Nora Müller

Glucocorticoids exert opposing effects on macrophage function dependent on their concentration

Glucocorticoids (GCs) are involved in the modulation of macrophage function and thereby control the hosts immune responses to pathogens. However, neither the role of hormone concentration nor the differential contribution of the glucocorticoid (GR) and the mineralocorticoid receptors (MR) to these activities are known. Here we show that low levels of corticosterone enhance NO production as well as mRNA expression of pro‐inflammatory cytokines, chemokines and enzymes required for mediator synthesis. In contrast, at high corticosterone concentrations macrophage function was strongly repressed. Importantly, inactivation of the GR by lentiviral delivery of siRNAs abrogated both the immunostimulatory and the immunosuppressive GC actions whereas inactivation of the MR had no effect. Furthermore, removal of endogenous GCs by adrenalectomy in vivo induced a preactivated state in macrophages that could be modulated by corticosterone. We conclude that GCs exert distinct effects on macrophage function dependent on their concentration, and that they primarily act through the GR despite concomitant expression of the MR.

Glucocorticoids engage different signal transduction pathways to induce apoptosis in thymocytes and mature T cells.

Glucocorticoids (GC) induce apoptosis in a variety of cells, but their exact mode of action is controversial. Although initiation relies on the GC receptor (GR) and de novo gene expression, the effector phase differs among cell types. Proteasomal degradation as well as caspase-3, - 8, and -9 activity are essential for GC-induced apoptosis in murine thymocytes, but the same enzymes are dispensable in splenic T cells. Live imaging by confocal microscopy revealed that lysosomal cathepsin B, an unrecognized component of this pathway to date, becomes rapidly activated in thymocytes after GC exposure. This is followed by leakage of cathepsin B into the cytosol, nuclear condensation, and processing of caspase-8 and -3. According to our model, activation of caspase-3 by caspase-9 in thymocytes occurs both directly as well as indirectly via a lysosomal amplification loop. Interestingly, acute T lymphoblastic leukemia cells depend on caspase activity to undergo GC-induced cell death similar to thymocytes. Collectively, the apoptotic program induced by GCs comprises cell type-specific as well as common features.

Impact of female sex hormones on the maturation and function of human dendritic cells.

Problem  During pregnancy, the immune and the endocrine system cooperate to ensure that the fetal allograft develops without eliciting a maternal immune response. This is presumably in part achieved by dendritic cells (DCs) that play a dominant role in maintaining peripheral tolerance. In this study, we investigated whether female sex hormones, such as human chorionic gonadotropin (hCG), progesterone (Prog), and estradiol (E2), which are highly elevated during pregnancy, induce the differentiation of DCs into a tolerance‐inducing phenotype.

Measles Virus Interacts with and Alters Signal Transduction in T-Cell Lipid Rafts

ABSTRACT By a contact-dependent surface interaction, the measles virus (MV) glycoprotein complex induces a pronounced inhibition of T-cell proliferation. We now show that MV directly interacts with glycosphingolipid-enriched membrane microdomains on human primary T cells and alters recruitment and segregation of membrane proximal signaling components. Contact-dependent interference with T-cell receptor-stimulated tyrosine phosphorylation and Ca mobilization is a late event seen 24 h after MV treatment. In contrast, stimulated recruitment of pleckstrin homology domain-containing proteins such as Akt and Vav is inhibited early after MV contact, as is segregation of the activated Akt kinase from rafts. Tyrosine phosphorylation of the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), p85, is apparently normal then, yet this protein fails to partition to the lipid raft fraction, and this is associated with stable expression of its negative regulator Cbl-b. Thus, by interaction with lipid rafts, MV contact initially targets recruitment of PI3K by preventing stimulated Cbl-b degradation and activation of PI3K-dependent signaling components.

