Optimization of a flow cytometric assay to evaluate the human neutrophil ability to phagocytose immune complexes via Fcγ and complement receptors.

INTRODUCTION: This study aims to optimize some experimental conditions of a flow cytometric assay to examine the human neutrophil ability to phagocytose immune complexes (ICs) via Fcγ and complement receptors (FcγR and CR, respectively). The parameters assessed were: number of cells, concentration of ICs, reaction time, pH and concentration of the Trypan Blue quenching solution. METHODS: Neutrophils were isolated from peripheral blood of healthy volunteers. Precipitated ICs composed of IgG and fluorescein isothiocyanate (FITC)-labeled ovalbumin, opsonized or not with serum complement, were used to trigger the neutrophil phagocytosis via FcγR, CR, and FcγR+CR. Fluorescence of the internalized ICs was measured by flow cytometry, after quenching the extracellular fluorescence with Trypan Blue. RESULTS: The optimal experimental conditions established for the phagocytosis assay were: 1 × 10(6) cells mL(-1) and 40 µg mL(-1) FITC-labeled ICs, incubated for 30 min, at 37°C, in 0.5 mL of reaction volume. Trypan Blue solution at 1.25 mg mL(-1) pH4.4 was the best fluorescence quencher of FITC-labeled ICs attached to the outer surface of neutrophils. DISCUSSION: The selected experimental conditions were viable to evaluate IC phagocytosis by neutrophils; they are also suitable to compare the efficiency of IC phagocytosis mediated by FcγR and CR classes of membrane receptors, alone or in combination. This method finds application in studies of (i) the receptor-specific phagocytic function of normal and pathogenic neutrophils as well as (ii) the impact of drugs and therapies on this essential effector function of neutrophils.

The flavonols quercetin, myricetin, kaempferol, and galangin inhibit the net oxygen consumption by immune complex-stimulated human and rabbit neutrophils.

Stimulated human neutrophils exhibit increased net oxygen consumption (NOC) due to the conversion of O2 into the superoxide anion by the NADPH oxidase enzymatic complex during the respiratory burst. In several inflammatory diseases, overproduction of these oxidants causes tissue damage. The present study aims to: (a) optimize the experimental conditions used to measure the NOC in serum-opsonized zymosan (OZ)- and insoluble immune complex (i-IC)-stimulated human and rabbit neutrophils; and (b) compare the effect of four flavonols (quercetin, myricetin, kaempferol, and galangin) on this activity. We used a Clark-type oxygen electrode to measure the NOC of stimulated neutrophils. Eliciting the neutrophil respiratory burst with OZ and i-IC yielded similar maximum O2 uptake levels within the same species, but the human neutrophil NOC was almost four times higher than the rabbit neutrophil NOC. The optimal experimental conditions established for both cell types were 4 x 10(6) neutrophils mL(-1), 2 mg mL(-1) OZ, and 240 microg mL(-1) i-IC. Upon stimulation with OZ or i-IC, the tested flavonols reduced the human and rabbit neutrophil NOC in the same order of potency--quercetin and galangin were the most and the least potent, respectively. These compounds were around four times more effective in inhibiting the rabbit as compared to the human neutrophil NOC, respectively. The four flavonols were not toxic to human or rabbit neutrophils. The experimental conditions used are suitable for both the determination of human and rabbit neutrophil NOC and for the assessment of the modulatory effects of natural compounds on these activities. The relationship between the level of NOC and the inhibitory potency of the flavonols suggests that rabbit neutrophils can be useful experimental models to predict the effect of drugs on immune complex-stimulated human neutrophils.

Flavonols modulate the effector functions of healthy individuals' immune complex-stimulated neutrophils: a therapeutic perspective for rheumatoid arthritis.

