Proteolipidic vectors for gene transfer to the lung.

In order to develop improved synthetic gene transfer vectors, we have synthesized bifunctional peptides composed of a DNA binding peptide (P2) and ligand peptides selected by the phage display technique on tracheal epithelial cells. We have evaluated the capacity of these peptides to enhance the gene transfer efficiency of the cationic lipid DOTAP to the mouse lung. To optimize the in vivo transfection efficiency, we first compared the efficiency of DOTAP to transfect the lung by either intravenous injection or aerosolization. We then tested DNA/Peptide/DOTAP complexes formed at different Peptide/DNA and DOTAP/DNA charge ratios. Under optimal conditions, precompaction of DNA by peptide P2 gave a higher expression in the mouse lung using the luciferase reporter gene than DOTAP/DNA complexes. A further increase of transfection efficiency was obtained with the bifunctional peptide P2-9. Experiments performed with the GFP reporter gene showed expression in the alveolar parenchyme.

Surfactant protein A and lipid are internalized via the coated-pit pathway by type II pneumocytes.

Surfactant protein (SP) A and SP-A-mediated lipid uptake by isolated type II cells were investigated with biochemical and morphological methods. Inhibition of coated-pit function by potassium depletion severely reduced both SP-A and SP-A-mediated lipid internalization. Lipid uptake in the absence of SP-A was not affected. With confocal laser scanning microscopy and immunoelectron microscopy, SP-A and lipid predominantly (60%) colocalized in intracellular vesicles carrying early endosomal markers (EEA1) 5 min after endocytosis but were negative for the late endosomal or lysosomal marker LAMP-1. As estimated by subcellular fractionation, at this time point, 23% of the internalized SP-A and 45% of internalized lipid were localized within light (<0.38 M sucrose) fractions, which contain lamellar bodies and are positive for EEA1. The remaining label was predominantly found within EEA1-positive and plasma membrane-containing subfractions (> or = 1 M sucrose). We suggest that in isolated type II cells in vitro, SP-A and lipid are taken up together via the coated-pit pathway and that at early time points, both components reside in the same early endosomal compartment.

Distinct changes in pulmonary surfactant homeostasis in common beta-chain- and GM-CSF-deficient mice.

Pulmonary alveolar proteinosis (PAP) is caused by inactivation of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or GM receptor common beta-chain (beta(c)) genes in mice [GM(-/-), beta(c)(-/-)], demonstrating a critical role of GM-CSF signaling in surfactant homeostasis. To distinguish possible phenotypic differences in GM(-/-) and beta(c)(-/-) mice, surfactant metabolism was compared in beta(c)(-/-), GM(-/-), and wild-type mice. Although lung histology in beta(c)(-/-) and GM(-/-) mice was indistinguishable, distinct differences were observed in surfactant phospholipid and surfactant protein concentrations and clearance from lungs of beta(c)(-/-) and GM(-/-) mice. At 1-2 days of age, lung saturated phosphatidylcholine (Sat PC) pool sizes were higher in wild-type, beta(c)(-/-), and GM(-/-) mice compared with wild-type adult mice. In wild-type mice, Sat PC pool sizes decreased to adult levels by 7 days of age; however, Sat PC increased with advancing age in beta(c)(-/-) and GM(-/-) mice. Postnatal changes in Sat PC pool sizes were different in GM(-/-) compared with beta(c)(-/-) mice. After 7 days of age, the increased lung Sat PC pool sizes remained constant in beta(c)(-/-) mice but continued to increase in GM(-/-) mice, so that by 56 days of age, lung Sat PC pools were increased three- and sixfold, respectively, compared with wild-type controls. After intratracheal injection, the percent recovery of [(3)H]dipalmitoylphosphatidylcholine and (125)I-recombinant surfactant protein (SP) C was higher in beta(c)(-/-) compared with wild-type mice, reflecting decreased clearance in the receptor-deficient mice. The defect in clearance was significantly more severe in GM(-/-) than in beta(c)(-/-) mice. The ratio of SP Sat PC to SP-A, -B, and -C was similar in bronchoalveolar lavage fluid (BALF) from adult mice of all genotypes, but the ratio of SP-D to Sat PC was markedly increased in beta(c)(-/-) and GM(-/-) mice (10- and 5-fold, respectively) compared with wild-type mice. GM-CSF concentrations were increased in BALF but not in serum of beta(c)(-/-) mice, consistent with a pulmonary response to the lack of GM-CSF signaling. The observed differences in surfactant metabolism suggest the presence of alternative clearance mechanisms regulating surfactant homeostasis in beta(c)(-/-) and GM(-/-) mice and may provide a molecular basis for the range in severity of PAP symptoms. surfactant metabolism; alveolar macrophage; granulocyte-macrophage colony-stimulating factor

