Surfactant therapy for acute lung injury and acute respiratory distress syndrome.

This article examines exogenous lung surfactant replacement therapy and its usefulness in mitigating clinical acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). Surfactant therapy is beneficial in term infants with pneumonia and meconium aspiration lung injury, and in children up to age 21 years with direct pulmonary forms of ALI/ARDS. However, extension of exogenous surfactant therapy to adults with respiratory failure and clinical ALI/ARDS remains a challenge. This article reviews clinical studies of surfactant therapy in pediatric and adult patients with ALI/ARDS, focusing on its potential advantages in patients with direct pulmonary forms of these syndromes.

Lung inflammation induced by endotoxin is enhanced in rats depleted of alveolar macrophages with aerosolized clodronate.

Clodronate liposomes were given to rats via intratracheal inhalation to investigate the importance of alveolar macrophages (AMs) in inhaled endotoxin-induced lung injury. When AM depletion was maximal (87% to 90%), rats were exposed to lipopolysaccharide (LPS) or saline. Neither clodronate nor saline liposomes induced an influx of neutrophils (PMNs) into the lungs. However, depleted LPS-exposed rats had 5- to 8-fold higher numbers of lavage PMNs and greater lavage cell reactive oxygen species release compared to undepleted rats. Although AM depletion by itself did not significantly increase inflammatory cytokine expression in lung tissue, LPS-induced message levels for interleukin (IL)-1alpha, IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha were approximately 2-fold higher in AM-depleted rats compared to undepleted rats. These results indicate that cells other than AMs can recruit inflammatory cells into the lungs during acute LPS-induced injury and that AMs play an important suppressive role in the innate pulmonary inflammatory response.

Efficient depletion of alveolar macrophages using intratracheally inhaled aerosols of liposome-encapsulated clodronate.

Rat alveolar macrophages (AMs) were depleted via intratracheal inhalation (ITIH) of clodronate-containing liposomes. AM depletion following ITIH delivery of clodronate liposomes was 33.2 +/- 14.2 on day 1, 88.1 +/- 6.2 on day 3, and 91.4 +/- 1.8 on day 4 relative to control rats given saline-containing liposomes. Almost all (approximately 99%) of the AMs remaining at the 3-day time point were peroxidase negative, suggesting that immature macrophages were not recruited from the circulation to replace those undergoing cell death on that day. Only 0.5% +/- 0.5% of bronchoalveolar lavage (BAL) cells were neutrophils at this time (normalized to controls). Whole-body inhalation did not induce as much AM depletion at 3 days (37.6% +/- 10.1%) and required larger amounts of liposome-encapsulated clodronate compared to ITIH. Intratracheal instillation (as opposed to inhalation) of clodronate liposomes produced a significant inflammatory response characterized by the influx of both polymorphonuclear neutrophils (PMNs) and macrophages. In subsequent pilot studies, the response to intratracheally instilled crystalline silica (75 microg) was found to be markedly reduced in rats depleted of AMs by the ITIH method. We conclude that ITIH of clodronate liposomes in rats is both efficient and useful for examining the role of AMs in pulmonary toxicology.

Surface activity of a synthetic lung surfactant containing a phospholipase-resistant phosphonolipid analog of dipalmitoyl phosphatidylcholine.

