Sunna Hauschildt

Diethyldithiocarbamate inhibits induction of macrophage NO synthase

We investigated whether sodium diethyldithiocarbamate (DETC), an inhibitor of the nuclear transcription factor kappa B (NFkappaB), modulates induction of NO synthase (NOS) in murine bone marrow‐derived macrophages. A short exposure (between 1 and 16 h) of L929‐cell medium‐preconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1–1 mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappaB is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of, the gene encoding inducible NOS.

The relationship between L-arginine-dependent nitric oxide synthesis, nitrite release and dinitrosyl-iron complex formation by activated macrophages

We identified the source of the nitrogen included into nitric oxide (NO) and studied the relationship between formation of NO, intracellular dinitrosyl ferrous iron complex (DNIC) and release of nitrite by murine bone-marrow-derived macrophages stimulated with E. coli lipopolysaccharide (LPS). NO was trapped in the cell membrane by iron-diethyldithiocarbamate complex (FeDETC) and was detected as a paramagnetic NOFe(DETC)2 complex by electron paramagnetic resonance (EPR) spectroscopy. Macrophages stimulated for 7 h up to 48 h with LPS and then incubated for 2 h with DETC exhibited an anisotropic EPR signal of axial symmetry with g-factor values g perpendicular = 2.035, g parallel = 2.02 and a triplet hyperfine structure (hfs) at g perpendicular characteristic for NOFe(DETC)2. In cells incubated with [15NG]L-arginine instead of [14NG]L-arginine the EPR signal of [15N]OFe(DETC)2 was detected with a doublet hfs at g perpendicular, indicating that NO was generated exclusively from the terminal guanidino-nitrogen of extracellular L-arginine. The ratio of NO formation and of nitrite release changed with time of exposure to LPS, nitrite exceeding NO at early stages of macrophage activation, and NO exceeding nitrite at later stages. DNIC with thiolate ligands (0.5 nmol/10(7) cells) was observed in stimulated macrophages not loaded with DETC. Furthermore, DNIC released from macrophages was trapped in the extracellular medium by bovine serum albumin (BSA) (1 nmol/10(7) cells per 2 h) by formation of a paramagnetic DNIC with BSA. DNIC release not only provides a route for iron loss from activated macrophages, but may also play a role in the cytotoxic and microbiostatic activity of macrophages.

pH-Dependent LAK Cell Cytotoxicity

In the microenvironment of many solid tumors the pH is considerably lower (mean pH between 6.6 to 7.2) than the pH in normal tissue (pH 7.0-7.5). Therefore, the influence of acidic pH on the cytotoxic activity of lymphokine-activated killer cells (LAK cells) after different culture periods was tested. K-562 human erythroleukemia cells were selected as target cells. Cell killing was measured using a two-color flow cytometric method. At physiological pH of 7.4, LAK cell-mediated cytotoxicity ranged from 15 to 48% (E:T ratio = 50:1). The specific lysis of target cells was considerably reduced (up to 70% inhibition of specific lysis) under acidic conditions (pH 6.8, 6.3, 5.8). This effect was independent of donors, duration of the culture period, and the E:T ratio in the cytotoxic assay. As pH gradients surrounding tumor cells may reach values below pH 6.0 at the cell surface, the pH-dependence of LAK cell cytotoxicity could at least partially explain the inhibition of the natural immune response in solid tumors. Therapeutic immunological strategies concerning the enhancement of the natural immune response like LAK cell and IL-2 immunotherapy including IL-2 gene therapy may only be successful if a simultaneous inhibition of the acidification process and an elevation of tumor pH is achieved.

Inhibitors of poly (ADP-ribose) polymerase suppress lipopolysaccharide-induced nitrite formation in macrophages

Stimulating bone marrow derived macrophages with LPS results in the induction of NO-synthase as measured by NO2- formation. Inhibitors of poly(ADP-ribose)polymerase, namely nicotinamide, 3-aminobenzamide and 3-methoxybenzamide, prevented NO2- formation in a dose dependent manner. Inhibition was most effective if the inhibitors were added at the same time as LPS. When added 10 h after exposure to LPS, a time at which expression of the enzyme had reached its maximum, no inhibition was observed. The inhibitors also blocked early events in activation such as protein and RNA-synthesis as well as DNA-synthesis. Thus prevention of NO2- formation may be related to inhibition of these events. Activation of macrophages by LPS was not accompanied by an increase but rather by a small decrease in ADP-ribosyltransferase activity. Whether this decrease plays a physiological role in activation needs further exploration.

Localization of the cell activator lipopeptide in bone marrow-derived macrophages by electron energy loss spectroscopy (EELS)

Synthetic lipopeptide analogues of bacterial lipoprotein constitute potent polyclonal activators for monocytes/macrophages and B lymphocytes. However, the fate of the lipopeptides after their interaction with target cells is as yet unknown. In order to follow the routes and to determine the distribution of the lipopeptide within macrophages after stimulation, we investigated lipopeptide-stimulated bone marrow-derived macrophages using the novel method of electron energy loss spectroscopy (EELS). Our results show that the lipopeptide was present in different compartments of the cell. The major amount of the activator was located within the cytoplasm and the plasma membrane, and minor quantities were detected within the nuclear membrane and the nucleus. The distribution of the lipopeptides varied depending on the duration of stimulation. Our results should help to elucidate the molecular mechanisms of macrophage stimulation by lipopeptides or other cell activators.

