Characterisation of lectin binding patterns of mouse bronchiolar and rat alveolar epithelial cells in culture

Lung epithelial cell differentiation pathways remain unclear. This is due in part to the plasticity of these cells and the lack of markers which accurately reflect their differentiation status. The aim of this study was to determine if lectin binding properties are useful determinants of functional differentiation status in vitro. Mouse Clara cells were cultured for 5 days. During this time, no alteration in differentiation was evident by electron microscopy. No significant alteration in binding reactivity of Bauhinia purpurea (BPA), Maclura pomifera (MPA), Concanavalin A, Wheat germ or Helix pomatia lectins occurred in cultures compared with Clara cells in mouse lung tissue. In contrast, nitrotetrazolium blue reductase activity and CC10 expression declined in culture. Rat type II cells were cultured for 8 days. Between days 0 and 4, the number of type II cells identified by electron microscopy was constant at 70–80%, decreasing to 8% by day 6. In contrast, by day 4, only 42% cells retained alkaline phosphatase activity. BPA and MPA reactivity was altered at day 0 and day 4 respectively, compared with cells in situ. Therefore, the reactivity of lectins analysed here does not reflect functional differentiation status of cultured mouse Clara cells. However, BPA and MPA reactivity may be a sensitive indicator of alterations in rat type II cell differentiation in vitro.     

Hydrogen ion currents in rat alveolar epithelial cells.

Alveolar epithelial cells isolated from rats and maintained in primary culture were studied using the whole-cell configuration of the "patch-clamp" technique. After other ionic conductances were eliminated by replacing permeant ions with N-methyl-D-glucamine methanesulfonate, large voltage-activated hydrogen-selective currents were observed. Like H+ currents in snail neurons and axolotl oocytes, those in alveolar epithelium are activated by depolarization, deactivate upon repolarization, and are inhibited by Cd2+ and Zn2+. Activation of H+ currents is slower in alveolar epithelium than in other tissues, and often has a sigmoid time course. Activation occurs at more positive potentials when external pH is decreased. Saturation of the currents suggests that diffusion limitation may occur; increasing the pipette buffer concentration from 5 to 120 mM at a constant pH of 5.5 increased the maximum current density from 8.7 to 27.3 pA/pF, indicating that the current amplitude can be limited in 5 mM buffer solutions by the rate at which buffer molecules can supply H+ to the membrane. These data indicate that voltage-dependent H+ currents exist in mammalian cells.     

Glucose-6-phosphate dehydrogenase in rat lung alveolar epithelial cells. An ultrastructural enzyme-cytochemical study

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway in carbohydrate metabolism, and it plays an important role in cell proliferation and antioxidant regulation within cells in various organs. Although marked cell proliferation and oxidant/antioxidant metabolism occur in lung alveolar epithelial cells, definite data has been lacking as to whether cytochemically detectable G6PD is present in alveolar epithelial cells. The distribution pattern of G6PD within these cells, if it is present, is also unknown. The purpose of the present study was to investigate the subcellular localization of G6PD in alveolar cells in the rat lung using a newly-developed enzyme-cytochemistry (copper-ferrocyanide) method. Type I cells and stromal endothelia and fibroblasts showed no activities. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of type II alveolar epithelial cells. The cytochemical controls ensured specific detection of enzyme activity. This enzyme may play a role in airway defense by delivering substances for cell proliferation and antioxidant forces, thus maintaining the airway architecture.     

Secretagogue-induced proteolysis of lung spectrin in alveolar epithelial type II cells.

Incubation of isolated rat alveolar epithelial type II cells with secretagogues (calcium ionophore, ATP or terbutaline) resulted in rapid proteolysis of lung spectrin and appearance of multiple proteolytic products which showed immunoreactivity with an antibody against human erythrocyte spectrin. These proteolytic products were similar to those generated from erythrocyte spectrin or cultured lung tumor cells (A549 cells) incubated with purified calpain. Furthermore, incubation of alveolar type II cells with a calpain-specific inhibitor modulated the secretagogue-induced proteolysis of lung spectrin. Thus, stimulation of secretion appeared to activate endogenous calpain in type II cells, suggesting that calpain-mediated proteolysis of a submembranous cytoskeletal protein could play an important role in the secretory process.     

