Mechanical forces modulate alveolar epithelial phenotypic expression.

Physical forces play an important role in modulating lung development, growth, compliance, differentiation and metabolism. Both tonic distension and phasic changes in volume occur during development and after birth. Morphometric studies have shown that alveolar epithelial cells are distended during lung expansion from functional residual capacity. In both in vivo and in vitro model systems, mechanical distension stimulates surfactant secretion. Drawing on the results of developmental anomalies and experiments in vivo, we and others have generated the underlying hypothesis that mechanical distension promotes expression of the type I cell phenotype and inhibits expression that of the type II; contraction has the opposite effects. The results of recent experiments, using both cultured type II cells from adult rodents and fetal lung explant tissue to test this hypothesis, provide support. The molecular and biochemical mechanisms by which physical forces affect lung functions are currently under investigation.

Identification of a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells.

Here we describe a monoclonal antibody (MMC4) that recognizes a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells in the lung, proximal tubule epithelial cells in the kidney, and villus epithelial cells in the small intestine. Biochemical analysis showed that the MMC4 antigen was sensitive to heating and proteinase K digestion and that it is distributed in the detergent-rich phase after Triton X-114 phase separation. These data suggest that the MMC4 antigen is an integral membrane protein. Glycerol gradient sedimentation identified two forms of the MMC4 antigen: one with a sedimentation coefficient of 10.1 and one with a sedimentation coefficient of 1.66, suggesting that the antigen may be part of a multiprotein complex. During rat development (fetal day 16 to adult), the MMC4 antigen increased 12-fold in the lung and 200-fold in the kidney. In the intestine, the MMC4 antigen increased 150-fold by neonatal day 1 and then decreased to adult values. Our data demonstrate that the MMC4 antigen is unlike known type II cell- and Clara cell-associated proteins. The MMC4 monoclonal antibody will be useful as a marker of epithelial cell phenotype in development and injury studies.

[Ca(2+)](i) oscillations regulate type II cell exocytosis in the pulmonary alveolus.

Pulmonary surfactant, a critical determinant of alveolar stability, is secreted by alveolar type II cells by exocytosis of lamellar bodies (LBs). To determine exocytosis mechanisms in situ, we imaged single alveolar cells from the isolated blood-perfused rat lung. We quantified cytosolic Ca(2+) concentration ([Ca(2+)](i)) by the fura 2 method and LB exocytosis as the loss of cell fluorescence of LysoTracker Green. We identified alveolar cell type by immunofluorescence in situ. A 15-s lung expansion induced synchronous [Ca(2+)](i) oscillations in all alveolar cells and LB exocytosis in type II cells. The exocytosis rate correlated with the frequency of [Ca(2+)](i) oscillations. Fluorescence of the lipidophilic dye FM1-43 indicated multiple exocytosis sites per cell. Intracellular Ca(2+) chelation and gap junctional inhibition each blocked [Ca(2+)](i) oscillations and exocytosis in type II cells. We demonstrated the feasibility of real-time quantifications in alveolar cells in situ. We conclude that in lung expansion, type II cell exocytosis is modulated by the frequency of intercellularly communicated [Ca(2+)](i) oscillations that are likely to be initiated in type I cells. Thus during lung inflation, type I cells may act as alveolar mechanotransducers that regulate type II cell secretion.

The effects of mechanical forces on lung functions.

The lung is a dynamic organ that is subjected to mechanical forces throughout development and adult life. This review article addresses the types of mechanical forces in the lung and their effects on development and normal lung functions. The effects of mechanical forces on the various different cell types of the lung are discussed, as are the mechanisms underlying mechanotransduction.

A novel alveolar type I cell-specific biochemical marker of human acute lung injury.

