Pathology of the surfactant system of the mature lung: second San Diego conference.

Knowledge of the surfactant system has grown immensely in the past decade. A variety of investigative strategies, including manipulation of surfactant protein gene expression in mice, has contributed dramatically to our understanding of the role of surfactant components in lung function. These approaches have fostered investigations that will further our knowledge of the role of lung surfactant in host defense and will provide information that should lead to improved strategies for the treatment of lung disease.

Surfactant replacement therapy.

Dysfunction of the surfactant system of the lung in the setting of acute lung injury (ALI) is likely to contribute to the pathophysiology of that syndrome. Multiple mechanisms, including injury to alveolar type II cells and inhibition by plasma proteins contribute to this loss of function. Similar injury occurs in animal models of acute lung injury and, in that setting, treatment with exogenous surfactant causes marked improvement in gas exchange. Clinical studies of surfactant treatment of ALI suggest benefit, and definitive phase III trials are now in progress.

Reperfusion lung injury after unilateral pulmonary artery occlusion.

OBJECTIVE: To test the hypothesis that reperfusion of the canine lung after 1 week of vascular occlusion results in acute injury of the reperfused lung with concurrent impairment in gas exchange. METHODOLOGY: In 11 conditioned dogs, the left pulmonary artery was completely occluded by a vascular clip placed at thoracotomy. One week later, at repeat thoracotomy, the clip was removed in six animals (reperfused group) but left in place in five (sham group). Bronchoalveolar lavage fluid (BAL) components, gas exchange, haemodynamics and histological alterations were examined. RESULTS: During occlusion, the mean pulmonary artery pressure and pulmonary vascular resistance increased significantly, and after 6 days there was a significant increase in ventilation to high ventilation perfusion ratio (V/Q) areas. With reperfusion, the previously occluded lung demonstrated, in comparison to the contralateral lung, a significant increase in BAL cellularity and neutrophil fraction, gross and histological evidence of oedema, and impaired surfactant activity. Shunt fraction, measured by the inert gas technique, also increased only after reperfusion, although mild hypoxaemia occurred in both groups. Endothelial abnormalities and perivascular oedema were noted in both groups, but were more marked in the reperfused lungs. CONCLUSION: Reperfusion of the canine lung after 1 week of complete occlusion resulted in evidence of mild acute lung injury. The aetiology of this injury was multifactorial.

Effect of recombinant SP-C surfactant in a porcine lavage model of acute lung injury.

Synthetic surfactants allow examination of the effects of specific components of natural surfactant. To determine whether surfactant containing apoprotein C, dipalmitoyl-phosphatidylcholine, phosphatidylglycerol, and palmitic acid restores gas-exchanging function in acute lung injury (ALI), we administered such surfactant (in doses of 50 or 100 mg/kg and in volumes from 1 to 6 ml/kg) or phospholipid (PL) alone, by intratracheal instillation, to pigs with ALI induced by massive saline lavage. Animals ventilated with 100% O(2) and receiving 1, 2, 4, or 6 ml/kg of 50 mg/kg recombinant surfactant apoprotein C (rSP-C) surfactant or 2 ml/kg of 50 mg/kg PL (control) had mean arterial PO(2) values, 4 h after treatment, of 230, 332, 130, 142, or 86 Torr, respectively. Animals receiving 1, 2, or 4 ml/kg of 100 mg/kg rSP-C surfactant or 2 ml/kg of 100 mg/kg PL (control) had mean arterial PO(2) values of 197, 214, 148, or 88 Torr, respectively. Surfactant PL distribution was homogeneous. Hyaline membrane formation was reduced in treated animals. Thus, in this model of ALI, rSP-C with PL has the capacity to improve gas exchange and possibly modify lung injury.

Effect of phosphocholine cytidylyltransferase overexpression on phosphatidylcholine synthesis in alveolar type II cells and related cell lines.

