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.     

Definition of an immunologic marker for type II pneumocytes

Based on our previous finding in lung parenchyma of high concentration of the shared epitopes of gp600, a well characterized kidney glycoprotein, we attempted to identify the anatomic site of these epitopes and characterize them biochemically. Affinity-purified polyclonal anti-gp600 antibody was used as the probe. Immunocytochemically in lung on light and electron microscopy the probe reacted exclusively with type II pneumocytes and no other lung cell. The reaction was also demonstrated on freshly isolated and cultured type II pneumocytes. Both approaches showed the reaction to localize on the cell membrane of type II pneumocytes. Immunoprecipitation of radiolabeled type II pneumocyte cell membranes identified two 270- to 290-kDa polypeptides as the reactive proteins. We conclude that the reactive epitopes for anti-gp600 in lung parenchyma are exclusively localized on type II pneumocytes and have a Mr of approximately 270 to 290 kDa and that anti-gp600 may be used as a specific immunologic marker for the type II pneumocytes. Finally, it is possible that the differences in the molecular forms of the cross-reactive proteins in lung and kidney identified in this report are the reason for the known non-nephritogenicity of rat lung for the induction of Heymann nephritis in rat.     

Morphological aspects of type II alveolar pneumocytes following treatment with puromycin in vivo

Ultrastructural modifications of type II pneumocytes (PNM-II) in mice were analysed 125 and 155 minutes after puromycin treatment (12 mg/100 gm at 0, 30, 60 and 90 minutes). A quantitative evaluation of the cell compartments was carried out and the inhibition of protein synthesis in PNM-II was monitored by light microscopic radioautography, following 3H-leucine injection. In electron micrographs, following a 125-minute puromycin treatment, the number and size of lamellar bodies, the precursors of lung surfactant material appeared markedly reduced. The multivesicular bodies (MVB), which are normally very frequent in PNM-II, had almost completely disappeared, as had composite bodies. Golgi saccules were dilated, while the area occupied by Golgi vesicles was enlarged. Observations following the 155-minute puromycin treatment showed a strong enhancement of these modifications. Smooth and coated vesicles of the Golgi area, as well as peroxisomes, did not appear modified by puromycin. Elongated zones of autophagy were more prevalent after 125-minute treatment than after the 155-minute one. Small bodies were frequently observed in the cytoplasm, near the Golgi zone. They were bounded by a smooth membrane and contained tiny vesicles and/or electron-dense lamellae similar to those present within the lamellar bodies. Parallel membranes formed folds, some of them in continuity with lamellar bodies, thus encircling portions of cytoplasm. These structures, which were few in number in controls, were very frequently observed in treated cells, mainly after the 125-minute treatment. These extensive alterations of PNM-II morphology appeared to be related to a disturbed production of pulmonary surfactant.     

The alveolar pores of Kohn in young postnatal rat lungs and their relation with type II pneumocytes.

In order to obtain more information on the development, morphology and function of the pores of Kohn, the lungs of Wistar rats are studied during their early postnatal period, up to 3 weeks of age, by scanning and transmission electron microscopy. The substantial development of the interalveolar pores on days 14 and 21 coincides with the period of septal rearrangement when secondary interalveolar septa become lengthened and thinner. The high frequency of transseptal type II pneumocytes from day 7 onwards, and their typical localization near the pores of Kohn at this period of lung development especially suggests that type II pneumocytes are engaged in the formation of the pores of Kohn. During early lung development, the pores of Kohn seem to serve as passageways for alveolar macrophages.     

