Septal Tips Research Articles

Galectin-1 in secondary alveolar septae of neonatal mouse lung

In mice, alveolarization occurs during postnatal days 4 through 12, when secondary alveolar septae create thin-walled alveoli in the distal lung. We hypothesized that genes predominantly expressed in newly forming secondary alveolar septae influence the process of alveolarization. To address this hypothesis, tips of secondary alveolar septae were isolated from sections of postnatal day 6 mouse lung tissue using laser capture microdissection. Total RNA was isolated and amplified from the dissected alveolar septal tips and from intact postnatal day 6 lung tissue. Gene expression in the samples was characterized using Affymetrix mouse U74AN2 GeneChips. Galectin-1 was an abundantly expressed transcript that was enriched in the alveolar septal tips compared with levels in the whole lung tissue. Galectins are beta-galactoside-binding proteins involved in the regulation of cell proliferation, differentiation, and apoptosis in fibroblasts, muscle cells and endothelial cells, cell types that are present in the alveolar wall. Immunostaining in postnatal day 6 lung tissue confirmed that galectin-1 protein is concentrated in the tips of secondary alveolar septae, predominantly in myofibroblasts. Fibroblasts isolated from day 6 neonatal mouse lung tissue contained galectin-1 protein. Real-time PCR demonstrated that galectin-1 mRNA levels in mouse lung tissue peak at postnatal day 6. Immunoblot analysis confirmed that peak levels of lung galectin-1 protein are found at postnatal days 6 to 12. The increased expression of galectin-1 at the site and time of ongoing alveolarization in the newborn mouse is suggestive that galectin-1 may play an important role in this critical aspect of lung development.

Disordered Pulmonary Myofibroblast Distribution and Elastin Expression in Preterm Infants with Ureaplasma Urealyticum Pneumonitis

Respiratory colonization of preterm infants with Ureaplasma urealyticum is a significant risk factor for bronchopulmonary dysplasia, a chronic lung disease characterized by arrest of alveolar development, variable interstitial fibrosis, and disordered elastic fibers in the distal airspaces. As indicated in previous studies, moderate to severe fibrosis is a hallmark of pathology in the Ureaplasma-infected preterm lung. To further characterize the preterm lung's response to Ureaplasma, lung specimens from 4 gestational controls (GC), 12 other pneumonia and 5 Ureaplasma-infected infants were analyzed by immunohistochemistry for alpha-smooth muscle actin (alphaSMA) and transforming growth factor beta1 (TGFbeta1), Hart's elastin staining, and in situ hybridization for tropoelastin (TE) expression. Cells positive for alphaSMA were observed in thickened, extensive bundles surrounding terminal airspaces in Ureaplasma and other pneumonia cases compared to individual myofibroblasts in GC. The myofibroblast pattern correlated with the severity of fibrosis, but not duration of ventilation. Transforming growth factor beta1 immunostaining was primarily localized to alveolar macrophages and was increased in Ureaplasma more than in other pneumonia cases. Elastic fibers and TE-expressing cells were spatially limited to emerging septal tips in GC. In pneumonia cases, increased deposition of elastic fibers was observed surrounding terminal airspaces, but TE expression was similar to GC. In Ureaplasma specimens, accumulation of elastic fibers correlated with duration of ventilation, and TE expression was extensive throughout the walls of terminal airspaces. These findings suggest that Ureaplasma is associated with alveolar macrophage TGFbeta1 immunostaining and myofibroblast proliferation contributing to abnormal septation, interstitial fibrosis, and a prolonged and strong elastogenic response in the preterm lung.

