Traffic volumes and respiratory health care utilization among residents in close proximity to the Peace Bridge before and after September 11, 2001.

A recent study based on data over a 10-year period (1991-2000) showed a positive association between health care utilization and prevalence of asthma, and commercial traffic at a U.S.-Canada border crossing. We wanted to determine whether decreases in total traffic would also be associated with decreases in health care utilization for respiratory illnesses. Following September 11, 2001, there was a 50% drop in total traffic at the Peace Bridge border crossing point between Buffalo, New York and Fort Erie, Ontario, Canada. To investigate the impact of such a traffic decline on health care utilization for respiratory illnesses, weekly respiratory admissions to Kaleida Health System, Western New York's largest health care provider were analyzed according to ICD9CM classification and compared with total weekly traffic volumes for 3-month periods in 2000 and 2001 (August, September, and October). The total number of patients admitted to hospital or seen in emergency departments for respiratory illnesses during the 3-month periods of both years was 5288. A 50% drop in total traffic following Labor Day and September 11, 2001, from week 4 to week 7 was found to be statistically significant (p = 0.031) when a one-way ANOVA was performed. Likewise, the drop in total respiratory cases approached statistical significance (p = 0.052) when a one-way ANOVA was conducted. The results suggest an association between decrease in traffic volumes with decrease in health care utilization for respiratory diseases. These results suggest that current levels of traffic may be impacting on the respiratory health of residents in the nearby community.

Patterns of inpatient and outpatient care for asthma in Erie and Niagara Counties, western New York State.

The prevalence of asthma, as well as the morbidity and mortality due to asthma, has increased in the United States, especially among poor minority subpopulations. The causes of these increases are complex and not well understood. Our findings from an analysis of emergency room (ER) visits and hospitalizations for Erie and Niagara Counties in western New York State for the period 1984-1991 provides important background to this problem. Of all respiratory disorders, asthma was the most frequent reason for ER visits and was second only to pneumonia as a reason for hospital admissions. In Erie County the hospitalization rates for asthma in two inner-city communities with predominantly minority populations were 1.48 and 2.09 times higher than those in the rest of the county. Furthermore, the hospitalization rates for these communities showed an increasing trend over the study period. Gender differences were also found. Boys age 0 to 9 years were hospitalized for asthma twice as often as girls. However, over 15 years of age, females had admission rates that were twice those of males. In contrast, hospitalization rates for pneumonia were equal for males and females, which would suggest gender differences particular to asthma. Hospitalizations for asthma in the western New York region cost an estimated $6,000,000 in 1990. We conclude that asthma is a major cause of morbidity in this region with excessive and increasing impact on inner-city communities.

The prevalence of asthma in children of elementary school age in western New York.

To determine the prevalence of caregiver-reported asthma in children 4 to 13 years old in metropolitan western New York State, surveys were conducted during 1997-1999 in the Buffalo, Niagara Falls, Iroquois, and Gowanda school systems. Questionnaires (3,889) were sent to the homes of elementary school children in nine schools in western New York. The caregivers were asked to complete a 13-item questionnaire for the child. Of the questionnaires, 60.5% (2,353/3,889) were completed. Of all children, 18% had physician-diagnosed asthma. Of children diagnosed with asthma, 86% were taking medication. Symptoms were consistent with suspected undiagnosed asthma for 13% of the children. Buffalo had the highest rate of diagnosed asthma (20%) for the age group. Gowanda had a prevalence of 18%, Iroquois 16%, and Niagara Falls 15%. Variations were observed in asthma prevalence rates among different racial/ethnic groups. In general, boys had a significantly (P = .001) increased odds of being asthmatic compared with girls. Overall, African-Americans and Hispanic/Latino children had significantly (P = .012 and P = .005, respectively) higher asthma prevalence rates, two to five times those of their Caucasian peers. In Gowanda, the prevalence of diagnosed asthma among Native American children was 23%, compared to 15% among Caucasian children. Of diagnosed Native American children, 71% were female. In Gowanda, a significant association (P = .007) of asthma among children in split-grade classes was observed compared to nonsplit grades. Of Native American children in split grades, 60% were diagnosed asthmatics. These observations reveal a high prevalence of asthma in the age group of 4 to 13 year olds in western New York. Local variations in potential triggers of asthma need to be considered when advising asthmatics. The results suggest that some grades have a disproportionate amount of children with asthma. The implications of asthma for children's early education need to be examined further.

