BACKGROUND:Peripheral blood monocytes may participate in allergic diseases by releasing inflammatory mediators. We have recently shown enhancement of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity in adherent human peripheral blood monocytes from allergic patients when immunoglobulin E (IgE) binds to its low affinity receptor. Interferon-gamma (IFN-gamma), a monocyte-activating lymphokine, has been shown to prime monocytes for superoxide anion release.OBJECTIVE:We hypothesized that IFN-gamma could directly activate blood monocyte superoxide anion release and evaluated its modulatory effect on IgE-induced superoxide anion release from those cells.METHODS:Seven patients with allergic asthma, 6 patients with allergic rhinitis, and five nonallergic controls were studied. We measured superoxide anion release from their blood monocytes using a chemiluminescence assay. Cells were either nonstimulated, or stimulated for 30 minutes with IFN-gamma and/or serum IgE.RESULTS:We found that IFN-gamma stimulated superoxide anion production and decreased the IgE-induced superoxide anion production after 30 minutes of IFN-gamma preincubation only in blood monocytes from patients with allergic asthma.CONCLUSIONS:These data suggest that IFN-gamma receptors may be increased and/or coupled to NADPH oxidase in allergic asthmatic patients.

Inflammatory processes in asthma are characterized by an infiltration of inflammatory cells including mononuclear phagocytes. It has been observed that mononuclear phagocytes, alveolar macrophages and blood monocytes, release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects. Chemiluminescence assays were developed to measure the superoxide anion and the other reactive oxygen species. The chemiluminescence response was first analysed with a luminometer, which made it possible to study cells in suspension before and after PMA-stimulation. Secondly a video-imaging camera was used in experiments on adherent cells before and after stimulation with PMA and/or specific stimulus IgE/anti-IgE. Both techniques showed that human alveolar macrophages, blood monocytes, PMN and lymphocytes were spontaneously primed in vivo and were more easily stimulated in asthma. Analysis of adherent cells in vitro may provide give information on the physiological condition of adherent cells in vivo.

There is increasing evidence to suggest that human blood polymorphonuclear neutrophils (PMNs) and monocytes play an important role in the inflammatory processes of asthma. In asthmatic patients, PMNs and monocytes were shown to be activated more than in healthy subjects. We investigated the capacity of these two cell populations to generate reactive oxygen species (ROS) in stable and unstable asthmatic patients. The two populations of asthmatic patients were identified by asthma activity, as expressed by clinical events occurring within 2 weeks prior to the study. Oxygen species formation was analysed for isolated purified PMNs and monocytes (Mos) by chemiluminescence (CL) using lucigenin and luminol as luminescent probes. CL was determined on nonstimulated and on phorbol myristate acetate (PMA)-stimulated cells. The stimulatability coefficient (PMA-stimulated/nonstimulated cell ratio) of each cell population was then calculated. Resting PMNs and Mos generated significantly greater amounts of ROS in stable asthmatic patients, and much more in unstable asthmatic patients, as compared to healthy subjects, both in lucigenin and luminol enhanced CL. Non O2.- ROS production from PMA-stimulated PMNs and Mos was identical in unstable asthmatic patients and in healthy subjects, whereas a significant decrease was observed in stable asthmatic patients, as assessed by luminol enhanced CL. PMA-stimulated cells showed no difference in O2.- generation, as assessed by lucigenin enhanced CL. However, the stimulatability coefficient of all asthmatic patients was always significantly lower than that of healthy subjects. These results suggest that there are differences in priming and stimulation of Ros production from PMNs and Mos between stable and unstable asthmatic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar macrophages (AM) may take part in the amplification of the inflammatory mechanism involved in asthma. During an asthma attack, mast cells and eosinophils release arachidonic acid derivative mediators of inflammation such as sulfidopeptide leukotrienes. Among them, LTC4 has been shown to be present in bronchoalveolar fluid. In asthmatic patients, we showed that the ability of AM to transform LTC4 into its derivatives LTD4 and LTE4 was related to the intensity of the local inflammation observed during endoscopy. AM from asthmatics incubated in the presence of LTC4 or LTE4, generated LTB4 and 5-HETE, which are potent chemoattractants. Nedocromil sodium (10(-4) M) decreased LTB4 releasability and intracellular 5-HETE concentrations in zymosan-stimulated AM from asthmatic patients, and was shown to decrease the LTC4 or LTE4-promoted formation of LTB4 and 5-HETE.

