Exposure Of Alveolar Macrophages Research Articles

Inhibition of Mouse Alveolar Macrophage Production of Tumor Necrosis Factor Alpha by Acute in vivo and in vitro Exposure to Tobacco Smoke

We investigated the effects of tobacco smoke exposure on the production of tumor necrosis factor alpha (TNF alpha) by alveolar macrophages (AM) in mice (C57BL/6). The results obtained are as follows: (1) In vivo tobacco smoke exposure caused a significant decrease in the production of TNF alpha by AM with the stimulation of lipopolysaccharide (LPS; control group: 19.32 +/- 5.52 U/ml, smoked group: 4.28 +/- 0.98 U/ml; p less than 0.05). (2) In vitro exposure of AM to tobacco smoke extracts (water-soluble extracts) also caused a decrease in the production of TNF alpha up to 93% of control with stimulation of LPS (p less than 0.05) without any decrease in cellular viability. We concluded that the production of TNF alpha by AM was impaired by smoking via direct action of the factors present in tobacco smoke.

In vitro study of gas effects on alveolar macrophages.

To evaluate the biological effects of gas pollutants on alveolar macrophages several in vitro systems have been developed. We described here an original method of cell culture in aerobiosis, which permitted direct contact between the atmosphere and the target cells. We studied the long term (24 h) and short term (30 min) effects of NO2 on alveolar macrophages. Our results demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either cell injury or cell activation associated with the release of various bioactive mediators (superoxide anion, neutrophil chemotactic activity). Cell culture in aerobiosis opens new ways for the research on the biological effects of gas pollutants.

Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases.

Mononuclear phagocytes have the capacity to directly participate in extracellular matrix turnover via secretion of neutral proteinases. We have studied the effects of in vivo and in vitro differentiation upon cellular content or secretion of a spectrum of neutral proteinases, along with a counter-regulatory metalloproteinase inhibitor (TIMP). We found 1) matrix-degradative serine proteinases (leukocyte elastase and cathepsin G) were lost during cellular maturation and/or differentiation; 2) the 92-kDa type IV/type V collagenase and TIMP were secreted earliest in mononuclear phagocyte differentiation, whereas stromelysin secretion was observed only by LPS-stimulated alveolar macrophages; 3) exposure of alveolar macrophages, but not monocytes, to phorbol esters and LPS resulted in markedly augmented secretion of all studied metalloproteinases and TIMP; 4) monocyte-derived macrophages partially (but not completely) mimicked the metalloproteinase secretory phenotype of alveolar macrophages; and 5) the secretory phenotype of alveolar macrophages for interstitial collagenase (but not TIMP) was largely lost during in vitro culture. These results underscore the complexity of the process of differentiation in human mononuclear phagocytes, and provide insights into the variable capacity of mononuclear phagocytes to degrade extracellular matrix components. Moreover, we anticipate that human mononuclear phagocytes at various stages of differentiation will provide a useful model system for study of the variable regulation of secretion of human matrix-degrading metalloproteinases.

Liposomes and pulmonary alveolar macrophages: functional and morphologic interactions.

In vitro toxicity of liposomes and their functional and morphologic interactions with rat pulmonary alveolar macrophage (AMs) were investigated using viability (trypan blue exclusion), phagocytic and killing activity (uptake and digestion of live S. cerevisiae), surface adherence, respiratory burst (nitro-blue tetrazolium reduction), and morphometry (computerized image analysis) as indicators. Liposome stability in physiologic solutions and uptake of liposome-encapsulated carboxyfluorescein (CF) by AMs was assessed by fluorescence spectroscopy and microscopy. Liposomes made from saturated phospholipids and cholesterol were stable, whereas liposomes consisting of unsaturated phospholipids without cholesterol lost 30% to 40% of their content over 24 h. However, CF uptake was highest with unsaturated phospholipid preparations, whereas uptake of the three other formulations was comparable. Although liposome exposure did not affect macrophage viability, a reduction in the number of phagocytizing macrophages to 73% of control was noted after 24-h incubation with the highest lipid concentration tested (10 mumol/ml). Phagocytic killing was similar under all circumstances observed. The fraction of intracellularly killed yeast ranged from 32% to 42% for both control and experimental samples. An increase in cell surface area from 166.1 +/- 39.9 microns 2 on day O (n = 709) to 196.3 +/- 57.6 microns 2 on day 1 (n = 516) and 211.2 +/- 48.0 microns 2 on day 4 (n = 834) was observed after liposome treatment. The corresponding average cell areas of control samples did not change during the observation period. There was no net cell loss of adherence from monolayers as determined by protein assay. The respiratory burst, indicating generation of intracellular superoxide, was also similar--84% to 92% of experimental and control cells under all conditions showed a strong nitro-blue tetrazolium reduction. In summary, in vitro exposure of AMs to large concentrations of liposomes, although producing an increase in macrophage size, was not associated with aberrant macrophage morphologic features, function, or toxicity for the parameters examined.

