Alveolar epithelial transport in the adult lung

The alveolar surface comprises >99% of the internal surface area of the lungs. At birth, the fetal lung rapidly converts from a state of net fluid secretion, which is necessary for normal fetal lung development, to a state in which there is a minimal amount of alveolar liquid. The alveolar surface epithelium facing the air compartment is composed of TI and TII cells. The morphometric characteristics of both cell types are fairly constant over a range of mammalian species varying in body weight by a factor of approximately 50,000. From the conservation of size and shape across species, one may infer that both TI and TII cells also have important conserved functions. The regulation of alveolar ion and liquid transport has been extensively investigated using a variety of experimental models, including whole animal, isolated lung, isolated cell, and cultured cell model systems, each with their inherent strengths and weaknesses. The results obtained with different model systems and a variety of different species point to both interesting parallels and some surprising differences. Sometimes it has been difficult to reconcile results obtained with different model systems. In this section, the primary focus will be on aspects of alveolar ion and liquid transport under normal physiologic conditions, emphasizing newer data and describing evolving paradigms of lung ion and fluid transport. We will highlight some of the unanswered questions, outline the similarities and differences in results obtained with different model systems, and describe some of the complex and interweaving regulatory networks.     

Developmental regulation of glucose transporters GLUT3, GLUT4 and GLUT8 in the mouse cerebellar cortex

Glucose uptake into the mammalian nervous system is mediated by the family of facilitative glucose transporter proteins (GLUT). In this work we investigate how the expression of the main neuronal glucose transporters (GLUT3, GLUT4 and GLUT8) is modified during cerebellar cortex maturation. Our results reveal that the levels of the three transporters increase during the postnatal development of the cerebellum. GLUT3 localizes in the growing molecular layer and in the internal granule cell layer. However, the external granule cell layer, Purkinje cell cytoplasm and cytoplasm of the other cerebellar cells lack GLUT3 expression. GLUT4 and GLUT8 have partially overlapping patterns, which are detected in the cytoplasm and dendrites of Purkinje cells, and also in the internal granule cell layer where GLUT8 displays a more diffuse pattern. The differential localization of the transporters suggests that they play different roles in the cerebellum, although GLUT4 and GLUT8 could also perform some compensatory or redundant functions. In addition, the increase in the levels and the area expressing the three transporters suggests that these roles become more important as development advances. Interestingly, the external granule cells, which have been shown to express the monocarboxylate transporter MCT2, express none of the three main neuronal GLUTs. However, when these cells migrate inwardly to differentiate in the internal granule cells, they begin to produce GLUT3, GLUT4 and GLUT8, suggesting that the maturation of the cerebellar granule cells involves a switch in their metabolism in such a way that they start using glucose as they mature.     

GLUT11, but not GLUT8 or GLUT12, is expressed in human skeletal muscle in a fibre type-specific pattern

Nine novel sugar transporter-like proteins have been discovered in the past 5 years. The mRNA for three of these, the glucose transporters (GLUT) GLUT8, GLUT11 and GLUT12, have been detected in human skeletal muscle. In the present study, we examined the pattern of expression and localization of the GLUT isoforms 8, 11 and 12 in human skeletal muscle using an immunohistochemical approach. Biopsies of human skeletal muscle from sedentary or trained healthy adults, from fetal muscle (24 weeks of gestation), from obese type-2 diabetic subjects, and from patients suffering from polymyositis or amyotrophic lateral sclerosis (ALS) were studied. GLUT8 and 12 immunoreactivity was below detection level in both developing and adult muscle fibres. GLUT11 immunoreactivity, however, was present in slow-twitch muscle fibres, but not in fast twitch fibres. Since, in contrast, GLUT4 was expressed in all investigated muscle fibres, the pattern of expression of GLUT11 differs from that of GLUT4, suggesting a specialized function for GLUT11 with a regulation independent from that of GLUT4. Obesity, type-2 diabetes, training, conditions of de- and reinnervation (ALS) and regeneration (polymyositis) failed to induce GLUT8 or -12 expression. Likewise, the fibre type-dependent pattern of GLUT11 immunoreactivity was unaltered. However, some slow muscle fibres lose their GLUT11 immunoreactivity under regeneration. Our results indicate that GLUT11 immunoreactivity, in contrast to that of GLUT4, is expressed exclusively in slow-twitch muscle fibres and is unaffected by physiological and pathophysiological conditions except in primary myopathy. GLUT8 and GLUT12 do not appear to be of importance in human muscle under physiological and pathophysiological conditions.     

