Free Alveolar Macrophages Research Articles

Inhalation toxicity study of disk-shaped potassium octatitanate particles (Terracess TF) in rats following 90 days of aerosol exposure

Since fibrous particles such as asbestos and some man-made fibers (MMF) have been known to produce carcinogenic or fibrogenic effects, disk-shaped potassium octatitanate (POT) particles (trade name: Terracess TF) were manufactured as nonfibrous particles. A 90-day inhalation toxicity study of Terracess TF was performed to evaluate comparative inhalation toxicity of the disk shape with a fibrous shape that was previously evaluated. Four groups of 20 male and 15 female rats each were exposed to Terracess TF aerosols at concentrations of 0, 2, 10, or 50 mg/m3 for 90 days. Ten male and 10 female rats per group were sacrificed at 90 days of exposure. After 90 days of exposure, 5 male rats per group were sacrificed at 3 wk of recovery period and 4–5 male rats per group or 5 female rats per group were sacrificed at 15 wk of recovery for lung clearance and histopathology. The mass median aerodynamic equivalent diameter (MMAED) of the aerosols of test materials ranged from 2.5 to 2.9 μm. There were no test-substance-related adverse effects on clinical observations. At the end of the 90-day exposure, a slight increase in lung-to-body weight ratios was observed at 50 mg/m3 in male but not in female rats. However, lung weights were within normal limits after 3- or 15-wk recovery periods. Microscopically, inhaled Terracess TF particles were mostly phagocytized by free alveolar macrophages (AMs) in the alveolar airspaces and alveolar walls maintained normal structure at 2 and 10 mg/m3. At 50 mg/m3, some alveoli were distended and filled with aggregates of particle-laden AMs. The alveolar walls showed slight type II pneumocyte hyperplasia, but neither proliferative inflammation nor alveolar fibrosis was present at 50 mg/m3. The clearance half-times for Terracess TF were estimated to be in the order of 6 to 9 mo for the 50-mg/m3 group and 2 to 3 mo for the 10- and 2-mg/m3 groups. The lung responses and lung clearance rate were comparable to those of “nuisance” type dusts at these concentrations. Based on interpretation that aggregated particle-laden AMs in alveoli was considered to be an early histopathological sign of lung overloading, an effect level was considered to be 50 mg/m3 and no-observed-adverse-effect level (NOAEL) was 10 mg/m3. This experiment clearly demonstrated that particle morphology was considered to be an important factor to determine inhaled particle toxicity.

Prenatal nicotine increases pulmonary alpha7 nicotinic receptor expression and alters fetal lung development in monkeys.

It is well established that maternal smoking during pregnancy is a leading preventable cause of low birth weight and prematurity. Less appreciated is that maternal smoking during pregnancy is also associated with alterations in pulmonary function at birth and greater incidence of respiratory illnesses after birth. To determine if this is the direct result of nicotine interacting with nicotinic cholinergic receptors (nAChRs) during lung development, rhesus monkeys were treated with 1 mg/kg/day of nicotine from days 26 to 134 of pregnancy. Nicotine administration caused lung hypoplasia and reduced surface complexity of developing alveoli. Immunohistochemistry and in situ alpha-bungarotoxin (alphaBGT) binding showed that alpha7 nAChRs are present in the developing lung in airway epithelial cells, cells surrounding large airways and blood vessels, alveolar type II cells, free alveolar macrophages, and pulmonary neuroendocrine cells (PNEC). As detected both by immunohistochemistry and by alphaBGT binding, nicotine administration markedly increased alpha7 receptor subunit expression and binding in the fetal lung. Correlating with areas of increased alpha7 expression, collagen expression surrounding large airways and vessels was significantly increased. Nicotine also significantly increased numbers of type II cells and neuroendocrine cells in neuroepithelial bodies. These findings demonstrate that nicotine can alter fetal monkey lung development by crossing the placenta to interact directly with nicotinic receptors on non-neuronal cells in the developing lung, and that similar effects likely occur in human infants whose mothers smoke during pregnancy.

Ontogeny of macrophage subpopulations and Ia-positive dendritic cells in pulmonary tissue of the rat

The development of macrophage subpopulations dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gut-associated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.