Respiratory tract responses to dust: Relationships between dust burden, lung injury, alveolar macrophage fibronectin release, and the development of pulmonary fibrosis

A multidisciplinary approach was used to investigate the responses of the respiratory tract to silica (SiO 2) or titanium dioxide (TiO 2). Rats were intratracheally instilled with 5–100 mg/kg of dust and bronchoalveolar lavage fluid (BALF) lactate dehydrogenase (LDH) and total protein (TP) and ex vivo alveolar macrophage (AM) fibronectin release assessed on Days 7, 14, and 28 after exposure. Lung dust burdens were determined on Days 1, 7, and 28 after instillation. Both dusts elicited dose-related increases in BALF LDH and TP, a response which was more pronounced and progressive with SiO 2. All doses of SiO 2 elicited persistent increases in AM fibronectin release. TiO 2 stimulated persistent increases in AM fibronectin release at ≥ 50 mg/kg, with transient or no effect at ≤ 10 mg/kg. Increased SiO 2 retention was observed for all doses and TiO 2 retention was increased only at doses ≥ 50 mg/kg. In vitro exposure of naive AM to SiO 2 or TiO 2 did not stimulate AM fibronectin release. Histopathology demonstrated fibrosis at all SiO 2 doses; only TiO 2 doses ≥ 50 mg/kg resulted in fibrosis. These results reveal an association between increased dust retention, lung injury, activation of AM fibronectin release, and the development of fibrosis. The magnitude and temporal pattern of responses clearly differentiated SiO 2 from TiO 2. The correlation of BALF markers of lung injury and increased AM fibronectin release with the development of fibrosis supports the use of these parameters as predictive biomarkers of dust-induced interstitial lung disease.

Effects of in vitro ozone exposure on peroxidative damage, membrane leakage, and taurine content of rat alveolar macrophages

Rat alveolar macrophages (AM) were isolated by pulmonary lavage, allowed to adhere to a tissue culture flask, and then exposed to 0.45 ± 0.05 ppm ozone. After exposures ranging from 0 to 60 min, the medium was decanted and cells were harvested. Cells were assayed for oxidant damage and media analyzed for leakage of intracellular components. Increasing length of exposure to ozone resulted in a decreased number of adherent AM and decreased cell viability. Resting and zymosan-stimulated chemiluminescence increased immediately after ozone exposure and reached a maximum at 15–30 min, then declined to initial levels after 60 min of ozone exposure. Lipid peroxidation and leakage of protein and K + ions increased with increasing length of exposure to ozone, while leakage of reduced and oxidized glutathione increased through 30 min, then declined (reduced) or leveled off (oxidized). Activity of the Na + K + ATPase decreased with time while intracellular taurine concentration exhibited an initial rise, peaked at 30 min, and then returned to the untreated level. Leakage of taurine into the medium increased with time of exposure, suggesting that exposure of AM to ozone results in a shift from bound to free intracellular taurine. These data indicate that in vitro exposure of AM to ozone results in a time-dependent alteration of cell function, membrane integrity, and viability.

Interactions between cyclic AMP stimulating drugs and PAF-acether in guinea-pig alveolar macrophages

Intra-tracheal administration of PAF-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to guinea-pigs induces a bronchoconstriction, which appears to depend on the cell population located in the bronchi, mainly alveolar macrophages [1]. In addition, the exposure of alveolar macrophages in vitro to PAF-acether results in an increased liberation of arachidonic acid (AA) from phosphatidylcholine and phosphatidyl-inositol, suggesting the activation o fPLA 2 or PLC in concert with diglyceride lipase activities [2]. Activation of macrophages is known to be suppressed by PGE 2 and /r agonists because of their ability to elevate intracellular cAMP (cyclic AMP) concentrations [3]. Considering these findings, and in view of the hypothetized role of PAF-acether and alveolar macrophages in asthma, we examined the possible involvement of cAMP levels for controlling the effects of PAF-acether in alveolar macrophages.

Relative effects of asbestos and wollastonite on alveolar macrophages.