己糖激酶2通过抑制线粒体凋亡通路减少LPS引起的人肺上皮BEAS-2B细胞凋亡

目的:探讨脂多糖(LPS)诱导人正常肺上皮BEAS-2B细胞凋亡的分子机制,并对己糖激酶2(HK2)在该效应中的作用进行分析。方法:采用不同浓度的LPS作用于BEAS-2B细胞建立损伤模型,CCK-8实验检测细胞存活率; Hoechst 33342染色及Annexin V/PI双染法分析细胞凋亡水平;通过使用线粒体凋亡通路抑制剂或者外在凋亡通路抑制剂鉴定细胞凋亡通路;在BEAS-2B细胞中转染HK2过表达质粒以验证HK2对上述效应的影响。Western blot法确认HK2过表达效果;免疫荧光实验检测HK2亚细胞定位。结果:CCK-8实验结果显示,LPS以时间和剂量依赖性方式降低BEAS-2B细胞活力; Hoechst 33342染色结果表明,给予LPS处理后的BEAS-2B细胞核出现固缩和碎裂;同时Annexin V/PI双染实验结果表明,处于凋亡状态的细胞由2. 89%增加至42. 4%,细胞凋亡率明显升高(P 0. 05)。线粒体凋亡通路执行蛋白caspase-9特异性抑制剂可显著抑制细胞凋亡,而caspase-8抑制剂却无此效应。在BEAS-2B细胞的凋亡过程中伴随着HK2的表达下调,而HK2过表达可以有效阻止以上事件的发生。结论:己糖激酶2可通过抑制线粒体凋亡通路减少LPS引起的人肺上皮细胞凋亡。     

Apical and basolateral conductance in cultured A6 cells

Confluent monolayers of the cultured renal distal tubule cell line (A6) were impaled with microelectrodes under short-circuit conditions. Specific membrane conductances were calculated from equivalent circuit equations. Transport properties of the apical and basolateral membranes were investigated during control conditions and short-term increases in basolateral potassium concentration [K⁺] from 2.5 to 20 mmol/l, with or without 0.5 mmol/l Ba²⁺ at the basolateral side. As in most other epithelia, the apical membrane represents the major resistive barrier. Transcellular, apical and basolateral membrane conductances (g _c, g _o and g _i respectively), obtained from 22 acceptable microelectrode studies, averaged 61, 80 and 292 S/cm², respectively. There was a highly significant correlation between short-circuit current (I _sc) and g _o, whereas g _i was unrelated to I _sc. The I _sc, which averaged 4.1 A/cm², was almost completely blocked by amiloride. This was associated with fast hyperpolarization; the intracellular potential (V _sc) increased from –69 to –83 mV and the fractional apical resistance rose to nearly 100%. Using the values of V _sc during amiloride at normal and high [K⁺], an apparent transference number for K⁺ at the basolateral membrane of 0.72 can be calculated. This value corresponds with the decrease in g _i to about 25% of the control values after blocking the K⁺ channels with Ba²⁺. The nature of the remaining conductance is presently unclear. The cellular current decreased during high [K⁺] and Ba²⁺, in part resulting from reduction of the electrochemical gradient for apical Na⁺ uptake due to the depolarization. In addition, g _o decreased to less than 40%, which is considerably lower than predicted by the constant-field equation; this might indicate voltage sensitivity of the apical Na⁺ permeability.     

