Reaction of alveolar macrophages to inhaled metal aerosols.

For more than a decade we have exposed rabbits to different metals, usually in soluble form, and investigated the effects on the lungs. The metal concentrations have been around 1 mg/m3,i.e., not more than a factor of 10 above occupational threshold limit values. The exposure periods have been 1-8 months (6 hr/day, 5 days/week). We have studied especially the morphology and function of alveolar macrophages (AM), the morphology of alveolar type I and type II epithelial cells, and analyzed lung phospholipids. Several metals produce specific, complex effects. For example, metallic and soluble nickel (NiCl2) increase both number and size of the type II cells, increase the production of surfactant, and affect morphology and function of AM. Cobalt (CoCl2) induces a different effect on type II cells from nickel, causing the formation of nodules in these cells. Trivalent chromium [Cr(NO3)3] does not affect either type II cells or the amount of surfactant significantly, but markedly affects AM. The administered metals affect AM both directly and indirectly. For example, nickel induces an increased production of surfactant, resulting in overfed AM with an increased metabolic activity. However, nickel also induces a direct decrease in the release of lysozyme activity by AM. Our results emphasize the complexity of the effects on the lungs of inhaled agents, which can act both directly and indirectly on AM.

Phagolysosomal pH in alveolar macrophages.

We studied phagolysosomal pH in alveolar macrophages (AM) using fluorescein-labeled yeast (FYP) and silica particles (FSP) as probes. Fluorescence intensities from the ingested test particles were measured on populations of AM using fluorescence spectrometry and on individual phagolysosomes using fluorescence microscopy. Measurements were performed on rabbit AM, which had been incubated with FYP or FSP (in vitro procedure). We also instilled FYP or FSP via the trachea into rabbit lungs and after 1 day, 1 week, 1 month, and 3 months lavaged the lungs and measured the pH in AM (in vivo procedure). Phagolysosomal pH was independent of the number and size of the fluorescent particles. Measurements of populations of AM with fluorescence spectrometry and of individual phagolysosomes with fluorescence microscopy gave similar average pH. For the FYP, pH decreased during the first day after lavage both in the in vitro and the in vivo procedures. For the FSP, pH was unchanged during the same period. After 1 day pH was similar for both particles. Electron microscopy showed a larger number of lysosomes in contact with phagosomes and a higher percentage of vacuolated phagosomes for FYP than for FSP. In the in vivo procedure, pH was unchanged at least up to 1 month, and this pH was lower than that in the in vitro procedure. The difference was probably due to conditions at the time of phagocytosis. Particles retained in the lung parenchyma were within AM, and their location within the AM appeared unchanged from 1 week up to 3 months.

Alveolar macrophages and lung lesions after combined exposure to nickel, cobalt, and trivalent chromium.

In earlier inhalation exposures of rabbits, nickel increased the production of surfactant by type II cells, with secondary effects on morphology and function of alveolar macrophages. Cobalt induced mainly a nodular growth pattern of the type II cells. Trivalent chromium seemed to impair the capacity of macrophages to catabolize surfactant but did not affect the type II cells. We exposed rabbits by inhalation to combinations of nickel (0.6 mg/m3 as NiCl2) and trivalent chromium [1.2 mg/m3 as Cr(NO3)3] (Ni-Cr), cobalt (0.5 mg/m3 as CoCl2) and nickel (0.5 mg/m3) (Co-Ni), or cobalt (0.5 mg/m3) and chromium (1.2 mg/m3) (Co-Cr) for 4 months, 5 days/week, 6 hr/day. Alveolar macrophages, alveolar type II cells, and lung content of phospholipids were determined. All combined exposures induced more pronounced lung lesions than exposures for each of the metals. Phospholipid concentrations were significantly higher. There were significantly higher percentages of macrophages filled with surfactant-like inclusions and a smooth surface. Accumulations of macrophages in alveoli were more widespread. Chromium potentiated the effects of nickel and cobalt on the type II cells, which led to secondary effects on the macrophages. Nickel potentiated the specific effects of cobalt, i.e., type II cell nodule formation. The result indicates that noxious effects could also be induced in man by combined exposure to nickel, cobalt, and trivalent chromium in concentrations similar to those occurring in some occupational settings.

Intraphagolysosomal pH in canine and rat alveolar macrophages: flow cytometric measurements.

