Portal de Periódicos da CAPES

Transgenic Overexpression of Granulocyte Macrophage-Colony Stimulating Factor in the Lung Prevents Hyperoxic Lung Injury

2003; Elsevier BV; Volume: 163; Issue: 6 Linguagem: Inglês

10.1016/s0002-9440(10)63594-8

1525-2191

Robert Paine, Steven E. Wilcoxen, Susan B. Morris, Cláudio Sartori, Carlos E. O. Baleeiro, Michael A. Matthay, Paul J. Christensen,

Respiratory Support and Mechanisms

Granulocyte macrophage-colony stimulating factor (GM-CSF) plays an important role in pulmonary homeostasis, with effects on both alveolar macrophages and alveolar epithelial cells. We hypothesized that overexpression of GM-CSF in the lung would protect mice from hyperoxic lung injury by limiting alveolar epithelial cell injury. Wild-type C57BL/6 mice and mutant mice in which GM-CSF was overexpressed in the lung under control of the SP-C promoter (SP-C-GM mice) were placed in >95% oxygen. Within 6 days, 100% of the wild-type mice had died, while 70% of the SP-C-GM mice remained alive after 10 days in hyperoxia. Histological assessment of the lungs at day 4 revealed less disruption of the alveolar wall in SP-C-GM mice compared to wild-type mice. The concentration of albumin in bronchoalveolar lavage fluid after 4 days in hyperoxia was significantly lower in SP-C-GM mice than in wild-type mice, indicating preservation of alveolar epithelial barrier properties in the SP-C-GM mice. Alveolar fluid clearance was preserved in SP-C-GM mice in hyperoxia, but decreased significantly in hyperoxia-exposed wild-type mice. Staining of lung tissue for caspase 3 demonstrated increased apoptosis in alveolar wall cells in wild-type mice in hyperoxia compared to mice in room air. In contrast, SP-C-GM mice exposed to hyperoxia demonstrated only modest increase in alveolar wall apoptosis compared to room air. Systemic treatment with GM-CSF (9 μg/kg/day) during 4 days of hyperoxic exposure resulted in decreased apoptosis in the lungs compared to placebo. In studies using isolated murine type II alveolar epithelial cells, treatment with GM-CSF greatly reduced apoptosis in response to suspension culture. In conclusion, overexpression of GM-CSF enhances survival of mice in hyperoxia; this effect may be explained by preservation of alveolar epithelial barrier function and fluid clearance, at least in part because of reduction in hyperoxia-induced apoptosis of cells in the alveolar wall. Granulocyte macrophage-colony stimulating factor (GM-CSF) plays an important role in pulmonary homeostasis, with effects on both alveolar macrophages and alveolar epithelial cells. We hypothesized that overexpression of GM-CSF in the lung would protect mice from hyperoxic lung injury by limiting alveolar epithelial cell injury. Wild-type C57BL/6 mice and mutant mice in which GM-CSF was overexpressed in the lung under control of the SP-C promoter (SP-C-GM mice) were placed in >95% oxygen. Within 6 days, 100% of the wild-type mice had died, while 70% of the SP-C-GM mice remained alive after 10 days in hyperoxia. Histological assessment of the lungs at day 4 revealed less disruption of the alveolar wall in SP-C-GM mice compared to wild-type mice. The concentration of albumin in bronchoalveolar lavage fluid after 4 days in hyperoxia was significantly lower in SP-C-GM mice than in wild-type mice, indicating preservation of alveolar epithelial barrier properties in the SP-C-GM mice. Alveolar fluid clearance was preserved in SP-C-GM mice in hyperoxia, but decreased significantly in hyperoxia-exposed wild-type mice. Staining of lung tissue for caspase 3 demonstrated increased apoptosis in alveolar wall cells in wild-type mice in hyperoxia compared to mice in room air. In contrast, SP-C-GM mice exposed to hyperoxia demonstrated only modest increase in alveolar wall apoptosis compared to room air. Systemic treatment with GM-CSF (9 μg/kg/day) during 4 days of hyperoxic exposure resulted in decreased apoptosis in the lungs compared to placebo. In studies using isolated murine type II alveolar epithelial cells, treatment with GM-CSF greatly reduced apoptosis in response to suspension culture. In conclusion, overexpression of GM-CSF enhances survival of mice in hyperoxia; this effect may be explained by preservation of