Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a ficoll‐hypaque gradient

Abstract

Polymorphonuclear neutrophils (PMN) have an important role in the host defence response to infection. These cells produce large amounts of reactive oxygen species (O(2).(-), H(2)O(2) and ONOO(-)) with microbicidal activity. PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density (Ficoll-Hypaque gradient and dextran), yielding a highly homogeneous cellular population. However, some cellular activation due to membrane perturbation is also expected. We studied how the production of reactive oxygen species and release of myeloperoxidase (MPO) from blood PMN are affected by the use of the Ficoll-Hypaque density gradient. PMN isolated by spontaneous sedimentation and total blood were used for comparisons. Lucigenin- and luminol-enhanced chemiluminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli. The release of MPO, estimated by the chemiluminescence of the luminol/H(2)O(2) reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B, was also higher for PMN isolated by a density gradient. In conclusion, it was shown that the PMN isolation procedure affects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results.

Increased whole blood chemiluminescence in patients with chronic renal failure independent of hemodialysis treatment

“…Perhaps other factors that are generated and/or accumulated in the blood and tissues of patients with renal failure, such as circulating adhesion molecules and advanced glycation end-products, can be responsible for enhanced LBCL [15,48]. Advanced glycation end-products are able to prime PMNs and monocytes to enhanced ROS production (via activation of cytosolic phospholipase A 2 ) upon stimulation with various agonists, including fMLP [35]. Due to their relatively high molecular weight, they are hardly dialyzable and, therefore, the increased ability of uremic phagocytes to produce ROS in response to agonist stimulation is not corrected by HD [22].…”

“…and that HD moderately primes cell response to fMLP, an analogue of bacterial chemotactic peptide. The advantage of the LBCL method in comparison to the commonly applied ROS generation in pure PMN and/or monocyte suspensions is that the assay can be performed just after blood collection, without the leukocyte isolation procedure, which can possibly alter the oxidative metabolism of blood phagocytes [35,47]. However, blood specimens for the LBCL assay should be diluted many times to avoid significant interference from erythrocytes, plasma proteins, and low-molecular--weight anti-oxidants, including uric acid [23,24].…”

Novel cell death program leads to neutrophil extracellular traps

“…As was also observed by others (Rebecchi et al, 2000), some neutrophil dextran/fi coll preparations were preactivated, which lead to high background of NETs in unstimulated controls. Interestingly, this isolation method may cause ROS production in neutrophils (Rebecchi et al, 2000). The data presented here was generated with neutrophils isolated by percoll density-gradient centrifugation.…”

“…Initial observations of NETs were from neutrophils isolated by a dextran/fi coll method (Weinrauch et al, 2002). As was also observed by others (Rebecchi et al, 2000), some neutrophil dextran/fi coll preparations were preactivated, which lead to high background of NETs in unstimulated controls. Interestingly, this isolation method may cause ROS production in neutrophils (Rebecchi et al, 2000).…”

Cross‐linking of neutrophil CD11b results in rapid cell surface expression of molecules required for antigen presentation and T‐cell activation

“…Others have also noted that neutrophils can be non‐specifically activated by Ficoll and/or dextran as assessed by increased myeloperoxidase release, increased expression of FMLP‐binding sites and generation of reactive O 2 species 22,23 . It was concluded by these workers that the use of purified neutrophils ‘may lead to misinterpretation of results’ 23 and/or ‘may lead to important differences of in vivo polymorphonuclear function being obscured’ 24 . The whole blood method described in this paper proved to be simple, rapid, reliable, inexpensive and requires only a small volume of blood per test.…”