Modulation of laminin integrin receptors in the postnatal and adult rat lung.

Recent studies have shown that type II pneumocytes, at birth and day 3 postnatally, have a diffuse distribution and localize at alveolar 'corners' between 3 and 7 days. Since alpha 3 beta 1 and alpha 6 beta 1 are laminin-binding receptors that are well expressed by rat type II alveolar epithelial cells, we postulated that they may play a role in the localization of the cells in the alveolus. To begin the evaluation of this hypothesis, we studied the temporal and spatial expression of the alpha 3, alpha 6, and beta 1 integrin subunit protein and mRNA in whole rat lungs during postnatal development by immunofluorescence, confocal microscopy, and Northern blot analysis. The temporal expression of proteins analyzed by immunochemistry, with integrin subunit specific antibodies, increased during the 3- to 7-day postnatal period and in adult lungs. Densitometric values of the alpha 3, alpha 6, and beta 1 mRNA expression, normalized to 28S rRNA, quadrupled from day 1 to day 3 postnatally. The mRNA expression of different integrin chains was elevated 1.5- to threefold from days 5 to 7 postnatally compared to day 1 levels. The alpha 3 and alpha 6 integrin subunit mRNA decreased to newborn levels in adult lungs, whereas the beta 1 integrin mRNA in adult lungs was expressed at approximately 50% of its level in newborn lungs. We postulate that the increases in alpha 3, alpha 6, and beta 1 integrin mRNA expression during the early neonatal period may be important for the spatial distribution of type II pneumocytes.

Adhesive characteristics of type II pneumocyte subpopulations from saline- and silica-treated rats.

Alveolar epithelial cells isolated from silica-treated rat lungs provide a system for the in vitro study of repair mechanisms. In studies of type II cell interactions with the extracellular matrix, we observed that type IIB pneumocytes from silica-treated rats adhered to tissue culture plastic more readily than do normal type II cells. This paper examines the adhesion characteristics of IIA and IIB cells and their modulation by divalent cations. We describe differences in the adhesive behavior of two subpopulations of freshly isolated type II pneumocytes from saline- and silica-treated rats. The observations have implications for repair and tissue remodeling in the lung.

An RGD containing peptide from HIV-1 Tat-(65-80) modulates protooncogene expression in human bronchoalveolar carcinoma cell line, A549.

Tat (transactivator of transcription) is essential for HIV-1 replication in vivo and in vitro. Tat-(65-80), an RGD containing domain, has been shown to regulate proliferative function of a variety of cell lines, including a human adenocarcinoma cell line, A549. The exact cellular and molecular mechanisms by which these effects are mediated, remain unknown. To evaluate the hypothesis that Tat-(65-80) modulates the expression of immediate early genes (IEG) c-jun, c-myc, c-fos and the tumor suppressor gene p53, serum starved A549 cells were incubated with Tat-(65-80) or heat-inactivated Tat-(65-80) at 10 ng/ml. Total cellular RNA was isolated from the cells at various time points (0-24 hours). In each case, 5 micrograms of RNA was reverse transcribed in 20 microliters of reaction volume. Equal amounts of cDNA were subjected to polymerase chain reaction (PCR) and analyzed by electrophoresis. The photographic negatives of the ethidium bromide stained gels were quantitated by densitometric scanning and normalized to corresponding beta-actin PCR products. Treatment with Tat-(65-80) showed a twofold induction of c-jun at 0.5 h. Peak expression occurred at 60 minutes and remained above baseline at 24 hours (h). c-myc was increased at 0.5 h, reached a twofold increase at 2 h and remained above baseline at 24 h. c-fos increased seven fold at 0.5 h and declined subsequently to baseline at 8 h. p-53 gene was reduced fivefold at 0.5 h and remained downregulated thereafter. These results show that Tat-(65-80) can modulate growth related genes in human lung epithelial cells.

Induction of interleukin-1 and interleukin-8 mRNAs and proteins by TGF beta 1 in rat lung alveolar epithelial cells.