A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats

Administration of the CD28 superagonistic antibody JJ316 is an efficient means to treat autoimmune diseases in rats, but the humanized antibody TGN1412 caused devastating side effects in healthy volunteers during a clinical trial. Here we show that JJ316 treatment of rats induced a dramatic redistribution of T lymphocytes from the periphery to the secondary lymphoid organs, resulting in severe T lymphopenia. Live imaging of secondary lymphoid organs revealed that JJ316 administration almost instantaneously (<2 minutes) arrested T cells in situ. This reduction in T cell motility was accompanied by profound cytoskeletal rearrangements and increased cell size. In addition, surface expression of lymphocyte function-associated antigen-1 was enhanced, endothelial differentiation sphingolipid G protein-coupled receptor 1 and L selectin levels were downregulated, and the cells lost their responsiveness to sphingosine 1-phosphate-directed migration. These proadhesive alterations were accompanied by signs of strong activation, including upregulation of CD25, CD69, CD134, and proinflammatory mediators. However, this did not lead to a cytokine storm similar to the clinical trial. While most of the early changes disappeared within 48 hours, we observed that CD4+CD25+FoxP3+ regulatory T cells experienced a second phase of activation, which resulted in massive cell enlargement, extensive polarization, and increased motility. These data suggest that CD28 superagonists elicit 2 qualitatively distinct waves of activation.

ORIGINAL ARTICLE: Impact of Female Sex Hormones on the Maturation and Function of Human Dendritic Cells

Problem  During pregnancy, the immune and the endocrine system cooperate to ensure that the fetal allograft develops without eliciting a maternal immune response. This is presumably in part achieved by dendritic cells (DCs) that play a dominant role in maintaining peripheral tolerance. In this study, we investigated whether female sex hormones, such as human chorionic gonadotropin (hCG), progesterone (Prog), and estradiol (E2), which are highly elevated during pregnancy, induce the differentiation of DCs into a tolerance‐inducing phenotype.

Measles Virus Contact with T Cells Impedes Cytoskeletal Remodeling Associated with Spreading, Polarization, and CD3 Clustering

CD3/CD28‐induced activation of the PI3/Akt kinase pathway and proliferation is impaired in T cells after contact with the measles virus (MV) glycoprotein (gp) complex. We now show that this signal also impairs actin cytoskeletal remodeling in T cells, which loose their ability to adhere and to promote microvilli formation. MV exposure results in an almost complete collapse of membrane protrusions associated with reduced phosphorylation levels of cofilin and ezrin/radixin/moesin (ERM) proteins. Consistent with their inability to activate Cdc42 and Rac1 in response to the ligation of CD3/CD28, T cells exposed to MV fail to acquire a morphology consistent with spreading and lamellopodia formation. In spite of these impairments of cytoskeleton‐driven morphological alterations, these cells are recruited into conjugates with dendritic cells as efficiently as control T cells. The signal elicited by MV, however, prevents T cells to polarize as documented by a failure to redistribute the microtubule organizing center toward the synapse. Moreover, CD3 cannot be efficiently clustered and redistributed to the central region of the immunological synapse. Thus, by inducing microvillar collapse and interfering with cytoskeletal remodeling, MV signaling disturbs the ability of T cells to adhere, spread, and cluster receptors essential for sustained T‐cell activation.

The glycoprotein-hormones activin A and inhibin A interfere with dendritic cell maturation