Intracellular localization of a lipopeptide macrophage activator: immunocytochemical investigations and EELS analysis on ultrathin cryosections of bone marrow-derived macrophages.

Synthetic lipopeptides, structurally derived from the N‐terminal part of bacterial lipoprotein, constitute macrophage and B‐lymphocyte activators. The molecular mechanism of macrophage activation by lipopeptides still remains unclear. The purpose of our study was to determine the route and kinetics of lipopeptide distribution in bone marrow‐derived macrophages. The intracellular localization of the C‐terminally biotinylated lipodipeptide Pam3Cys‐Ser was investigated on ultrathin cryosections using the biotinstreptavidin‐gold system. Our findings indicate that the lipopeptide penetrates the plasma membrane and can already be found within the cytoplasm, the nuclear membrane, and within the nucleus after 2 min of stimulation. The pattern of lipopeptide distribution obtained 2 min after stimulation resembles that obtained after longer incubation times (8 and 20 min). Correlating distribution patterns were observed when using the method of electron energy loss spectroscopy (EELS). These findings are a clear indication for the rapid uptake of lipopeptides into eukaryotic cells, and are of importance for further studies of the immunostimulating properties of the bacterial lipopeptides and vaccines derived therefrom.

Interaction of the lymphoid cell line BCL1 with lipopeptide analogues of bacterial lipoprotein: electron energy loss spectroscopy (EELS) as a novel method to detect the distribution of the activator within the cells.

The lipopeptide Pam3Cys-Ser, a synthetic analogue of the N-terminal part of bacterial lipoprotein, constitutes a potent activator for B lymphocytes, monocytes/macrophages and several lymphoid cell lines. We applied the novel method of electron energy loss spectroscopy (EELS) to determine, after stimulation, the distribution of the activator within the cell compartments of the lipopeptide sensitive cell line BCL1. Our results show that the lipopeptide, 20 min after the addition to the cell culture, was found at different locations within the cell: A major amount of the mitogen was found in the plasma membrane. Remarkably, considerable amounts of the activator were also found on the cytoplasm, the nuclear membrane, and the nucleus. After 24 h, a substantial amount of the lipopeptide was still present within the cells. These findings should help to elucidate the molecular mechanism of lymphocyte stimulation by lipopeptides. The novel method of EELS, which was demonstrated here using lipopeptides as examples, constitutes a valuable tool of localizing any given compounds such as growth factors or drugs within cells.

Role of proteinkinase C and phosphatidylinositol metabolism in lipopeptide-induced leukocyte activation as signal transducing mechanism

Synthetic lipopeptides are potent B-lymphocyte and macrophage activators. The role of phosphatidylinositol metabolism and proteinkinase C in lipopeptide induced leukocyte activation were investigated. In murine B-lymphocytes and in bone marrow derived macrophages, lipopeptide failed to induce phosphatidylinositol breakdown, whereas in the macrophage cell line P388D1 formation of inositolphosphates was increased. Translocation of proteinkinase C from a cytosolic to a membrane compartment was only observed in the cell line P388D1 indicating that in the other cells tested lipopeptide acts via different signal transduction pathways.

Modulation of protein kinase C activity by NaF in bone marrow derived macrophages

Stimulation of murine bone marrow derived macrophages with NaF, prelabeled with [1‐14C]oleate and [3H]inositol, increased the production of inositol phosphates and the release of 1,2‐[14C]diacylglycerol (DAG). Moreover, NaF also induced activation of protein kinase C. These results indicate that bone marrow derived macrophages exhibit a phosphatidyl‐4,5‐bisphosphate phospholipase C activity, sensitive to NaF, which might be modulated by G‐proteins. Activation of protein kinase C could have been mediated by NaF‐induced release of DAG.

Phosphatidylinositol Metabolism and Protein Kinase C Activation in Leukocytes by Lipopeptides

The synthetic lipopeptide Pam3Cys-Ser-Ser-Asn-Ala, an analogue of the N-terminal part of bacterial lipoprotein, constitutes a potent macrophage and B lymphocyte activator. In the macrophage cell-line P388D1 Pam3Cys-Ser-Ser-Asn-Ala stimulated phosphoinositol turnover, whereas in small resting lymphocytes no enhanced turnover was observed. Upon lipopeptide stimulation, a translocation of PKC from the cytosol to the plasma membrane was found in the cell line P388D1 but not in lymphocytes. Substituting lipopeptide for diacylglycerol shows that Pam3Cys-Ser-Ser-Asn-Ala leads to an activation of protein kinase C at Ca2+ concentrations of 0.5 mM. Thus, mitogenic lipopeptides constitute novel tools for investigating the molecular mechanism of transmembrane signaling in leukocyte activation.