Klaus Schweizer

Induction of apoptosis in human lymphocytes treated with Viscum album L. is mediated by the mistletoe lectins.

Viscum album L. (VAL) is a phytopreparation used in adjuvant cancer therapy with both immunostimulatory and DNA stabilizing properties at low drug concentrations and cytostatic/cytotoxic properties at higher concentrations. The present work examines the cytotoxic effects of VAL extracts produced from mistletoes grown on different host trees and of purified toxic proteins from VAL, such as the D-galactose-specific lectin I (ML I), the N-acetyl-D-galactosamine-specific ML II and ML III, and crude viscotoxins towards cultured human lymphocytes. The decrease in the number of cultured lymphocytes and blast cells treated with whole plant extracts from VAL was host tree-specific. Nevertheless, there was no close correlation to the content of MLs or viscotoxins. Using the purified proteins, it became obvious that the cell killing was mediated by the induction of apoptosis, as measured by the appearance of a hypodiploid DNA peak using flow cytometry. ML III was the most effective to induce apoptosis, followed by ML II and ML I, while the viscotoxins and oligosaccharides from VAL did not. By measuring the surface expression of IL-2R alpha chains, transferrin receptors and APO-1/Fas molecules on non-apoptotic T cells, no significant changes were observed at low ML concentrations (1 ng/ml), but their decrease at higher ones. Our findings suggest that there might be at least two different ways of cell killing operative in VAL-mediated cytotoxicity: (a) the typical apoptotic cell death with the appearance of hypo-diploid nuclei, and (b) a direct or indirect killing by damaging the cell membrane with subsequent influx of Ca2+ and of the DNA intercalating dye propidium iodide and cell shrinkage. These effects might not be exclusive, as they probably occur simultaneously.

A co-stimulatory signal through ICAM-β 2 integrin-bindingpotentiates neutrophil phagocytosis

The β2 integrin LFA–1 (lymphocyte function associated antigen; CD11a/CD18) is the common ligand for the intercellular adhesion molecules (ICAMs). Integrins support cell function by providing co-stimulatory second signals that are a precondition for full cell activation first described for ICAM–1–binding to LFA–1 in lymphocytes. Integrins can also serve to activate functions associated with distinct subunits of other integrins. In addition to LFA–1, neutrophils express the β2 integrin Mac–1 (CD11b/CD18; CR3) that apparently contains multiple sites that bind invading microbes directly or through surface–fixed C3 (ref. 6), resulting in activation of the phagocyte function. Expression of the LFA–1 counter–receptor ICAM–1 on endothelial cells occurs only at the site of inflammation. Therefore, in neutrophils, ICAM–1 ligand binding could, as with lymphocytes, also play a part as a co–stimulatory signal to induce full phagocytotic function. We show that in neutrophils, the LFA–1 ligand interaction is the stimulatory signal to express full phagocytotic activation. This is best demonstrated by the rapid association of Streptococcus pyogenes with neutrophils, followed by ingestion, strong oxidative–burst induction and enhanced killing of these bacteria, which are well–known for their resistance to human neutrophil defense. These findings may contribute to the development of therapeutic strategies targeting the modulation of ICAM–1–leukocyte interaction.

Differences in the apoptosis-inducing properties of Viscum album L. extracts.

Viscum albumL.(mistletoe) extracts are widely used in adjuvant cancer therapy. In contrast to purified components, such as mistletoe lectins and viscotoxins, whole plant extracts of mistletoe resulted in DNA stabilization in cyclophosphamide- treated lymphocytes but also provided cytotoxicity in tumour cells and lymphocytes.The killing capacities of mistletoe extracts were host tree-specific and not correlated with mistletoe lectin or viscotoxin content. In human lymphocytes, only mistletoe lectins induced a pathway of apoptotic killing. Within 72 h, the lectin B chains also Increased the number of lymphocytes undergoing apoptosis. This finding suggests that inhibition of protein synthesis by the A chain of the hololectin may accelerate a receptor-mediated killing pathway induced by the B chains. An unexpected finding was related to the mistletoe-mediated killing, which was more effective against CD8+T cells with an activated phenotype than CD19+ B cells and CD4+T cells. In vitro treatment of human neutrophils with mistletoe resulted in a slight decrease of phagocytosis and burst activity.The observed dose-dependent occurrence of two neutrophil subsets with different burst activities indicates differences in their susceptibility to mistletoe and suggests the implication of an induction of the apoptotic killing pathway.

Effects of Viscum album L. on cyclophosphamide-treated peripheral blood mononuclear cells in vitro : sister chromatid exchanges and activation/proliferation marker expression

Based on recently published data, Viscum album L. (VAL) extracts have been shown to provide a DNA stabilizing effect which seems to be restricted to the peripheral blood mononuclear cells (PBMC). We have now investigated whether VAL exerts effects of cellular protection for phytohemagglutinin-activated PBMC treated with cyclophosphamdie (CP) in vitro. The addition of VAL resulted in a slight reduction of CP-induced sister chromatid exchanges of cultured PBMC from healthy individuals. The incubation with CP significantly reduced the expression of the low affinity interleukin-2 receptor (IL-2R alpha chain) and of transferrin receptor (TfR) on PHA-stimulated T lymphocytes. The addition of 10 micrograms/ml VAL was protective against the CP-induced depression of IL-2R alpha chain and TfR expression on these cells. The simultaneous addition of CP and purified VAL components, such as ML I, ML II/III, and viscotoxins did not significantly change expression of IL-2R alpha chain and TfR on T cells. Thus, so far undefined VAL components might be responsible for the observed protection effects of the whole plant extract. The results presented here should encourage investigation of this drug, which might become an interesting adjuvant in cancer therapy.

