Altered regulation of surfactant phospholipid and protein A during acute pulmonary inflammation.

Biochemical changes in the pulmonary surfactant system caused by exposure to toxicants are often accompanied by an influx of inflammatory cells into the lungs. We have investigated the possibility that the inflammatory and surfactant biochemical effects might be connected. Co-treatment with dexamethasone, a synthetic anti-inflammatory glucocorticoid, mitigated the increases in free cells and total intracellular surfactant phospholipid normally seen in animals given silica alone, suggesting a relationship between the free cell population of the alveoli and the surfactant system during alveolitis. Furthermore, we have investigated whether induction of the surfactant system is a universal response to alveolar inflammation. Inflammation was induced in the lungs by intratracheal injections of titanium dioxide, silica, bleomycin or lipopolysaccharide (LPS) suspended in isotonic saline. Inflammatory cell and surfactant responses were measured at 3 days and 14 days following injection. There was a distinct alveolar inflammatory cell profile following administration of each agent, at each time point, indicating a dynamic inflammatory cell population during the course of the study. Furthermore, surfactant phospholipid and protein A (SP-A) pools exhibited unique responses to the inflammatory agents. Only silica-treated lungs maintained elevated levels of surfactant phospholipids and SP-A throughout the course of the experiment. We conclude that both the surfactant components and the inflammatory cell population of the alveoli undergo dynamic changes following treatment with these inflammatory agents and that activation of the surfactant system is not a universal response to alveolar inflammation, since surfactant components were not always elevated during times of increased alveolar cellularity. The unique inflammatory cell infiltrate elicited by silica is of particular interest in that surfactant components were elevated throughout the course of the experiment in this group. Indeed, we have shown that the size of the intracellular pool of surfactant is directly proportional to the number of polymorphonuclear leukocytes but not alveolar macrophages or lymphocytes in the alveoli following silica treatment. Finally, our data suggest that the phospholipid and SP-A components of surfactant respond differentially to the pulmonary toxicants in this study.

Production of low molecular weight cadmium-binding proteins in rabbit lung following exposure to cadmium chloride.

Low molecular weight cadmium-binding proteins were studed in lung tissue from rabbits exposed to aerosols of CdCl2. Lungs obtained from animals exposed by inhalation to aerosols of 800 or 1600 micrograms/m3 CdCl2 for 2-hr periods/day, every other day for a 5-day period, were found to contain at least three low molecular weight cadmium-binding proteins, two of which were similar electrophoretically and spectrally to rabbit liver metallothionein. The third protein(s), which accounted for the majority of the cadmium in the soluble fraction of the tissue, did not bind to an anionic exchange gel and did not appear to be a polymerized form of metallothionein. Translocation studies of lung cadmium suggest a long half-life for cadmium in lung tissue following inhalation exposure, due perhaps to the high affinity of cadmium for specific lung cadmium-binding proteins. A small but significant redistribution of lung cadmium did occur to both kidney and liver tissue with time.

The pulmonary extracellular lining.

The extracellular lining of the lungs is reviewed. The pulmonary extracellular lining is a complex mixture of phospholipids, proteins and carbohydrates which is absolutely essential for the maintenance of normal pulmonary functions such as gas exchange. Without the lining the lungs would collapse. Alterations in the pulmonary extracellular lining may underlie some disease conditions induced by toxic agents, especially those which interfere with the formation of pulmonary surfactant. The extracellular lining could be used to detect and monitor damage and disease caused by agents toxic to the lungs. The lining contains many hydrolytic enzymes which may act to detoxify certain toxic agents such as those which contain ester groups. The pulmonary extracellular lining could play a significant role mediating the toxic action of inhaled agents as well as the removal of those agents from the lungs.

Hypertrophy and hyperplasia of alveolar type II cells in response to silica and other pulmonary toxicants.

Alveolar Type II cells serve two major functions in the lung, both of which are essential for the preservation of normal lung function. First, Type II cells synthesize and secrete pulmonary surfactant, and second, they function as progenitor cells for maintaining the alveolar epithelium. The Type II cell population of the lung is quite sensitive to the deposition of toxicants in the distal lung, responding in two principal ways. Damage to the Type I epithelium stimulates Type II cells to proliferate and subsequently differentiate to replace the injured Type I cells. Second, a portion of the Type II cell population may become hypertrophic. Both of these events are frequent findings in the diseased or damaged lung. The Type II cell changes are often associated with increases in surfactant pools. In those cases where ultrastructural characteristics of hypertrophic Type II cells were examined, the appearance of these cells was consistent with that of an activated cell type. Alterations in the lamellar body compartment are a common finding in hypertrophic Type II cells, with increases in both lamellar body size and number. It is likely that the hypertrophic, or activated, Type II cells account for the increased levels of surfactant found in the lungs after exposure to a variety of toxic agents. We examined, in detail, Type II cell hyperplasia and hypertrophy induced by silica deposition. Both Type II cell hyperplasia and hypertrophy were prominent responses. The proliferative response led to an approximate doubling of the number of Type II cells in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretory cells of the peripheral pulmonary epithelium as targets for toxic agents.