In vivo autoradiographic demonstration of beta-adrenergic binding sites in adult rat type II alveolar epithelial cells

Adult male rats were injected intravenously with the muscarinic binding probe 3H-Quinuclidinyl benzilate (QNB) or the beta-adrenergic probe 3H-dihydroalprenolol (DHA). Other rats were pre-treated with an intraperitoneal injection of a 500-fold excess of L-isoproterenol prior to the DHA. Light microscopic autoradiography of 0.5 micron sections of lung from the QNB group demonstrated very little labelling even after 6 months of exposure. In contrast, trachealis smooth muscle from these animals contained substantial labelling. Autoradiographs of lung from rats injected with DHA demonstrated labelling which was well localized over alveolar septa and concentrated over the cytoplasm of type II cells. Quantitative analysis of labelling in the DHA groups indicated a significant reduction of labelling in animals treated with L-isoproterenol prior to DHA, in both the alveolar parenchyma in general and over type II cells. The results of this study provide morphologic evidence for the uptake and specific binding of beta-adrenergic antagonists by the adult lung in vivo, while failing to demonstrate similar binding of a muscarinic probe. In addition, the results demonstrate specific beta-adrenergic receptors on type II cells in vivo and substantiate the view of a direct effect of beta-adrenergic agonists on alveolar type II cells.     

Purinoceptor agonists stimulate phosphatidylcholine secretion in primary cultures of adult rat type II pneumocytes

We examined the effect of purinoceptor agonists on phosphatidylcholine secretion in primary cultures of type II pneumocytes from adult rats. Surfactant is a major product of the type II cell and phosphatidylcholine is its principal component. Adenosine, AMP, ADP and ATP stimulated phosphatidylcholine secretion in a concentration-dependent manner. At the optimum concentration (1 mM), adenosine and AMP stimulated phosphatidylcholine secretion more than 2-fold, while ATP stimulated 5-fold and ADP almost 7-fold. Because of the magnitude of the response it is tempting to speculate that secretion of surfactant may be under purinoceptor regulation. None of these agents influenced cellular phosphatidylcholine synthesis or lactate dehydrogenase release into the medium, so the effects were primarily on secretion and were not secondary to effects on synthesis or cell damage. Non-metabolizable analogs of adenosine, 5'-N-ethyl-carboxyamidoadensoine (NECA) and L-N6-phenylisopropyladenosine (L-PIA), stimulated secretion to the same extent as adenosine and the effect of NECA was antagonized by 8-phenyltheophylline, suggesting a P1 purinoceptor-mediated mechanism. The stimulatory effect of ATP was diminished by alpha, beta-methylene ATP but only slightly by 8-phenyltheophylline, suggesting that, although part of the ATP effect could be explained by catabolism to adenosine, the P2 purinoceptor may also be involved in regulation of surfactant secretion.     

Human dendritic cell lysosome-associated membrane protein expressed in lung type II pneumocytes

Human dendritic cell LAMP (hDC-LAMP) is a unique member of the lysosome-associated membrane protein (LAMP) family with a tissue distribution initially described as restricted to major histocompatibility class II (MHC II) compartments of activated DC before the translocation of MHC II to the cell surface [Immunity 9 (1998) 325]. In this report, we show that hDC-LAMP is also expressed by lung type II pneumocytes, another cell type with constitutive expression of MHC II. A recombinant hDC-LAMP protein and a monospecific anti-hDC-LAMP polyclonal antibody were prepared. The antibody reacted specifically with hDC-LAMP sequences of hDC-LAMP protein expressed in transfected cells and with a 54 kDa protein of normal human lung tissue with properties corresponding to those of transgene expressed hDC-LAMP. Immunohistochemical analysis of hDC-LAMP in human lung showed its presence in alveolar type II epithelial cells (type II pneumocytes) as well as in cells in the interfollicular area of bronchus-associated lymph nodes, where interdigitating DCs are concentrated, and with lesser staining of alveolar macrophages. The native protein contained approximately 16% carbohydrates, most of which are sialyl N-linked oligosaccharides, with an acidic isoelectric point (pI 4.8). The restricted localization of this protein to lung type II pneumocytes and DCs is in contrast to hLAMP-1, which was present in many cell types of the lung and lymph node. Type II pneumocytes are known to express MHC II and the abundant expression of hDC-LAMP in these cells as well as in DCs suggests its possible relationship to specific MHC II related function(s) of DC and type II pneumocytes.     

