Effects of alveolar surfactant aggregates on T-lymphocyte proliferation

The effects of alveolar large aggregate (LA) and small aggregate (SA) surfactant subfractions isolated from healthy adult rats on mitogen-stimulated proliferative responses of human peripheral blood mononuclear cells (PBMC) was examined. Various concentrations of total surfactant suppressed proliferation of stimulated lymphocytes by up to 95% of mitogen-stimulated cells alone. LA subfractions of total surfactant had no effect on proliferation, whereas SA significantly enhanced the lymphocyte proliferation at lower concentrations (7.8 microg/ml) compared to mitogen-stimulated cells alone. Higher concentrations of SA (62.5 microg/ml) inhibited lymphocyte proliferation. This concentration-dependent effect of SA on proliferation of PBMC was also present when cells were stimulated with various lectins including anti-CD3, concanavalin A and phytohemagglutinin. Analysis of the supernatant of mitogen-stimulated cell cultures treated with inhibitory concentrations of SA showed decreased amounts of interleukin (IL)-2, compared to cells alone, which could be reversed by adding exogenous IL-2 to the cell cultures with the SA. These results suggest that alveolar surfactant subfractions have distinct functions within the alveoli, both biophysically and with respect to their effects on the host's immunomodulatory responses.     

Step response of lung surface-to-volume ratio by light-scattering stereology

By means of backscattered light from a pointlike source on the pleural surface, we investigated the dynamic behavior of the surface-to-volume ratio (S/V) in excised dog lobes subjected to small volume steps both in and out on both the inflation and deflation limb of standard pressure-volume maneuvers. The technique utilizes the established correlation of the pattern of backscattered light with morphometric mean linear intercept and is suitable for dynamic studies. We hypothesized that 1) there would be a difference in the timing of stress relaxation or recovery between alveolar septa and the fibromuscular tissue in the alveolar duct that would reveal itself as a temporally changing S/V after a step-volume change and 2) that geometric hysteresis (looping of S/V with volume), as seen with large volume excursion histories, would be similarly present in small tidal volume loops. Our experimental results contradicted both hypotheses. In particular, we found virtually no change in S/V after a step-volume change, even in the presence of substantial stress adaptation. In addition, when geometric hysteresis of small loops was present, it was always in the sense opposite to the geometric hysteresis of large loops. We conclude that 1) there is a functional "matching" of the stress-adaptive timing between alveolar septa and ductal mouths and 2) during small volume looping, the stress hysteresis (looping of stress with volume) in the ductal tissue may be larger than that of the septa, including surface tension.     

Effects of alveolar pressure on lung angiotensin-converting enzyme function in vivo

Angiotensin-converting enzyme lines the luminal surface of pulmonary capillary endothelial cells. The metabolism of its synthetic substrate, 3H-benzoyl-L-phenylalanyl-L-alanyl-L-proline ([3H]BPAP) has been used as an indicator of pulmonary microvascular function. Because the flow-volume status of the pulmonary capillaries is dependent on intra-alveolar pressure, we have studied the effects of airway pressure on endothelial plasmalemmal angiotensin-converting enzyme function in rabbit lungs in vivo. Static inflation of the lungs to a pressure of 0 or 5 Torr did not change percent transpulmonary metabolism and Amax/Km ratio (defined as E X Kcat/Km and thus, under normal conditions, an indirect measure of perfused endothelial luminal surface area) compared with control measurements during conventional mechanical ventilation. When the inflation pressure was increased to 10 Torr, percent metabolism of [3H]BPAP remained unaltered but Amax/Km decreased to 60% of the control value. This decrease was in close relation to the decrease in pulmonary blood flow. Addition of 5 cmH2O positive end-expiratory pressure (PEEP) to the mechanical ventilation also decreased Amax/Km values and pulmonary blood flow but did not influence percent metabolism of [3H]BPAP. These results suggest that the detected alterations in apparent enzyme kinetics were more likely due to hemodynamic changes than to alterations in angiotensin-converting enzyme function. Thus high static alveolar pressures as well as PEEP probably reduced the fraction of perfused microvessels as reflected in changes in Amax/Km ratios. This information should prove useful in interpreting the response of pulmonary endothelial enzymes to injury.     