Disaturated phosphatidylcholine (DSPC) is the predominate phospholipid component of lung surfactant. In the alveolar type II cell, the cytidine diphosphocholine (CDP-choline) pathway is the major biosynthetic pathway for DSPC. To investigate the hypothesis that phosphocholine cytidylyltransferase (CT) is the rate-limiting enzyme in the CDP-choline pathway, rat alveolar type II cells or lung tumor-derived cell lines (A549 or H441) with type II cell features were transfected with CT complementary DNA (cDNA). Cell fractions were subsequently assayed for CT protein and activity, and cell rates of DSPC synthesis were determined. In all cases, cell CT protein and activity were increased after transfection with CT cDNA but not after control transfection. Rat type II cells, but not A549 or H441 cells, increased the rate of DSPC synthesis after transfection with CT cDNA. Exposure of type II cells transfected with CT cDNA to palmitic acid resulted in a further increase in CT protein and activity. Exposure to dexamethasone resulted in increased CT protein and activity and increased synthesis of DSPC. The results confirm that CT has a rate-limiting and regulatory role in the synthesis of type II cell DSPC, and raise possibilities for novel therapeutic interventions.

Analyzing the time and value of housestaff inpatient work.

OBJECTIVE: To determine time allocation and the perceived value to education and patient care of the weekday activities of internal medicine housestaff on inpatient rotations and to compare the work activities of interns and residents. DESIGN: An observational study. We classified activities along five dimensions (association, location, activity, time, and value), developed a computer-assisted self-interview survey, and demonstrated its face and content validity, internal consistency, and interrater reliability. Subjects were assigned survey computers for 5 consecutive weekdays over a 24-week period, into which they entered data when prompted several times a day. SETTING: The medical service of a university-affiliated Veterans Administration Medical Center. PARTICIPANTS: Sixty housestaff (36 interns, 24 residents) rotating on the inpatient wards. MEASUREMENTS AND MAIN RESULTS: We analyzed activities according to content (direct patient care, indirect patient care, education), association, and location. Likert-scale ratings of perceived value to education and patient care were also obtained. Housestaff provided complete responses to 3,812 (95%) of 3,992 prompts by a median of 11 seconds; 93% of responses were logically consistent across the measured dimensions. Housestaff spent more time in indirect patient care (56%) than in direct patient care (14%) or educational activities (45%). Formal educational activities had the highest educational value (66 on 0-100 scale), and direct care had the highest value to patient care (81). Over 30% of time was spent in administrative activities, which had low educational value(40). Compared with residents, interns allocated significantly less time to educational activities (38% vs 57%) and more time to lower-value activities such as documentation (19% vs 12%). CONCLUSIONS: Improved data collection methods demonstrate that housestaff in our program, particularly interns, spend much of their workday in activities that are low in educational and patient care value. Selective elimination or delegation of such activities would preserve higher-value experiences during reductions in overall inpatient training time. Planners can use automated random sampling to guide the rational redesign of housestaff work.

Sustained submaximal exercise does not alter the integrity of the lung blood-gas barrier in elite athletes.

The extreme thinness of the pulmonary blood-gas barrier results in high mechanical stresses in the capillary wall when the capillary pressure rises during exercise. We have previously shown that, in elite cyclists, 6-8 min of maximal exercise increase blood-gas barrier permeability and result in higher concentrations of red blood cells, total protein, and leukotriene B4 in bronchoalveolar lavage (BAL) fluid compared with results in sedentary controls. To test the hypothesis that stress failure of the barrier only occurs at the highest level of exercise, we performed BAL in six healthy athletes after 1 h of exercise at 77% of maximal O2 consumption. Controls were eight normal nonathletes who did not exercise before BAL. In contrast with our previous study, we did not find higher concentrations of red blood cells, total protein, and leukotriene B4 in the exercising athletes compared with control subjects. However, higher concentrations of surfactant apoprotein A and a higher surfactant apoprotein A-to-phospholipid ratio were observed in the athletes performing prolonged exercise, compared with both the controls and the athletes from our previous study. These results suggest that, in elite athletes, the integrity of the blood-gas barrier is altered only at extreme levels of exercise.

Intense exercise impairs the integrity of the pulmonary blood-gas barrier in elite athletes.