IL-4 increases surfactant and regulates metabolism in vivo.

Mice that express interleukin (IL)-4 in Clara cells (CCSP-IL-4) develop chronic airway inflammation and an alveolar proteinosis-like syndrome. To identify the role of IL-4 in surfactant homeostasis, we measured lipid and protein metabolism in the lungs of CCSP-IL-4 mice in vivo. Alveolar saturated phosphatidylcholine (Sat PC) pools were increased 6.5-fold and lung tissue Sat PC pools were increased 4. 8-fold in the IL-4 transgenic mice. Whereas surfactant protein (SP) A was increased proportionately to Sat PC, SP-D was increased approximately 90-fold in the IL-4 mice compared with wild-type mice and was associated with 2.8-fold increase in SP-D mRNA. The incorporation of palmitate and choline into Sat PC was increased about twofold in CCSP-IL-4 mice. Although trace doses of radiolabeled Sat PC were cleared from the air spaces and lungs of CCSP-IL-4 mice more slowly than in wild-type mice, net clearance of Sat PC from the lungs of CCSP-IL-4 mice was sixfold higher in the IL-4 mice than in wild-type mice because of the larger Sat PC pool sizes. Expression of IL-4 in Clara cells increased surfactant lipid synthesis and clearance, establishing a new equilibrium with increased surfactant pools and an alveolar proteinosis associated with a selective increase in SP-D protein, demonstrating a previously unexpected effect of IL-4 in pulmonary surfactant homeostasis.

Dosing and delivery of a recombinant surfactant in lung-injured adult sheep.

The purpose of this study was to evaluate a surfactant based on a recombinant surfactant protein-C (rSP-C) at three different doses (25, 100, and 200 mg lipid/kg) in the saline lavage adult sheep model of acute lung injury. All three doses resulted in significant improvements in gas exchange, although the 100 and 200 mg/kg doses were superior to the 25 mg/kg dose. There were no significant differences in effect of the 100 and 200 mg/kg doses. In addition, the physiologic efficacy and lobar surfactant distribution patterns were similar when two different surfactant delivery methods were compared. This comparison involved administering the surfactant directly into each lobe under bronchoscopic guidance, versus instilling the surfactant through an endotracheal tube into the lungs. However, the former technique took significantly longer to perform (24.5 +/- 3.3 min versus 11.6 +/- 2.5 min, p < 0.05) and required a skilled bronchoscopist. In conclusion, rSP-C surfactant was effective in improving gas exchange in this model of lung injury, although higher doses were required for optimal responses. The bronchoscopic administration technique produced results similar to those of the tracheal instillation method, but had some disadvantages that may limit the widespread clinical use of this technique in patients with lung injury.

Surfactant protein-C in ventilated premature lamb lung.

