Susanne Berchtold

CD83 on dendritic cells: more than just a marker for maturation

CD83 has been known for a long time to be one of the best markers for mature dendritic cells (DCs). Studies with herpes simplex virus type 1 (HSV-1)-infected DCs, whereby the viral infection leads to the degradation of CD83, as well as investigations inhibiting CD83 mRNA transport, have provided evidence that CD83 might also be important for DC biology. Recently, we have shown that the soluble extracellular CD83 domain inhibits DC-mediated T-cell proliferation, representing the first report describing a functional role for CD83.

Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4.

Glucocorticoid excess induces hyperglycemia, which may result in diabetes. The present experiments explored whether glucocorticoids trigger apoptosis in insulin-secreting cells. Treatment of mouse β-cells or INS-1 cells with the glucocorticoid dexamethasone (0.1 μmol/l) over 4 days in cell culture increased the number of fractionated nuclei from 2 to 7 and 14%, respectively, an effect that was reversed by the glucocorticoid receptor antagonist RU486 (1 μmol/l). In INS-1 cells, dexamethasone increased the number of transferase-mediated dUTP nick-end labeling–staining positive cells, caspase-3 activity, and poly-(ADP-) ribose polymerase protein cleavage; decreased Bcl-2 transcript and protein abundance; dephosphorylated the proapoptotic protein of the Bcl-2 family (BAD) at serine155; and depolarized mitochondria. Dexamethasone increased PP-2B (calcineurin) activity, an effect abrogated by FK506. FK506 (0.1 μmol/l) and another calcineurin inhibitor, deltamethrin (1 μmol/l), attenuated dexamethasone-induced cell death. The stable glucagon-like peptide 1 analog, exendin-4 (10 nmol/l), inhibited dexamethasone-induced apoptosis in mouse β-cells and INS-1 cells. The protective effect of exendin-4 was mimicked by forskolin (10 μmol/l) but not mimicked by guanine nucleotide exchange factor with the specific agonist 8CPT-Me-cAMP (50 μmol/l). Exendin-4 did not protect against cell death in the presence of cAMP-dependent protein kinase (PKA) inhibition by H89 (10 μmol/l) or KT5720 (5 μmol/l). In conclusion, glucocorticoid-induced apoptosis in insulin-secreting cells is accompanied by a downregulation of Bcl-2, activation of calcineurin with subsequent dephosphorylation of BAD, and mitochondrial depolarization. Exendin-4 protects against glucocorticoid-induced apoptosis, an effect mimicked by forskolin and reversed by PKA inhibitors.

Human monocyte derived dendritic cells express functional P2X and P2Y receptors as well as ecto-nucleotidases

We investigated the expression and function of P2 receptors and ecto‐nucleotidases on human monocyte derived dendritic cells (DC). In addition we analyzed the effect of extracellular ATP on the maturation of DC. By RT‐PCR, DC were found to express mRNA for several P2X (P2X1, P2X4, P2X5, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y10, P2Y11) receptors. As shown by FURA‐2 measurement, triggering of P2 receptors resulted in an increase in free intracellular Ca2+. In combination with Tumor necrosis factor‐α, ATP increased the expression of the DC surface markers CD80, CD83 and CD86 indicating a maturation promoting effect. DC expressed the ecto‐apyrase CD39 and the ecto‐5′‐nucleotidase CD73 as demonstrated by RT‐PCR. Extracellular ATP was rapidly hydrolyzed by these ecto‐enzymes as shown by separation of 3H‐labeled ATP metabolites using a thin layer technique. These data suggest that ATP acts as a costimulatory factor on DC maturation.

Signaling Lymphocytic Activation Molecule Is Expressed on Mature CD83+ Dendritic Cells and Is Up-Regulated by IL-1β

Signaling lymphocyte activation molecule (SLAM), a 70-kDa costimulatory molecule that mediates CD28-independent proliferation of T cells and IFN-γ production, has been identified on human T cells, immature thymocytes, and a subset of B cells. We have found that SLAM is expressed on mature but not immature dendritic cells (DC). However, the SLAM-associated protein, is missing in DC. SLAM surface expression is strongly up-regulated by IL-1β. Addition of IL-1β to the DC maturation mixture also increases the stimulatory properties of DC. These findings provide a new marker for DC maturation and help to explain two areas of DC biology. First, SLAM is a receptor for the measles virus, previously shown to infect DC. Second, SLAM could possibly contribute to the enhanced immunostimulatory functions of DC that are observed following the addition of IL-1.

Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC) and represents the best marker for mature DC. Here, we report the cloning of the cDNA encoding mouse CD83 (mCD83) from a murine bone marrow‐derived DC (BM‐DC) cDNA library. DNA sequence analysis revealed a 196 amino acid protein including a signal peptide of 21 amino acids which shares 63% amino acid identity with hCD83. Using Northern blot analyses, mCD83 mRNA was found to be strongly expressed in mouse BM‐DC and its expression was upregulated following stimulation with LPS or TNF‐α. Transfection experiments using COS‐7 cells revealed that mCD83 is glycosylated. Furthermore, the extracellular CD83 domain was recombinantly expressed in Escherichia coli and one‐dimensional NMR data strongly support that the protein is structurally folded.

Yersinia enterocolitica Targets Cells of the Innate and Adaptive Immune System by Injection of Yops in a Mouse Infection Model

Yersinia enterocolitica (Ye) evades the immune system of the host by injection of Yersinia outer proteins (Yops) via a type three secretion system into host cells. In this study, a reporter system comprising a YopE-β-lactamase hybrid protein and a fluorescent staining sensitive to β-lactamase cleavage was used to track Yop injection in cell culture and in an experimental Ye mouse infection model. Experiments with GD25, GD25-β1A, and HeLa cells demonstrated that β1-integrins and RhoGTPases play a role for Yop injection. As demonstrated by infection of splenocyte suspensions in vitro, injection of Yops appears to occur randomly into all types of leukocytes. In contrast, upon infection of mice, Yop injection was detected in 13% of F4/80+, 11% of CD11c+, 7% of CD49b+, 5% of Gr1+ cells, 2.3% of CD19+, and 2.6% of CD3+ cells. Taking the different abundance of these cell types in the spleen into account, the highest total number of Yop-injected cells represents B cells, particularly CD19+CD21+CD23+ follicular B cells, followed by neutrophils, dendritic cells, and macrophages, suggesting a distinct cellular tropism of Ye. Yop-injected B cells displayed a significantly increased expression of CD69 compared to non-Yop-injected B cells, indicating activation of these cells by Ye. Infection of IFN-γR (receptor)- and TNFRp55-deficient mice resulted in increased numbers of Yop-injected spleen cells for yet unknown reasons. The YopE-β-lactamase hybrid protein reporter system provides new insights into the modulation of host cell and immune responses by Ye Yops.

Latent Membrane Protein 1 of Epstein-Barr Virus Induces CD83 by the NF-κB Signaling Pathway

ABSTRACT Epstein-Barr virus (EBV) infects human resting B cells and transforms them in vitro into continuously growing lymphoblastoid cell lines (LCLs). EBV nuclear antigen 2 (EBNA2) is one of the first viral proteins expressed after infection. It is able to transactivate viral as well as cellular target genes by interaction with cellular transcription factors. EBNA2 target genes can be studied easily by using an LCL (ER/EB2-5) in which wild-type EBNA2 is replaced by an estrogen-inducible EBNA2. Since the cell surface molecule CD83, a member of the immunoglobulin superfamily and a marker for mature dendritic cells, appeared on the surface of ER/EB2-5 cells within 3 h after the addition of estrogen, we analyzed the regulation of CD83 induction by EBV in more detail. Despite its rapid induction, CD83 turned out to be an indirect target gene of EBNA2. We could show that the viral latent membrane protein 1 (LMP1) is responsible for the induction of CD83 by using an LCL expressing a ligand- or antibody-inducible recombinant nerve growth factor receptor-LMP1 fusion protein. The inducibility of the CD83 promoter by LMP1 was mediated by the activation of NF-κB, as seen by use of luciferase reporter assays using the CD83 promoter and LMP1 mutants. Additionally, fusion constructs of the transmembrane domain of LMP1 and the intracellular signaling domain of CD40, TNF-R1, and TNF-R2 likewise transactivated the CD83 promoter via NF-κB. Our studies show that CD83 is also a target of the NF-κB signaling pathway in B cells.

