Günther Böck

Activation of PDGF receptor α in vascular smooth muscle cells by mechanical stress

Hypertension increases mechanical force on the arterial wall by as much as 30%, resulting in marked alterations in signal transductions and gene expression in vascular smooth muscle cells (VSMCs) that contribute to matrix protein synthesis, cell proliferation, and differentiation. How the mechanical stimuli are converted into a biological signal in cells has yet to be studied. We investigated the role of both cyclic strain and shear stresses in initiating the cellular signaling on cultured VSMCs and found that mechanical forces evoked activation of mitogen‐activated protein kinases, followed by enhanced DNA binding activity of transcription factor AP‐1. Physical forces rapidly induced phosphorylation of platelet‐derived growth factor receptor (PDGFR) α, an activated state. When GRB2, an adapter protein, was immunoprecipitated from treated VSMCs followed by Western blot analysis with anti‐phosphotyrosine, ‐PDGFRα, and ‐GRB2 antibodies, respectively, phosphotyrosine positive staining was observed on PDGFRα bands of the same blot in stretch‐stressed VSMCs, supporting the mechanical stress‐induced activation of PDGFRα. Conditioned medium from stretch‐stressed VSMCs did not result in PDGFRα phosphorylation, and antibodies binding to all forms of PDGFs did not block stress‐induced PDGFRα activation. Thus, mechanical stresses may directly perturb the cell surface or alter receptor conformation, thereby initiating signaling pathways normally used by growth factors.—Hu, Y., Böck, Güunther, Wick, G., Xu, Q., Activation of PDGF receptor α in vascular smooth muscle cells by mechanical stress. FASEB J. 12, 1135–1142 (1998)

Puma cooperates with Bim, the rate-limiting BH3-only protein in cell death during lymphocyte development, in apoptosis induction.

The physiological role of B cell lymphoma 2 (Bcl-2) homology 3–only proteins has been investigated in mice lacking the individual genes identifying rate-limiting roles for Bim (Bcl-2–interacting mediator of cell death) and Puma (p53–up-regulated modulator of apoptosis) in apoptosis induction. The loss of Bim protects lymphocytes from apoptosis induced by cytokine deprivation and deregulated Ca++ flux and interferes with the deletion of autoreactive lymphocytes and the shutdown of immune responses. In contrast, Puma is considered the key mediator of p53-induced apoptosis. To investigate the hypothesis that Bim and Puma have overlapping functions, we generated mice lacking both genes and found that bim−/−/puma−/− animals develop multiple postnatal defects that are not observed in the single knockout mice. Most strikingly, hyperplasia of lymphatic organs is comparable with that observed in mice overexpressing Bcl-2 in all hemopoietic cells exceeding the hyperplasia observed in bim−/− mice. Bim and Puma also have clearly overlapping functions in p53-dependent and -independent apoptosis. Their combined loss promotes spontaneous tumorigenesis, causing the malignancies observed in Bcl-2 transgenic mice, but does not exacerbate the autoimmunity observed in the absence of Bim.

Human renal‐cell carcinoma tissue contains dendritic cells

Immune surveillance of cancer requires antigen‐presenting cells which activate T cells specific for tumor‐associated antigens. We show here that substantial numbers of dendritic cells, which are the most potent antigen‐presenting cells, emigrate from renal‐tumor explants in organ culture. Tumor‐derived dendritic cells presented with all characteristics of mature dendritic cells. Dendritic cells could be identified by typical cytoplasmic projections (=veils). They expressed high levels of MHC products and of the co‐stimulator CD86 (B7‐2). Dendritic cells expressed the CD45RO isoform but not CD45RA. The most important point was that up to 9% of the emigrating leukocytes expressed the CD83 antigen, a specific marker for mature dendritic cells. CD83+ cells were approximately 40‐fold enriched in the tumor tissue as compared to the peripheral blood. In contrast to cultured blood dendritic cells, tumor‐emigrant dendritic cells had a reduced potential to capture soluble antigen, as shown by the exclusion of fluoresceinated Dextran molecules. Finally, in mixed leukocyte reactions, tumor‐derived dendritic cells were able to stimulate naive T cells from cord blood, which is a unique feature of dendritic cells. This study demonstrates that genuine dendritic cells reside in or infiltrate renal‐cell carcinoma tissue. The failure of patients with renal‐cell carcinoma to mount an anti‐tumor immune response despite the presence of professional antigen‐presenting cells in the tumor tissue suggests that tumor‐associated dendritic cells are suppressed in situ, in a similar way to that described for tumor‐infiltrating lymphocytes.

