Transmembrane potential changes during phagocytosis in rat alveolar macrophages

Studies were carried out to measure changes in the transmembrane potential of rat alveolar macrophages during exposure of the cells to zymosan particles or to the membrane perturbant, phorbol-12-myristate-13-acetate (PMA), and to determine if changes in membrane potential are related to superoxide anion release. Exposure of the cells to either zymosan or PMA leads to membrane depolarization, which precedes superoxide anion release. Furthermore, the magnitude of the depolarization is dependent upon the concentration of either zymosan or PMA. During exposure of the alveolar macrophages to increasing levels of zymosan, there is an increase in the amount of superoxide released as well as an increase in the magnitude of the depolarization. Incubation of the cells in medium containing 150 mM K+, a medium which causes membrane depolarization, leads to superoxide release from resting cells and a decrease in the amount of superoxide released from cells exposed to zymosan. These results indicate that release of superoxide anion from rat alveolar macrophages is related to membrane depolarization and suggest that the transmembrane potential change may act as a signal to initiate the phagocytotic responses of the cells.     

Transmembrane potential of isolated rat alveolar type II cells

Type II cells were isolated from rat lungs by elastase digestion and purified by centrifugal elutriation. The fluorescent dye, Di-S-C3(5), was used as a probe to monitor transmembrane potential (Em) of cells suspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered medium. With this technique, the Em of type II cells was estimated to be -27 +/- 2 mV. This resting Em is very close to the equilibrium potential for chloride (-21 mV), which suggests that chloride is passively distributed in type II cells. The resting Em of type II cells is more dependent on the extracellular concentration of potassium (K+) than on external sodium (Na+); i.e., the membrane depolarizes as external sodium is replaced by potassium, suggesting that in unstimulated type II cells the membrane is more permeable to potassium than to sodium. In addition, the resting potential appears to be due, in part, to the activity of a ouabain-sensitive, Na-K pump, which acts to hyperpolarize type II cells. Addition of a membrane perturbant, phorbol myristate acetate (PMA, 10 micrograms/ml), to a type II cell suspension results in an increase in oxygen consumption and membrane depolarization. Both of these responses are sodium dependent and thus appear to be linked to a PMA-induced increase in sodium permeability.     

Unique opsonic requirements of rat pulmonary alveolar macrophages

Rat pulmonary alveolar macrophages (PAM) were compared with peripheral blood neutrophils for their ability to killEscherichia coli andStaphylococcus aureus in vitro. A wide range of opsonic conditions were studied, including from 10 to 40% autologous serum, alveolar lavage fluid (ALF), concentrated ALF supernatant, and several bacterial preopsonization procedures. PAM killed at most 41% of the initialE. coli inoculum in the presence of 40% serum and ALF. PAM killed only 5% of the initial inoculum ofS. aureus with 40% serum preopsonization, 10% serum plus ALF in the reaction mixture. No improvement in killing of either organism was seen after addition of ALF concentrated supernatant to the assay. In contrast to PAM, neutrophils killed more than 90% of the initial inoculum of either organism in the presence of 10% serum. The results imply that additional opsonic or physical factors in vivo are necessary for maximum PAM microbicidal function.     

Effects of neonatal monosodium-L-glutamate treatment on rat alveolar macrophages

Treatment of neonatal rats with repeated doses of monosodium-L-glutamate resulted in changes of morphology and function of alveolar macrophages recovered from adult female rats. Numerous cellular lipid vacuoles and lamellar structures were observed in these alveolar macrophages under transmission electron microscopy. There was approximately 50% increase of cellular total lipid content measured by Oil-Red-O staining and colorimetric method. The yeast phagocytosis and intracellular killing ability, as well as the inhibitory effect on the growth of tumor cells were reduced in alveolar macrophages from neonatal MSG-treated rats.     

