Hansjörg Hauser

Review: Activities of IRF-1

Interferon (IFN) regulatory factor-1 (IRF-1) was isolated by virtue of its affinity to specific DNA sequences in the IFN-β promoter that mediate virus responsiveness. IRF-1 was the first factor identified of the IRF family and was most extensively characterized at the molecular level. Also, its physiologic role in host defense against pathogens, tumor prevention, and development of the immune system was investigated in detail. Even though some of the functions first associated with IRF-1 were later found to be mediated in part or predominantly by other activators of the IRF family of transcription factors, IRF-1 has remained a central paradigm in the transcriptional regulation of the IFN response.

Dicistronic transcription units for gene expression in mammalian cells

Dicistronic vectors utilizing the internal ribosomal entry site sequence of poliovirus as the intercistronic region were constructed for gene expression in mammalian cells. We have developed two monocistronic expression vectors which facilitate the creation of dicistronic expression plasmids. The dicistronic expression plasmids encode transcription units which allow the coordinated translation of the two genes. Using internal luciferase and secreted alkaline phosphatase, we show the correlated expression of both reporter genes and expression levels comparable to those achieved by the respective monocistronic expression vectors.

Physical association between STAT1 and the interferon‐inducible protein kinase PKR and implications for interferon and double‐stranded RNA signaling pathways

The interferon‐inducible double‐stranded RNA protein kinase PKR controls protein synthesis through the phosphorylation of eukaryotic translation initiation factor (eIF)‐2. In addition to its demonstrated role in translational control, several reports have suggested a transcriptional role for PKR. Here we report that PKR is involved in IFN‐ and dsRNA‐signaling pathways by modulating the function of the signal transducer and activator of transcription STAT1. We also show that PKR associates with STAT1 in mouse and human cells. The association is not a kinase–substrate interaction since STAT1 phosphorylation is not modified by PKR in vitro or in vivo. In addition, the formation of the PKR–STAT1 complex is not dependent upon the enzymatic activity of PKR but does require the dsRNA‐binding domain of PKR. Moreover, there is a concomitant decrease in PKR–STAT1 interaction and increase in STAT1 DNA binding in response to IFNs or dsRNA. These findings suggest that PKR plays an important role in IFN and dsRNA‐signaling pathways by modulating the transcriptional function of STAT1.

Vectors for efficient expression in mammalian fibroblastoid, myeloid and lymphoid cells via transfection or infection

We have constructed two related types of multi-cloning mammalian expression vectors. The first, pMPSVEH/HE, carries the promoter of the myeloproliferative sarcoma virus (MPSV). This promoter was found to be stronger than both the SV40 early and the trans-activated human immunodeficiency virus promoters in many cell lines including human and rodent fibroblastoid, lymphoid or myeloid cells. The other, pBEH/HE, carries the simian virus 40 (SV40) early promoter and origin of replication. This offers the possibility of encapsidation in SV40 pseudovirions and subsequent gene transfer into, e.g., hemopoietic cells, via infection. The usefulness of the expression systems was tested with a number of genes and cell lines.

Genetic optimization of recombinant glycoprotein production by mammalian cells

Genetically modified mammalian cells are the preferred system for the production of recombinant therapeutic glycoproteins. Other applications include engineering of cell lines for drug screening and cell-based therapies, and the construction of recombinant viruses for gene therapy. This article highlights contemporary core genetic technologies and emerging strategies for genetically engineering mammalian cells for optimal recombinant-protein expression.

Specifity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines

The polymerase chain reaction (PCR) amplification was used for the detection of mycoplasma contamination in 42 continuous cell lines. Using the microbiological cultivation on agar as the reference method, 29 cell lines were regarded as positive and 13 cell lines as negative. The double-step PCR analysis employed nested primers that anneal to gene sequences coding for the evolutionarily conserved 16 S rRNA of some 25 different mycoplasma species (including the ones most commonly found in cell cultures). In terms of the positivity or negativity of mycoplasma infection the results were identical for the agar assay and PCR amplification. All positive cell lines displayed distinct, unequivocal, objectively discernible bands on agarose gels while the non-infected specimens showed no DNA amplification. A simultaneously performed comparison with four other commonly used detection methods (DNA-RNA hybridization in solution, DAPI DNA fluorescence staining, immunostaining with a monoclonal antibody and an ELISA) showed that PCR produced significantly less false-negative or false-positive results than all the other methods. Furthermore, in dilution experiments, PCR correctly detected the infecting mycoplasmas at the lowest level of 1/10(4) whereas the other assays were less sensitive. It is concluded that double-step PCR employing nested primers is superior to other mycoplasma detection methods in many respects: simplicity and speed, high specificity and extreme sensitivity, objectivity and accuracy.

Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: The role of NF-κB-repressing factor (NRF) in basal repression of the hiNOS gene

Although nuclear factor (NF)-κB plays a central role in mediating cytokine-stimulated human inducible nitric-oxide synthase (hiNOS) gene transcription, very little is known about the factors involved in silencing of the hiNOS promoter. NF-κB-repressing factor (NRF) interacts with a specific negative regulatory element (NRE) to mediate transcriptional repression of certain NF-κB responsive genes. By sequence comparison with the IFN-β and IL-8 promoters, we identified an NRE in the hiNOS promoter located at −6.7 kb upstream. In A549 and HeLa human cells, constitutive NRF mRNA expression is detected by RT-PCR. Gel shift assay showed constitutive NRF binding to the hiNOS NRE. Mutation of the −6.7-kb NRE site in the hiNOS promoter resulted in loss of NRF binding and increased basal but not cytokine-stimulated hiNOS transcription in promoter transfection experiments. Interestingly, overexpression of NRF suppressed both basal and cytokine-induced hiNOS promoter activity that depended on an intact cis-acting NRE motif. By using stably transformed HeLa cells with the tetracycline on/off expression system, reduction of cellular NRF by expressing antisense NRF increased basal iNOS promoter activity and resulted in constitutive iNOS mRNA expression. These data demonstrate that the transacting NRF protein is involved in constitutive silencing of the hiNOS gene by binding to a cis-acting NRE upstream in the hiNOS promoter.

IFN-Stimulated Gene 15 Is Synergistically Activated Through Interactions Between the Myelocyte/Lymphocyte-Specific Transcription Factors, PU.1, IFN Regulatory Factor-8/IFN Consensus Sequence Binding Protein, and IFN Regulatory Factor-4: Characterization of a New Subtype of IFN-Stimulated Response Element

Type I IFNs cause the induction of a subset of genes termed IFN-stimulated genes (ISGs), which harbor a specific DNA element, IFN-stimulated response element (ISRE). This ISRE confers the responsiveness to the IFN signal through the binding of a family of transcription factors designated IFN regulatory factors (IRFs). Some IRFs can bind to the DNA alone, such as IRF-1, which elicits transcriptional activation, or IRF-2, which leads to transcriptional repression. In addition, these factors associate with IRF-8/IFN consensus sequence binding protein (ICSBP), an immune cell-restricted IRF, and the assembled heterocomplexes lead to synergistic repression of ISRE elements. ISG15 is a prototype ISG that contains a well-characterized ISRE. Here we show that PU.1, an ETS member essential for myeloid/lymphoid cell differentiation, forms heterocomplexes with the immune-restricted IRFs, IRF-8\/ICSBP and IRF-4, which lead to transcriptional activation of ISG15. These data allowed the characterization of a subset of ISREs designated ETS/IRF response element (EIRE), which are differentially regulated in immune cells. EIREs are unique in their ability to recruit different factors to an assembled enhanceosomes. In nonimmune cells the factors will mainly include IRF members, while cell type-restricted factors, such as PU.1, IRF-8\/ICSBP, and IRF-4, will be recruited in immune cells. IRF heterocomplex formation leads to transcriptional repression, and conversely, PU.1/IRFs heterocomplex formation leads to transcriptional activation. The fact that IRF-8\/ICSBP is an IFN-γ-induced factor explains why some of the EIREs are also induced by type II IFN. Our results lay the molecular basis for the unique regulation of ISGs, harboring EIRE, in immune cells.

Hepatic dendritic cell subsets in the mouse

The CD11c+ cell population in the non‐parenchymal cell population of the mouse liver contains dendritic cells (DC), NK cells, B cells and T cells. In the hepatic CD11c+ DC population from immunocompetent or immunodeficient [recombinase‐activating gene‐1 (RAG1)–/–] C57BL/6 mice (rigorously depleted of T cells, B cells and NK cells), we identified a B220+ CD11cint subset of ‘plasmacytoid’ DC, and a B220– CD11c+ DC subset. The latter DC population could be subdivided into a major, immature (CD40lo CD80lo CD86lo MHC class IIlo) CD11cint subset, and a minor, mature (CD40hi CD80hi CD86hi MHC class IIhi) CD11chi subset. Stimulated B220+ but not B220– DC produced type I interferon. NKT cell activation in vivo increased the number of liver B220– DC three‐ to fourfold within 18 h post‐injection, and up‐regulated their surface expression of activation marker, while it contracted the B220+ DC population. Early in virus infection, the hepatic B220+ DC subset expanded, and both, the B220+ as well as B220– DC populations in the liver matured. In vitro, B220– but not B220+ DC primed CD4+ or CD8+T cells. Expression of distinct marker profiles and functions, and distinct early reaction to activation signals hence identify two distinct B220+ and B220– subsets in CD11c+ DC populations freshly isolated from the mouse liver.

GARP: a key receptor controlling FOXP3 in human regulatory T cells

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4+CD25hi T (Treg) cells. Based on transcriptional profiling of ex vivo activated Treg and helper CD4+CD25− T (Th) cells we have identified GARP (glycoprotein‐A repetitions predominant), LGALS3 (lectin, galactoside‐binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human Treg cell function, which are induced upon T‐cell receptor stimulation. Retroviral overexpression of GARP in antigen‐specific Th cells leads to an efficient and stable re‐programming of an effector T cell towards a regulatory T cell, which involves up‐regulation of FOXP3, LGALS3, LGMN and other Treg‐associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down‐regulation of GARP in Treg cells significantly impaired the suppressor function and was associated with down‐regulation of FOXP3. Moreover, down‐regulation of FOXP3 resulted in similar phenotypic changes and down‐regulation of GARP. This provides compelling evidence for a GARP‐FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor‐β induced Treg cells as we show here that the latter do not up‐regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in Treg cells following T‐cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.