8 Galectin-1 is Located in Fibroblasts of Neonatal Mouse Lung and is up-Regulated by Retinoic Acid

Background: Our previous work demonstrated that galectin-1 is enriched in the tips of developing secondary alveolar septa in neonatal mouse lung. Thus, we hypothesize that galectin-1 is a regulator and/or marker of alveologenesis, a process that is impaired in infants with bronchopulmonary dysplasia.Objective: In order to further elucidate the role of galectin-1 in distal lung development, we characterized the localization of galectin-1 in the developing lung and evaluated the effects of retinoic acid, a known stimulator of alveolar formation, on galectin-1 expression.Design/Methods: the lungs of postnatal day six mice were inflation fixed and immunostained for galectin-1, surfactant protein B (a marker of type II alveolar epithelial cells) and endoglin (an endothelial cell marker). In addition, fibroblasts were isolated from postnatal lung tissue, maintained in vitro, then treated with retinoic acid at a 1 micromolar concentration for 24 hours. Immunoblot analysis was used to evaluate the levels of galectin-1 protein in the cultured lung fibroblasts.Results: The pattern of galectin-1 immunostaining in neonatal mouse lung differs markedly from that of surfactant protein B and endoglin, demonstrating that galectin-1 is localized to neither the type II alveolar cell nor the microvascular endothelium. The galectin-1 stained cells are located in the center of the secondary septal tips, making it likely that the galectin-1 is contained in fibroblasts. Fibroblasts isolated from neonatal mouse lung were found to contain galectin-1 protein, and galectin-1 levels were increased after treatment of the fibroblasts with retinoic acid.Conclusions: In the neonatal mouse lung, galectin-1 is localized in secondary septal wall fibroblasts, a cell type which is important in the pathogenesis of bronchopulmonary dysplasia. The up-regulation of galectin-1 expression in lung fibroblasts represents a potential mechanism by which retinoic acid stimulates alveologenesis, since galectin-1 has been shown to promote the growth and differentiation of smooth muscle cells and endothelial cells, which are both important components of the developing alveolar wall.

Galectin-1 Expression is Increased in The Secondary Alveolar Tips of Mouse Lung.

Bronchopulmonary dysplasia, a common complication of prematurity, is characterized by inadequate alveolarization. The process of alveolarization, by which the lung forms mature gas-exchanging units, is not well understood. In mice, alveolarizaion occurs during postnatal days four through twelve, when the formation of secondary septa creates thin walled alveoli. The purpose of this study was to investigate the genes involved in this process. RNA was isolated from dissected tips of secondary septa and whole lung tissue of day six postnatal mouse lung. The tips of secondary septa were obtained via laser capture microscopy of frozen sections. Total RNA was isolated from the tip and whole lung samples and amplified in the same manner. Affymetrix gene profiling was then performed, using mouse U74Av2 GeneChips. The signal for galectin-1 mRNA was six fold higher in the secondary septal tips than in whole lung tissue (p<0.05). Galectins, or S-type lectins, are beta-galactoside-binding proteins involved in the regulation of cell proliferation, differentiation and apoptosis. Galectin-1 is a homodimer of two 14 kDa subunits. It is the most abundant galectin in the lung. To confirm the relative abundance of galectin-1 in secondary septal tips versus whole lung, immunostaining of sections of day six postnatal and adult mouse lung tissue was performed. Staining for galectin-1 was concentrated in the tips of the secondary septa in the day six postnatal tissue. Furthermore, staining was dramatically increased in the day six mouse lung tissue when compared to staining levels in the adult lung sections. Immunoblot analysis of lung homogenates obtained at different stages of lung development demonstrated that a peak of galectin-1 expression occurs at postnatal days six and twelve, corresponding to the time of alveolarization. The increased expression of galectin-1 at the site and time of ongoing alveolarization suggests that it may play a role in this important aspect of lung development. Supported by NIH grants HL-62861 and HL-07638.

Interaction of Integrin Receptors with Extracellular Matrix is Involved in Trophoblast Giant Cell Migration in Bovine Placentomes

Integrins are heterodimeric glycoproteins involved in cell–cell and cell–extracellular matrix adhesion and signal transduction. We evaluated the distribution and the putative role of integrin receptors and extracellular matrix (ECM) proteins during trophoblast giant cell (TGC) migration and fusion with uterine epithelial cells in the cow. Placentomes from 24 cows, covering day 80 to day 270 of gestation, were used for indirect immunohistochemistry against integrin subunits α 1, α 2, α 3, α 4, α 5, α 6, α v, β 1, β 3, β 4and ECM proteins collagen type I and IV, fibronectin, laminin. The basement membranes of fetal and maternal epithelia and endothelia were immunoreactive for laminin, fibronectin and collagen IV. Collagens I and IV were found in maternal stroma, while fibronectin was present in fetal and maternal stroma. The integrin subunits α 2, α 6and β 1were observed in basal aspects of fetal and maternal epithelial and endothelial cells. Additionally, the α 6and β 1integrin subunits were colocalized with laminin on TGC. The integrin α 2subunit was also found on TGC, but localized with a strong gradient to the basal side. Cells of the maternal connective tissue, including endothelium, expressed α 1, α 2, α 3, α 5, α 6, α v, β 3and β 4. The expression of α 2, α 5, α v, β 3and β 4occured mainly in the septal tips. Cells of the fetal mesenchyme were positive for integrin subunits α 1, α 2, α 3, α 4, α 5, α 6, and β 1. Our results indicate that α 2β 1collagen and α 6β 1laminin receptors anchor epi- and endothelial cells to basement membranes. We suggest that TGC migrate along a matrix of laminin and maintain cell–cell contact with mononuclear trophoblast cells via α 2β 1heterodimers. Integrins in maternal stroma and fetal mesenchyme may be involved in the regulation of proliferation and differentiation of maternal septa and fetal villi.