alveolar epithelial barrier function and fluid clearance, at least in part because of reduction in hyperoxia-induced apoptosis of cells in the alveolar wall. Exposure of mammals to high concentrations of oxygen for prolonged periods results in noncardiogenic pulmonary edema, acute lung injury, and eventually, death. Hyperoxia-induced lung injury has become a well-recognized animal model for studies of the pathophysiology of acute lung injury. Studies of the mechanisms of hyperoxic lung injury have focused primarily on two cells. Early studies emphasized the role of pulmonary capillary endothelial cells.1Folz R Piantadosi C Crapo J Oxygen toxicity.in: Crystal R West J Barnes P Weibel E The Lung: Scientific Foundations. Lippincott-Raven, Philadelphia1997: 2713-2722Google Scholar After early endothelial cell injury, there is accumulation of inflammatory cells within the vascular space, and leak of protein-rich fluid into the interstitium. More recent studies have focused on the role of alveolar epithelial cells in these models. The alveolar epithelium provides a very tight barrier to fluid entry into the alveolar space.2Gorin AB Stewart PA Differential permeability of endothelial and epithelial barriers to albumin flux.J Appl Physiol Respir Environ Exer Physiol. 1979; 47: 1315-1324PubMed Google Scholar Alveolar epithelial cells also actively remove salt and water from the distal airspaces to maintain gas exchange.3Matthay MA Folkesson HG Clerici C Lung epithelial fluid transport and the resolution of pulmonary edema.Physiol Rev. 2002; 82: 569-600Crossref PubMed Scopus (563) Google Scholar After prolonged hyperoxic stress alveolar epithelial cell function is severely impaired, with decreased barrier function,4Holm BA Notter RH Siegle J Matalon S Pulmonary physiological and surfactant changes during injury and recovery from hyperoxia.J Appl Physiol. 1985; 59: 1402-1409Crossref PubMed Scopus (123) Google Scholar diminished active fluid reabsorption capacity and Na/K ATPase activity,5Olivera WG Ridge KM Sznajder JI Lung liquid clearance and Na,K-ATPase during acute hyperoxia and recovery in rats.Am J Respir Crit Care Med. 1995; 152: 1229-1234Crossref PubMed Scopus (103) Google Scholar and altered surfactant protein and lipid accumulation.4Holm BA Notter RH Siegle J Matalon S Pulmonary physiological and surfactant changes during injury and recovery from hyperoxia.J Appl Physiol. 1985; 59: 1402-1409Crossref PubMed Scopus (123) Google Scholar, 6Minoo P King RJ Coalson JJ Surfactant proteins and lipids are regulated independently during hyperoxia.Am J Physiol. 1992; 263: L291-L298PubMed Google Scholar, 7Nogee LM Wispe JR Clark JC Weaver TE Whitsett JA Increased synthesis and mRNA of surfactant protein A in oxygen-exposed rats.Am J Respir Cell Mol Biol. 1989; 1: 119-125Crossref PubMed Scopus (37) Google Scholar It is likely that both necrosis and apoptosis of alveolar wall cells contribute to hyperoxic lung injury.8Barazzone C Horowitz S Donati YR Rodriguez I Piguet PF Oxygen toxicity in mouse lung: pathways to cell death.Am J Respir Cell Mol Biol. 1998; 19: 573-581Crossref PubMed Scopus (230) Google ScholarThe pathophysiological mechanisms involved in oxygen-induced lung injury are complex.1Folz R Piantadosi C Crapo J Oxygen toxicity.in: Crystal R West J Barnes P Weibel E The Lung: Scientific Foundations. Lippincott-Raven, Philadelphia1997: 2713-2722Google Scholar A variety of experimental manipulations can influence the outcome ofextended hyperoxia in animal models. Maneuvers that directly increase pulmonary antioxidant defenses9Ilizarov AM Koo HC Kazzaz JA Mantell LL Li Y Bhapat R Pollack S Horowitz S Davis JM Overexpression of manganese superoxide dismutase protects lung epithelial cells against oxidant injury.Am J Respir Cell Mol Biol. 2001; 24: 436-441Crossref PubMed Scopus (60) Google Scholar, 10Simonson SG Welty-Wolf KE Huang YC Taylor DE Kantrow SP Carraway MS Crapo JD Piantadosi CA Aerosolized manganese SOD decreases hyperoxic pulmonary injury in primates. I. Physiology and biochemistry.J Appl Physiol. 1997; 83: 550-558PubMed Google Scholar, 11Welty-Wolf KE SS Huang YC Kantrow SP Carraway MS Chang LY Crapo JD Piantadosi CA Aerosolized manganese SOD decreases hyperoxic pulmonary injury in primates. II. Morphometric analysis.J Appl Physiol. 