Intracellular dissolution of inhaled inorganic particles is an important clearance mechanism of the lung and occurs in phagolysosomal vacuoles of phagocytes. Flow cytometric measurements of intraphagolysosomal pH in alveolar macrophages (AM) obtained from beagle dogs, Wistar rats, and from a baboon were made using fluorescein isothiocyanate-labeled amorphous silica particles (FSP). AM were obtained by bronchoalveolar lavage. FSP were phagocytized by AM in cell suspensions incubated in full media for 24 hr up to 6 days. Dual laser flow cytometry was performed and six-parameter list mode data were recorded from forward scatter, side scatter, and fluorescence intensities at 530 nm excited at 457 nm and 488 nm as well as logarithmic fluorescence intensity at wavelengths 630 nm excited at 488 nm. In this way it was possible to discriminate viable AM with phagocytized FSP from lysing AM with phagocytized FSP and from cells without FSP and from free FSP. Viable cells were distinguished from lysing cells by staining with propidium iodide immediately before the flow cytometric measurement. A calibration curve for the pH value was determined from FSP suspended in buffered media at pH values ranging from 3.5 to 7.5. First flow cytometrical results indicated that after an incubation time of 24 hr, the mean intraphagolysosomal pH of viable AM was 4.7 +/- 0.3 for dogs and 5.1 +/- 0.5 for rats. The intraphagolysosomal pH of the baboon AM was 4.5.

Phagolysosomal pH and dissolution of cobalt oxide particles by alveolar macrophages.

We studied phagolysosomal pH in rabbit alveolar macrophages (AM) incubated with 0-15 microM chloroquine. There was a dose-related increase in pH with chloroquine concentration. Electron microscopy showed that chloroquine increased lysosomal size. In a second experiment we studied dissolution of radiolabeled cobalt oxide particles by rabbit AM, phagolysosomal pH, and lysosomal size. The cells were incubated for 2 days with 0, 2.5, and 10 microM chloroquine. Size and pH increased with chloroquine concentration. Dissolution of cobalt particles by the AM did not clearly change with pH. In a third experiment, dissolution of cobalt oxide particles in 0.1 M acetate buffer in saline with pH 4.0, 5.0, and 6.0 was studied. At the same pH, dissolution in acetate buffer was faster than in the AM, and the dissolution appeared to decrease faster with increasing pH than in the AM. A simple model for dissolution of a particle in a phagolysosome was proposed. This model predicts the types of differences in dissolution between AM and buffered saline.

Regional deposition of 3.6-micron particles and lung function in asthmatic subjects.

In a group of moderately severe asthmatic subjects, regional deposition of 3.6-microns (aerodynamic diameter) monodispersed Teflon particles labeled with 111In was studied twice. The particles were inhaled with maximally deep inhalation at 0.5 l/s. Lung retention was measured at 0, 6, 24, and 48 h by use of a profile scanner equipped with two 13 x 5-cm NaI crystals. The retentions at 24 (Ret24) and 48 h were highly correlated (r = 0.96 with a slope of the regression line close to 1). There was a poor correlation between retention at 6 h and Ret24 (r = 0.54). The Ret24 values at the two exposures were well correlated (r = 0.86). There were significant correlations between airway resistance as well as single-breath nitrogen test phase III and Ret24 (r = 0.70 and 0.67, respectively). The correlation between single-breath nitrogen test phase III and Ret24 persisted also when only subjects within a narrow interval of airway resistance were included. The study indicates that regional deposition can be studied by measurements of Ret24 in subjects with moderately severe asthma and that it is dependent on changes in both large and small airways.

On the function of cilia in the female reproductive tract.

A recently discovered syndrome is characterized by congenital immotility of the cilia. It consists of chronic infections in the respiratory system, male infertility, and, in about one-half of the cases, situs inversus. The syndrome thus includes Kartagener's syndrome. The syndrome provides a unique opportunity to gain insight into the role of the cilia in the human body. Five women evidently suffering from this syndrome were examined. They had no gynecologic disorders. Three of the five women had tried to become pregnant and two have suceeded. A review of the literature indicates that men with Kartagener's syndrome are usually infertile, whereas the women are not. This fact and the data presented herein constitute strong evidence that ciliary motility is not essential for female fertility. More conclusive proof could be obtained if any of the affected women would consent to ultrastructural investigation of the oviductal cilia.

