Tracing surfactant transformation from cellular release to insertion into an air-liquid interface.

Pulmonary surfactant is secreted by alveolar type II cells as lipid-rich, densely packed lamellar body-like particles (LBPs). The particulate nature of released LBPs might be the result of structural and/or thermodynamic forces. Thus mechanisms must exist that promote their transformation into functional units. To further define these mechanisms, we developed methods to follow LBPs from their release by cultured cells to insertion in an air-liquid interface. When released, LBPs underwent structural transformation, but did not disperse, and typically preserved a spherical appearance for days. Nevertheless, they were able to modify surface tension and exhibited high surface activity when measured with a capillary surfactometer. When LBPs inserted in an air-liquid interface were analyzed by fluorescence imaging microscopy, they showed remarkable structural transformations. These events were instantaneous but came to a halt when the interface was already occupied by previously transformed material or when surface tension was already low. These results suggest that the driving force for LBP transformation is determined by cohesive and tensile forces acting on these particles. They further suggest that transformation of LBPs is a self-regulated interfacial process that most likely does not require structural intermediates or enzymatic activation.

Pulmonary surfactant function studied with the pulsating bubble surfactometer (PBS) and the capillary surfactometer (CS).

Two instruments, the pulsating bubble surfactometer (PBS) and the capillary surfactometer (CS), were constructed for a study of pulmonary surfactant's physical properties. The instruments study spherical surfaces as in alveoli (PBS) and cylindrical surfaces as in terminal conducting airways (CS). Phospholipids, pulmonary surfactant's main components, are amphiphilic and, therefore, spontaneously form a film at air-liquid interfaces. When the film in the PBS is compressed to a reduced area during 'expiration', the molecules come closer together. Thereby, a high surface pressure develops, causing surface tension to be reduced more than bubble radius. If these conditions, observed with the PBS are analogous in lungs, alveolar stability would be promoted. The CS was developed for a study of how surfactant has ability to maintain patency of narrow conducting airways. Provided adsorption is extremely fast, a surfactant film will line the terminal conducting airway as soon as liquid blocking the airway has been extruded. During expiration that film will develop high surface pressure (=low surface tension). This will counteract the tendency for liquid to accumulate in the airway's most narrow section. If surfactant is dysfunctioning, liquid is likely to accumulate and block terminal airways. Airway resistance would then increase, causing FEV(1) to be reduced.

Role of interferon gamma in the pathogenesis of primary respiratory syncytial virus infection in BALB/c mice.

Immunologic mechanisms are thought to contribute to the pathogenesis of respiratory syncytial virus (RSV) bronchiolitis in humans. RSV-infected BALB/c mice exhibit tachypnea and signs of outflow obstruction, similar to symptoms in humans. Interferon gamma (IFNgamma) has been found to be the predominant cytokine produced in humans and mice with RSV infection. We therefore undertook this study to evaluate the role of IFNgamma in the development of respiratory illness in RSV-infected mice. BALB/c mice were infected with RSV, and lung function was assessed by plethysmography. Bronchoalveolar lavage (BAL) fluids were analyzed for the concentration of interferon gamma (IFNgamma) and the presence of inflammatory cells, and lung tissue sections were examined for histopathologic changes. The role of IFNgamma was further addressed in studies of IFNgamma knock-out mice (IFNgamma(-/-)) and of mice depleted of IFNgamma by in vivo administration of a neutralizing antibody. After infection, mice developed respiratory symptoms that were strongly associated with the number of inflammatory cells in BAL, as well as with the concentrations of IFN-gamma. Both IFN-gamma(-/-) mice and mice treated with anti-IFNgamma developed more extensive inflammation of the airways than control mice. However mice lacking IFNgamma exhibited less severe signs of airway obstruction. Together these data suggest a protective role of IFNgamma in RSV infection in terms of limiting viral replication and inflammatory responses but also a pathogenic role in causing airway obstruction.

Altered airway surfactant phospholipid composition and reduced lung function in asthma.