1997; 83: 559-568PubMed Google Scholar are protective; manipulations of pulmonary inflammation, including pretreatment with lipopolysaccharide,12Frank L Summerville J Massaro D Protection from oxygen toxicity with endotoxin. Role of the endogenous antioxidant enzymes of the lung.J Clin Invest. 1980; 65: 1104-1110Crossref PubMed Scopus (133) Google Scholar tumor necrosis factor,13Tsan MF White JE Santana TA Lee CY Tracheal insufflation of tumor necrosis factor protects rats against oxygen toxicity.J Appl Physiol. 1990; 68: 1211-1219Crossref PubMed Scopus (63) Google Scholar or interleukin (IL)-1,14Tsan MF Lee CY White JE Interleukin 1 protects rats against oxygen toxicity.J Appl Physiol. 1991; 71: 688-697Crossref PubMed Scopus (51) Google Scholar or transgenic expression of IL-1115Waxman AB Einarsson O Seres T Knickelbein RG Warshaw JB Johnston R Homer RJ Elias JA Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation.J Clin Invest. 1998; 101: 1970-1982Crossref PubMed Scopus (152) Google Scholar or IL-1316Corne J Chupp G Lee CG Homer RJ Zhu Z Chen Q Ma B Du Y Roux F McArdle J Waxman AB Elias JA IL-13 stimulates vascular endothelial cell growth factor and protects against hyperoxic acute lung injury.J Clin Invest. 2000; 106: 783-791Crossref PubMed Scopus (138) Google Scholar can protect rodents from death because of extended hyperoxia. Growth factors for alveolar epithelial cells17Panos RJ BP Simonet WS Rubin JS Smith LJ Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats.J Clin Invest. 1995; 96: 2026-2033Crossref PubMed Scopus (219) Google Scholar and pulmonary endothelial cells16Corne J Chupp G Lee CG Homer RJ Zhu Z Chen Q Ma B Du Y Roux F McArdle J Waxman AB Elias JA IL-13 stimulates vascular endothelial cell growth factor and protects against hyperoxic acute lung injury.J Clin Invest. 2000; 106: 783-791Crossref PubMed Scopus (138) Google Scholar also can render mice tolerant of levels of oxygen that are normally lethal.Granulocyte macrophage-colony stimulating factor (GM-CSF) is a potent growth factor originally recognized for its effects on survival, proliferation, maturation, and differentiation of hematopoietic cells. It is now clear that pulmonary GM-CSF plays a central role in homeostasis in the normal lung. Gene-targeted mice deficient in GM-CSF (GM-CSF−/− mice) develop alveolar proteinosis18Dranoff G Crawford AD Sadelain M Ream B Rashid A Bronson RT Dickersin GR Bachurski CJ Mark EL Whitsett JA Mulligan RC Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis.Science. 1994; 264: 713-716Crossref PubMed Scopus (747) Google Scholar because of abnormal surfactant turnover.19Ikegami M Ueda T Hull W Whitsett JA Mulligan RC Dranoff G Jobe AH Surfactant metabolism in transgenic mice after granulocyte macrophage-colony stimulating factor ablation.Am J Physiol. 1996; 270: L650-L658PubMed Google Scholar These mice demonstrate increased susceptibility to pneumonia with bacteria20LeVine AM Reed JA Kurak KE Cianciolo E Whitsett JA GM-CSF-deficient mice are susceptible to pulmonary group B streptococcal infection.J Clin Invest. 1999; 103: 563-569Crossref PubMed Scopus (151) Google Scholar and fungi21Paine III, R Preston AM Wilcoxen S Jin H Siu BB Morris SB Reed JA Ross G Whitsett JA Beck JM Granulocyte-macrophage colony-stimulating factor in the innate immune response to Pneumocystis carinii pneumonia in mice.J Immunol. 2000; 164: 2602-2609Crossref PubMed Scopus (129) Google Scholar because of impaired alveolar macrophage function. Expression of GM-CSF exclusively in the lungs of GM-CSF−/− mice is sufficient to restore normal surfactant metabolism and normal host defense function.20LeVine AM Reed JA Kurak KE Cianciolo E Whitsett JA GM-CSF-deficient mice are susceptible to pulmonary group B streptococcal infection.J Clin Invest. 1999; 103: 563-569Crossref PubMed Scopus (151) Google Scholar, 21Paine III, R Preston AM Wilcoxen S Jin H Siu BB Morris SB Reed JA Ross G Whitsett JA Beck JM Granulocyte-macrophage colony-stimulating factor in the innate immune response to Pneumocystis carinii pneumonia in mice.J Immunol. 