Gregor Rothe

Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescin.

Hydroethidine (HE) and 2′,7′‐dichlorofluorescin (DCFH) were used for the flow cytometric measurement of reactive oxygen metabolites in leukocytes. Hydroethidine and DCFH were both rapidly oxidized in a cell‐free cuvette assay to ethidium bromide (EB) and 2′,7′‐dichlorofluorescein (DCF) by H2O2 and peroxidase, but not by H2O2 alone, while only HE was oxidized by KO2, a source of O‐ 2. Quiescent lymphocytes, monocytes, and neutrophils spontaneously oxidized HE to EB, while DCFH was only oxidized to a low degree. Neutrophils increased 6.9‐fold in EB red fluorescence and 12.5‐fold in DCF green fluorescence during the respiratory burst induced by phorbol 12‐myristate 13‐acetate or 6.1‐fold and 4.7‐fold, respectively, during the respiratory burst induced by Escherichia coll bacteria. The HE or DCFH oxidation during the respiratory burst, unlike the spontaneous HE oxidation, was not inhibitable by 10 mM NaN. indicating a non‐mitochondrial source of cellular oxidants during the respiratory burst such as NADPH oxidase, which produces O‐ 2. The oxidation of DCFH, but not of HE, was decreased in stimulated neutrophils, which were simultaneously loaded with HE and DCFH. Intracellular DCFH oxidation induced by incubation of resting neutrophils with extracellular H2O2 was not influenced by the presence of HE. This indicates that HE is oxidized at an earlier step in the reactive oxygen metabolism of neutrophils than DCFH, i.e., by early oxygen metabolites like O2 ‐, while DCFH is oxidized in part by H2O2 and phagosomal peroxidases. The differential oxidation of HE and DCFH during simultaneous cellular staining permits the analysis of up to three functionally different neutrophil populations in septic patients. This is of interest for the determination of disease‐related alterations of oxygen metabolism in quiescent and stimulated leukocytes.

Modulation of intracellular formation of reactive oxygen intermediates in peritoneal macrophages and microglia/brain macrophages by propentofylline.

Ischemia-induced nerve cell death can partly be prevented by propentofylline, a pharmacon structurally related to xanthine derivates that interacts with the neuromodulatory function of endogenous adenosine. To evaluate a possible mechanism of neuroprotection by propentofylline, we studied its effect on the cellular production of reactive oxygen intermediates in microglial cells, which under pathological conditions can differentiate into brain macrophages, in comparison to peritoneal macrophages. Using a flow cytometric assay, we determined the intracellular formation of reactive oxygen intermediates by measuring the oxidation of the membrane-permeable and nonfluorescent dihydrorhodamine 123 to the cationic and intracellularly trapped, green fluorescent rhodamine 123 in single viable cells. Propentofylline at the therapeutic concentration of 50 μM completely inhibited the Ca2+-dependent Con A-induced increase in the production of reactive oxygen intermediates in peritoneal macrophages. In isolated and cultured microglial cells, which have a high spontaneous respiratory burst activity, the spontaneous production of reactive oxygen intermediates was reduced by ∼30%. A phorbol 12-myristate 13-acetate-induced rise in the respiratory burst activity could not be inhibited by propentofylline in either cell type. An increased generation of reactive oxygen intermediates is thought to contribute to nerve cell death after brain ischemia, edema, and neurodegenerative diseases like Alzheimers disease. These pathological conditions are all accompanied by an activation of microglial cells. We therefore suggest that the neuroprotective properties of propentofylline might in part be due to a modulation of the microglial production of potentially harmful reactive oxygen intermediates.

Inhibition of the oxidative burst response of N-formyl peptide-stimulated neutrophils by serum amyloid-A protein

Strong binding of the acute phase protein serum amyloid-A (SAA) to human neutrophils was found using flow cytometry. This binding was shown to be functionally relevant with respect to the oxidative burst reaction assayed on N-formyl peptide-stimulated neutrophils by the intracellular oxidation of non-fluorescent dihydrorhodamine to fluorescent rhodamine 123. The results show reduction of the oxidative burst response by isolated SAA (and recombinant SAA2). Inhibition was also demonstrated by acute phase as compared to normal human serum. This inhibitory effect was abolished by the purified monoclonal anti-amyloid A antibody mc29, strongly suggesting that SAA counteracts neutrophil responses to cytokines or bacterial products. This newly recognized function of SAA may help to prevent oxidative tissue destruction.

Respiratory burst activity in brain macrophages: a flow cytometric study on cultured rat microglia

A new flow cytometric method for the investigation of the respiratory burst of macrophages/ microglia isolated from neonatal rat brain has been established. Respiratory burst activity was measured quantitatively in single viable cells by the intracellular oxidation of non‐fluorescent dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123. Cultured microglia exhibited high spontaneous respiratory burst activity already before stimulation. After maximal stimulation with phorbol myristate acetate, DHR oxidation rose by 40–95%. The respiratory burst activity in resident or inflammatory, i.e. thioglycolate elicited, peritoneal macrophages was significantly lower than in cultured brain macrophages suggesting a high potential of microglia for oxidative tissue destruction.