Morphology of aging lung in F344/N rat: alveolar size, connective tissue, and smooth muscle cell markers.

This study investigated the morphological changes of lungs in F344/N rats (9-36 months old). We initially examined general and quantitative morphological changes, and then we used immunohistochemistry to detect distributional changes in collagen subtypes (types I, III, and IV) and smooth muscle cell (SMC) markers (alpha-smooth muscle actin (ASMA), gamma-smooth muscle actin (GSMA), desmin, and vimentin) in the lungs. In 24-month-old rats, alveolar ducts and alveolar sacs were enlarged, and alveoli were wider and shallower than in younger animals. In old rats (>/=27 months), terminal and respiratory bronchioles and alveolar ducts were dilated and alveoli were more extended than in 24-month-old rats. No age-related distributional changes were observed for collagen types I, III, and IV as revealed by immunohistochemistry, or elastin as revealed by resorsin fuchsin. SMCs in the extra- and intrapulmonary bronchi were immunoreactive for ASMA, GSMA, and desmin, but not for vimentin at all ages. In old rats (>/=27 months), SMCs were loosely arranged in comparison with younger animals, and stainability for GSMA and desmin was decreased. In the respiratory bronchioles and alveolar ducts, a few cells immunoreactive for ASMA and vimentin were observed in the smooth muscle aggregations of the alveolar orifice in rats younger than 12 months. In older rats (>20 months), cells immunoreactive for ASMA and vimentin were increased in septal tips. In conclusion, extension of distal airways and immunohistochemical changes of SMC markers in F344/N rat lungs were evident by approximately 24 months of age, but there was no apparent change in connective tissue morphology.

Expression of betaig-h3 by human bronchial smooth muscle cells: localization To the extracellular matrix and nucleus.

Bronchial smooth muscle cells play a central role in normal lung physiology by controlling airway tone. In addition, airway smooth muscle hyperplasia and hypertrophy are important factors in the pathophysiology of asthma. In this study, expression of betaig-h3, a recently identified component of the extracellular matrix (ECM), was investigated in primary human bronchial smooth muscle (HBSM) cells. Northern blot analysis demonstrated that treatment of cultured HBSM cells with transforming growth factor-beta1 resulted in a 4- to 5-fold increase in the steady-state level of betaig-h3 messenger RNA. Western blot analysis of secreted proteins using monospecific antibodies generated against peptide sequences found in the N- and C-terminal regions of the protein identified several isoforms having apparent mass of 70-74 kD. Immunohistochemical analysis of human lung localized betaig-h3 to the vascular and airway ECM, and particularly to the septal tips of alveolar ducts and alveoli, suggesting that it may have a morphogenetic role. Analysis of cultured HBSM cells identified betaig-h3 in both the ECM as well as the cytoplasm, and surprisingly also in the nucleus. These results demonstrate that betaig-h3 is produced by resident lung cells, is a component of lung ECM, and may play an important role in lung structure and function. The presence of this protein in nuclei suggests that it may have regulatory functions in addition to its role as a structural component of lung ECM.

Induction of tenascin in rat lungs undergoing bleomycin-induced pulmonary fibrosis.

Lung injury induced by bleomycin is associated with early inflammation and subsequent excessive deposition of extracellular matrix. In the present study, we investigated the expression of extracellular matrix glycoprotein tenascin (TN) during pulmonary injury induced by bleomycin. After the initial lung injury induced by intratracheal bleomycin instillation, TN and collagen type III (COL III) mRNAs were greatly induced. The pattern of induction of TN was distinct from that of COL III. TN was primarily induced during the early inflammatory phase, whereas the increase in COL III synthesis continued during the reparative phase. The induction and localization of TN mRNA during bleomycin-induced pulmonary injury were also examined by in situ hybridization. TN mRNA was focally induced in rat lungs 3 days after bleomycin administration. Induction of TN mRNA was spatially restricted in the areas of tissue inflammation. The interstitial cells in alveolar septal walls and secondary septal tips in the areas of tissue damage were the major source of TN mRNA production. Expression of TN mRNA was decreased as the inflammation attenuated and development of fibrosis proceeded. Immunocytochemical analyses of TN protein distribution in the lung yielded corroborative results. Immunoreactive TN protein was found in a patchy distribution in alveolar septal walls and secondary septal tips in the areas of damaged tissues. This study demonstrated that TN is a unique early-response extracellular matrix component to bleomycin-induced pulmonary injury and is induced at the sites of the inflammation, suggesting a potential role of TN as a modulator of pulmonary inflammation and repair.