Elemental analysis of lung tissue particles and intracellular iron content of alveolar macrophages in pulmonary alveolar proteinosis

Background

Pulmonary alveolar proteinosis (PAP) is a rare disease occurred by idiopathic (autoimmune) or secondary to particle inhalation. The in-air microparticle induced X-ray emission (in-air micro-PIXE) system performs elemental analysis of materials by irradiation with a proton microbeam, and allows visualization of the spatial distribution and quantitation of various elements with very low background noise. The aim of this study was to assess the secondary PAP due to inhalation of harmful particles by employing in-air micro-PIXE analysis for particles and intracellular iron in parafin-embedded lung tissue specimens obtained from a PAP patient comparing with normal lung tissue from a non-PAP patient. The iron inside alveolar macrophages was stained with Berlin blue, and its distribution was compared with that on micro-PIXE images.

Results

The elements composing particles and their locations in the PAP specimens could be identified by in-air micro-PIXE analysis, with magnesium (Mg), aluminum (Al), silicon (Si), phosphorus (P), sulfur (S), scandium (Sc), potassium (K), calcium (Ca), titanium (Ti), chromium (Cr), copper (Cu), manganase (Mn), iron (Fe), and zinc (Zn) being detected. Si was the major component of the particles. Serial sections stained by Berlin blue revealed accumulation of sideromacrophages that had phagocytosed the particles. The intracellular iron content of alveolar macrophage from the surfactant-rich area in PAP was higher than normal lung tissue in control lung by both in-air micro-PIXE analysis and Berlin blue staining.

Conclusion

The present study demonstrated the efficacy of in-air micro-PIXE for analyzing the distribution and composition of lung particles. The intracellular iron content of single cells was determined by simultaneous two-dimensional and elemental analysis of paraffin-embedded lung tissue sections. The results suggest that secondary PAP is associated with exposure to inhaled particles and accumulation of iron in alveolar macrophages.     

Localization of thyroid hormone in subpopulations of rat alveolar macrophages

Summary Radioimmunoassay and immunocytochemical staining methods were used to study the distribution of thyroid hormone in rat alveolar macrophages. The cells were fractionated into six subpopulations by Percoll density gradient. Positive immunoreactive tri-iodothyronine (T₃) was observed in all subpopulations of macrophages, especially in low-density (1.040 and 1.050 g cm^–3) groups, by avidin-biotin-peroxidase immunostaining techniques. The macrophages also showed various patterns of cellular T₃ stainability. Results from radioimmunoassay of macrophage extracts also demonstrated that macrophages of low density had a higher level of total T₃ than those of higher densities (1.060 g cm^–3).     

Flow cytometric analyses of lectin binding to rat alveolar macrophages

The binding characteristics of ten FITC-labeled plant lectins (Con-A, MPA, BPA, PNA, WGA, SBA, UEA-I, DBA, GS-I, GS-II) to lavaged rat alveolar macrophages were assessed by flow cytometry. The alveolar macrophages (AM) were incubated with varying concentrations of each lectin in a pinocytosis-inhibiting buffer. In addition to measuring lectin-associated green fluorescence, the electronic cell volumes and axial light loss characteristics of the AM were also measured flow cytometrically. These latter parameters were found to be good indicators of cell agglutination caused by some of the lectins, and, in conjunction with green fluorescence measurements, usefully serve to determine optimal or nonagglutinating lectin concentrations for flow cytometric studies. With the exception of UEA-I, all of the lectins examined bound to AM, although a wide range of binding was observed among the lectins. At subagglutinating concentrations, Con A, MPA, BPA, PNA, WGA, SBA, and GS-I bound to the AM with unimodal patterns. Histograms of lectin-associated fluorescence intensity obtained with DBA clearly presented a pattern consistent with a more complex, bimodal distribution of labeled AM, suggesting the presence of at least two subset populations. The low-intensity distribution of AM represented congruent to 70% of the cells, while the more strongly labeled subset represented congruent to 15% of the parent AM population. The remaining balance of the AM was identified as another subpopulation by the failure to detectably bind to the DBA. While GS-II bound to all of the AM, this lectin labeled about 5% of the cells much more intensely than the bulk of the population. Thus, two subset populations of AM could be resolved according to their differing avidities for the GS-II lectin.     

