Pulmonary Surfactant Preserves Viability of Alveolar Type II Cells Exposed to Polymyxin B In Vitro

Background

Exogenous surfactant derived from animal lungs is applied for treatment of surfactant deficiency. By means of its rapid spreading properties, it could transport pharmaceutical agents to the terminal air spaces. The antimicrobial peptide Polymyxin B (PxB) is used as a topical antibiotic for inhalation therapy. Whereas it has been shown that PxB mixed with surfactant is not inhibiting surface activity while antimicrobiotic activity is preserved, little is known concerning the effects on synthesis of endogenous surfactant in alveolar type II cells (ATIIC).

Objective

To investigate ATIIC viability and surfactant-exocytosis depending on PxB and/or surfactant exposure.

Methods

ATIIC were isolated from rat lungs as previously described and were cultivated for 48 h. After incubation for a period of 1–5 h with either PxB (0.05 or 0.1 mg/ml), modified porcine surfactant (5 or 10 mg/ml) or mixtures of both, viability and exocytosis (spontanously and after stimulation) were determined by fluorescence staining of intracellular surfactant.

Results

PxB 0.1 mg/ml, but not porcine surfactant or porcine surfactant plus PxB reduces ATIIC-viability. Only PxB alone, but not in combination with porcine surfactant, rapidly reduces fluorescence in ATIIC at maximum within 3 h, indicating stimulation of exocytosis. Subsequent ionomycin-stimulation does not further increase exocytosis of PxB incubated ATIIC. In presence of surfactant, stimulating effects of PxB and ionomycin on exocytosis are reduced.

Conclusion

PxB alone shows negative effects on ATIIC, which are counterbalanced in mixtures with surfactant. So far, our studies found no results discouraging the concept of a combined treatment with PxB and surfactant mixtures.     

Persurf,a New Method to Improve Surfactant Delivery: A Study in Surfactant Depleted Rats

Purpose

Exogenous surfactant is not very effective in adults with ARDS, since surfactant does not reach atelectatic alveoli. Perfluorocarbons (PFC) can recruit atelectatic areas but do not replace impaired endogenous surfactant. A surfactant-PFC-mixture could combine benefits of both therapies. The aim of the proof-of-principal-study was to produce a PFC-in-surfactant emulsion (Persurf) and to test in surfactant depleted Wistar rats whether Persurf achieves I.) a more homogenous pulmonary distribution and II.) a more homogenous recruitment of alveoli when compared with surfactant or PFC alone.

Methods

Three different PFC were mixed with surfactant and phospholipid concentration in the emulsion was measured. After surfactant depletion, animals either received 30 ml/kg of PF5080, 100 mg/kg of stained (green dye) Curosurf™ or 30 ml/kg of Persurf. Lungs were fixated after 1 hour of ventilation and alveolar aeration and surfactant distribution was estimated by a stereological approach.

Results

Persurf contained 3 mg/ml phospholipids and was stable for more than 48 hours. Persurf-administration improved oxygenation. Histological evaluation revealed a more homogenous surfactant distribution and alveolar inflation when compared with surfactant treated animals.

Conclusions

In surfactant depleted rats administration of PFC-in-surfactant emulsion leads to a more homogenous distribution and aeration of the lung than surfactant alone.     

Mitochondrial ATPase activity and membrane fluidity changes in rat liver in response to intoxication with Buckthorn (Karwinskia humboldtiana)

Background

Karwinskia humboldtiana (Kh) is a poisonous plant of the rhamnacea family. To elucidate some of the subcellular effects of Kh toxicity, membrane fluidity and ATPase activities as hydrolytic and as proton-pumping activity were assessed in rat liver submitochondrial particles. Rats were randomly assigned into control non-treated group and groups that received 1, 1.5 and 2 g/Kg body weight of dry powder of Kh fruit, respectively. Rats were euthanized at day 1 and 7 after treatment.

