Burst Of Alveolar Macrophages Research Articles

Effects of coarse chalk dust particles (2.5-10 μm) on respiratory burst and oxidative stress in alveolar macrophages.

The main aim of the present study was to examine in vitro responses of rat alveolar macrophages (AMs) exposed to coarse chalk dust particles (particulate matter in the size range 2.5-10 μm, PM(coarse)) by respiratory burst and oxidative stress. Chalk PM(coarse)-induced respiratory burst in AMs was measured by using a luminol-dependent chemiluminescence (CL) method. Also, the cell viability; lactate dehydrogenase (LDH) release; levels of cellular superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and acid phosphatase (ACP); plasma membrane ATPase; and extracellular nitric oxide (NO) level were determined 4 h following the treatment with the different dosages of chalk PM(coarse). The results showed that chalk PM(coarse) initiated the respiratory burst of AMs as indicated by strong CL, which was inhibited by diphenyleneiodonium chloride and L-N-nitro-L-arginine methyl ester hydrochloride. It suggested that chalk PM(coarse) induced the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in AMs. This hypothesis was confirmed by the fact that chalk PM(coarse) resulted in a significant decrease of intracellular SOD, GSH, ACP, and ATPase levels and a notable increase of intracellular CAT, MDA content, and extracellular NO level, consequently leading to a decrease of the cell viability and a increase of LDH release. It was concluded that AMs exposed to chalk PM(coarse) can suffer from cytotoxicity which may be mediated by generation of excessive ROS/RNS. Graphical Abstract The possible mechanism of coarse chalk particles-induced adverse effects in AMs.

Investigation of fine chalk dust particles’ chemical compositions and toxicities on alveolar macrophages in vitro

The aim of the study is to investigate chemical compositions of fine chalk dust particles (chalk PM2.5) and examine their adverse effects on alveolar macrophages (AMs) in vitro. Morphologies and element concentrations of individual chalk particles were analyzed by using the quantitative energy-dispersive electron probe X-ray microanalysis (ED-EPMA). The oxidative response of AMs and the potential to generate nitric oxide (NO) by luminol-dependent chemiluminescence (CL) and nitrate reductase method were assessed 4h following the treatment of AMs with differing dosages of fine chalk particles, respectively. Oxidative stress and cytotoxicity elicited by chalk PM2.5 were also examined. The results showed that fine chalk particles were mainly composed of gypsum, calcite, dolomite and a little amount of organic adhesives. Exposure to chalk PM2.5 at 100μgmL−1 or 300μgmL−1 significantly increased intracellular catalase, malondialdehyde, and NO levels and decreased superoxide dismutase level in AMs, leading to leakage of lactate dehydrogenase (LDH) and reduction of the cell viability. Furthermore, luminol-dependent CL from respiratory burst in AMs was enhanced. It was suggested that chalk PM2.5 could make oxidative damages on AMs and result in cytotoxicity, being likely attributed to excessive reactive oxygen species or reactive nitrogen species induced by mixture of fine gypsum and calcite/dolomite particles.

Respiratory burst in alveolar macrophages exposed to urban particles is not a predictor of cytotoxicity

We examined the utility of respiratory burst measurements in alveolar macrophages to assess adverse cellular changes following exposure to urban particles. Cells were obtained by bronchioalveolar lavage of Fisher 344 rats and exposed (0–100μg/well) to urban particles (EHC-93, SRM-1648, SRM-1649, PM2.5), the soluble (EHC-93sol) and insoluble (EHC-93insol) fractions of EHC-93 (EHC-93tot), mineral particles (TiO2, SiO2) and metal oxides (iron III oxide, iron II/III oxide, copper II oxide, nickel II oxide). The particle-induced respiratory burst was measured by chemiluminescence for 2h after the addition of particles. The cells were then stimulated with phorbol 12-myristate 13-acetate (PMA), yeast Zymosan fragments (Zymosan), or lipopolysaccharide plus interferon-gamma (LPS/IFN-γ) and the stimulant-induced respiratory burst was measured. Independently of the potential of particles to induce directly a respiratory burst, exposure to most particles attenuated the subsequent stimulant-induced burst.The notable exception was SiO2, which produced a strong respiratory burst upon contact with the macrophages and enhanced the subsequent response to PMA or LPS/IFN-γ. Based on the degree of inhibition of the stimulant-dependent respiratory burst, particles were clustered into groups of high (SRM-1649, iron III oxide), intermediate (EHC-93tot, EHC-93insol, SRM-1648, VERP, iron II/III oxide, copper II oxide), and low (EHC-93sol, SiO2, TiO2 and nickel II oxide) potency. Across these clusters, the potency of the particles to inhibit the stimulant-dependent respiratory burst showed poor correlation with cytotoxicity determined by XTT reduction assay.