The extracellular lining of the pulmonary peripheral airways is of vital importance to the lung. In this report, some aspects of the pulmonary extracellular lining and the epithelial cells believed to be responsible for its formation and secretion have been briefly reviewed. The influence of a number of toxic agents on the extracellular lining either directly or via those cells involved in its formation indicates that the extracellular lining may be important in understanding numerous toxic agent interactions with the lung.

Pathways of differentiation of airway epithelial cells.

The question being examined is whether one or more morphologically distinct cell types can be identified in the conducting airways of adult rabbits possessing stem cell functions. The term "stem cell" is used to denote cells with extensive self-replicating potential and the ability to produce differentiated progeny. According to various models of cell renewal in the conducting airways that have been proposed over the years, two different cell types have to be regarded as primary candidates for the stem cell: basal cells and some type of secretory cells. The question is complicated by the fact that significant differences exist between species in the distribution and morphological characteristics of airway cell types. In addition, different airway segments may or may not be occupied by different populations of stem cells. Previously, investigators have addressed the problem by studying normal cell regeneration or injury induced cell regeneration in vivo in the whole animal. We decided to attempt a different approach, namely, to separate specific cell types and to study the proliferation and differentiation capacity of such cell isolates using in vitro and in vivo cell culture techniques. Our studies lead us to conclude that the conducting airways of adult rabbits contain at least two distinct cell populations endowed with stem cell potential, namely basal cells and bronchiolar Clara cells. From that it follows that the trachea and bronchi, on one hand, and the bronchioles, on the other hand, are occupied by two different stem cell populations governing renewal of the epithelial lining.

Ultrastructural immunohistochemical localization of Clara cell secretory protein in pulmonary epithelium of rabbits.

Highly purified Clara cells (93 +/- 3%) isolated from the lungs of rabbits were used to produce an antiserum against Clara cell secretory proteins. This antiserum was used to identify and study the biosynthesis and secretion of [35S]methionine-labeled proteins from isolated Clara cells. The antiserum recognized one major secretory protein with apparent molecular weight of 6 kDa and reacted weakly with a higher molecular weight protein of about 180 kDa. Biosynthesis and secretion of these proteins was not detected in preparations of isolated alveolar type II cells or alveolar macrophages. Immunocytochemical localization of the antigen with colloidal gold indicated a dual localization in bronchiolar Clara cells. Gold labeling was found over the osmiophilic secretory granules of Clara cells and smooth endoplasmic reticulum. In tracheal Clara cells, labeling was found mostly in association with secretory granules and relatively little in association with the smooth endoplasmic reticulum. Labeling was also found over the lamellar bodies of type II cells, although the reaction was weak. Labeling of ciliated cells, alveolar type I cells, capillary endothelial cells, and alveolar macrophages was not distinguishable from background. These data indicate that Clara cells of both the bronchioles and trachea of rabbits synthesize and secrete the low molecular weight protein previously called Clara cell secretory protein (CCSP). This antigen does not belong to that group of surfactant proteins whose molecular weights range from 26 to 40 kDa.

Induction and suppression of hepatic and extrahepatic microsomal foreign-compound-metabolizing enzyme systems by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on a number of hepatic and extrahepatic foreign-compound-metabolizing enzyme systems in microsomes from rats, rabbits and guinea pigs were investigated. Following TCDD treatment, the N-demethylation of benzphetamine, aminopyrine and ethylmorphine was suppressed in hepatic microsomes from male but not from female rats. However, both cytochrome P-450 and benzpyrene hydroxylase were significantly stimulated in hepatic microsomes from both male and female rats at doses as small as 1 mug TCDD/kg body weight. The inductive effect on rat hepatic microsomal enzymes was considerably more persistent than the suppressive effect. Following a single oral dose of 25 mug TCDD/kg weight, benzpyrene hydroxylase of male rat liver microsomes remained significantly elevated for 73 days but the suppression of benzphetamine N-demethylase had gone after 35 days. The induction of benzpyrene hydroxylase in male rat liver microsomes by TCDD was independent of the age of the rat and the levels to which this enzyme was increased was similar in male rats of all ages. However, the suppression of benzphetamine N-demethylase in male rat liver microsomes was age related: the suppression was seen only in adult animals and in the very young (10 days old) the enzyme was actually induced by TCDD. Inductive effects appeared in both smooth and rough-surfaced hepatic microsomes from male rats but the suppression of N-demethylation occurred only in the smooth-surfaced microsomes (SER). In microsomes from extrahepatic tissues of the rat, induction of mixed-function oxidases (MFOs) by TCDD occurred only in the kidney. However, UDPglucuronyltransferase was induced in microsomes from lung, kidney, intestine and brain but not testes. The response in the rabbit and guinea pig to TCDD differed considerably from that in the rat. Benzpyrene hydroxylase was unaffected in hepatic microsomes from the guinea pig and actually suppressed in microsomes from rabbit liver. Benzphetamine N-demethylase was also suppressed in rabbit liver microsomes. Glucuronyl-transferase was unaffected by TCDD in microsomes from liver, lung or kidney of the rabbit and guinea pig. The only lung enzyme responsive to TCDD was biphenyl 4-hydroxylase of the rabbit and guinea pig. Suppression was not observed in any of the extraheptic tissues studied and may be confined to only certain hepatic systems.