Results

Rats under Kh treatment at all dose levels tested, does not developed any neurologic symptoms. However, we detected alterations in membrane fluidity and ATPase activity. Lower dose of Kh on day 1 after treatment induced higher mitochondrial membrane fluidity than control group. This change was strongly correlated with increased ATPase activity and pH gradient driven by ATP hydrolysis. On the other hand, membrane fluidity was hardly affected on day 7 after treatment with Kh. Surprisingly, the pH gradient driven by ATPase activity was significantly higher than controls despite an diminution of the hydrolytic activity of ATPase.

Conclusions

The changes in ATPase activity and pH gradient driven by ATPase activity suggest an adaptive condition whereby the fluidity of the membrane is altered.     

A neurotoxic phospholipase a(2) impairs yeast amphiphysin activity and reduces endocytosis

Background

Presynaptically neurotoxic phospholipases A₂ inhibit synaptic vesicle recycling through endocytosis.

Principal Findings

Here we provide insight into the action of a presynaptically neurotoxic phospholipase A₂ ammodytoxin A (AtxA) on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin.

Conclusions

We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein–protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A₂ work can open new ways to regulate endocytosis.     

In Vitro Surfactant and Perfluorocarbon Aerosol Deposition in a Neonatal Physical Model of the Upper Conducting Airways

Objective

Aerosol delivery holds potential to release surfactant or perfluorocarbon (PFC) to the lungs of neonates with respiratory distress syndrome with minimal airway manipulation. Nevertheless, lung deposition in neonates tends to be very low due to extremely low lung volumes, narrow airways and high respiratory rates. In the present study, the feasibility of enhancing lung deposition by intracorporeal delivery of aerosols was investigated using a physical model of neonatal conducting airways.

Methods

The main characteristics of the surfactant and PFC aerosols produced by a nebulization system, including the distal air pressure and air flow rate, liquid flow rate and mass median aerodynamic diameter (MMAD), were measured at different driving pressures (4–7 bar). Then, a three-dimensional model of the upper conducting airways of a neonate was manufactured by rapid prototyping and a deposition study was conducted.

Results

The nebulization system produced relatively large amounts of aerosol ranging between 0.3±0.0 ml/min for surfactant at a driving pressure of 4 bar, and 2.0±0.1 ml/min for distilled water (H₂Od) at 6 bar, with MMADs between 2.61±0.1 µm for PFD at 7 bar and 10.18±0.4 µm for FC-75 at 6 bar. The deposition study showed that for surfactant and H₂Od aerosols, the highest percentage of the aerosolized mass (∼65%) was collected beyond the third generation of branching in the airway model. The use of this delivery system in combination with continuous positive airway pressure set at 5 cmH₂O only increased total airway pressure by 1.59 cmH₂O at the highest driving pressure (7 bar).

Conclusion

This aerosol generating system has the potential to deliver relatively large amounts of surfactant and PFC beyond the third generation of branching in a neonatal airway model with minimal alteration of pre-set respiratory support.     

The Idiopathic Pulmonary Fibrosis Honeycomb Cyst Contains A Mucocilary Pseudostratified Epithelium

Background

We previously identified a MUC5B gene promoter-variant that is a risk allele for sporadic and familial Idiopathic Pulmonary Fibrosis/Usual Interstitial Pneumonia (IPF/UIP). This allele was strongly associated with increased MUC5B gene expression in lung tissue from unaffected subjects. Despite the strong association of this airway epithelial marker with disease, little is known of mucin expressing structures or of airway involvement in IPF/UIP.

Methods

Immunofluorescence was used to subtype mucus cells according to MUC5B and MUC5AC expression and to identify ciliated, basal, and alveolar type II (ATII) cells in tissue sections from control and IPF/UIP subjects. Staining patterns were quantified for distal airways (Control and IPF/UIP) and in honeycomb cysts (HC).

Results

MUC5B-expressing cells (EC) were detected in the majority of control distal airways. MUC5AC-EC were identified in half of these airways and only in airways that contained MUC5B-EC. The frequency of MUC5B+ and MUC5AC+ distal airways was increased in IPF/UIP subjects. MUC5B-EC were the dominant mucus cell type in the HC epithelium. The distal airway epithelium from control and IPF/UIP subjects and HC was populated by basal and ciliated cells. Most honeycombing regions were distinct from ATII hyperplasic regions. ATII cells were undetectable in the overwhelming majority of HC.