The transforming growth factor-beta (TGF-beta) has been shown to increase in lung injury and in fibrotic states of the lung. In the current study, we sought to investigate whether TGF beta 1 induced the expression of IL-1 alpha and IL-8 in rat alveolar epithelial cells. We evaluated TGF beta 1, IL-1 alpha, and IL-8 expression by immunofluorescence in silica-injured and saline-treated control rat lungs. Antibodies to IL-1 alpha, IL-8, and TGF beta 1 showed intense staining in silica-injured lungs as compared to saline-instilled lungs. Primary isolated type II cells from silica-injured lungs showed increased expression of IL-1 alpha as compared to saline-instilled lungs. To evaluate the effects of TGF beta 1, we treated an immortalized rat type II cell-derived cell line (LM5) with 100 pg/ml of TGF beta 1 in serum-free medium for 0-24 hours and analyzed the expression of IL-1 alpha and IL-8 mRNAs and proteins using semiquantitative RT-PCR, Northern blot analysis, Western blot analysis, and immunohistochemistry. Densitometric analysis of Northern blots showed modest constitutive expression of IL-1 alpha gene in untreated control LM5 cells. TGF beta 1 treatment resulted in an increase in IL-1 alpha mRNA, that reached maximum levels (4-fold) by 2 hours and remained elevated for 4-16 hours, with a subsequent decline by 24 hours. Similarly, Northern blot and RT-PCR analysis demonstrated that TGF beta 1 treatment resulted in maximum induction of IL-8 mRNA (6- 8.5-fold) within 1-4 hours. The levels remained elevated for up to 24 hours afterwards. Western blot analysis results further confirmed the expression of both IL-1 alpha and IL-8 proteins by LM5 cells. TGF beta 1 treatment resulted in increased expression of both IL-1 alpha and IL-8 proteins. Immunofluorescence studies demonstrated increased staining of IL-1 alpha by TGF beta 1 as compared to untreated cells. These results suggest that TGF beta 1 may regulate IL-1 alpha and IL-8 expression in alveolar epithelial cells and contribute to polymorphonuclear leukocyte recruitment and lung injury in clinical states with increased TGF beta 1.

Monocyte chemoattractant protein-1 in chronic proliferative immune complex nephritis.

In rats with chronic serum sickness, proliferative immune complex glomerulonephritis progresses in three discrete stages, designated mild, moderate, and severe. One distinguishing immunopathologic feature, the progressive increase in the number of glomerular macrophages, is closely correlated with decreasing kidney function. We hypothesized that monocyte chemoattractant protein-1, a beta-subfamily chemokine with potent monocyte-specific chemotactic activity, might contribute to this macrophage accumulation. Immunohistochemical methods were used to identify monocyte chemoattractant protein-1 in kidney tissue sections. Total RNA was extracted from the kidneys of rats at each stage of chronic serum sickness, and age-matched controls, and Northern blot analysis was performed with a rat monocyte chemoattractant protein-1 cDNA probe. Tissue staining localized monocyte chemoattractant protein-1 to the glomerular capillary wall and mesangium in chronic serum sickness. Minimal quantities of monocyte chemoattractant protein-1 mRNA were detected in the kidneys of normal control rats, with marked increases in mRNA as chronic serum sickness nephritis progressed to the moderate stage. There was then an apparent decrease in monocyte chemoattractant protein-1 mRNA in the severe stage. The degree of protein staining and mRNA levels paralleled each other. We conclude that monocyte chemoattractant protein-1 is a potentially important chemotactic agent in chronic serum sickness nephritis.

Distribution of alveolar type II cells in neonatal and adult rat lung revealed by RT-PCR in situ.

Type II pneumocytes in newborn lungs are more uniformly distributed, whereas in adult lungs they are located at alveolar corners. We used morphometry and reverse transcription-polymerase chain reaction in situ hybridization of surfactant protein C mRNA to determine the patterns of type II cell distribution in random lung sections from Sprague-Dawley rats at various neonatal stages and adults. There was a progressive increase in the percentage of type II cells at alveolar corners from 30% at 1 day to 51, 62, 78, and 81% at 3, 5, and 7 days old and adult rats, respectively. There were statistically significant differences (P < 0.001) in the localization of type II cells from the nearest alveolar corner in the 1-day-old compared with 7-day-old and adult rat lungs. These results show that rat type II cells localize to the alveolar corners within the first 7 days postnatally and provide a system for study of factors that regulate alveolar epithelial cell distribution.

Alcohol inhibits lipopolysaccharide-induced tumor necrosis factor alpha gene expression by peripheral blood mononuclear cells as measured by reverse transcriptase PCR in situ hybridization.