Effects of pulmonary surfactant and surfactant protein A on phagocytosis of fractionated alveolar macrophages: relationship to starvation.

Pulmonary surfactant isolated from bronchoalveolar lavage fluid of rat lung contained a high content of surfactant protein A (SP-A) in starved for 2 days compared to fed controls, but this phenomena returned to baseline following more than 4 days starvation. As determined by immunoperoxidase staining of lung sections using SP-A antibody, SP-A could be consistently observed in nonciliated bronchiolar (Clara) cells, alveolar type II cells and some alveolar macrophages (AM). Fc receptor-mediated phagocytosis of AM was enhanced by SP-A, which was dependent on the dosis and reached a maximum at 10 micrograms of SP-A/ml. Antibody to SP-A completely inhibited the enhanced response of phagocytosis. When exposed AM subpopulations, separated into four fractions (I, II, III and IV) by discontinuous Percoll gradient, to SP-A or pulmonary surfactant prepared from rats fed and starved for 2 days enhanced their phagocytic activity in high dense cells (III and IV), particularly to SP-A and pulmonary surfactant from rats starved for 2 days. Whereas little change in lower dense fractions (I and II) were seen in all exposures except for SP-A that enhanced the cells of fraction II. These results supported the concept that pulmonary surfactant and its apoprotein, SP-A, are a factor to regulate lung defense system including activation of AM that undergo different processes following starvation.     

Effects of Perfluorocarbons on surfactant exocytosis and membrane properties in isolated alveolar type II cells

Background

Perfluorocarbons (PFC) are used to improve gas exchange in diseased lungs. PFC have been shown to affect various cell types. Thus, effects on alveolar type II (ATII) cells and surfactant metabolism can be expected, data, however, are controversial.

Objective

The study was performed to test two hypotheses: (I) the effects of PFC on surfactant exocytosis depend on their respective vapor pressures; (II) different pathways of surfactant exocytosis are affected differently by PFC.

Methods

Isolated ATII cells were exposed to two PFC with different vapor pressures and spontaneous surfactant exocytosis was measured. Furthermore, surfactant exocytosis was stimulated by either ATP, PMA or Ionomycin. The effects of PFC on cell morphology, cellular viability, endocytosis, membrane permeability and fluidity were determined.

Results

The spontaneous exocytosis was reduced by PFC, however, the ATP and PMA stimulated exocytosis was slightly increased by PFC with high vapor pressure. In contrast, Ionomycin-induced exocytosis was decreased by PFC with low vapor pressure. Cellular uptake of FM 1-43 - a marker of membrane integrity - was increased. However, membrane fluidity, endocytosis and viability were not affected by PFC incubation.

Conclusions

We conclude that PFC effects can be explained by modest, unspecific interactions with the plasma membrane rather than by specific interactions with intracellular targets.     

Changes in surfactant pools after a physiological increase in alveolar surfactant