Kinetics of uptake and distribution of arachidonic acid by rat alveolar macrophages

The time course of uptake and distribution of 3H-arachidonic acid (3H-AA) into rat alveolar macrophage phospholipid pools was examined. Macrophages incubated with exogenous 3H-AA in RPMI-1640 containing 0.1% bovine serum albumin (BSA), incorporated this radiolabel into phosphatidylcholine and phosphatidylinositol (PI) with plateaus reached within 2 to 4 hours, which remained relatively constant for up to 18 hours. Incorporation of 3H-AA into phosphatidylethanolamine was small, but continued to increase for 14 hours. Analysis of phosphate content in phospholipid pools revealed that treatment with exogenous 5 nM arachidonic acid had no effect upon pool sizes, but there was a selective incorporation of 3H-AA into PI. Cells were incubated with 3H-AA in RPMI alone or medium containing either 0.2% lactalbumin, fetal calf serum at variable concentrations, 10% Nu Serum, or 0.1% BSA. Incubation of macrophages with 3H-AA in RPMI alone or containing 0.2% lactalbumin, resulted in approximately 70% of the radiolabel taken up by the cells being incorporated into triglyceride. The addition of BSA to RPMI-1640 medium was found to facilitate selective uptake of 3H-AA into phospholipids. Approximately 70% of incorporated 3H-AA was releasable through the action of exogenous phospholipase A2.     

Kinetics of uptake and distribution of arachidonic acid by rat alveolar macrophages

The time course of uptake and distribution of ³H-arachidoni acid (³H-AA) into rat alveolar macrophage phospholipid pools was examined. Macrophages incubated with exogenous ³H-AA in RPMI-1640 containing 0.1% bovine serum albumin (BSA), incorporated this radiolabel into phosphatidylcholine and phosphatidylinositol (PI) with plateau reached within 2 to 4 hours, which remained relatively constant for up to 18 hours. Incorporation of ³H-AA into phospholipid pools revealed that treatment with exogenous 5 nM arachidonic acid had no effect upon pool sizes, but there was a selective incorporation if ³H-AA into PI. Cells were incubated with ³H-AA in RPMI alone or medium containing either 0.2% lactalbumin, fetal calf serum at variable concentrations, 10% Nu, Serum, or 0.1% BSA. Incubation of macrophages with ³H-AA in RPMI alone or containing 0.2% lactalbumin, resulted in approximately 70% of the radiolabel taken up by the cells being incorporated into triglyceride. The addition of BSA to RPMI-1640 medium was found to facilitate selective uptake of ³H-AA into phospholipids. Approximately 70% of incorporated ³H-AA was releseable through the action of exogenous phospholipase A₂.     

Zinc hydroxide stimulates superoxide production by rat alveolar macrophages.

The effect of zinc hydroxide on superoxide (O2-) production by rat alveolar macrophages was determined by chemiluminescence and by cytochrome c reduction. Zinc ions had no effect on the chemiluminescence of unstimulated alveolar macrophages. By contrast, zinc hydroxide (ZnOH2), a neutralized form of zinc ions, increased the chemiluminescence level and O2- release. Increased O2- release was inhibited by pertussis toxin, isoquinoline sulfonamide and pretreatment with EGTA. These findings indicate that zinc hydroxide formation from zinc compounds can stimulate the O2- production by alveolar macrophages by receptor-mediated and Ca(2+)-dependent process.     

Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages

Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP-A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a (51)Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 +/- 2.5, whereas the addition of rat SP-A (5 microg/ml) significantly reduced the cytotoxic index to 16.6 +/- 1.2 (P < 0. 001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody or D-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica.     

Solubility of nickel oxide particles in various solutions and rat alveolar macrophages

The solubility of five types of commercial nickel oxide particles was determined in different types of solutions, including distilled water, physiological saline, buffered saline, and tissue-culture medium, in order to estimate their solubility in the human respiratory tract. In addition, we examined the solubility of the two types of particles that were the most and least soluble particles of the above five types of nickel oxide, in rat alveolar macrophages cultured in vitro. The solubility of the nickel oxide particles in these solutions varied remarkably with their types, suggesting that, even though they are called as “nickel oxide,” their solubilities are different among the manufacturer and the product lot. Their solubilities were also influenced by the types of solution and the existence of carbon dioxide in the atmosphere. The solubility of nickel oxide particles in the alveolar macrophages was significantly larger than that observed in the culture medium without macrophages, but smaller than that observed in the distilled water. These results suggest that the actual solubility of nickel oxide particles in the respiratory tract may be difficult to estimate by the conventional solubility analysis method using distilled water, and that the enhancement of particle dissolution by the alveolar macrophages and the depression of particle solubility by the coexisting salt and carbon dioxide should be taken into consideration for the accurate estimation.     

