Chemical characterization of specific pulmonary macrophage cell surface antigen.
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Biomedical subjects
J D Brain
Publications and source records attributed to J D Brain.
Airway macrophages. The importance of the fixation method.
Two methods of fixation, intravascular perfusion and intratracheal instillation, were compared with regard to the number, distribution, and appearance of airway and alveolar macrophages. With instillation of fixative, the number of airway macrophages present was reduced to only 32.6% of that seen after intravascular perfusion. Macrophages may have important physiologic and pathologic roles in the airways. The method of fixation is crucial for preservation of their numbers and location.
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Monoclonal antibody to an alveolar macrophage surface antigen in hamsters.
A conventionally produced antibody specific for hamster lung macrophages was prepared by immunizing guinea pigs with lung macrophages from LSH (inbred) hamsters. Specificity was achieved by absorbing the resulting serum with hamster blood cells and peritoneal macrophages. This antibody was used to precipitate antigen from detergent lysates of hamster lung macrophages. To produce monoclonal antibodies, F1 hybrids of Balb C X C57Bl6 mice were immunized with these immunoprecipitates. Fusion of splenic lymphocytes from these mice with NS-1 myeloma cells produced 4 hybrid cell lines. Subcloning yielded 18 lines producing antibody reacting only with lung macrophages, and 2 lines secreting nonreactive antibody. Screening used lung and peritoneal macrophages and an ELISA assay. Using gel electrophoresis and lysates of 125I-labeled lung macrophages, all 18 lines reacted with the same antigen, a protein of 102,000 daltons. Subclasses of these monoclonal antibodies included IgG2b kappa and IgG1 kappa. Quantitative ELISA assays showed that the antibody reacted with lung macrophages of LSH and LVG (outbred) hamsters, but not with hamster resident peritoneal macrophages, spleen cells, or bone marrow cells. The antibody did not cross-react with lung or resident peritoneal macrophages from mice, rats, or guinea pigs. By flow cytometry, no reaction was detected with resident, thioglycollate-elicited, or BCG-stimulated peritoneal macrophages. When frozen sections of lung and other organs were examined by indirect immunofluorescence and immunoperoxidase methods, only alveolar macrophages were stained.
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Correlation between the behavior of magnetic iron oxide particles in the lungs of rabbits and phagocytosis.
Five New Zealand white male rabbits were exposed (30 min; 300 mg/m3) to a submicrometric magnetic iron oxide aerosol (gamma-Fe2O3) produced by burning iron pentacarbonyl in a reducing atmosphere. After aerosol inhalation, an external magnetic field was applied to the rabbits to magnetize and align the ferrimagnetic particles within their lungs. After removal of the external field, a remanent magnetic field was detectable at the body surface. Using a flux-gate magnetometer probe in an enclosure shielded against external magnetic noise, the peak remanent field after magnetization was measured periodically during the next 6 weeks. After each magnetization, the strength of the remanent field decayed rapidly with time (relaxation). The mechanism responsible is particle rotation caused by tissue, cell, organelle, or Brownian movement. The rate of relaxation changed with time after particle inhalation, especially during the first day; changes in the relaxation rate correlated with an estimate of in situ particle phagocytosis during that time. Analysis of pulmonary lavage fluid from 15 rabbits into which radioactive gold-198 had been intratracheally instilled showed that, at 1 hr after instillation, 27% of the gold had been phagocytized, whereas at 16 hr 91% had been ingested. The strength of the magnetic field immediately after each magnetization (that is, before relaxation) was used to estimate the amount of iron oxide in the lungs. At 1 day after exposure, 96.8 +/- 8.8% (mean +/- standard error) of the initial dust was still present; at 10 days, 67.9 +/- 16.2%; and at 40 days, 16.0 +/- 4.6%. It is concluded that ferrimagnetic particles can serve as an easily measured, long-lasting marker that can be used for noninvasive studies of clearance and of particle phagocytosis and as a probe for intracellular processes such as organelle motion.
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Alterations in alveolar macrophages in hamsters developing pulmonary fibrosis.