The Adp-stimulated Nadph Oxidase Activates The Ask-1/mkk4/jnk Pathway In Alveolar Macrophages

The role of H2O2 as a second messenger in signal transduction pathways is well established. We show here that the NADPH oxidase-dependent production of and H2O2 or respiratory burst in alveolar macrophages (AM) (NR8383 cells) is required for ADP-stimulated c-Jun phosphorylation and the activation of JNK1/2, MKK4 (but not MKK7) and apoptosis signal-regulating kinase-1 (ASK1). ASK1 binds only to the reduced form of thioredoxin (Trx). ADP induced the dissociation of ASK1/Trx complex and thus resulted in ASK1 activation, as assessed by phosphorylation at Thr845, which was enhanced after treatment with aurothioglucose (ATG), an inhibitor of Trx reductase. While dissociation of the complex implies Trx oxidation, protein electrophoretic mobility shift assay detected oxidation of Trx only after bolus H2O2 but not after ADP stimulation. These results demonstrate that the ADP-stimulated respiratory burst activated the ASK1–MKK4–JNK1/c-Jun signaling pathway in AM and suggest that transient and localized oxidation of Trx by the NADPH oxidase-mediated generation of H2O2 may play a critical role in ASK1 activation and the inflammatory response.

Priming of alveolar macrophage respiratory burst by H(2)O(2) is prevented by phosphatidylcholine-specific phospholipase C inhibitor Tricyclodecan-9-yl-xanthate (D609).

The respiratory burst in alveolar macrophages is enhanced in vitro by pre-exposure to nontoxic concentrations of hydroperoxides before stimulation by an agonist, which may represent a feed-forward regulatory mechanism. Tricyclodecan-9-yl-xanthate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), suppresses this priming of the respiratory burst by pre-exposure to H(2)O(2) in NR8383 alveolar macrophages (up to 100 microM D609, 400 nmol of H(2)O(2) added to 5 x 10(6) cells 15 min before stimulation with ADP). D609 has potential as an antioxidant due to its dithiocarbonate functional group that allows it to slowly react with H(2)O(2) and rapidly reduce cytochrome c, which interferes with a common assay for the respiratory burst. Nonetheless, the antioxidant properties of D609 do not account for its inhibition of priming of the respiratory burst by H(2)O(2). Reduction of nitro blue tetrazolium is the basis for an assay for superoxide production with which D609 does not interfere. With this assay, it was found that D609 does not inhibit the respiratory burst per se, but prevents its enhancement by pre-exposure to H(2)O(2). Consistent with a role of diacylglycerol generation by phospholipase C, this enhancement was mimicked by pre-exposure to phorbol ester. In contrast with priming, receptor-mediated stimulation of the respiratory burst depends on the better characterized phosphatidylinositol-specific phospholipase C. Priming of the respiratory burst by H(2)O(2) joins the list of inflammatory responses that are inhibited by D609. Nevertheless, the results herein indicate that caution should be exercised in the interpretation of the effects of D609 to consider both antioxidant effects and inhibition of PC-PLC.

Vitronectin protects alveolar macrophages from silica toxicity.