Surface activity and sensitivity to inhibition from phospholipase A2 (PLA2), lysophosphatidylcholine (LPC), and serum albumin were studied for a synthetic C16:0 diether phosphonolipid (DEPN-8) combined with 1.5% by weight of mixed hydrophobic surfactant proteins (SP)-B/C purified from calf lung surfactant extract (CLSE). Pure DEPN-8 had better adsorption and film respreading than the major lung surfactant phospholipid dipalmitoyl phosphatidylcholine and reached minimum surface tensions <1 mN/m under dynamic compression on the Wilhelmy balance and on a pulsating bubble surfactometer (37 degrees C, 20 cycles/min, 50% area compression). DEPN-8 + 1.5% SP-B/C exhibited even greater adsorption and had overall dynamic surface tension lowering equal to CLSE on the bubble. In addition, films of DEPN-8 + 1.5% SP-B/C on the Wilhelmy balance had better respreading than CLSE after seven (but not two) cycles of compression-expansion at 23 degrees C. DEPN-8 is structurally resistant to degradation by PLA2, and DEPN-8 + 1.5% SP-B/C maintained high adsorption and dynamic surface activity in the presence of this enzyme. Incubation of CLSE with PLA2 led to chemical degradation, generation of LPC, and reduced surface activity. DEPN-8 + 1.5% SP-B/C was also more resistant than CLSE to direct biophysical inhibition by LPC, and the two were similar in their sensitivity to biophysical inhibition by serum albumin. These findings indicate that synthetic surfactants containing DEPN-8 combined with surfactant proteins or related synthetic peptides have potential utility for treating surfactant dysfunction in inflammatory lung injury.

Content-dependent activity of lung surfactant protein B in mixtures with lipids.

The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B approximately SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels < or =0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions <1 mN/m in pulsating bubble studies at low phospholipid concentration (1 mg/ml). Mixtures of PPL or SL with SP-B (0.5%) also had minimum surface tensions <1 mN/m at 1 mg/ml, whereas DPPC/SP-B (0.5%) reached <1 mN/m at 2.5 mg/ml. Physiological activity also was strongly dependent on SP-B content. The ability of instilled SL/SP-B mixtures to improve surfactant-deficient pressure-volume mechanics in excised lavaged rat lungs increased as SP-B content was raised from 0.1 to 0.75% by weight. This study emphasizes the crucial functional activity of SP-B in lung surfactants. Significant differences in SP-B content between exogenous surfactants used to treat respiratory disease could be associated with substantial activity variations.

High-yield purification of lung surfactant proteins sp-b and sp-c and the effects on surface activity.

Several protocols for purification of milligram quantities of lung surfactant proteins (SP)-B and SP-C were studied for separation efficiency and surface activity of the isolated proteins recombined with synthetic phospholipids (SPL). SP-B and SP-C were obtained from calf lung surfactant extract by C8 chromatography with isocratic elution by either of three solvent systems: 7:1:0.4 MeOH/CHCl(3)/5% 0.1 M HCl (solvent A), 7:1 MeOH/CHCl(3)+ 0.1% TFA (solvent B), and 7:1:0.4 MeOH/CHCl(3)/H(2)O + 0.1% TFA (solvent C). Solvents A and C yielded pure apoprotein in a single pass, with estimated total protein recoveries of >85 and >90%, respectively. Solvent B was less effective in purifying SP-B and SP-C, had a lower recovery efficiency, and gave isolates with less surface activity. Mixtures of SPL plus SP-B eluted with solvents A and C adsorbed to equilibrium surface tensions of 21-22 mN/m and reached minimum surface tensions <1 mN/m during dynamic cycling. Mixtures of SPL with SP-C obtained with solvents A and C had equilibrium surface tensions of 26-27 mN/m and minimum dynamic values of 2-7 mN/m. The ability to obtain milligrams of virtually lipid-free SP-B and SP-C in a single column pass will facilitate research on their biological, structural, and biophysical properties.

Concentration-dependent, temperature-dependent non-Newtonian viscosity of lung surfactant dispersions.