Rheumatoid arthritis (RA) patients usually exhibit immune complex (IC) deposition and increased neutrophil activation in the joint. In this study, we assessed how four flavonols (galangin, kaempferol, quercetin, and myricetin) modulate the effector functions of healthy individuals' and active RA patients' IC-stimulated neutrophils. We measured superoxide anion and total reactive oxygen species production using lucigenin (CL-luc)- and luminol (CL-lum)-enhanced chemiluminescence assays, respectively. Galangin, kaempferol, and quercetin inhibited CL-lum to the same degree (mean IC50=2.5 µM). At 2.5 µM, quercetin and galangin suppressed nearly 65% CL-lum of active RA patients' neutrophils. Quercetin inhibited CL-luc the most effectively (IC50=1.71±0.36 µM). The four flavonols diminished myeloperoxidase activity, but they did not decrease NADPH oxidase activity, phagocytosis, microbial killing, or cell viability of neutrophils. The ability of the flavonols to scavenge hypochlorous acid and chloramines, but not H2O2, depended on the hydroxylation degree of the flavonol B-ring. Therefore, at physiologically relevant concentrations, the flavonols partially inhibited the oxidative metabolism of IC-stimulated neutrophils without affecting the other investigated effector functions. Using these compounds to modulate IC-mediated neutrophil activation is a promising safe therapeutic strategy to control inflammation in active RA patients.

Patients with systemic sclerosis present increased DNA damage differentially associated with DNA repair gene polymorphisms.

OBJECTIVE: Patients with systemic sclerosis (SSc) exhibit increased toxicity when exposed to genotoxic agents. In our study, we evaluated DNA damage and polymorphic sites in 2 DNA repair genes (XRCC1 Arg399Gln and XRCC4 Ile401Thr) in patients with SSc. METHODS: A total of 177 patients were studied for DNA repair gene polymorphisms. Fifty-six of them were also evaluated for DNA damage in peripheral blood cells using the comet assay. RESULTS: Compared to controls, the patients as a whole or stratified into major clinical variants (limited or diffuse skin involvement), irrespective of the underlying treatment schedule, exhibited increased DNA damage. XRCC1 (rs: 25487) and XRCC4 (rs: 28360135) allele and genotype frequencies observed in patients with SSc were not significantly different from those observed in controls; however, the XRCC1 Arg399Gln allele was associated with increased DNA damage only in healthy controls and the XRCC4 Ile401Thr allele was associated with increased DNA damage in both patients and controls. Further, the XRCC1 Arg399Gln allele was associated with the presence of antinuclear antibody and anticentromere antibody. No association was observed between these DNA repair gene polymorphic sites and clinical features of patients with SSc. CONCLUSION: These results corroborate the presence of genomic instability in SSc peripheral blood cells, as evaluated by increased DNA damage, and show that polymorphic sites of the XRCC1 and XRCC4 DNA repair genes may differentially influence DNA damage and the development of autoantibodies.

Afrormosin, an Isoflavonoid from Amburana cearensis A. C. Smith, Modulates the Inflammatory Response of Stimulated Human Neutrophils.

Isoflavones are phytoestrogens known by their anti-inflammatory, antioxidant and immunomodulatory properties. Presently, there is no information on whether afrormosin, an isoflavone from Amburana cearensis A.C. Smith (Fabaceae), has some effect on the inflammatory response from stimulated human neutrophils. Thus, the aim of this study was to evaluate the anti-inflammatory and antioxidant potentials of afrormosin on human neutrophils. Neutrophils (2.5 × 10(6) cells/mL) were incubated with afrormosin (3.35-335.2 µM) prepared from a product isolated from Amburana cearensis A.C. Smith with a 78.5% degree of purity and stimulated by the addition of cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate-13-acetate (PMA). Afrormosin inhibited the neutrophil degranulation induced by fMLP (10.47-335.2 µM) or PMA (0.33-167.6 µM), myeloperoxidase activity (3.3-335.2 µM), TNF-α secretion (16.7-335.2 µM) and the reactive oxygen species (ROS) generation (16.7-335.2 µM). On the other hand, afrormosin did not show any effect either on elastase or as a free radical scavenger. These data suggest that afrormosin modulates intermediary steps of the neutrophil ROS generation process. In addition, the modulatory effect of afrormosin on human neutrophil degranulation seems to be directed towards PMA-induced activation, indicating a potent inhibition of the protein kinase C activity. This study provided evidence, for the first time, to support the anti-inflammatory and antioxidant activities of afrormosin, creating novel insights into the pharmacological actions of this natural isoflavone.