Gap junction-microtubule associations in rat alveolar epithelial cells

Connexin 43 (Cx43) is a predominant gap junction (GJ) protein expressed by alveolar epithelial cells (AEC) in primary cell culture. Cx43 trafficking, assembly, and turnover are regulated by multiple mechanisms, including those mediated by integrins, by extracellular matrix, and by the cytoskeleton. Immunocytochemical double labeling demonstrates association of microtubules with internalization of Cx43-positive GJ plaques. Antibodies against the alpha 5-integrin subunit block cell-matrix interactions without effect on tubulin expression, whereas inhibition of MAP kinase kinase by PD-98059 reduces tubulin expression, based on both Western blot and immunostaining. To examine direct association of microtubules (MT) with GJ plaques, we treated day 3 AEC for 0.5-24 h with colchicine, an inhibitor of tubulin polymerization. After 60 min, MTs were disassembled, whereas Western blot analysis showed no change in tubulin expression. In parallel, colchicine initiated redistribution of immunopositive Cx43 from the membrane to the cytosol. These observations support the premise that direct association of the cytoskeleton with gap junctions plays a significant role in regulation of Cx43 expression and distribution through integrin-mediated signal transduction pathways.     

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.     

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.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Lectin binding to parietal cells of human gastric mucosa

A light microscopic and ultrastructural analysis of lectin receptors on parietal cells from human gastric mucosa was performed utilizing 12 biotinylated lectins in conjunction with an avidin-biotin-peroxidase complex. Peanut agglutinin conjugated directly to peroxidase was also used. Several fixatives and fixation regimens were evaluated for optimal preservation of parietal cell saccharide moieties. Formalin proved to be the most practical fixative for light microscopic studies. A periodate-lysine-paraformaldehyde (PLP) combination provided good preservation of lectin binding capacity but yielded relatively poor ultrastructure. Conversely, glutaraldehyde provided excellent preservation of ultrastructure but a somewhat diminished lectin binding activity, which was overcome by using long incubation times and high concentrations of reagents. Parietal cells reacted strongly with Bandieraea simplicifolia, Dolichos biflorus, peanut agglutinin, and soybean agglutinin (all specific for galactosyl/galactosaminyl groups) and weakly with Ulex europaeus (specific for fucose). At the light microscopic level a beaded, perinuclear staining pattern was observed which, ultrastructurally, corresponded to an intense staining of intracytoplasmic canaliculi. The membranes of the intracytoplasmic canaliculi were characterized by an abundance of galactosyl residues, a paucity of fucosyl groups, and a lack of mannosyl and glucosyl residues. The biochemical and physiological significance of these findings is discussed.     

Lectin binding ability of B-chronic lymphocytic leukaemia cells.

The binding of five fluorescein-labelled lectins: peanut agglutinin (PNA), lentil agglutinin (LEN), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and asparagus pea agglutinin (ASP) to human B-cell chronic lymphocytic leukaemia (B-CLL) and B lymphocytes of normal donors was studied. The specificity of the fluorescence was demonstrated by inhibition with appropriate saccharides. The proportion of B cells was estimated using anti-B cell monoclonal antibody. Both leukaemic and normal B cells showed the binding ability of all except of one (ASP) studied lectins. We have found the differences in surface carbohydrate patterns between B-CLL and normal B lymphocytes. B-CLL cells showed the considerably lower ability to bind SBA and slightly higher expression of PNA and LEN receptors in comparison to normal B cells. The analysis of WGA binding allowed for recognizing two groups of CLL patients: one with high and the second one with low WGA receptor expression. The double marker studies revealed that B cells could simultaneously react with anti-B cell monoclonal antibody and fluorochrome labelled lectins.     

Invited review: biophysical properties of sodium channels in lung alveolar epithelial cells.

Amiloride-sensitive sodium channels in the lung play an important role in lung fluid balance. Particularly in the alveoli, sodium transport is closely regulated to maintain an appropriate fluid layer on the surface of the alveoli. Alveolar type II cells appear to play an important role in this sodium transport, with the role of alveolar type I cells being less clear. In alveolar type II cells, there are a variety of different amiloride-sensitive, sodium-permeable channels. This significant diversity appears to play a role in both normal lung physiology and in pathological states. In many epithelial tissues, amiloride-sensitive epithelial sodium channels (ENaC) are formed from three subunit proteins, designated alpha-, beta-, and gamma-ENaC. At least part of the diversity of sodium-permeable channels in lung arises from the assembling of different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. This leads to epithelial tissue in the lung, which has enormous flexibility to alter the magnitude and regulation of salt and water transport. In this review, we discuss the biophysical properties and occurrence of these various channels and some of the mechanisms for their regulation.     