Currently there is no recognized biochemical or molecular marker for human parenchymal lung injury analogous to markers for acute myocardial injury. Injury to the alveolar epithelial barrier is of central importance in the pathogenesis of and recovery from acute lung injury. In animal models, an alveolar type I cell-specific protein, RTI(40), has been shown to be an accurate marker of alveolar epithelial damage. We now report that HTI(56), a novel apical plasma membrane protein specific to the human type I cell, is a biochemical marker for lung injury. Using a sensitive, quantitative, light-based ELISA, we measured HTI(56) in pulmonary edema fluid from 15 patients with a clinical diagnosis of acute lung injury and 12 control patients with hydrostatic (cardiogenic) pulmonary edema. HTI(56) was also measured in plasma from these two groups and from 11 normal volunteers. The amount of HTI(56) was 4. 3-fold higher (p < 0.0001) in alveolar edema fluid and 1.4-fold higher (p < 0.05) in plasma from the patients with acute lung injury, compared with patients with hydrostatic pulmonary edema. To our knowledge, this study is the first to utilize a specific marker of alveolar epithelial damage in human disease and demonstrates the feasibility of using a blood test to detect lung parenchymal damage.

Mechanical distention modulates alveolar epithelial cell phenotypic expression by transcriptional regulation.

The development of a normal pulmonary alveolar epithelium, essential for gas exchange, is critical for the successful adaptation to extrauterine life. From observations of natural and experimental developmental abnormalities, it has been hypothesized that mechanical factors may play a role in regulating differentiation of the pulmonary alveolar epithelium. To test this hypothesis directly, we have investigated the in vitro effects of mechanical distention on the expression of specific markers for the type I and type II cell phenotypes. Fetal rat lung (18-d) explants were mechanically distended in culture for 18 h. Mechanical distention caused an increase in RTI 40 messenger RNA (mRNA), a marker of the type I cell phenotype, of 10.6 times (n = 3, P < 0.05) that of undistended controls. In contrast, mechanical distention resulted in a decrease in mRNA content of two markers of the type II cell phenotype, surfactant protein (SP)-B and SP-C. SP-B was reduced to 10 +/- 9% (n = 3, P < 0.005) and of SP-C to 12 +/- 7% (n = 3, P < 0.0001) of undistended controls. Mechanical distention had no effect on content of mRNA for SP-A or 18S ribosomal RNA. Examined by nuclear run-on assays, mechanical distention caused changes in transcriptional rates of RTI 40, SP-B, and SP-C. These data show that mechanical distention stimulates expression of a type I cell marker and inhibits expression of markers for the type II phenotype; these effects occur at least in part at the transcriptional level. These studies support the hypothesis that mechanical distention of fetal lung tissue stimulates expression of the type I cell phenotype and inhibits expression of the type II phenotype.

HTI56, an integral membrane protein specific to human alveolar type I cells.

The alveolar epithelium is composed of two morphologically distinct types of cells, Type I and Type II cells. The thin cytoplasmic extensions of Type I cells cover more than 95% of the internal surface area of the lungs. Type I cells provide the very short diffusion pathway essential for gas exchange. Because there were no biochemical markers specific for human Type I cells, we developed a strategy to produce a monoclonal antibody (MAb) specific for human Type I cells. Isolated human lung cells were used as immunogens; >5000 clones from seven fusions were screened to identify an MAb specific for a 56-kD protein of Type I cells, HTI56. By Western blotting, HTI56 is unique to the lung. By immunoelectron microscopy, it is localized to the Type I cell apical plasma membrane. The pI of HTI56 is 2.5-3.5. HTI56 is glycosylated and has the biochemical characteristics of an integral membrane protein. HTI56 is detectable by Week 20 of gestation and its expression increases in fetal lung explant culture. HTI56 should be useful as a marker for human Type I cells both morphologically and biochemically. It may also be useful in studies of disease and as a marker for lung injury.

Characterization of the gene and promoter for RTI40, a differentiation marker of type I alveolar epithelial cells.