3-Phenylcoumarin derivatives selectively modulate different steps of reactive oxygen species production by immune complex-stimulated human neutrophils.

Immune complex (IC) deposition in tissues triggers the release of harmful oxidant and lytic compounds by neutrophils. We examined how ten 3-phenylcoumarin derivatives affect the reactive oxygen species (ROS) production by IC-stimulated human neutrophils. Most of the 3-phenylcoumarins inhibited the luminol-enhanced chemiluminescence (CL-lum) more strongly than they inhibited the lucigenin-enhanced chemiluminescence (CL-luc), without clear signs of toxicity. The most effective CL-lum inhibitors, 6,7-dihydroxy-3-[3',4'-methylenedioxyphenyl]-coumarin (5) and 6,7-dihydroxy-3-[3',4'-dihydroxyphenyl]-coumarin (19), also inhibited myeloperoxidase activity more potently and had higher hypochlorous acid scavenging ability, but did not affect the NADPH-oxidase activity. The type, number, and position of the substituent influenced the pharmacological effects of 3-phenylcoumarins; however, the structural requirements for CL-lum and CL-luc inhibition were a little different. Compounds 5 and 19 are promising prototypes of therapeutic molecules to modulate ROS production by neutrophils in IC-mediated inflammatory diseases.

Evaluation of the Potential of Brazilian Propolis against UV-Induced Oxidative Stress.

This study investigated the potential use of topically and orally administered propolis extracts to prevent UV irradiation-induced oxidative stress in skin. The results illustrated that green propolis extract (GPE) contained greater amounts of polyphenols, coumaric acid, drupanin, baccharin and artepillin C than did brown propolis extract (BPE). GPE showed higher antioxidant activity than BPE when the IC(50) (concentration that caused 50% inhibition) values were compared. Interesting, the oral treatment of hairless mice demonstrated a recovery of 30.0% for GPE and 22.8% for BPE with respect to UV irradiation-induced GSH depletion. The topical pretreatment of animals with both propolis extract solutions recovered around 14.0% of the depleted GSH. However, the employed treatments did not inhibit the increase of cutaneous proteinase secretion/activity caused by irradiation. These findings indicate that despite differences in composition and antioxidant properties, GPE and BPE both successfully prevent UV-induced GSH depletion in vivo and are both promising antioxidant systems against oxidative stress in skin. Based on these findings, complementary studies should be performed to enhance our understanding of the protective effects of propolis extracts in skin.

Modulatory effects of rutin on biochemical and hematological parameters in hypercholesterolemic Golden Syrian hamsters.

Flavonoids have been reported to exhibit several pharmacological properties, mainly in cardiovascular and inflammatory diseases. In the present study, we observed that rutin, a known glycosylated flavonoid isolated from Dimorphandra mollis, had a lowering effect on plasma triglyceride levels of diet-induced hypercholesterolemic Golden Syrian hamsters, but did not change total cholesterol and high-density lipoprotein cholesterol levels. Moreover, high-fat or rutin supplemented diets showed no immunotoxic effects, since no significant changes were observed on total white blood cells, granulocytes and mononuclear cells, as well as on the neutrophil apoptosis degree, when compared to untreated animals. Therefore, rutin seems to be a selective and non-toxic modulator of hypercholesterolemia, which can be promising for the development of new drugs.

Modulatory effects of rutin on biochemical and hematological parameters in hypercholesterolemic Golden Syrian hamsters

Flavonoids have been reported to exhibit several pharmacological properties, mainly in cardiovascular and inflammatory diseases. In the present study, we observed that rutin, a known glycosylated flavonoid isolated from Dimorphandra mollis, had a lowering effect on plasma triglyceride levels of diet-induced hypercholesterolemic Golden Syrian hamsters, but did not change total cholesterol and high-density lipoprotein cholesterol levels. Moreover, high-fat or rutin supplemented diets showed no immunotoxic effects, since no significant changes were observed on total white blood cells, granulocytes and mononuclear cells, as well as on the neutrophil apoptosis degree, when compared to untreated animals. Therefore, rutin seems to be a selective and non-toxic modulator of hypercholesterolemia, which can be promising for the development of new drugs.