We recently showed that alcohol significantly suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production by whole blood and total mononuclear cells from healthy subjects as measured by bioassay. In the current study, we further examined the effect of alcohol on LPS-induced TNF-alpha gene expression by semiquantitative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybridization methods. Peripheral blood mononuclear cells were cultured with LPS (10 micrograms/ml) for 4 to 8 h with or without different concentrations of ethanol (0.1, 0.2, and 0.3% [vol/vol]). Total RNA from treated and untreated cultures was extracted and used for solution PCR analysis. Treated and untreated cells were subjected to both conventional in situ hybridization and RT-PCR in situ hybridization. In solution RT-PCR in vitro analysis, alcohol significantly suppressed TNF-specific message. In conventional in situ hybridization, the effect of alcohol on TNF-alpha gene expression was poorly detected. However, when cells were subjected to RT-PCR prior to in situ hybridization, cells treated with alcohol significantly suppressed expression of the message for TNF-alpha. These studies confirm our earlier finding that alcohol suppressed the production of TNF-alpha by LPS-induced whole blood cells and peripheral blood mononuclear cells. Furthermore, these studies also demonstrate that the RT-PCR in situ technique is a powerful tool for detecting and amplifying specific genes in whole cells when limited numbers of cells are available for RNA extraction.

Differential modulation of integrin receptors and extracellular matrix laminin by transforming growth factor-beta 1 in rat alveolar epithelial cells.

The transforming growth factors-beta (TGFs-beta) family of genes plays important roles in cell growth and differentiation in many cell types. TGF beta modulates the synthesis and accumulation of extracellular matrix (ECM) components and the expression of cell surface receptors for ECM components. TGF beta is increased in alveolar lining fluid during inflammatory reactions of the lung and has been identified in alveolar epithelial cells of developing lungs and hyperplastic type II cells during repair. However, little is known about how TGF beta may regulate expression of extracellular matrix proteins and ECM receptors in lung alveolar epithelial cells. Laminin, a major glycoprotein component of epithelial basement membrane, is synthesized and secreted by alveolar epithelial cells. To study the effects of TGF beta on modulation of laminin and its integrin receptors alpha 6 beta 1 and alpha 3 beta 1 in lung alveolar epithelial cells, a rat alveolar type II cell-derived cell line, LM5, was incubated with TGF beta 1 (0-100 pg/ml) in serum-free medium for 0-16 h. We examined the expression of integrin subunits and laminin beta 2 chain (s-laminin) mRNAs and protein expression. By Northern blot analysis, TGF beta 1 induced dose-dependent increases in alpha 6 and beta 1 mRNA levels. TGF beta 1 also increased the expression of laminin beta 2 chain mRNA at 12-16 h poststimulation. In contrast, TGF beta 1 decreased alpha 3 mRNA expression. Immunoprecipitation studies of TGF beta 1-treated cells showed increased surface expression of both alpha 6 and beta 1 protein while surface expression of the alpha 3 integrin subunit was decreased. The same treatment resulted in increased laminin protein expression. These data suggest that TGF beta 1 may regulate alveolar epithelial cell differentiation in part through its modulation of integrins and laminin chains.

Divalent cation-dependent regulation of rat alveolar epithelial cell adhesion and spreading.

Interactions between cells and extracellular matrix are in large part mediated by integrins in divalent cation-dependent processes. Integrins are important for cell differentiation, proliferation, and migration during development and repair of diverse tissue types. The roles played by integrin adhesion receptors in the lung are just beginning to be investigated. It is plausible that integrins play a central role in mediating lung basement membrane influences on alveolar epithelial type II cell localization and differentiation. The current studies were carried out to determine the patterns of alveolar epithelial cell adherence and spreading on different substrata and their divalent cation and RGD requirements. We utilized a rat type II cell-derived cell line, LM5, and a human alveolar cell carcinoma cell line A549. Both cell types showed similar responses to divalent cations. Adhesion and spreading on different extracellular matrix components had different divalent cation requirements. Mn2+ enhanced adhesion and spreading on fibronectin (FN), type IV collagen, and laminin, but not on type I collagen or plastic. Mn(2+)-enhanced cell adhesion to FN was RGD-dependent and partially inhibited by an anti-alpha 5 integrin antibody. Small increases in Ca2+ concentration (0.1-0.5 mM), but not Mg2+, suppressed Mn(2+)-mediated adhesion and spreading. Thus, variations in the relative divalent cation concentrations in the vicinity of the integrin-ligand complex may modulate the receptor-acceptor interactions. These results support the view that alterations in extracellular divalent cations are important regulators of alveolar epithelial cell interactions with lung basement membrane.