Pulmonary surfactant in bronchoalveolar lavage fluid (BALF) and induced sputum from adults with stable asthma (n = 36) and healthy controls (n = 12) was analyzed for phospholipid and protein compositions and function. Asthmatic subjects were graded as mild, moderate, or severe. Phospholipid compositions of BALF and sputum from control subjects were similar and characteristic of surfactant. For asthmatic subjects, the proportion of dipalmitoyl phosphatidylcholine (16:0/16:0PC), the major phospholipid in surfactant, decreased in sputum (P < 0.05) but not in BALF. In BALF, mole percent 16:0/16:0PC correlated with surfactant function measured in a capillary surfactometer, and sputum mole percent 16:0/16:0PC correlated with lung function (forced expiratory volume in 1 s). Neither surfactant protein A nor total protein concentration in either BALF or sputum was altered in asthma. These results suggest altered phospholipid composition and function of airway (sputum) but not alveolar (BALF) surfactant in stable asthma. Such underlying surfactant dysfunction may predispose asthmatic subjects to further surfactant inhibition by proteins or aeroallergens in acute asthma episodes and contribute to airway closure in asthma. Consequently, administration of an appropriate therapeutic surfactant could provide clinical benefit in asthma.

Surfactant function affected by airway inflammation and cooling: possible impact on exercise-induced asthma.

Pulmonary surfactant maintains patency of narrow conducting airways. An inflammation, with a leakage of plasma proteins into the airway lumen, causes surfactant to lose some of this ability. Will a lowering of temperature aggravate the deteriorating effect of an inflammation? Calf lung surfactant extract (CLSE) with proteins added was studied with a capillary surfactometer (CS) at temperatures of 25-42 degrees C. BALB/c mice were infected with respiratory syncytial virus (RSV). Six days later the lungs were lavaged and the surfactant in the lavage fluid was studied with the CS at temperatures of 25-42 degrees C. Lavage fluid from allergen challenged asthmatics was examined for its content of surfactant inhibitors at reduced temperatures. It was shown that CLSE with proteins gradually lost its ability to maintain patency as the temperature was lowered. Lavage fluid from the RSV infected mice showed a similar dysfunction at low temperatures. Lavage fluid from the airways of human asthmatics, when challenged with antigen but not with saline, contained agents inhibiting surface activity, particularly at reduced temperatures. Airway inflammation causes surfactant to lose its ability to maintain patency, particularly as the temperature is reduced. That might be a reason for the increased airway resistance observed in asthma patients hyperventilating in cold weather.

Pseudomonas aeruginosa from patients with cystic fibrosis affects function of pulmonary surfactant.

Patients with cystic fibrosis are severely affected by an infection with Pseudomonas aeruginosa, a microbe known to synthesize phospholipase C. This study was designed to determine whether that enzyme would affect the function of pulmonary surfactant phospholipids. Mucoid and nonmucoid strains of P. aeruginosa, freshly obtained from patients with cystic fibrosis, were cultured for 12 h on agar plates. The bacteria were suspended in saline solution and then pelleted by centrifugation. The supernatant was used to dilute the surfactant preparation, calf lung surfactant extract, from 35 to 2 mg/mL. Surfactant function, before and after incubation, was examined with a capillary surfactometer, an instrument specifically developed for an evaluation of the ability of surfactant to maintain patency of a narrow glass tube, simulating a terminal conducting airway. Phospholipid hydrolysis was also evaluated biochemically by determining the total content of phospholipids in surfactant before and after incubation. In five experiments, the lipids were separated with thin-layer chromatography, and the phosphorus content was determined in the diacylphosphatidylcholine band before and after incubation for 6, 24, and 48 h. Capillary openness and phospholipid concentration decreased as enzyme concentration and time of incubation increased (p<0.0001). Linear regression showed a significant correlation between time of capillary openness and phospholipid concentration (r = 0.957; p<0.0001). Calf lung surfactant extract hydrolysis was catalyzed by extracts of the bacteria, particularly the nonmucoid, analogous to the catalysis observed with phospholipase C. Surfactant hydrolysis catalyzed by enzymes from P. aeruginosa might severely affect surfactant function provided enzyme concentration is high and time of incubation is long.

Antigen-induced airway inflammation in atopic subjects generates dysfunction of pulmonary surfactant.