The pulmonary lipofibroblast (lipid interstitial cell) and its contributions to alveolar development.

The pulmonary lipofibroblast is located in the alveolar interstitium and is recognizable by its characteristic lipid droplets. During alveolar development it participates in the synthesis of extracellular matrix structural proteins, such as collagen and elastin, and as an accessory cell to the type II pneumocyte, in the synthesis of surfactant. The lipofibroblast contains cortical contractile filaments and is thereby related to the contractile interstitial cells that are normally found at the alveolar septal tips and after lung injury. The morphologic, immunologic, and biochemical characteristics of the lipofibroblast and its probable physiologic functions are reviewed. The retinoid and lipid metabolism of the lipofibroblast is compared with that of the hepatic lipocyte and the adipocyte. Although the functions of the lipofibroblast remain incompletely characterized, this cell type is emerging as an important contributor to pulmonary alveolar septal development.

Pulmonary Lipocytes in the Production of Interstitial Fibrosis. † 1598

Pulmonary interstitial fibrosis is an important component of the pathology of bronchopulmonary dysplasia (BPD). There is increasing evidence to support the role of infection, especially sepsis, in the pathogenesis of BPD. We have investigated the effects of combining chronic oxygen toxicity and lipopolysaccharide endotoxin in rats. Our hypothesis is that endotoxin activates pulmonary macrophages in an O2 damaged lung, which in turn activates pulmonary lipocytes to release stored retinoids and, with chronicity, initiates production of TGF-b by these cells. In the liver paradigm of fibrosis, this is followed by development of receptors for PDGF on lipocytes leading to autocrine proliferation, transformation into myofibroblasts and the production of cross-linked collagens. Rats treated with 100% O2 for 36 hours, 90% O2 for 36 hours, 80% O2 for 6 weeks and IP endotoxin for 3 days beginning at 72 hours, showed early immunostaining of lung epithelium, lung lipocytes and of liver lipocytes with TGF-β3, which disappeared with chronicity by 36 days. PDGF immunostaining also appeared early in lung and liver lipocytes and persisted for 36 days. Alpha smooth muscle actin, which appears with lipocyte transformation to myofibroblasts,showed immunoreactivity in cells around respiratory bronchioles and at septal tips, increasing greatly with chronicity. The presence of collagen in alveolar walls and septal tips also increased with time as shown by van Gieson staining and by electron microscopy. We suggest that activation of pulmonary lipocytes is an important component of the production of cross-linked collagen and its deposition in pulmonary interstitial fibrosis.

The Effect of the Angiotensin Converting Enzyme (ACE) Inhibitors Captopril and Enalapril on the Development of Oxygen Induced Lung Injury in the Rat.† 1563

Certain ACE inhibitors can decrease pulmonary and renal fibrosis resulting from exposure to ionizing radiation, possibly related to their intrinsic free radical scavenging properties. This study was designed to determine if ACE inhibitors can alter the pattern of lung injury and fibrosis induced by prolonged hyperoxia. Sixty male Sprague-Dawley rats were randomized into four groups: control (receiving neither oxygen nor drug), oxygen exposed controls(receiving no drug), oxygen exposed Captopril treated (a thiol containing ACE inhibitor), and oxygen exposed Enalapril treated (a non-thiol containing ACE inhibitor). Rats were exposed to 98-100% oxygen for 36 hours; 90% oxygen for 36 hours then maintained in 80% oxygen until their sacrifice on study day 5, 25, or 48. Captopril (400mg/L) and Enalapril (260mg/L) were provided continuously via drinking water. Samples of blood, lung and liver were examined for histologic and biochemical evidence of injury. Weight gain was decreased in all oxygen exposed animals regardless of drug treatment. Histologic evidence of acute oxygen toxicity (pulmonary edema) was present in all oxygen exposed animals regardless of therapy. Intensity of immunostaining for α-SMA (a marker of transformed myofibroblasts) seen around respiratory bronchioles, in alveolar walls and at septal tips was decreased with chronic ACE inhibitor treatment. Immunostaining of lipocytes and type II alveolar epithelial cells with PCNA (a marker of the S1 phase of cellular division) was prominent in all oxygen exposed animals on day five, but decreased at later time points. Intensity and distribution of immunostaining for PDGF and fibronectin was not altered by drug treatment. Immunostaining of liver and lung lipocytes and large airway epithelium with TGFβ decreased with chronic ACE inhibitor treatment. Staining for collagen deposition and elastic fibers around respiratory bronchioles, in alveolar walls and at septal tips was less prominent in ACE inhibitor treated animals at 45 days. We suggest that ACE inhibitors may decrease the long term effects of hyperoxia in rats through their effect on TGFβ production.