The bulk shear viscosities of aqueous dispersions of lavaged calf lung surfactant (LS) and its chloroform:methanol extract (CLSE) were measured as a function of concentration, shear rate and temperature. At 10-mg phospholipid per milliliter, dispersions of LS and vortexed CLSE in 0.15 M NaCl (saline) had low viscosities near 1 cp over a range of shear rates from 225 to 1125 s(-1). Lung surfactant viscosity increased with phospholipid concentration and became strongly non-Newtonian with higher values at low shear rates. At 37 degrees C and 40 mg/ml, LS and vortexed CLSE in saline had viscosities of 38 and 34 cp (77 s(-1)) and 12 and 7 cp (770 s(-1)), respectively. Viscosity values for LS and CLSE were dependent on temperature and, at fixed shear, were lower at 23 degrees C than at 37 or 10 degrees C. Hysteresis was also present in viscosity measurements depending on whether shear rate was successively increased or decreased during study. Addition of 5 mM Ca(2+) at 37 degrees C markedly reduced CLSE viscosity at all shear rates and decreased LS viscosity at low shear rates. Dispersion by sonication rather than vortexing increased the viscosity of CLSE at fixed shear, while synthetic phospholipids dispersed by either method had low, relatively Newtonian viscosities. The complex viscous behavior of dispersions of LS and CLSE in saline results from their heterogeneous aggregated microstructure of phospholipids and apoproteins. Viscosity is influenced not only by the aggregate surface area under shear, but also by phospholipid-apoprotein interactions and aggregate structure/deformability. Similar complexities likely affect the viscosities of biologically-derived exogenous surfactant preparations administered to patients in clinical surfactant therapy.

Pulmonary inflammation disrupts surfactant function during Pneumocystis carinii pneumonia.

During Pneumocystis carinii pneumonia (PCP) in mice, the degree of pulmonary inflammation correlates directly with the severity of lung function deficits. Therefore, studies were undertaken to determine whether the host inflammatory response contributes to PCP-related respiratory impairment, at least in part, by disrupting the pulmonary surfactant system. Protein and phospholipid content and surfactant activity were measured in the lavage fluid of infected mice in either the absence or presence of an inflammatory response. At 9 weeks postinfection with P. carinii, nonreconstituted SCID mice exhibited no signs of pulmonary inflammation, respiratory impairment, or surfactant dysfunction. Lavage fluid obtained from these mice had protein/phospholipid (Pr/PL) ratios (64% +/- 4.7%) and minimum surface tension values (4.0 +/- 0.9 mN/m) similar to those of P. carinii-free control mice. However, when infected SCID mice were immunologically reconstituted, an intense inflammatory response ensued. Pr/PL ratios (218% +/- 42%) and minimum surface tension values (27.2 +/- 2.7 mN/m) of the lavage fluid were significantly elevated compared to those of the lavage fluid from infected, nonreconstituted mice (P < 0.05). To examine the specific role of CD8(+) T-cell-mediated inflammation in surfactant dysfunction during PCP, mice with defined T-cell populations were studied. P. carinii-infected, CD4(+)-depleted mice had elevated lavage fluid Pr/PL ratios (126% +/- 20%) and elevated minimum surface tension values (16.3 +/- 1.0 mN/m) compared to normal mice (P < 0.05). However, when infected mice were additionally depleted of CD8(+) cells, Pr/PL ratios were normal and surfactant activity was improved. These findings demonstrate that the surfactant pathology associated with PCP is related to the inflammatory process rather than being a direct effect of P. carinii. Moreover, CD8(+) lymphocytes are involved in the mechanism leading to surfactant dysfunction.

Acid aspiration increases sensitivity to increased ambient oxygen concentrations.

Previously we have demonstrated that prolonged exposure to 100% ambient oxygen leads to a marked loss in functional lung volume and lung compliance, hypoxemia, and surfactant system abnormalities similar to acute respiratory distress syndrome (ARDS). However, 50% oxygen administration is believed to be safe in most clinical settings. In the present study, we have evaluated the effects of a 24-h exposure to 50% oxygen in rabbits immediately following experimental gastric acid aspiration. Mild hypoxemia, but no changes in mortality, lung volume, lung compliance, surfactant metabolism, or edema formation occurred after 24 h of normoxia postacid aspiration. Conversely, a relatively short (24-h) exposure to 50% oxygen after acid aspiration results in increased pulmonary edema, physical signs of respiratory distress, and mortality, as well as decreased arterial oxygenation, lung volume, lung compliance, and type II alveolar cell surfactant synthesis. These results suggest that acid aspiration alters the "set point" for oxygen toxicity, possibly by "priming" cells through activation of inflammatory pathways. This pathogenic mechanism may contribute to the progression of aspiration pneumonia to ARDS.

Combined-modality therapy with inhaled nitric oxide and exogenous surfactant in term infants with acute respiratory failure.

