Site specificity of surfactant protein expression in airways of baboons during gestation

BACKGROUND: There are disparate reports concerning the presence of surfactant proteins in the airways of lung. The recent finding of SP-A in tracheobronchial epithelium and submucosal glands in lungs from second trimester humans has renewed interest in potential new functions of surfactant in lung biology. METHODS: In situ hybridization studies were done to determine the distribution of SP-A, SP-B, and SP-C in baboon lung specimens from 60, 90, 120, 140, 160, and 180 (term) days of gestation and adults. Lungs from gestation controls were obtained at the time of hysterotomy and adult lungs at necropsy. Riboprobes used for in situ hybridization contained the entire coding regions for human SP-A, SP-B, and SP-C. RESULTS: At 60 days, SP-C mRNA expression was evident in focal portions of primitive tubular epithelium but not bronchi. This distal pattern of SP-C mRNA expression persisted and was present in some epithelial cells of respiratory bronchioles at term. At 90 days, SP-A mRNA expression was present in the epithelium of trachea and large bronchi. SP-B mRNA expression was found in small bronchi, bronchioles, and distal tubular epithelium at 120 days of gestation. SP-A mRNA bronchiolar localization became evident at 140 days of gestation and alveolar type 2 cellular expression at 160 days of gestation. Abrupt transitions of surfactant protein expression were identified (e.g., SP-A mRNA-positive cells in the epithelium of large bronchi with adjoining SP-B mRNA expression in small bronchi and bronchioles). CONCLUSIONS: Findings in the baboon indicate that there are well-delineated sites of surfactant protein mRNA expression in bronchial and bronchiolar epithelia. mRNA expressions of SP-A and SP-B are present in both bronchial and bronchiolar epithelium but at different sites, whereas SP-C expression is seen in loci of epithelial cells in respiratory bronchioles.     

Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide

Neonatal respiratory function depends on the development of a well-formed pulmonary capillary bed. Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell growth and angiogenesis. High levels of VEGF protein and messenger RNA (mRNA) have been detected in the developing lung, suggesting that VEGF plays a role in the development of the pulmonary capillary bed. To begin to understand the role of VEGF in human lung development, we explored the regulation of VEGF gene expression and the localization of VEGF protein and mRNA in a model of the developing human lung. VEGF protein and mRNA were detected in midtrimester human fetal lung tissue, and their levels increased with time in explant culture. VEGF protein and mRNA were increased by the maintenance of human fetal lung explants in 2% O2 environments compared with 20% O2 environments. VEGF mRNA levels were found to be increased by cyclic adenosine monophosphate (cAMP) in explants that were incubated in 20% O2, but not in those incubated in 2% O2. Immunostaining for VEGF protein demonstrated localization primarily in airway epithelial cells in midtrimester human fetal lung tissue. Immunostaining for VEGF increased with incubation of human fetal lung explants in 2% and 20% O2. Interestingly, VEGF protein was localized primarily in the basement membrane subjacent to airway epithelial cells after 4 d of incubation in 20% O2. Incubation of tissues in the presence of dibutyryl cAMP resulted in an increase in immunostaining for VEGF, primarily in the basement membranes of prealveolar ducts in 20% O2-treated tissues. In situ hybridization studies indicated that VEGF mRNA was present in both mesenchymal cells and airway epithelial cells. These data suggest that VEGF gene expression is regulated by both oxygen and cAMP in the developing human lung. The detection of VEGF mRNA and protein in distal airway epithelial cells and the detection of VEGF protein in the basement membrane subjacent to the airway epithelial cells suggest that translocation of VEGF protein occurs after its synthesis in the epithelium. Localization of VEGF to the basement membrane of airway epithelial cells may be important for directing capillary development in the human lung.     

Cholinergic dependence of a cortical neuronal mechanism that supports Pavlovian eyeblink conditioning

Mucin glycoproteins play a key role in the normal function of the airway epithelium. We examined the expression of mucin genes, MUC3, 4, 5AC, 5B, 6, 7, and 8 in human fetal tissues to establish the localization and age of onset of expression of each mucin gene during human development. We detected expression of MUC4, 5AC, 5B, and 7 in the mid-trimester airway epithelium but did not detect expression of MUC3, 6, or 8. MUC4 was expressed in the trachea and large airways in the majority of cells in the airway epithelium. Expression of MUC5AC was only seen in individual goblet cells in the trachea, while MUC5B was expressed in the surface epithelium of the trachea at 13 wk but was largely restricted to submucosal glands by 23 wk of gestation.     

