Surfactant protein A regulates surfactant phospholipid clearance after LPS-induced injury in vivo

Previous in vitro studies have suggested that surfactant protein A (SP-A) may play a role in pulmonary surfactant homeostasis by mediating surfactant secretion and clearance. However, mice made deficient in SP-A [SP-A (-/-) animals] have relatively normal levels of surfactant compared with wild-type SP-A (+/+) animals. We hypothesize that SP-A may play a role in surfactant homeostasis after acute lung injury. Bacterial lipopolysaccharide was instilled into the lungs of SP-A (-/-) mice and SP-A (+/+) mice to induce injury. Surfactant phospholipid levels were increased 1.6-fold in injured SP-A (-/-) animals, although injury did not alter [3H]choline or [14C]palmitate incorporation into dipalmitoylphosphatidylcholine (DPPC), suggesting no change in surfactant synthesis/secretion 12 h after injury. Clearance of [3H]DPPC from the lungs of injured SP-A (-/-) animals was decreased by approximately 40%. Instillation of 50 microg of exogenous SP-A rescued both the clearance defect and the increased phospholipid defect in injured SP-A (-/-) animals, suggesting that SP-A may play a role in regulating clearance of surfactant phospholipids after acute lung injury.     

Effects of prenatal ethanol exposure on the lungs of postnatal lambs

Prenatal ethanol exposure increases collagen deposition and alters surfactant protein (SP) expression and immune status in lungs of near-term fetal sheep. Our objectives were to determine 1) whether these prenatal effects of repeated gestational ethanol exposure persist after birth and 2) whether surfactant phospholipid composition is altered following prenatal ethanol exposure. Pregnant ewes were chronically catheterized at 90 days of gestational age (DGA) and given a 1-h daily infusion of ethanol (0.75 g/kg, n = 9) or saline (n = 7) from 95 to 135 DGA; ethanol administration ceased after 135 DGA. Lambs were born naturally at full term (146 ± 0.5 DGA). Lung tissue was examined at 9 wk postnatal age for alterations in structure, SP expression, and inflammation; bronchoalveolar lavage fluid was examined for alterations in surfactant phospholipid composition. At 134 DGA, surfactant phospholipid concentration in amniotic fluid was significantly reduced (P < 0.05) by ethanol exposure, and the composition was altered. In postnatal lambs, there were no significant differences between treatment groups in birth weight, postnatal growth, blood gas parameters, and lung weight, volume, tissue fraction, mean linear intercept, collagen content, proinflammatory cytokine gene expression, and bronchoalveolar lavage fluid surfactant phospholipid composition. Although SP-A, SP-B, and SP-C mRNA levels were not significantly different between treatment groups, SP-D mRNA levels were significantly greater (P < 0.05) in ethanol-treated animals; as SP-D has immunomodulatory roles, innate immunity may be altered. The adverse effects of daily ethanol exposure during late gestation on the fetal lung do not persist to 2 mo after birth, indicating that the developing lung is capable of repair.     

Improved lung preservation relates to an increase in tubular myelin-associated surfactant protein A

Background

Declining levels of surfactant protein A (SP-A) after lung transplantation are suggested to indicate progression of ischemia/reperfusion (IR) injury. We hypothesized that the previously described preservation-dependent improvement of alveolar surfactant integrity after IR was associated with alterations in intraalveolar SP-A levels.

Methods

Using immuno electron microscopy and design-based stereology, amount and distribution of SP-A, and of intracellular surfactant phospholipids (lamellar bodies) as well as infiltration by polymorphonuclear leukocytes (PMNs) and alveolar macrophages were evaluated in rat lungs after IR and preservation with EuroCollins or Celsior.

Results

After IR, labelling of tubular myelin for intraalveolar SP-A was significantly increased. In lungs preserved with EuroCollins, the total amount of intracellular surfactant phospholipid was reduced, and infiltration by PMNs and alveolar macrophages was significantly increased. With Celsior no changes in infiltration or intracellular surfactant phospholipid amount occurred. Here, an increase in the number of lamellar bodies per cell was associated with a shift towards smaller lamellar bodies. This accounts for preservation-dependent changes in the balance between surfactant phospholipid secretion and synthesis as well as in inflammatory cell infiltration.

Conclusion

We suggest that enhanced release of surfactant phospholipids and SP-A represents an early protective response that compensates in part for the inactivation of intraalveolar surfactant in the early phase of IR injury. This beneficial effect can be supported by adequate lung preservation, as e.g. with Celsior, maintaining surfactant integrity and reducing inflammation, either directly (via antioxidants) or indirectly (via improved surfactant integrity).     