MAGE-A3 is a frequent tumor antigen of metastasized melanoma.

MAGE-3 or MAGE-A3 is one of the best-characterized tumor antigens. Due to its tumor-restricted expression pattern and its recognition by both cytotoxic and helper T cells it constitutes a promising tumor antigen for anticancer immunotherapy, notably of malignant melanoma. Surprisingly, however, only very limited information is available on the frequency and consistency of its expression in metastatic melanoma lesions. We have now investigated the presence of MAGE-A3 mRNA in 316 tumor samples from 147 melanoma patients by RT-PCR. MAGE-A3 mRNA was detectable in 62% of metastases, and expression did not depend on the site of the metastases (skin, lymph node, and internal organs), age, sex, or duration of disease. Southern blot hybridization of the PCR product enhanced sensitivity of detection, and 26% more samples (13/50 samples tested) scored positive, indicating an even higher MAGE-A3 mRNA frequency than determined by simple ethidium bromide gel analysis. In 62 patients, we were able to investigate MAGE-A3 expression in several metastases from the same patient, and unexpectedly, both MAGE-A3-positive and MAGE-A3-negative metastases were found in 32% of these patients (20 of 62). Immunohistochemistry (using mAb 57B) demonstrated that the expression pattern was usually also heterogeneous with positively and negatively stained tumor cells within one metastasis. However, most (90%) of the metastases (47/52) gave a partially positive signal. Taken together, MAGE-A3 is a common and frequent tumor antigen in metastasized melanoma, but its expression is often heterogeneous.

Cloning and Characterization of the Promoter Region of the Human CD83 Gene

Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells (DC). To get insight into the regulation of hCD83 expression, we cloned a 3037 bp fragment up-stream of the translation initiation codon. Deletion mutants were constructed revealing highest promoter activity in the -261 fragment containing four SP1 binding sites and one NF-kappaB element. Electrophoretic mobility shift assays demonstrated the specific interaction of NF-kappaB factors with the NF-kappaB element as well as specific binding of SP1 and SP3 to the SP1 binding site. The hCD83 promoter was inducible by TNF-alpha. This inducibility was strictly dependent on the intact NF-kappaB element.

Innate immune defense defines susceptibility of sarcoma cells to measles vaccine virus based oncolysis

ABSTRACT The oncolytic potential of measles vaccine virus (MeV) has been demonstrated in several tumor entities. Here, we investigated the susceptibility of eight sarcoma cell lines to MeV-mediated oncolysis and found five to be susceptible, whereas three proved to be resistant. In the MeV-resistant cell lines, we often observed an inhibition of viral replication along with a strong upregulation of the intracellular virus-sensing molecule RIG-I and of the interferon (IFN)-stimulated gene IFIT1. Not only expression of IFIT1 but also phosphorylation of IFN-stimulated Stat1 took place rapidly and were found to be persistent over time. In contrast, susceptible cell lines showed a much weaker, delayed, or completely missing expression of IFIT1 as well as a delayed or only transient phosphorylation of Stat1, whereas exogenic stimulation with beta interferon (IFN-β) resulted in a comparable profound activation of Stat1 and expression of IFIT1 in all cell lines. Pretreatment with IFN-β rendered three of the susceptible cell lines more resistant to MeV-mediated oncolysis. These data suggest that differences in the innate immune defense often account for different degrees of susceptibility of sarcoma cell lines to MeV-mediated oncolysis. From a therapeutic perspective, we were able to overcome resistance to MeV by increasing the multiplicity of infection (MOI) and by addition of the prodrug 5-fluorocytosine (FC), thereby exploiting the suicide gene function of virotherapeutic vector MeV-SCD armed with the SCD fusion protein, which consists of yeast cytosine deaminase and yeast uracil phosphoribosyltransferase.