Pregnancy-specific alterations in the expression of the insulin-like growth factor system during early placental development in the ewe

The placenta is recognized as an important determinant of fetal growth rate, yet the factors regulating its proliferation remain poorly understood. Components of the insulin-like growth factor (IGF) system were localized in the ovine uterus using in situ hybridization between days 13-55 of gestation, the period of implantation and placentome formation. IGF-II messenger RNA (mRNA) expression was intense in the fetal mesoderm, particularly at the tips of the invading placentome villi. Moderate levels of IGF-II mRNA were also observed in the maternal caruncular stroma. In contrast, expression of IGF-1 mRNA was low (compared to estrous levels) and ubiquitous decreasing as gestation advanced. IGF-binding protein-2 (IGFBP-2 mRNA was not detected until day 29 of gestation, when it appeared restricted to the dense caruncular-like stroma lining the luminal epithelium, colocalized with IGFBP-4. High concentrations of IGFBP-4 mRNA expression were also found in the placentome capsule. IGFBP-3 mRNA expression was intense in the luminal epithelium between days 13-15 of gestation. Subsequently, levels in this region dropped significantly (P < 0.001). IGFBP-3 mRNA expression was also high in the maternal placentome villi, where photographic emulsions localized expression to blood vessel walls. Peak expression of IGF type 1 receptor (IGF-1R) mRNA was found in the deep uterine glands, with intermediate expression in the superficial uterine glands. Moderate expression of IGF-1R mRNA was initially recorded in caruncular stroma, but levels in this region decreased significantly (P < 0.001) to below the detection limit of the technique after interdigitation by the fetal allantochorion. Furthermore, IGF-1R mRNA could not be detected in any fetal placentome tissue. This study, therefore, has established the pattern of expression of the IGFs, IGF-1R, and three of the IGFBPs during establishment of the ovine placenta. It will form the basis for future work to investigate how this system is regulated and to determine the role of the IGFs in placental development.     

Transgenic mice overexpressing the complement inhibitor crry as a soluble protein are protected from antibody-induced glomerular injury

We report that matrilysin, a matrix metalloproteinase, is constitutively expressed in the epithelium of peribronchial glands and conducting airways in normal lung. Matrilysin expression was increased in airway epithelial cells and was induced in alveolar type II cells in cystic fibrosis. Other metalloproteinases (collagenase-1, stromelysin-1, and 92-kD gelatinase) were not produced by normal or injured lung epithelium. These observations suggest that matrilysin functions in injury-mediated responses of the lung. Indeed, matrilysin expression was increased in migrating airway epithelial cells in wounded human and mouse trachea. In human tissue, epithelial migration was reduced by > 80% by a hydroxamate inhibitor, and in mouse tissue, reepithelialization in trachea from matrilysin-null mice was essentially blocked. In vivo observations and cell culture studies demonstrated that matrilysin was secreted lumenally by lung epithelium, but upon activation or while migrating over wounds, some matrilysin was released basally. The constitutive production of matrilysin in conducting airways, its upregulation after injury, its induction by alveolar epithelium, and its release into both lumenal and matrix compartments suggest that this metalloproteinase serves multiple functions in intact and injured lung, one of which is to facilitate reepithelialization.     

Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells

We compared the expression of human alpha- and beta-defensins by various human tissues. mRNA for alpha-defensins HNP1-3, abundant in bone marrow, was detected in peripheral blood leukocytes, spleen and thymus by RT-PCR, which revealed alpha-defensins HD5 and HD6 only in the small intestine. In contrast, the pancreas and kidney expressed high levels of hBD-1 and lower levels of this beta-defensin were found in many organs by RT-PCR (salivary gland > trachea > prostate and placenta > thymus, testis, small intestine). hBD-1 mRNA was produced constitutively by cultured normal human epithelial cells derived from the trachea, bronchi, small airways and the mammary gland. These largely non-overlapping tissue distributions of human alpha- and beta-defensins suggest that hBD-1 may be positioned to defend epithelial cells and mucosae from infection, whereas expression of HNP1-3 in neutrophils and HD5 and HD6 in Paneth cells allows these alpha-defensins to participate in systemic and small intestinal host defenses, respectively.     