Natural protection from apoptosis by surfactant protein A in type II pneumocytes

Surfactant-associated protein A (SP-A) is a component of pulmonary surfactant that binds to a specific receptor (SPAR) on the surface of type II alveolar cells of the lung and regulates gene expression and surfactant secretion. Previously we have shown that activation of SPAR by SP-A binding initiates a signal through pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of phosphatidylinositol 3-kinase (PI3K). In other cell types, cytokines that activate the PI3K signaling pathway promote cell survival. Therefore we investigated whether there was an effect of SP-A on apoptosis as measured by DNA laddering, FACS analysis, TUNEL assay, and annexin V binding. SP-A protected primary cultures of rat type II alveolar cells against the apoptotic effects of etoposide and UV light and also protected the H441 human Clara lung tumor cell line against staurosporine-induced apoptosis. The protective effects of SP-A were abrogated by inhibition of either tyrosine-specific protein kinase activity or PI3K. SP-A/SPAR interaction thus initiates a signaling pathway that regulates apoptosis in type II cells. These findings may be important in understanding the pathogenesis of acute lung injury and pulmonary tumorigenesis and may suggest new therapeutic options.     

Corticosteroid receptor in type II pneumocytes of the rat

Glucocorticoid receptor in rat type II pneumocytes has been characterized. The Scatchard plot analysis of 3H-dexamethasone binding to type II cells showed a single class of binding sites. The apparent Kd of 3H-dexamethasone binding by a whole cell assay was 9.1 nM and the maximal binding capacity was 78.0 f mol/10(6) cells (0.31 pmol/mg cytosol protein).     

Localization of epidermal-type fatty acid binding protein in alveolar macrophages and some alveolar type II epithelial cells in mouse lung

Almost all alveolar macrophages in the mouse lung were strongly immunoreactive for epidermal-type fatty acid binding protein. At the electron microscope level, the immunoreactive material was localized diffusely in the cytoplasm but not within the nucleus. A certain number of alveolar type II epithelial cells were also immunoreactive for the protein with variable immuno-intensity, while a substantial number of the type II cells were immunonegative. No immunoreactive interstitial fibroblasts were encountered. Based on the present findings, possible roles of epidermal-type fatty acid binding protein in the host-defence mechanism played by alveolar macrophages are suggested.     

Stimulation of surfactant secretion by vasopressin in primary cultures of adult rat type II pneumocytes

The current study examined the effect of vasopressin on the secretion of phosphatidylcholine, the principal component of pulmonary surfactant, from adult rat alveolar type II pneumocytes in primary culture. Vasopressin stimulated secretion in a time- and dose-dependent manner. At a concentration of 10 nM, vasopressin stimulated release by 6-fold over the basal secretory rate. The concentration producing half the maximal response for vasopressin-induced secretion was 0.4 nM. The stimulation of phosphatidylcholine release by vasopressin was duplicated by the vasopressin fragment, amino acids 4 through 9. [Lys8]vasopressin and the selective vasopressin-2 agonist [deamino-8-D-Arg]vasopressin did not stimulate surfactant secretion effectively. The vasopressin- and fragment-induced secretion was inhibited by the vasopressin-1 receptor antagonist d(CH2)5TDAVP and the protein kinase C inhibitor, tetracaine, but not by the beta-adrenergic antagonist alprenolol. Vasopressin did not activate adenylate cyclase, which suggests that stimulation by vasopressin was independent of cyclic AMP. When vasopressin and isoproterenol were added concomitantly, the effects on phosphatidylcholine secretion were additive. This suggests that these two secretagogues operate via separate mechanisms.     