Os flavonóides possuem diversas propriedades farmacológicas, principalmente nas doenças cardiovasculares e inflamatórias. No presente estudo, observamos que a rutina, um conhecido flavonóide glicosilado isolado da Dimorphandra mollis, diminuiu o nível de triglicerídeos plasmáticos em hamsters Golden Syrian hipercolesterolêmicos sem alterar os níveis de colesterol total e colesterol HDL. Além disso, observamos que dietas hipercolesterolêmicas ou suplementadas com rutina não apresentaram efeito imunotóxico, uma vez que nenhuma alteração significativa foi observada nos leucócitos totais, granulócitos e células mononucleares, bem como no grau de neutrófilos em apoptose, quando comparado com animais não tratados. Portanto, a rutina parece ser um modulador seletivo e não tóxico da hipercolesterolemia, o que pode ser promissor para o desenvolvimento de novos fármacos.

Inhibition of immune complex-mediated neutrophil oxidative metabolism: a pharmacophore model for 3-phenylcoumarin derivatives using GRIND-based 3D-QSAR and 2D-QSAR procedures.

In this study, twenty hydroxylated and acetoxylated 3-phenylcoumarin derivatives were evaluated as inhibitors of immune complex-stimulated neutrophil oxidative metabolism and possible modulators of the inflammatory tissue damage found in type III hypersensitivity reactions. By using lucigenin- and luminol-enhanced chemiluminescence assays (CL-luc and CL-lum, respectively), we found that the 6,7-dihydroxylated and 6,7-diacetoxylated 3-phenylcoumarin derivatives were the most effective inhibitors. Different structural features of the other compounds determined CL-luc and/or CL-lum inhibition. The 2D-QSAR analysis suggested the importance of hydrophobic contributions to explain these effects. In addition, a statistically significant 3D-QSAR model built applying GRIND descriptors allowed us to propose a virtual receptor site considering pharmacophoric regions and mutual distances. Furthermore, the 3-phenylcoumarins studied were not toxic to neutrophils under the assessed conditions.

Superoxide anion production by neutrophils is associated with prevalent clinical manifestations in systemic lupus erythematosus.

To determine the relation between neutrophil function and the clinical characteristics of systemic lupus erythematosus (SLE), the superoxide anion (O2-) production by neutrophils, mediated by FcgammaR and FcgammaR/CR cooperation, was studied in 64 SLE patients classified according to their prevalent clinical manifestations. Three clinically distinct patterns were designated: (1) manifestations associated with the occurrence of cytotoxic antibodies (SLE-I group); (2) manifestations associated with circulating immune complexes (IC; SLE-II group), and (3) manifestations associated with IC and cytotoxic antibodies (SLE-III group). O2- production was evaluated by a lucigenin-dependent chemiluminescent assay in neutrophils stimulated with IC-IgG opsonized or not with complement. No difference in O2- production was observed when neutrophil responses from healthy controls were compared to the unclassified patients. However, when the SLE patient groups were considered, the following differences were observed: (1) SLE-I neutrophils showed lower O2- production mediated by the IgG receptor (FcgammaR) with the cooperation of complement receptors (FcgammaR/CR) than observed in the SLE-II, SLE-III, and healthy groups; (2) neutrophils from the SLE-II group showed a decreased [Formula: see text] production mediated by FcgammaR/CR compared to the SLE-III group, (3) SLE-III neutrophils produced more O(2)(-) than neutrophils from the SLE-II and control groups, and (4) CR showed inefficiency in mediating the O2- production by neutrophils from the SLE-I group. Comparative experiments on the kinetics of chemiluminescence (CL; Tmax and CLmax) disclosed differences only for the SLE-I group. Taken together, these results suggest that differences in oxidative metabolism of neutrophils mediated by FcgammaR/CR may reflect an acquired characteristic of disease associated with distinct clinical manifestations.