The blood-gas barrier must be very thin to allow gas exchange and it is therefore subjected to high mechanical stresses when the capillary pressure rises. In some animals, such as the thoroughbred race-horse during intense exercise, the stresses are so large that the capillaries fail and bleeding occurs. We tested the hypothesis that, in elite human athletes, the high capillary pressure that occurs during severe exercise alters the structure and function of the blood-gas barrier. We performed bronchoalveolar lavage (BAL) in six healthy athletes, who had a history suggestive of lung bleeding, 1 h after a 7-min cycling race simulation and four normal sedentary control subjects who did not exercise before BAL. The athletes had higher (p < 0.05) concentrations of red blood cells (0.51 x 10(5) versus 0.01 x 10(5).ml-1), total protein (128.0 versus 94.1 micrograms/ml), albumin (65.6 versus 53.0 micrograms/ml), and leukotriene B4 (LTB4) (243 versus 0 pg/ml) in BAL fluid than control subjects. The proportion of neutrophils was similar in athletes and control subjects but the proportion of lymphocytes in BAL fluid was reduced (p < 0.05). There were no differences in levels of surfactant apoprotein A, tumor necrosis factor bioactivity, lipopolysaccharide, or interleukin-8 (IL-8) between groups. These results show that brief intense exercise in athletes with a history suggestive of lung bleeding alters blood-gas barrier function resulting in higher concentrations of red cells and protein in BAL fluid. The lack of activation of proinflammatory pathways (except LTB4) in the airspaces supports the hypothesis that the mechanism for altered blood-gas barrier function is mechanical stress.

Human SP-A locus: allele frequencies and linkage disequilibrium between the two surfactant protein A genes.

Two surfactant protein A (SP-A) genes and several alleles for each SP-A locus have been previously described. In this report we investigate the potential usefulness of the SP-A loci as markers for genetic studies. We establish conditions that allow the identification of alleles with very similar sequences; We also determine the degree of polymorphism for each SP-A locus: The heterozygosity and polymorphism information content (PIC) values for the SP-A1 locus are 0.63 and 0.55, respectively, and for the SP-A2 locus are 0.50 and 0.56. In the course of these studies, we identify one new SP-A2 allele and show that the SP-A1 and SP-A2 loci are in linkage disequilibrium (P < 0.000001). We also identify 19 of the 20 possible haplotypes in a population of n = 239. Nine of the observed haplotypes reach statistical significance (P < 0.01) in this population, and the segregation of two haplotypes (6A2/1A0 and 6A4/1A) without recombination is verified in a family pedigree. These data together indicate that both SP-A loci are sufficiently polymorphic to be good markers for use in genetic studies. Furthermore, the finding of one novel allele suggests that additional unknown SP-A alleles are yet to be found.

Inhibition of the human neutrophil respiratory burst by native and synthetic surfactant.

Production of oxygen radicals by phagocytic cells and loss of surfactant function have each been implicated in the pathogenesis of acute lung injury. Therapeutic administration of exogenous surfactant to injured lungs in which neutrophils are the dominant cell type has been proposed. To understand the role of surfactant in modulating pulmonary inflammation and the impact of surfactant supplementation on diseased lungs, we studied the effect of native porcine and synthetic surfactant preparations on human neutrophil respiratory burst oxidase activity in vitro. We found that surfactant inhibited neutrophil superoxide production induced by either receptor-mediated [formylmethionylleucylphenylalanine (fMLP)] or non-receptor-mediated [phorbol myristate acetate (PMA)] agonists with an IC50 of approximately 0.015 mg phospholipid/ml for porcine surfactant or approximately 0.050 mg phospholipid/ml for synthetic surfactant. Surfactant had no effect on detection of superoxide generation in a noncellular system using xanthine and xanthine oxidase and only minimally inhibited superoxide generation by neutrophils that had been fully stimulated by prior exposure to PMA. There was no effect of surfactant on neutrophil calcium mobilization in response to fMLP, on lactoferrin release in response to PMA, or on membrane protein kinase C activity in response to PMA. Suspensions of dipalmitylphosphatidylcholine alone had no effect on neutrophil superoxide production. Taken together, these findings indicate that certain components of lung surfactant may effect relatively late steps in the activation of the respiratory burst or may alter subsequent steps involved in the assembly of the respiratory burst oxidase.

Effect of N-nitroso-N-methylurethane on gas exchange, lung compliance, and surfactant function of rabbits.