PIP: A recently discovered syndrome is characterized by congenital immotility of the cilia. It consists of chronic infections in the respiratory system, male infertility, and in about 1/2 of the cases, situs inversus. The syndrome thus includes Kartagener's syndrome. The syndrome provides a unique opportunity to gain insight into the role of the cilia in the human body. 5 women evidently suffering from this syndrome were examined. They had no gynecologic disorders. 3 of the 5 women have tried to become pregnant and 2 have succeeded. A review of the literature indicates that men with Kartagener's syndrome are usually infertile, whereas the women are not. This fact and the data presented therein constitute strong evidence that ciliary motility is not essential for female fertility. More conclusive proof could be obtained if any of the affected women would consent to ultrasturctural investigation of the oviductal cilia.

Are alveolar macrophages translocated to the lymph nodes?

Hilar lymph nodes from 5 rabbits exposed for 3 months (5 days/week, 6 h/day) to metallic nickel dust (1.2 mg/m3) and from 2 controls were studied with electron microscopy. Some lymph node macrophages were filled with laminated bodies and had a vesiculated cytoplasm, a well developed endoplasmic reticulum and a few of them contained nickel particles as well. This means that they appeared identical to macrophages found in alveoli in nickel exposed rabbits. We propose that there is a transport of alveolar macrophages to the lymph nodes in nickel exposed rabbits.

Decreased level of lysozyme in rabbit lung lavage fluid after inhalation of low nickel concentrations.

Six rabbits were exposed for 4 months (5 days/week, 6 h/day) and 6 rabbits for 8 months to approx. 0.1 mg/m3 of metallic nickel dust (U.S. threshold limit value (TLV) 1 mg/m3). Another 8 rabbits were exposed for 4-6 weeks (5 days/week, 6 h/day) to 0.3 mg/m3 (as Ni) of nickel chloride (U.S. TLV 0.1 mg/m3). After exposure lungs were lavaged. Concentration of lysozyme in the lavage fluid was estimated with the lyso-plate technique (agar plates with heat-killed Micrococcus lysodeikticus) after macrophages had been removed. All 3 exposed groups had markedly lower concentrations of lysozyme than corresponding controls. Mean values in controls and exposed rabbits were: for 4 months metallic nickel dust exposure 2.3 and less than or equal to 0.04 microgram/ml; for 8 months metallic nickel dust exposure 1.4 and less than or equal to 0.5 microgram/ml; and for nickel chloride exposure 1.9 and less than or equal to 0.4 microgram/ml.

Morphology and release of lysozyme following exposure of rabbit lung macrophages to nickel or cadmium in vitro.

Lung macrophages lavaged from 7 rabbits were incubated with 0, 3, 6, 12 and 24 micrograms/ml of nickel as NiCl2 and macrophages from 4 rabbits were incubated with 0, 0.1, 1, 3 and 6 micrograms/ml of cadmium as CdCl2. After 2 days lysozyme activity in the medium in which the macrophages were cultivated, was estimated using a technique with agar plates prepared with heat-killed Micrococcus lysodeikticus. Macrophage morphology was examined by scanning and transmission electron microscopy. For nickel there was a dose-related inverse relationship between the lysozyme activity and concentration of nickel. Many macrophages exposed to the higher nickel concentrations had a rounded form and, thus, the surface area of each cell which came in contact with the glass appeared to be less than that for control macrophages. There was, however, no increase in the number of macrophages detached from their glass support. Cadmium exposure did not influence lysozyme levels of activity, in spite of morphological indications of cell toxicity. From the present study we conclude that the decreased lysozyme activity seen previously in vivo after nickel inhalation is likely to be due to a direct effect of nickel ions on macrophages and that the increased lysozyme activity seen in vivo after cadmium inhalation is probably a secondary effect, subsequent to inflammation.

Increased airway responsiveness after skin sensitisation to 3-carene, studied in isolated guinea pig lungs.