A Mn(2+)-enhanced, RGD-dependent adhesion technique for isolation of adult rat type II alveolar epithelial cells for immediate functional studies.

This report describes a Mn(2+)-enhanced, RGD-dependent adhesion technique for isolation of adult rat type II cells for immediate functional studies. Lung cells were dissociated by 30 U/ml porcine pancreatic elastase and 50 micrograms/ml trypsin instilled in the airways. Macrophages were selectively removed by adhesion on purified normal goat IgG-coated petri dishes. Type II cells were isolated by adhesion for 45 min, on ProNectin-F-coated dishes in the presence of 0.5 mM Mn2+. The adherent type II cells were then detached with 0.025% trypsin, 2 mM EDTA in Hepes-buffered saline, pH 7.4. The technique yielded 1.5 to 1.7 x 10(7) (n = 8) cells from a 150- to 200-g rat. Greater than 90% of the cells were pure type II cells as judged by tannic acid staining and immunostaining with monoclonal antibody 4AmAb, which recognizes pneumocin, a type II cell marker. The technique reduced the time required for cell isolation from the current 16 to 24 h to 2 to 2.5 h, using commonly available laboratory equipment and reagents. Cells isolated by the procedure were used to study cell adhesion and spreading on purified extracellular matrix components in the presence of different divalent cations. Mn2+, Co2+, Ni2+, and Mg2+ enhanced adhesion of freshly isolated type II cells to fibronectin and ProNectin-F, while Ca2+ did not promote type II cell adhesion on these substrata. RGDS peptide at 1 mg/ml concentration inhibited the divalent cation-enhanced cell adhesion.

Matrix-driven pneumocyte differentiation.

This commentary presents evidence in support of a hypothesis that adult mammalian alveolar epithelial basement membrane possesses functional and structural domains that determine sites at which type I and type II cells localize. The hypothesis provides a framework for understanding how, after normal repair of the epithelium, a constant ratio of type I and type II cells, and the localization of the cell types is maintained.

Isolation and partial characterization of pneumocin, a novel apical membrane surface glycoprotein marker of rat type II cells.

Rat alveolar type II pneumocytes, in situ, label with Maclura pomifera agglutinin (MPA), a plant lectin that recognizes alpha-galactosyl oligosaccharide residues of glycoproteins and glycolipids. To study the glycoproteins recognized by the lectin, MPA lectin affinity chromatography was used to isolate a novel glycoprotein, pneumocin, from type II and whole rat lung cell membranes. Pneumocin isolated from adult rat lungs was a non-disulfide-linked sialoglycoprotein with an Mr of 165 kD. Asparagine-linked oligosaccharides contributed 5 to 10% to the Mr. Two-dimensional chymotryptic peptide maps of pneumocin isolated from whole lung membranes and type II cells were similar. The glycoprotein partitioned in the detergent phase on Triton X-114 phase separation. Murine monoclonal antibodies developed against the purified glycoprotein localized on apical membranes of type II pneumocytes in situ. The antibodies did not label type I cells or lamellar bodies but labeled luminal surfaces of vesicular structures of type II cells. Isolated type II cells labeled with antibodies after 1 d in culture but showed significantly less staining of cells after 4 d of culture. These observations demonstrate that pneumocin is a cell surface sialoglycoprotein marker of type II cells. Western blot analysis of liver and kidney cell membranes suggest that related glycoproteins may also be present in those tissues. The isolation technique and monoclonal antibodies should permit further characterization and functional studies of the glycoprotein.

Identification of pneumocin, a developmentally regulated apical membrane glycoprotein in rat lung type II and Clara cells.