Isolation of alveolar epithelial cells from lung tissue obtained at autopsy

Summary Lung alveolar epithelial cells have been studied in a variety of laboratory animal models, and studies of human alveolar epithelial cells are important for comparison to information obtained from animal studies. Autopsy material is a source of human cells for study. Studies of human autopsy material revealed variables that negatively affected the yield of viable cells. For specimens from adults, these included death greater than 12 h before cell isolation, obvious severe lung fibrosis, longstanding metabolic disorders, and lung congestion indicated by weight of the right middle lobe greater than 150 g. Samples from children yielded significant numbers of viable cells up to 18 h after death. For 17 specimens that conformed to the above criteria, approximately 8.5×10⁶ alveolar cells were obtained per gram of tissue (tissue weights ranged from 30 to 108 g) using a procedure involving instillation of proteases into the airways. The cells could be further fractionated, and 10 to 15% of the mixed cells obtained were type II pneumocytes. Analysis of NADPH cytochrome-c-reductase distribution in subcellar fractions provided evidence that the cells obtained were intact. Phospholipid enzyme activities and synthetic activity were within the ranges previously found in laboratory studies of freshly obtained animal lungs. These results suggest that significant numbers of viable and functional human lung cells, including type II pneumocytes, can be obtained from autopsy material. This research was supported by a grant (HL 33083) from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Summary Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair. The 16-day fetal tissue is mitotically active, does not exhibit a well defined monolayer, and demonstrates weak fluorescence binding for WGA, Con A and RCA. Conversely, SBA binding is readily detected on many cell surfaces. By 19 days in utero, the endothelial monolayers becomes organized and cell proliferation greatly diminishes. WGA, Con A and RCA now exhibit binding similar to that seen in the adult tissue. SBA binding is not detected at this time. Thus, changes in lectin binding during wound repair of the adult rat corneal endothelium mimic changes in lectin binding seen during the development of the tissue.Supported by grant EY-06435 from The National Institutes of Health     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Class II molecules on rat alveolar type II epithelial cells

Class II (Ia) molecules of the major histocompatibility complex are important in the presentation of antigen to T cells and in the regulation of the immune response. Recent studies have suggested that many epithelial cell types can express class II molecules. We examined rat alveolar type II epithelial cells, a cell which can synthesize and secrete pulmonary surface-active material, for the expression of class II antigens. Using an indirect immunofluorescent technique with a mouse anti-rat class II monoclonal antibody (OX-4), the majority of type II cells isolated from pathogen-free Sprague-Dawley rats expressed Ia antigens as determined by fluorescent microscopy and cell sorter analysis. In culture, the Ia expression was lost from type II cells. The addition of recombinant interferon-gamma to cultures of type II cells induced the expression of class II antigens. These findings suggest that class II antigen expression on type II cells may have relevance to immune responses occurring in the lung.     

Insulin binding to rat intestinal epithelial cells following partial small-bowel resection

Partial (60%) resection of rat small bowel was performed in order to obtain a model of intestinal mucosal hyperplasia for studying specific insulin binding. The affinity, but not the binding capacity, of insulin receptors in the adaptive mucosa decreased three and seven days following enterectomy. This modification took place only in crypt cells but not in mature villous cells. Since plasma insulin levels were not altered by the surgical manipulation, the observed decrease of insulin binding could not be related to regulation by insulin concentration. These results do not support a trophic role of insulin on intestinal mucosa and appear to be more a consequence of the hyperactive status of proliferation and differentiation at the mucosat level.     

Lectin binding sites inParamecium tetraurelia cells

Summary Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis.When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in 6–10 vacuoles, which are several m in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (other-wise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts).Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of tips), while ConA (fluorescence concentrated over bodies) and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable matrix.Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function.Abbreviations AED aminoethyldextran - BSE backscatter electrons - ConA Concanavalin A - DAB 3,3-diaminobenzidine - EM electron microscope - FITC fluorescein-isothiocyanate - kD kiloDalton - ME mercaptoethanol - MIP membrane-intercalate particle - M_r apparent molecular weight - PAGE polyacrylamide-gel-electrophoresis - PAS periodic acid Schiff - pI isoelectric point - POX peroxidase - RCA II Ricinus communis agglutinin II - SDS sodium dodecylsulphate - SEM scanning electron microscope - WGA wheat germ agglutinin     

Lectin binding in the diabetic rat kidney

Summary In this study metal-conjugated concanavalin A (Con A) andBandieraea simplicifolia isolectin II (BSA II) have been applied to sections from kidneys of controls rats and rats which had untreated diabetes for 70 days or for 200 days. Lectin binding was measured by atomic absorption spectrophotometric analysis of ferritin-iron or hemocyanin-copper.Con A binding increased significantly with diabetes; was totally blocked by alpha-D-mannoside; was not inhibited by fructose lysine; and was enhanced by NaHB₄ preincubation. BSA II binding also increased significantly with diabetes.