In an effort to understand the processes that establish and maintain the differentiated state of the alveolar epithelium, we have analyzed the gene for rat type I cell 40 kD protein (RTI40), an apical integral plasma membrane protein expressed in type I but not type II alveolar epithelial cells. The RTI40 gene spans 35 kilobase pairs; it contains 6 exons and at least 6 rat Identifier repetitive elements. Three exons encode the predicted RTI40 extracellular domain and one encodes the single transmembrane spanning domain. The final exon encodes one amino acid followed by a stop codon. RTI40 gene transcription starts downstream from a TATA homology, which is immediately adjacent to putative binding sites for thyroid transcription factor 1 and Sp1. In H441 cell transfections, mutagenesis of a 5'-flanking fragment (-2496 to +104) revealed two regions that contribute to promoter activity: -1247 through -795 and -163 through -81. Heterologous promoter fusion experiments suggest that a cooperative interaction between these regions activates transcription. In transfected type II cells, deletion across the proximal region produced a 6-fold drop in promoter activity, whereas deletion across the distal region was without apparent effect. These results provide a foundation to analyze further the factors that govern alveolar epithelial cell phenotype.

Highly water-permeable type I alveolar epithelial cells confer high water permeability between the airspace and vasculature in rat lung.

Water permeability measured between the airspace and vasculature in intact sheep and mouse lungs is high. More than 95% of the internal surface area of the lung is lined by alveolar epithelial type I cells. The purpose of this study was to test whether osmotic water permeability (Pf) in type I alveolar epithelial cells is high enough to account for the high Pf of the intact lung. Pf measured between the airspace and vasculature in the perfused fluid-filled rat lung by the pleural surface fluorescence method was high (0.019 +/- 0.004 cm/s at 12 degrees C) and weakly temperature-dependent (activation energy 3.7 kcal/mol). To resolve the contributions of type I and type II alveolar epithelial cells to lung water permeability, Pf was measured by stopped-flow light scattering in suspensions of purified type I or type II cells obtained by immunoaffinity procedures. In response to a sudden change in external solution osmolality from 300 to 600 mOsm, the volume of type I cells decreased rapidly with a half-time (t1/2) of 60-80 ms at 10 degrees C, giving a plasma membrane Pf of 0.06-0.08 cm/s. Pf in type I cells was independent of osmotic gradient size and was weakly temperature-dependent (activation energy 3.4 kcal/mol). In contrast, t1/2 for type II cells in suspension was much slower, approximately 1 s; Pf for type II cells was 0.013 cm/s. Vesicles derived from type I cells also had a very high Pf of 0.06-0.08 cm/s at 10 degrees C that was inhibited 95% by HgCl2. The Pf in type I cells is the highest measured for any mammalian cell membrane and would account for the high water permeability of the lung.

Mechanical distension modulates pulmonary alveolar epithelial phenotypic expression in vitro.

The pulmonary alveolar epithelium is composed of two distinct types of cells, type I and type II cells, both of which are critical for normal lung function. On the basis of experiments of both nature and in vivo studies, it has been hypothesized that expression of the type I or type II phenotype is influenced by mechanical factors. We have investigated the effects of mechanical distension on the expression of specific markers for the type I and type II cell phenotypes in cultured alveolar type II cells. Rat alveolar type II cells were tonically mechanically distended in culture. Cells were analyzed for a marker for the type I phenotype (rTI40, an integral membrane protein specific for type I cells) and for markers for the type II phenotype [surfactant protein (SP) A, SP-B, and SP-C] as well as for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mechanical distension caused a 68 +/- 25% (n = 3) increase in mRNA content of rTI40 relative to undistended controls. In contrast, mechanical distension resulted in a decrease in mRNA content of SP-B to 35 +/- 19% (n = 3) and of SP-C to 20 +/- 6.7% (n = 3) of undistended controls. There was no effect on mRNA content of SP-A or GAPDH. The differences in mRNA content of SP-B and SP-C were found to be primarily due to changes at the transcriptional level by nuclear run-on assays. The effects on rTI40 appear to be due to posttranscriptional events. These data show that mechanical distension influences alveolar epithelial phenotypic expression in vitro, at least in part, at the transcriptional level.

Purification and analysis of RTI40, a type I alveolar epithelial cell apical membrane protein.