If pulmonary surfactant develops a dysfunction, its ability to maintain patency of narrow conducting airways diminishes, which is likely to cause an increased airway resistance. We hypothesized that antigen challenge will cause inflammation in the conducting airways and that this will cause a surfactant dysfunction. Twenty atopic patients underwent bronchoalveolar lavage (BAL) 5 min and 48 h after challenge with antigen in one segment and with saline solution in another. BAL fluid (BALF) cell count, differential, and proteins were determined. Surfactant function was studied with a capillary surfactometer (CS), an instrument specifically designed to evaluate surfactant's ability to maintain patency. Eosinophils increased 80-fold 48 h after antigen challenge and total protein increased from 84 to 241 micrograms/ml (median values). BALF surfactant lost part of its ability to maintain openness of the capillary, from 68.8% to 14.0% (p < 0.05). Protein concentration negatively correlated with percent openness (r = -0.62, p = 0.005). We conclude that the antigen challenge resulted in an inflammatory reaction that caused pulmonary surfactant to lose some of its ability to maintain airway patency and speculate that surfactant dysfunction is probably an important factor contributing to increased airway obstruction in allergen-induced exacerbation of asthma.

Dysfunction of pulmonary surfactant in asthmatics after segmental allergen challenge.

Increased airway resistance in asthma may be partly due to poor function of pulmonary surfactant. This study investigated the inflammatory changes of bronchoalveolar lavage fluid (BALF) and the performance of BALF surfactant in healthy control subjects (n = 9) and patients with mild allergic asthma (n = 15) before and after segmental challenge. BALF was obtained for baseline values, and 24 h after challenge with saline solution in one lung segment and with allergen in another. Cell counts, phospholipid and protein concentrations, and ratios of small to large surfactant aggregates (SA/LA) were analyzed. Surface tension was determined with a pulsating bubble surfactometer, and the ability of the BALF surfactant to maintain airway patency was assessed with a capillary surfactometer. Baseline values of control subjects and asthmatics were not different. Challenge with saline and antigen raised total inflammatory cells in both control subjects and asthmatics. Allergen challenge of asthmatics, but not of healthy volunteers, significantly increased eosinophils, proteins, SA/ LA, and surface tension at minimum bubble size, and diminished the time the capillary tube is open. In conclusion, allergen challenge in asthmatics induced surfactant dysfunction, probably mainly because of inhibiting proteins. During an asthma attack, narrow conducting airways may become blocked, which might contribute to an increased airway resistance.

Breathing and pulmonary surfactant function in mice 24 h after ozone exposure.

The aim of this study was to determine whether an acute ozone exposure affects breathing, and the ability of pulmonary surfactant to maintain the patency of terminal conducting airways. BALB/c mice were exposed to ozone (1 part per million (ppm)) for 2, 4, 6, and 8 h. They were examined with plethysmography and with bronchoalveolar lavage (BAL) 24 h later. The BAL fluid was analysed for the presence of inflammatory cells and concentrations of proteins and phospholipids. Surfactant in the remaining BAL fluid was concentrated five-times and examined with a capillary surfactometer (CS). The surfactant was then washed with a large volume of saline solution which was removed following centrifugation. Already, after a 2 h ozone exposure, the respiratory frequency increased from 297+/-6 to 386+/-11 breaths x min(-1) (p<0.0001). Pressure amplitude per breath diminished (p<0.001), indicating a reduced tidal volume. A highly significant surfactant dysfunction was observed with the CS (p<0.0001), although phospholipids increased. However, proteins also increased (p<0.0001) and they or other water-soluble inhibitors apparently caused the surfactant dysfunction since, when they were removed with a washing procedure, the surfactant's normal ability to maintain patency was restored. The acute ozone exposure affected breathing and caused an airway inflammation. The inflammatory proteins or other water-soluble inhibitors reduced the surfactant's ability to secure airway patency.

Ozone affects breathing and pulmonary surfactant function in mice.

