Phorbol Ester-stimulated Neutrophils Research Articles

The Fluorescein-derived Dye Aminophenyl Fluorescein Is a Suitable Tool to Detect Hypobromous Acid (HOBr)-producing Activity in Eosinophils

The specific detection of peroxidase activity in human granulocytes is essential to elucidate their role in innate immune responses, immune regulation, and inflammatory diseases. The halogenating activity of myeloperoxidase in neutrophils can be determined by the novel fluorescent probe aminophenyl fluorescein (APF). Thereby non-fluorescent APF is oxidized by HOCl to form fluorescein. We successfully verified that APF equally detects the hypobromous acid (HOBr)-producing activity of eosinophil granulocytes. This was revealed by three different approaches. First, we investigated the conversion of non-fluorescent APF into fluorescein by HOCl and HOBr by means of fluorescence and mass spectrometry approaches. Thereby comparable chemical mechanisms were observed for both acids. Furthermore in vitro kinetic studies were used to detect the halogenating activity of myeloperoxidase and eosinophil peroxidase by using APF. Here the dye well reflected the different substrate specificities of myeloperoxidase and eosinophil peroxidase regarding chloride and bromide. Finally, peroxidase activities were successfully detected in phorbol ester-stimulated neutrophils and eosinophils using flow cytometry. Thereby inhibitory studies confirmed the peroxidase-dependent oxidation of APF. To sum up, APF is a promising tool for further evaluation of the halogenating activity of peroxidases in both neutrophils and eosinophils.

The Effects of Photodynamic Therapy on Human Neutrophil Migration Using Bacteriochlorin a

Bacteriochlorin a photodynamic therapy (BCA-PDT) caused inhibition of interleukin (IL)-8-activated neutrophil migration, at concentrations that did not induce membrane damage. Random migration and migration induced by other chemoattractants were also inhibited, indicating that the effect of BCA-PDT was not at the level of chemoattractant-receptor interaction but down stream. The BCA-PDT completely blocked superoxide production of phorbol ester-stimulated neutrophils indicating that superoxide production by neutrophils present in the tumor before and during BCA-PDT is not the cause of inactivation of tumor cells. Both type I and type II quenchers prevented inhibition by BCA-PDT but only in electroporated cells. Confocal laser scanning microscopy showed that the fluorescence of BCA was located inside the cell. These results show that the effects of BCA-PDT are intracellular and of a mixed type I/type II character and that the neutrophils present in the tumor during illumination probably do not contribute to tumor eradication by releasing reactive oxygen species.

Neutrophil-mediated activation of mitoxantrone to metabolites which form adducts with DNA.

In this study we have shown that phorbol ester-stimulated human neutrophils are able to oxidatively activate mitoxantrone and result in covalent incorporation of the drug into cellular DNA. The use of the myeloperoxidase inhibitor sodium azide confirmed that the activation and covalent binding of mitoxantrone to cellular DNA was due to its metabolism by the haem enzyme myeloperoxidase. Phorbol ester-stimulated neutrophils were also able to oxidatively metabolise mitoxantrone and facilitate extracellular covalent binding of the drug to calf thymus DNA. These results suggest that myeloperoxidase may contribute to the mode of action of mitoxantrone.

Deferoxamine interferes with adhesive functions of activated human neutrophils.

Deferoxamine is a potent chelator of ferric iron. Past studies have shown that deferoxamine interferes with acute inflammatory tissue injury in a number of animal models. In cell culture, it inhibits neutrophil-medicated killing of endothelial cells. Both the animal model and cell culture data are thought to reflect the capacity of deferoxamine to interfere with the superoxide anion- and and ferric iron-dependent reduction of hydrogen peroxide to the hydroxyl radical (Fenton Reaction). The present study describes a second mechanism by which deferoxamine may interfere with the acute inflammatory response. Here it is shown that deferoxamine has the capacity to inhibit neutrophil adhesion to lung epithelial cells and vascular endothelial cells. Adhesion of phorbol ester-stimulated neutrophils to both cell types is reduced by 70-80%. The inhibitory effects are reversible and are overcome when ferric iron is present along with deferoxamine in a 2:1 molar ratio. Concentrations of deferoxamine that prevent neutrophil adhesion also prevent neutrophil-mediated killing of the same target cells. In contrast, deferoxamine does not significantly inhibit activation-induced up-regulation of neutrophil surface adhesion structures (CD11b/CD18) and does not prevent binding of a monoclonal antibody that recognizes beta 2 integrins in the high-affinity state. Release of proteolytic enzymes from activated cells is also not significantly inhibited by deferoxamine. Taken together, these data indicate that deferoxamine modulates neutrophil adhesive functions associated with the activated state. The ability of deferoxamine to interfere with neutrophil binding to target cells may contribute to its anti-inflammatory activity.