RTI40 is a 40-42 kDa protein that, within the lung, is specific to the apical plasma membrane of the rat alveolar type I cell. Type I cells cover greater than 95% of the internal surface area of the lung. In this report, we describe some of the physical properties of RTI40, and its purification to homogeneity. By liquid phase isoelectric focusing, the pI of the protein is 3.0+/-0.5. In two-dimensional immunoblots, there is a 1.0 pH unit charge train, suggesting post-translational modification of the protein. We have purified the protein to homogeneity by the following method. A membrane preparation from perfused rat lungs was extracted with detergent and applied to an ion-exchange column. Immunoreactive fractions from the column were pooled, dialyzed and further fractionated by reverse phase high performance liquid chromatography (HPLC). Essentially all the antigenicity was recovered in one protein peak that was homogeneous both by spectral analysis and silver-stained polyacrylamide gels. Because the purified protein was N terminus blocked, we cleaved the protein with CNBr and fractionated peptide fragments by reverse phase HPLC. Fractions were pooled and concentrated. Direct amino acid sequencing of the major peptide fragment yielded a 15 amino acid peptide homologous to a mouse osteoblast protein, OTS-8. Analysis of purified RTI40 shows that the protein contains glycan, some of which is sialic acid. Characterization of RTI40 should facilitate future studies of the functional properties of RTI40.

Loss of immunoreactivity for RTI40, a type I cell-specific protein in the alveolar epithelium of rat lungs with bleomycin-induced fibrosis.

After lung injury, the epithelial cells lining the alveolar surface in rat lung show an altered distribution of several membrane proteins. Pulmonary fibrosis was induced by intratracheal administration of bleomycin into the lung of rats and the distribution of RTI40, a recently detected alveolar epithelial type I cell antigen, was examined, as well as the relationship between RTI40 and a type I cell-specific antigen recognized by the monoclonal antibody MEP-1 and the type I cell-binding lectin Bauhinia purpurea in serial sections and double stainings. Loss of RTI40 protein was observed in fibrotic lungs, particularly in areas with obliteration of alveoli. Pre-embedding immunoelectron microscopy confirmed this observation by detection of RTI40 protein in the alveolar lumen. Western blot analysis revealed elevated levels of RTI40 in the bronchoalveolar fluid of bleomycin-treated rats with a maximum at day 7 after treatment. Twenty-eight days after bleomycin application, the bronchoalveolar fluid contained three times the amount of RTI40 x mg protein(-1) of control lungs, as determined by semiquantitative dot blot. These results suggest RTI40 as a tool for the evaluation of alveolar epithelial type I cell behaviour during re-epithelialization processes.

Lipoprotein-stimulated surfactant secretion in alveolar type II cells: mediation by heterotrimeric G proteins.

Low- and high-density lipoproteins (LDL and HDL, respectively) stimulate alveolar type II cells to secrete surfactant. Increases in phosphoinositide hydrolysis, cytosolic Ca2+, and membrane-associated protein kinase C activity precede LDL- and HDL-stimulated secretion. We report three lines of evidence supporting the hypothesis that Gi mediates LDL- and HDL-stimulated surfactant secretion and signal transduction in type II cells. First, pertussis toxin (PTX) inhibited secretion stimulated by the apolipoprotein ligands for either the LDL receptor or the HDL binding protein. Second, PTX inhibited protein kinase C activity in cell membranes stimulated by LDL or HDL. Third, treatment of cell membranes with LDL or HDL inhibited PTX-catalyzed labeling of substrates corresponding in molecular mass to Gi alpha. These observations suggest that receptor-mediated activation of Gi is required for LDL- and HDL-stimulated secretion and that LDL and HDL activate Gi. These studies in type II cells are the first to support the hypothesis that Gi mediates the effects of LDL or HDL on important phenotype-specific functions of differentiated cells.

Maintenance of the differentiated type II cell phenotype by culture with an apical air surface.