The effect on breathing of BALB/c mice immediately following ozone exposure (2 ppm) for 0, 2, 4, 6, and 8 h was studied with a whole body plethysmograph. Whether such exposure affected the normal function of pulmonary surfactant of maintaining airway patency was evaluated with a capillary surfactometer. Respiratory rate in mice that were not exposed was 358+/-16 (mean+/-S.E.) breaths/min and decreased to 202+/-10 after 6 h exposure. The mean pressure change caused by breathing diminished significantly, indicating a reduced tidal volume. BAL fluid from controls maintained patency for 88+/-2% of the study time, 120 s, implying a good surfactant function, but the ozone exposure caused the surfactant to lose its capability of maintaining patency (P < 0.0001). This decaying surfactant function of the BAL fluid coincided with an increasing protein concentration in the fluid of exposed animals (1.46+/-0.14 mg/ml in the 8-h group) as compared to controls (0.44+/-0.04 mg/ml, P < 0.0001). It is concluded that leakage of plasma proteins into the airway lumen was probably the main reason for the surfactant dysfunction, which may have contributed to the altered breathing pattern.

Respiratory syncytial virus affects pulmonary function in BALB/c mice.

BALB/c mice inoculated intranasally with respiratory syncytial virus (RSV) were studied in a whole-body plethysmograph to determine if signs of respiratory illness similar to those observed in human infants could be detected. Also, responsiveness to methacholine was assessed. RSV-infected mice showed significantly higher respiratory rates than did controls (409.2 vs. 305.2 breaths/min, P < .0001). Significantly increased airway responsiveness to methacholine was noted, infected mice responding to a 100-fold lower dose than controls (P = .003). Together, these data provide the first objective evidence of respiratory illness in the mouse model of RSV infection, which enhances the value of this model for evaluating effects of vaccines, antivirals, and other drugs acting on respiratory tract disease caused by RSV.

Whole-body plethysmography, does it measure tidal volume of small animals?

A whole-body plethysmograph was used for mice. The increase in pressure caused by each inhalation was equivalent to the increase that could be calculated to result from heating and humidification of the inhaled air. However, comprehending that a drop in temperature and humidity would cause an abrupt pressure decline during exhalation was difficult. Pressure changes in the plethysmograph were also studied with an artificial chest, modeling the respiratory mechanics, but without the "inhaled" air being heated or humidified. The "chest" consisted of a metal bellows oscillated by a stepper motor 25 to 175 times per minute. Hereby air (0.05 to 0.20 mL) moved in and out of the bellows. The air passed through a polyethylene tube, the length of which was proportional to "airway resistance" and varied from 5 to 35 cm. It was found that the pressure oscillation was affected not only by "tidal volume" of the mechanical chest but also by "respiratory rate" and by "airway resistance." We concur with previous investigators that the plethysmograph pressure reflects alveolar pressure and that fluctuations cannot be explained by changes in temperature and humidity. Accordingly, tidal volume can only be qualitatively and not quantitatively assessed.

Surfactant dysfunction develops in BALB/c mice infected with respiratory syncytial virus.

Recent reports suggest an important role for pulmonary surfactant in maintaining the patency of narrow conducting airways. The hypothesis that surfactant dysfunction is an important factor in respiratory syncytial virus (RSV) infection was tested in a mouse model. Mice, inoculated with either a low or a high dose of RSV, were subjected to bronchoalveolar lavage (BAL), and the fluids were analyzed for percentage of inflammatory cells and concentrations of proteins and phospholipids. After concentration of the surfactant by centrifugation, its function was analyzed with a capillary surfactometer. RSV infection resulted in a dose-dependent disruption of surfactant function (p < 0.0001). BAL fluid supernatants were added to calf lung surfactant extract (CLSE) to examine whether surfactant inhibiting agents were present. Indeed, BAL fluid supernatants of RSV-infected mice disrupted the normal function of calf lung surfactant extract in a dose dependent way (p < 0.0001), indicating the presence of inhibitors. Protein concentrations were increased in BAL fluids of RSV-infected mice versus control mice (p < 0.0001), and were inversely related to surfactant function (r = -0.44, p = 0.0004), suggesting an inhibitory effect of proteins. Protein concentration also correlated with the percentage of inflammatory cells (r = 0.51, p = 0.004). Phospholipid concentrations were not affected by the RSV infection. The results of these studies strongly suggest that a disruption of pulmonary surfactant function, most likely due to inhibition from inflammatory proteins, is important for the pathophysiology of RSV infection.

Dysfunction of guinea-pig pulmonary surfactant and type II pneumocytes after repetitive challenge with aerosolized ovalbumin.