The application of visual cell profiles in the study of premalignant atypias in sputum

Because the diagnosis of squamous-cell carcinoma of the lung by routine cytologic screening of sputum is often inconclusive, our laboratory is studying the use of cytomorphologic profiles as a reliable diagnostic aid. This study reports the analysis of the profiles of 75 subjects, both cigarette-smoking volunteers and hospitalized patients suspect for lung cancer. Twenty-five of the subjects had been classified as having squamous metaplasia, 25 as having atypias and 25 as having squamous-cell carcinoma. Four slides were made for each subject, with 100 random fields viewed on each slide. The frequency of free alveolar macrophages, metaplastic squamous cells, epithelial atypical cells and malignant squamous cells were noted for each field. The results indicated that, although there were large differences between individual profiles within each diagnostic category, there were significant differences between the average profiles for each diagnostic category. Furthermore, although there were differences in cell frequencies from slide to slide and within slides, the differences were constant across diagnostic categories. The results are supportive of the progressive-atypia hypothesis for squamous-cell carcinoma of the lung and provide a baseline for comparison with subsequent individual profiles.

Biological Effects of Mount Saint Helens Volcanic Ash on Cultured Human Alveolar Macrophages

Free alveolar macrophages (FAMs) obtained by bronchoalveolar lavage from healthy nonsmoking volunteers were incubated with varying concentrations (0–300 μg/ml) of Mt. Saint Helens volcanic ash obtained from either Portland, Oregon, or Pullman, Washington, to assess the cytotoxic effects of the ash on human lung cells. Trypan dye exclusion techniques were employed for assessment of cell viability. Following the initial 24 hour culture with the Portland ash samples, decreased viability was observed at all ash concentrations (P < 0.001 in all instances), and further decreases in viability were noted at 48 and 72 hours for all concentrations of ash tested (P < 0.001 in all instances). When the Pullman, Washington, ash sample was evaluated, a decrease in cell viability was noted for the 300 μg/ml concentration (P < 0.017) after the initial 24 hours in culture. Further decreases in cell viability were noted only when cells were cultured for longer time intervals (48 and 72 hours) (P < 0.05 in all instances). Differences in cellular response to the 2 ash samples were further investigated by exposing FAMs from a single individual to the 2 different types of ash. These studies demonstrated similar cytotoxic effects of the 2 ash samples at all concentrations and times tested (P < 0.30 in all instances) with the exception of the 100 μg/ml concentrations at 72 hours (P < 0.020). These data suggest that the differences observed between the 2 types of ash in the independent studies are probably related to interindividual variation in FAM response to the ash rather than to differences in the cytotoxicities of the 2 ash samples. Cytotoxicity of the volcanic ash was also compared with other environmentally relevant airborne particulates, such as amosite and chrysotile asbestos, as well as amorphous and crystalline silica. These results demonstrated an intermediate cytotoxic effect of the ash between innocuous amorphous silica and the very cytotoxic chrysotile asbestos. The affinity for volcanic ash to adsorb tritiated benzo(a)pyrene (3H-BaP) was also compared with that of amorphous silica and amosite asbestos. These studies demonstrate that volcanic ash has intermediate adsorption qualities (4.3 ± 0.1; pmoles 3H-BaP adsorbed/μg particulate ± SD) between those of amorphous silica (1.9 ± 1.0) and amosite asbestos (7.8 ± 1.2) (P < 0.05 in all instances). These data suggest volcanic ash exhibits moderate biological properties compared with those of other environmentally important airborne particulates. Whether in vitro studies reflect in vivo response of human lung cells to the ash cannot be determined at this time, and follow-up of assessment of individuals exposed to the ash will be required to assess its long-term effects on pulmonary tissue.

The role of alveolar macrophages in surfactant turnover. An experimental study with metabolite VIII of bromhexine (Ambroxol).

To study the functional activity of alveolar macrophages in surfactant turnover experiments on rats were carried out with stimulation of surfactant production by Ambroxol® (metabolite VIII of bromhexine). It was shown that after Ambroxol® treatment alveolar macrophages contained abundant phospholipids, predominantly of the surfactant system, in their cytoplasm. Enzyme histochemical studies showed increased activities ofβ-glucuronidase and acid phosphatase in the cytoplasm of these macrophages. The number of free alveolar macrophages in bronchial lavage increased more than threefold. These findings demonstrate that alveolar macrophages play an important role in surfactant turnover by phagocytosis of this material.

