呼吸道上皮中的短暂扩充细胞——clara细胞

clara细胞为一类无纤毛、无粘液,而有着丰富分泌颗粒的呼吸道上皮细胞。clara细胞的功能为分泌蛋白、表达细胞色素氧化酶、对外源物的生物转换作用,以及作为呼吸道中的短暂扩充细胞来修复受损的呼吸道上皮。随着对干细胞、肿瘤干细胞及所处壁龛的深入研究,其在呼吸道上皮中的更新、修复及肿瘤发生中的作用也愈来愈受到重视,并为肿瘤的治疗研究带来了前景。     

A two-receptor pathway for catabolism of Clara cell secretory protein in the kidney

Clara cell secretory protein (CCSP) is a transport protein for lipophilic substances in bronchio-alveolar fluid, plasma, and uterine secretion. It acts as a carrier for steroid hormones and polychlorinated biphenyl metabolites. Previously, the existence of receptors for uptake of CCSP.ligand complexes into the renal proximal tubules had been suggested. Using surface plasmon resonance analysis, we demonstrate that CCSP binds to cubilin, a peripheral membrane protein on the surface of proximal tubular cells. Binding to cubilin results in uptake and lysosomal degradation of CCSP in cultured cells. Surprisingly, internalization of CCSP is blocked not only by cubilin antagonists but also by antibodies directed against megalin, an endocytic receptor that does not bind CCSP but associates with cubilin. Consistent with a role of both receptors in renal uptake of CCSP in vivo, patients deficient for cubilin or mice lacking megalin exhibit a defect in tubular uptake of the protein and excrete CCSP into the urine. These findings identify a cellular pathway consisting of a CCSP-binding protein (cubilin) and an endocytic coreceptor (megalin) responsible for tissue-specific uptake of CCSP and associated ligands.     

Distribution of Clara cell secretory protein expression in the tracheobronchial airways of rhesus monkeys

Clara cell secretory protein (CCSP) is a protective lung protein that is believed to have antioxidant, immunomodulatory, and anticarcinogenic properties; to be present in all adult mammals; and to be well conserved in rodents, humans, and nonhuman primates. The rationale for this study is to define the distribution and abundance of CCSP in the airway epithelium and lavage fluid of the adult rhesus monkey and to provide information for evaluating CCSP as a marker of Clara cells and as a biomarker of lung health. Lung tissue and lavage fluid from 3-yr-old rhesus monkeys were examined using histopathology and immunohistochemistry. Proximal bronchi, midlevel bronchi, and terminal/respiratory bronchioles were compared for immunohistochemical localization of CCSP in three-dimensional whole mounts as well as in paraffin and Araldite sections. Immunoreactive CCSP was found in nonciliated cells throughout the airway epithelium. Proximal and midlevel airways had the highest labeling. CCSP decreased in distal airways, and respiratory bronchioles had little to no CCSP. CCSP in the most distal airways was in tall cuboidal cells adjacent to the pulmonary artery. Although a large number of cells were present in the terminal bronchioles that would be classified as Clara cells based on morphology (nonciliated cells with apical protrusions), only a small number stained positively for immunoreactive CCSP. Semiquantitative analysis of Western blots indicated that changes in lavage CCSP are consistent with, and may be predictive of, overall CCSP levels in the airway epithelium in this primate species that is phylogenetically similar to humans.     

Airway regeneration: the role of the Clara cell secretory protein and the cells that express it

Clara cell secretory protein (CCSP) is one of the most abundant proteins in the airway surface fluid, and has many putative functions. Recent advances in the field of stem cells and lung regeneration have identified potentially new roles of CCSP and CCSP-expressing cell populations in airway maintenance, repair and regeneration. This review focuses on the airway regenerative potential of CCSP and the cells that express this protein. The use of this protein or CCSP-expressing cells as an indication of biologic processes that contribute to lung injury or repair is highlighted.     

IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 stimulates inflammatory and remodeling responses and contributes to the pathogenesis of human airways disorders. To further understand the cellular and molecular events that mediate these responses, we characterized the effects of IL-13 on monocyte chemotactic proteins (MCPs) and compared the tissue effects of transgenic IL-13 in mice with wild-type (+/+) and null (-/-) CCR2 loci. Transgenic IL-13 was a potent stimulator of MCP-1, -2, -3, and -5. This stimulation was not specific for MCPs because macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, MIP-3alpha, thymus- and activation-regulated chemokine, thymus-expressed chemokine, eotaxin, eotaxin 2, macrophage-derived chemokines, and C10 were also induced. The ability of IL-13 to increase lung size, alveolar size, and lung compliance, to stimulate pulmonary inflammation, hyaluronic acid accumulation, and tissue fibrosis, and to cause respiratory failure and death were markedly decreased, whereas mucus metaplasia was not altered in CCR2(-/-) mice. CCR2 deficiency did not decrease the basal or IL-13-stimulated expression of target matrix metalloproteinases or cathepsins but did increase the levels of mRNA encoding alpha1-antitrypsin, tissue inhibitor of metalloproteinase-1, -2, and -4, and secretory leukocyte proteinase inhibitor. In addition, the levels of bioactive and total TGF-beta(1) were decreased in lavage fluids from IL-13 transgenic mice with -/- CCR2 loci. These studies demonstrate that IL-13 is a potent stimulator of MCPs and other CC chemokines and document the importance of MCP-CCR2 signaling in the pathogenesis of the IL-13-induced pulmonary phenotype.     

B cell induction of IL-13 expression in NK cells: role of CD244 and SLAM-associated protein

NK cells are an important component of the innate immune system that can also interact with B cells in a mutually productive manner. We have previously shown that activated B cells can induce NK cells to up-regulate their secretion of IFN-gamma. In this study, we show that B cells, and, particularly, marginal zone B cells, can, in addition, induce NK cells via direct cell-cell interactions to express mRNA encoding the Th2 cytokine IL-13. The induction of NK cell IL-13 mRNA expression requires the ligation of the CD244 receptor by the CD48 ligand on B cells via signaling pathways that depend upon expression of the X-linked lymphoproliferative disease gene product, SH2D1A/DSHP/SAP (SLAM-associated protein, or SAP) in NK cells. Thus, the positive signals attributed to the B cell activation of CD244 on murine NK cells appears to be more similar to the activity of CD244 on human cells. The induction of IL-13 mRNA by B cells may account for the effect of NK cells on the generation of Th2-type responses in the presence of some adjuvants.     

Ca signaling and fluid secretion by secretory cells of the airway epithelium

Cytoplasmic Ca²⁺ is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca²⁺ in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca²⁺. Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca²⁺ as a second messenger. Changes in intracellular Ca²⁺ concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca²⁺-activated K⁺ channels and Cl⁻ channels. We also review evidence of interactions of Ca²⁺ signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca²⁺ signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways.     

In vitro translation of rabbit lung Clara cell secretory protein mRNA

The major secretory product of Clara cells is a low molecular weight protein (CCSP) whose extracellular function, at this time, is not known. The primary translation product of its mRNA is a protein with molecular weight approximately 1 kD greater than that of the native secreted protein (6.0 kD). The primary translation product is not detected in incubated lung tissue, only the secretory protein is found. The primary translation product is trypsin sensitive whereas the secretory protein is not. Cell free translation of the mRNA in the presence of microsomes results in cleavage of the signal peptide and the appearance of the lower molecular weight trypsin-resistant secretory protein. These data indicate that the low molecular weight Clara cell secretory protein is synthesized as a larger, trypsin sensitive, protein. Passage of the protein into the cisternae of the endoplasmic reticulum results in loss of the signal peptide and alterations to the tertiary structure of the protein rendering it trypsin insensitive.     

The role of IL-13 in established allergic airway disease

The effectiveness of targeting IL-13 in models where airway hyperresponsiveness (AHR) and airway inflammation have already been established is not well-described. We investigated the effects of blocking IL-13 on the early and late phase airway responses and the development of AHR in previously sensitized and challenged mice. BALB/cByJ mice were sensitized (days 1 and 14) and challenged (days 28-30) with OVA. Six weeks later (day 72), previously sensitized/challenged mice were challenged with a single OVA aerosol and the early and late phase response and development of AHR were determined. Specific in vivo blockade of IL-13 was attained after i.p. injection of a soluble IL-13Ralpha2-IgG fusion protein (sIL-13Ralpha2Fc) on days 71-72 for the early and late responses and on days 71-73 for the development of AHR. sIL-13Ralpha2Fc administration inhibited the late, but not early, phase response and the OVA challenge-induced changes in lung resistance and dynamic compliance; as well, sIL-13Ralpha2Fc administration decreased bronchoalveolar lavage eosinophilia and mucus hypersecretion following the secondary challenge protocols. These results demonstrate that targeting IL-13 alone regulates airway responses when administrated to mice with established allergic airway disease. These data identify the importance of IL-13 in the development of allergen-induced altered airway responsiveness following airway challenge, even when administered before rechallenge of mice in which allergic disease had been previously established.     

Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium

Toll-like receptors (TLRs) are critical for the recognition of inhaled pathogens that deposit on the airway epithelial surface. The epithelial response to pathogens includes signaling cascades that activate the EGF receptor (EGFR). We hypothesized that TLRs communicate with EGFR via epithelial signaling to produce certain innate immune responses. Airway epithelium expresses the highest levels of TLR2, TLR3, TLR5, and TLR6, and here we found that ligands for these TLRs increased IL-8 and VEGF production in normal human bronchial epithelial cells. These effects were prevented by treatment with a selective inhibitor of EGFR phosphorylation (AG-1478), a metalloprotease (MP) inhibitor, a reactive oxygen species (ROS) scavenger, and an NADPH oxidase inhibitor. In an airway epithelial cell line (NCI-H292), TNF-alpha-converting enzyme (TACE) small interfering RNA (siRNA) was used to confirm that TACE is the MP involved in TLR ligand-induced IL-8 and VEGF production. We show that transforming growth factor (TGF)-alpha is the EGFR ligand in this signaling cascade by using TGF-alpha neutralizing antibody and by showing that epithelial production of TGF-alpha occurs in response to TLR ligands. Dual oxidase 1 (Duox1) siRNA was used to confirm that Duox1 is the NADPH oxidase involved in TLR ligand-induced IL-8 and VEGF production. We conclude that multiple TLR ligands induce airway epithelial cell production of IL-8 and VEGF via a Duox1--> ROS--> TACE--> TGF-alpha--> EGFR phosphorylation pathway. These results show for the first time that multiple TLRs in airway epithelial cells produce innate immune responses by activating EGFR via an epithelial cell signaling cascade.     

Modeling the airway epithelium in allergic asthma: Interleukin-13-induced effects in differentiated murine tracheal epithelial cells

Summary Mucous cells of the airway epithelium play a crucial role in the pathogenesis of human inflammatory airway diseases. Therefore, it is of importance to complement in vivo studies that use murine models of allergic asthma with in vitro mechanistic studies that use murine airway epithelial cells, including mucus-containing cells. In this study, we report the development and characterization of an in vitro culture system for primary murine tracheal epithelial (MTE) cells comprising ciliated cells and a substantial number of mucous cells. The increase in mucous cell number over that observed in the native murine airway, or in previously described murine cultures, creates a culture intermediate between the in vivo murine airway epithelium and in vitro cultures of human airway epithelial cells. To establish the usefulness of this culture system for the study of epithelial effects during inflammatory airway diseases, the cells were exposed to interleukin (IL)-13, a central inflammatory mediator in allergic asthma. The IL-13 induced two characteristic epithelial effects, proliferation and modulation of MUC5AC gene expression. There was a concentration dependence of these events, wherein high concentrations of IL-13 (10 ng/ml) induced proliferation, whereas lower concentrations (1 ng/ml) increased MUC5AC mRNA (where mRNA is messenger RNA). Interestingly, these effects occurred in an inverse manner, with the high concentration of IL-13 also provoking a significant decrease in MUC5AC gene expression. Thus, MTE cells cultured in this manner may provide an important link between experimental findings from animal models of allergic asthma and their application to human disease.     

Critical role for IL-13 in the development of allergen-induced airway hyperreactivity

Airway hyperresponsiveness to a variety of specific and nonspecific stimuli is a cardinal feature of asthma, which affects nearly 10% of the population in industrialized countries. Eosinophilic pulmonary inflammation, eosinophil-derived products, as well as Th2 cytokines IL-13, IL-4, and IL-5, have been associated with the development of airway hyperreactivity (AHR), but the specific immunological basis underlying the development of AHR remains controversial. Herein we show that mice with targeted deletion of IL-13 failed to develop allergen-induced AHR, despite the presence of vigorous Th2-biased, eosinophilic pulmonary inflammation. However, AHR was restored in IL-13(-/-) mice by the administration of recombinant IL-13. Moreover, adoptive transfer of OVA-specific Th2 cells generated from TCR-transgenic IL-13(-/-) mice failed to induce AHR in recipient SCID mice, although such IL-13(-/-) Th2 cells produced high levels of IL-4 and IL-5 and induced significant airway inflammation. These studies definitively demonstrate that IL-13 is necessary and sufficient for the induction of AHR and that eosinophilic airway inflammation in the absence of IL-13 is inadequate for the induction of AHR. Therefore, treatment of human asthma with antagonists of IL-13 may be very effective.     