BACKGROUND: Asthma symptoms may partially be caused by a surfactant dysfunction. The inflammatory reaction, so characteristic of asthma, involves a protein invasion of airways which harmfully affects the surfactant function. However, mild asthma attacks might also impede the surfactant synthesis in alveolar type II cells. OBJECTIVE: The present study evaluates the hypothesis that type II pneumocyte metabolic function might be disturbed in a model of mild asthma. METHODS: Immunized, as well as not immunized control guinea-pigs, were challenged three times at two-day intervals with 0.04% ovalbumin aerosol. Bronchoalveolar lavage (BAL) was performed one day after the last challenge and the fluid was evaluated for surface activity, and content of phospholipids and proteins. Alveolar type II cells were isolated and their ability to incorporate a 3H labeled surfactant precursor was evaluated. RESULTS: BAL fluid from immunized and challenged animals showed less surface activity (P < 0.01) when compared with BAL fluid from controls, not immunized but challenged. Most likely the reduced surface activity was caused by a 74% increase in the protein concentration (P < 0.05). Isolated type II cells from immunized and challenged animals had 33% less phospholipids than cells from controls (P < 0.05), and phosphatidylcholine synthesis was reduced 35% (P < 0.05). CONCLUSION: These results suggest that the synthesis, intra-cellular storage, and biophysical activity of surfactant are decreased in an intermittent and mild form of asthma.

Surface properties after a simulated PLA2 hydrolysis of pulmonary surfactant's main component, DPPC.

The inflammation, so conspicuous in cases of respiratory distress, pneumonia, and asthma, is associated with an airway invasion of plasma proteins and a release into the airway lumen of phospholipase A2 (PLA2). This enzyme catalyzes hydrolysis of surfactant phospholipids, the most abundant and important of which is dipalmitoylphosphatidylcholine (DPPC). Its hydrolysis yields equimolar proportions of lysophosphatidylcholine and palmitic acid (LPC/PA). Exact quantification of DPPC hydrolysis is complicated. Consequently, it was decided to simulate hydrolysis whereby DPPC (3 mg/ml) was gradually replaced with LPC/PA (3 mg/ml), yielding seven different grades of simulated hydrolysis: 0, 17, 33, 50, 67, 83, and 100%. Surface properties of the seven mixtures were examined with various concentrations of albumin added. The Bubble Surfactometer was used to study the surfactant film that is given time to develop at a spherical air-liquid interface. A Capillary Surfactometer was used to evaluate surface properties required for airway patency. It was found, using both of these instruments that the surface activity improved as the simulated hydrolysis of DPPC to LPC/PA increased toward 100%, where the activity was maximal. With the Bubble Surfactometer, surface activity of LPC/PA, 3 mg/ml, improved as albumin concentration increased, and when it reached 15 mg/ml, surface tension became 0 mN/m after only 2 min. With the Capillary Surfactometer, requiring a much faster film adsorption, albumin had an opposite effect. LPC/PA alone maintained patency 100% of the time studied, while even a minimal addition of albumin inhibited surfactant function.

Increased airway resistance due to surfactant dysfunction can be alleviated with aerosol surfactant.

To investigate the contribution of pulmonary surfactant to a low airflow resistance through narrow conducting airways, a system was developed with which it was possible to determine the resistance meeting a steady flow of air at 0.5 mL/min. The airflow, delivered by an infusion pump, entered the extreme periphery of a conducting airway in an excised rat lung and exited through the trachea. The resistance was determined by measuring the pressure of the air entering the lung. If the airway remained open, the pressure was only slightly above zero; when a blocking liquid column formed in the lumen of the airway, the pressure increased rapidly but dropped abruptly as the liquid was pushed away into a wider airway section. When endogenous pulmonary surfactant was removed with a saline lavage, the airway was blocked almost constantly by an endless re-formation of liquid columns. Consequently, during a 4-min period of pressure recording, free airflow was observed only rarely. However, administration of aerosol surfactant increased the duration of free airflow in relation to the volume administered. After an injection of 80 mL of aerosol surfactant, the airway stayed open 89 +/- 3% of the 4-min recording time compared with only 28 +/- 5% when the same volume of air (80 mL) without surfactant had passed through the airway (p < 0.0001). We conclude that surfactant contributes to a free airflow through conducting airways and may have an important role in the maintenance of low airway resistance.