Tenascin in rat lung development: in situ localization and cellular sources.

Tenascin (TN) is a hexameric extracellular matrix glycoprotein that may play an important role during lung development. TN protein is temporally and spatially restricted during lung organogenesis. The temporo-spatial and cellular expression of TN mRNA in lung remains unclear. Localization of message expression of TN in rat lung tissue was first investigated by using in situ hybridization performed with an antisense RNA probe. TN mRNA was present primarily within the mesenchyme of day 16 gestational age fetal rat lung tissue, whereas immunoreactive TN protein was found along the basement membrane. In postnatal day 3 rat lung tissue, TN mRNA was detected along alveolar septal walls and was concentrated at secondary septal tips. Expression of TN message was consistent with localization of immunoreactive TN protein. Accumulation of TN mRNA in alveolar septal tips suggests that mesenchyme may be the major source of TN mRNA. To investigate the cellular source of TN in rat lung, we studied the expression of TN in cultured rat lung fibroblasts, endothelial cells, and alveolar epithelial cells. Two TN isoforms having molecular mass of 230 and 180 kDa were in conditioned medium and in cellular extracts of lung fibroblasts and endothelial cells. TN was secreted and deposited in the extracellular matrix closely associated with the surface of lung fibroblasts and endothelial cells. Lung alveolar epithelial cells showed undetectable or barely detectable amounts of TN. These studies demonstrated that TN isoforms are expressed not only by lung fibroblasts but also by lung endothelial cells. The unique spatial localization of TN mRNA during lung development and expression of TN by different lung cell types suggested TN may be involved in matrix organization and cell-cell interactions during lung development.

Collagen mRNA content and distribution in the lungs of rats exposed to ozone.

cDNAs, synthesized by the polymerase chain reaction (PCR) technique, were used to quantify mRNA concentrations for the alpha 1 (I) and alpha 1 (III) chains of collagen types I and III and for beta-actin in the lungs of rats exposed to either filtered air or to 1.2 ppm of ozone. The alpha 1 (I) procollagen mRNA concentration was increased by about 30% in the lungs of rats exposed to ozone, while the concentrations of the mRNAs for alpha 1 (III) procollagen and for beta-actin were the same in the lungs of control and ozone-exposed animals. The lungs from the rats exposed to 1.2 ppm of ozone preferentially synthesized type I collagen as compared with controls. Sites of increased expression of the alpha 1 (I) procollagen mRNA were detected by in situ hybridization in lung sections embedded in paraffin prepared from rats exposed either to filtered air or to ozone. The lungs from rats treated with ozone focally expressed increased amounts of alpha 1 (I) procollagen mRNA in the lung parenchyma at the septal tips and the bronchiole-alveolar duct junctions. They also showed an apparent diffuse increase in alpha 1 (III) procollagen mRNA expression. We conclude that exposure of rats to high concentrations of ozone causes a specific increase in the lung content of mRNA for the major chain of type I collagen.

Distribution of Manganese Superoxide Dismutase mRNA in Normal and Hyperoxic Rat Lung