Mutagenesis by metal-induced oxygen radicals.

To assess the contribution of reactive oxygen species (ROS) to metal-induced mutagenesis, we have determined the spectrum of mutations in the lacZ alpha gene after exposure of M13mp2 DNA to Fe2+, Cu2+, and Ni2+. With iron and copper ions, mutations are clustered and are predominantly single-base substitutions. Fe, Cu, and phorbol ester-stimulated neutrophils also produced tandem double CC-->TT mutations. This mutation may provide a marker for the role of oxidative damage in carcinogenesis. Mutagenesis by Ni2+ required the complexing of the metal to a tripeptide and the addition of H2O2. To assess the contribution of ROS in mammalian cells, we determined the spectrum of mutations produced when purified DNA polymerases-alpha and -beta synthesized DNA using a template that had been damaged by ROS. The mutation spectra produced by the two polymerases indicates that these enzymes substitute different nucleotides opposite the same lesions.

DNA damage induced by phorbol ester-stimulated neutrophils is augmented by extracellular cofactors. Role of histidine and metals.

Activated neutrophils cause extensive DNA damage in neighboring nonphagocytic cells. To determine whether compounds in the extracellular milieu participate in the DNA damage process, murine neutrophils were cocultivated with target tumor cells in media of varying composition. Using the alkaline elution assay, it was found that the level of strand breaks induced was significantly higher (2.8-fold) in complex cell culture media than in minimal phosphate-buffered saline. Addition of amino acids in general and of histidine in particular increased the level of damage nearly to that observed in complete media (2.7- and 2.1-fold, respectively). The histidine stimulation was concentration-dependent and reached a maximum at 100-400 microM. The mechanism whereby this occurred is not proven but probably derived from chelation of metals and participation in a site-specific Fenton reaction. Addition of the cell-impermeable chelator EDTA dramatically inhibited induction of strand breaks by neutrophils in complete media and prevented the enhancement of damage induced by histidine in phosphate-buffered saline. None of the effects on neutrophil-induced damage could be attributed to modulation of the oxidative burst activity of the cells (O2- and H2O2 production). Histidine also enhanced induction of strand breaks by reagent H2O2. However, EDTA had no effect or actually increased the level of damage induced by both a bolus of H2O2 and a flux of H2O2 generated by glucose oxidase. The cell-permeable chelator o-phenanthroline inhibited both neutrophil- and H2O2-induced damage. The results indicate that secondary reactions involving extracellular amino acids and metals contribute significantly to neutrophil-induced DNA damage to neighboring cells. Moreover, the data show that the mechanism whereby neutrophils induce this damage cannot be attributed solely to secretion of H2O2.

Protection against lethal hyperoxia by tracheal insufflation of erythrocytes: role of red cell glutathione.

Intact erythrocytes placed into the tracheobronchial tree of hyperoxic rats dramatically improved their chances for survival. Over 70 percent of the animals so treated survived more than 12 days during continuous exposure to 95 percent oxygen, whereas all of the control animals died within 96 hours. Lungs from erythrocyte-protected rats showed almost none of the morphologic damage suffered by untreated animals. Erythrocytes containing cyanomethemoglobin were as beneficial as normal erythrocytes, but cells in which glutathione was partially blocked were significantly less protective. Analogous results were obtained in vitro: 51Cr-labeled target cells released 70 to 90 percent of their label when exposed briefly to hydrogen peroxide or to toxic oxygen species generated by phorbol ester-stimulated neutrophils. Addition of intact erythrocytes decreased release by approximately 75 percent, but significantly less than this if red blood cell glutathione was partially blocked. These results suggest that insufflated erythrocytes, through their recyclable glutathione, protect rats from toxic oxygen species engendered by hyperoxia.