Inhalation of 3-carene has been shown to induce bronchoconstriction in concentrations not far from the threshold limit value. In this study, one group of guinea-pigs were sensitised by dermal exposure to 3-carene according to the modified Cumulative Contact Enhancement Test protocol and another group of animals was used as controls. Lungs from the skin-sensitised and control guinea-pigs were perfused with diluted autologous blood (13 ml blood/87 ml buffer) and exposed to 3-carene at an air concentration of 3000 mg/m(3). In both groups there was a reduction in compliance and conductance but this reduction was significantly (P<0.05) more pronounced (2.5-3 times) in lungs obtained from sensitised animals than from control animals. In a previous study with similar design, but with plain buffer instead of diluted autologous blood as perfusate, we found no statistically significant difference in lung bronchoconstriction. Thus, it is concluded that skin sensitisation can increase lung reactivity to 3-carene and that important mediators of this effect seem to be present in the blood.

Does airway responsiveness increase after skin sensitisation to 3-carene: a study in isolated guinea pig lungs.

Guinea pigs were sensitised by dermal exposure to 3-carene according to the modified cumulative contact enhancement test (CCET) protocol. Lungs from sensitised and non-sensitised animals were then perfused with buffer and exposed for a period of 10 min to two different air concentrations of 3-carene, 600 and 3000 mg/m3. 3-Carene caused a statistically significant bronchoconstriction even at the relatively low concentration of 600 mg/m3 and the constriction was dose dependent. 600 mg/m3 of 3-carene caused a reduction of 19% in conductance capacity and 16% in compliance capacity. 3000 mg/m3 of 3-carene decreased lung compliance and conductance by 43 and 31%, respectively. The lungs from sensitised animals tended to show a greater response than lungs obtained from control animals. The lower concentration of 3-carene is close to and may even be below, occupational limit values in Sweden, Germany and USA.

Alveolar macrophage function in nickel dust exposed rabbits.

8 rabbits were exposed to metallic nickel dust (2 mg/m3, of which about half was respirable) for 4 weeks. The lungs were lavaged and the macrophages were collected. In comparison with 8 control rabbits, a significant increase was noted in the nickel exposed rabbits as concerned the weight and density of the lungs, the size variation of the lung cells, the phagocytosis of silver coated particles, and the metabolic activity as measured by NBT reduction. The last mentioned increase was recorded during basal conditions as well as during phagocytosis. The NBT reduction during phagocytosis was significantly correlated with the degree of phagocytosis of silver coated particles in both control and exposed rabbits. It is suggested that the exposure to nickel dust has unspecifically activated the macrophages perhaps by increased production of phospholipids.

Increased airway responsiveness of a common fragrance component, 3-carene, after skin sensitisation--a study in isolated guinea pig lungs.

Lungs from skin-sensitised and non-sensitised guinea pigs were exposed via the airways to 3-carene (1900 mg/m3) and perfused with buffer containing either autologous plasma or lymphocytes. The experiments were performed in order to investigate the importance of blood components for the increased lung responsiveness seen in skin-sensitised animals. A reduction in lung function was noted in all lungs during 3-carene exposure. There was no difference in the 3-carene response between lungs from skin-sensitised animals versus lungs from non-sensitised animals when the perfusion buffer contained lymphocytes. However, when plasma diluted with buffer was used as perfusion medium, there was a significant enhancement in the response in lungs from sensitised versus lungs from non-sensitised animals. This implies that skin sensitisation increases lung responses to inhaled 3-carene and those components in plasma, and not the lymphocyte fraction, contributes to the observed increased lung responsiveness.

Phagolysosomal pH in alveolar macrophages from smokers and non-smokers.

1. Alveolar macrophages efficiently phagocytize particles deposited in the alveolar part of the lung. Phagolysosomal pH in alveolar macrophages from rabbits, dogs, baboons and guinea pigs is around 5 pH units. A low pH is of importance for clearance of inorganic particles and probably also for defence against bacteria and fungi. 2. Phagolysosomal pH in alveolar macrophages from smokers and non-smokers was measured using fluorescein-conjugated amorphous silica particles as a probe. pH was the same in both groups and similar to earlier studies on alveolar macrophages from animals. 3. The morphological picture between alveolar macrophages from smokers and non-smokers differed clearly with a high number of dense inclusions of great variation in size and shape in macrophages from smokers. Ingested test particles were located in all types of inclusions. 4. We conclude that phagolysosomal pH in human alveolar macrophages is similar to that in several animal species and that it is rather insensitive to influence from the environment.