We have used previously characterized models to investigate the reuptake of surfactant from the alveolus. In model 1, rats were swum in a water bath at 33 degrees C for 30 min, which increased tidal volume (VT) approximately 300% and frequency 60%; they were then allowed to rest for up to 4 h. In model 2, rats were exposed to 5% CO2-13% O2-82% N2 for 24 h, which increased both VT and frequency approximately 200%; these rats were then rested for up to 24 h. In both models we harvested a tissue fraction (lamellar bodies, lb) and two alveolar fractions--tubular myelin rich (alv-1) and tubular myelin poor (alv-2). Immediately after swimming, lb-dipalmitoylphosphatidylcholine (DPPClb) was 18% below the control of 0.94 +/- 0.037 (SE) mg/g wet lung (n = 24 rats; P less than 0.05); this returned to control by 2 h. Whereas DPPCalv-1 was constant at all time points, DPPCalv-2 was increased 50% above the control of 2.68 +/- 0.085 mg/g dry lung (n = 27 rats; P less than 0.001) immediately and up to 1 h after swimming. It returned to control levels between 2 and 3 h. After gas exposure, DPPC in lb, alv-1, and alv-2 was 33, 64, and 89%, respectively, above controls. All three fractions had normalized after 24 h. Our results demonstrate marked differences in the response of the surfactant system to acute and more prolonged stimuli. Of particular interest was the constancy of alv-1 with swimming, suggesting that it may be the controlled variable. However, the system appeared to be reset by prolonged hyperpnea, a process that may involve an increase in synthesis of surfactant.     

Effect of oxygen and hypergravitation on alveolar surfactant

The effect of prolonged exposure (up to 66 hours) to pure oxygen breathing and to short (5-minute) oxygen breathing combined with acceleration (+5Gz) on the surface tension and surface potential of the alveolar washout of the albino rat lungs was determined. Both experimental conditions produced atelectasis and a decrease of the surfactant surface activity. Possible mechanisms of shifts of the surfactant activity under hyperoxia only, and hyperoxia with accelerations are discussed.     

In vivo and in vitro uptake of surfactant lipids by alveolar type II cells and macrophages

The uptake of fluorescent-labeled liposomes (with a surfactant-like composition) by alveolar macrophages and alveolar type II cells was studied using flow cytometry, in vivo by instillation of the labeled liposomes in the trachea of ventilated rats followed by isolation of the alveolar cells and determination of the cell-associated fluorescence, and in vitro by incubation of isolated alveolar cells with the fluorescent liposomes. The results show that the uptake of liposomes by the alveolar cells is time and concentration dependent. In vivo alveolar macrophages internalize more than three times as many liposomes as alveolar type II cells, whereas in vitro, the amount of internalized liposomes by these cells is approximately the same. In vitro, practically all the cells (70-75%) internalize liposomes, whereas in vivo only 30% of the alveolar type II cells ingest liposomes vs. 70% of the alveolar macrophages. These results indicate that in vivo, only a small subpopulation of alveolar type II cells is able to internalize surfactant liposomes.     

Exogenous surfactant changes the phenotype of alveolar macrophages in mice

Alveolar macrophages are essential for the maintenance of surfactant homeostasis. We asked whether surfactant treatment would change alveolar macrophage number and whether the alveolar macrophage phenotype would become activated or apoptotic when challenged in vivo with exogenous surfactant. Surfactant pool size in mice was increased by repetitive surfactant treatments containing 120 mg/kg (110 micromol/kg) saturated phosphatidylcholine. The number of alveolar macrophages recovered by alveolar lavage decreased after the first dose by 49% and slightly increased after the second and third doses. Up to 28.5% of the macrophages became large and foamy, and their appearance normalized within 12 h. Surfactant treatment did not increase the percent of apoptotic or necrotic cells. The alveolar macrophages were not activated as indicated by no change in expression of CD14, CD16, CD54, CD95, and scavenger receptor class A types I and II after surfactant treatment. Surfactant treatment in healthy mice transiently changed the phenotype of alveolar macrophages to large and foamy without indications of changes in the surface markers characteristic of activation.     

Lipids in the lungs and alveolar surfactant in anaphylactic shock

The process of anaphylactoid response of rats to introduction of egg protein is associated with a decrease of the pulmonary surfactant surface activity. The factors of metabolic surfactant inactivation are as follows: protein accumulation, the disturbance of lipids transport between pulmonary cells and alveolar surface, change in fatty-acidic composition of surfactant phospholipids. The isolation of arachidonic acid from surfactant phospholipids in anaphylactoid shock is an evidence for the participation of the pulmonary surface-active phase in the process of biosynthesis of the lipid mediators in respiratory organs.     