Asbestos body phagocytosis by human free alveolar macrophages

Phagocytosis of asbestos bodies by human free alveolar macrophages (FAMs) was documented employing light microscopy. This process was more carefully studied utilizing scanning electron microscopy (SEM) which demonstrated morphological and surface membrane changes in FAMs following phagocytosis of asbestos bodies. FAM viability was also evaluated following 24–72-h incubation of cells with asbestos bodies at a final concentration of 250 μg/ml. Slight, but significant, cytotoxicity was observed following the initial 24-h culture period ( P = 0.032, paired, 2-tailed t-test). No further cytotoxicity was observed, however, when cells were further incubated for 48-h and 72-h intervals ( P > 0.05 in all instances). These studies demonstrate asbestos bodies are readily phagocytized by cultured FAMs, and are only slightly cytotoxic to these human lung cells.

Desquamative Interstitial Pneumonitis: Cellular Phase of Fibrosing Alveolitis

Since the original description of desquamative interstitial pneumonitis in 1967, several investigators have questioned the specificity of the disease. The concept of desquamative interstitial pneumonitis and usual fibrosing interstitial pneumonitis as two manifestations of a nonspecific disease spectrum has been proposed. Twenty-six patients with desquamative interstitial pneumonitis were evaluated with respect to clinicopathologic correlates. Biopsies were reviewed without knowledge of the patients' clinical course. The absolute histopathologic criteria for the diagnosis were (1) intra-alveolar accumulations of free alveolar cells with PAS-positive diastaseresistant cytoplasmic granules; (2) mononuclear cell interstitial inflammation; and (3) absence of necrosis, hyaline membranes, intra-alveolar fibrosis, asbestos bodies, and birefringent crystalline dust material. Transmission electron microscopic studies revealed the dominant alveolar lining cell to be the granular pneumocyte, with a prominent population of free alveolar macrophages. Patients were segregated into group 1 (cellular phase) and 2 (cicatrized phase). Although favorable short-term responses to corticosteroid therapy were observed in both groups, long-term responses were variable. Apparent complete remissions were noted in both groups.

Inhaled feed particles in experimental pigs with and without vitamin A deficiency.

An attempt was made to experimentally reproduce an unusual respiratory disease of pigs fed a dusty mixture of finely ground peas, wheat, and barley. Groups of vitamin-A-deficient piglets from this herd were either given dry feed and daily exposure to dust from the feed or were given wet feed. Vitamin-A-fortified pigs of another stock were also given the dry feed and dust exposure. The upper and lower respiratory tracts, renal pelves, and interlobular ducts of salivary glands were studied histologically. The distinguishing features of lungs were starch particles from feed (up to 31.2 mum in diameter) in all lungs (but more in lungs of pigs given dry feed), free alveolar macrophages, giant cell formations, and mild degenerative processes of respiratory epithelium. The epithelium of most renal pelves was marked by mucinous degeneration.

Dehydration and susceptibility to toxic chemicals.

Deprivation of water for 48 hours increased mortality of rats and mice injected with water soluble compounds of lead, antimony, and methacholine chloride but did not greatly alter susceptibility to lipid soluble trichloroethylene, benzene, and parathion. The brain appeared resistant to prolonged dehydration when tested by the hypothalamic self-stimulation method, Cilia-mucus clearance in chick trachea and the number of free alveolar macrophages recoverable from rat lungs were reduced by 24 to 48 hour water deprivation. In vitro metabolism of hexobarbital by liver microsomal enzymes was affected by water and food deprivation. These animal experiments suggest that dehydration may slier the susceptibility of man to toxic foreign chemicals.

Dehydration and recovery of free alveolar macrophages.