Corneal epithelial stem cells in health and disease

The cornea on the front surface of the eye provides our window to the world. Maintenance of corneal transparency is dependent on the integrity and functionality of the outermost corneal epithelium which itself is maintained throughout life by a population of limbal epithelial stem cells (LESC). If this adult stem cell population is depleted by injury or disease, the transparency of the cornea and therefore vision is threatened. LESC deficiency results in corneal opacification, inflammation, vascularization, and severe discomfort. Cultured LESC therapy is one of only several examples of the successful use of an adult stem cell therapy in patients. Hence, the ready accessibility of a transparent stem cell niche and the clinical precedence for use of stem cell therapy make the cornea a unique and excellent model for the study of adult stem cells in health and disease. This review will discuss our current understanding of LESC biology, pathology, and therapeutic application.     

新型隐球菌与肺泡上皮细胞的体外相互作用

目的研究新型隐球菌与肺泡上皮细胞的体外相互作用,探讨隐球菌肺部感染的发病机制。方法体外培养Ⅱ型肺泡上皮细胞A549(ATCC CCL-185),检测新型隐球菌2种变种对细胞的时间/浓度黏附率、通过率;检测新型隐球菌对细胞的损伤作用;透射电镜观察相互作用的超微结构。结果2种变种的新型隐球菌可以对A549细胞产生黏附与侵袭,黏附率与侵袭率呈现时间依赖性;同时还可以使A549细胞凋亡率升高,对其造成损伤,这与菌体的活力相关。超微结构可见隐球菌与肺泡上皮细胞的黏附与侵袭过程。2种变种之间在黏附率、通过率及对细胞的损伤作用方面差异无统计学意义。结论活的隐球菌黏附与侵袭肺泡上皮细胞是隐球菌感染肺部的重要条件,不同变种对肺部的易感性可能不存在差异。进一步明确二者的作用机制对隐球菌的发病机制研究具有重要意义。     

Glucose homeostasis across human airway epithelial cell monolayers: role of diffusion, transport and metabolism

Glucose in airway surface liquid (ASL) is maintained at low concentrations compared to blood glucose. Using radiolabelled [³H]-d-glucose and [¹⁴C]-l-glucose, detection of d- and l-glucose by high-performance liquid chromatography and metabolites by nuclear magnetic resonance, we found that glucose applied to the basolateral side of H441 human airway epithelial cell monolayers at a physiological concentration (5 mM) crossed to the apical side by paracellular diffusion. Transepithelial resistance of the monolayer was inversely correlated with paracellular diffusion. Appearance of glucose in the apical compartment was reduced by uptake of glucose into the cell by basolateral and apical phloretin-sensitive GLUT transporters. Glucose taken up into the cell was metabolised to lactate which was then released, at least in part, across the apical membrane. We suggest that glucose transport through GLUT transporters and its subsequent metabolism in lung epithelial cells help to maintain low glucose concentrations in human ASL which is important for protecting the lung against infection.     

人支气管上皮细胞恶性转化中凋亡敏感性及bcl-2的变化

目的: 揭示细胞凋亡敏感性在人癌细胞体外恶性转化动态过程中的变化。方法:用已获部分恶性的SV₄₀T转染的人支气管上皮细胞系M为材料, 通过凋亡TDT原位标记、染色体FISH、RNA及蛋白检测等技术,研究同一株细胞恶性转化过程中凋亡及bcl-2、P53基因的变化。结果:恶性转化的M后代细胞较未转化的M前代细胞对顺铂诱发细胞凋亡现象不敏感;bcl-2基因的转录和表达在M后代细胞明显高于M前代细胞,而且在M细胞恶性转化进程中呈积累现象;P53蛋白在M前、后代细胞均表达。结论:bcl-2过表达使细胞对凋亡敏感性下降在人支气管上皮细胞恶性转化中起重要作用;P53蛋白的灭活不是此SV₄₀T转染细胞恶性转化进程中bcl-2表达积累的主要原因。     

Glucose transporters GLUT4 and GLUT8 are upregulated after facial nerve axotomy in adult mice

