Ralf Ross

Compartmentalized production of CCL17 in vivo: strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen.

Dendritic cells (DCs)* fulfill an important regulatory function at the interface of the innate and adaptive immune system. The thymus and activation-regulated chemokine (TARC/CCL17) is produced by DCs and facilitates the attraction of activated T cells. Using a fluorescence-based in vivo reporter system, we show that CCL17 expression in mice is found in activated Langerhans cells and mature DCs located in various lymphoid and nonlymphoid organs, and is up-regulated after stimulation with Toll-like receptor ligands. DCs expressing CCL17 belong to the CD11b+CD8−Dec205+ DC subset, including the myeloid-related DCs located in the subepithelial dome of Peyers patches. CCL17-deficient mice mount diminished T cell–dependent contact hypersensitivity responses and display a deficiency in rejection of allogeneic organ transplants. In contrast to lymphoid organs located at external barriers of the skin and mucosa, CCL17 is not expressed in the spleen, even after systemic microbial challenge or after in vitro stimulation. These findings indicate that CCL17 production is a hallmark of local DC stimulation in peripheral organs but is absent from the spleen as a filter of blood-borne antigens.

Diminished contact hypersensitivity response in IL-4 deficient mice at a late phase of the elicitation reaction.

Contact hypersensitivity (CHS) is thought to depend on the activation of T cells of Th1 and/or Tc1 type. The role of Th2/Tc2 cells in the contact allergic reaction is not clear. The aim of this study was to analyse the functional contribution of Th2/Tc2 cells in CHS using the interleukin‐4 (IL‐4) deficient mouse model. Interleukin‐4 deficient (IL4T) and control (wt) mice were sensitized by epicutaneous application of 2,4‐dinitrofluorobenzene. The ear swelling response measured 24 h after challenge was similar in IL4T and control mice. However, from 48 h onwards, ear swelling values were significantly reduced in IL4T mice. The stimulatory capacity of freshly isolated as well as 3‐day cultured epidermal cells, prepared from IL4T and wt mice, for allogeneic T cells in a primary and secondary response, was comparable. The reduced number of T cell receptor (TCR) γδ+ cells observed in epidermal sheets prepared from IL4T mice was not responsible for the decreased ear swelling response in IL4T mice, because the use of TCR δ deficient mice lacking TCR γδ+ cells revealed a down‐regulatory role of this cell population in the CHS response. The data indicate that the effector stage of the CHS response can be subdivided into two phases. The first phase proceeds efficiently in IL‐4 deficient mice indicating the dependence on Th1/Tc1 cells, while the second phase does not develop in mice lacking IL‐4, suggesting the possibility that Th2/Tc2 cells intensify the reaction.

The Human Fascin Gene Promoter Is Highly Active in Mature Dendritic Cells Due to a Stage-Specific Enhancer

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.

The role of NO in contact hypersensitivity

Contact dermatitis or contact hypersensitivity (CHS) is a common T lymphocyte-mediated allergic disease characterized by local inflammatory skin reactions following contact with small reactive compounds called haptens. In common with other allergic processes, the development of contact dermatitis proceeds in two phases: a sensitization phase which occurs on first exposure to allergen, and an elicitation phase which occurs on subsequent exposure when the clinical manifestations of the disease are observed. This process is hapten-specific. While the pathophysiology of the sensitization phase is well characterized, our understanding of the elicitation phase is still incomplete, including the relative contribution of the different effector cells and mediators involved. Here we summarize current knowledge of the contribution of nitric oxide (NO) to skin inflammation with special focus on CHS. A number of inflammatory stimuli trigger expression of NO in human and animal skin, and topical application of an NO-releasing cream results in inflammation. Moreover, expression of the inducible isoform of nitric oxide synthase (iNOS) is induced in CHS and iNOS inhibitors injected intradermally suppress CHS responses. However, iNOS-deficient mice develop an aggravated CHS response late in the elicitation phase, suggesting that NO is involved in downregulation of CHS. Based on these data, we propose a comprehensive model of the role of NO in CHS.