The collagen-like region of surfactant protein A (SP-A) is required for correction of surfactant structural and functional defects in the SP-A null mouse

Pulmonary surfactant isolated from gene-targeted surfactant protein A null mice (SP-A(-/-)) is deficient in the surfactant aggregate tubular myelin and has surface tension-lowering activity that is easily inhibited by serum proteins in vitro. To further elucidate the role of SP-A and its collagen-like region in surfactant function, we used the human SP-C promoter to drive expression of rat SP-A (rSPA) or SP-A containing a deletion of the collagen-like domain (DeltaG8-P80) in the Clara cells and alveolar type II cells of SP-A(-/-) mice. The level of the SP-A in the alveolar wash of the SP-A(-/-,rSP-A) and SP-A(-/-,DeltaG8-P80) mice was 6.1-and 1.3-fold higher, respectively, than in the wild type controls. Tissue levels of saturated phosphatidylcholine were slightly reduced in the SP-A(-/-,rSP-A) mice compared with SP-A(-/-) littermates. Tubular myelin was present in the large surfactant aggregates isolated from the SP-A(-/-,rSP-A) lines but not in the SP-A(-/-,DeltaG8-P80) mice or SP-A(-/-) controls. The equilibrium and minimum surface tensions of surfactant from the SP-A(-/-,rSP-A) mice were similar to SP-A(-/-) controls, but both were markedly elevated in the SP-A(-/-,DeltaG8-P80) mice. There was no defect in the surface tension-lowering activity of surfactant from SP-A(+/+,DeltaG8-P80) mice, indicating that the inhibitory effect of DeltaG8-P80 on surface activity can be overcome by wild type levels of mouse SP-A. The surface activity of surfactant isolated from the SP-A(-/-,rSP-A) but not the SP-A(-/-,DeltaG8-P80) mice was more resistant than SP-A(-/-) littermate control animals to inhibition by serum proteins in vitro. Pressure volume relationships of lungs from the SP-A(-/-), SP-A(-/-,rSP-A), and SP-A(-/-,DeltaG8-P80) lines were very similar. These data indicate that expression of SP-A in the pulmonary epithelium of SP-A(-/-) animals restores tubular myelin formation and resistance of isolated surfactant to protein inhibition by a mechanism that is dependent on the collagen-like region.     

Inhaled nitric oxide increases surfactant protein gene expression in the intact lamb

Inhaled nitric oxide (iNO) is used to treat a number of disease processes. Although in vitro data suggest that nitric oxide (NO) alters surfactant protein gene expression, the effects in vivo have not been studied. The objective of this study was to evaluate the effects of iNO on surfactant protein (SP)-A, -B, and -C gene expression in the intact lamb. Thirteen 4-wk-old lambs were mechanically ventilated with 21% oxygen and received iNO at 40 ppm (n = 7) or vehicle gas (n = 6) for 24 h. Peripheral lung biopsies were obtained at 0, 12, and 24 h and analyzed for surfactant mRNA, protein, and total DNA content. Inhaled NO increased SP-A and SP-B mRNA content by 80% from 0 to 12 h and by 78 and 71%, respectively, from 0 to 24 h. There was an increase in SP-A and SP-B protein content by 45% from 0 to 12 h, and a decrease by 70 and 65%, respectively, from 0 to 24 h. DNA content was unchanged. The mechanisms and physiological effects of these findings warrant further investigation.     

Surfactant protein A increases matrix metalloproteinase-9 production by THP-1 cells

Matrix metalloproteinase (MMP)-9 from alveolar macrophages is a major source of elastolytic activity in the lung. It is increased in the bronchoalveolar lavage fluid of patients with emphysema. Although the importance of macrophage-derived elastolytic activity in the pathogenesis of emphysema is well established, questions remain about MMP-9 regulation and activity. Because surfactant protein A (SP-A) is capable of modulating other functions of human monocytic cells, we hypothesized that SP-A may regulate MMP-9 expression. Vitamin D3-differentiated THP-1 cells and peripheral blood mononuclear cells were stimulated in vitro with several concentrations of SP-A for different incubation times. MMP-9 mRNA expression was measured by dot-blot analysis, gelatinolytic activity in the medium was determined by gel zymography, protein expression was determined by ELISA, and a specific MMP-9 activity assay was used to measure the state of activation of this enzyme in the cell supernatants. SP-A induced the expression of MMP-9 in both cell types, the effect was time and dose dependent, and MMP-9 was released in its zymogen form. On the basis of results of neutralizing antibody studies, we believe that SP-A action is mediated through Toll-like receptor-2. Even though the biological meaning of these findings remains to be elucidated, these observations suggest the presence of a novel, locally controlled mechanism by which MMP-9 levels may be regulated in alveolar macrophages. We speculate that SP-A may influence the protease/antiprotease balance in the lungs of patients with quantitative and/or qualitative changes in surfactant constituents favoring an abnormal breakdown of extracellular matrix components.     