Rabbit alveolar macrophages were exposed in culture to chrysotile asbestos, wollastonite, or latex, and the effects on various biochemical and physiological parameters related to cellular viability and fibrogenicity were determined. Exposure of alveolar macrophages to asbestos, wollastonite, or latex for 3 d has no effect on oxygen consumption or cellular volume. However, treatment of alveolar macrophages with as little as 25 micrograms asbestos/ml for 1 d increases lysosomal enzyme release and decreases membrane integrity, i.e., decreases trypan blue exclusion and increases leakage of cytosolic enzymes. In contrast, exposure of alveolar macrophages to wollastonite or latex at 250 micrograms/ml does not induce lysosomal enzyme release or alter membrane integrity even after 3 d of exposure in culture. These data suggest that chrysotile asbestos damages rabbit alveolar macrophages, while wollastonite, a potential substitute for asbestos, is far less cytotoxic.

Response of bovine alveolar macrophages in vitro to welding fume particles.

Alveolar macrophages (AM) from bovine lungs were exposed in culture to manual metal are (MMA) welding fume particles, chromium (Cr), UICC chrysotile A or anatase for 17-20 hr. All the welding particle samples were more cytotoxic to AM than to anatase. Particles from the welding of mild steel with a rutile-coated electrode were less cytotoxic than those produced with a basic-coated electrode. Particles from the welding of stainless steel were slightly more cytotoxic, and much of this activity was probably due to CrVI. Selective release of N-acetyl-beta-glucosaminidase (beta-NAG) was only detected after exposure of AM to chrysotile. Supplementation of the incubation medium with 10% serum increased the viability of all exposed AM cultures, an effect not produced by serum albumin alone. Incubation of particle samples with dipalmitoyl phosphatidylcholine (DPPC) prior to addition to AM reduced the cytotoxicity of the "rutile" welding particles and of chrysotile.

Response of Bovine Alveolar Macrophages in Vitro to Welding Fume Particles

Alveolar macrophages (AM) from bovine lungs were exposed in culture to manual metal are (MMA) welding fume particles, chromium (Cr), UICC chrysotile A or anatase for 17-20 hr. All the welding particle samples were more cytotoxic to AM than to anatase. Particles from the welding of mild steel with a rutile-coated electrode were less cytotoxic than those produced with a basic-coated electrode. Particles from the welding of stainless steel were slightly more cytotoxic, and much of this activity was probably due to CrVI. Selective release of N-acetyl-beta-glucosaminidase (beta-NAG) was only detected after exposure of AM to chrysotile. Supplementation of the incubation medium with 10% serum increased the viability of all exposed AM cultures, an effect not produced by serum albumin alone. Incubation of particle samples with dipalmitoyl phosphatidylcholine (DPPC) prior to addition to AM reduced the cytotoxicity of the "rutile" welding particles and of chrysotile.

The effect of human alveolar macrophages on the bactericidal capacity of neutrophils.

Human alveolar macrophages (AMs) were obtained by bronchoscopy from 11 healthy adult subjects and placed into tissue culture for 24 hr. Brief preexposure (15 min) of human neutrophils to AM culture supernatants led to a greater than twofold increase in neutrophil killing of a serum-resistant strain of Pseudomonas aeruginosa (P less than 0.02). No increase in phagocytosis of 35S-labeled Pseudomonas could be detected for neutrophils preexposed to AM supernatants. However, upon exposure to bacteria, neutrophils preincubated with AM supernatants generated significantly more (P less than 0.05) superoxide anion than controls. This suggested that AM supernatants enhanced neutrophil oxidative bactericidal capacity. The material in AM supernatants which enhanced neutrophilic killing of Pseudomonas was less than 10,000 daltons in mass and heat-stable (56 C for 30 min). Release of this material was partially inhibitable by exposure of AMs to cycloheximide in tissue cultures. These data suggest that an AM-mediated amplification of neutrophil bactericidal capacity might be important in the defense of the human lung.

Biosynthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) from 1-alkyl-2-acyl-sn-glycero-3-phosphocholine by rat alveolar macrophages. Phospholipase A2 and acetyltransferase activities during phagocytosis and ionophore stimulation.