Endothelin in human nasal mucosa

Endothelin (ET), a potent vasoconstrictor and bronchoconstrictor peptide synthesized by endothelial and epithelial cells, was examined for its potential functions in human inferior turbinate nasal mucosal tissue by four techniques: (1) immunoreactive ET was localized in the mucosa by immunohistochemistry; (2) receptors for ET were identified by autoradiography employing [125I]ET; (3) ET-1 mRNA was localized by in situ hybridization; and (4) the secretory functions of ET were examined by the release of mucous and serous cell products after the addition of ET to human nasal turbinates in short-term cultures. Specific ET-1-immunoreactive material was found most extensively in small muscular arteries and in serous cells in submucosal glands. ET-1 was also found to a lower extent in the walls of venous sinusoids. [125I]ET-1 binding sites were localized by autoradiography to submucosal glands and to venous sinusoids and small muscular arterioles. mRNA for ET-1 was found most extensively in the venous sinusoids and to a lesser extent in small muscular arteries. In mucosal explant cultures, ET-1 and ET-2 stimulated lactoferrin and mucous glycoprotein release from serous and mucous cells, but ET-3 was inactive. The observations indicate that in the human nasal mucosa, ET is present in the vascular endothelium and the serous cells in submucosal glands and acts on glandular ET receptors to induce both serous and mucous cell secretion. It is also likely that ET plays a role in the regulation of vasomotor tone.     

Distribution of casein-like proteins in various organs of rat

Casein-like proteins were detected in various organs of rat by use of a specific antiserum raised against rat milk caseins. The antiserum specifically recognized alpha 1-, alpha 2-, beta-, and gamma-caseins in rat milk by Western blot analysis, whereas no immunoreactive band was observed in sera of rat and fetal bovine and in bovine caseins. Immunohistochemical studies of this antiserum on formalin-fixed mammary glands showed that immunoreactive caseins were localized to the apical portion of the cytoplasm in lactating mammary epithelial cells and in the luminal secretion, which indicates a directional secretion of caseins to the lumen by the mammary epithelial cells. With this antiserum, immunoreactive substances were detected in various organs, including the pancreatic ducts and islets of Langerhans, the secretory ducts of salivary glands, zona fasciculata cells and ganglion cells of adrenal gland, distal tubules and convoluted collecting tubules of kidney, epithelial cells of bronchioles and large pneumocytes of the lung, hair follicles, sebaceous glands, and the prickle cell layer of skin, uterine glands and epithelium of the endometrium, hepatic bile ducts, and brain. In Western blot analysis, major immunoreactive substances in the above organ extracts showed a similarity in molecular weight to alpha 2-casein of rat milk. Skin was the only tissue that expressed both alpha 2- and beta-caseins. There were no other immunoreactive bands with similarity to beta- and gamma-caseins in the other organ extracts, but higher molecular weight immunoreactive bands (> 100 kD) were detected in some organ extracts, such as salivary gland, kidney, liver, lung, and uterus. These findings suggest that the alpha 2-casein-like substance is localized not only in the mammary gland but also in a variety of organs and may play an important role as a functional molecule in those organs.     

Recombinant uracil phosphoribosyltransferase from the thermophile Bacillus caldolyticus: expression, purification, and partial characterization

The hydrophobic surfactant protein C (SP-C) is known to modulate the biophysical properties of surfactant phospholipid. Although SP-C mRNA has been demonstrated in human fetal lung, there is limited information regarding developmental expression and processing of proSP-C protein. Two epitope-specific human proSP-C antisera, anti-hCPROSP-C (His59-Ser72) and anti-hCTERMSP-C (Gly162-Gly175), were generated to complement previously produced anti-NPROSP-C (Met10-Gln23) for the study of proSP-C expression in human fetal lung. Western blotting and immunocytochemistry detected expression of proSP-C protein by 12-16 wk of gestation. ProSP-C immunoreactivity of preculture lung, limited to expression of proSP-C21 in airway epithelial cells, was markedly enhanced by culture of lung explants in dexamethasone. To examine synthesis of proSP-C, homogenates from explants were labeled with 35S-Met/Cys for 0.5-4 h. Immunoprecipitation with anti-NPROSP-C detected 35S-proSP-C21 by 30 min and, after 2 h of labeling, there was a 15-fold increase in 35S-proSP-C21 in dexamethasone-treated lungs versus controls. Synthesis of proSP-C21 was followed by the appearance of a 24-kD form and smaller processing intermediates including 6-10-kD forms. Posttranslational processing of proSP-C21 was not observed in control explants. SP-C(6-10) were not recognized by either anti-CPROSP-C or anti-hCTERMSP-C. These results indicate that low level expression of proSP-C protein first occurs in epithelial cells early in the second trimester and that expression can be enhanced by dexamethasone. Initial posttranslational processing of human proSP-C involves modification of proSP-C21 to SP-C24 and subsequent proteolysis of C-terminal propeptide domains. We speculate that absence of low Mr intermediates in unstimulated second trimester fetal lung tissue reflects developmental and glucocorticoid dependent regulation of proSP-C21 synthesis and posttranslational processing.     