Hamsters treated with intratracheally instilled bleomycin (0.16 U/100 g) followed by a 72-hr exposure to 70% oxygen develop a slowly progressive interstitial pneumonitis with fibrosis. Lung lavage was performed during fibrogenesis at 30, 60, and 120 days after treatment. The number of macrophages recovered was increased at all of these times. Macrophages were evaluated using flow cytometry and a monoclonal antibody specific for a surface antigen present on mature lung macrophages but deficient in younger cells. The mean density of antigenic sites per cell surface area was significantly lower than control values at the three times studied (-24.7, -20.0, and -20.9%). Thus, a significant fraction of macrophages present in this model of progressive pulmonary fibrosis are immature. The in vivo uptake of radioactive colloidal gold by the pulmonary macrophages was also determined. Thirty days after treatment, macrophage endocytosis of colloidal gold was reduced by 22% of control. The total number of harvested macrophages increased twofold, however, a change that usually increases endocytotic rates. It is concluded that macrophages are increased during fibrogenesis and that this increase is caused by a continued influx of new macrophages. In addition, the phagocytic function of these macrophages is less efficient.
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Differences in effects of immediate and delayed hyperoxia exposure on bleomycin-induced pulmonary injury.
Several reports have suggested that patients treated with bleomycin may be at greater risk of developing respiratory failure when exposed to elevated concentrations of oxygen. We studied the interactions of bleomycin and hyperoxia in Syrian golden hamsters. Animals were instilled intratracheally with bleomycin at a dose of 0.5 unit/100 g of body weight, followed immediately by exposure to 70% oxygen for 72 hours. Mortality was 90% in these hamsters, compared to 15% in an age-matched control group treated with bleomycin alone. Postmortem studies revealed that pathologic changes were confined to the lungs which showed severe, hemorrhagic, diffuse alveolar damage. To determine the effect of delaying exposure to hyperoxia, bleomycin at a dose of 0.5 unit/100 g of body weight was instilled and animals were kept in room air for 1 and 2 months before exposure to 70% or 100% oxygen for 72 hours. No significant increase in mortality or interstitial pneumonitis and fibrosis was seen in these groups during or after the hyperoxic exposures. Mortality in controls treated with saline and hyperoxia was zero. We conclude that simultaneous treatment with bleomycin and hyperoxia results in a synergistic effect on mortality and on the development of pulmonary fibrosis. However, there is no synergism if the hyperoxic exposure is delayed for at least 1 month following bleomycin treatment.
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Correlation of lung macrophage age and surface antigen in the hamster.
A monoclonal antibody specific for a surface antigen found on hamster lung macrophages has been produced. Macrophages obtained from LSH Syrian golden hamsters by pulmonary lavage have varying amounts of this antigen on their surface. We compared the age of alveolar macrophages (using 3H-thymidine) with the amount of surface antigen. Lung macrophages were obtained by repeated saline lavage at 1, 3, 5, and 10 days after 3H-thymidine injection. Monoclonal antibody was then reacted with these cells followed by fluorescein isothiocyanate-conjugated protein A. Cell size and fluorescence were analyzed by flow cytometry. A wide range of fluorescent intensity was observed; the cells were sorted into four subpopulations (SPs). SP1 had the lowest fluorescence per cell, and SP4 had the highest. The sorted cells were placed on glass slides, and autoradiographs were made. The percentage of labeled macrophages in each SP was determined. At 1 day after thymidine injection, cells with a paucity of antigen (SP1) were the most highly labeled; 12.5% of SP1 macrophages were labeled, but only 1.4 and 1.1% of SP3 and SP4 were labeled, respectively. The labeling was relatively even in all four SPs at 3 days, but at 5 days the labeling of cells in SP2 and SP3 was highest. By day 10, labeled macrophages had large amounts of surface antigen and were in SP3 and SP4. These findings suggest that pulmonary macrophages that have recently synthesized DNA lack surface antigen. As time passes, cells mature and more antigen is acquired. The amount of surface antigen reflects cell age and provides a useful tool to isolate and study macrophage SPs.
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Lactate dehydrogenase isoenzymes in hamster lung lavage fluid after lung injury.