Silicosis is an interstitial lung disease caused by the inhalation of crystalline silicon dioxide. Current concepts suggest that a crucial step in the development of silicosis is silica-induced injury of alveolar macrophages (AM). The adhesive protein vitronectin is a natural constituent of the lung, in which its function is largely unexplored. This study investigated a possible role for vitronectin in protecting AM from silica exposure. In this study, the concentration of vitronectin was shown to be increased in the bronchoalveolar lavage fluid of silica-treated rats. Vitronectin affinity for silica was shown both in vitro and in vivo by immunostaining. Vitronectin reduced silica-induced injury to cultured AM as determined with the (51)Cr release assay. Vitronectin reduced silica-induced free radical production as determined with a cell-free thiobarbituric acid assay. Additionally, vitronectin reduced the silica-induced respiratory burst in AM as determined with chemiluminescence. This study suggests that vitronectin may protect AM during the initial exposure to silica.

Modulation of the alveolar macrophage superoxide production by protein phosphorylation.

Stimulation of alveolar macrophages (AM) with adenosine-5-diphosphate (ADP) results in transient production of superoxide anion radical (O2.-; superoxide) and H2O2 in a metabolic event known as the respiratory burst. Initiation of the respiratory burst appears to depend on activation of protein kinase activity, whereas protein phosphatases might involved in termination of the burst. The involvement of protein kinase C was suggested by inhibition by bisindolylmaleimide I (GF 109203X), a relatively specific inhibitor. KN-62, an inhibitor of calcium-calmodulin protein kinase II, also partly inhibited the respiratory burst stimulated by ADP and phorbol esters. The role of protein phosphatases in termination of the ADP-stimulated respiratory burst of AM was examined with calyculin A (CA) (25-75 nM) or okadaic acid (OA) (1-5 microM), two inhibitors of protein phosphatase 1 and 2a (PP1;PP2a). A dose-dependent prolongation of the respiratory burst was observed in the presence of these inhibitors. CA and OA also markedly enhanced the rate of superoxide production stimulated by ADP, consistent with involvement of PP1/PP2a in regulating both the rate of activation and timing of termination. Treatment of AM with cyclosporin A (CsA) (1-50 microM), an inhibitor of the calcium-dependent protein phosphatase 2b (PP2b), stimulated superoxide production by itself and significantly prolonged the duration of ADP-stimulated superoxide production. CsA, however, did not increase the ADP-stimulated rate of superoxide production. Thus, PP1/PP2a appear to be the primary phosphatases for controlling the intensity of the respiratory burst during receptor-elicited superoxide production in AM, whereas PP1/PP2a and PP2b play a role in turning off the respiratory burst.

Interaction of alveolar macrophages from pulmonary patients and healthy subjects with liposomes

Formation of primary metabolites of oxidative burst in alveolar macrophages from healthy subjects and patients with chronic lung diseases is studied using the method of luminoldependent chemiluminescence. Incubation with liposomes composed from phosphatidylcholine and a phosphatidylcholine-fatty acid esters mixture induces similar activation of oxidative burst in alveolar macrophages from healthy subjects and patients with lung diseases.

Effect of the pH and Biochemical Milieu of the Lung on Release of Iron from the Surface of Respirable Industrial Fibres and Ability to Cause a Respiratory Burst in Alveolar Macrophages

Effect of the pH and Biochemical Milieu of the Lung on Release of Iron from the Surface of Respirable Industrial Fibres and Ability to Cause a Respiratory Burst in Alveolar Macrophages

Biomaterial-induced dysfunction in the capacity of rabbit alveolar macrophages to kill Staphylococcus epidermidis RP12.

The effect of poly(methyl methacrylate) (PMMA), titanium alloy, and silicone discs on the capacity of rabbit alveolar macrophages (AM) to kill RP12 strain of Staphylococcus epidermidis (RP12) was studied in vitro. When freshly harvested AM were preincubated with PMMA discs for 3 h and subsequently assayed for RP12 killing, there was no change in the RP12 killing capacity of AM. However, when AM were incubated with PMMA discs for 6 or 18 h at 37 degrees C in 5% CO2, the RP12 killing capacity of AM was reduced to 15% and 4%, respectively. Preincubation of AM with titanium alloy for 6 h reduced RP12 killing capacity of AM to 30%, and to 21% in 18-h incubation. Silicone discs did not affect the RP12 killing by AM at 6 h of preincubation, but reduced RP12 killing (35%) by AM when preincubated for 18 h. Preincubation of AM with PMMA discs for 3 or 6 h did not affect the level of PMA-elicited oxidative burst of AM as measured by a luminol-enhanced chemiluminescent assay. Superoxide dismutase, which eliminated the oxidative burst of AM by 90%, did not affect the RP12 killing by AM.