Ethanolic crude extract and flavonoids isolated from Alternanthera maritima: neutrophil chemiluminescence inhibition and free radical scavenging activity.

Extracts from Alternanthera maritima are used in Brazilian folk medicine for the treatment of infectious and inflammatory diseases. Bioassay-guided fractionation of A. maritima aerial parts yielded an ethanolic crude extract, its butanolic fraction and seven isolated flavonoids (two aglycones, two O-glycosides and three C-glycosides) with antioxidative activity. The ability of these samples to scavenge enzymatically generated free radicals (luminol-horseradish peroxidase-H2O2 reaction) and inhibit reactive oxygen species (ROS) production by opsonized zymosan-stimulated human neutrophils (PMNLs) was evaluated by chemiluminescence methods. In both assays, the butanolic fraction was significantly more active than the ethanolic crude extract, the flavonoid aglycones had high inhibitory activities and the C-glycosylated flavonoids had no significant effect even at the highest concentration tested (50 micromol/L). However, the O-glycosylated flavonoids inhibitory effects on chemiluminescence were strongly dependent on the chemical structure and assay type (cellular or cell-free system). Under the conditions tested, active samples were not toxic to human PMNLs.

Neutrophil effector functions triggered by Fc-gamma and/or complement receptors are dependent on B-ring hydroxylation pattern and physicochemical properties of flavonols.

Tissue damage in autoimmune diseases involves excessive production of reactive oxygen species (ROS) triggered by immune complexes (IC) and neutrophil (PMN) interactions via receptors for the Fc portion of IgG (FcgammaR) and complement receptors (CR). Modulation of both the effector potential of these receptors and ROS generation may be relevant to the maintenance of body homeostasis. In the present study, the modulatory effect of four flavonols (myricetin, quercetin, kaempferol, galangin) on rabbit PMN oxidative metabolism, specifically stimulated via FcgammaR, CR or both classes of receptors, was evaluated by luminol- and lucigenin-dependent chemiluminescence assays. Results showed that flavonol inhibitory effect was not dependent on the cell membrane receptor class stimulated but related to the lipophilicity of the compounds (their apparent partition coefficient values were obtained by high-performance liquid chromatography), and was also inversely related to the number of hydroxyl groups in the flavonol B ring and the ROS-scavenger activity (assessed by the luminol--H2O2--horseradish peroxidase reaction). Under the experimental conditions the flavonols tested were not toxic to PMNs (evaluated by lactate dehydrogenase release and trypan blue exclusion) and did not interfere with IC-induced phagocytosis (evaluated by transmission electron microscopy). Our results suggested that inhibition of IC-stimulated PMNs effector functions by the flavonols tested herein was the result of cooperation of different cellular mechanisms.

Inhibition of horseradish peroxidase catalytic activity by new 3-phenylcoumarin derivatives: synthesis and structure-activity relationships.

Twenty hydroxylated and acetoxylated 3-phenylcoumarins were synthesized, and the structure-activity relationships were investigated by evaluating the ability of these compounds to modulate horseradish peroxidase (HRP) catalytic activity and comparing the results to four flavonoids (quercetin, myricetin, kaempferol and galangin), previously reported as HRP inhibitors. It was observed that 3-phenylcoumarins bearing a catechol group were as active as quercetin and myricetin, which also show this substituent in the B-ring. The presence of 6,2'-dihydroxy group or 6,7,3',4'-tetraacetoxy group in the 3-phenylcoumarin structure also contributed to a significant inhibitory effect on the HRP activity. The catechol-containing 3-phenylcoumarin derivatives also showed free radical scavenger activity. Molecular modeling studies by docking suggested that interactions between the heme group in the HRP active site and the catechol group linked to the flavonoid B-ring or to the 3-phenyl coumarin ring are important to inhibit enzyme catalytic activity.

Sesquiterpene lactones from Lychnophora pohlii: neutrophil chemiluminescence inhibition and free radical scavenger activity.