OBJECTIVE: To report cases of neonates successfully treated with both exogenous surfactant and inhaled nitric oxide (INO). DESIGN: Retrospective chart review of full term infants treated between January and May 1999 in the neonatal intensive care unit of The Children's Hospital at Strong, University of Rochester, Rochester, New York. PATIENTS: Three full-term infants treated with surfactant and INO were identified. Each infant had severe acute respiratory failure (as a result of severe aspiration syndromes) and a clinical diagnosis of pulmonary hypertension and parenchymal lung disease in the absence of congenital malformations. INTERVENTIONS: One infant received INO (20-40 ppm) followed by exogenous surfactant (100mg/kg); the other two received surfactant followed by INO. MAIN RESULTS: All three infants exhibited a favorable response to treatment with these agents in terms of improved arterial oxygenation as summarized by oxygenation index and all survived to discharge home without referral for extracorporeal membrane oxygenation. CONCLUSIONS: No adverse interactions were observed related to INO plus surfactant therapy. The responses of these critically ill infants were consistent with the hypothesis that the actions of INO in dilating the pulmonary microvasculature and of exogenous surfactant in stabilizing and recruiting alveoli are complementary and may lead to additive clinical benefits. These case results suggest that more extensive clinical studies are warranted for combined-modality therapy with INO and exogenous surfactant in patients with the acute respiratory distress syndrome.

The reaction of thiolates with 2,3-dibromo-1-propanol revisited: application to the synthesis of bis(fattyalkylthio)propanols.

This work compares two reaction schemes for preparing 2,3-bis(fattyalkylthio)-1-propanols for further synthetic adaptation as hydrophobic analogs of lung surfactant phosphatidylcholines. An attempt to prepare 2,3-bis(fattyalkylthio)-1-propanols based on the previously published methods of Bell and co-workers (B.R. Ganong, C.R. Loomis, Y.A. Hannun, R.M. Bell, 1986. Proc. Natl. Acad. Sci. USA 83, 1184-1188; B.R. Ganong, R.M. Bell, 1987. Methods Enzymol. 141, 313-320; J.P. Walsh, L. Fahrner, R.M. Bell, 1990. J. Biol. Chem. 265, 4374-4381) was found to give the rearranged 1,3-bis(fattyalkylthio)-2-propanols as major products. As a reliable alternative, the reaction of ethyl 2,3-dibromopropionate with 2 equivalents of long chain sodium n-alkanethioates gave the corresponding ethyl 2,3-bis(n-alkylthio)propionates, which were then reduced with LiAlH4 to yield the desired 2,3-bis(fattyalkylthio)-1-propanols. Both 13C and 1H NMR spectroscopy were used to differentiate the two possible 1,3- and 2,3-dithio substituted alcohol products and to rigorously assign their structures.

Effect of hydrophobic surfactant peptides SP-B and SP-C on binary phospholipid monolayers. I. Fluorescence and dark-field microscopy.

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.

Multiple mechanisms of lung surfactant inhibition.

We studied the mechanisms by which C16:0 lysophosphatidylcholine (LPC) and albumin inhibit the surface activity of calf lung surfactant extract (CLSE) by using a pulsating bubble apparatus with a specialized hypophase exchange system, plus adsorption and Wilhelmy balance measurements. In the absence of inhibitors, CLSE (1 mg phospholipid/mL) reached minimum surface tension (gamma(min)) < 1 mN/m within 5 min of bubble pulsation at 20 cycles/min at 37 degrees C. Mixtures of CLSE:LPC had impaired surface activity depending on LPC content: gamma(min) was raised to 5 mN/m by 14 wt % LPC, to 15 mN/m by 25-30 wt% LPC, and to >20 mN/m (67 wt % LPC), even at high CLSE concentrations (3 and 6 mg phospholipid/mL). In contrast, inhibition of CLSE by albumin was more easily abolished when surfactant concentration was raised. Mixtures of albumin (3 mg/mL) and CLSE (1 mg phospholipid/mL) had gamma(min) >20 mN/m, but normal values of gamma(min) < 1 mN/m were reached at higher CLSE concentration (3 mg phospholipid/mL) even when albumin concentration was increased 8-fold to 24 mg/mL. In hypophase exchange studies, LPC, but not albumin, was able to penetrate preformed CLSE surface films and raise gamma(min) CLSE surface films with gamma(min) < 1 mN/m were isolated by an initial hypophase exchange with saline, and a second exchange with an LPC-containing hypophase raised gamma(min) to approximately 10 mN/m. CLSE surface films retained the ability to reach gamma(min) < 1 mN/m in analogous hypophase exchange studies with albumin. The ability of LPC to penetrate surface films of CLSE, although albumin could not, was also demonstrated in adsorption experiments in a Teflon dish, where diffusion was minimized by subphase stirring. Wilhelmy balance experiments also demonstrated that LPC could mix and interact with CLSE or dipalmitoyl phosphatidylcholine in solvent-spread surface films. The ability of LPC or other cell membrane lipids to penetrate interfacial films and raise gamma(min) even at high surfactant concentration may increase their inhibitory actions during acute lung injury.

