Isolation of healthy individuals' and rheumatoid arthritis patients' peripheral blood neutrophils by the gelatin and Ficoll-Hypaque methods: comparative efficiency and impact on the neutrophil oxidative metabolism and Fcγ receptor expression.

In vitro assessment of the functional responses of leukocytes sometimes requires their isolation from blood, joint and tissues. In this study, we compared the efficiency of two procedures - the gelatin method and Ficoll-Hypaque density centrifugation gradient - to isolate peripheral blood neutrophils of healthy individuals and patients with active rheumatoid arthritis (RA). We also assessed whether these procedures affect the neutrophil activation status. Both purification procedures were concluded in 90min, and yielded cell populations with similar degrees of purity (80-90%), number of neutrophils (1-2×10(6) cells per mL of blood), and viability (97-100%). In vitro neutrophil priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the reactive oxygen species producing ability of the cells stimulated with n-formyl-methionyl-leucyl-phenylalanine (n-fMLP), soluble immune complexes (s-ICs), and insoluble immune complexes (i-ICs). Isolated neutrophils not treated with GM-CSF responded to n-fMLP and i-IC, but not to s-IC. Almost all of the neutrophils (98-100%) purified by both methods expressed FcγRII/CD32 and FcγRIII/CD16, but they did not express significant levels of FcγRI/CD64. Similar results were obtained for healthy individuals' and RA patients' neutrophils. In summary, the gelatin method was comparable to Ficoll-Hypaque gradient in terms of purity, yield, and viability of the neutrophil preparations. Both methods neither primed or activated the neutrophils, nor affected their functional responsiveness. Therefore, both methods are suitable to isolate peripheral blood neutrophils of healthy individuals and RA patients.

Antioxidant activity and cytotoxicity as mediators of the neutrophil chemiluminescence inhibition by butylated hydroxytoluene.

The tissue damage found in some inflammatory and autoimmune diseases has been shown to be mediated by an increased activation of neutrophil effector functions. In this study, we investigated the inhibitory effect of the phenolic compound butylated hydroxytoluene (BHT) on reactive oxygen species (ROS) generation by opsonized zymosan-stimulated neutrophils, assessed by luminol- and lucigenin-enhanced chemiluminescence (CL-lum and CL-luc, respectively), and some aspects of its mechanism of action. BHT showed concentration-dependent: (a) inhibitory effect on CL-lum and CL-luc; (b) cytotoxic effect, expressed by increased lactate dehydrogenase leakage by the cells; (c) interaction with neutrophil membranes; (d) ROS scavenger activity. These biological effects were observed in the same range of concentrations (0-5 x 10(-5) mol/l). Taken together, the results suggest that inhibition of neutrophil chemiluminescence by BHT was a result of multiple mechanisms, especially a cytotoxic effect probably mediated by BHT interaction with neutrophils membranes, and the ROS scavenging effect.

Effect of rutin on polymorphonuclear leukocytes oxidative metabolism in hypercholesterolemic Golden Syrian hamsters: evaluation by chemiluminescence and flow cytometry.

Atherosclerosis has been described as an inflammatory disease in which polymorphonuclear leukocytes (PMNLs) seem to be involved. These cells may induce atherosclerotic lesions by releasing reactive oxygen species (ROS) and a sort of pro-inflammatory mediators. In this study, the PMNL oxidative metabolic status of Golden Syrian hamsters fed a normal diet (ND), or a high-fat diet (10% coconut oil plus 0.2% cholesterol) supplemented (R-HCD) or not (HCD) with 0.1% (w/w) rutin was evaluated after 120 days of treatment. PMNL oxidative metabolism was assessed by whole blood luminol-enhanced chemiluminescence and 2',7'-dichlorofluorescein diacetate-dependent flow cytometry. The results obtained by both methods were similar and showed no significant changes in ROS generation by PMNLs in blood samples from HCD or R-HCD animals when compared to ND. Furthermore it was shown that rutin supplementation did not significantly affect plasma lipid and lipoprotein levels in the hypercholesterolemic animals characterized by significantly increased total plasma cholesterol, triglycerides and low- and high-density lipoprotein cholesterol levels. The results suggest that in this model atherosclerosis development is not related to circulating PMNL activation and rutin supplementation has no immunomodulatory or hypocholesterolemic effects.

Immunomodulatory effects of methoxylated flavonoids from two Chromolaena species: structure-activity relationships.

Excessive generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) is involved in the pathology of many inflammatory diseases. Compounds isolated from natural sources with antioxidant activity can be helpful to inhibit and/or modulate the oxidative damage associated with PMNL-derived ROS. The present study investigated the relationship between the chemical structure of five methoxylated flavonoids, isolated from Chromolaena hirsuta and Chromolaena squalida, and their inhibitory activity on ROS generation by opsonized zymosan-stimulated PMNL. The antioxidant efficacy of the studied flavonoids, assessed by luminol-dependent chemiluminescence, was dependent on the position and number of methoxy and hydroxy groups.

Inhibitory activity of flavonoids from Lychnophora sp. on generation of reactive oxygen species by neutrophils upon stimulation by immune complexes.

Formation of circulating immune complexes (ICs) is essential for clearance of invading agents. In some circumstances ICs might deposit on host tissues, leading to an inflammatory process that involves massive activation of neutrophils (PMNs), release of reactive oxygen species (ROS) and lysosomal enzymes and damage to the host tissue. Extracts of plants from Lychnophora sp. are used in Brazilian folk medicine as antiinflammatory agents. In this study, we evaluated the effect of eight flavonoids isolated from L. granmongolense, L. salicifolia and L. ericoides on the generation of ROS by rabbit PMNs stimulated with two kinds of ICs: particles of serum-opsonized zymosan (OZ) and insoluble ICs (ICIgG). ROS production was measured by chemiluminescence (CL) assay. We observed that 5- and 7- dihydroxylated compounds at 5 micromol/L inhibited almost totally ICIgG- and OZ-triggered luminol-CL and OZ-triggered lucigenin-CL. The degree of inhibitory effect among the other flavonoids was different, depending on the kind of ICs used to trigger ROS generation by PMNs and the number and position of methoxy groups. Moreover, under the conditions assessed, the studied flavonoids were not toxic to the rabbit PMNs. These results suggest that the actions of flavonoids on ROS generation by stimulated PMNs are highly dependent on their structures.