Free Alveolar Cells Research Articles

Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung.

Earlier work from this laboratory showed that abnormal fibroblast phenotypes isolated from fibrotic human lung produce factor(s) capable of inducing apoptosis and necrosis of alveolar epithelial cells in vitro [B. D. Uhal, I. Joshi, A. True, S. Mundle, A. Raza, A. Pardo, and M. Selman. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L819-L828, 1995]. To determine whether epithelial cell death is associated with proximity to abnormal fibroblasts in vivo, the spatial distribution of epithelial cell loss, DNA fragmentation, and myofibroblasts was examined in the same tissue specimens used previously for fibroblast isolation. Paraffin sections of normal and fibrotic human lung were subjected to in situ end labeling (ISEL) of fragmented DNA and simultaneous immunolabeling of alpha-smooth muscle actin (alpha-SMA); replicate samples were subjected to electron microscopy and detection of collagens by the picrosirius red technique. Normal human lung exhibited very little labeling except for positive alpha-SMA immunoreactivity of smooth muscle surrounding bronchi and vessels. In contrast, fibrotic human lung exhibited moderate to heavy ISEL of interstitial, cuboidal epithelial, and free alveolar cells. ISEL of the alveolar epithelium was not distributed uniformly but was most intense immediately adjacent to underlying foci of alpha-SMA-positive fibroblast-like interstitial cells. Both electron microscopy and picrosirius red confirmed epithelial cell apoptosis, necrosis, and cell loss adjacent to foci of collagen accumulation surrounding fibroblast-like cells. These results demonstrate that the cuboidal epithelium of the fibrotic lung contains dying as well as proliferating cells and support the hypothesis that alveolar epithelial cell death is induced by abnormal lung fibroblasts in vivo as it is in vitro.

Impaired Regulation of Surfactant Phospholipid Metabolism in the Isolated Rat Lung after Nitrogen Dioxide Inhalation

Various drugs have been shown to stimulate surfactant phospholipid metabolism. Particularly β-adrenergic agonists play an important role under physiologic conditions. For the first time we have studied whether nitrogen dioxide (NO2) inhalation alters β-adrenergic regulation of surfactant phospholipid metabolism in the model of the isolated lung. Rats were continuously exposed in vivo to a 5 ppm NO2-containing atmosphere for 48 hr. The lungs were isolated and perfused in presence of the β-adrenergic agonist dopexamine and surfactant metabolism was studied in three lung compartments: (1) lung lavage, (2) lung tissue, and (3) lavagable free alveolar cells. We found that (1) in normal rat lungs dopexamine increased the incorporation of palmitate and choline from the perfusate into lung lavage phospholipids. In nitrogen dioxide exposed rat lungs β-adrenergic stimulation did not cause an increase in precursor incorporation. No significant difference in unstimulated precursor incorporation was found for normal and NO2-exposed rat lungs. (2) Lung tissue from rats exposed to NO2 showed a decreased precursor incorporation into disaturated phosphatidylcholine due to an augmented cellular pool size. (3) Lavagable alveolar cells showed an increased palmitate uptake after nitrogen dioxide inhalation and by β-adrenergic stimulation. From these data we conclude that nitrogen dioxide inhalation impairs the β-adrenergic regulation of surfactant phospholipid metabolism. Moreover these data underline the importance of β-adrenergic agonists in surfactant metabolism.

Particle distribution in lung and lymph node tissues of rats and dogs and the migration of particle‐containing alveolar cells in vitro

Whole rat lungs and individual dog lung lobes were instilled either with low numbers (10(7)) or high numbers (10(9)) of fluorescent polystyrene microspheres (PLM), or with saline alone. Particle distributions in dog and rat lung lobes and tracheobronchial lymph nodes (TBLN) were studied up to several weeks after particle instillations using methacrylate-embedded tissues and epifluorescence light microscopy. Free alveolar cells were obtained from rats and dogs by lung lavage 1 or 7 d after particle instillations. Lavaged cells were tested for directed migration toward the chemoattractant N-formylmethionyl-leucyl-phenylalanine (FMLP). Random migration in the absence of the FMLP was used as a control. The dog lung interstitium contained many more particles than did the rat lung interstitium, and particle numbers in interstitial and TBLN cells of dogs were higher than in those of rats. FMLP enhanced the number of migrating cells about twofold. Increasing particle numbers in lavaged phagocytes (greater than 10 particles/phagocyte in dogs; greater than 20 particles/phagocyte in rats) decreased their ability to migrate. The higher fractions of particles in the dog lung interstitium are thought to be an important reason for prolonged retention and increased TBLN transport of deposited particles in dogs as compared with rats. Our results suggest that cell mobility is lost after ingestion of high numbers of particles, and that this occurs earlier with dog than with rat lung cells.