Flavonoids are known to reduce reactive oxygen species released by polymorphonuclear neutrophils (PMNs) in vitro. We have studied the effects of S5682 (Daflon 500 mg), a purified flavonoid fraction composed of 90% diosmin and 10% hesperidin. S5682 produced a dose-dependent inhibition of the luminol chemiluminescence (CL) induced by phorbol myristate acetate on PMNs (IC50 = 5 x 10(-5) M), with no effect on superoxide anion (O2.-) formation and on cellular superoxide dismutase activity as determined by lucigenin-amplified CL. The CL results were confirmed by the hydrogen peroxide (H2O2) determination showing that S5682 reduced H2O2 formed through either PMN stimulation (IC50 = 1.6 x 10(-6) M) or an in vitro enzymatic mechanism (IC50 = 2 x 10(-6) M). S5682 inhibited luminol-dependent CL induced by H2O2 (IC50 = 5 x 10(-6) M). However, O2 was not formed from H2O2 in contact with S5682 and the UV spectrum of this compound was not modified. In contrast, S5682 inhibited luminol-dependent CL induced by H2O2 in the presence of horseradish peroxidase (IC50 = 3 x 10(-6) M), and the UV spectrum of S5682 was modified. Luminol-dependent CL induced by hypochlorite (OCl- 10(-5) M) was also inhibited by S5682 (IC50 = 7 x 10(-5) M). This inhibitory effect was similar to that of sodium azide on myeloperoxidase activity. Moreover, OCl- 5 x 10(-4) M also altered the UV spectrum of S5682 10(-4) M. These results indicate that S5682 could be active on the H2O2-OCl(-)-myeloperoxidase system.

In the present study we measured the inhibition by 34 compounds, either flavonoids or related substances, of the release of reactive oxygen species by human neutrophils after stimulation by three agents: the bacterial peptide N-fMetLeuPhe (FMLP), the protein kinase C activator phorbol myristate acetate (PMA) or opsonized zymosan (OZ), using two chemiluminescent probes, lucigenin or luminol in the presence or absence of horseradish peroxidase (HRP). The data matrix (34 x 7) was submitted to multivariate analysis: first, a correspondence factorial analysis to uncover levels of correlation among the biochemical parameters and the specificity of action of the test-compounds and second, a minimum spanning tree analysis that classified the chemical structures into a network describing both specificity and amplitude of the inhibition of the chemiluminescence response. The major conclusions of the analyses were: (a) opposition between inhibition of poly-morphonuclear leukocytes (PMNs) stimulated by FMLP and of PMNs stimulated by PMA or OZ implying that, for the molecules under study, there was a fundamental difference in the manner in which this inhibition occurred and, conversely, a difference in the nature of the stimulatory action of these activators. Molecules lacking hydroxyl groups on ring B, i.e. chrysin, chalcone, flavone and galangin, molecules glycosylated in position 7, i.e. hesperidin and naringin and ring B mono-hydroxylated molecules were, for the most part, at the origin of this dichotomy and might interfere with the membrane FMLP receptor; (b) a marked difference in chemiluminescence inhibition in the presence or absence of HRP that can be explained by the differential action of catechins compared to flavone and flavonol derivatives; (c) a similarity in biological profile between non-flavonoids such as chalcone and phloretin and low mean-activity flavonoids such as chrysin and galangin and between the non-flavonoid curcumin and the highly active flavonoid isorhamnetin; (d) a reaffirmation of the importance of ring A (C 5,7) and ring B (C 3',4') dihydroxylation, ring C (C 3) hydroxylation, but also of the presence of a methoxy group on ring B in engendering high potency. This potency is generally decreased by C2-C3 saturation and by glycosylation. The most active molecules identified in this study provide valuable information for the selection of simpler molecules (e.g. metabolites accounting for the potency of orally administered flavonoids) for further structure-activity relationship (SAR) studies that could lead to the design of novel drugs or prodrugs.

The inhibition of 5-lipoxygenase could be involved in the mechanism of action of methotrexate (MTX). We studied 8 patients with active rheumatoid arthritis (RA) immediately before and the day after the first dose of MTX (10 mg intramuscularly). Leukotriene B4 (LTB4) formation by polymorphonuclear leukocytes was significantly decreased (32%, p less than 0.01). This essentially involved released LTB4. A slight decrease was also obtained in omega-oxidation products. Similar results were obtained for plasma LTB4 (30%, p less than 0.02). A non-significant decrease in 5-HETE was noted. Conversely, 12-HETE was not modified. Our results suggest MTX has an effect on the 5-lipoxygenase pathway, particularly at the LTA4 epoxide hydrolase step, since 5-HETE and 6-trans-LTB4 isomers are not involved.