Postnatal stimulation of hepatic microsomal enzymes following administration of TCDD to pregnant rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) administered to pregnant rats at 3 mu-g/kg as a single oral dose during early, middle, or late gestation caused marked elevations of some maternal hepatic microsomal enzymes for at least 10 weeks after treatment. This dose was not teratogenic and fetal rates of glucuronidation of testosterone and p-nitrophenol (PNP) were unaffected. Increases in fetal liver benzpyrene hydroxylase (BPH) activities were evident during late gestation although cytochrome P-450 and cytochrome b-5 contents were unchanged. The offspring of pregnant rats administered TCDD had markedly elevated hepatic PNP UDP-glucuronyltransferase (UDPGT) BPH, and microsomal cytochrome contents whereas the perinatal development of testosterone UDPGT was unchanged. PNP glucuronidation attained a maximal 8-fold increase above controls by 3 weeks after birth and activities were twice that of controls 8 weeks after birth (adults). Maximal increases in benzpyrene hydroxylation rates occurred one day after birth when in the prenatally exposed group activities were approximately 20 times higher than controls. Foster mother experiments demonstrated that the postnatal inductive effect resulted both from exposure of newborns to TCDD via maternal milk and the activation of an inducing mechanism occurring after birth. Tese data demonstrate that multiple factors are responsible for the induction of hepatic microsomal enzymes in the newborn following administration of TCDD to pregnant rats.

Quantitation of specific proteins in polyacrylamide gels by the elution of Fast Green FCF.

The quantitation of proteins in polyacrylamide gels stained with Fast green FCF has been investigated using a modification of the elution technique originally described by Fenner et al. (Fenner, C., Traut, R.R., Mason, D.T. and Wikman-Coffelt, J. (1975) Anal. Biochem. 63, 595--602) for Coomassie Blue and adapted by Medugorac (Medugorac, I. (1979) Basic Res. Cardiol. 74, 406--416) for use with proteins stained with Fast Green FCF. The elution of dye from stained protein was accomplished using 1.0 M NaOH instead of aqueous pyridine as required by the original method. The primary advantages of our modification are that the time required for protein quantitation has been considerably reduced and the use of toxic organic solvents has been eliminated. We have investigated the applicability of the method of several different proteins and our results indicate: (a) The quantity of Fast Green FCF eluted from specific proteins is proportional to the quantity of protein applied to the gel, but varies for each individual protein. (b) The method allows quantitation over a very wide range of protein (1--800 micrograms). (c) Quantitation of protein is independent of the width of the stained bands as well as acrylamide concentration. (d) The method is applicable to gels of many types including disc, slab and continuous gradient gels. (e) Protein can be estimated from the patterns obtained by two-dimensional polyacrylamide gel electrophoresis. (f) The presence of Triton X-100 in gel and protein sample does not affect quantitation; the method is applicable to gels containing SDS provided that SDS is removed prior to staining. (g) Precipitation of protein with 12.5% TCA following electrophoresis does not interfere with quantitation. (h) The reproducibility of the technique is excellent, with standard deviations being less than 10% of the mean in all cases. This method appears highly versatile but requires appropriate standards for the quantitation of individual proteins.

Phospholipid exchange between subcellular organelles of rabbit lung.

A soluble protein fraction (PLEP) prepared from rabbit lung can catalyze the exchange of phospholipids between subcellular organelles of the lung and between these subcellular organelles and synthetic liposomes. Phospholipid exchange between microsomes and synthetic liposomes and between mitochondria and synthetic liposomes was stimulated 8-fold and 2.5-fold, respectively, in the presence of the protein fraction. Lung exchange protein could also catalyzed phospholipid exchange between subcellular organelles of the liver and synthetic liposomes. Phospholipid transfer between microsomes and lamellar bodies of the lung was stimulated 2-fold by the exchange protein. Both radiolabeled phosphatidylcholine (PC) and phosphatidylinositol (PI) were transferred from 32P-labeled microsomes to lamellar bodies, but the exchange protein exhibited no transfer activity for phosphatidylglycerol (PG) and that for phosphatidylethanolamine (PE) was insignificant compared to the transfer activity for phosphatidylcholine and phosphatidylinositol. While the physiological role of the phospholipid exchange proteins in the lung is unknown, it is possible that they participate in the distribution of the newly synthesized phospholipids from the site of synthesis to lamellar bodies and other membrane compartments of cells.