2000; 164: 2602-2609Crossref PubMed Scopus (129) Google Scholar, 22Huffman JA Hull WM Dranoff G Mulligan RC Whitsett JA Pulmonary epithelial cell expression of GM-CSF corrects the alveolar proteinosis in GM-CSF-deficient mice.J Clin Invest. 1996; 97: 649-655Crossref PubMed Scopus (232) Google Scholar, 23Ikegami M Jobe AH Huffman Reed JA Whitsett JA Surfactant metabolic consequences of overexpression of GM-CSF in the epithelium of GM-CSF-deficient mice.Am J Physiol. 1997; 273: L709-L714PubMed Google Scholar, 24Paine III, R Morris SB Jin H Wilcoxen SE Phare SM Moore BB Coffey MJ Toews GB Impaired functional activity of alveolar macrophages from GM-CSF-deficient mice.Am J Physiol. 2001; 281: L1210-L1218Google Scholar, 25Shibata Y Berclaz PY Chroneos ZC Yoshida M Whitsett JA Trapnell BC GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU. 1.Immunity. 2001; 15: 557-567Abstract Full Text Full Text PDF PubMed Scopus (454) Google ScholarIn addition to its predicted effects on alveolar macrophages, GM-CSF is a potent growth factor for alveolar epithelial cells. In vitro, GM-CSF is a mitogen for rat26Huffman Reed JA Rice WR Zsengeller ZK Wert SE Dranoff G Whitsett JA GM-CSF enhances lung growth and causes alveolar type II epithelial cell hyperplasia in transgenic mice.Am J Physiol. 1997; 273: L715-L725PubMed Google Scholar or murine (unpublished observations) alveolar epithelial cells in primary culture. The present study was undertaken to determine whether overexpression of GM-CSF in the lung would impact the response of mice to hyperoxia. We now report that SP-C-GM mice are relatively tolerant of extended hyperoxia that is lethal to wild-type mice. This enhanced survival is a consequence of diminished apoptosis and preservation of alveolar epithelial cell function in the face of hyperoxic stress.Materials and MethodsAnimalsWild-type C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Bitransgenic mice in which GM-CSF is expressed exclusively in the lungs, were generated from GM-CSF-deficient mice by transgenic expression of a chimeric gene containing GM-CSF under the SP-C promoter (SP-C-GM mice).22Huffman JA Hull WM Dranoff G Mulligan RC Whitsett JA Pulmonary epithelial cell expression of GM-CSF corrects the alveolar proteinosis in GM-CSF-deficient mice.J Clin Invest. 1996; 97: 649-655Crossref PubMed Scopus (232) Google Scholar The specificity of the SP-C promoter results in targeted expression of GM-CSF exclusively by type II alveolar epithelial cells of these SP-C-GM mice. Founder SP-C-GM mice were kindly provided by Dr. J Whitsett (Children's Hospital, Cincinnati, OH). Although GM-CSF is not detectable by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage (BAL) fluid of normal wild-type mice, BAL fluid GM-CSF concentration is >100 pg/ml in the SP-C-GM mice.21Paine III, R Preston AM Wilcoxen S Jin H Siu BB Morris SB Reed JA Ross G Whitsett JA Beck JM Granulocyte-macrophage colony-stimulating factor in the innate immune response to Pneumocystis carinii pneumonia in mice.J Immunol. 2000; 164: 2602-2609Crossref PubMed Scopus (129) Google Scholar All mice were housed in microisolator cages under laminar flow hoods in an isolation room of the Animal Care Facilities at the University of Michigan and the Ann Arbor Veterans Affairs Medical Center. Mice were supplied with autoclaved bedding, food, and water. The Animal Care Committees at the University of Michigan and the Ann Arbor Veterans Affairs Medical Center approved all procedures.In Vivo Exposure of Mice to Hyperoxia and Treatment with rmGM-CSFFor exposure to hyperoxia, mice in microisolator cages were placed in a Plexiglas chamber (Reming BioInstruments Inc., Redfield, NY). Mice continued to receive food and water ad libitum. Oxygen from an H cylinder was administered into the chamber under control of a Pro-ox device (Reming BioInstruments Inc.) that continuously measured oxygen tension and adjusted flow to maintain a steady-state fraction of oxygen of >95%. In selected experiments mice were treated with rmGM-CSF (9 μg/kg/day; R&D Systems, Minneapolis, MN) or an equal volume of mouse serum, given by subcutaneous injection. Mice were treated daily for 4 days during exposure to hyperoxia.BAL Fluid AnalysisMice were euthanized with pentobarbital and the lungs perfused via the right ventricle until the effluent was free of blood. The trachea was