Epithelial marker genes are expressed in cultured embryonic rat lung and in vivo with similar spatial and temporal patterns

Explants of embryonic lung are often used to characterize lung growth, bronchial tree pattern, and cell differentiation. Most investigators culture lungs for 3-7 days in defined media lacking, e.g., added growth factors or hormones. If growth and differentiation are comparable to that in vivo, these cultures show considerable promise for identifying developmental regulatory molecules and target genes, and for elucidating molecular responses. We used in situ hybridization and RT-PCR to compare times and sites of expression of mRNAs of six epithelial genes in cultured and uncultured fetal rat lungs. These genes, expressed in distal lung of adult rats, are surfactant proteins (SP) A, B, and C; LAR, a receptor-type tyrosine phosphatase; Clara cell secretory protein (CC10, CCSP); and T1alpha. SP-A, SF-B, LAR, and CC10 are expressed by both Clara and Type II cells in adult animals. SP-C and T1alpha are unique markers for Type II and Type I cells, respectively. SP-C, LAR, and T1alpha are expressed before the lung is explanted (Day 13.5); SP-A, -B, and CC10 mRNAs are first detected later. The onset of expression is similar in vivo and in vitro. Although the patterns of expression differ for each mRNA, their sites of expression in culture match those in vivo relative to the bronchial tree. The explanted embryonic lung appears to be an excellent experimental model.     

Development of PGP 9.5- and calcitonin gene-related peptide-like immunoreactivity in organ cultured fetal rat lungs

Knowing that small-granule endocrine cells develop in organ cultured fetal lungs, we investigated whether the cells produce regulatory peptides in vitro, and if sufficient amounts appear to permit using the cultures as an experimental system for physiological study of secretory mechanisms. The paired lungs from 14-day and 15-day fetal rats were organ cultured for 1-8 days and examined daily for development of immunoreactivity against marker proteins and regulatory peptides associated with small-granule endocrine cells and nerves. They proved reactive against protein gene product 9.5 (PGP) and calcitonin-gene related peptide (CGRP) but not against calcitonin or neurofilament protein 200 K, although positive controls were obtained for these substances in lungs from postnatal animals. Initially PGP-like immunoreactivity is associated with cell bodies and processes of neuroblasts which run medial to the bronchial axis on day 14 and are increasingly prevalent on day 15. In 15-day explants PGP becomes detectable after a day in vitro in rare "clear cell" precursors of small-granule cells located in the epithelium lining proximate parts of the lungs, although in 14-day explants comparable reactivity is not seen until the third day (14 + 3 days). In culture PGP-positive neuroblasts increase in number, and nerve processes gradually extend down the airway to encircle the sleeve of smooth muscle that develops as the bronchial tree expands. Concurrently, the initially small clusters of small-granule cells increase in size, and new ones appear in the airway lining. By 15 + 5 days they extend to the boundary between a taller, more proximal epithelium and a glycogen-rich cuboidal layer that lines one or two most-distal generations of branches. Thereafter, the trachea and central, cartilage-bound segments of the primary bronchi mainly contain solitary endocrine cells and the more peripheral lung a mixture of single cells and clusters, much as in near-term lungs in vivo. At this stage PGP-positive nerves extend as far as the entrances of the terminal sacs, and most are distributed to the airway muscle plexus. Exceptionally, they may innervate a small-granule cell cluster, converting it into a neuroepithelial body. CGRP-like immunoreactivity initially appears in small-granule cells of 15 + 2-day cultures but does not develop in ganglion cells or nerves. It localizes to endocrine cells at all conducting airway levels, increasing in staining intensity and accounting for most if not all of the PGP-positive population between 15 + 4 - 15 + 8 days.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular localization of insulin-like growth factor II mRNA in the human fetus and the placenta: detection with a digoxigenin-labeled cRNA probe and immunocytochemistry