Detection of metallothionein in bronchoalveolar cells and lavage fluid following repeated cadmium inhalation

Metallothionein (MT) levels were measured by radioimmunoassay in bronchoalveolar cells and fluids derived from rat lungs following 0, 8, 12, 18, or 24 exposures to Cd acetate aerosols. Alveolar cell metallothionein levels increased from a base line value of 0.35 micrograms MT/mg protein in control rats to a value of 21.73 micrograms MT/mg protein in rats exposed 24 times to Cd aerosols. Cell-free lavage fluid levels of metallothionein also increased in response to frequency of Cd exposure but were 5 to 10-fold lower than in the free alveolar cells. Gel chromatographic analysis of free alveolar cells from Cd-exposed animals revealed the presence of a 11,600-Da Cd-binding protein with an absorption maximum of 254 nm. Metallothionein analyses of purified populations of alveolar macrophages and polymorphonuclear leukocytes from exposed animals demonstrated that macrophages but not polymorphonuclear leukocytes contained metallothionein.

Relationship of silica and bacillus calmette-guerin exposures to humoral and cellular immune functions

The consequences of silica preexposures were studied on subsequent bacillus Calmette-Guerin (BCG) pulmonary infections. Silica had a variable effect on colonization by BCG depending on organ and number of BCG treatments. Silica preexposures resulted in increased numbers of free alveolar cells subsequent to BCG infections, with an initially rapid increase of neutrophils. Both silica and two BCG treatments were capable of decreasing the animals' splenic response to stimulations with Escherichia coli aerosols, and BCG greatly enhanced antibody-dependent cell-mediated cytotoxicity in silica preexposed mice. 5'-Nucleotidase activity was depressed by both silica and BCG in pulmonary washout cells indicating that inhalation of these substances may have a stimulating effect on the alveolar macrophages.

Structure-function correlation of early stages of lung injury induced by intratracheal bleomycin in the rabbit.

After measurement of lung volume, pressure-volume relationship of the lungs, flow-volume relationship, and single-breath diffusing capacity, the effect of endotracheal injection of bleomycin (10 micrograms/kg) on structural and functional changes was assessed in 21 rabbits and compared with 8 rabbits who received endotracheal injection of saline. Lung function was reassessed in the control rabbits at 1, 3, and 5 wk, and they were then killed. The bleomycin-treated rabbits were divided into 3 groups and retested at 1 (n = 7), 3 (n = 6), and 5 (n = 8) wk. Each group of animals was killed after the functional assessment, and after fixation the lungs were examined morphometrically. The degree of fibrosis in the bleomycin-treated animals was small even at 5 wk. Volume densities of pulmonary structures, alveolar wall thickness, airway diameters, airway inflammation scores, and fibrosis scores were determined and their correlation with the functional measurements was assessed. Lung volume subdivisions, pressure-volume characteristics, and diffusing capacity for carbon monoxide were significantly reduced, and the resistance of the upstream segment of airways was significantly increased after the bleomycin injection. Changes in lung volume correlated significantly with the volume densities of free alveolar cells, nonparenchymal tissue, and alveolar wall thickness. The exponent K, derived by fitting an exponential function to the pressure-volume data, which is indicative of lung elastic behavior, did not correlate with any morphometric index of interstitial disease. The diffusing capacity for CO was significantly correlated with the volume densities of alveolar wall, nonparenchymal tissue, and alveolar wall thickness, but these correlations became nonsignificant when the diffusing capacity was corrected for alveolar volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of bleomycin lung toxicity by N-acetyl cysteine in the rat

N-acetyl cysteine (NAC) has recently been shown to have antioxidant properties, and since bleomycin produces pulmonary damage via free oxygen radical toxicity, the possible protective effect of NAC on bleomycin lung toxicity was investigated. Rats received saline (n = 7), NAC (n = 6), bleomycin (n = 7) or bleomycin and NAC (n = 6) by direct intratracheal injection. Seven days later the animals were killed and the lungs processed for histology or morphometry. All rats treated with bleomycin only had typical changes of bleomycin lung toxicity whereas the animals treated with bleomycin and NAC had minimal pathology. The control animals had normal lungs. These results were confirmed by morphometry which demonstrated significantly higher volume densities (p less than .01) of alveolar wall and free alveolar cells in the bleomycin group compared to the other 3 groups. It is concluded that NAC inhibits bleomycin lung toxicity when administered by direct intratracheal injection.