cannulated and the lungs were lavaged with a total of 3 ml of phosphate-buffered saline (PBS) in aliquots of 0.5 ml. The lavage aliquots for each mouse were pooled and the cell-free supernatant was collected after centrifugation (400 × g). The concentration of murine albumin in the BAL fluid was determined by ELISA (Bethyl Laboratories, Montgomery, TX).Alveolar Fluid ResorptionThe rate of alveolar fluid clearance was determined from the change throughout time in concentration of radiolabeled albumin that had been instilled into the distal airspaces via the trachea, using standard methods adapted for mice.27Fukuda N Folkesson HG Matthay MA Relationship of interstitial fluid volume to alveolar fluid clearance in mice: ventilated vs. in situ studies.J Appl Physiol. 2000; 89: 672-679Crossref PubMed Scopus (77) Google Scholar, 28Ma T Fukuda N Song Y Matthay MA Verkman AS Lung fluid transport in aquaporin-5 knockout mice.J Clin Invest. 2000; 105: 93-100Crossref PubMed Scopus (254) Google Scholar Baseline alveolar fluid clearance was determined in SP-C-GM and wild-type mice. Alveolar fluid clearance was also measured in both groups of mice after 80 hours of exposure to hyperoxia.Preparation of InstillateThe instillate consisted of 5 g/100 ml bovine serum albumin (Sigma Chemical Co., St. Louis, MO) in Ringer's lactate adjusted to 330 mOsm/kg H2O with NaCl to be isosmolar with mouse plasma, and 0.1 μCi of 131I-labeled albumin (Merck-Frost, Montreal, Canada) as the labeled alveolar protein tracer.Surgical PreparationMice were euthanized by an overdose of pentobarbital sodium (200 mg/kg i.p.). The trachea was dissected and cannulated with a 20-gauge, trimmed Angiocath plastic needle (Becton Dickinson, Sandy, UT). The lungs were kept inflated with 5 cmH2O of continuous positive airway pressure and oxygenated with 100% oxygen throughout the experiment. The body temperature was maintained at 37 to 38°C with an external lamp, as in previous studies.General ProtocolIn all studies, 13 ml/kg of the instillate was delivered throughout 30 seconds into both lungs through the tracheal cannula. After 2 and 30 minutes, an alveolar fluid sample (0.05 to 0.10 ml) was aspirated with a 1-ml syringe directly connected to the 20-gauge angiocath. The aspirate was weighed and the radioactivity measured in a γ-counter. Alveolar fluid clearance (percent of instilled fluid volume) was calculated by measuring the increase in tracer-labeled albumin (131I-albumin) concentration in the instilled solution. Because the initial volume of the instilled solution and the initial and final radioactivity of the samples were known, alveolar fluid clearance could be determined by using the following mass-balance equation: alveolar fluid clearance = (1 − radioactivity in the instilled sample/radioactivity in the final sample) × 100, where alveolar fluid clearance is expressed as mean ± SD percentage of the initial volume of instillate that was cleared from the distal air spaces during the 30 minutes. Using this same protocol, pulmonary edema induced by hyperoxia-mediated acute lung injury was also quantified by calculating the decrease of the radioactivity (because of dilution) from the initial instillate in the sample collected 2 minutes after the instillation.Lung Histology and Staining for Caspase 3At appropriate time points, mice were euthanized with pentobarbital and the lungs perfused via the right ventricle until the effluent was free of blood. For selected photomicrographs, lungs were fixed in 2% glutaraldehyde, dehydrated in ethanol, and infiltrated and embedded in Polybed resin (Ernest F. Fullan, Inc., Schenectady, NY). Semithin (1 μm) plastic sections were prepared and stained with toluidine blue stain. For histological scoring of lung injury, the lungs were removed and inflated first with air, then with neutral buffered formalin. Paraffin-embedded tissue blocks were sectioned and stained with hematoxylin and eosin. The extent of histological injury was scored on a scale of 0, no injury; 1, minimal abnormality on searching; 2, mild alveolar wall edema, few red cells; 3, severe edema of the alveolar wall, mild alveolar exudates, red cells in the alveolar space; or 4, extensive alveolar exudates, obvious alveolar wall