BackgroundPregnancy represents an exclusive situation in which the immune and the endocrine system cooperate to prevent rejection of the embryo by the maternal immune system. While immature dendritic cells (iDC) in the early pregnancy decidua presumably contribute to the establishment of peripheral tolerance, glycoprotein-hormones of the transforming growth factor beta (TGF-beta) family including activin A (ActA) and inhibin A (InA) are candidates that could direct the differentiation of DCs into a tolerance-inducing phenotype.MethodsTo test this hypothesis we generated iDCs from peripheral-blood-monocytes and exposed them to TGF-beta1, ActA, as well as InA and Dexamethasone (Dex) as controls.ResultsBoth glycoprotein-hormones prevented up-regulation of HLA-DR during cytokine-induced DC maturation similar to Dex but did not influence the expression of CD 40, CD 83 and CD 86. Visualization of the F-actin cytoskeleton confirmed that the DCs retained a partially immature phenotype under these conditions. The T-cell stimulatory capacity of DCs was reduced after ActA and InA exposure while the secretion of cytokines and chemokines was unaffected.ConclusionThese findings suggest that ActA and InA interfere with selected aspects of DC maturation and may thereby help preventing activation of allogenic T-cells by the embryo. Thus, we have identified two novel members of the TGF-beta superfamily that could promote the generation of tolerance-inducing DCs.

MIC-1 (a multifunctional modulator of dendritic cell phenotype and function) is produced by decidual stromal cells and trophoblasts

BACKGROUND Macrophage inhibitory cytokine-1 (MIC-1) is a multifunctional cytokine produced in high amounts by placental tissue. Inhibiting trophoblast invasion and suppressing inflammation through inhibition of macrophage activation, MIC-1 is thought to provide pleiotropic functions in the establishment and maintenance of pregnancy. So far, little is known about the decidual cell subsets producing MIC-1 and the effect of this cytokine on dendritic cells (DCs), which are known to play a distinct role in the development of pro-fetal tolerance in pregnancy. METHODS To identify the decidual cell types expressing and secreting MIC-1, immunohistochemical staining, PCR experiments, western blot analysis and ELISAs were performed. Immature DCs (iDCs) were generated from peripheral blood-derived monocytes and differentiated in the presence of MIC-1 or dexamethasone (Dex) for control. Migratory and proliferative activity of DCs after MIC-1 exposure was investigated by migration and proliferation assay. Cytokine secretion after MIC-1 exposure was tested in isolated uNK cells, isolated CD14+ monocytes, monocyte-derived iDCs and mature DCs. Subsequently, the phenotype of DCs was studied using FACS analysis. To test the T-cell stimulatory capacity of pre-incubated DCs, mixed lymphocyte reaction was applied. Finally, the expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) after the exposure of MIC-1 to maturing DCs was analysed by western blot. RESULTS Immunohistochemical staining, PCR and western blot experiments demonstrated that MIC-1 is mainly expressed by trophoblast cells and decidual stromal cells. Analysis of the MIC-1 secretion of decidual cell types by ELISA again characterized trophoblast and stromal cells as main producers. The migratory activity of iDCs was significantly induced by MIC-1. No changes in proliferative activity of DCs were observed after MIC-1 pre-incubation. The secretion of pro- or anti-inflammatory cytokines was not affected significantly by MIC-1. Studying the phenotype of DCs after MIC-1 exposure by FACS analysis, we observed that MIC-1 suppresses the expression of typical maturation molecules such as CD25 and CD83 as well as of CD86 during cytokine-induced DC maturation similar to Dex. In addition, T-cell stimulatory capacity of DCs was significantly reduced after MIC-1 exposure. MIC-1 was also able to increase slightly the expression of IDO (a key immunomodulatory enzyme promoting periphereal tolerance) in maturing DCs. CONCLUSIONS We have identified MIC-1 as a novel factor (secreted by decidual cells in early pregnancy) that could promote the increase of a tolerogenic subtype of DC in decidua.

CD9 clustering and formation of microvilli zippers between contacting cells regulates virus-induced cell fusion.

Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9‐specific monoclonal antibody, inhibits the release of HIV‐1 and canine distemper virus (CDV)‐ but not measles virus (MV)‐induced cell–cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop of CD9, induces rapid relocation and clustering of CD9 in net‐like structures at cell–cell contact areas. High‐resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9‐associated proteins β1‐integrin and EWI‐F were co‐clustered with CD9 at cell–cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within CD9 clusters, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell–cell fusion by controlling the access of the fusion machinery to cell contact areas.