Peripheral nerve axotomy in adult mice elicits a complex response that includes increased glucose uptake in regenerating nerve cells. This work analyses the expression of the neuronal glucose transporters GLUT3, GLUT4 and GLUT8 in the facial nucleus of adult mice during the first days after facial nerve axotomy. Our results show that whereas GLUT3 levels do not vary, GLUT4 and GLUT8 immunoreactivity increases in the cell body of the injured motoneurons after the lesion. A sharp increase in GLUT4 immunoreactivity was detected 3 days after the nerve injury and levels remained high on Day 8, but to a lesser extent. GLUT8 also increased the levels but later than GLUT4, as they only rose on Day 8 post‐lesion. These results indicate that glucose transport is activated in regenerating motoneurons and that GLUT4 plays a main role in this function. These results also suggest that metabolic defects involving impairment of glucose transporters may be principal components of the neurotoxic mechanisms leading to motoneuron death.     

Dendritic cells and airway epithelial cells at the interface between innate and adaptive immune responses

Because they can recognize and sample inhaled allergens, dendritic cells (DC) have been shown to be responsible for the initiation and maintenance of adaptive Th2 responses in asthma. It is increasingly clear that DC functions are strongly influenced by a crosstalk with neighboring cells like epithelial cells. Whereas the epithelium was initially considered only as a barrier, it is now seen as a central player in controlling the function of lung DCs through release of innate cytokines-promoting Th2 responses. Clinically relevant allergens, as well as known environmental and genetic risk factors for allergy and asthma, often interfere directly or indirectly with the innate immune functions of airway epithelial cells and DC. A better understanding of these interactions might lead to a better prevention and ultimately to new treatments for asthma.     

人偏肺病毒感染人肺上皮细胞后Toll样受体的表达及其功能

目的 观察人偏肺病毒(human metapneumoviras)感染人肺上皮细胞后Toll样受体(TLR)表达变化及其信号通路的功能,探讨hMPV诱导气道炎症的部分机制.方法 hMPV感染体外培养的人肺上皮细胞株A549,检测病毒在A549细胞中的生长曲线,并通过RT-PCR,real-timeRT-PCR方法检测细胞TLR mRNA的表达,ELISA检测细胞培养上清IFN-α及TNF-α的表达.结果 (1)hMPV可在A549细胞中复制,感染后3 d达高峰10~(5.2)TCID_(50)/ml.(2)RT-PCR结果提示:hMPV感染A549细胞6 h后大部分TLR的表达均上调.(3)定量PCR结果提示:hMPV感染A549细胞后TLR3-4、TLR7-9的表达增高,且有时间依赖性,而紫外灭活的hMPV刺激细胞后TLR表达无明显变化.(4)ELISA结果提示hMPV感染后24 h,IFN-α及TNF-α的表达均明显升高.结论 人偏肺病毒感染A549细胞后可上调TLR表达,其诱导的炎性反应与部分TLR介导的信号转导途径有关.     

Distinct transport characteristics of basolateral peptide transporters between MDCK and Caco-2 cells

The cellular polarity of [14C]glycylsarcosine (Gly-Sar) transport in Madin-Darby canine kidney (MDCK) cells was compared with that in the human intestinal cell line Caco-2. In MDCK cells, [14C]Gly-Sar accumulation was greater at the basolateral side than at the apical side, and there was little net transcellular transport. In Caco-2 cells, [14C]Gly-Sar accumulation was greater at the apical side and unidirectional transcellular transport occurred from the apical to basolateral side. Efflux of [14C]Gly-Sar from MDCK cells to either side was negligible, whereas that from Caco-2 cells was significantly faster to the basolateral side. The basolateral peptide transporter in MDCK cells possessed a similar substrate specificity, but a much higher substrate affinity, than that in Caco-2 cells. The basolateral peptide transporter in MDCK cells did not accumulate Gly-Sar during hypertonic stress. These findings indicate that the basolateral peptide transporter in MDCK cells is involved in the cellular uptake of small peptides, but not in the extrusion of small peptides to the extracellular space.     