The study of differentiated functions of alveolar type II cells has been hampered because of the lack of good in vitro systems. We report that culture of type II cells on collagen gels with an apical surface exposed to air promotes expression of differentiated type II cell characteristics. Cells cultured in this manner are cuboidal, contain lamellar bodies, and produce tubular myelin; in addition, they secrete phosphatidylcholine in response to exogenous ATP. Cultures contain mRNA for surfactant proteins A, B, and C and surfactant proteins A, B, and D. In contrast, when type II cells are cultured with an apical surface exposed to liquid rather than to air, the cells are squamous, do not express surfactant proteins or their respective mRNA, and do not contain lamellar bodies or produce tubular myelin. Type II cells cultured on plastic for 7 days, which no longer express mRNA for surfactant proteins, can be induced to express these mRNA by changing culture conditions to that of an air surface. The culture system described in this paper should be useful for studies of surfactant metabolism, regulation of alveolar epithelial phenotypic expression, and the processing of transiently expressed transgenes.

Nitric oxide attenuates lung endothelial injury caused by sublethal hyperoxia in rats.

Inhaled nitric oxide (NO) has been shown to prevent oxidant-induced lung injury in isolated-perfused lung models, whereas NO-derived oxidants may contribute to acute lung injury secondary to hyperoxia. Whether inhaled NO improves or contributes to oxidant-mediated lung injury may depend on the timing of NO administration or how lung injury is assessed. The objective of these studies was to determine whether inhaled NO (20 ppm) was protective or harmful to the different lung barriers when it was administered with 95% O2 for 60 h in Sprague-Dawley rats by measuring fluid transport and permeability to protein across the lung endothelium and the alveolar epithelium. Inhaled NO significantly attenuated the O2-mediated lung endothelial injury and abolished the increase in the bronchoalveolar lavage fluid content of rTI40, a specific and sensitive marker of alveolar epithelial type I cell injury, that occurs secondary to hyperoxia. In conclusion, inhaled NO administered with high concentrations of O2 may protect the lung endothelium and the alveolar epithelium against O2-mediated injury.

Effects of mechanical factors on growth and maturation of the lung in fetal sheep.

Previous fetal studies indicated that endocrine factors control surfactant maturation, whereas mechanical forces affect lung growth, but not surfactant. We altered mechanical forces in fetal sheep lungs at 100-108 days gestation by tracheal ligation (TL, n = 15, 7 successful studies) to accelerate lung growth, transection of cervical spinal cord (TCSC, n = 17, 6 successful studies) to produce lung hypoplasia, or sham operation (n = 11, 6 successful studies). The reasons for the high mortality rates are not known. At delivery (130-142 days), groups were similar in gestational age, weight, and cortisol. Effects on lung growth were similar to, but effects on surfactant differed from, previous reports. TL increased lung growth but decreased saturated phosphatidylcholine (SatPC) and surfactant protein (SP)A and apparently decreased SP-B and relative numbers of alveolar type II cells (based on immunohistochemical studies of 1 animal in each group); TCSC had opposite effects. In contrast to a previous study (J. A. Kitterman, G. C. Liggins, G. A. Campos, J. A. Clements, C. S. Forster, C. H. Lee, and R. K. Creasy, J. Appl. Physiol, 51: 384-390, 1981), SatPC did not correlate with cortisol. We conclude that altering mechanical forces in fetal lung affects not only lung growth but also surfactant maturation and possibly alveolar epithelial differentiation and disturbs the normal correlation between cortisol and surfactant. Associated changes in insulin-like growth factor I (IGF-I; increased by TL, P = 0.003) suggest a possible role for IGF-I in these effects.

Biochemical detection of type I cell damage after nitrogen dioxide-induced lung injury in rats.