Altered oxidative responses and antibacterial activity of adult rabbit alveolar macrophages exposed to poly(methyl methacrylate)

The effect of poly(methyl methacrylate) (PMMA) on the oxidative responses and antibacterial activity of adult rabbit alveolar macrophages (AM) was studied. PMMA beads ( ca. 0.3 μm diameter) elicited an acute respiratory burst within 6–8 min after the addition of the beads. In contrast, Teflon ® beads of comparable size ( ca. 0.2 μm diameter) did not elicit an oxidative burst of AM. An oxidative response was elicited only by those PMMA samples that had affinity for AM adherence. Incubation of AM with PMMA beads reduced the subsequent phorbol myristate acetate (PMA)-elicited oxidative burst by more than 80%. The Staphylococcus epidermidis —RP12 killing capacity of AM was greatly increased when PMMA beads ( ca. 0.3 μm) were added to the challenge dose of bacteria. Pre-incubation of freshly harvested AM with PMMA beads, which greatly reduced subsequent PMA-elicited chemiluminescent (CL) responses did not significantly affect the RP12 killing capacity of AM. Our data also suggest that killing of the RP12 strain of S. epidermidis does not involve reactive oxygen intermediates.

Selective down-regulation of alveolar macrophage oxidative response to opsonin-independent phagocytosis.

We have compared the oxidative response of alveolar macrophages (AM) during opsonin-dependent and independent phagocytosis by using multiparameter flow cytometry. The respiratory burst of AM during phagocytosis was quantitated by the intracellular oxidation of the nonfluorescent precursors dichlorofluorescin diacetate (DCFH) or hydroethidine (HE, a reduced precursor of ethidium) to their fluorescent (oxidized) counterparts. After loading freshly isolated normal hamster AM with DCFH or HE, red or green fluorescent beads, respectively, were added to the shaking cell suspensions. Ingestion of opsonized particles by AM caused a marked increase in oxidation of both DCFH and HE proportional to the number of beads ingested. In contrast, uptake of one to three unopsonized particles per cell led to inhibition of oxidative activity compared to control cells incubated without particles. AM ingesting four or more unopsonized particles showed some increase in oxidative metabolism, but far less than that with identical numbers of particles in opsonin-dependent ingestion. Similar results were obtained using fluorescent labeled staphylococcal bacteria. Using three-color flow cytometry to study cells ingesting both types of particles, cells first ingesting unopsonized beads were also found to have an inhibited oxidative response to subsequently ingested opsonized particles. The mitochondrial poison antimycin inhibited most of the intracellular oxidative response to either type of phagocytosis. The remaining antimycin-insensitive, membrane derived respiratory burst of AM was also substantially diminished after phagocytosis of unopsonized particles vs similar numbers of opsonized particles. The greatly increased mitochondrial respiration in AM during phagocytosis of opsonized particles may be related to bactericidal mechanisms. Killing of ingested Staphylococcus by AM was markedly impaired in the presence of antimycin. The results suggest that AM may ingest the numerous, unopsonized inert particles that are inhaled without generation of potentially toxic oxygen metabolites, while retaining the capacity to undergo a respiratory burst after ingesting opsonized particles and bacteria. The mechanism(s) for this distinct response may include generation of an inhibitor of intracellular oxidative metabolism.