Although surfactants containing only lipids and surfactant protein C (SP-C) or SP-C analogs can be effective for the treatment of surfactant deficiency in animal models, there is no information concerning the alveolar or lung clearance of SP-C. Because the other lipid and protein components of surfactant are cleared very slowly from the preterm lung, we hypothesized that SP-C also would be cleared slowly. Therefore, we compared the losses of iodinated native SP-C (nSP-C) and a recombinant SP-C analog (rSP-C, phenylalanines in positions 4 and 5 and isoleucine in position 32 of the human sequence) to [14C]dipalmitoylphosphatidylcholine (DPPC) after airway administration at birth of trace or treatment doses of surfactant given to preterm lambs. In preterm lambs given trace doses at 134-136 d gestation, alveolar [14C]DPPC and [125I]rSP-C decreased to 14.7% recovery for DPPC and 8.3% recovery for rSP-C after 2 h ventilation. There was no loss of [14C]DPPC from the total lungs (alveolar wash + lung tissue), and approximately 20% of the [125I]rSP-C was lost from the lungs. For 128 d gestational age lambs treated with 100-mg/kg doses of surfactants containing nSP-C or 2% rSP-C, the alveolar and total lung recoveries for [125I]nSP-C or [125I]rSP-C were equivalent to that of [14C]DPPC after 5 h ventilation. These results demonstrate that nSP-C and rSP-C have alveolar clearances and accumulations into preterm lung tissue that are similar to those of DPPC.

Clearance of SP-C and recombinant SP-C in vivo and in vitro.

Surfactant protein (SP) C metabolism was evaluated in vivo by measurements of the clearance of bovine native SP-C (nSP-C) and a recombinant SP-C (rSP-C) in rabbits and mice and in vitro by the uptake into MLE-12 cells. rSP-C is the 34-amino acid human sequence with phenylalanine instead of cysteine in positions 4 and 5 and isoleucine instead of methionine in position 32. Alveolar clearances of iodinated SP-C and rSP-C after tracheal instillation were similar and slower than those for dipalmitoyl phosphatidylcholine (DPC) in the rabbit. nSP-C and rSP-C were cleared from rabbit lungs similarly to DPC, each with a half-life (t1/2) of approximately 11 h. In mice, the clearance of rSP-C from the lungs was slower (t1/2 28 h) than the clearance of DPC (t1/2 12 h). Liposome-associated dinitrophenyl-labeled rSP-C was taken up by MLE-12 cells, and the uptake was inhibited by excess nSP-C. The pattern of inhibition of dinitrophenyl-rSP-C uptake by SP-B, but not by SP-A, was similar to that previously reported for nSP-C. Clearance kinetics of nSP-C were similar to previous measurements of pulmonary clearance of SP-B in rabbits and mice. rSP-C has clearance kinetics and uptake by cells similar to those of nSP-C.

Effects of ventilation style on surfactant metabolism and treatment response in preterm lambs.

We investigated whether the style of ventilation would influence respiratory physiology or surfactant metabolism in surfactant-treated preterm lambs. Preterm lambs were delivered at 131 +/- 1 d gestation and treated with an organic solvent extract of sheep surfactant (100 mg/kg). The lambs were randomized to ventilation peiods of 2 h, 5 h, 10 h, or 24 h, and to ventilation with a low rate (15 breaths/min) and high VT (15 ml/kg), with a high rate (50 breaths/min) and low VT (8 ml/kg), or with high-frequency oscillatory ventilation (HFOV). Gas exchange and lung volumes were similar across time and for the different ventilation styles. Saturated phosphatidylcholine (SatPC) in alveolar lavage was lower for the HFOV group than for the other ventilation groups at 10 h and 24 h. The rate of loss of surfactant protein B (SP-B) from these preterm animals' lungs was slow and not influenced by ventilation style. The percentages of surfactants in large-aggregate forms were not changed by style of ventilation, and the large-aggregate surfactants had excellent function when tested in surfactant-deficient preterm rabbits. Alveolar lavage protein was low (30 ml/kg), and tissue hyaluronan did not change with time or ventilation style. In preterm lambs ventilated without causing injury, the extreme styles of ventilation examined in the study had minimal effects on lung function, surfactant function, or surfactant metabolism.