Methotrexate (MTX) has been proved to be effective in rheumatoid arthritis (RA). The mechanism of action of MTX in this disease remains unelucidated but may involve inhibition of the enzyme 5-lipoxygenase. Arachidonic acid metabolites were studied in eight patients with active RA immediately prior to and 24 hours after the first intramuscular injection of 10 mg MTX. None of the patients were taking corticosteroids. Nonsteroidal antiinflammatory drugs were withdrawn four days before the study. Reverse phase high-performance liquid chromatography was used to quantitate metabolites produced by 5-lipoxygenase (5-LO) and 12-LO in plasma (full spectrum of blood cells) and purified neutrophils (PN) after stimulation with calcium ionophore A 21387 (50 muM for 30 minutes and 5 muM for 5 minutes, respectively). LTB4 production by PNs was significantly decreased (- 32 %, p < 0,01) 24 hours after MTX administration. A moderate (- 17 %), nonsignificant (NS) fall in LTB4 omega-oxidation products (wP) was seen. Production of 5-HETE was also slightly decreased (15 %, NS). Findings in plasma were comparable, with a significant decrease in total LTB4 (- 29.8 %, p < 0,01) and moderate falls in wP (- 18.8 %, NS) and in 5-HETE production (- 17 %, NS). Production of 12-HETE was unchanged. These findings suggest that MTX in a single dose is responsible for a decrease in the synthesis of LTB4 and 5-LO products in neutrophils and other blood cells in RA patients but does not affect 12-LO activity.

Among the cells which participate in amplification of the local inflammatory reaction in asthma, neutrophils (PMN) are pro-inflammatory cells that can generate inflammatory mediators and arachidonic acid derivatives in particular. In asthmatic patients (AP) with attacks, the capacity of blood PMN to produce 5-lipoxygenase metabolites was investigated and compared to the response in healthy subjects (HS). PMN from 6 AP and from 6 HS were stimulated by calcium ionophore A23187 and arachidonate 5-lipoxygenase metabolites were analyzed by reverse-phase HPLC. LTB4, 6-trans LTB4, omega OH-LTB4 and 5-HETE were identified. In AP, total LTB4 synthesis was enhanced as compared to synthesis with PMN in HS. But the total 5-HETE synthesis by PMN from AP was decreased. Thus, the inflammatory potential of PMN from AP was enhanced in comparison to HS. The anti-inflammatory effect of nedocromil sodium (NS) was studied in the 5-lipoxygenase metabolism of arachidonic acid. NS (10(-4) mol/l) inhibited total LTB4 synthesized by PMN in AP but not in HS. We conclude that NS affects leukotriene synthesis only in cells with enhanced inflammatory potential.

Methotrexate (MTX) has been proved to be effective in rheumatoid arthritis (RA). The mechanism of action of MTX in this disease remains unelucidated but may involve inhibition of the enzyme 5-lipoxygenase. Arachidonic acid metabolites were studied in eight patients with active RA immediately prior to and 24 hours after the first intramuscular injection of 10 mg MTX. None of the patients were taking corticosteroids. Nonsteroidal antiinflammatory drugs were withdrawn four days before the study. Reverse phase high-performance liquid chromatography was used to quantitate metabolites produced by 5-lipoxygenase (5-LO) and 12-LO in plasma (full spectrum of blood cells) and purified neutrophils (PN) after stimulation with calcium ionophore A 21387 (50 microM for 30 minutes and 5 microM for 5 minutes, respectively). LTB4 production by PNs was significantly decreased (-32%, p < 0.01) 24 hours after MTX administration. A moderate (-17%), nonsignificant (NS) fall in LTB4 omega-oxidation products (wP) was seen. Production of 5-HETE was also slightly decreased (-15%, NS). Findings in plasma were comparable, with a significant decrease in total LTB4 (-29.8%, p < 0.01) and moderate falls in wP (-18.8%, NS) and in 5-HETE production (-17%, NS). Production of 12-HETE was unchanged. These findings suggest that MTX in a single dose is responsible for a decrease in the synthesis of LTB4 and 5-LO products in neutrophils and other blood cells in RA patients but does not affect 12-LO activity.