Rats fed dry chow diet were deprived of wafer for 24 or 48 hours. Free alveolar cells were recovered from the excised lungs by repeated instillation and recovery of saline. The number of cells per gram lung weight harvested from dehydrated rats was significantly below control values and was closely related to the duration of wafer deprivation and loss of body weight. This decrease in cell recovery was not primarily attributable to reduction in food intake which accompanies water deprivation, since rats allowed wafer but restricted to the same food intake did not show as significant a decrease as occurred with dehydration. Free alveolar cells are considered lung macrophages which are responsible for clearance of particulates and microorganisms reaching the deep lung tissue. These experiments indicate that dehydration may affect this important mechanism.

Identification of cells labeled with tritiated thymidine in the pulmonary alveolar walls of the mouse.

A detailed investigation with both the light and the electron microscope was carried out to determine what kinds of cells in the pulmonary alveoli are capable of cell division. The dividing cells were labeled with tritiated thymidine, and their behavior was studied for 30 days. It was shown that the number of labeled cells decreased by more than one-half between the fifth and tenth days, and decreased further between the tenth and twentieth, and the twentieth and thirtieth days. Electron microscopy revealed that the labeled cell population included all kinds of cells normally found in the alveolar walls. The largest group of labeled cells were leukocytes in the vascular system. The large decrease in the number of labeled cells between the fifth and tenth days was shown to be related to decrease in the number of labeled leukocytes in the capillaries of the lung. Endothelial cells and type 2 alveolar cells were classified as stem-type, renewing cell populations. The free alveolar macrophages were classified...

Radioautographic study of human pulmonary tissues.

The radioautograms of human lungs were studied after <i>in vitro</i> incubation with tritiated thymidine. Our materials consisted of the lungs of 12 normal foetuses (crown-rump length varied from 7·5 to 16·5 cm.) and of 11 adults (32 to 68 years of age). In the foetal lungs the uptake of tritiated thymidine was highest in the epithelium surrounding small tubular spaces, forerunners of future bronchial epithelium, and in the undifferentiated mesenchyme. The percentage of labelled cells was scarce in the alveolar wall cells of adults (0·05% to 0·65%), was unrelated to age, and appeared to be most frequent in phagocytic elements (so-called alveolar cells). There was an uptake of tritiated thymidine in scattered free alveolar macrophages and in a few interstitial cells. The media and adventitia of blood vessels did not show labelling in most cases. The labelling of epithelium of bronchioles and respiratory bronchioles varied from 0·20% to 0·70%. The findings of other investigators in the study of cell turnover in the lungs of laboratory animals are discussed and compared with the data obtained in the present study.

Electron Microscopic Study of the Phagocytosis Process in Lung

Diluted India ink was instilled into the nasal cavity of mice and the lungs of some animals were fixed with osmium tetroxide at various intervals after one instillation. The lungs of other animals were fixed after 4, 7, 9, 16, or 18 daily instillations. The India ink was found to be phagocytized almost exclusively by the free alveolar macrophages. A few particles are occasionally seen within thin portions of alveolar epithelium, within the "small" alveolar epithelial cells, or within occasional leukocytes in the lumina of alveoli. The particles are ingested by an invagination process of the plasma membrane resulting in the formation of intracellular vesicles and vacuoles. Ultimately large amounts of India ink accumulate in the cell, occupying substantial portions of the cytoplasm. The surfaces of phagocytizing macrophages show signs of intense motility. Their cytoplasm contains numerous particles, resembling Palade particles, and a large amount of rough surfaced endoplasmic reticulum. These structures are interpreted as indicative of protein synthesis. At the level of resolution achieved in this study the membranes of this reticulum appear as single dense "lines." On the other hand, the plasma membrane and the limiting membranes of vesicles and of vacuoles often exhibit the double-line structure typical of unit membranes (Robertson, 37). The inclusion bodies appear to be the product of phagocytosis. It is believed that some of them derive from the vacuoles mentioned above, and that they correspond to similar structures seen in phase contrast cinemicrographs of culture cells. Their matrix represents phagocytized material. Certain structures within this matrix are considered as secondary and some of these structures possess an ordered form probably indicative of the presence of lipid. The possible origin and the fate of alveolar macrophages are briefly discussed.

The ultrastructure of mouse lung: the alveolar macrophage.

Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1(1/2) hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small ( approximately 6 mmicro) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.