Excessive production of reactive oxygen species (ROS) by polymorphonuclear leucocytes (PMNLs) is thought to contribute to the pathology of many inflammatory diseases. Sesquiterpene lactones (STLs) seem to be important contributors to the anti-inflammatory activity of many species of Lychnophora (Asteraceae), which have been widely used in Brazilian folk medicine because of this pharmacological property. In this study, the inhibitory effects of three STLs isolated from Lychnophora pohlii (lychnopholide, centratherin and goyazensolide) on rabbit PMNL oxidative burst were evaluated by the luminol-enhanced chemiluminescence (CL-lum) assay. All STLs tested showed concentration-dependent inhibitory activity on CL-lum but were not cytotoxic to PMNLs (evaluated by lactate dehydrogenase release) under the assessed conditions. Moreover, goyazensolide, the most active STL, had no free radical scavenger property, as assessed by 1,1-diphenyl-2picrylhydrazyl radical assay, and had no inhibitory effect on the luminol-horseradish peroxidase-hydrogen peroxide chemiluminescence. Taken together, the results of this investigation suggest that the concomitant presence of methacrylate ester and hydroxyl groups contributes to a high inhibitory effect on PMNL oxidative metabolism. This effect was not mediated by free radical scavenger or cytotoxic effects, but probably by inhibition of enzymes involved in the signal transduction pathways of the ROS generation process.

In vitro antioxidant and in vivo photoprotective effects of an association of bioflavonoids with liposoluble vitamins.

A new tendency in cosmetic formulations is the association of botanical extracts and vitamins to improve skin conditions by synergic effects. The objective of this study was to determine the antioxidant activity of associated bioflavonoids, retinyl palmitate (RP), tocopheryl acetate (TA) and ascorbyl tetraisopalmitate (ATIP), as well as their photoprotective effects in preventing increased erythema, transepidermal water loss (TEWL) and sunburn cell formation in hairless mouse skin. The antioxidant activity of solutions containing the association or each substance separately was evaluated in vitro by a chemiluminescence assay. The photoprotective effect was evaluated by means of in vivo tests. Dorsal skin of hairless mice was treated daily by topical applications for 5 days with formulations containing or not containing (vehicle) the flavonoid-vitamins association (5%). The skin was irradiated (UVA/B) 15 minutes after the last application. The results showed that bioflavonoids had in vitro antioxidant properties and also that when they were associated with vitamins their antioxidant activity was more pronounced. On the other hand, erythema and UV damage to the permeability barrier function (TEWL) was not significantly reduced by previous treatment with the flavonoid-vitamin-association formulations, when compared to the irradiated vehicle-treated area. However, the treatment protected the skin from UV damage because it reduced the number of sunburn cells, when compared to the vehicle-treated area. Finally, the association of vitamins and bioflavonoids added to a dermocosmetic formulation showed a relevant biological activity in terms of photoprotection, because the association of bioflavonoids and vitamins acted by different mechanisms, such as antioxidation and absorption of UV radiation, which suggests its use in antiaging and photoprotective products.

Receptores para imunoglobulina G (FcyR) / Receptors for immunoglobulin G (FcyR)

Os receptores para IgG (Fc'gama'R) fazem uma importante ligação entre as respostas imunes humoral e celular. Estes receptores medeiam várias respostas biológicas tais como: fagocitose, endocitose, captura e clearance de imunocomplexos, citotoxicidade e liberação de mediadores inflamatórios. Os Fc'gama'R humanos pertencem à superfamília das imunoglobulinas e três classes principais destes receptores são descritas, as quais apresentam várias isoformas. Estas moléculas diferem quanto à afinidade e especificidade para os isotipos de IgG, distribuição celular, sinalização intracelular e pesos moleculares. Além disso, o polimorfismo genético é responsável por variações entre os indivíduos. A diversidade estrutural e funcional dos Fc'gama'R faz destas moléculas importantes alvos para a imunoterapia. A ativação e desativação de células efetoras via Fc'gama'R pode ser explorada para o desenvolvimento de novas terapias para o câncer, doenças infecciosas e desordens auto-imunes. Esta revisão descreve detalhes estruturais e funcionais, relevância clínica e alguns usos terapêuticos dos Fc'gama'R

Evaluation of the anti-inflammatory and antioxidant activities of di-C-glucoflavones from Lychnophora ericoides (Asteraceae).