Lactate Dehydrogenase Isoenzymes in Hamster Lung Lavage Fluid after Lung Injury. Beck, B. D., Gerson, B., Feldman, H. A., and Brain, J. D. (1983). Toxicol. Appl. Pharmacol. 71, 59-71. Lactate dehydrogenase (LD) levels and isoenzyme patterns were determined in the cell-free supernatant fractions of lung lavage fluid from hamsters exposed to alpha-quartz, iron oxide, Triton X-100, 100% O2, or 200 ppm SO2. The isoenzyme patterns were compared to those derived from hamster lung homogenates, serum, polymorphonuclear neutrophils (PMNs), pulmonary macrophages, and red blood cells. The isoenzyme patterns from alpha-quartz- and iron oxide-exposed animals resembled each other and were similar to that of PMNs. In contrast, the pattern seen after Triton X-100 exposure was similar to those of whole lung homogenates and of red blood cells. A 96-hr exposure to 100% O2 yielded an LD isoenzyme pattern in lung lavage fluid similar to that of serum. Exposure to SO2 did not alter LD levels, showing that upper airways damage is not reflected by changes in LD in lung lavage fluid. We conclude that LD isoenzyme patterns of lung lavage fluid can be used to differentiate among types of pulmonary injury and may help identify the sites of injury.
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Uptake of latex particles by macrophages: characterization using flow cytometry.
Flow cytometry can be used to characterize the uptake of particles by small samples of pulmonary macrophages both quickly and accurately. We found that there was a linear relationship between the number of fluorescent latex particles and the fluorescent intensity associated with each cell up to 47 particles/cell. Macrophages lavaged from the lungs of Syrian golden hamsters were metabolically and functionally stable for 3 h of incubation; the average intracellular concentrations of Na+ and K+ were 29 +/- 2 and 158 +/- 6 mM, respectively. Particle uptake was significantly inhibited by removing divalent cations with ethylenediaminetetraacetic acid and lowering the incubation temperature (37 degrees C) to 24 and 3 degrees C. The cell-to-cell variability in the uptake of particles was greater than the Poisson distribution would predict based on the mean number of particles per cell.
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Uptake of latex particles by pulmonary macrophages: role of calcium.
The uptake of latex particles by both viable and fixed hamster pulmonary macrophages was calcium and trypsin sensitive. Particle uptake did not stimulate the uptake of 45Ca. However, when 45Ca uptake was stimulated with A23187, particle uptake was inhibited. When cobalt was added with A23187, the uptake of 45Ca was inhibited and particle uptake returned to control levels. A23187, cytochalasin B, and A23187 plus cytochalasin B all reduced particle uptake to the same extent. Although both A23187 and ouabain produced similar changes in the intracellular levels of Na+ and K+, only A23187 inhibited particle uptake. We conclude that extracellular Ca2+ promotes particle-cell binding through its interaction with a trypsin-sensitive receptor in the pulmonary macrophage membrane. In contrast, elevated intracellular Ca2+ levels inhibit particle ingestion but not attachment.
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Anesthesia alters the pattern of aerosol retention in hamsters.
General anesthesia was used to produce nonventilated areas of the lung, and aerosol inhalation was used to locate these areas, assuming that no aerosol deposits in a nonventilated region. Male Syrian golden hamsters were anesthetized with pentobarbital sodium (90 mg/kg), which reduced respiratory frequency, tidal volume, minute volume, and O2 consumption to 61, 41, 24, and 36%, respectively, of the corresponding awake levels. Awake and anesthetized hamsters were exposed to the aerosol for 30 min; then the lungs were excised, dried at total lung capacity, sliced into sections, and dissected into pieces. Autoradiographs were made of slices, and the activity and weight of pieces were determined. The evenness index (EI), a measure of the uniformity of retention, was calculated for each piece. With complete uniformity of retention, all EI's would be 1.0. In awake animals, only 0.2% (by wt) of the lungs had little or no retention (EI's less than 0.20). More particles deposited in the apex than in the base of the lungs. General anesthesia for extended periods of time with no deep breaths alters ventilation and therefore the distribution of aerosol retention. Many regions of the lungs in the anesthetized animals received few or no particles (11.6% of lungs had EI less than 0.20); however, no consistent pattern was observed in the location of these areas from animal to animal. The apex-to-base gradient for retention in these animals was also reversed. Radioactive aerosols can be used as probes to indicate the extent and distribution of nonventilated areas in the lungs.
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Behavior of magnetic particles in hamster lungs: estimates of clearance and cytoplasmic motility.
