Expression and localization of lung surfactant protein B in Eustachian tube epithelium

Surfactant protein (SP) B is an essential component of the pulmonary surfactant complex, which participates in reducing the surface tension across the alveolar air-liquid interface. The Eustachian tube (ET) connects the upper respiratory tract to the middle ear, serving as an intermittent airway between the pharynx and the middle ear. Recently, we described the expression of SP-A and SP-D in the ET, suggesting their role in middle ear host defense. Our present aim was to detect whether the expression of SP-B is evident in the porcine ET. With Northern blot analysis, RT-PCR, and in situ hybridizations, SP-B mRNA was identified and localized in the ET epithelium. The cellular localization of SP-B was revealed with immunohistochemistry, electron microscopy, and immunoelectron microscopy. The protein was found in the secretory granules of epithelial cells and also attached to the microvilli at the luminal side of these cells. The SP-B immunoreactivity of aggregates isolated from ET lavage fluid was similar to that isolated from bronchoalveolar lavage fluid. We conclude that there are specialized cells in the ET epithelium expressing and secreting SP-B and propose that SP-B may facilitate normal opening of the tube and mucociliary transport.     

Expression and localization of the thromboxane A2 receptor in human nasal mucosa

Thromboxane A2 (TXA2) has been thought a potent mediator involved in allergic rhinitis, because TXA2 was recovered from the nasal lavage fluid of allergic rhinitis patients after allergen provocation and TXA2 receptor antagonists relief nasal allergic symptoms. In order to clarify the expression of TXA2 receptor in human nasal mucosa, we investigated TXA2 receptor mRNA expression and its protein localization by polymerase chain reaction (PCR) and immunohistochemistry, respectively. Human turbinates were obtained after turbinectomy from 10 patients with nasal obstruction refractory to medical therapy. RT-PCR analysis of total RNA from nasal mucosa demonstrated the expression of TXA2 receptor alpha mRNA. The immunohistochemical studies revealed that anti-TXA2 receptor alpha antibody labeled vascular smooth muscle cells, vascular endothelial cells, epithelial cells and submucosal glands in the nasal mucosa. The results may have an important clinical implication for understanding the role of TXA2 receptor on upper airway diseases such as allergic rhinitis and non-allergic rhinitis.     

Expression and subcellular localization of Spred proteins in mouse and human tissues

Spred-1 and Spred-2 (Sprouty-related protein with an EVH1 domain) are recently described members of the EVH1 (Ena/VASP-homology domain 1) family. Both Spred-1 and Spred-2 are membrane-associated substrates of receptor tyrosine kinases and they act as negative regulators of the Ras pathway upon growth factor stimulation. Since the Spred family members seem to exert overlapping molecular functions, the isotype-specific function of each member remains enigmatic. To date, no comprehensive expression profiling of Spred proteins has been shown. Therefore, we compared mRNA and protein expression patterns of Spred-1 and Spred-2 systematically in mouse organs. Furthermore, we focused on the tissue-specific expression of Spred-2 in adult human tissues, the subcellular localization, and the potential role of Spred-2 in the organism. Our studies show that expression patterns of Spred-1 and Spred-2 differ markedly among various tissues and cell types. In mouse, Spred-1 and Spred-2 were found to be expressed predominantly in brain, whereas Spred-2 was found to be more widely expressed in various adult tissues than Spred-1. In humans, Spred-2 was found to be strongly expressed in glandular epithelia and, at the subcellular level, its immunoreactivity was associated with secretory vesicles. Using confocal microscopy we found Spred-2 to be strongly colocalized with Rab11 and, to a lesser extent, with Rab5a GTPase, an observation that was not made for Spred-1. We conclude that the two members of the recently discovered Spred protein family, Spred-1 and Spred-2, show a highly specific expression pattern in various tissues reflecting a specific physiological role for the individual Spred isoforms in these tissues. Furthermore, it becomes most likely that Spred-2 is involved in the regulation of secretory pathways.     