Aerobic metabolism requires a continuous oxygen supply, which in turn can form partially reduced species (free radicals) that damage cellular components. To prevent this, organisms have elaborate free radical-scavenging defenses that include the superoxide dismutases. The lungs are unique in their role as an oxygen-gathering system, making these defenses critical to lung integrity. Manganese superoxide dismutase (Mn-SOD) levels increase in rats exposed to sublethal doses of hyperoxia and correlate with the development of tolerance to higher levels of hyperoxia. Although pulmonary Mn-SOD protein and mRNA levels both change with hyperoxia, the timing and levels differ dramatically. Lung heterogeneity makes extrapolation of data from whole tissue homogenates or cultures difficult. In this study, in situ hybridization of Mn-SOD in the lungs of adult rats exposed to air or to 85% O2 for 3 days was performed. In animals exposed to either air or 85% O2, Mn-SOD transcripts were present in arterioles, the septal tips of alveolar ducts, alveolar type II cells, and mesothelial cells. Hyperoxic lung had an intense, continuous labeling of the pleura that was distinctly greater than the intermittent labeling of the pleura found in control animals. The high level of expression of Mn-SOD mRNA in alveolar duct septal tips in both control and O2-exposed animals may be secondary to increased aerobic activity in these regions, which contain collagen and elastin and are important stress-bearing elements in the lung. Alveolar type II cells are metabolically active secretory cells and thus may experience increased endogenously generated oxidative stress. Pleural effusions are common after hyperoxic exposures, suggesting damage to the mesothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of injury and microdosimetry of ozone in the ventilatory unit of the rat.

The distribution of ozone-induced injury across ventilatory units of the lungs was determined and compared with the predicted distribution of ozone dose across the same units to evaluate dose-response relationships. Sprague-Dawley rats were exposed to either 0.98 ppm ozone 8 h/day for 90 days or to filtered air only. En bloc microdissection was used to identify and isolate in longitudinal profile the bronchiole-alveolar duct junction, first pair of alveolar duct generations, and intervening bifurcation ridge. The first alveolar outpocketing along the bronchiolar wall of each isolation was used to identify the center of a series of concentric arcs radiating outward at 100-microns intervals across each ventilatory unit. The intercept lengths of each arc with the tissue of alveolar septal tips (edges) and alveolar walls were measured and expressed as a function of distance into the ventilatory unit. Relative ozone dose across the ventilatory unit was estimated using the geometry of the tracheobronchial tree and the volume and surface area distribution within individual ventilatory units. This mathematical model of ozone dose demonstrated a high degree of correlation to this measured tissue injury response. The findings of this study demonstrate that microdosimetry and microtoxicology can be used to determine dose-response relationships within the ventilatory unit and to assess questions of tissue sensitivity in ozone-induced lung injury.

Distal airway remodeling in rats chronically exposed to ozone.

The morphologic changes in the centriacinar region of lungs from 14 rats exposed to either filtered air (8) or 0.95 ppm ozone (6) 8 hours daily for 90 days were examined morphologically and morphometrically. Rats were killed with an overdose of sodium pentobarbital administered intraperitoneally, the trachea cannulated, and thoracic viscera and lungs removed from the chest. Lungs were fixed via intratracheal instillation of a paraformaldehyde/glutaraldehyde cacodylate buffered fixative at 30 cm water pressure. Lung volumes were determined by weight displacement and the left lung lobe was sectioned transversely into 12 slabs. Three slabs from the cranial, middle, and caudal thirds of the lobe were embedded in paraffin and studied by light microscopy. With these sections, we estimated the volume of proximal bronchiole, terminal bronchiole, respiratory bronchiole, and combined alveolar duct/sac within the lung. Bronchioles dissected from preselected regions of the right middle lobe were studied by transmission electron microscopy. Dissected terminal airways were sectioned in a longitudinal plane through their midlumen. From these dissected airways, 4 subregions of the centriacinus were then examined: (1) terminal bronchiole, (2) respiratory bronchiole, (3) centriacinar alveolar duct wall, and (4) centriacinar alveolar septa. The results of this study showed that after chronic ozone exposure, there was a 13 to 21% decrease in terminal bronchiole luminal diameter but no significant change in total terminal bronchiole volume. The most notable change was a 3.4-fold increase in respiratory bronchiole volume. Analysis of measured volume and diameter changes in the terminal bronchiole, coupled with volume increases in respiratory bronchiole, support the conclusion that respiratory bronchiole (RB) is formed from the centriacinar alveolar duct. Morphologic parameters supporting this conclusion included the presence of fused basement membrane beneath reactive bronchiolar epithelium in the RB, the presence of similar basal laminar changes in both the RB and proximal alveolar duct septal tips, and the observation that most severe epithelial damage and inflammation occurred in the most proximal alveolar duct rather than in the terminal bronchiole. A major conclusion of this study is that the focus of the most severe injury within the acinus appears to shift distally as respiratory bronchiole segments are formed. Hence most of the damage occurs at the tips of alveolar septa at the respiratory bronchiole-alveolar duct junction.