Additivity of protein and nonprotein inhibitors of lung surfactant activity.

This study examined the degree of additivity of several physiologically relevant protein and nonprotein inhibitors in impairing the surface activity of whole and extracted calf lung surfactant (LS and CLSE) on a pulsating bubble apparatus at 37 degrees C. Inhibitors investigated were albumin, hemoglobin, C16:0 and C18:1 lysophosphatidylcholine (LPC), oleic acid (OA), palmitoleic acid (PA), arachidonic acid (AA), and mixed red blood cell membrane lipids (RBCML). In the absence of inhibitors, LS (0.5 mg/ml) and CLSE (0.75 mg/ml) reached minimum surface tensions < 1 mN/m within 5 min of bubble pulsation (20 cycles/min, 50% area compression). Each inhibitor acting alone was able to reduce the surface activity of LS and CLSE, either raising minimum surface tension or increasing the time course of surface tension lowering or both. Several combinations of inhibitors exhibited additivity in impairing LS or CLSE activity at a lower concentration in mixtures than when present alone (albumin plus either C16:0 LPC, C18:1 LPC, or RBCML; hemoglobin plus either C16:0 LPC, C18:1 LPC, RBCML, PA, OA, or AA). The degree of additivity, however, was typically small in terms of the magnitude of reduction in inhibitor concentration or the rise in minimum surface tension relative to the effects of the most severe single inhibitor present. Substantial synergy was not found for any of the combinations of protein and nonprotein inhibitors investigated. Mixtures of albumin with PA or AA actually had a reduced inhibitory effect on LS and CLSE activity compared with the free fatty acids alone, apparently because of albumin binding of these molecules. In all cases, the detrimental effects of mixed inhibitors on LS and CLSE activity were reversed at increased surfactant concentration. These results indicate that surfactant dysfunction in acute respiratory distress syndrome (ARDS) could be increased in severity by interactions between some inhibitory substances, but that supplementation with exogenous CLSE would be effective in reversing inactivation by the mixtures of blood proteins, membrane lipids, and fatty acids studied.

Comparison of two strategies for surfactant prophylaxis in very premature infants: a multicenter randomized trial.