Transcriptional targeting of dendritic cells in gene gun-mediated DNA immunization favors the induction of type 1 immune responses

Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection of the skin. We transcriptionally targeted transgene expression to DC using vectors containing the murine fascin promoter (pFascin) to control antigen production and compared the immune response elicited with conventional DNA immunization using plasmid constructs with the ubiquitously active CMV promoter (pCMV). Biolistic transfection with pFascin initiated a marked type 1 immune response characterized by the occurrence of a large population of IFN-gamma-producing T helper (Th) cells in spleen and draining lymph nodes. Consistently, immunoglobulin production was dominated by IgG2a antibodies. In contrast, the humoral response after repeated administration of pCMV was strongly enhanced and characterized by a type 2-like isotype pattern (IgG1 > IgG2a). Cytokine production analysis in vitro indicated compartmentalization of the immune response, revealing large numbers of IL-4-producing Th cells in the lymph nodes and dominant presence of IFN-gamma-producing Th cells in the spleen. Biolistic transfection with pFascin, like immunization with pCMV, led to potent induction of cytotoxic T cells as was assessed by JAM test. Thus gene gun immunization with plasmids that focus transgene expression and antigen production specifically to DC propagates type 1-biased cellular immune responses.

Differential Regulation of CCL22 Gene Expression in Murine Dendritic Cells and B Cells

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.

Complex Evolution of Tandem-Repetitive DNA in the Chironomus thummi Species Group

Abstract. The subspecies Chironomus thummi thummi and C. t. piger display dramatic differences in the copy number and chromosomal localization of a tandemly repeated DNA family (Cla elements). In order to analyze the evolutionary dynamics of this repeat family, we studied the organization of Cla elements in the related outgroup species C. luridus. We find three different patterns of Cla element organization in C. luridus, showing that Cla elements may be either strictly tandem-repetitive or be an integral part of two higher-order tandem repeats (i.e., Hinf[lur] elements, Sal[lur] elements). All three types of Cla-related repeats are localized in the centromeres of C. luridus chromosomes. This suggests that the dispersed chromosomal localization of Cla elements in C. t. thummi may be the result of an amplification and transposition during evolution of this subspecies.

Maturation of epidermal Langerhans cells: increased experssion of β‐and γ‐actin isoforms as a basis of specialized cell functions

Epidermal Langerhans cells (LC) represent immature dendritic cells. During in vitro culture in the presence of keratinocytes they mature into potenet immunostimulatory cells for naive T cells. This process is thought to simulate in vivo maturation of LC following activation by antigen contect. Maturation of LC is accompanied by morphological alteration. Applying a differential screening procedure we isolated differentially expressed cDNAs involved in the maturation events including cDNAs of the cytoskeletal actin isoforms β‐and γ‐actin. Stronger signals with hybridization probes derived from cultured LC compared with probes derived from freshly isolated LC indicate upregulation of actin expression. Upregulated expression of actin was confirmed by RT‐PCR, Western blot and immunofluorescence analysis. Staining with flurescence‐labelled and increase in F‐actin levels in cultured LC. Thus our data show that maturation of LC, which involves formation of dendritic structures and movement of formerly immobile cell, is accompanied by augmented expresion of actin and formation of additional actin filaments. Furthermore, actin mRNA, often used as reference to assess mRNA amounts for Northern blotting or competitive RT‐PCR because of its high and ubiquitous expression is an inappropriate standard for the analysis of LC and DC.

Isolation of Differentially Expressed Genes in Epidermal Langerhans Cells

Epidermal Langerhans cells (LC) represent immature dendritic cells (DC) resident in the skin, which are not yet able to prime naive T cells1. In vitro cultivation of LC in the presence of keratinocytes, supplying survival and differentiation signals, induces maturation events in LC2. These are highlighted by the downregulation of the biosynthesis of MHC class II molecules3, by the upregulation of the surface expression of adhesion and costimulatory molecules like CD80, CD86, CD54 and CD584,5, and by the acquisition of a potent immunostimulatory capacity for T cells6. Mature LC are potent inducers of naive T cells. Thus LC represent an ideal model system to investigate the maturation of DC (reviewed in 7).