disruption. Sections were scored by an observer blinded to the identity of the sections. Injury scores are presented as median values. Staining for activated caspase 3 to detect apoptotic cells was performed according to the manufacturer's protocol (R&D Systems) on deparaffinized lung sections that had been fixed in paraformaldehyde (4%).Measurement of Relative Apoptosis and of Vascular Endothelial Growth Factor (VEGF) in Lung HomogenatesMice were euthanized and the pulmonary circulation perfused with saline until the effluent was free of blood. The lungs were removed from the proximal airways with sharp scissors and homogenized using a Tissue Tearor (Biospec Products, Inc.). After removal of debris by low-speed centrifugation, histone-associated DNA was determined by ELISA (Cell Death ELISA; Roche, Indianapolis, IN) as a measure of apoptosis. VEGF protein in the lung homogenate was measured by ELISA (R&D Systems).Isolation and Culture of Murine Alveolar Epithelial CellsMurine type II alveolar epithelial cells were isolated by the method of Corti and colleagues.29Corti M Brody AR Harrison JH Isolation and primary culture of murine alveolar type II cells.Am J Respir Cell Mol Biol. 1996; 14: 309-315Crossref PubMed Scopus (274) Google Scholar Mice were sedated with pentobarbital, secured to a dissecting board and exsanguinated by cutting the inferior vena cava. The anterior thoracic wall was removed and the left ventricle was cut with sharp scissors. The pulmonary vasculature was perfused with normal saline via a direct right ventricular puncture until the lungs were visually free of blood. The trachea was cannulated with a 20-gauge intravenous catheter secured with a suture. The lungs were filled with 1 to 2 ml of Dispase (Worthington Biochemical Corp., Lakewood, NJ) via a syringe connected to the tracheal catheter. Low-melt agarose (1%, 0.45 ml prewarmed to 45°C) was infused via the tracheal catheter and the lungs were suspended in ice-cold PBS for 2 minutes to allow the agarose to harden in the airways. The lungs then were immersed in Dispase (2 ml) at room temperature for 45 minutes. The lungs then were chilled again briefly in ice-cold PBS and transferred to a sterile Petri dish containing Dulbecco's modified Eagle's medium with 0.01% DNase. Using forceps, lung tissue was teased away from the airways, which were removed. The media and lung tissue were transferred to a trypsinizing flask and gently stirred with a magnetic stir bar for 10 minutes. The suspension was filtered successively through 100-, 40-, and 25-μm nylon mesh filters to create a single cell suspension. The cells were collected by centrifugation, counted, and resuspended in Dulbecco's modified Eagle's medium with biotinylated anti-CD-32 (FcγR) (0.65 μg/million cells) and anti-CD-45 (common leukocyte antigen) (1.5 μg/million cells). After incubation at 37°C for 30 minutes, the cells were pelleted, counted, resuspended in 7 ml of Dulbecco's modified Eagle's medium, and added to prewashed streptavidin-coated magnetic particles. The mixture was incubated for 30 minutes at room temperature with gentle rocking. The tube then was attached to a magnetic separator for 15 minutes to remove bone marrow-derived cells. Cells not bound with magnetic particles were recovered from the tube with a glass pipette, pelleted, and suspended in culture media. Viability was >97% by trypan blue exclusion. The cells were plated overnight in 60-mm culture plates. The nonadherent cells, including type II alveolar epithelial cells, were recovered and counted. Viability was >97% by trypan blue exclusion. These cells were >95% vimentin-negative. The percentage of vimentin-positive cells is similar to what we have achieved using a widely accepted isolation of rat type II cells described by Dobbs and colleagues.30Dobbs LG Gonzalez R Williams MC An improved method for isolating type II cells in high yield and purity.Am Rev Respir Dis. 1986; 134: 141-145PubMed Google Scholar For suspension culture, cells were suspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin, and streptomycin, and cultured in Teflon containers at 1 × 106 cells/ml at 37°C in an atmosphere of 7% CO2 in air.Statistical MethodsSurvival data are compared using chi-square analysis. In