Expression of 15-lipoxygenase-1 in human nasal epithelium: its implication in mucociliary differentiation

15-lipoxygenase-1 (15-LO-1) is involved in the differentiation of human tracheobronchial epithelial cells. Here, we investigated the relation between 15-LO-1 expression and the differentiation of human nasal epithelium. In retinoic acid (RA)-sufficient culture media, 15-LO-1 expression in normal human nasal epithelial cell time-dependently increased, but its expression was undetectable in RA-deficient culture media. Moreover, in RA-deficient culture media, IL-4 at 1 ng/ml concentration time-dependently induced 15-LO-1 expression. In addition, MUC8 gene expression, a marker of mucociliary differentiation, was up-regulated by 15-LO-1, which was itself induced by IL-4. In murine nasal mucosa, the expression of leukocyte type-12-LO, a functional equivalent of 15-LO-1, reduced after postnatal day 7. Our findings suggest that 15-LO-1 is related to the differentiation of human nasal epithelium, and that it may mediate the mucociliary differentiation of human nasal epithelium.     

Lysozyme localization in human gastric and duodenal epithelium

Summary The distribution of lysozyme in normal gastric and duodenal mucosa was studied by light- and electronmicroscopic immunocytochemical techniques (direct enzyme-labeled antibody method).In the duodenal mucosa, lysozyme was found in the Paneth cells and the epithelial cells of Brunner's glands. Electron-microscopically, lysozyme was found in rough endoplasmic reticulum and perinuclear spaces, which were assumed to be protein-synthesizing organelles, and also in the secretory granules of Paneth cells. Additionally, lysozyme was detected in the stomach in mucinous granules and in some parts of the rough endoplasmic reticulum within the epithelial cells of the pyloric glands, the mucous neck cells of the fundic glands, and in several surface epithelial cells of the plyoric and fundic regions.This suggests that some quantity of lysozyme in gastrointestinal secretion originates from the gastric and duodenal glands, and that it acts as a defense mechanism in the gastrointestinal tract.     

The proteins of human lung surfactant

Human pulmonary surfactant was purified from bronchoalveolar lavage of patients. The proteins present in surfactant were analyzed by SDS-polyacrylamide gel electrophoresis into serum and non-serum components. One non-serum surfactant protein (Mr = 43 000) was then identified in the 100 000 X g supernatant of a lung homogenate on the basis of phospholipid binding. This lung protein was purified and partially characterized. The presence of 3-methyl histidine and reaction in Western blot analysis with antibody against chicken muscle actin both strongly suggested that the 43 000 Da protein of human surfactant is indeed cytoplasmic actin. It is proposed that this surfactant protein is involved in the secretion and not necessarily in the function of surfactant.     

Cytokeratin expression in human respiratory epithelium of nasal polyps and turbinates

The cytokertatins in respiratory epithelial cells (REC) of human nasal polyps and turbinates were analyzed by immunohistochemistry. Cytokeratin 19 (CK19) was present in all REC, CK5 and 14 were expressed primarily in basal cells, and CK7, 8, and 18 were found in suprabasal cells. Differences in cytoplasmic locations were also apparent among the individual cytokeratins. CK13 was not detected in any REC of these tissues. The results indicate the profile of cytokeratins in REC of human nasal polyps and turbinates is essentially identical to that of REC in the more distal respiratory tract.     

Metabolism and absorption enhancement of methionine enkephalin in human nasal epithelium

The objective of this study was to investigate absorption enhancing approaches for systemic delivery of methionine enkephalin via the nose. Absorption promotion of methionine enkephalin in the presence of protease inhibitors (bestatin, puromycin) and absorption enhancers (glycocholate, dimethyl-beta-cyclodextrin) were investigated in human nasal epithelium. Co-administration of the peptide with protease inhibitors and absorption enhancers resulted in a remarkable increase in Met-Enk permeation (4- to 94-fold). The increase was proportional to transepithelial resistance reduction and permeation of paracellular marker dye. Perturbation of the epithelial tight junctions seen in vitro may not occur in vivo due to mucus protection and mucociliary clearance.     

Lung surfactant proteins in the early human placenta

Pulmonary surfactant is a complex mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D). The biological functions of SP-A and SP-D are primarily twofold, namely surfactant homeostasis and host defense. The hydrophobic surfactant proteins, SP-B and SP-C, are required for achieving the optimal surface tension reducing properties of surfactant by promoting the rapid adsorption of surfactant phospholipids along the alveolar surface. Despite the promising findings, only little is known about the extrapulmonary distribution of these proteins. Therefore, in this study, the presence of SP-A, SP-B, SP-C and SP-D in early human placenta has been investigated. First-trimester placental tissues (22–56 days) were obtained from women undergoing curettage during normal pregnancies. In parallel tissue sections, vimentin, cytokeratin-7 and CD-68 immunostainings were used for the identification of mesenchymal cells, trophoblast cells and Hofbauer cells, respectively. According to immunohistochemistry (IHC) results, SP-A, SP-B, SP-C and SP-D immunoreactivities with different staining intensities were observed in trophoblastic layers of chorionic villous tree, trophoblastic cell columns, stromal cells, Hofbauer cells, angiogenic cell cords and vascular endothelium. Fetal hematopoietic cells showed a variable staining pattern for all four surfactant proteins ranging from none to strong intensity. Western blotting of tissue extracts confirmed our IHC results. The presence of surfactant glycoproteins in early human placenta may yield a very important feature of surfactants during first trimester and enables further studies of the role of surfactants in various pregnancy complications.     