Pulmonary surfactant given prophylactically alleviates an asthma attack in guinea-pigs.

BACKGROUND: Previous studies have indicated that the increased airway resistance that develops in asthma may partly be due to a surfactant dysfunction. If so, it might be possible to alleviate the acute signs following an allergen challenge by prophylactically instilling into the airways a well functioning pulmonary surfactant. OBJECTIVE: The study was planned and enacted to test the above hypothesis. METHODS: The lung function (airway resistance, tidal volume, minute ventilation, and dynamic compliance) of 22 immunized guinea-pigs was studied for 30 min following a challenge. Ten of the animals had received a tracheal instillation of 0.5 mL calf lung surfactant extract (CLSE, 35 mg/mL) prior to the challenge. RESULTS: The animals receiving the dose of 17.5 mg surfactant were less affected by the challenge than were the controls. Only one of them died following the challenge, whereas four of the 12 controls succumbed. Lung function was significantly less affected among the nine surviving animals treated with surfactant prior to the challenge than among the eight surviving controls (P < 0.01) and also their blood gases (pCO2 and pO2) were less influenced (P < 0.05). CONCLUSION: The study indicated that the symptoms developing after a challenge, which to some extent simulate those of asthma, can be alleviated by a prophylactic airway instillation of pulmonary surfactant.

Surfactant dysfunction develops when the immunized guinea-pig is challenged with ovalbumin aerosol.

BACKGROUND: The cause of the airway resistance developing during an asthma attack is not completely understood. Besides bronchospasm and airway oedema a surfactant dysfunction has been suggested as a reason for an increased airway resistance. OBJECTIVE: This paper aims at examining if indeed surfactant dysfunction develops when an asthma attack is induced in guinea-pigs. METHODS: Guinea-pigs, immunized against ovalbumin and then challenged (by inhaling the antigen) underwent lung function tests (n = 7) and were compared with seven animals challenged, but not immunized. Lung lavage was carried out in three groups of guinea-pigs: controls, never immunized nor challenged (n = 7), not immunized but challenged (n = 6), immunized and challenged, no lung function test (n = 6). After concentrating the lavage fluid 10 times the surface activity was evaluated with the pulsating bubble surfactometer. The fluid's concentration of phospholipids and proteins was determined as was the phospholipid composition. RESULTS: The 19 immunized and challenged animals all developed severe respiratory distress, six so seriously that they died. Lung function tests showed significantly increased airway resistance and decreased tidal volume, minute volume, and dynamic compliance. Surface activity of lavage fluid from immunized and challenged animals was significantly reduced when compared with fluid from control animals (P < 0.01). Immunization and challenge had no effect on the lavage fluid's phospholipid concentration or composition, but the proteins were at a higher concentration than in the fluid of the controls (P < 0.01). CONCLUSION: Proteins leaking into the airways inhibited the surfactant. This, in turn might have caused conducting airways to become blocked by liquid columns, which would increase airway resistance.

Pulmonary surfactant maintains patency of conducting airways in the rat.

The hypothesis was tested that after extrusion of the liquid columns that often block the lumen of conducting airways, the latter will remain open because of well-functioning pulmonary surfactant preventing the liquid columns from returning. The extirpated lungs of 22 Wistar rats were studied. Via a tracheal tube a very fine catheter (PE 10) was inserted and advanced until it pierced the pleura. It was extracted until only 2 mm remained in the lung parenchyma. A pressure transducer measured the resistance that met a steady flow of air through the series of tubes: the PE 10 tube, the conducting airway of the lung, and the tracheal tube. The airway resistance was studied for 240 s after three airway flushings, two with saline solution and one with calf lung surfactant extract (CLSE), 3 mg/ml. The pressure recording showed that a low pressure, indicating airway patency, occurred for only 31 +/- 8 s (mean +/- SEM) after the first saline flush, and for 26 +/- 8 s after the second. After the CLSE flush the airway remained open for 174 +/- 12 s, which indicated a significantly reduced resistance (p < 0.0001). The results imply that well-functioning pulmonary surfactant is required for a low airway resistance.