Effect of bronchial mucus of bronchial asthma patients and chronic bronchitis on ciliary activity of ciliated epithelial cells

M. V. Lomonosov Moscow State University. Institute of Immunology, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR I. P. Ashmarin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 9, pp. 321–323, September, 1990.     

The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung epithelial cells

Cobalt is a toxic metal used in various industrial applications leading to adverse lung effects by inhalation. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt‐induced toxicity in human lung cells, especially normal lung epithelial cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in normal primary human lung epithelial cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration‐dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble and particulate cobalt induced similar cytotoxicity while soluble cobalt was more genotoxic than particulate cobalt. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung epithelial cells. Environ. Mol. Mutagen. 57:282–287, 2016. © 2016 Wiley Periodicals, Inc.     

Electron microscopic demonstration of Langerhans cells in human tonsillar epithelium

Langerhans cells are believed to play an important role in cutaneous immune responses. The tonsils likewise have been regarded as lymphoid tissue performing critical functions in the immune system. The present study was undertaken to determine whether Langerhans cells are present in tonsillar epithelium. Samples of epithelium from four tonsils were processed and examined with an electron microscope. Cells morphologically typical of Langerhans cells were found to be present in the tonsillar epithelium.     

Immunolocalization of CD1d in human intestinal epithelial cells and identification of a beta2-microglobulin-associated form

In order to better understand the role of intestinal CD1d, we sought to define the cellular localization and further characterize the biochemical structure of CD1d in human intestinal epithelial cells (IEC). Using a CD1d-specific rabbit anti-gst-CD1d antibody, immunoprecipitation of radiolabeled cell surface proteins detected a previously identified 37 kDa protein as well as a 48-50 kDa protein which were confirmed by Western blotting with a CD1d-specific mAb, D5. Immunoprecipitation of protein lysates with the CD1d-specific mAb, D5 and 51.1.3, and the beta2-microglobulin (beta2m)-specific mAb, BBM.1, followed by N-glycanase digestion and Western blotting with the D5 mAb showed that the 48-50 kDa protein was a beta2m-associated, CD1d glycoprotein. CD1d was immunolocalized to the apical and lateral regions of native small and large intestinal IEC as defined by confocal laser microscopy using the D5 mAb and the rabbit anti-gst-CD1d antibody. In addition, a large apical intracellular pool of CD1d was identified. Identical observations were made with polarized T84 cells. Selective biotin labeling of apical and basolateral cell surfaces followed by immunoprecipitation with the D5 mAb, N-glycanase digestion and avidin blotting confirmed the presence of glycosylated CD1d on both cell surfaces and immunolocalization of the 37 kDa non-glycosylated form of CD1d to the apical cell surface. These studies show that CD1d is located in an ideal position for luminal antigen sampling and presentation to subjacent intraepithelial lymphocytes.     

Relationship of mesenchymal changes to alveolar epithelial cell differentiation in fetal rat lung

Summary The influence of mesenchymal components on epithelial cell differentiation in fetal lung has mostly been studied in vitro. Here, the relationship of direct epithelial — mesenchymal cell interactions and of matrix changes beneath epithelial cells to the development of Type 2 and Type 1 epithelium is examined in vivo. In late gestation, as epithelial division slows, these cells come in close apposition to fibroblasts. In some places extracellular filaments connect these different cell types, often bridging the basal lamina. In other regions, direct cell-cell contact is made at membrane structures resembling gap junctions which connect fibroblasts to the cuboidal epithelium which develops characteristics of Type 2 cells. After day 20, as endothelial cell proliferation increases, epithelial cells lying directly over the endothelium do not contact fibroblasts. These cells lose lamellar bodies as they flatten out to become Type 1 cells lying on a fused basal lamina made by epithelium and endothelium. The results provide in vivo evidence that Type 2 cell morphology and function is influenced by direct contact with underlying fibroblasts and collagen fibrils. Differentiation to Type 1 epithelium appears to be modulated by capillary growth, either through loss of epithelial contact with the fibroblast and its products, or through an effect of an endothelial matrix component.This research project was supported by a grant from the Medical Research Council of Canada