Imipramine and chloroquine induced alterations in phospholipid content of rat lung

The influence of the amphiphilic drugs imipramine (150 mg/kg b. w./day) and chloroquine (75 mg/kg b. w./day) on the phospholipid (PL) -metabolism of rat lung, its PL-content and PL-composition were measured in the cell free lung lavage fluid (alveolar surfactant), in the free alveolar cells (mainly alveolar macrophages), and in the residual lung tissue. In addition to the long-term administration (15 applications during a period of 3 weeks), the influence of short-term administration (2 or 4 applications, resp., during a period of 2 or 4 days) was examined. The alveolar macrophages show the largest increase in PL-content. As revealed by its composition the stored PL are of surfactant origin. In chloroquine treated rats the number of macrophages is increased as well. The concentration of stored PL is higher in macrophages of imipramine treated rats. The excessive accumulation of PL in this animal group possibly impairs the clearance function of alveolar macrophages. This is suggested to be the reason for the accumulation of alveolar surfactant in imipramine treated rats. The influence of the drugs on the PL-content of the residual lung tissue was weak. The results of this study show that amphiphilic drugs cause an accumulation of surfactant-PL within the alveolar macrophages and can promote the alveolar surfactant content even after short-term application. The role of alveolar macrophages in alveolar surfactant catabolism is demonstrated.

Abnormal alveolar cells in monocrotaline induced pulmonary hypertension.

Monocrotaline given to rats causes lung injury which is followed by pulmonary hypertension. A large, abnormal intraalveolar cell has been repeatedly observed. The purpose of the present study was to define the nature of the cell and to determine how it related to the lung injury. As observed by electron microscopy, the intraalveolar cell contained numerous large, dense granules, as well as lipid whorls, extensive Golgi complexes, and many small vesicles. These cells appeared to be macrophages, possibly altered by ingestion of lipid-like material which was free in the alveolar space. Enlarged type II pneumonocytes and mast cells were seen in the alveolar walls but their appearance differed from that of the free alveolar cells. The electron microscopic findings plus the presence of Fc and C3b receptors on the cells indicated they were alveolar macrophages. When the severity of pulmonary hypertension was varied by a) giving monocrotaline to rats of varying age, b) by varying the dose of monocrotaline, or c) by varying the time interval after monocrotaline administration, the number of abnormal alveolar macrophages related to the severity of the pulmonary hypertension. When we reduced the number of circulating leukocytes by whole body radiation, monocrotaline administration was followed both by accelerated pulmonary hypertension and increased numbers of abnormal alveolar macrophages. The abnormal macrophage appeared to be a marker of the monocrotaline induced pulmonary hypertension. However, neither the abnormal macrophage nor the monocrotaline injury appeared to depend on circulating leukocytes.

A method for obtaining swine alveolar macrophages by segmental pulmonary lavage

A technique for obtaining alveolar macrophages (AM) from anesthetized swine is described. Animals were intubated and segmental pulmonary lavage was performed utilizing a double lumen catheter (DLC). An average of 98% of the initial 100 ml lavage fluid was recovered with a typical yield of 1 × 10 8 free alveolar cells (FAC). AM were then separated from other FAC by their adherence to plastic. The final adherent cell population consisted of >95% macrophage as determined by morphology and non-specific esterase activity. The technique described had no adverse effects on the animals even when repeated on the same animal several days later.

Enumeration and Ultrastructure of C4-Producing Free Alveolar Cells from Guinea Pig Lung

Free alveolar cells from guinea pig lung producing the fourth emoponent of C (C4) were identified, enumerated, and characterized by using anti-C4 Fab-peroxidase conjugates in conjunction with transmission electron microscopy. The C4-producing cell population consisted of: 1) alveolar macrophages (AM); 2) less well differentiated phagocytes similar in morphology to exudate macrophages; and 3) weakly phagocytic secretory cells with numerous profiles of rough-surfaced endoplasmic reticulum (ER). Internal immunolabeling allowed the visualization of C4 in the ER, perinuclear space, and Golgi complex of producer cells and its release at cell surfaces; synthesis of C4 in vitro was sensitive to inhibitors both of protein synthesis and messenger RNA function. The percentage of free alveolar cells from normal animals competent for C4 production as indicated by cell surface immunolabeling was approximately 1% of the total cells obtained by lavage. Transnasal infection with Listeria monocytogenes, generation of a pulmonary granulomatous reaction by i.v. injection of heat-killed BCG, and aerosol infection of nonvaccinated animals with Myco-bacterium tuberculsois each resulted in an increase in numbers of AM and exudate macrophage-like free alveolar cells competent for C4-production.

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.

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.

Effect of sulphur dioxide on the enzyme activity of the alveolar macrophage of rats.

Adult rats were exposed to sulphur dioxide to induce hypersecretion of mucus and so increase the amount of mucus reaching the alveoli. The comparative activity of four lysosomal hydrolytic enzymes was studied histochemically in free alveolar cells, thought to be macrophages, from the lungs of the exposed and control animals. Although the activity of β-glucuronidase, β-galactosidase, and N-acetyl-β-glucosaminidase appeared increased in some free alveolar cells in the peribronchiolar region, a marked increase of acid phosphatase activity was seen in the free alveolar cells throughout the lung parenchyma. It is suggested that in rats acid phosphatase in alveolar macrophages is connected with the catabolism and removal of mucopolysaccharide and increases in response to the extra amount of mucus reaching the alveoli after respiratory irritation with sulphur dioxide.