Structure and surface energy of the surfactant layer on the alveolar surface

The surface energy of the alveolar surfactant layer is determined in the scope of a modification of the structural model of Larsson et al. [(1999) J Disp Sci Technol 20:1-12], according to which this layer is built up of a lipid monolayer adsorbed at the hypophase/air interface and supported by a network of lipid bilayers immersed into the hypophase, i.e., the alveolar liquid. Formulae are derived for the dependence of the specific surface energy of the surfactant layer on the distance between the bilayers constituting the layer. It is shown that at equilibrium this energy can have values comparable with or less than 1 mJ/m2 needed for normal functioning of the alveolus during the respiration cycle. The specific surface energy of the surfactant layer with monolayer-bilayer structure can have such low values only if the layer is of optimal thickness and if the specific line energy of the monolayer-bilayer contact lines is negative and that of the bilayer-bilayer contact lines is positive. It is found that in dynamic regime the change in the specific surface energy of the alveolar surfactant layer with bilayer-monolayer structure is in qualitative agreement with that determined experimentally during lung inflation and deflation.     

Protein composition of rabbit alveolar surfactant subfractions

The goal of this investigation was to characterize the proteins in subfractions of alveolar surfactant obtained by lung lavage and separated by differential centrifugation. It was previously demonstrated that the material in the more sedimentable fraction, which was enriched in tubular-myelin and was surface-active may be a precursor to the less sedimentable, vesicular, inactive material [1]. Separation of the proteins by polyacrylamide gel electrophoresis showed that the more sedimentable subfractions and rabbit surfactant isolated by conventional methods contained proteins with molecular weights comparable to those previously reported for alveolar surface active material (approximately 36 000 and 10 000). The less sedimentable subfractions contained less of these proteins. Immunoblots with anti-dog surfactant apoprotein antibodies, which cross-react with rabbit proteins, supported these observations. Immunoblots also showed that all of the subfractions contained serum proteins and secretory IgA, with the less sedimentable subfractions containing more secretory IgA. These results suggested that changes in protein composition may accompany functional changes in surfactant in the alveoli.     

Characteristics of the antioxidant system of alveolar surfactant in immediate-type allergies

The antioxidant system (glutathione peroxidase, glutathione reductase, superoxide dismutase, total antioxidant activity) of the lung surfactant has been studied for and intensity of peroxidation in that surfactant after administration of sensitizing and resolving doses of the allergen to animals. An increase in the amount of lipid peroxidation products as well as in activity of superoxide dismutase followed by a fall of gamma-glutamyl transpeptidase activity was observed in the lung surfactant 3 and 12 days after introduction of a sensitizing dose of the allergen. Intensification of 5-lipoxygenase activity and accumulation of malonic dialdehyde in the lung surfactant under the anaphylactic shock were accompanied by inhibition of activity of the glutathione-dependent antioxidant system glutathione reductase and glutathione peroxidase) as well as by a fall of antioxidative activity of the surfactant. The data obtained have evidenced for a imbalance between the induction and metabolism systems of lipid hydroperoxides in the respiratory organs under immediate allergies.     

Regulation of surfactant secretion in alveolar type II cells

Molecular mechanisms of surfactant delivery to the air/liquid interface in the lung, which is crucial to lower the surface tension, have been studied for more than two decades. Lung surfactant is synthesized in the alveolar type II cells. Its delivery to the cell surface is preceded by surfactant component synthesis, packaging into specialized organelles termed lamellar bodies, delivery to the apical plasma membrane and fusion. Secreted surfactant undergoes reuptake, intracellular processing, and finally resecretion of recycled material. This review focuses on the mechanisms of delivery of surfactant components to and their secretion from lamellar bodies. Lamellar bodies-independent secretion is also considered. Signal transduction pathways involved in regulation of these processes are discussed as well as disorders associated with their malfunction.