Human lung surfactant protein A exists in several different oligomeric states: oligomer size distribution varies between patient groups.

BACKGROUND: Lung surfactant protein A (SP-A) is a complex molecule composed of up to 18 polypeptide chains. In vivo, SP-A probably binds to a wide range of inhaled materials via the interaction of surface carbohydrates with the lectin domains of SP-A and mediates their interaction with cells as part of a natural defense system. Multiplicity of lectin domains gives high-affinity binding to carbohydrate-bearing surfaces. MATERIALS AND METHODS: Gel filtration analyses were performed on bronchoalveolar lavage (BAL) fluid samples from three patient groups: pulmonary alveolar proteinosis (n = 12), birch pollen allergy (n = 11), and healthy volunteers (n = 4). Sucrose density gradient centrifugation was employed to determine molecular weights of SP-A oligomers. SP-A was solubilized from the lipid phase to compare oligomeric state with that of water soluble SP-A. RESULTS: SP-A exists as fully assembled complexes with 18 polypeptide chains, but it is also consistently found in smaller oligomeric forms. This is true for both the water- and lipid-soluble fractions of SP-A. CONCLUSION: The three patient groups analyzed show a shift towards lower oligomeric forms of SP-A in the following sequence: healthy-pulmonary alveolar proteinosis-pollen allergy. Depolymerization would be expected to lead to loss of binding affinity for carbohydrate-rich surfaces, with loss or alteration of biological function. While there are many complex factors involved in the establishment of an allergy, it is possible that reduced participation of SP-A in clearing a potential allergen from the lungs could be an early step in the chain of events.     

Surfactant in respiratory distress syndrome and lung injury

A deficiency in alveolar surfactant due to immaturity of alveolar type II epithelial cells causes respiratory distress syndrome (RDS). In contrast to animals, the fetal maturation of surfactant in human lungs takes place before term, exceptionally large quantities of surfactant accumulating in the amniotic fluid. The antenatal development of surfactant secretion is very variable but corresponds closely to the risk of RDS. The variation in SP-A and SP-B genes, race, sex and perinatal complications influence susceptibility to RDS. Surfactant therapy has improved the prognosis of RDS remarkably. Abnormalities in alveolar or airway surfactant characterize many lung and airway diseases. In the acute respiratory distress syndrome, deficiencies in surfactant components (phospholipids, SP-B, SP-A) are evident, and may be caused by pro-inflammatory cytokines (IL-1, TNF) that decrease surfactant components. The resultant atelectasis localizes the disease, possibly allowing healing (regeneration, increase in surfactant). In the immature fetus, cytokines accelerate the differentiation of surfactant, preventing RDS. After birth, however, persistent inflammation is associated with low SP-A and chronic lung disease. A future challenge is to understand how to inhibit or redirect the inflammatory response from tissue destruction and poor growth towards normal lung development and regeneration.     

Pulmonary surfactant and inflammation in septic adult mice: role of surfactant protein A.

Surfactant alterations, alveolar cytokine changes, and the role of surfactant protein (SP)-A in septic mice were investigated. Sepsis was induced via cecal ligation and perforation (CLP). Septic and sham mice were euthanized at 0, 3, 6, 9, 12, 15, and 18 h after surgery. Mice deficient in SP-A and mice that overexpressed SP-A were euthanized 18 h after surgery. In wild-type, sham-operated mice, surfactant pool sizes were similar at all time points, whereas in the CLP groups there was a significant decrease in small-aggregate surfactant pool sizes beginning 6 h after CLP. Interleukin-6 concentrations in bronchoalveolar lavage fluid from septic animals increased from 6 to 18 h after surgery. Identical surfactant alterations and concentrations of cytokines were observed in septic mice that were SP-A deficient or that overexpressed SP-A. In conclusion, alterations of pulmonary surfactant and alveolar cytokines occur simultaneously, 6 h after a systemic insult. In addition, we did not detect a role for SP-A in regulating surfactant phospholipid pool sizes or pulmonary inflammation in septic mice.     