Chapter 29 Flow Cytometric Determination of Cysteine and Serine Proteinase Activities in Living Cells with Rhodamine 110 Substrates

Publisher Summary Rhodamine 110 (R110) protease substrates are advantageous for cytometric single-cell protease activity measurements because of specific cleavage, low toxicity, absent fluorescence background, autoaccumulation due to positive electrical charge at physiological pH, and pH-independent fluorescence. Besides cysteine and serine proteinase substrates, additional R110 substrates for the determination of leucine and phenylalanine aminopeptidases (metalloproteinase) and cathepsin D (aspartic proteinase) have been recently synthesized. The intracellular cathepsin D measurement is performed as the coupled enzyme reaction with cathepsin D endopeptidase cuts followed by aminopeptidase digestion of remaining residues until liberation of free R110. Coupled protease reaction, at least in principle, will allow the specific determination of many other intracellular endopeptidases. Altogether, the cytometric use of R110 proteinase substrates will open up a new area of studies on the mechanism and regulation of cellular protein and peptide catabolism.

Biochemical Parameters of Cell Function

Cellular activation is characterized by early changes in functional parameters such as depolarization of the plasma membrane potential [1], increase in the cytosolic free calcium concentration [2], alkalinization of intracellular pH [3], increased mitochondrial charge [4], production and release of reactive oxidants [5], changes in cellular glutathione content [6], and activation and release of lysosomal proteases [7, 8]. These parameters are sensitive indicators of stimulus-coupled biochemical processes even before protein synthesis, gene transcription, or cellular proliferation is induced.

Activation of Neutrophils by Tumor Necrosis Factor-α During Sepsis

Endotoxin exposure of macrophages during the onset of bacterial infections leads to the sequential production of the cytokines tumor necrosis factor-α (TNF-α), interleukin-1β and interleukin-6 [1]. These cytokines are thought to enhance the local anti-bacterial immune response. TNF-α, the most potent modulator of neutrophil function, induces in vitro the inhibition of neutrophil Chemotaxis [2], the expression of CD11/CD18 integrins resulting in the adherence of neutrophils to matrix proteins with the release of oxidants [3] and specific granule constituents [4], extravasation [5, 6], and increased phagocytosis [7].

The Oxidative Burst Reaction of Neutrophils Is Reduced by the Acute Phase Serum Amyloid-A Protein

The acute-phase reaction is considered to represent an adaptation of the organism toward meeting such life-threatening incidents as injury, necrosis, inflammation, sepsis, and tumor spread. The acute-phase reaction is induced by cytokines mainly from activated monocytes and macrophages and is characterized by changes in the concentration of definite plasma proteins, called acute-phase proteins. The concentration of such positive acute-phase proteins as α1-proteinase inhibitor, complement factors, fibrinogen, haptoglobin, C-reactive protein, and serum amyloid-A protein (SAA) is increased, in contrast to a decrease seen in such negative acute-phase proteins as albumin or transthyretin [1].

FLOW CYTOMETRIC ANALYSIS OF PROTEASE ACTIVITIES IN VITAL CELLS AND AUTOMATED CLASSIFICATION OF LIST MODE DATA WITH THE DIAGNOSI PROGRAM

Huomscence in situ hybridization is a useful method m study the expression of mRNA in heterogeneous samples. We adapted the FiSH method on cell suspension for flo~v cytometry (FCM) analysis to investigate the polyA-RNA content in HL-60 leukemic cell line. Oligo-d6oxythymidine (odT)i

Detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin B and L.

was coupled via its pbosphamide derivative to FITC with a spacer ann of. two carbons. This compound was purified and used as a probe for FISH detection of poly-A RNA . The cell DNA was then stained with propidium iodide (Pl) and cell suspension analysed by FCM. The specifity for poly-A hybridization was checked by competitive hybridization in the presence of an excess of free poly-A or poly-U. Both polynuclootides abolished fluorescent labelling of the cells by FITCodT. Labelling was also inhibited by previous treatment of the cellls by RNase. The method was used to follow the kinetic of poly-A RNA in HL.60 cells treated by lug/mi of actinomycin D. A 60% decrease in the poly.A RNA was found as soon as 3h of ureatment. In the same ~ime, only a 20% of total RNA (as measured using acridine orange and FCM) w:is detected. After 24h of treatment, 80~ of poly-A RNA were lost by the cells while 60% of total RNA were preserved. Biparametric analysis of FITC-OdT/Pi labelled cells showed that the poly-A RNA content varied during the cell cycle. This FISH method, associated with flow cytometry, could then be a useful tool to study the effect of drugs on the RNA metabolism independently of the position of the cells into the cell cycle.