Pneumocin (Mr, 165 kD) is a recently identified apical membrane surface sialoglycoprotein marker of type II pneumocytes. A murine monoclonal IgG1 subclass-producing clone 4A (4A mAb), which was developed against the purified pneumocin, and recognized pneumocin on Western blots of adult rat lung homogenates, was used to study expression of the glycoprotein in developing rat lungs. Pneumocin localized to apical membranes of late fetal, neonatal, and adult rat type II pneumocytes as well as Clara cells in situ, by immunofluorescence and immunoelectron microscopy. Faint immunofluorescence was observed in 17-d fetal lungs. However, 19-d fetal lungs showed intense immunofluorescence with the antibody. On immunoelectron microscopy, apical membranes of 19-d fetal and adult rat lung type II cells were labeled by 4A mAb, but type I cells were not stained. On Western blots, amounts of pneumocin increased up to the fourth day after birth, when near-adult levels were attained. Lower molecular weight forms (Mr, 80 to 90 kD) were recognized in 17-d fetal lung. These bands decreased in amount with a corresponding increase in the 165-kD band that was typically observed in adult lungs. Immunoglobulins that were eluted from polyvinylidene difluoride strips containing the 165-kD band recognized the Mr 80 to 90 kD bands and 50-kD component, suggesting that fetal forms of the protein shared an epitope in common with the adult pneumocin. Reactivity of the glycoprotein with 4A mAb was destroyed by enzymatic digestion with trypsin and staphylococcal V8 protease. These data demonstrate that pneumocin is a developmentally regulated apical membrane marker of differentiated type II and Clara cells.(ABSTRACT TRUNCATED AT 250 WORDS)

An in vitro model for polymorphonuclear-leukocyte-induced injury to an extracellular matrix. Relative contribution of oxidants and elastase to fibronectin release from amnionic membranes.

Alteration of the extracellular matrix by inflammatory cells is believed to be important in both lung injury and the subsequent restoration of lung architecture. Here we describe the results of the interaction between an acellular human amnionic membrane model and stimulated human polymorphonuclear neutrophils (PMN) in vitro. Polymorphonuclear neutrophil suspensions were placed on one surface of the amnion, and either the chemotactic peptide FMLP or the cell membrane activator phorbol myristate acetate (PMA) was placed on the opposite side of the amnion. Stroma and basement membrane sides of the amnion were separately exposed to the PMN. The PMN suspension was removed and centrifuged, and the supernatant was assayed for superoxide anion (O2-.) and for elastase activity. Injury to the acellular amnion was evaluated by transmission electron microscopy and by measurement of fibronectin (FN) released from the membrane matrix. Although both stimulants cause a concentration-dependent release of O2-., only PMA stimulated elastase release. These effects were similar when either the stroma or the basement membrane side was exposed to PMN. PMA-stimulated cells and supernatants from PMA-stimulated cells caused solubilization of membrane at different incubation times. Electron microscopy confirmed the disruption of the basement membrane of the amnion by PMA-stimulated PMN. Oxidant scavengers (SOD and catalase) did not prevent matrix degradation, and elastase inhibition by a specific chloromethylketone inhibitor diminished FN release on both sides of the amnion by activated PMN supernatants, but only on the basement membrane side by intact PMN. We conclude that in this model, elastase rather than oxygen radicals solubilizes FN from the matrix.

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro. A novel system for type II pneumocyte culture.

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.

An acellular human amnionic membrane model for in vitro culture of type II pneumocytes: the role of the basement membrane in cell morphology and function.

To determine whether a preformed basement membrane contributes to the maintenance of morphology and function of type II pneumocytes, we cultured isolated adult rat type II pneumocytes on the basement membrane and stromal surfaces of an acellular human amnionic membrane and on plastic. The presence of lamellar bodies on transmission electron microscopy and epithelial morphology in culture and a characteristic phospholipid profile after incubation with 3H-acetate identified the cells as type II. When type II cells were cultured on a preexisting basement membrane, they formed a well-organized monolayer with polarity, centrally located surface microvilli, and more basally located nuclei. Individual cells maintained a cuboidal morphology for 8-10 days. Intracellularly, there were numerous mitochondria, endoplasmic reticulum (ER), and lamellar bodies. The cells secreted a new basal lamina of their own. When cultured on the stromal side of the amnion, the cells became flattened within 48-60 hours, formed small lamellar bodies, and had scanty surface microvilli; they formed clumps and appeared less ordered. These cells did not secrete a visible basement membrane, and the majority detached from the stromal surface after 7-8 days in culture. In addition, culture on the basement membrane aspect of the amnion prevented the rapid decline in the percentage of 3H-acetate label incorporated in phosphatidylcholine after 72 hours of culture. We conclude that a preformed basement membrane influences the function and morphology of type II pneumocytes, organizes them into a monolayer in culture, and influences deposition of a visible basal lamina. Thus, the acellular human amnion provides an excellent model for the systematic study of basement membrane influence on the biology and pathology of these cells.