Polarized light microscopy reveals physiological and drug-induced changes in surfactant membrane assembly in alveolar type II pneumocytes

In alveolar type II (AT II) cells, pulmonary surfactant (PS) is synthetized, stored and exocytosed from lamellar bodies (LBs), specialized large secretory organelles. By applying polarization microscopy (PM), we confirm a specific optical anisotropy of LBs, which indicates a liquid-crystalline mesophase of the stored surfactant phospholipids (PL) and an unusual case of a radiation-symmetric, spherocrystalline organelle. Evidence is shown that the degree of anisotropy is dependent on the amount of lipid layers and their degree of hydration, but unaffected by acutely modulating vital cell parameters like intravesicular pH or cellular energy supply. In contrast, physiological factors that perturb this structure include osmotic cell volume changes and LB exocytosis. In addition, we found two pharmaceuticals, Amiodarone and Ambroxol, both of which severely affect the liquid-crystalline order. Our study shows that PM is an easy, very sensitive, but foremost non-invasive and label-free method able to collect important structural information of PS assembly in live AT II cells which otherwise would be accessible by destructive or labor intense techniques only. This may open new approaches to dynamically investigate LB biosynthesis - the incorporation, folding and packing of lipid membranes - or the initiation of pathological states that manifest in altered LB structures. Due to the observed drug effects, we further suggest that PM provides an appropriate way to study unspecific drug interactions with alveolar cells and even drug-membrane interactions in general.     

Biochemical evidence for the presence of arginine decarboxylase activity in Trypanosoma cruzi.

Trypanosoma cruzi was found to release 14CO2 from radiolabeled arginine, and this effect was inhibited by either DL-alpha-difluoromethylarginine or monofluoromethylagmatine, both specific inhibitors of arginine decarboxylase (ADC). Furthermore, agmatine, which can be derived metabolically only by ADC-mediated arginine decarboxylation, was produced when T. cruzi was incubated with radiolabeled arginine, and agmatine production was inhibited in the presence of DL-alpha-difluoromethylarginine. These results constitute direct biochemical evidence for the presence in T. cruzi of ADC, an enzyme that does not occur in mammalian cells.     

The effect of anti-oxidants and medium composition on isolation and culture of alveolar type II pneumocytes

Alveolar type II pneumocytes were isolated from adult male rabbits and were placed in primary culture. The presence of anti-oxidants throughout the isolation procedure, particularly ascorbic acid and glutathione, was found to enhance in vitro attachment efficiency of cells. The use of a culture medium substituting D-valine for L-valine also significantly enhanced attachment efficiency. Although these cells do not ordinarily proliferate in culture, a low-serum medium containing insulin, transferrin, selenium and hydrocortisone allowed limited proliferation in addition to promoting dome formation in culture.     

Nitric oxide decreases surfactant protein gene expression in primary cultures of type II pneumocytes

Inhaled nitric oxide (NO) is a selective pulmonary vasodilator effective in treating persistent pulmonary hypertension in newborns and in infants following congenital heart disease surgery. Recently, multiple in vivo and in vitro studies have shown a negative effect of NO on surfactant activity as well as surfactant protein gene expression. Although the relationship between NO and surfactant has been studied previously, the data has been hard to interpret due to the model systems used. The objective of the current study was to characterize the effect of NO on surfactant protein gene expression in primary rat type II pneumocytes cultured on a substratum that promoted the maintenance of type II cell phenotype. Exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP), decreased surfactant protein (SP)-A, (SP)-B, and (SP)-C mRNA levels in type II pneumocytes in a time- and dose-dependent manner. The effect was mediated in part by an increase in endothelin-1 secretion and a decrease in the intracellular messenger, phosphorylated ERK1/2 mitogen-activated protein kinases (MAPK). Exposing type II pneumocytes to endothelin-1 receptor antagonists PD-156707 or bosentan before exposure to SNAP partially prevented the decrease in surfactant protein gene expression. The results showed that NO mediated the decrease in surfactant protein gene expression at least in part through an increase in endothelin-1 secretion and a decrease in phosphorylated ERK1/2 MAPKs.