Distinct effects of SP-A and SP-B on endocytosis of SP-C by pulmonary epithelial cells.

Binding and endocytosis of surfactant protein (SP) C were assessed in a mouse pulmonary adenocarcinoma cell line, MLE-12, and in isolated rat pulmonary type II epithelial cells. Binding and uptake of SP-C were detected using fluorescently labeled SP-C and dinitrophenyl-labeled SP-C (DNP-SP-C). Endocytosis of DNP-SP-C was visualized by immunocytochemistry and light microscopy. Endocytosis of DNP-SP-C occurred in MLE-12 cells, pulmonary type II epithelial cells, and NIH/3T3 cells, indicating that uptake of SP-C does not have an absolute requirement for a cell-specific receptor. After 30-60 min at 37 degrees C, DNP-SP-C was concentrated in large intracellular bodies in MLE-12 cells. Endocytosis of DNP-SP-C by MLE-12 cells or type II epithelial cells was decreased by SP-B or SP-B and SP-A together. SP-A alone did not inhibit DNP-SP-C uptake. Endocytosis of DNP-SP-C was inhibited by a 10-fold excess of lipid vesicles containing SP-C but not by a 10-fold excess of lipid alone. The inhibitory effect of SP-B on SP-C uptake may play a role in maintaining surface-active material at the air-liquid interface.

Anti-HER2 immunoliposomes for targeted therapy of human tumors.

Anti-HER2 immunoliposomes (ILs) have been constructed by conjugation of Fab' fragments of recombinant humanized monoclonal antibody rhuMAbHER2 to small sterically stabilized unilamellar liposomes, to create a targeted drug delivery vehicle for the treatment of HER2 (c-erbB-2, neu)-overexpressing cancers. Parameters affecting in vitro binding and internalization of ILs include liposome composition, Fab' linkage site and Fab' density. Anti-HER2 ILs have been constructed to optimize intracellular drug delivery. Doxorubicin (dox)-loaded ILs are highly stable and exhibit prolonged circulation in rats. In nude mice bearing HER2-overexpressing tumor xenografts, anti-HER2 ILs administered i.v. resulted in efficient tumor localization, with penetration of the ILs throughout the tumor mass and accumulation within tumor cells. In contrast, non-targeted liposomes resulted in extracellular tumor accumulation only. In multiple HER2-overexpressing human breast tumor xenograft models, treatment with dox-loaded anti-HER2 ILs produces significantly increased antitumor cytotoxicity as compared to free dox or dox-loaded non-targeted liposomes and significantly less systemic toxicity than free dox. To explore further the intracellular delivery advantages of ILs, anti-HER2 ILs bearing cationic lipids are being developed for nucleic acid delivery. These cationic immunoliposomes mediate efficient and specific transfection of target cells with reporter genes, as well as intracellular delivery of labeled oligonucleotides. Thus, anti-HER2 ILs represent an efficient and feasible strategy to achieve targeted intracellular delivery of therapeutic agents.

Alveolar-to-vascular leakage of surfactant protein A in ventilated immature newborn rabbits.

We measured alveolar-to-vascular leakage of surfactant protein A (SP-A) in immature newborn rabbits delivered at a gestational age of 27 days. Experimental animals received, via a tracheal cannula, 2 ml/kg of a mixture of modified porcine surfactant (Curosurf, 80 mg/ml) and human recombinant SP-A (4 mg/ml). Littermate controls received the same volume of human SP-A in saline (4 mg/ml). After 30 min of artificial ventilation with a frequency of 40/min and an inspiration time of either 0.75 or 0.45 s, blood was sampled from the right ventricle and the lungs were lavaged. The content of human SP-A in serum and lung lavage fluid was determined with ELISA kits, and the alveolar-to-vascular leak expressed as the quotient of total SP-A in serum and lavage fluid. The leak in control animals amounted to about 2% of SP-A in lung wash and was several times higher in these animals than in those receiving surfactant. The leak was of the same order irrespective of whether the animals were ventilated with long or short inspiration time. We speculate that serum levels of SP-A may reflect the degree of lung injury in various forms of respiratory failure.