INTRODUCTION: Previous trials of surfactant therapy in premature infants have demonstrated a survival advantage associated with prophylactic therapy as an immediate bolus, compared with the rescue treatment of established respiratory distress syndrome. The optimal strategy for prophylactic therapy, however, remains controversial. When administered as an endotracheal bolus immediately after delivery, surfactant mixes with the absorbing fetal lung fluid and may reach the alveoli before the onset of lung injury. This approach, however, causes a brief delay in the initiation of standard neonatal resuscitation, including positive pressure ventilation, and is associated with a risk for surfactant delivery into the right main stem bronchus or esophagus. As an alternative approach, surfactant prophylaxis may be administered in small aliquots soon after resuscitation and confirmation of endotracheal tube position. Although this strategy has substantial logistical advantages in clinical practice, its efficacy has not been established. OBJECTIVE: The purpose of this study was to determine whether the established benefits of the immediate bolus strategy for surfactant prophylaxis could still be achieved using a postventilatory aliquot strategy after initial standard resuscitation and stabilization. DESIGN: Multicenter randomized clinical trial with patients randomized before delivery to immediate bolus or postventilatory aliquot therapy. PARTICIPANTS: Inborn premature infants delivered to mothers at an estimated gestational age of 24[0/7] to 28[6/7] weeks. INTERVENTIONS: Those infants who were randomized to the immediate bolus strategy were intubated as rapidly as possible after birth, and a 3-mL intratracheal bolus of calf lung surfactant extract (Infasurf) was administered before the initiation of positive pressure ventilation. Those infants who were randomized to the postventilatory aliquot strategy received standard resuscitation measures with intubation by 5 minutes of age, if not required earlier. At 10 minutes after birth, 3 mL of surfactant was administered in 4 divided aliquots of 0.75 mL each. Patients in both groups were eligible to receive up to three additional doses of surfactant as rescue therapy in the neonatal intensive care unit, if needed. OUTCOME MEASURES: The primary outcome variable was survival to discharge to home. Secondary variables included neonatal complications and requirement for oxygen therapy at 36 weeks' postmenstrual age. RESULTS: Among three centers, 651 infants were enrolled and randomized before delivery. Survival to discharge to home was similar for the two strategies for surfactant therapy as prophylaxis: 76% for the immediate bolus group and 80% for the postventilatory aliquot group. In a secondary analysis, the rate of supplemental oxygen administration at 36 weeks' postmenstrual age was 18% for the immediate bolus group and 13% for the postventilatory aliquot group. CONCLUSIONS: Survival to discharge to home was similar with immediate bolus and postventilatory aliquot strategies for surfactant prophylaxis. Because of its logistical advantages in the delivery room and its beneficial effects on prolonged oxygen requirements, we recommend the postventilatory aliquot strategy for surfactant prophylaxis of premature infants delivered before 29 weeks' gestation.

Primary importance of zwitterionic over anionic phospholipids in the surface-active function of calf lung surfactant extract.

The relative contributions of zwitterionic and anionic phospholipids to the surface-active function of calf lung surfactant extract (CLSE) were assessed by measurements of surface properties in vitro and pressure-volume (P-V) mechanics in excised rat lungs in situ. Surface activity and mechanical effects were compared for chromatographically purified CLSE subfractions containing the complete mix of phospholipids (PPL) or modified phospholipids depleted in anionic components (mPPL), alone or combined with 1.3% (by weight) of hydrophobic surfactant proteins (SP-B and SP-C). Surface pressure-time (pi-t) adsorption isotherms at 37 degrees C were very similar for dispersions of PPL and mPPL in a Teflon dish with a stirred subphase to minimize diffusion resistance. Combination of either PPL or mPPL with hydrophobic SP substantially improved adsorption, but mixtures of PPL:SP and mPPL:SP had only small differences in pi-t isotherms and reached the same final equilibrium pi of approximately 47 mN/m achieved by CLSE. Surface pressure-area (pi-A) isotherms and maximum surface pressures were also very similar for spread films of PPL versus mPPL and PPL:SP versus mPPL:SP on the Wilhelmy balance (23 degrees C and 37 degrees C). Respreading based on pi-A isotherm area calculations was slightly better in surface-excess films of PPL versus mPPL and PPL:SP versus mPPL:SP, but differences were minor and were smaller at 37 degrees C than at 23 degrees C. Overall dynamic surface activity in oscillating bubble studies was not significantly different for PPL versus mPPL or for PPL:SP versus mPPL:SP, and the latter two mixtures both reached minimum surface tensions < 1 mN/m (37 degrees C, 20 cycles/min, 0.5 mM phospholipid). Dispersions of PPL:SP, mPPL:SP, and CLSE were also not significantly different in improving P-V mechanics almost to normal when instilled in lavaged, excised rat lungs at 37 degrees C (30 mg/2.5 ml saline). These data suggest that zwitterionic phospholipids have a major role over anionic phospholipids in interacting with hydrophobic SP in the adsorption, dynamic surface tension lowering, film respreading, and pulmonary mechanical activity of the hydrophobic components of calf lung surfactant in CLSE.