OBJECTIVES: To define the effect of N-nitroso-N-methyl-urethane (NNNMU) on pulmonary gas exchange, compliance and the biochemical and functional properties of the lung surfactant system. DESIGN: Four days after inducing lung injury, gas exchange and pulmonary compliance were studied and a bronchoalveolar lavage was taken. SETTING: Experimental laboratory of a university department of medicine, division of pulmonary and critical care medicine. ANIMALS: Ten rabbits after they had received an injection of NNNMU and five control animals. INTERVENTIONS: Controlled mechanical ventilation and bronchoalveolar lavage. MEASUREMENTS AND RESULTS: Measurements of gas exchange (using the multiple inert gas elimination technique), hemodynamics and pulmonary compliance were performed during ventilatory and hemodynamic steady state. A bronchoalveolar lavage (BAL) was taken after sacrificing the animal. BAL samples were processed for cell count and biochemical and functional surfactant analysis. Animals injected with NNNMU developed mild, but significant reduction in PaO2, while maintaining eucapnia during spontaneous air breathing. V/Q distributions and arterial blood gases were similar in all animals when ventilated mechanically with a fixed tidal volume. Compliance of the lung and phospholipid levels in lavage of NNNMU animals was significantly lower than in control animals (CON). Function of surfactant recovered from animals receiving NNNMU was decreased significantly where compared to CON. Thus, NNNMU resulted in a lowered lavage surfactant phospholipid content, impaired surfactant function, decreased compliance and hypoxemia during spontaneous ventilation. However, gas exchange was similar to that of control animals during mechanical ventilation. CONCLUSION: We conclude that NNNMU-induced gas exchange abnormalities present after 4 days are mild and are reversed by fixed volume mechanical ventilation despite marked alteration in surfactant function and lung compliance. These observations further define properties of a lung injury model that is of value in the study of surfactant replacement.

Parathyroid hormone-related protein, an autocrine regulatory factor in alveolar epithelial cells.

Alveolar epithelial cells in vivo, primary cultures of adult rat type II cells, and human A549 alveolar carcinoma cells express parathyroid hormone-related protein (PTHrP). Here we demonstrated that type II cells and A549 cells also express the PTHrP receptor and that they exhibit differentiation-related responses to the amino-terminal PTHrP fragment, PTHrP-(1-34). PTHrP receptor expression in A549 cells was shown by detection of a 0.3-kb reverse transcriptase polymerase chain reaction product formed by primers specific for PTHrP receptor. In situ hybridization studies localized the site of production of PTHrP and PTHrP receptor mRNA in rat lung cells with morphology and location typical of type II cells. Primary cultures of such type II cells also expressed PTHrP receptor mRNA. Incubation with PTHrP-(1-34) stimulated disaturated phosphatidylcholine (DSPC) synthesis in A549 cells and increased the release of newly synthesized DSPC by cultured type II cells and A549 cells. In addition, PTHrP-(1-34) increased the number of lamellar bodies per type II cell and increased their expression of alkaline phosphatase in a dose-dependent manner. Thus PTHrP-(1-34) promoted a differentiated type II cell phenotype. Since cultured type II cells, alveolar epithelial cells in vivo, and A549 cells express PTHrP and the PTHrP receptor, PTHrP-(1-34) may be an autocrine regulatory factor in type II cells and lung cancer cells.

Inhibitory effect of porcine surfactant on the respiratory burst oxidase in human neutrophils. Attenuation of p47phox and p67phox membrane translocation as the mechanism.

Surfactant has been shown to inhibit the production of reactive oxygen intermediates by various cells including alveolar macrophages and peripheral blood neutrophils. Superoxide O2-. production by the respiratory burst oxidase in isolated plasma membranes prepared from PMA-treated human neutrophils was significantly attenuated by prior treatment with native porcine surfactant. The effect was concentration dependent with half-maximal inhibition seen at approximately 0.050 mg surfactant phospholipid/ml. Kinetic analyses of the membrane-bound enzyme prepared from neutrophils stimulated by PMA in the presence or absence of surfactant demonstrated that surfactant treatment led to a decrease in the maximal velocity of O2-. production when NADPH was used as substrate, but there was no effect on enzyme substrate affinity. Immunoblotting studies demonstrated that surfactant treatment induced a decrease in the association of two oxidase components, p47phox and p67phox, with the isolated plasma membrane. In contrast, surfactant treatment of the cells did not alter the phosphorylation of p47phox. A mixture of phospholipids (phosphatidylcholine and phosphatidylglycerol in a 7:3 ratio) showed similar inhibition of the PMA-induced O2-. generation. Taken together, these data suggest the mechanism of surfactant-induced inhibition of O2-. production by human neutrophils involves attenuation of translocation of cytosolic components of the respiratory burst oxidase to the plasma membrane. The phospholipid components of surfactant appear to play a significant role in this mechanism.

Fractal analysis of surfactant deposition in rabbit lungs.