Conclusions

The distal airway contains a pseudostratified mucocilary epithelium that is defined by basal epithelial cells and mucus cells that express MUC5B predominantly. These data suggest that the HC is derived from the distal airway.     

Effect of Surfactant and Partial Liquid Ventilation Treatment on Gas Exchange and Lung Mechanics in Immature Lambs: Influence of Gestational Age

Objectives

Surfactant (SF) and partial liquid ventilation (PLV) improve gas exchange and lung mechanics in neonatal RDS. However, variations in the effects of SF and PLV with degree of lung immaturity have not been thoroughly explored.

Setting

Experimental Neonatal Respiratory Physiology Research Unit, Cruces University Hospital.

Design

Prospective, randomized study using sealed envelopes.

Subjects

36 preterm lambs were exposed (at 125 or 133-days of gestational age) by laparotomy and intubated. Catheters were placed in the jugular vein and carotid artery.

Interventions

All the lambs were assigned to one of three subgroups given: 20 mL/Kg perfluorocarbon and managed with partial liquid ventilation (PLV), surfactant (Curosurf®, 200 mg/kg) or (3) no pulmonary treatment (Controls) for 3 h.

Measurements and Main Results

Cardiovascular parameters, blood gases and pulmonary mechanics were measured. In 125-day gestation lambs, SF treatment partially improved gas exchange and lung mechanics, while PLV produced significant rapid improvements in these parameters. In 133-day lambs, treatments with SF or PLV achieved similarly good responses. Neither surfactant nor PLV significantly affected the cardiovascular parameters.

Conclusion

SF therapy response was more effective in the older gestational age group whereas the effectiveness of PLV therapy was not gestational age dependent.     

Ultrastructural changes of the intracellular surfactant pool in a rat model of lung transplantation-related events

Background

Ischemia/reperfusion (I/R) injury, involved in primary graft dysfunction following lung transplantation, leads to inactivation of intra-alveolar surfactant which facilitates injury of the blood-air barrier. The alveolar epithelial type II cells (AE2 cells) synthesize, store and secrete surfactant; thus, an intracellular surfactant pool stored in lamellar bodies (Lb) can be distinguished from the intra-alveolar surfactant pool. The aim of this study was to investigate ultrastructural alterations of the intracellular surfactant pool in a model, mimicking transplantation-related procedures including flush perfusion, cold ischemia and reperfusion combined with mechanical ventilation.

Methods

Using design-based stereology at the light and electron microscopic level, number, surface area and mean volume of AE2 cells as well as number, size and total volume of Lb were determined in a group subjected to transplantation-related procedures including both I/R injury and mechanical ventilation (I/R group) and a control group.

Results

After I/R injury, the mean number of Lb per AE2 cell was significantly reduced compared to the control group, accompanied by a significant increase in the luminal surface area per AE2 cell in the I/R group. This increase in the luminal surface area correlated with the decrease in surface area of Lb per AE2. The number-weighted mean volume of Lb in the I/R group showed a tendency to increase.

Conclusion

We suggest that in this animal model the reduction of the number of Lb per AE2 cell is most likely due to stimulated exocytosis of Lb into the alveolar space. The loss of Lb is partly compensated by an increased size of Lb thus maintaining total volume of Lb per AE2 cell and lung. This mechanism counteracts at least in part the inactivation of the intra-alveolar surfactant.     

Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus

Background

Influenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes.

Methods

Alveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively.

Results

We found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels.

Conclusions

Our results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.     

How Pig Sperm Prepares to Fertilize: Stable Acrosome Docking to the Plasma Membrane

Background

Mammalian sperms are activated in the oviduct. This process, which involves extensive sperm surface remodelling, is required for fertilization and can be mimicked under in vitro fertilization conditions (IVF).

