturation of dendritic cells.

A key feature of maturation of dendritic cells is the down-regulation of antigen-processing and up-regulation of immunostimulatory capacities. To study the differential expression of transcription factors in this process, we investigated the nuclear translocation and DNA binding of Rel/NF-kappaB and octamer factors during in vitro generation and maturation of dendritic cells compared with macrophage development. RelB was the only factor strongly up-regulated during the generation of both immature dendritic cells and macrophages. Cytokine-induced maturation of dendritic cells resulted in an increase in nuclear RelB, p50, p52, and especially c-Rel, whereas cytokine-treated macrophages responded poorly. This up-regulation of NF-kappaB factors did not correlate with lower levels of cytosolic NF-kappaB inhibitors, the IkappaBs. One IkappaB, Bcl-3, was strongly expressed only in mature dendritic cells. Furthermore, generation and maturation of dendritic cells led to a continuous down-regulation of the octamer factor Oct-2, whereas monocytes and macrophages displayed high Oct-2 levels. A similar pattern of maturation-induced changes in transcription factor levels was found in cultured murine epidermal Langerhans cells, suggesting a general physiological significance of these findings. Finally, this pattern of differential activation of Rel and octamer factors appears to be suitable in determining the maturation stage of dendritic cells generated by treatment with different cytokine combinations in vitro. (Blood. 2000;95:277-285)

Full length cDNA of rat RT1.DMa and RT1.DMb and expression of RT1.DM genes in dendritic and Langerhans cells.

MHC encoded DM heterodimers and classical MHC class II complexes meet in an endosomal/lysosomal compartment where DM heterodimers support peptide loading of MHC class II. Studies on peptide loading of rat class II and on peptide persistence in cells of the dendritic lineage prompted us to establish full length cDNA clones coding for the subunits alpha and beta of rat DM molecules as well as a mAb directed against the luminal moiety of the beta subunit. Here we describe the establishment of the first full length cDNA clones of rat RT1.DMa and RT1.DMb. The mode of expression of RT1.DM at the transcript level in bone marrow culture-derived dendritic cells, in Langerhans cells and in a number of additional accessory cells is reported. The beta protein was identified in detergent lysates of RT1.DM expressing cells by Western blot analysis using a newly established monoclonal antibody directed against the luminal part of RT1.DMbeta.

Uptake of microparticle-adsorbed protein antigen by bone marrow-derived dendritic cells results in up-regulation of interleukin-1 alpha and interleukin-12 p40/p35 and triggers prolonged, efficient antigen presentation.

Dendritic cells synthesize and express major histocompatibility complex (MHC) class II peptide-binding elements constitutively and, therefore, belong to the category of professional antigen-presenting cells. Unlike other cells that show constitutive class II expression, such as B cells and certain T cell clones, dendritic cells possess the unique capacity to activate naive T cells. Using dendritic cells generated in vitro by culture of mouse bone marrow in the presence of low doses of recombinant mouse granulocyte/macrophage colony-stimulating factor, we found that discrete maturation stages of these cells can be distinguished which were correlated with defined functional capabilities. The striking observation was the presence of a progenitor dendritic cell expressing low levels of class II which, unlike its differentiated counterpart in vitro, possessed pronounced phagocytic activity. Adding protein antigen to dendritic cells in a particle-adsorbed form, as compared to a soluble form, we demonstrate that phagocytosis of the particle-adsorbed protein by progenitor dendritic cells involves an activation event. This is evidenced by the de novo synthesis of transcripts of interleukin-1 alpha and interleukin-12 p40/p35 as well as transcripts of MHC class II. Most importantly, an augmented and prolonged antigen-presentation capacity was observed when the antigen was given in particle-adsorbed instead of soluble form. These findings indicate that progenitor dendritic cells are functionally more flexible and potent than fully differentiated dendritic cells and that they play a crucial role in antigen presentation. It is suggested that these findings will open up new possibilities to devise strategies for vaccine development.

Modulation of accessory cell function of immortalized bone marrow-derived macrophages by granulocyte/macrophage colony-stimulating factor.

To generate cloned macrophage populations with sensitivity towards granulocyte/macrophage colony-stimulating factor (GM-CSF), bone marrow-derived macrophages (BMM phi) were immortalized by transformation with SV40. A panel of transformed clones was established. The majority of clones represented independently derived transformants, as evidenced by restriction fragment length polymorphism using genomic DNA digested with EcoRI and TaqI and the 5.2 kb SV40 DNA for hybridization analysis. The cells belong to the macrophage lineage according to several criteria, e.g. the presence of nonspecific esterase, their phagocytic capacity and their morphology. Many clones were potent antigen-presenting cells (APC), without exogenous stimulation. Two clones, which did not act efficiently as APC when used untreated, were positively responsive to GM-CSF. GM-CSF stimulation of both clones resulted in potent APC capacity. I-A alpha, I-A beta and gamma chain-specific transcripts were observed upon stimulation with GM-CSF, corresponding to detectable levels of class II surface display as revealed by cytofluorometric analysis. Thus the macrophage clones established will allow dissection of the differential effects of GM-CSF on the parameters of antigen presentation.

Dendritic cells from mouse bone marrow: in vitro differentiation using low doses of recombinant granulocyte-macrophage colony-stimulating factor.

Dendritic cells (DC) are potent stimulator cells that are crucially involved in primary T cell responses. Since DC comprise a minor population in lymphoid cell suspensions tedious and time consuming procedures are required for their preparation. This work outlines an in vitro culture system that promotes the differentiation of DC from unfractionated mouse bone marrow (BM) cells in the presence of low doses of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Unlike co-induced BM-macrophages the outgrowing BM-DC express high levels of MHC class II molecules; they are negative for specific and nonspecific esterase and are nonphagocytic. A rapid one step purification procedure employing immunomagnetic bead selection yielded up to 95% BM-DC enriched cell fractions. The bead-selected BM-DC were powerful inducers of the allogeneic mixed leukocyte reaction. Thus, our findings demonstrate that low dose rGM-CSF-driven in vitro culture of BM cells provides convenient access to substantial numbers of DC and will greatly facilitate their further exploration.