Bacillus Calmette‐Guérin mycobacteria stimulate human blood dendritic cells

Bacillus Calmette‐Guérin (BCG) mycobacteria have been used as adjuvant in the active immunotherapy of various human cancers. In addition, dendritic cells, which are the most potent antigen‐presenting cells, have been shown to be capable of initiating anti‐tumor immune responses. Here we investigated the effects of BCG on dendritic cells cultured from human blood. Addition of BCG resulted in rapid homotypic adhesion of dendritic cells. Moreover, BCG concentrations ranging from 104 to 106 bacteria/ml enhanced expression of the dendritic‐cell‐maturation antigen CD83 and of the T‐cell co‐stimulator CD86 (B7‐2) in a dose‐dependent manner. Concomitant with the increase of CD83 and CD86 expression, the cells lost the ability to capture soluble antigens, as determined by the exclusion of fluoresceinated Dextran molecules. Strikingly, the same dosages of BCG‐bacteria stimulated TNF‐α‐gene transcription and TNF‐α‐protein release from dendritic cells in a dose‐dependent fashion. BCG infection of dendritic cells in the presence of a neutralizing antibody directed against TNF‐α inhibited CD83 expression by more than 50% indicating that the BCG‐induced maturation of dendritic cells was at least partially mediated by dendritic‐cell‐derived TNF‐α. The finding that BCG activates the most potent antigen‐presenting cells reveals a plausible immunological mechanism of the occasionally observed anti‐tumor activity of BCG.

Monocyte-derived dendritic cells release neopterin

Increased neopterin concentrations in body fluids are found in diseases associated with activated, cell‐mediated immunity including infections, autoimmune diseases, and certain malignancies. Monocytes/macrophages are known to secrete large amounts of neopterin upon stimulation with interferon‐γ (IFN‐γ). Ontogenetically, the major part of dendritic cells (DC) belongs to the myeloid lineage. Therefore, we investigated whether cultured monocyte‐derived DC can elaborate neopterin. Cells were treated with cytokines in the presence or absence of monocyte‐conditioned medium as a maturation stimulus. DC secreted an average 3.5 nmol/l neopterin. In response to IFN‐γ, cells significantly increased their output of neopterin. In distinction to monocytes/macrophages, neopterin production in DC was highly sensitive to IFN‐α and IFN‐β. Further, lipopolysaccharides (LPS) enhanced neopterin synthesis, whereas tumor necrosis factor α, interleukin (IL)‐1β, IL‐2, IL‐10, and IL‐18 were ineffective. Simultaneously, tryptophan degradation by induction of indoleamine (2,3)‐dioxygenase (IDO) was tested in stimulated cells. Our results showed that IFN‐γ as well as LPS are inducers of IDO in DC.

Dendritic cells generated from blood precursors of chronic myelogenous leukemia patients carry the Philadelphia translocation and can induce a CML-specific primary cytotoxic T-cell response.

Dendritic cells (DC) are professional antigen‐presenting cells specialized in the initiation of primary immune responses. We were interested to know whether mature DC can be grown in vitro from peripheral blood mononuclear cells (PBMC) of patients with chronic myelogenous leukemia (CML), and whether they carry the Philadelphia (Ph) translocation. Using a method recently described, DC were generated from PBMC precursors of 12 patients with CML using GM‐CSF, IL‐4, and monocyte‐conditioned medium. DC exhibited the typical morphology with thin cytoplasmatic processes and expressed high levels of MHC class II, CD86, and CD83 typical for mature DC. After sorting with the monoclonal antibody CD83, a cell population of more than 95% CD83 positive cells was obtained. The presence of the Ph translocation was analyzed in these cells, in PBMC, lymphoblastoid cell lines (LCL), and in phytohemagglutinin (PHA)‐induced T blasts from the same patients by fluorescence in situ hybridization (FISH). In contrast to all other cells analyzed, the vast majority of DC (95.9 ± 0.7%) displayed the Ph translocation, irrespective of disease stage or therapy. PBMC were predominantly positive for the Ph chromosome (67.6 ± 7.3%), whereas only 11.4 ± 1% of the B cells and 4.4 ± 1.1% of the PHA blasts carried the Ph translocation. Using such leukemic DC as antigen‐presenting cells, a primary CML‐directed cytotoxic immune response in vitro was obtained, as shown by the specific recognition of Ph chromosome positive cells. We conclude that DC can be generated from blood progenitors of CML patients in vitro and exhibit, to a large extent, the Ph translocation. Such DC, which in a preliminary experiment have been able to induce a primary CML‐directed cytotoxic immune response in vitro, might be ideal candidates for adoptive immunotherapy either by direct transfer of DC for in vivo generation of a T‐cell response or by in vitro generation of CML‐specific cytotoxic autologous or HLA‐matched normal T‐cell clones for use in vivo. Genes Chromosomes Cancer 20:215–223, 1997.

CD4+CD8+TCRlow thymocytes express low levels of glucocorticoid receptors while being sensitive to glucocorticoid‐induced apoptosis

While signaling by either the TCR or glucocorticoid receptor (GR) can induce apoptosis in thymocytes, recent studies have shown that combining these signals results in survival of CD4+CD8+ thymocytes. Although glucocorticoids (GC) in this way may directly affect T cell selection, no data are available addressing GR expression in thymocyte subsets and in individual cellswithin subsets. We studied GR expression by combining immunofluorescence cell surface staining for CD4, CD8 and TCR with intracellular staining of GR in four‐color cytometry. Significant differences of GR expression were observed in various thymocyte subsets, although a homogeneous distribution of GR expression in individual thymocyte subsets emerged. The highest GR expression was found in CD4–CD8–TCR– thymocytes, and decreased during development via the CD4–CD8+TCR– subpopulation into the CD4+CD8+TCRlow subset. Interestingly, the latter population, although expressing less than half the GR density of CD4–CD8–TCR– cells, is the most sensitive subset to GC‐in‐duced apoptosis. Up‐regulation of TCR expression by the CD4+CD8+TCRlow subset to CD4+CD8+TCRhigh cells was accompanied by a parallel increase in GR expression. The latter finding and the presence of a homogeneous distribution of GR in each thymocyte subset provides an experimental basis for the concept that GR can antagonize TCR‐mediated signals at a constant rate relative to TCR expression.