Methodology/Principal Findings

Here we demonstrate that such treatments caused stable docking and priming of the acrosome membrane to the apical sperm head surface without the emergence of exocytotic membrane fusion. The interacting membranes could be isolated as bilamellar membrane structures after cell disruption. These membrane structures as well as whole capacitated sperm contained stable ternary trans-SNARE complexes that were composed of VAMP 3 and syntaxin 1B from the plasma membrane and SNAP 23 from the acrosomal membrane. This trans-SNARE complex was not observed in control sperm.

Conclusions/Significance

We propose that this capacitation driven membrane docking and stability thereof is a preparative step prior to the multipoint membrane fusions characteristic for the acrosome reaction induced by sperm-zona binding. Thus, sperm can be considered a valuable model for studying exocytosis.     

Determination of EGFR Endocytosis Kinetic by Auto-Regulatory Association of PLD1 with μ2

Background

Upon ligand binding, cell surface signaling receptors are internalized through a process tightly regulated by endocytic proteins and adaptor protein 2 (AP2) to orchestrate them. Although the molecular identities and roles of endocytic proteins are becoming clearer, it is still unclear what determines the receptor endocytosis kinetics which is mainly regulated by the accumulation of endocytic apparatus to the activated receptors.

Methodology/Principal Findings

Here we employed the kinetic analysis of endocytosis and adaptor recruitment to show that μ2, a subunit of AP2 interacts directly with phospholipase D (PLD)1, a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). We also demonstrate that the PLD1-μ2 interaction requires the binding of PLD1 with phosphatidic acid, its own product.

Conclusions/Significance

These results suggest that the temporal regulation of EGFR endocytosis is achieved by auto-regulatory PLD1 which senses the receptor activation and triggers the translocation of AP2 near to the activated receptor.     

Effect of Atomoxetine on Hyperactivity in an Animal Model of Attention-Deficit/Hyperactivity Disorder (ADHD)

Background

Hyperactivity related behaviors as well as inattention and impulsivity are regarded as the nuclear symptoms of attention-deficit/hyperactivity disorder (ADHD).

Purpose

To investigate the therapeutic effects of atomoxetine on the motor activity in relation to the expression of the dopamine (DA) D2 receptor based on the hypothesis that DA system hypofunction causes ADHD symptoms, which would correlate with extensive D2 receptor overproduction and a lack of DA synthesis in specific brain regions: prefrontal cortex (PFC), striatum, and hypothalamus.

Methods

Young male spontaneously hypertensive rats (SHR), animal models of ADHD, were randomly divided into four groups according to the daily dosage of atomoxetine and treated for 21 consecutive days. The animals were assessed using an open-field test, and the DA D2 receptor expression was examined.

Results

The motor activity improved continuously in the group treated with atomoxetine at a dose of 1 mg/Kg/day than in the groups treated with atomoxetine at a dose of 0.25 mg/Kg/day or 0.5 mg/Kg/day. With respect to DA D₂ receptor immunohistochemistry, we observed significantly increased DA D₂ receptor expression in the PFC, striatum, and hypothalamus of the SHRs as compared to the WKY rats. Treatment with atomoxetine significantly decreased DA D₂ expression in the PFC, striatum, and hypothalamus of the SHRs, in a dose-dependent manner.

Conclusion

Hyperactivity in young SHRs can be improved by treatment with atomoxetine via the DA D2 pathway.     

Disaturated-phosphatidylcholine and Surfactant protein-B turnover in human acute lung injury and in control patients

Background

Patients with Adult Respiratory Distress Syndrome (ARDS) and Acute Lung Injury (ALI) have low concentrations of disaturated-phosphatidylcholine and surfactant protein-B in bronchoalveolar lavage fluid. No information is available on their turnover.

Objectives

To analyze disaturated-phosphatidylcholine and surfactant protein-B turnover in patients with ARDS/ALI and in human adults with normal lungs (controls).

Methods

²H₂O as precursor of disaturated-phosphatidylcholine-palmitate and 1¹³C-Leucine as precursor of surfactant protein-B were administered intravenously to 12 patients with ARDS/ALI and to 8 controls. Disaturated-phosphatidylcholine and surfactant protein-B were isolated from serial tracheal aspirates, and their fractional synthetic rate was derived from the ²H and ¹³C enrichment curves, obtained by gas chromatography mass spectrometry. Disaturated-phosphatidylcholine, surfactant protein-B, and protein concentrations in tracheal aspirates were also measured.