Immunohistochemical localization of secretory proteins in the endometrial epithelium of the rabbit

Summary Proteins of uterine fluid and lung homogenates of the rabbit were separated by gel and ion exchange chromatography. Purified protein fractions were used for immunisation and antiserum production. By means of several absorptions, six monospecific antisera against uteroglobin and five other proteins were obtained. Using immunohistochemistry, four of them could be localised in the uterine epithelium from oestrus and the first and the seventh day post coitum, and also in the blastocyst. The present study indicates the involvement of different endometrial cells in the synthesis and release of the various proteins of uterine secretion.Supported by grant Ki 154/6-7 from the Deutsche Forschungsgemeinschaft     

Ion transporting proteins of human bronchial epithelium

The electrolyte transport system across human airway epithelium followed by water movement is essential for the normal mucociliary clearance that allows the maintenance of the aseptic condition of the respiratory tract. The function of epithelial cells is to control and regulate ionic composition and volume of fluids in the airways. Various types of proteins taking part in assuring effective ions and water transport in apical and basolateral membranes of the airway epithelium have been found (e.g., CFTR, ENaC, CaCC, ORCC, potassium channels, NaKATPase, aquaporins). The paper reviews the current state of the art in the field of ion channels, transporters, and other signaling proteins identified in the human bronchial epithelium.     

Intracellular localization of processing events in human surfactant protein B biosynthesis

Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to alveolar type 2 cell phenotype. Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa SP-B. To examine the intracellular sites of SP-B processing, we carried out immunofluorescence cytochemistry and inhibitor studies on human fetal lung in explant culture and isolated type 2 cells in monolayer culture using polyclonal antibodies to human SP-B(8) (Phe(201)-Met(279)) and specific epitopes within the N- (NFProx, Ser(145)-Leu(160); NFlank Gln(186)-Gln(200)) and C-terminal (CFlank, Gly(284)-Ser(304)) propeptides of pro-SP-B. Fluorescence immunocytochemistry using epitope-specific antisera showed colocalization of pro-SP-B with the endoplasmic reticulum resident protein BiP. The 25-kDa intermediate was partially endo H-sensitive, colocalized with the medial Golgi resident protein MG160, and shifted into the endoplasmic reticulum in the presence of brefeldin A, which interferes with anterograde transport from endoplasmic reticulum to Golgi. The 9-kDa intermediate colocalized in part with MG160 but not with Lamp-1, a transmembrane protein resident in late endosomes and lamellar bodies. Brefeldin A induced a loss of colocalization between MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear tubular array. In pulse-chase studies, brefeldin A blocked all processing of 42-kDa pro-SP-B whereas similar studies using monensin blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We conclude that: 1) the first enzymatic cleavage of pro-SP-B to the 25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2) cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies.     

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.     

Interleukin-13 stimulates production of nitric oxide in cultured human nasal epithelium

The diversity and extent of signaling functions of nitric oxide (NO) in cell physiology as well as its presence and influence as a common component of ambient air pollution and tobacco smoke are gaining increasing research attention relative to both health and disease. While cellular NO production is typically associated with inflammatory cells and processes, the airway epithelium particularly of the paranasal sinuses, has been documented to be a rich source of excreted NO. Inasmuch as excreted NO derives from both mucosal and inflammatory cell sources, distinguishing the individual contribution of these compartments to total excreted cellular NO is potentially problematic. We simulated an inflammatory mucosal environment by stimulating human nasal epithelial cultures with interleukin-13 (IL-13), a mediator produced by eosinophils in asthma, allergic rhinitis, and sinusitis. While a consistent baseline of NO excretion in control cultures was documented, widely variable individual responses to IL-13 exposure were observed in companion cultures maintained under identical conditions and tested at the same time. These studies suggest that cellular NO excretion by the healthy epithelial mucosa is subject to considerable individual variability and may be significantly elevated among some individuals in the presence of IL-13 stimulation.