Strontium Levels in Different Causes of Death: Diagnostic Efficacy in Drowning

Trace element determination can be applied in forensic medicine to diagnose the cause of death. Drowning is the second leading cause of death from unintentional injury. Despite the many diagnostic methods used, the post-mortem diagnosis of drowning continues to be one of the most difficult in forensic pathology. Strontium is a highly sensitive marker of water aspiration in a liquid medium rich in this metal. The aims of this study were to confirm the diagnostic value of strontium in cases of drowning compared with other causes of death, to analyse factors that could affect its concentration and to ascertain the sensitivity and specificity of strontium in right and left ventricles and peripheral serum for the post-mortem diagnosis of drowning. We studied 120 cadavers selected from medico-legal autopsies with different causes of death. Strontium (Sr) levels were measured in the serum (left and right ventricles and peripheral vein) of all cadavers and, in the case of drowning, in the water medium itself, by using Zeeman AAS. Our results confirm the usefulness of blood Sr levels for diagnosing seawater and freshwater drowning, although great care should be exercised in the latter case.     

L-Arg对LPS诱导的急性肺损伤大鼠肺表面活性物质和肺泡巨噬细胞功能的影响

目的:探讨L-精氨酸(L-Arg)对脂多糖(LPS)诱导的急性肺损伤大鼠肺表面活性物质和肺泡巨噬细胞功能的影响。方法:舌下静脉注射脂多糖(LPS)复制肺损伤模型。健康雄性SD大鼠48只,随机分为对照组、模型组(LPS组)和L-Arg治疗组(L-Arg组)(n=16)。分别于给予LPS 3 h或6 h后给予生理盐水(对照组及LPS组,ip)和L-Arg(500 mg/kg ip)(L-Arg治疗组),治疗3 h。原位杂交法(ISH)检测肺组织中肺表面活性蛋白A(SP-A)mRNA的表达;测定肺泡灌洗液(BALF)中的总蛋白(TP)。体外分离培养大鼠肺泡巨噬细胞,以LPS(终浓度10 mg/L)处理巨噬细胞,观察L-Arg对肺泡巨噬细胞的影响。结果:与对照组比较,大鼠肺损伤后SP-A mRNA表达减弱,BALF中TP增多(P<0.01)。肺损伤3 h用L-Arg治疗3 h后,SP-A mRNA阳性细胞表达明显增强,BALF中TP较LPS组相同时间点明显降低(P<0.05,P<0.01),肺损伤减轻。体外实验中,与正常对照组相比,LPS组细胞培养上清中乳酸脱氢酶(LDH)、一氧化氮(NO)、肿瘤坏死因子-α(TNFα-)和白细胞介素-6(IL-6)浓度明显增高(P<0.01);L-Arg明显减少LPS所致的LDH的释放,降低TNFα-和IL-6浓度。结论:L-Arg可减轻内毒素性肺损伤,此机制可能与增强SP-AmRNA表达有关;LPS可刺激巨噬细胞分泌促炎因子和NO,L-Arg可抑制LPS对巨噬细胞的作用。     

Surfactant protein A inhibits the non-enzymatic lipid peroxidation of porcine lung surfactant.

The ability of surfactant protein A (SP-A) to inhibit the ascorbate-Fe(2+) induced lipid peroxidation of polyunsaturated fatty acids found in porcine lung surfactant (surfacen) was assessed by measuring the light emission - chemiluminescence during a 180-min incubation period at 37 degrees C. The light emission (chemiluminescence) was concentration dependent. Changes in the fatty acid composition of surfacen were observed when the lung surfactant was incubated in an ascorbate-Fe(2+) system. The main polyunsaturated fatty acids C18:2 n6 and C20:4 n6 found in the lung surfactant decreased considerably after a 180-min lipid peroxidation process. Native SP-A isolated from pig lungs inhibited oxidation of surfactant long chain polyunsaturated fatty acids, mainly arachidonic acid, in a dose-dependent fashion that was half-maximal (60% inhibition) at a concentration of 2.0 microg/ml and almost complete (73.6% inhibition) at 4.0 microg/ml, as indicated by inhibition of light emission and fatty acid composition analysis. At the highest concentration of lung SP-A used a very good correlation between the protection of the most polyunsaturated fatty acids and inhibition of light emission was observed.     

Inhibitory effects of surfactant protein A on surfactant phospholipid hydrolysis by secreted phospholipases A2

Hydrolysis of surfactant phospholipids by secreted phospholipases A(2) (sPLA(2)) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA(2)-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA(2)-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA(2) and the consequences of sPLA(2)-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA(2)-X activity, but fails to interfere with that of sPLA(2)-V. Moreover, in vitro inhibition of sPLA(2)-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA(2)-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA(2)-IIA activity. Administration of sPLA(2)-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA(2) activity, which may play a protective role by maintaining surfactant integrity during lung injury.     