Ferrimagnetic particles suspended in saline were instilled intratracheally into the lungs of Syrian golden hamsters. The particles were magnetized and aligned by applying an external magnetic field. Upon removal of the external field, the particles produced a remanent magnetic field from the lungs which decayed due to random misalignment of the particles (relaxation). Magnetization and relaxation measurements were performed immediately after instillation, then repeatedly during the first 24 h, and finally at intervals of several days up to 30 days after the instillation. The size of the initial remanent magnetic field immediately following each external magnetization is a measure of the amount of iron oxide in the lungs. It decreased with time, reflecting particle clearance. The rate of relaxation increased steeply during the first 12 h after the instillation and decreased slowly between the 5th and 30th day. Changes in the location of particles from extracellular to intracellular sites and movements from ectoplasmic to endoplasmic sites within cells may be responsible for the observed changes in relaxation rates with time.
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Treatment of murine cytomegalovirus pneumonia with acyclovir and interferon.
A murine model of lethal cytomegalovirus pneumonia following intratracheal inoculation of normal mice was used to study the effects of antiviral therapy with acyclovir (ACV) and interferon (IF), alone and in combination. Five hundred units of fibroblast IF administered intraperitoneally the day prior to infection and 100 mg/kg/day ACV administered intraperitoneally for 7 days beginning within 3 h of infection were both highly effective in preventing mortality. Compared to the condition of untreated animals, each drug reduced viral titers in lung homogenates of the treated animals 7 days after infection. However ACV, unlike IF, diminished viral growth in the lung at 14 days, prevented viral dissemination to spleens and salivary glands in some animals, and significantly protected animals from reactivation of virus during immunosuppression 2 months after infection. The combination of ACV + IF offered no increased antiviral activity compared to ACV alone and failed to protect animals from reactivation. In these experiments the overall antiviral activity of ACV alone was greater than IF and the combination of ACV + IF.
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Retention of inhaled particles in hamsters with pulmonary fibrosis.
Aerosol retention was studied in hamsters 30, 60, and 90 days after the initiation of interstitial pulmonary fibrosis by a combination of bleomycin (bleo), 0.16 U/100 g body weight given intratracheally, and O2, for 72 h. Groups of bleo-O2-treated and control animals were exposed (awake) for 25 min to a 99mTc-labeled insoluble aerosol (activity median aerodynamic diameter, 0.45 micron; geometric standard deviation, 1.3). Within 5 min after exposure, the hamsters were killed and their lungs were excised and dried at total lung capacity, and sliced into 1-mm sections. Slices were dissected into pieces, and an evenness index (EI) was calculated for each piece (formula: see text). With uniformity of retention, all Els would be 1. The distribution of Els in control animals had a mean of 1.0 and a SD of 0.27; 0% of the Els were less than or equal to 0.20. Total retention diminished and was less uniform in bleo-O2-treated animals. At 30 days, the SD increased to 0.62, and 6% of the Els were less than or equal to 0.20. At 60 and 90 days, nonuniformity decreased but was still greater than that in the control animals (SD60 = 0.42, SD90 = 0.36). When examined histologically, individual pieces with low Els had more disease than those with high Els. Local decreases in compliance caused by fibrosis may have altered regional ventilation and retention. Our data also correlate with the progression of fibrosis from a focal lesion at 30 days to a more diffuse lesion at 90 days.
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Relative toxicity of inhaled metal sulfate salts for pulmonary macrophages.
The effects of metal sulfate aerosols on respiratory defense mechanisms in hamsters were studied. Pulmonary macrophage phagocytic rates were measured by determining the in vivo uptake of radioactive colloidal gold (198Au) 1, 24, or 48 h after a single 4-h exposure. The concentrations of sulfate aerosols causing a 50% inhibition in pulmonary macrophage endocytosis (EC50) were determined. When hamsters were exposed for 4 h to cupric sulfate (greater than or equal to 4.8 mg/m3), zinc sulfate (greater than or equal to 3.1 mg/m3), ferric sulfate (greater than or equal to 7.8 mg/m3), or zinc ammonium sulfate (greater than or equal to 10.0 mg/m3), macrophage endocytosis was significantly reduced 1 h after exposure compared with that in unexposed control animals. Although the response was variable, 24 h after exposures to the higher sulfate concentrations the percent of gold ingested by pulmonary macrophages remained depressed. By 48 h, the rate of macrophage endocytosis in hamsters had returned to normal control values except in hamsters exposed to 4.8 mg/m3 cupric sulfate or 9.8 mg/m3 ferric sulfate. These hamsters showed significant increases in phagocytosis. The EC50 values in milligrams of sulfate per cubic meter for cupric sulfate, zinc sulfate, ferric sulfate, and zinc ammonium sulfate were 2.7, 4.5, 7.5, and 17.9, respectively. These results are negatively correlated with the ranking of sulfates using the criteria of relative irritant potency, as measured by increases in pulmonary flow resistance. Thus, rankings of related chemical structures are not absolute. Their relative toxicities vary depending on the end point selected.