The effect of exogenous surfactant in the treatment of acute lung injury may depend on homogeneity of distribution of the material delivered. Analyses of distribution rely on sectioning the lung, determining surfactant concentration for each piece, and describing the variation in that value. Results of such analyses are influenced by how finely the lung is sectioned. We have reanalyzed data from prior experiments to determine whether the distribution of administered surfactant is fractal, that is, is independent of the scale of measurement. Lungs from animals receiving surfactant radiolabeled with [3H]dipalmitoylphosphatidylcholine were cut into 108 pieces, and the normalized radioactivity in each piece was determined. Sectioning of the lungs into different numbers of pieces (n = 2, 6, 12, 18, 36, 54, or 108) was simulated, and corresponding radioactivity contents were calculated. The coefficient of variation (CV) of these normalized values was then calculated for each scale of measurement (expressed as relative piece volume), and ln(CV) was plotted as a function of the logarithm of relative piece volume. These relationships were linear (average correlation coefficient = 0.96) for all animals, consistent with CV being a fractal property. We conclude that the intrapulmonary distribution of surfactant may be fractal and is therefore a property of the lung. This study demonstrates the utility of fractal analysis in describing the pulmonary distribution of substances introduced via the airway.

Exposure of the hydrophobic components of porcine lung surfactant to oxidant stress alters surface tension properties.

We have tested the hypothesis that oxidation of lung surfactant results in loss of surface tension lowering function. Porcine lung surfactant was exposed to conditions known to cause lipid peroxidation (0.2 mM FeCl2 + 0.1 mM H2O2 or 5 microM CuCl2). Lipid peroxidation was verified by detection of conjugated dienes, thiobarbituric acid reactive substances, fluorescent products, hydroxy alkenals, and loss of unsaturated fatty acids. Exposed samples had significantly diminished surface tension lowering ability in vitro as measured in a bubble surfactometer. Samples exposed to FeCl2 + H2O2 had significantly diminished surface tension lowering ability in vivo as indicated by their reduced ability to improve lung compliance of surfactant-deficient fetal rabbits. Oxidation of phospholipid mixtures with surface tension lowering activity and containing unsaturated acyl groups resulted in partial loss of activity as determined in vitro. These results suggest that the effect of oxidants on lung surfactant function is due, in part, to effects on the phospholipid components and that acute pulmonary inflammation accompanied by oxygen radical production may result in surfactant lipid peroxidation and loss of surface tension lowering function.

Acute effects of a single dose of porcine surfactant on patients with the adult respiratory distress syndrome.

In an attempt to restore functional surfactant to the lungs of patients with the adult respiratory distress syndrome (ARDS), we have treated six patients within the first 2 days of the onset of ARDS with a single dose of hydrophobic components of porcine surfactant. Surfactant (4 g in 50 ml) delivered via a bronchoscope in aliquots to each of the lobar bronchi was well tolerated and caused a modest transient improvement in gas exchange. No significant changes in chest radiograph or lung compliance were detected. Analysis of bronchoalveolar lavage (BAL) fluid showed no change in albumin, alpha-1-proteinase inhibitor specific activity, or cell count. Bronchoalveolar lavage phospholipid concentrations were elevated 3 h after surfactant administration relative to preadministration levels and fell by 24 h. In addition, in two patients we found reduced inhibition of surfactant function in BAL after surfactant replacement. These observations suggest a role for surfactant replacement in the treatment of patients with ARDS and support the need for continuing investigation.

H2O2 increases expression of pulmonary artery endothelial cell platelet-derived growth factor mRNA.

Endothelial cells subjected to cell injury are capable of producing platelet-derived growth factor (PDGF), a mitogen for the stimulation of fibroblast and smooth muscle cell proliferation. Cultured bovine pulmonary artery endothelial cells were exposed to low concentrations of H2O2 for 30 min. Total cell RNA was isolated and subjected to Northern analysis with use of a v-sis PDGF cDNA probe. Results demonstrate a fourfold increase in cell PDGF mRNA immediately after exposure of bovine pulmonary artery endothelial cells to 50 microM H2O2. Evidence of expression of PDGF was sought in samples of cell supernatant collected 48 h after exposure. No evidence of PDGF activity or PDGF antigen could be demonstrated in those supernatants. Although the biologic activities of PDGF suggest that PDGF production by endothelial cells may contribute to the pulmonary pathology associated with acute lung injury, our results suggest that posttranscriptional events may prevent expression of PDGF under the experimental conditions of this investigation.