Changes in surfactant protein gene expression in a neonatal rabbit model of hyperoxia-induced fibrosis.

Lung injuries, including bronchopulmonary dysplasia, alter the surfactant system. We developed a newborn rabbit model of acute, followed by chronic, hyperoxic injury to study surfactant protein (SP) gene expression. Initial litters were exposed to >95% O2 until 50% died (LD50; 7-11 days old). Subsequent litters were exposed to >95% O2 for 8 days, followed by 60% O2 until 22-36 days. Controls were exposed to room air. LD50 animals displayed acute pulmonary inflammation, edema, protein leak, and surfactant dysfunction. These changes resolved, and fibrosis developed by 22 days. Whole lung SP-A mRNA expression (measured by membrane hybridization) was twice control levels at 4 days of >95% O2, with specific elevations in terminal bronchioles and type II cells at 4 days and the LD50 by in situ hybridization. Whole lung SP-B and SP-C mRNA were unchanged from control throughout exposure. However, in situ hybridization showed elevations in SP-B and SP-C mRNA in type II cells in inflamed areas at the LD50. SP mRNA alterations resolved by 22-36 days. The surfactant system recovers from acute hyperoxic injury, despite continued 60% O2 exposure.

Differential activity and lack of synergy of lung surfactant proteins SP-B and SP-C in interactions with phospholipids.

This study shows that the hydrophobic lung surfactant proteins (SP)-B and SP-C are not synergistic in enhancing functionally relevant surface behaviors in films and dispersions with phospholipids, and that SP-B is more effective than SP-C in facilitating surface activity. Purified bovine SP-B, SP. C, or SP-B/C (1/1 by wt) were combined with chroma tographically purified calf lung surfactant phospholipids (PPL), or with dipalmitoyl phosphatidylcholine (DPPC) or complex phospholipid mixtures containing 75% or 50% DPPC (75% SPL or 50% SPL). Adsorption was consistently better in corresponding mixtures of phospholipids plus SP-B versus SP-C, but was not improved further by substitution of SP-B/C for SP-B. Interfacial films of DPPC or SPL plus 1.3% SP-B or 1.3% SP-C had improved respreading compared to phospholipids alone (Wilhelmy balance, 23 degrees C and 37 degrees C), but substitution of mixed SP-B/C for either pure apoprotein did not increase respreading further. Surface-excess films of phospholipids plus SP-B had higher maximum surface pressures, or maintained a high maximum pressure through more consecutive compressions, than corresponding films with SP-C. Dispersions of phospholipids plus 1.3% SP-B or mixed (1/1) SP-B/C (2.6%) rapidly lowered surface tension to < 1 mN/m in oscillating bubble studies (20 cpm, 37 degrees C), while corresponding dispersions containing SP-C reduced surface tension more slowly or reached higher minima. Mixtures of 50% SPL with SP-B versus SP-C were also better able to resist inhibition by serum albumin in bubble and adsorption studies, and inhibition resistance was not significantly improved in mixtures containing 2.6% SP-B/C (1/1) versus 1.3% SP-B. The lack of synergy in hydrophobic apoprotein function, coupled with the greater effectiveness of SP-B in improving phospholipid adsorption, dynamic surface activity, and inhibition resistance, suggests that mixtures of phospholipids plus SP-B or related peptides may be particularly relevant its clinical exogenous surfactants.

Acylation of pulmonary surfactant protein-C is required for its optimal surface active interactions with phospholipids.