The Brazilian medicinal plant Lychnophora ericoides is commercially available as an analgesic and anti-inflammatory agent. The phytochemical investigation of the leaf polar extract yielded 6,8-di-C-beta-glucosylapigenin (1) and the new compound 6,8-di-C-beta-glucosylchrysin (2). 6,8-Di- C-beta-glucosylapigenin (1) showed significant anti-inflammatory activity in the carrageenan-induced rat paw edema. We did not observe any statistical difference between the two compounds (1 and 2) in inhibiting chemiluminescence in opsonized zymosan-stimulated polymorphonuclear leukocytes, suggesting that the anti-inflammatory property of 6,8-di- C-beta-glucosylapigenin (1) is not related to its antioxidant activity.

Application of the chemiluminescence systems to evaluate the role of Fcgamma and complement receptors in stimulating the oxidative burst in neutrophils.

We have established a luminol- and a lucigenin-dependent CL methods to investigate the role of the receptors for Fc portion of IgG (FcgammaR) and/or complement receptors (CR) in mediating the oxidative burst in neutrophils from systemic lupus erythematosus (SLE) patients compared with healthy controls. In the luminol-CL system, all the reactive oxygen species (ROS) are responsible for light production, whereas in the lucigenin-CL system, only the first ROS generated, converts the lucigenin into an unstable intermediate molecule, which also emits light. First, neutrophils from healthy controls and SLE patients were stimulated with different IC opsonized or not with complement from normal human serum (NHS) or SLE serum, in presence of 10(-4) M luminol. This method was able to differentiate the role of the FcgammaR, CR and FcgammaR/CR co-operation in mediating the oxidative burst, as well as show that the oxidative burst mediated by these receptors was reduced in neutrophils from SLE patients. Second, neutrophils from healthy controls and SLE patients were stimulated with different IC, opsonized or not with NHS, in presence of 10(-3) M lucigenin. In this case, the lucigenin-CL system was also able to differentiate the role of FcgammaR and FcgammaR/CR co-operation, as well as show differences among healthy controls and two different groups of SLE patients according to their clinical manifestations. In conclusion, we have established two sensitive CL systems to study the role of FcgammaR and/or CR in stimulating the oxidative burst of neutrophils, which can be applied in monitoring the involvement of these receptors in the immunopathogenesis of SLE.

A simple method to study the activity of natural compounds on the chemiluminescence of neutrophils upon stimulation by immune complexes.

INTRODUCTION: Neutrophils (PMNs) are the main effector cells involved in the immune response to microorganisms. However, in various noninfectious states, such as autoimmune and immune complex (ICs) diseases, ICs are found to be deposited in various organs, leading to recruitment and activation of PMNs at these sites of deposition. Consequently, reactive oxygen species (ROS) and lysosomal enzymes are extensively released by activated PMNs into the extracellular milieu, leading to host tissue injury. METHODS: In the present study, we discuss some experimental conditions of a luminol-enhanced chemiluminescence (LECL) assay to study the effect of natural compounds on the production of ROS by rabbit PMNs stimulated with precipitated ICs. Moreover, we evaluated the activities of quercetin and 7-allyloxycoumarin on this ROS-producing system and their toxicity to PMNs. RESULTS: Both compounds had concentration-dependent inhibitory effects on LECL. Quercetin at concentration of 5 micromol/l inhibited 94.5+/-1.0% of LECL, whereas 7-allyloxycoumarin at concentration of 200 micromol/l inhibited 53.8+/-2.4% of LECL. Neither compound was toxic to PMNs under the tested conditions. DISCUSSION: The proposed method may be useful for the screening of nontoxic compounds that can modulate ROS production by IC-stimulated PMNs. Special attention should be devoted to natural compounds from higher plants, since their potential as sources of new drugs is still largely unexplored.