Human alveolar macrophages (AM) from bronchoalveolar lavage of asthmatic patients (AP) and healthy volunteers (HS) were compared for their respective capacities to produce lipoxins and leukotrienes when stimulated by calcium ionophore A23187 with or without 15(S)-HETE. The metabolites were analyzed using an isocratic RP-HPLC system and their formation profiles evaluated on the basis of chromatographic behaviour, UV spectral characteristics and co-elution with synthetic standards. Without 15-HETE, AM from AP produced more LTB4 and 5-HETE than those from HS. In the presence of 15-HETE, human AM were able to produce 5,15-diHETE and lipoxins. Moreover, the total amount of lipoxins synthesized by AM from AP was 2 fold higher than that synthesized by AM from HS, thus showing an enhanced cell activation via the 5-lipoxygenase (5-LO) pathway. These results presented AM as in vitro 15-HETE metabolizing cells and suggested some hypothesis about human AM 5-LO regulation mechanism. The enhanced 5-LO activity in AM from AP suggested that in vivo they could participate in cell to cell interaction mechanisms involved in inflammatory lung diseases and might also take up and transform 15-HETE predominantly released by airway epithelial cells.

Polymorphonuclear neutrophils (PMN) generate 5-HETE which can be retained within cells as free metabolites or esterified into cellular lipids. Since this metabolite has been shown to have certain inflammatory properties, we compared the generation and distribution profile of 5-HETE in A 23187-stimulated PMN from asthmatic patients (AP) and normal subjects (NS). 5-HETE was analyzed using RP-HPLC. After 5 min, total 5 HETE generation was similar in the two populations. However, esterified 5-HETE was significantly enhanced in AP (72 +/- 3% versus 47 +/- 2% of the total synthesis, p less than 0.005), whereas intracellular free 5-HETE was decreased (13 +/- 3% versus 37 +/- 4%, p less than 0.005) and similar low release was observed. Kinetic studies showed that PMN from AP esterified 5-HETE more rapidly and to a greater extent than PMN from NS. By contrast, more intracellular free 5-HETE was recovered in PMN from NS. Esterification seems to be the major pathway of 5-HETE metabolism in PMN from AP. Moreover, we showed that most of the 5-HETE added exogenously was esterified into cellular lipids. In these experimental conditions, PAF-induced migration of PMN was increased. The enhanced ability of PMN to migrate could be due to the increase of 5-HETE esterification process.

Besides eosinophils, inflammatory processes in asthma are characterized by an infiltration of inflammatory cells, including mononuclear phagocytes, such as alveolar macrophages (AM) and blood monocytes, in the airways. Monocyte activation has been observed in the blood after exercise or allergen-induced asthma. Stimulated AM in chronic and stable asthmatic patients have been shown to release oxygen species. We thus investigated the intensity of the activation of monocytes from 18 asthmatic patients compared with 18 healthy subjects. Oxygen species release was analyzed for monocytes in suspension by chemiluminescence using a luminometer and for monocytes maintained in adherence using conventional assay and video imaging camera. Circulating blood monocytes in suspension from asthmatic patients and control subjects showed the same baseline free radical release. Monocytes in suspension from asthmatic patients were more stimulatable by PMA: specifically, monocytes release more H2O and peaks of O2-. are sooner; moreover, peaks of total free radical release are higher, and this plateau is sustained. Compared with monocytes from control subjects, those from asthmatic patients evaluated after adherence show a higher baseline for O2-. and higher total free radical release. Monocytes from asthmatic patients spontaneously release more O2-. over time in nonstimulated cells and release more O2-. with PMA stimulation; they show the same peak level total free radical release as those from control subjects after stimulation. SOD activity analysis on adherent monocytes was lower in asthmatic compared with control subjects. These data show that monocytes from asthmatic patients were activated compared with control monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism involved in amplification of the local inflammatory process, characteristic of asthma, was investigated through the role of human alveolar macrophages. During asthma attacks, mast cells and eosinophils are known to be activated in order to release arachidonic acid derived inflammation mediators such as sulfidopeptide leukotrienes. It is now known that these metabolites, particularly leukotriene C4, are present in bronchoalveolar lavage from asthmatic patients. Alveolar macrophages, recovered by bronchoalveolar lavage and purified by adherence, are able to transform LTC4 into LTE4. In four asthmatic patients with severe local inflammation as determined by fibrobronchoscopy, these phagocytes, incubated in the presence of LTC4, also generated LTB4 and 5-HETE, which remained within the cells. These preliminary results are discussed relative to amplification of the local process, involving cooperation between the different cells involved in airway responsiveness.