This study investigates the importance of thioester-linked acyl groups in lung surfactant protein C (SP-C) in facilitating interactions with phospholipids that yield functionally important surface active behaviors. Native SP-C, palmitoylated at cysteine residues at positions 5 and 6, was isolated from bovine lung surfactant by liquid chromatography. Deacylated SP-C (dSP-C), unchanged in composition and sequence from SP-C but having a decreased alpha-helical content in films with dipalmitoyl phosphatidylcholine (DPPC) of 52 versus 70%, was obtained by treatment with 0.1 M sodium carbonate buffer at pH 10. Surface activity was studied for SP-C and dSP-C combined with column-purified phospholipids (PPL) from calf lung surfactant or with synthetic phospholipids (DPPC or a synthetic phospholipid mixture (SPL) containing 50:35:15, DPPC:egg phosphatidylcholine:egg phosphatidylglycerol). Interfacial measurements included surface pressure time adsorption isotherms for dispersed surfactants with diffusion minimized, dynamic surface pressure area isotherms and respreading for films in the Wilhelmy balance, and overall surface tension lowering at physiologic cycling rate in oscillating bubble experiments. Dispersions of PPL:SP-C and SPL:SP-C rapidly adsorbed to high equilibrium surface pressures of 47-48 mN/m, significantly better than corresponding dispersions containing dSP-C. The adsorption of PPL:dSP-C was essentially unchanged from that of PPL alone, and the adsorption of SPL:dSP-C was improved only slightly over SPL alone. In Wilhelmy balance studies, dynamic respreading was significantly improved over phospholipids alone in films of SP-C plus PPL, SPL, or DPPC. Respreading was improved less markedly by dSP-C in corresponding films with SPL or DPPC and not at all in films with PPL. Maximum surface pressures were also higher in cycled films of SP-C versus dSP-C combined with PPL or SPL. In bubble experiments (37 degrees C, 20 cycles/min), dispersions of PPL:SP-C and SPL:SP-C reached low minimum surface tensions of <1 and 5 mN/m, respectively, whereas PPL:dSP-C and SPL:dSP-C only reached minima of approximately 20 mN/m as did PPL and SPL alone. Acylation in SP-C is crucial for its interactions with phospholipids over the full spectrum of adsorption and dynamic surface behaviors important for lung surfactant.

Content of dipalmitoyl phosphatidylcholine in lung surfactant: ramifications for surface activity.

The content of dipalmitoyl phophatidylcholine (DPPC) in the phosphatidylcholine (PC) fraction of calf lung surfactant extract (CLSE) is measured by gas chromatography (GC) and estimated from the widely used osmium tetroxide assay for disaturated phosphatidylcholine (DSPC). The surface-active properties of model phospholipid/apoprotein surfactants with varying DPPC content are also defined and compared relative to CLSE. GC analysis of fatty acids in PC isolated from CLSE indicated a possible range of 30 to 65% for DPPC content depending on C16:0 fatty chain mismatching, and further studies using phospholipase A2 treatment indicated an actual DPPC content < or = 41%. The osmium tetroxide assay gave a very high value of 70% for the DSPC content of surfactant PC, and experiments with synthetic phospholipids demonstrated that this assay responded inappropriately in the presence of monounsaturated PC, leading to falsely elevated DSPC values. The influence of DPPC content on adsorption and film behavior was investigated in model surfactants containing 40, 60, and 80% DPPC (DPPC/egg PC/egg PG, 40:50:10, 60:30:10, and 80:10:10 by mol) combined with 1.3% hydrophobic surfactant protein (SP)-B and -C. The biophysical properties of the model surfactant with 40% DPPC were found to be closer to CLSE than those of mixtures with 60 or 80% DPPC. The adsorption of dispersions containing 40% DPPC with 1.3% SP-B, C was almost identical to CLSE and was improved in rate and magnitude compared with the mixtures with higher DPPC content (60 or 80%). In Wilhelmy balance studies of cycled films, respreading was increased and maximum surface pressure was decreased for the 40% versus higher DPPC content mixtures, again approaching CLSE in behavior. All synthetic phospholipid (SPL):SP mixtures lowered surface tension to < 1 mN/m in oscillating bubble studies at physiologic cycling rate (20 cpm), but the 40% DPPC mixture had a time dependent most closely matching that of CLSE. Our measured DPPC content near 40% for lung surfactant PC, and the similarly high activity of a related synthetic phospholipid/apoprotein model mixture, suggest that exogenous surfactants with relatively low DPPC contents might be important for future study and development.