Viscum album L. Extracts reduce sister chromatid exchanges in cultured peripheral blood mononuclear cells

Increasing concentrations of Viscum album L. extracts were shown to significantly reduce sister chromatid exchange (SCE) frequency of phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) of healthy individuals. This decrease of SCE could not be explained either by changes in lymphocyte subpopulations, by cytostatic effects of the drug or by accelerated proliferation of PHA-stimulated PBMC. Currently, no other cells tested have shown this effect. One therapeutic effect of these anti-mutagenic drugs could be a stabilisation of mononuclear blood cell DNA.

Effects of a phytopreparation from Helleborus niger on immunocompetent cells in vitro.

Extracts of Helleborus species are used as phytopreparations with immunostimulatory properties in Romanian traditional medicine. In Germany, Helleborus niger is used in homeopathy and as an adjuvant therapy in the treatment of tumor patients in anthroposophical medicine. In vitro application of an aqueous extract from Helleborus niger resulted in a slight induction of sister chromatid exchanges (SCE) in cultured peripheral blood mononuclear cells (PBMC) from healthy individuals, an effect associated with a slight increase of the [3H]thymidine uptake in the DNA of isolated lymphocytes. Since the cytokines interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha were reported to increase the number of SCE, we measured the concentrations of these cytokines in the supernatants of cultured PBMC treated with the plant extract. Here, no significant changes were observed as compared with the controls, but a trend to higher supernatant concentrations of TNF-alpha in six out of ten individuals was noted. Compared with lymphocytes treated with the alkylating substance, cyclophosphamide, the increase of the SCE levels induced by the plant extract is weak. The relevance of this DNA destabilizing property remains to be clarified.

ACTIVATION OF GRANULOCYTES BY PHORBOL-12-MYRISTATE-14-ACETATE (PMA) ENHANCES PHAGOCYTOSIS OF Streptococcus pyogenes

The expression of the M protein is thought to be responsible for the ability of Streptococcus pyogenes to resist phagocytosis by human polymorphonuclear leukocytes (PMNL)6. Recently M-like proteins (Mrp) were shown to contribute to this major virulence mechanism10.

Flow cytometric cerebrospinal fluid analysis in children

Flow cytometry (FC) is of increasing importance for the analysis of cerebrospinal fluid (CSF) lymphocytes because of its ability to detect a large spectrum of cellular characteristics (granularity, volume, surface antigen expression) even in small amounts of cells. Data on CSF FC in children are very limited. Here, we summarize our 3-year experience of CSF FC routinely performed in pediatric patients with assumed inflammatory central nervous system (CNS) disease. Among 109 samples sent for analysis, flow cytometric detection of major leukocyte subsets was possible in 78% (85 out of 109), which exceeds the 31% rate of our retrospective microscopic pediatric control group. Apart from physiologic lymphocytes (100%) or monocytes (48%), 11 out of these 85 samples showed granulocytes, two showed proliferated monocytes, and nine displayed proliferated lymphocytes. In most children, the proliferated lymphocytes consisted of a polyclonal population of CD4+ and CD8+ T cells. Compared with literature data, eight children showed abnormally composed lymphocyte subsets (surface antigen expression) within the main lymphocyte population. However, none of these changes was specific for distinct diseases or allowed a distinction between patients with and without primary inflammatory processes. These data suggest that CSF FC may be the most effective modality to differentiate major CSF leukocyte subsets. At present, further differentiation of distinct cell populations, such as proliferated lymphocytes, is of limited clinical impact. This may, however, gain increasing interest in the future.

Sister chromatid exchange-inducing DNA lesions and depression of activation markers on the surface of cultured peripheral blood mononuclear cells after the addition of streptococcal pyrogenic exotoxins A and C.

Cultivation of peripheral blood mononuclear cells (PBMC) in the presence of streptococcal pyrogenic exotoxins (SPE) A and C resulted in a significant induction of sister chromatid exchange (SCE)-inducing DNA lesions. Concomitantly, the expression of interleukin-2 receptor α chain (IL-2Rα chain), transferrin receptor (TfR), and major histocompatibility complex class II molecule HLA-DR on the surface of phytohemagglutinin-activated T cells from whole blood culture cells (WBCC) significantly decreased within 72 h, that is at least two cell cycles, whereas unstimulated T cells from WBCC did not express these markers but had lost their CD3 molecules, an effect reported to precede apoptosis as part of a T cell inactivation pathway. However, no apoptotic cells were observed within a cultivation period of 120 h. We observed clearcut differences in the responses towards SPE A in WBCC and isolated lymphocytes, since SPE A-treated lymphocytes showed an increase in the [3H]-thymidine incorporation and did express IL-2Rαchain and TfR on their cell surface. Regardless of the precise underlying mechanism, T cells from WBCC seem to be in a state of functional incompetence. The data presented here are the first to provide strong evidence that streptococcal toxins produce SCE-inducing DNA lesions in PBMC, an effect that might contribute to the process of immune cell lethality in streptococcal toxic shock-like syndrome and could be of pivotal importance in the pathogenesis of severe streptococcal disease.

Recombinant Human Granulocyte Colony–Stimulating Factor Does Not Influence Distribution or Sister Chromatid Exchange Frequency of Proliferating Mononuclear Cells of the Peripheral Blood

Phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC) were treated with increasing concentrations of filgrastim, the unglycosylated methionine granulocyte colony-stimulating factor of man (rhG-CSF), and cultured for 72 h. There were no impaired proliferation or differentiation of proliferating PBMC, no impaired expression of activation markers such as the low-affinity interleukin 2 receptor and transferrin receptor, and no induction of sister chromatid exchanges. Under these conditions, no effects of a general DNA destabilization of peripheral blood leukocytes was observed. Thus, longterm administration of therapeutical concentrations of rhG-CSF should not produce severe mutagenic effects.