other experiments, data are expressed as mean ± SEM and compared by t-test (two groups), or by analysis of variance with the Neuman-Keuls multiple range test (more than two groups). Ordinal data are evaluated by the Kruskal-Wallis test. All tests are performed using the InStat software program (version 3.01; GraphPad Software, San Diego, CA). Data are considered statistically significant if P values are 95% oxygen, 100% of the mice had died by day 6 in hyperoxia (Figure 1). Mice surviving beyond day 3 were clearly impaired, with limited movement, hunched backs, and burrowing behavior. In contrast, SP-C-GM mice demonstrated remarkable tolerance to hyperoxia. All of the SP-C-GM mice remained alive after 6 days in >95% oxygen, when control mice were all dead. After 10 days in hyperoxia, 70% of the SP-C-GM mice remained alive. The survivors were active, moving about the cages in no apparent distress, and continuing to take food and water. Thus, transgenic overexpression of GM-CSF in the alveolar space resulted in significantly enhanced tolerance of hyperoxic stress.Pulmonary Histology of Wild-Type and SP-C-GM Mice Exposed to HyperoxiaPhotomicrographs of semithin sections of representative lungs from wild-type and SP-C-GM mice after 4 days in hyperoxia are shown in Figure 2. As described previously by Huffman Reed and colleagues,26Huffman Reed JA Rice WR Zsengeller ZK Wert SE Dranoff G Whitsett JA GM-CSF enhances lung growth and causes alveolar type II epithelial cell hyperplasia in transgenic mice.Am J Physiol. 1997; 273: L715-L725PubMed Google Scholar at baseline, the SP-C-GM mice had increased numbers of alveolar macrophages, and qualitatively increased numbers of type II alveolar epithelial cells compared to wild-type mice. After 4 days in hyperoxia wild-type mice developed modest alveolar exudate and some blebbing of cells along the alveolar wall, best seen under high magnification (Figure 2C). These changes were less evident in SP-C-GM mice in hyperoxia (Figure 2D). To provide a semiquantitative assessment of histological evidence of lung injury, paraffin sections were scored for parameters of lung injury. The median score for mice of both strains in normoxia was 0. Histological measures of injury after hyperoxia were relatively modest in wild-type mice, and were significantly reduced in SP-C-GM mice (Figure 2E). Thus, overexpression of GM-CSF resulted in decreased histological evidence of lung injury after exposure to hyperoxia.Figure 2.Histological appearance of lungs after 4 days in hyperoxia. After 4 days in an atmosphere of >95% oxygen mice were euthanized and semithin sections of lung were prepared and stained with toluidine blue for light microscopy. A and C: Representative sections of lungs of wild-type mice. B and D: Representative sections from SP-C-GM mice. E: The extent of histological injury in the lungs of wild-type (cross-hatched bar) and SP-C-GM (solid bar) mice that had been in hyperoxia for 4 days was scored on paraffin-embedded sections, as described in Materials and Methods, on a scale of 0 to 4 by two observers blinded to the identity of the sections. The median score for mice of both strains in normoxia was 0. Ordinal data are presented as median values. *, P < 0.05 versus wild type by Student's t-test. Original magnifications: ×40 (A and B); ×100 (C and D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Leak of Albumin into the Alveolar Space after Exposure to HyperoxiaMurine albumin was measured in BAL fluid from wild-type and SP-C-GM mice in normoxia and after exposure to hyperoxia (Figure 3). In wild-type mice, albumin in the BAL fluid, as an indication of increased protein permeability across the alveolar epithelium, was significantly increased after 4 days in hyperoxia. In contrast, BAL fluid albumin remained near normoxic levels in SP-C-GM mice exposed to hyperoxia. Thus, hyperoxia resulted in noncardiogenic pulmonary edema in wild-type mice, while SP-C-GM mice were protected from protein leak across the alveolar wall in response to this stress.Figure 3.Effect of overexpression of GM-CSF on BAL fluid albumin concentration in hyperoxia. Wild-type and SP-C-GM mice were placed in an atmosphere of >95% oxygen. After 2 or 4 days the concentration of mu