IGF-II plays a major role in the regulation of human fetal growth and development. However, more extensive information on the cellular sites of IGF-II synthesis in the fetus would provide more insight into its role in fetal organogenesis. Thus we have determined the sites of IGF-II synthesis in 18-26-wk gestation human fetal tissues using in situ hybridization with a digoxigenin-labeled cRNA probe to localize IGF-II mRNA in fetal liver, kidney, adrenal gland, cerebral cortex, costal cartilage, skeletal muscle, and lung, and in placental tissue. In human fetal tissues it has to date been impossible to clearly assign IGF-II mRNA to epithelial cells of entodermal origin. Besides their already known localization in cell matrix and a variety of mesodermal cell types, strong IGF-II mRNA-positive signals were detected in epithelial cells in the liver (hepatocytes), bronchial and bronchiolar epithelium, undifferentiated renal tubular epithelium, mature glomerular epithelium, pelvic urothelium, and adrenal epithelial cells of the zona persistens. To identify the cellular location of immunoreactive IGF-II, we also performed immunocytochemical studies in tissues of the same fetuses. Every tissue studied except the cerebral cortex contained immunoreactive cells; however, immunostaining was generally weaker than in situ hybridization signals. Our data show that the distribution of IGF-II in human fetal tissue is much more widespread than hitherto thought. A digoxigenin-labeled detection system for IGF-II is more capable of detecting the cellular expression pattern of IGF-II than radioactive probes and is suitable for analysis of routinely prepared paraffin-embedded material.     

Clearance of lead-212 ions from rabbit bronchial epithelium to blood

The absorption of 212Pb ions from bronchial epithelium to blood has been investigated in anaesthetized rabbits. The 212Pb ions were introduced by intubation either into the trachea or into smaller, more distal bronchi. Removal from lung was followed by external gamma-counting. Mucociliary clearance to the GI tract was blocked by tracheostomy. Two distinct phases of clearance from bronchial epithelium to blood were observed. Approximately 20% of deposited 212Pb is rapidly absorbed with a half-time of about 4 min, the remainder with a biological half-time of about 9 h, irrespective of the site of instillation in the bronchial tree. Two hours after deposition, the 212Pb remaining in lung was found to be partitioned between mucus and the bronchial epithelium, with a substantial but minor fraction in the epithelium. Uptake of 212Pb in the skeleton was estimated to be about 20% of the 212Pb entering the blood circulation. Removal by the kidneys, at 25%, was comparable with skeletal uptake. These results are compared with previously published work using rodents, dogs and man which demonstrated either rapid or slow absorption but not both phases occuring together.     

Distribution of fibroblast growth factor (FGF)-2 and FGF receptor-1 messenger RNA expression and protein presence in the mid-trimester human fetus

Fibroblast growth factors (FGF) are known to have key roles in embryonic growth and morphogenesis, but their presence and contributions to fetal development are unclear. In particular, little information exists as to the relevance of FGF and their specific receptors to human fetal development. We studied the anatomical distribution of messenger RNA encoding FGF-2 and one of its high affinity receptors, FGFR1, using in situ hybridization in a variety of human fetal tissues in early second trimester. Corresponding protein distributions were determined by immunohistochemistry. Both FGF-2 and FGFR1 mRNA and proteins were found to be present in every organ and tissue examined, but with defined cellular localizations. In skeletal muscle, both FGF-2 and FGFR1 mRNA and peptides were present in differentiated fibers, and both co-localized to proliferating chondrocytes of the epiphyseal growth plate. FGF-2 and FGFR1 mRNA and peptides were also present within cardiac or gastrointestinal smooth muscle. Within the gastrointestinal tract FGF-2 mRNA and peptide were located in the submucosal tissue, whereas FGFR1 was expressed within the overlying mucosa. Similarly, in skin, FGF-2 was expressed within the dermis whereas FGFR1 mRNA and peptide were most apparent in the stratum germinativum of the epidermis. In kidney and lung, FGFR1 mRNA was located in the tubular and alveolar epithelia respectively, whereas FGF-2 was expressed in both epithelial and mesenchymal cell populations. Both growth factor and receptor were widespread in both neuroblasts and glioblasts in the cerebral cortex of the brain. Immunoreactivity for FGF-2 and FGFR1 was seen in all vascular endothelial cells of major vessels and capillaries. Within the skin, kidney, lung, and intestine FGF-2 immunoreactivity was found in basement membranes underlying epithelia, and was associated with the extracellular matrix and plasma membranes of many cell types. The results show that FGF-2 and one of its receptors are widely expressed anatomically in the mid-trimester human fetus.     

Comparative evaluation of the sensitivity to Mycoplasma pneumoniae of organ cultures of the trachea and lungs of certain animals

A possibility was shown of cultivation of M. pneumoniae in the organ cultures of the trachea and the lungs of a pig embryo. 5-7-day guinea pigs, and 3-5-day chicks. The lung and tracheal explants of chicks possessed the greatest sensitivity to M. pneumoniae. M. pneumoniae strains were isolated from the nasopharyngeal smears and washings from the bronchi obtained in bronchoscopy of patients suffering from chronic pneumonia, with the use of organ culture of chick trachea. Explains of chick trachea could serve as a medium for the isolation of M. pneumoniae from the patients.