SP-A is necessary for increased clearance of alveolar DPPC with hyperventilation or secretagogues

The role of surfactant protein-A (SP-A) in pulmonary uptake and metabolism of [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) was studied in SP-A gene-targeted mice (SP-A -/-). Unilamellar liposomes were instilled into the trachea of anesthetized mice. Uptake was measured as dpm in lungs plus liver and kidney for in vivo experiments and in lungs and perfusate for isolated lung experiments. [(3)H]DPPC uptake increased with CO(2)-induced hyperventilation in wild-type mice (SP-A +/+) but was unchanged in SP-A -/-. Secretagogue treatment approximately doubled the uptake of [(3)H]DPPC in isolated lungs from SP-A +/+ but had no effect in SP-A -/-. Lungs degraded 23 +/- 1.2% of internalized [(3)H]DPPC in SP-A +/+ and 36 +/- 0.6% in SP-A -/-; degradation increased with 8-bromoadenosine 3',5'-cyclic monophosphate in SP-A +/+ but was unchanged in SP-A -/-. Activity of lysosomal-type phospholipase A(2) (PLA(2)) was significantly greater in lungs from SP-A -/- compared with SP-A +/+. Thus SP-A is necessary for lungs to respond to hyperventilation or secretagogues with increased DPPC uptake and also modulates the PLA(2)-mediated degradation of internalized DPPC.     

Decreased surfactant proteins in lambs with pulmonary hypertension secondary to increased blood flow

Infants with increased pulmonary blood flow secondary to congenital heart disease suffer from tachypnea, dyspnea, and recurrent pulmonary infections. We have recently established a model of pulmonary hypertension secondary to increased pulmonary blood flow in lambs after in utero placement of an aortopulmonary vascular graft. The purpose of the present study was to utilize our animal model to determine the effects on the expression of surfactant proteins A (SP-A), B (SP-B), and C (SP-C). At age 4 wk, SP-A mRNA content in lambs decreased to 61.4 +/- 8% of age-matched control value (n = 5; P < 0.05). In addition, SP-A protein content was decreased to 50 +/- 12% of control value (n = 6; P < 0.0001). Although we did not observe statistically significant changes in SP-B mRNA content, SP-B protein was decreased to 74 +/- 25% of control value (n = 4; P < 0.02). There was no difference in SP-C mRNA. These data show that in a model of congenital heart disease with pulmonary hypertension secondary to increased pulmonary blood flow, there is a decrease in SP-A gene expression as well as a decrease in SP-A and SP-B protein contents.     

Surfactant protein-A plays an important role in lung surfactant clearance: evidence using the surfactant protein-A gene-targeted mouse

Previous studies with the isolated perfused rat lung showed that both clathrin- and actin-mediated pathways are responsible for endocytosis of dipalmitoylphosphatidylcholine (DPPC)-labeled liposomes by granular pneumocytes in the intact lung. Using surfactant protein-A (SP-A) gene-targeted mice, we examined the uptake of [(3)H]DPPC liposomes by isolated mouse lungs under basal and secretagogue-stimulated conditions. Unilamellar liposomes composed of [(3)H]DPPC: phosphatidylcholine:cholesterol:egg phosphatidylglycerol (10:5:3:2 mol fraction) were instilled into the trachea of anesthetized mice, and the lungs were perfused (2 h). Uptake was calculated as percentage of instilled disintegrations per minute in the postlavaged lung. Amantadine, an inhibitor of clathrin and, thus, receptor-mediated endocytosis via clathrin-coated pits, decreased basal [(3)H]DPPC uptake by 70% in SP-A +/+ but only by 20% in SP-A -/- lung, data compatible with an SP-A/receptor-regulated lipid clearance pathway in the SP-A +/+ mice. The nonclathrin, actin-dependent process was low in the SP-A +/+ lung but accounted for 55% of liposome endocytosis in the SP-A -/- mouse. With secretagogue (8-bromoadenosine 3',5'-cyclic monophosphate) treatment, both clathrin- and actin-dependent lipid clearance were elevated in the SP-A +/+ lungs while neither pathway responded in the SP-A -/- lungs. Binding of iodinated SP-A to type II cells isolated from both genotypes of mice was similar indicating a normal SP-A receptor status in the SP-A -/- lung. Inclusion of SP-A with instilled liposomes served to "rescue" the SP-A -/- lungs by reestablishing secretagogue-dependent enhancement of liposome uptake. These data are compatible with a major role for receptor-mediated endocytosis of DPPC by granular pneumocytes, a process critically dependent on SP-A.