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Comparative toxicology of the respiratory tract.
It is difficult to extrapolate observations and results from one species to another and from animals to humans because of interspecies differences. A complete and systematic description of such differences among commonly used laboratory animals is lacking. We have reviewed the relevance of deposition, clearance, and the type and magnitude of biological response to inhaled aerosols. Current predictions on the probability of deposition of inhaled aerosols differ, but the fraction of aerosol that is actually deposited in the respiratory tract appears independent of body size. Different species of animals breathing the same aerosol do not receive identical lung doses, and thus exposure concentration is not an adequate description of lung dose. Parameters that affect the magnitude of local doses include changes in ventilation, collection efficiency, lung anatomy, and clearance mechanisms. Apart from these variations, the interspecies differences in substance metabolism and innate biological responsiveness make it unlikely that the extent of lung damage will be identical even in cases where lung doses are equal. We need a comprehensive view of species differences with predictive power.
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Effects of exercise on particle deposition in Syrian golden hamsters.
The effects of exercise and its associated increase in ventilation on the deposition of inhaled particles were investigated. Both total retention and patterns of distribution of a 99mTc sulfur colloid aerosol (activity median aerodynamic diameter, 0.38 micron; geometric standard deviation, 1.35) were measured in male Syrian golden hamsters. Animals were either anesthetized, resting, or exercising on a treadmill during a 15-min aerosol exposure. Each hamster's oxygen consumption (VO2) was continuously monitored during the exposure; immediately after, the animal was killed. The lungs were excised, inflated, and dried in a microwave oven. The rigid lungs were sliced and dissected in a predetermined way so that retention at specific locations could be compared. The radioactivity and weight of 40 pieces from each of 12 hamsters were measured. The uniformity of deposition was described by an evenness index (EI) for each piece: EI = (cpm/g)piece/(cpm/g) whole lung. With theoretical uniformity of retention, all EI values should be 1.0. During aerosol exposure, the exercising group had a VO2 of 5.0 +/- 0.6 (SD) ml STPD/min/100 g, which was 2 times the resting group (2.5 +/- 0.4) and 4 times the anesthetized group (1.2 +/- 0.2). The total retention of particles in the lungs increased in a parabolic manner as a function of VO2; the exercising animals had a retention 6 times greater than the anesthetized animals. The increased retention in running hamsters may reflect either increased ventilation alone or increased collection efficiency. Each animal's activity level also affected local distribution of particles in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)
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Progressive pulmonary fibrosis in hamsters.
The concomitant treatment of hamsters with bleomycin and hyperoxia results in a synergistic development of pulmonary injury. We exposed hamsters for 72 hr to 70% oxygen following a single intratracheal instillation of bleomycin (0.16 U/100 g body weight). Groups of 10 animals were killed at 3, 6, 10, 30, 60, 90, and 120 days after instillation for histopathologic and morphometric assessment. Diffuse alveolar damage developed acutely. At 30 days, the intense acute cellular infiltrate had subsided, leaving a focal interstitial pneumonitis. Morphometric quantitation at 10 days revealed that 33.5 +/- 5.3% (x +/- SE) of the lung was diseased; there was apparent healing by 30 days, when 10.5 +/- 2.0% of the lung was diseased. However, progression to diffuse pneumonitis with fibrosis was seen at 60, 90, and 120 days, when 30.2 +/- 4.9%, 38.5 +/- 5.8%, and 38.8 +/- 4.5% of the lung was diseased, respectively. In vivo pulmonary function studies on treated animals at 25 and 55 days showed decreasing dynamic compliance and increased minute ventilation, which corroborates the presence of interstitial fibrosis. We conclude that simultaneous treatment of hamsters with bleomycin and hyperoxia results in interstitial fibrosis with a distribution and progression that mimics human pulmonary fibrosis. This model appears ideally suited for the study of progressive fibrosis and will be useful when development of a widely distributed lesion is crucial.
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