Rat alveolar and interstitial macrophages in the fibrosing stage following quartz exposure.

Exposure to quartz induces pulmonary inflammation and development of fibrosis. In order to study the fibrosing process, we investigated morphology, function and phenotype of alveolar (AMs) and interstitial (IMs) macrophages at an early stage of fibrosis in rats. Rats were exposed by intratracheal instillations of 10 mg quartz (n=8) or saline (n=8) and studied 3 months later. AMs were obtained by bronchoalveolar lavage and IMs by mechanical fragmentation, followed by enzymatic digestion of lung tissue. Histology revealed subacute silicosis, with early focal fibrosis and alveolar lipoproteinosis. AM quartz exposure increased phagocytic activity and expression of major histocompatibility complex (MHC) Ia antigens, the latter being associated with cellular antigen presenting capacity. IM had an even more pronounced expression of MHC than AM after quartz exposure. Both macrophage fractions had a higher expression of OX-42 (complement receptor 3, CR3) than controls, but the increase in the IM fraction might be explained by the remaining AM in the IM fraction. Exposed AM adhered less to extracellular matrix components (vitronectin and fibronectin) than controls. In contrast, the adhesion of IM to vitronectin increased after exposure. Besides increased adhesion, the effects on IM were scarce. Our results therefore do not support the hypothesis that IM has a key role in the process of inflammation, including fibrosis.

Human alveolar deposition of 4 micron teflon particles.

On two occasions ten healthy men inhaled with maximally deep inhalations at 0.5 1/sec a test aerosol of 4 micron teflon particles tagged with 111In. Radioactivity in the lungs was measured a few min, 24 hr, and 2, 3, and 4 days after inhalation. Retention decreased greatly between the measurements performed a few min and 24 hr after inhalation, but was similar in measurements 1 to 4 days after inhalation. Among individuals, retention varied considerably 1 to 4 days after inhalation (by a factor of 2), but the retention in each individual subject was uniform. Measuring the retention of a radioactively tagged test aerosol 1 day after inhalation, and regarding this retention as alveolar deposition, might be of value in preventive measures against certain toxic dusts.

Increased mucociliary transport by adrenergic stimulation.

Tracheobronchial clearance of inhaled 6mum Teflon particles (density 2 gm/cu cm) tagged with technetium 99m was studied in healthy subjects by external measurement of the radioactivity in the lungs for two hours. Clearance, salivary secretion, blood pressure, and heart rate were determined in eight subjects after double-blind, subcutaneous administration of 0.25 mg terbutaline sulfate, a beta-adrenoceptor stimulating compound and vehicle, respectively, in a crossover study. Terbutaline produced a marked increase in clearance and a slight increase in heart rate, but had no important effect on salivary secretion or blood pressure. The result indicates that the blood concentrations of catecholamines might be of importance for the regulation of the mucociliary transport rate.

Lung clearance of 4-micron particles coated with silver, carbon, or beryllium.

Lung clearance in rabbits during the first week after inhalation of 4-micron teflon particles tagged with chromium-51 and coated with silver, carbon, or beryllium was studied by external measurements of the radioactivity in the lungs. Ten rabbits were exposed to silver- and carbon-coated particles on two separate occasions within 3 weeks. Clearances of the two types of particles were quite similar. Clearance in eight other rabbits that had inhaled silver-coated particles was quite similar to clearance in eight rabbits that had inhaled beryllium-coated particles. The result is regarded as evidence that alveolar macrophages do not play an active role in removing intact particles from the lung the first week after inhalation.

RBDATA-EULEP: providing information to improve internal dosimetry.

The overall aim of the concerted action RBDATA-EULEP is to provide information to improve the assessments of intakes of radionuclides and of the resulting doses. This involves a review of the behaviour of radionuclides following intake, and the transfer of expertise on methodology by organising small training workshops. The main activity is the development of an electronic database, effectively an annotated bibliography, but the electronic format used facilitates extension, updating and information retrieval. It consists of linked tables of references and experiments, with details and comments on the materials, procedures and results. By June 2002 it contained information on 524 inhalation, 282 ingestion and 164 injection experiments from 391 references. It will be extended, and Internet access provided. Prospective users include groups developing standards for internal dosimetry, scientists conducting research on radionuclide biokinetics and health physicists assessing the consequences of accidental intakes.