We have previously shown that injury to lung epithelial type I cells can be detected biochemically by measuring the airway fluid content of a type I cell-specific protein, rTI40, in a model of severe acute lung injury [M. C. McElroy, J.-F. Pittet, S. Hashimoto, L. Allen, J. P. Wiener-Kronish, and L. G. Dobbs. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L181-L186, 1995]. The first objective of the present study was to evaluate the utility of rTI40 in the assessment of alveolar injury in a model of milder acute lung injury. Rats were exposed to 18 parts/ million NO2 for 12 h; control rats received filtered air for 12 h. In NO2-exposed rats, the total amount of rTI40 in bronchoalveolar fluid was elevated 2-fold compared with control values (P < 0.001); protein concentration was 8.5-fold of control values (P < 0.001). The increase in rTI40 was associated with morphological evidence of injury to type I cells limited to the proximal alveolar regions of the lung. The second objective was to correlate the severity of alveolar type I cell injury with functional measurements of lung epithelial barrier integrity. NO2 inhalation stimulated distal air space fluid clearance despite a significant increase in lung endothelial and epithelial permeability to protein. These data demonstrate that rTI40 is a useful biochemical marker for mild focal injury and that exposure to NO2 alters lung barrier function. Taken together with our earlier studies, these results suggest that the quantity of recoverable rTI40 can be used as an index of the severity of damage to the alveolar epithelium.

Evidence for G beta gamma-mediated cross-talk in primary cultures of lung alveolar cells. Pertussis toxin-sensitive production of cAMP.

In the presence of activated Gs alpha, the beta gamma complex of heterotrimeric G proteins (beta gamma) stimulates adenylyl cyclase (AC) in membranes prepared from cells expressing recombinant AC II or AC IV. Conditional stimulation of AC by beta gamma has been hypothesized to integrate cross-talk between Gs- and non-Gs-coupled regulation of cellular cAMP (Tang, W. J., and Gilman, A. G. (1991) Science 254, 1500-1503). Although observations in cells expressing recombinant receptors, G alpha s, and AC support this hypothesis (Federman, A. D., Conklin, B. R., Schrader, K. A., Reed, R. R., and Bourne, H. R. (1992) Nature 356, 159-161), this mechanism has not been investigated in differentiated cells. Expression of AC II has been reported only in lung, olfactory, and brain tissue. We found that rat lung alveolar type II cells express AC II and IV. Therefore, we hypothesized that beta gamma conditionally stimulates AC in type II cells. Consistent with this hypothesis, we found that the alpha 2-adrenergic agonist UK14304 did not affect basal cAMP in type II cells but potentiated terbutaline-stimulated cAMP accumulation. Treatment of cells with pertussis toxin partially inhibited terbutaline-stimulated cAMP accumulation and completely inhibited the effects of UK14304. In type II cell membranes, purified beta gamma tripled the terbutaline-stimulated increase in AC activity. In contrast, beta gamma inhibited AC activity in the absence of terbutaline. The addition of purified Go alpha blocked beta gamma-induced effects. In summary, type II cells expressing endogenous AC II and IV regulate cAMP accumulation and AC activity in a manner consistent with conditional stimulation by beta gamma. These observations support the overall hypothesis that conditional stimulation of AC by beta gamma integrates cross-talk between signal transduction systems in differentiated cells.

A type I cell-specific protein is a biochemical marker of epithelial injury in a rat model of pneumonia.

In this study we determined whether the alveolar fluid content of a specific epithelial type I cell protein, rTI40, can be used as a biochemical marker for lung injury. A model of alveolar epithelial injury was developed by instilling Pseudomonas aeruginosa bacteria (PA103) into the airspaces of anesthetized, ventilated rats. After 6 h, the alveolar fluid content of rTI40 from PA103-treated rats was increased over 80-fold in comparison to alveolar fluid from control rats (P < 0.05). This increase in rTI40 correlated with both morphological evidence of injury to alveolar epithelial type I cells and increased permeability of the alveolar epithelium to protein tracers. In contrast, the lactate dehydrogenase activity of alveolar fluid from PA103-treated rats was elevated only threefold over control values at 6 h (P < 0.05). In a second study using a less injurious strain of P. aeruginosa (PA103 exsA::omega), the alveolar fluid content of rTI40 was the same as control values. These findings indicate that the alveolar fluid content of a type I cell-specific protein can be used as a sensitive and specific biochemical marker of type I cell injury.