Clearance of surfactant protein A from rabbit lungs.

Surfactant protein A (SP-A) is a major surfactant protein with multiple biophysical, metabolic, and host defense functions. To further characterize its metabolism in vivo, we measured clearance of SP-A from adult rabbit lungs. Trace amounts of [125I]SP-A radiolabeled by the Bolton-Hunter method and mixed with [3H]dipalmitoylphosphatidylcholine (DPPC) were given intratracheally via a bronchoscope to rabbits. Groups of five to six animals were studied 10 min to 16 h after labeled surfactant administration. After collection of alveolar washes, lamellar bodies were isolated from lung tissue. Macrophages also were isolated from alveolar washes. [125I]SP-A was cleared more rapidly than DPPC from the airspaces. Both [125I]SP-A and [3H]DPPC were lost exponentially from the total lungs, with half-life values of 6.5 h for SP-A and 12 h for DPPC (P < 0.01). In macrophages, the highest radioactivities for SP-A and DPPC were at 10 to 45 min and the radiolabels subsequently disappeared similarly. In lamellar bodies, 125I and 3H radioactivities per mumol saturated phosphatidylcholine (Sat PC) increased in parallel and were highest at 2 h. Whereas radiolabeled lipids were recovered in lamellar bodies for up to 16 h, iodinated SP-A was lost, indicating less recycling of SP-A than DPPC. We previously showed independent pathways of SP-A and Sat PC secretion in rabbits. These results demonstrate the different clearance kinetics of these two principle components of surfactant.

Effect of endocytosis inhibitors on alveolar clearance of albumin, immunoglobulin G, and SP-A in rabbits.

Protein in the alveolar space may be cleared by endocytosis and degradation inside alveolar epithelial cells, by transcytosis across the alveolar epithelium, or by restricted diffusion through the epithelium. The relative contributions of these three pathways to clearance of large quantities of protein from the air spaces is not known. This study investigated the effects of monensin and nocodazole, agents which inhibit endocytosis in cell culture, on alveolar epithelial protein transport in anesthetized rabbits. There was evidence that monensin and nocodazole inhibited endocytosis by the alveolar epithelium in vivo. Nocodazole increased the number of vesicles in the alveolar epithelium and capillary endothelium. Monensin increased vesicle density in the endothelium. These results suggested that the inhibitors disrupted microtubules or interrupted cellular membrane traffic in the lung. Both inhibitors decreased lung parenchymal uptake of immunoreactive human albumin from the air spaces. Monensin and nocodazole inhibited albumin uptake in cultured alveolar type II cells. Monensin increased the amount of 125I-labeled surfactant protein A associated with the lungs, compared with the quantity remaining in the air space 2 h after instillation. Although the drugs decreased alveolar epithelial protein uptake, they did not decrease alveolar clearance of 125I-labeled immunoglobulin G or 131I-labeled albumin in anesthetized rabbits. Thus monensin- and nocodazole-sensitive protein-uptake pathways do not account for most alveolar protein clearance when the distal air spaces are filled with a protein solution.

Uptake of pulmonary surfactant protein C into adult rat lung lamellar bodies.