Level of expression of IL-13R alpha 2 impacts receptor distribution and IL-13 signaling

IL-13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2. IL-4Ralpha and IL-13Ralpha1 form a heterodimeric signaling receptor for IL-13. In contrast, IL-13Ralpha2 binds IL-13 with high affinity but does not signal. IL-13Ralpha2 exists on the cell surface, intracellularly, and in soluble form, but no information is available regarding the relative distributions of IL-13Ralpha2 among these compartments, whether the compartments communicate, and how the relative expression levels impact IL-13 responses. Herein, we investigated the distribution of IL-13Ralpha2 in transfected and primary cells, and we evaluated how the total level of IL-13Ralpha2 expression impacted its distribution. Our results demonstrate that the distribution of IL-13Ralpha2 is independent of the overall level of expression. The majority of the IL-13Ralpha2 protein existed in intracellular pools. Surface IL-13Ralpha2 was continually released into the medium in a soluble form, yet surface expression remained constant supporting receptor trafficking to the cell surface. IL-13Ralpha2 inhibited IL-13 signaling proportionally to its level of expression, and this inhibition could be overcome with high concentrations of IL-13.     

IL-13 and IL-4 promote TARC release in human airway smooth muscle cells: role of IL-4 receptor genotype

The chemokine thymus- and activation-regulated chemokine (TARC) induces selective migration of Th2, but not Th1, lymphocytes and is upregulated in the airways of asthmatic patients. The purpose of this study was to determine whether human airway smooth muscle (HASM) cells produce TARC. Neither IL-4, IL-13, IL-1beta, IFN-gamma, nor TNF-alpha alone stimulated TARC release into the supernatant of cultured HASM cells. However, both IL-4 and IL-13 increased TARC protein and mRNA expression when administered in combination with TNF-alpha but not IL-1beta or IFN-gamma. Macrophage-derived chemokine was not expressed under any of these conditions. TARC release induced by TNF-alpha + IL-13 or TNF-alpha + IL-4 was inhibited by the beta-agonist isoproterenol and by other agents that activate protein kinase A, but not by dexamethasone. To determine whether polymorphisms of the IL-4Ralpha have an impact on the ability of IL-13 or IL-4 to induce TARC release, HASM cells from multiple donors were genotyped for the Ile50Val, Ser478Pro, and Gln551Arg polymorphisms of the IL-4Ralpha. Our data indicate that cells expressing the Val50/Pro478/Arg551 haplotype had significantly greater IL-13- or IL-4-induced TARC release than cells with other IL-4Ralpha genotypes. These data indicate that Th2 cytokines enhance TARC expression in HASM cells in an IL-4Ralpha genotype-dependent fashion and suggest that airway smooth muscle cells participate in a positive feedback loop that promotes the recruitment of Th2 cells into asthmatic airways.     

Expression of myoferlin in human airway epithelium and its role in cell adhesion and zonula occludens-1 expression

Background

Normal airway epithelial barrier function is maintained by cell-cell contacts which require the translocation of adhesion proteins at the cell surface, through membrane vesicle trafficking and fusion events. Myoferlin and dysferlin, members of the multiple-C2-domain Ferlin superfamily, have been implicated in membrane fusion processes through the induction of membrane curvature. The objectives of this study were to examine the expression of dysferlin and myoferlin within the human airway and determine the roles of these proteins in airway epithelial homeostasis.

Methods

The expression of dysferlin and myoferlin were evaluated in normal human airway sections by immunohistochemistry, and primary human airway epithelial cells and fibroblasts by immuno blot. Localization of dysferlin and myoferlin in epithelial cells were determined using confocal microscopy. Functional outcomes analyzed included cell adhesion, protein expression, and cell detachment following dysferlin and myoferlin siRNA knock-down, using the human bronchial epithelial cell line, 16HBE.

Results

Primary human airway epithelial cells express both dysferlin and myoferlin whereas fibroblasts isolated from bronchi and the parenchyma only express myoferlin. Expression of dysferlin and myoferlin was further localized within the Golgi, cell cytoplasm and plasma membrane of 16HBE cells using confocal micrscopy. Treatment of 16HBE cells with myoferlin siRNA, but not dysferlin siRNA, resulted in a rounded cell morphology and loss of cell adhesion. This cell shedding following myoferlin knockdown was associated with decreased expression of tight junction molecule, zonula occludens-1 (ZO-1) and increased number of cells positive for apoptotic markers Annexin V and propidium iodide. Cell shedding was not associated with release of the innate inflammatory cytokines IL-6 and IL-8.

Conclusions/Significance

This study demonstrates the heterogeneous expression of myoferlin within epithelial cells and fibroblasts of the respiratory airway. The effect of myoferlin on the expression of ZO-1 in airway epithelial cells indicates its role in membrane fusion events that regulate cell detachment and apoptosis within the airway epithelium.