Respiratory burst in alveolar macrophages of diabetic rats

Bactericidal ability of alveolar macrophages is depressed in rats with diabetes mellitus. To define the mechanism of this abnormality, we measured the parameters of respiratory burst in alveolar macrophages, peripheral blood monocytes, and neutrophils of rats 8 wk after the induction of diabetes by streptozocin. Superoxide anion (O2-.) generation during basal conditions and after stimulation with phorbol myristate acetate (PMA) was measured as superoxide dismutase-inhibitable cytochrome c reduction. NADPH, the principal substrate for NADPH-oxidase-dependent O2-. generation, was measured in the alveolar macrophages and quick-frozen lungs by the enzyme-cycling method. O2-. generation after PMA was significantly lower in the alveolar macrophages of diabetics than in the controls (14.4 +/- 2.0 nmol.10(6) cells-1.20 min-1 vs. 26.2 +/- 1.9, P less than 0.05). Conversely the peripheral blood monocytes of diabetics demonstrated an enhanced O2-. production after PMA stimulation. There was no significant difference in the neutrophil O2-.-generation between the groups. The alveolar macrophage NADPH (control 0.44 +/- 0.15 nmol/10(6) cells vs. diabetic 0.21 +/- 0.04, P less than 0.05) and lung tissue NADPH levels (control 81.4 +/- 16.3 nmol/g dry wt vs. diabetic 35.8 +/- 20.5, P less than 0.05) were significantly lower in the diabetics than in the controls. These data indicate that the O2-.-generating capacity of alveolar macrophages is markedly depressed in diabetes, whereas their precursors, monocytes, are primed to generate O2-. with PMA stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of free radicals from freshly fractured silica dust. Potential role in acute silica-induced lung injury.

Data presented here indicate that freshly fractured silica exhibits surface characteristics and biologic reactivity distinct from aged silica, and on this basis we propose that these surface features may lead to enhanced manifestations of lung injury. Grinding of silica produces approximately 10(18) Si and Si-O (silicon-based) radicals per gram of dust on the particulate surface which are characterized by an electron spin resonance (ESR) spectrum centered around g = 2.0015. These silicon-based radicals react with aqueous media to produce OH radicals, which are demonstrable using a DMPO spin trap. The concentration of silicon-based radicals in silica decreases with aging in air and exhibits a half-life of approximately 30 h, whereas its ability to generate OH radicals in aqueous solution decreases with a half-life of approximately 20 h. However, on storage in aqueous media, the concentration of silicon-based radicals and the dust's ability to generate OH radicals decrease significantly within a few minutes. Freshly ground silica is also more biologically reactive than aged silica, because freshly crushed silica activates a greater respiratory burst in alveolar macrophages than aged silica, i.e., storage of ground dust in air decreases silica-induced superoxide anion secretion, hydrogen peroxide release, and NBT reduction by 25%, 68%, and 43%, respectively. Furthermore, compared to aged silica, freshly ground silica exhibits a greater cytotoxic effect on cellular membrane integrity, i.e., a 1.5-fold increase in LDH release from macrophages, a 36-fold increase in hemolytic activity, and a three-fold increase in the ability to induce lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine-coated particles generate Superoxide (O−2) and chemiluminescence in alveolar macrophages

WE have recently shown that histamine bound to red cells forms ‘rosettes’ with various guinea pig leukocytes and that these ‘histamine receptors’ are particularly well expressed on the alveolar macrophage1. As the lung is a major site of histamine storage and high concentrations of histamine are present in the sputum in certain respiratory diseases such as chronic bronchitis and asthma2,3, we have performed experiments to determine whether free histamine, or histamine bound to particles, interacts with its membrane receptor to initiate the respiratory burst in alveolar macrophages. Activation of the respiratory burst in phagocytic cells is achieved by a number of agents, both soluble and particulate, many of which bind to the plasma membrane (for example, particles opsonised with complement4) through cell surface receptors. This suggests that there may be a close association between ‘membrane recognition’ of certain exogenous stimuli and the enzyme which initiates the primary oxygen-consuming reaction (also thought to be membrane bound and probably ‘NADPH oxidase’5). In the experiments reported here Superoxide anion formation (O−2) and chemiluminescence have been measured as these are recognised features of the respiratory burst of phagocytic cells5.