Previous studies have provided evidence that a large proportion of secreted surfactant lipids is taken up from the alveolar air space by type II cells, incorporated into lamellar bodies, and resecreted. Our goal was to characterize the clearance of exogenously administered recombinant surfactant protein C (SP-C) and to determine if SP-C is taken up by type II cells and incorporated into lamellar bodies. SP-C was radiolabeled by alkylation with [3H]iodoacetic acid and retained its ability to enhance phospholipid adsorption to an air-liquid interface. A mixture of 100 micrograms phospholipid radiolabeled with [14C]dipalmitoylphosphatidylcholine and 10 micrograms SP-C was instilled into the lungs of spontaneously breathing anesthetized adult rats. At later times, the lungs were lavaged and subcellular organelles were isolated. The radioactivity of both phospholipids and SP-C (expressed as disintegrations per minute per microgram phospholipid) in lamellar body fractions increased up to 4 h postinstillation and began to decline after approximately 4 h. The results of this study suggest that SP-C and dipalmitoylphosphatidylcholine are taken up promptly from the alveolar air space and are incorporated into lamellar bodies with time courses that do not differ greatly.

Interaction of C1q receptor with lung surfactant protein A.

Earlier we reported the purification of C1q receptor (C1qR) from U937 cells and human tonsil lymphocytes (Malhotra, R. and Sim, R. B., Biochem. J. 1989. 218: 625) and showed that C1qR interacts with the ligands C1q, mannose-binding protein, conglutinin and lung surfactant protein A (SP-A) (Malhotra, R., Thiel, S., Reid, K. B. M. and Sim, R. B., J. Exp. Med. 1990. 172: 955). C1qR was characterized as an acidic glycoprotein, which, when solubilized, exists as a dimer of Mr 115,000 under non-denaturing conditions. In this article we provide evidence for binding of radioiodinated SP-A to U937 cells and show that binding of radioiodinated SP-A to U937 cells is specific, saturable, salt dependent and is inhibited by purified C1qR and by C1q. The interaction of SP-A with U937 cells was found to up-regulate the surface expression of C1qR. Incubation of SP-A with U937 cells at 37 degrees C for 80 min was found to increase the receptor number per cell. Increase in receptor number was inhibited in the presence of sodium azide and monensin. Incubation of cells with calcium ionophore A23187 induced increased surface expression in the absence of SP-A. The results indicate that interaction of SP-A with U937 cells triggers the expression of an intracellular pool of C1qR.

Distribution of endotracheally instilled surfactant protein SP-C in lung-lavaged rabbits.

In lung-lavaged surfactant-deficient rabbits (n = 6) requiring artificial ventilation, porcine surfactant was instilled endotracheally. This resulted in improvement of lung function so that the animals could be weaned off artificial ventilation. The animals were killed 4 1/2 h after surfactant administration and the porcine surfactant protein was localized in the lung with a MAb. We found surfactant protein in all lobes of the lung but the distribution was not homogeneous. Surfactant protein C was found in less than 15% of the alveolar spaces and in less than 1% of the bronchi.

Lung surfactant-associated proteins and type IV collagen share common epitopes. An immunocytochemical demonstration.

Among the surfactant-associated proteins (SP-A) characterized so far, there is a group of glycoproteins 26 to 34 kDa that features collagenlike sequences near their N-terminal end. We herein report the cross-reactivity of a rabbit polyclonal antibody to EHS tumor-derived type IV collagen towards rat SP-A. Rat lung tissues were processed for the localization of both type IV collagen and SP-A by high-resolution immunocytochemistry, applying the protein A-gold technique with specific antibodies. In addition to the various basal laminae of the pulmonary tissue, the antitype IV collagen antibody labeled the surfactant material found in alveolar spaces and macrophages, as well as in type II pneumocytes. The surfactant nature of the alveolar material labeled by the antiserum to type IV collagen was confirmed by the positive labeling obtained using an antibody to SP-A. This antibody labeled specifically the alveolar surfactant material, without binding any basal laminae. Several control experiments demonstrated the specificity of each labeling. These results were further supported by immunoblot experiments on nitrocellulose membrane. These findings thus provide further support to the existence of collagenlike sequences on SP-A, and further demonstrate that this structural similarity with collagens can lead to some cross-antigenicity.