1-Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-acyl-GPC) comprises 11% of the total phospholipids of rat alveolar macrophages. This endogenous pool of alkylacyl-GPC was prelabeled by incubating the macrophages with [1,2-3H]alkyllyso-GPC (54 Ci/mmol), which enters the cells and is acylated. The effect of various stimuli on the synthesis and release into the media of labeled alkylacetyl-GPC (platelet-activating factor) from the cells was used to establish the role of inactive alkylacyl-GPC as a precursor of the biologically active derivative. A phagocytic agent (zymosan, 100 micrograms/ml) and an ionophore (A23187, 2 microM) stimulated the release of both alkylacetyl-GPC and alkyllyso-GPC into the media at the expense of cellular alkylacyl-GPC. Phospholipase A2 activity (at pH 4.5 and in 1 mM EDTA) was also increased in the media. The stimulatory effect of zymosan and the ionophore on alkylacetyl-GPC release was prevented by mepacrine (0.1 mM), an agent that inhibits the release of fatty acids from phospholipids. These data indicate that phospholipase activity is required for the biosynthesis of alkylacetyl-GPC. However, since the inhibitory effect of mepacrine was not apparent when acetate was present, it appears that the acetylation step is rate limiting. Exposure of alveolar macrophages in culture to zymosan or A23187 stimulated acetyltransferase activity 250-300%. In contrast, phorbol myristate acetate (1.6 microM), which stimulated the accumulation of lysophospholipids but not the level of alkylacetyl-GPC in the media, did not substantially increase acetyltransferase activity. We conclude that alkylacyl-GPC serves as a precursor of alkylacetyl-GPC and that the production of this potent mediator by rat alveolar macrophages can be stimulated by agents that affect phospholipase A2 and acetyltransferase activities. The latter enzyme appears to have a regulatory function in the biosynthesis of alkylacetyl-GPC.

Role of macrophage activation and interferon in the resistance of alveolar macrophages from infected mice to influenza virus.

Lung macrophages from uninfected CD1 mice support the replication of influenza viruses (H1N1 and H0N1), but the cells from influenza-infected mice do not. The possible mechanisms of this resistance were investigated. Murine macrophages were "activated" in vitro with lipopolysaccharide and lymphokines, and in both cases activation was associated with resistance of cells to infection with influenza virus. Exposure of alveolar macrophages in vitro to 500 U of purified type I interferon per ml enhanced cell spreading and Fc receptor-mediated phagocytosis, suggesting macrophage activation, and protected the cells against infection with influenza virus. Alveolar macrophages were also protected by a soluble factor in the bronchoalveolar washings from influenza-infected mice. This effect was not virus specific and was abolished by anti-interferon serum.

Effect of in vitro exposure to hydrogen sulfide on rabbit alveolar macrophages cultured on gas-permeable membranes

Rabbit alveolar macrophages (AM) were cultured on gas-permeable membranes (Petriperm dishes) and standard 25-cm 2 plastic tissue-culture flasks (Falcon Plastics) and exposed to H 2S (50–60 ppm). Exposure of AM to approximately 54 ppm of H 2S for 24 hr during culture on gas-permeable membranes reduced the percentage of AM that phagocytized 2.02-μm polyvinyl toluene beads to 5% of control values. In contrast, AM cultured on plastic tissue-culture flasks showed no decrease in phagocytic ability when exposed to similar concentrations of H 2S. Unexposed AM were equally phagocytic under both culture systems. A decrease in phagocytic capability was most pronounced under continuous exposure regimens. Above a critical combination of H 2S concentration and duration of exposure, cessation of exposure did not halt a time-dependent degradation of phagocytic ability. Scanning electron microscopy of cultured AM exposed for 24 hr to 54 ppm H 2S revealed a general decrease in cell-surface ruffling and a pronounced loss of pseudopodia. Transmission electron microscopy of AM cultures exposed to 54 ppm H 2S for 24 hr revealed extensive disruption of interior organelles, cytoplasmic membrane degradation, and sharply reduced cell-surface ruffling.

Reactive forms of oxygen and chemiluminescence in phagocytizing rabbit alveolar macrophages

Chemiluminescence (CL), superoxide anion (O2-) production, and particle uptake were measured to determine the role of antibacterial substances in the chemiluminescent response associated with phagocytosis in rabbit alveolar macrophages (AM). Exposure of AM to zymosan particles induced both CL and the production of extracellular O2-. CL is inhibited by superoxide dismutase, an enzyme which catalyzes the conversion of O2- to hydrogen peroxide (H2O2), by catalase, an enzyme which destroys H2O2, and by the hydroxyl radical (.OH) scavengers, benzoate and ethanol. Superoxide dismutase and catalase probably exert their effects in the extracellular fluid. CL can also be produced by the addition of NaO2 or H2O2 to zymosan in a noncellular system. The chemiluminescent response occurs before particle uptake is complete, which also indicates that CL occurs in the extracellular fluid. These results suggest that CL induced by zymosan in AM is due to the extracellular reaction between various reactive forms of oxygen and zymosan.