Dendritic cells are key players of the immune system as they efficiently induce primary immune responses by activating naive T cells. We generated human dendritic cells from CD14+ blood precursors and investigated expression of the actin-bundling protein fascin during maturation by western blotting, immunofluorescence, and cytofluorometry. Cells obtained by culture of CD14+ blood precursors in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, which were only weakly positive for the maturation marker CD83, expressed low amounts of fascin. Addition of a cytokine cocktail including tumor necrosis factor alpha, interleukin-1beta, interleukin-6, and prostaglandin E2 induced maturation of the cells and enhanced fascin expression in parallel with CD83 expression. Isolated mature CD83+ cells displayed especially high fascin levels on western blots, as did gated CD83+ dendritic cells in cytofluorometry. Dendritic cells generated from CD34+ blood precursors expressed high levels of fascin as well. Confocal microscopy revealed that location of fascin within the cell was restricted to the area of the submembranous actin cytoskeleton and to the dendritic processes. Suppression experiments using antisense constructs of fascin hint at a retarded morphologic maturation of dendritic cells, supporting the view that fascin expression is pivotal for dendrite formation. Our data suggest that fascin could serve as a marker molecule to monitor the maturation state of in vitro generated dendritic cells for use in clinical trials.

Dendritic cells (DC), regarded as the most efficient APCs of the immune system, are capable of activating naive T cells. Thus, DC are primary targets in immunotherapy. However, little is known about gene regulation in DC, and for efficient transcriptional targeting of human DC, a suitable promoter is still missing. Recently, we successfully used the promoter of the murine actin-bundling protein fascin to transcriptionally target DC by DNA vaccination in mice. In this study, we report on isolation of the human fascin promoter and characterization of its regulatory elements. The actively expressed gene was distinguished from a conserved inactive genomic locus and a continuous region of 14 kb covering the gene and 3 kb of 5′-flanking sequences was subcloned, sequenced, and analyzed for regulatory elements. Regulatory sequences were found solely in the 5′-flanking promoter region. The promoter exerted robust activity in DC and a fascin-positive neuronal cell line, but not in the fascin-negative cells tested. Notably, promoter activity in DC markedly increased with maturation of DC. By progressive 5′ deletion, we identified a core promoter region, harboring a putative GC box, a composite cAMP responsive element/AP-1 binding site and a TATA box. By internal deletion, we demonstrated functional importance of either regulatory element. Furthermore, we identified a more distal stage-specific enhancer region also containing silencer elements. Taken together, the human fascin promoter allows for transcriptional targeting of mature DC and represents a promising tool for immunotherapy. To our knowledge, this study reports for the first time on promoter activity in human monocyte-derived DC.

Epidermal Langerhans cells represent an immature population of dendritic cells, not yet able to prime naïve T cells. Following in vitro culture Langerhans cells mature into potent immunostimulatory cells. We constructed a representative cDNA library of in vitro matured murine Langerhans cells. Applying a differential screening procedure 112 differentially expressed cDNA clones were isolated. Thirty-six clones represented cDNA fragments of the same gene, identifying it to be the most actively expressed gene induced in maturing Langerhans cells. A full-length cDNA was sequenced completely. The open reading frame codes for a protein of 92 amino acids containing a leader peptide of 24 amino acids, yielding a mature protein of 7.8 kDa molecular weight. Database searches revealed 99.4% sequence identity on the nucleotide level to the recently described mouse CC chemokine ABCD-1, as well as 74% sequence identity to the human CC chemokine, the macrophage-derived chemokine/stimulated T cell chemotactic protein. Expression was analyzed by reverse transcriptase-polymerase chain reaction on a large panel of cell types. Unlike the macrophage-derived chemokine, expression was not detected in macrophages stimulated by various cytokines. Expression is restricted to cultured Langerhans cells, in vitro cultured dendritic cells, and lipopolysaccharide-activated B cells. Recombinant protein was expressed in the yeast Pichia pastoris and purified to homogeneity. Whereas no chemotactic activity was observed in chemotaxis assays for naïve T cells, B cells, cultured dendritic cells, and Langerhans cells, a strong chemoattractant activity was exerted on activated T cells. Thus, production of this chemokine by dendritic cells may be essential for the establishment and amplification of T cell responses.

The activated T cell-attracting CC chemokine CCL22 is expressed by stimulated B cells and mature dendritic cells (DC). We have cloned and sequenced the complete mouse gene, including 4 kb of the 5′-flanking promoter region, and detected two distinct sites for initiation of transcription by 5′-RACE. Reporter gene assays indicate that the promoter reflects the specificity of the endogenous gene. Within the proximal promoter region, we identified potential binding sites for NF-κB, Ikaros, and a putative GC box. All three regions bind proteins. The NF-κB site was shown to specifically bind NF-κB subunits p50 and p65 from nuclear extracts of LPS-stimulated B cells, B cell line A20/2J, TNF-α-stimulated bone marrow-derived DC, and DC line XS106. Furthermore, promoter activity was affected by targeted mutagenesis of the NF-κB site and transactivation with p50 and p65. The region harboring the putative Ikaros site contributes to promoter activity, but the binding protein does not belong to the Ikaros family. The GC box was shown to specifically bind Sp1 using extracts from LPS-stimulated B cells and A20/2J but not from DC and DC line XS106. Additionally, Sp1 transactivated the promoter in A20/2J but not in XS106 cells, and mutation of the Sp1 site diminished transactivation. Furthermore, binding of the protein complex at the GC box is required for NF-κB activity, and the spatial alignment of the binding sites is of critical importance for promoter activity. Thus, identical and distinct proteins contribute to expression of CCL22 in DC and B cells.

The analysis of differential gene expression has become increasingly important in recent years. Typically, differentially expressed genes are identified in a primary screening procedure, yielding candidate genes whose differential expression has to be verified. We provide a highly sensitive, efficient and nonradioactive differential screening procedure to analyze numerous candidate genes in a single step. This comprises labeling of poly(A)+ RNA of the cell types analyzed with DIG Chem-Link and differential hybridization to the candidate genes fixed on dot blots. DIG Chem-Link allows, to our knowledge, for the first time efficient and direct nonradioactive labeling of RNA in vitro. Advantages of this method include extremely short exposure times and the feasibility to re-use the probes after prolonged storage. Using this procedure, we isolated several genes that are differentially expressed in maturing Langerhans cells.