A human leukaemia cell line - HL-60 - can be differentiated into neutrophils or macrophages and both differentiation processes are accompanied by changes of the lipid composition. Various methods were described for the extraction of lipids from cellular systems, but only two of them were applied to the HL-60 cell line so far. In this study we compared five selected extraction methods for the lipid extraction from HL-60 cells with regard to their qualitative analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS): chloroform/methanol at volume ratios 2:1 and 1:2, isopropanol/ chloroform, isopropanol/hexane and butanol. In addition, the cholesterol and phospholipid concentrations in organic extracts were measured by colorimetric assays. Results can be summarized as follows: For the analysis of polar phospholipids obtained from HL-60 cells by MALDI-TOF MS, a chlorofom/methanol (1:2) or isopropanol/chloroform mixture or butanol can be applied as extraction systems. On the other hand, if one would like to analyze changes in triacylglycerols, then chloroform/methanol (2:1) would be the method of choice.
Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils. Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils.
A small library of peptide analogues of the chemotactic tripeptide For-Met-Leu-Phe-NH2 modified by substitution of Leu at position 2 by three different fluorinated amino acids varying in content of fluorine, length of the fluorinated side chain, and alkylation degree at the alpha-carbon atom was synthesized. The influence of the fluorine substitution on the biological activity was investigated by measuring the oxidative activity of neutrophils using a luminol-dependent chemiluminescence assay.
Background/Aim: Polymorphonuclear leukocytes (neutrophils) release a variety of toxic agents – proteins and reactive oxygen species (ROS) – that are used to inactivate foreign microorganisms in the non-specific immune response. This study was undertaken to compare intracellular signalling pathways that lead to the ROS production as well as degranulation of azurophilic granules of human fMet-Leu-Phe/cytochalasin B stimulated neutrophils. Methods: Luminol-amplified chemiluminescence was used for monitoring the oxidative activity of human neutrophils in the presence of various inhibitors. The elastase activity was assessed in the neutrophil supernatant as a marker for degranulation of azurophilic granules. Results: Tested inhibitors of enzymes of signalling cascades showed the same effect on the ROS production and on the activity of elastase released from neutrophils. The only difference was obtained with staurosporine: it inhibited the chemiluminescence response, but increased the elastase release. Conclusion: Early signalling pathways leading to the ROS production and the degranulation are ubiquitous in human neutrophils. They are branching most probably at the point of the phosphatidic acid production by phospholipase D. A protein kinase activated by this lipid second messenger might play a central regulatory role in human neutrophils.
It has already been suggested that phosphatidic acids (PAs) play an important role in the regulation of signaling pathways involved in the production of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs). The present study was performed to elucidate the effects of extracellularly added PA-- 1,2-distearoyl- (DSPA) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphate (SAPA)--on the ROS production and on the elastase release by human PMNs. ROS production was monitored by luminol-amplified chemiluminescence and the elastase activity was measured in the supernatant of the PA-stimulated human PMNs by colorimetric assay. Obtained effects were compared with those of cells stimulated by either a chemotactic tripeptide, phorbol ester or calcium ionophore. Our results show that long-chain PAs at concentrations higher than 3 x 10(-5) mol/l stimulate the ROS production by human PMNs, whereas they were ineffective in promoting the elastase release. The chemiluminescence pattern of the SAPA-stimulated cells exhibited a biphasic curve, whereas cell stimulation with DSPA resulted in a monophasic chemiluminescence curve. Stimulation of the ROS production by PAs in dependence of the fatty acid composition required the activity of protein kinases.
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