Results

1) Surfactant protein-B turned over at faster rate than disaturated-phosphatidylcholine both in ARDS/ALI patients and in controls. 2) In patients with ARDS/ALI the fractional synthesis rate of disaturated-phosphatidylcholine was 3.1 times higher than in controls (p < 0.01), while the fractional synthesis rate of surfactant protein-B was not different. 3) In ARDS/ALI patients the concentrations of disaturated-phosphatidylcholine and surfactant protein-B in tracheal aspirates were markedly and significantly reduced (17% and 40% of the control values respectively).

Conclusions

1) Disaturated-phosphatidylcholine and surfactant protein-B have a different turnover both in healthy and diseased lungs. 2) In ARDS/ALI the synthesis of these two surfactant components may be differently regulated.     

Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging

Background

The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.

Methodology

To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.

Principal Findings

We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.

Conclusions/Signficance

Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.     

Enhanced clathrin-dependent endocytosis in the absence of calnexin

Background

Calnexin, together with calreticulin, constitute the calnexin/calreticulin cycle. Calnexin is a type I endoplasmic reticulum integral membrane protein and molecular chaperone responsible for the folding and quality control of newly-synthesized (glyco)proteins. The endoplasmic reticulum luminal domain of calnexin is responsible for lectin-like activity and interaction with nascent polypeptide chains. The role of the C-terminal, cytoplasmic portion of calnexin is not clear.

Methodology/Principal Findings

Using yeast two hybrid screen and immunoprecipitation techniques, we showed that the Src homology 3-domain growth factor receptor-bound 2-like (Endophilin) interacting protein 1 (SGIP1), a neuronal specific regulator of endocytosis, forms complexes with the C-terminal cytoplasmic domain of calnexin. The calnexin cytoplasmic C-tail interacts with SGIP1 C-terminal domains containing the adaptor complexes medium subunit (Adap-Comp-Sub) region. Calnexin-deficient cells have enhanced clathrin-dependent endocytosis in neuronal cells and mouse neuronal system. This is reversed by expression of full length calnexin or calnexin C-tail.

Conclusions/Significance

We show that the effects of SGIP1 and calnexin C-tail on clathrin-dependent endocytosis are due to modulation of the internalization of the receptor-ligand complexes. Enhanced clathrin-dependent endocytosis in the absence of calnexin may contribute to the neurological phenotype of calnexin-deficient mice.     

Duffy antigen receptor for chemokines mediates chemokine endocytosis through a macropinocytosis-like process in endothelial cells

Background

The Duffy antigen receptor for chemokines (DARC) shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.

Methodology/Principal Findings

We investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated ¹²⁵I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. ¹²⁵I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression. ¹²⁵I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF) enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.

Conclusions/Significance

These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.     

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).     

Sequential analysis of surfactant,lung function and inflammation in cystic fibrosis patients

Background

In a cross-sectional analysis of cystic fibrosis (CF) patients with mild lung disease, reduced surfactant activity was correlated to increased neutrophilic airway inflammation, but not to lung function. So far, longitudinal measurements of surfactant function in CF patients are lacking and it remains unclear how these alterations relate to the progression of airway inflammation as well as decline in pulmonary function over time.

Methods

As part of the BEAT trial, a longitudinal study to assess the course of airway inflammation in CF, we studied lung function, surfactant function and endobronchial inflammation using bronchoalveolar lavage fluid from 20 CF patients with normal pulmonary function (median FEV₁ 94% of predicted) at three times over a three year period.

Results

There was a progressive loss of surfactant function, assessed as minimal surface tension. The decline in surfactant function was negatively correlated to an increase in neutrophilic inflammation and a decrease in lung function, assessed by FEV₁, MEF_75/25%VC, and MEF_25%VC. The concentrations of the surfactant specific proteins A, C and D did not change, whereas SP-B increased during this time period.

Conclusion

Our findings suggest a link between loss of surfactant function driven by progressive airway inflammation and loss of small airway function in CF patients with limited lung disease.