Association of chlorphentermine with phospholipids in rat alveolar lavage materials, alveolar macrophages and type II cells

Administration of chlorphentermine to rats leads to an increase in the phospholipid content of pulmonary surfactant materials and alveolar macrophages. It is known that this drug binds to pure phospholipids and prevents their degradation by phospholipases. Therefore, experiments were carried out to determine if chlorphentermine binds to surfactant phospholipids in vitro and to measure the in vivo association of drug with phospholipids in alveolar lavage materials from rats injected with [14C]chlorphentermine. The presence of chlorphentermine in alveolar macrophages, type II cells and other small pneumocytes (a population of lung cells which does not include alveolar macrophages or type II cells) from treated animals was also assessed. Binding of the drug to surfactant phospholipids, as measured with the fluorescent probe, 1-anilino-8-naphthalene sulfonate, occurs in vitro and does not differ in various subfractions of alveolar lavage materials isolated by differential centrifugation. Following daily administration of chlorphentermine to rats for 3 days, the drug appears to be associated with surfactant phospholipids such that the molar ratio is 1:100 (chlorphentermine/phospholipid). Chlorphentermine is also associated with alveolar macrophages (molar ratio, 1:18) and type II cells (molar ratio, 1:33). Not much drug is associated with the population of other lung cells (molar ratio, 1:333). In alveolar macrophages, approx. 70% of the drug seems to be bound to phospholipid and/or sequestered in subcellular organelles. However, only 20% of the chlorphentermine is bound and/or sequestered in type II cells. The results of these experiments suggest that following chlorphentermine administration, the drug is associated with phospholipids in acellular pulmonary lavage materials, alveolar macrophages and type II cells. This drug-phospholipid interaction may impair phospholipid degradation and lead to a phospholipidosis in surfactant materials and alveolar macrophages.     

Influence of hypoxia on 5-lipoxygenase pathway in rat alveolar macrophages

The effect of hypoxia was studied on the ionophore A23187-induced leukotriene production by rat alveolar macrophages. The production of LTB4 and LTC4 decreased with reducing oxygenation without change of cell viability. The synthesis of 5-HETE increased during hypoxia and the total production of LTB4, LTC4 and 5-HETE, the major metabolites of the 5-lipoxygenase pathway in rat alveolar macrophages, was equal during normoxia and hypoxia. Arachidonate release and LTA4-converting into LTB4 and LTC4 was unaffected by hypoxia. LTB4- and LTC4-degradating activities were not affected by hypoxia. These results suggest that LTA4 synthase reaction of leukotrienes biosynthesis might be suppressed by hypoxia.     

A homologue of platelet-derived growth factor produced by rat alveolar macrophages

Rat alveolar macrophages secrete a growth factor that renders rat lung fibroblasts competent to initiate DNA synthesis in vitro in the presence of platelet-poor plasma. This biological activity resembles that of platelet-derived growth factor (PDGF). After separation from putative associated binding proteins by chromatography under acidic conditions, the macrophage-derived factor exhibited a relative molecular weight similar to that of highly purified human PDGF. The factor bound to a monospecific antibody to human PDGF and thus could be quantitated in an enzyme immunoassay for PDGF. It competed with radiolabeled human PDGF for receptor sites for PDGF on rat lung fibroblasts, and binding to these receptor sites could be specifically inhibited by anti-PDGF. These data strongly support the view that the factor derived from rat alveolar macrophages is homologous to human PDGF and is similar to human macrophage-derived PDGF-like growth factor. Furthermore, we have demonstrated that the lung contains both an effector cell (pulmonary macrophage) and a potential target cell (interstitial fibroblast) for this cytokine. Therefore the rat appears to be an appropriate animal model in which to study macrophage-derived PDGF-like growth factors as mediators of proliferation in pulmonary fibrogenesis.