Neopterin and 7,8-dihydroneopterin induce apoptosis in the rat alveolar epithelial cell line L2

The neopterin derivatives, neopterin and 7,8‐dihydroneopterin, modulate the cellular oxidant‐antioxidant balance as well as the expression of the inducible nitric oxide synthase (iNOS) gene. Since apoptosis can be induced by reactive oxygen intermediates and nitric oxide (NO) we investigated whether these neopterin derivatives induce apoptotic cell death. As model we selected the rat alveolar epithelial cell line L2. 24 h incubation of neopterin (1–1000 μM) as well as 7,8‐dihydroneopterin (1–1000 μM) resulted in a significant increase of percent apoptotic cells (measured by FACS analysis). Coincubation of both pteridines with the cytomix (interferon‐γ plus tumor necrosis factor‐α) led to a significantly higher apoptosis than the cytomix alone. In contrast to the cytomix, no iNOS gene expression and no NO release could be detected after incubation with neopterin or 7,8‐dihydroneopterin. We conclude that neopterin and 7,8‐dihydroneopterin are per se inducers of apoptosis which is not mediated by nitric oxide. This may be of importance in inflammatory pulmonary diseases associated with an activation of the cellular immune system.

HIV-1 gp41 contains two sites for interaction with several proteins on the helper T-lymphoid cell line, H9.

ObjectiveTo characterize putative binding sites for HIV-1 gp41 to H9 cells. DesignBased on accumulating evidence in the literature that HIV-1 can bind to cell surfaces independent of CD4, we attempted to clarify whether gp41, the transmembrane HIV-1 protein, contributes to CD4-independent binding. We therefore tested binding of gp41 to cells. MethodsUsing fluorescence-activated cell-sorter analysis, we examined the binding of recombinant gp160 (rgp160) and soluble gp41 (sgp41; Env amino acids 539–684) to H9 cells, and located the putative binding site(s) of gp41 by inhibition using 16 HIV-1 Env peptides. Putative HIV-1 receptor proteins in H9 cell lysates were Western blotted and precipitated using sgp41. Resultssgp41 bound to the CD4+ H9 cells and rgp160 bound to H9 cells independent of gp120-binding sites on CD4 molecules. Two gp41 peptides (Env amino acids 591—605 and 651—665) inhibited the binding of sgp41 to H9 cells. Four bands, of 37, 40, 55 and 97 kD, were blotted in H9 cell lysates, and three bands, 40, 97 and 108 kD, were observed in the precipitation analysis using lysates of 125l-surface-labelled H9 cells and sgp41 attached to sepharose beads. ConclusionsHIV-1 gp41 contains two putative binding sites to H9 cells. These sites may be located within Env amino acids 591–605 (ERYLKDQQLLGIWGC) and 651–665 (TLLEESQNQQEKNEQ). Using two different techniques, five proteins (37, 40, 55, 97 and 108 kD) were identified in H9 lysates as possible candidates for gp41 binding.

Repression of BIRC5/survivin by FOXO3/FKHRL1 sensitizes human neuroblastoma cells to DNA damage-induced apoptosis.

The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway regulates survival and chemotherapy resistance of neuronal cells, and its deregulation in neuroblastoma (NB) tumors predicts an adverse clinical outcome. Here, we show that inhibition of PI3K-PKB signaling in human NB cells induces nuclear translocation of FOXO3/FKHRL1, represses the prosurvival protein BIRC5/Survivin, and sensitizes to DNA-damaging agents. To specifically address whether FKHRL1 contributes to Survivin regulation, we introduced a 4-hydroxy-tamoxifen-regulated FKHRL1(A3)ERtm allele into NB cells. Conditional FKHRL1 activation repressed Survivin transcription and protein expression. Transgenic Survivin exerted a significant antiapoptotic effect and prevented the accumulation of Bim and Bax at mitochondria, the loss of mitochondrial membrane potential as well as the release of cytochrome c during FKHRL1-induced apoptosis. In concordance, Survivin knockdown by retroviral short hairpin RNA technology accelerated FKHRL1-induced apoptosis. Low-dose activation of FKHRL1 sensitized to the DNA-damaging agents doxorubicin and etoposide, whereas the overexpression of Survivin diminished FKHRL1 sensitization to these drugs. These results suggest that repression of Survivin by FKHRL1 facilitates FKHRL1-induced apoptosis and sensitizes to cell death induced by DNA-damaging agents, which supports the central role of PI3K-PKB-FKHRL1 signaling in drug resistance of human NB.